Basics of Biotechnology: textbook 9786010442306

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AL-FARABI KAZAKH NATIONAL UNIVERSITY

B.K. Zayadan L.B. Dzhansugurova S.K. Turasheva

BASICS OF BIOTECHNOLOGY Textbook Stereotypical publication

Almaty “Qazaq University” 2020

UDC 60(075) LBC 30.16я73 Z 38 Recommended for publication by the decision of the Academic Council of the Faculty of Biology and Biotechnology, Editorial and Publishing Council of Al-Farabi Kazakh National University (protocol No.4 from April 16, 2019 y.); Educational and methodical association on groups of specialties «Natural sciences», «Humanities», «Social sciences, economics and business», «Engineering and technology» and «Arts» of Republican educational-methodical Council on the basis of Аl-Farabi Kazakh National University (protocol No.1 from March 2, 2019 y.) Peer reviewers: Dr., Professor K. Zhambakin Dr., Professor D. Jussupova Dr., Professor I. Savicskaya

Z 38

Zayadan B.K. Basics of Biotechnology: textbook / B.K. Zayadan, L.B. Dzhansugurova, S.K. Turasheva. – Ster. pub. – Almaty: Qazaq University, 2020. – 428 p. ISBN 978-601-04-4230-6 This book represents the application of biotechnology in life, shows examples, explains the methods of growing and spreading of living organisms, explains how to obtain and clean the necessary objects of biotechnology. The aim of this book is to familiarize students with the main principles and tendencies of biotechnology. Basics of Biotechnology is intended for the Bachelor students of 5В070100-Bio­tech­ nology specialty.

UDC 60(075) LBC 30.16я73 ISBN 978-601-04-4230-6

© Zayadan B.K., Dzhansugurova L.B., Turasheva S.K., 2020 ©Al-Farabi KazNU, 2020

INTRODUCTION Biotechnology (Greek “bios” – life, “techne” – art, “logos” – science) is new branch which helps to obtain important economically beneficial products, strains of microorganisms, new species of plants and animals, helps to increase the population through the artificial conditions. Biotechnology is the application of scientific techniques used to modify and improve plants, animals, and microorganisms to enhance their value. Biotechnology is the area of knowledge that allows us to obtain products useful to people by the controlled cultivation of organisms – food, forages, medications, various raw materials, forms of nitrogen, safety measures for plants and animals. Also, biotechnology provides methods that help to utilize various types of organic waste. The most economically developed countries such as China, USA, Japan, France, Germany, Holland, Great Britain and others widely use and apply biotechnological methods in science. The present stage of scientific and technical progress is characterized by the revolutionary changes in biology, which becomes the leader of natural sciences. Biology has reached the molecular and subcellular level, the methods of allied sciences (physics, chemistry, mathematics, cybernetics, etc.) and system approaches are intensively used in it. Rapid development of the complex of biological profile with the expansion of the practical sphere of their application is also conditioned by the social and economic needs of society. The problems of deficiency of food resources (especially proteins), environmental pollution, shortages of raw materials and energy resources, development of new diagnostic tools and treatment cannot be solved by traditional methods. Therefore, there was an acute need for the development and application of fundamentally new methods and Technologies. An important role in solving a complex of these problems is given to biotechnology, within the framework of which the targeted use of biological systems and processes in various spheres of human activity is carried out. The use of scientific achievements and practical advances in biotechnology is closely connected with fundamental research and is realized at the highest level of modern science.

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PART I MICROBIAL BIOTECHNOLOGY

6

Part I. Microbial biotechnology

CHAPTER

1

HISTORY OF MICROBIAL BIOTECHNOLOGY Biotechnology has a long history and its roots are traced back to the ancient times of human life, when biotechnology had not yet been accepted and developed as a science. Approximately 7000 years BC, the Sumerians and Babylonians used to convert sugar into alcohol. By 4000 years BC, the Egyptians used leaven-containing yeast to improve bread quality. Moreover, the ancients knew how to use bacteria and molds for vinegar and cheese production. The Chinese used molds as antibiotics for treatment of purulent wounds about 500 years BC. This first period, from several thousand years to 150 years ago, is known as the ancient or traditional industrial microbiological period, when cultures of microorganisms were used in nonsterile conditions to make products. Approximately 150 years ago, Louis Pasteur proved the microbial source of fer­ mentation and established industrial microbiology as a science based on the scientific prin­ciples. During World War I, Chaim Weizmann used Clostridium bacterium for pro­ duc­tion of acetone and butanol. Then Aspergillus mold was used to produce a citric acid. Using Alexander Fleming’s discovery of antibiotic properties of Penicillium mold, Florey and Chain were able to prepare a pure form of penicillin during the Second World War. In the 1940s, Waksman discovered some aminoglycoside antibiotics like streptomycin and neomycin. In the late 1950s and 1960s, microorganisms were used to produce amino acids and unicellular proteins. This second period, from 150 years to 40 years ago, is known as the classic industrial microbiological period, when pure cultures of microorganisms were used in sterile conditions for products manufacturing. Furthermore, the microbial strains were improved by classic genetical methods such as protoplasm fusion and mutagenesis with physical and chemical mutagens (Glazer and Nikaido 2007). The third period, which began in the 1970s and continues to the present time, is known as the modern industrial microbiology or microbial biotechnology. The prominent features of this period are the use of recombinant DNA or genetic engineering methods for the improvement of industrial strains and production of recombinant proteins. In some sources, the term “biotechnology” is incorrectly used instead of genetic engineering or modification. This mistake originated in the United States where several years ago new genetic methods were considered as awful and de6

Chapter 1. History of microbial biotechnology

7

monic procedures. Therefore, the term “biotechnology” was used instead of genetic engineering and production of transgenic organisms to reduce worry and diversion of public opinion. Later, the media and politicians used the term and, thus, it entered legislation and government documents. Humans have been using genetic modification in selective breeding of plants and animals for higher productivity over tens of thousands of years. For more than 50 years, classic methods such as protoplasm fusion and mutagenesis have been used for genetic modification of organisms. Genetic engineering and its equivalent terms, genetic modification or genetic manipulations advanced the molecular biology techniques that have been used since the 1970s (Figure 1).

Figure 1. Timeline of industrial microbiology and microbial biotechnology

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Part I. Microbial biotechnology

The term “biotechnology” was first used by Karl Ereky in 1917, when molecular genetics and genetic engineering had not yet been discovered. In 1919, Ereky described biotechnology as a technology based on converting raw materials into useful products, in his book entitled “Biotechnology of Meat, Fat and Milk Production in Agricultural Large-scale Farm”. Later, many definitions were proposed for biotechnology. Application of biological systems, living organisms, or derivatives for production or modification of products is the most comprehensive definition for biotechnology. Biotechnology is not only a science or a set of methods, it is also the interdisciplinary science that encompasses microbiology, plant and animal science, biochemistry, cellular and molecular biology, genetic modification, and engineering fields with biological perspectives such as mechanics, electronics, information technology, robotics, and others. The European Federation of Biotechnology considers biotechnology in two categories “traditional or old” and “new or modern” biotechnology. Several thousand years before, traditional methods were used to produce beverages, foods, and dairy products in traditional or old biotechnology, which is the equivalent of traditional industrial microbiology and classic industrial microbiology. New methods of genetic engineering, which were used from the 1970s to the early 1980s, began to develop and change the traditional biotechnology to new or modern biotechnology, which is the equivalent of modern industrial microbiology or microbial biotechnology. Today, the third wave of biotechnology, known as industrial biotechnology or white biotechnology is expanding. It has made considerable progress in comparison with the second wave, namely red biotechnology or medical biotechnology and the first wave, namely, green biotechnology or agricultural biotechnology. Industrial biotechnology uses biological systems, especially microorganisms, in industrial fermentation processes to produce large quantities of pure materials and energy including alcohols, organic acids, amino acids, vitamins, solvents, antibiotics, biopolymers, biopesticides, enzymes, alkaloids, steroids, and others. Industrial biotechnology is based on biological catalysts and fermentation technology, and it is associated with advances in molecular genetics, protein engineering, and metabolic engineering of microorganisms and cells. Recently, new methods of metabolic engineering, industrial systems biology, bioinformatics, X-omics such as genomics, metagenomics, transcriptomics, proteomics, metabolomics, fluxomics, and even nanobiotechnology, have been used to find and modify microorganisms with industrial capacity and their valuable products. Сontrol questions: 1. What is biotechnology? 2. The role of Louis Pasteur’s research in modern biotechnology 3. Alexander Fleming’s discovery 4. When was the term biotechnololgy first used? Who coined this term? 5. When was the genetic engineering science discovered?

Chapter 2. Objects of biotechnology and their application

CHAPTER

9

2

OBJECTS OF BIOTECHNOLOGY AND THEIR APPLICATION Biotechnological objects are placed at different stages of the organization: a) subcellular structures (viruses, plasmids, DNA of mitochondria and chloroplasts, nuclear DNA); b) bacteria and cyanobacteria; c) fungi; d) algae; e) protozoa; f) cultures of plant and animal cells; Bacteria fulfill various biotechnological functions. They are used in production of various substances: Acetum (Gluconobacter suboxidans), lactic drinks and products (Lactobacillus, Leuconostoc) as well as microbial insecticides (Bacillus thuringiensis) and herbicides, proteins (Methylomonas), vitamins (Clostridium – Riboflavinum); waste treatment, production of fertilizers, dissolvents and organic acids, biogas and photohydrogenium. Such property of some bacteria as ability to fix atmospheric nitrogen is widely used in the world. Biotechnological functions of fungi are various. They are used for making such products as: 1) Antibiotics (Penicillins, Streptomycetes, Cephalosporins); 2) Gibberellins and cytotoxins (Fusarium and Botrytis cinerea); 3) Carotenoids (for example, the astaxanthin giving a red-orange shade to the pulp of salmons is produced by Rhaffia rhodozima which are added to a forage on fish factories); 4) Protein (Candida, Saccharomyces lipolitica); 5) Cheeses like Roquefort cheese and Camembert (Penicills); 6) Soy sauce (Aspergillus oryzae). Algae are generally used for receiving protein. Very perspective cultures of unicellular algae with highly productive strains are Chlorella and Scenedesmus species. Their biomass, after processing, is used as addition in cattle diets and in 9

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Part I. Microbial biotechnology

the food industry. One of the most valuable products produced from red algae is the agar – the polysaccharide present in algae’s membrane consisting of agarose and agaropectin. Its quantity reaches 30-40% of the alga weight. Alga is the only source of agar, agaroid, carrageenan, alginates. Biomass of protozoa contains up to 50% of protein. It has high biological value because it contains all essential amino acids, and the content of free amino acids in it is 10 times more than in the biomass of microalga, bacteria and meat. It demonstrates good opportunities of application of the protozoa as a source of fodder protein. Euglena is one of the most perspective sources of fodder protein. Plants are producers of many BAS (biologically active substances) – the compounds capable to exert impact on biological processes in the organism. Cardiac glycosides, saponins, sterols, carotenoids, polyphenols, alkaloids, vitamins, quinones and substances having specific aroma, taste and color refer to such compounds. Biologically active substances belong to products of secondary metabolism that are called secondary metabolites or by-products of biosynthesis. More than 100 thousands of secondary metabolites are produced by plants. Many of them are used in the pharmacological, cosmetic, food industry and are considered as economically important products. 2.1 Basic nature of cells All living things are composed of cells; there are two basic types of cells: prokaryotic cells and eukaryotic cells. Figure 2 shows the main features of typical cells of the two types. The parts of the cell will be described briefly beginning from its outer part. Cell wall: Prokaryotic cell wall contains glycopeptides; they are absent in eukaryotic cells. Cell walls of eukaryotic cells contain chitin, cellulose and other sugar polymers. They provide rigidity where cell walls are present.

Prokaryotic cell Eukaryotic cell Figure 2. Prokaryotic cell and Eukaryotic cell

Chapter 2. Objects of biotechnology and their application

11

Cell membrane: cell membrane encloses the cell, it includes a double phospholipids layer, not passive barrier, which enables the cell to select the metabolites that want to excrete waste products or to accumulate necessary substances. Ribosomes participate in synthesis of proteins and consist of two sub-units. Prokaryotic ribosomes are 70S ribosomes that have two sub-units: 30S (small) and 50S (large) sub-units. Eukaryotic ribosomes are 80S ribosomes and have 40S (small) and 60S (large) sub-units. (The S unit is Svedberg unit; this is a measure of sedimentation rate of particles in the ultracentrifuge, the sedimentation rate is proportional to the size of particles. Svedberg units are not additional units – two sub-units can have Svedberg values that do not add to the entire ribosome). The prokaryotic 30S sub-unit is constructed from a 16S RNA molecule and 21 polypeptide chains, while the 50S sub-unit is constructed from two RNA molecules, 5S and 23S, respectively, and 34 polypeptide chains. Mitochondria are membrane-enclosed structures where in aerobic eukaryotic cells the processes of oxidative phosphorylation and respiration occur in the release of energy. Prokaryotic cells lack mitochondria and the processes of energy release take place in the cell membrane. Nuclear membrane surrounds the nucleus in eukaryotic cells, but is absent in prokaryotic cells. In prokaryotic cells, only one single circular macromolecule of DNA constitutes the hereditary apparatus or genome. Eukaryotic cells have DNA spread in several chromosomes. Nucleolus is a structure within the eukaryotic nucleus for the synthesis of ribosomal RNA. Ribosomal proteins are synthesized in the cytoplasm transported into the nucleolus and combine with the ribosomal RNA to form small and large subunits of eukaryotic ribosome. Then, they are exported into the cytoplasm where they combine to form the intact ribosome. 2.2 Classification of living organisms: three domains of living organisms Classification of living organisms has evolved over time. The earliest classification of living things subdivided them into two categories: plants and animals. When in the middle of 16th century the microscope was invented, it was firstly used for observation of microorganisms. Then, living things were divided into plants, animals and protista (microorganisms) visible only with the microscope. This classification subsisted from 1866 to the 1960s. From the 1960s and the 1970s Whittaker’s division of living things into five groups was the accepted grouping of the living things. The basis for the classification was the cell-type: prokaryotic or eukaryotic; the organizational level: single-celled or multi-cellu-

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Part I. Microbial biotechnology

lar, the nutritional type: heterotrophy and autotrophy. Based on these characteristics, Whitakker divided living things into five groups: protista (protozoa and algae), monera (bacteria), fungi, plants and animals. The current classification of living things is based on the Carl Woese work of the University of Illinois. Earlier classifications were largely based on morphological characteristics, cell type; with our deeper knowledge of molecular basis of cell function, today’s classification is based on the sequence of ribosomal RNA (rRNA) in the 16S of the small sub-unit (SSU) of the prokaryotic ribosome, and the 18S ribosomal unit of eukaryotes. It is used for the following reasons: 16S (or 18S) rRNA is necessary for ribosome, important organelle found in all living things (i.e. it is universally distributed); its function is identical in all ribosomes. The changes of sequence were very slowly with evolution, and it contains changeable and stable sequences that enable the comparison of closely and distantly related species. The classification tries to link all living things with the evolution from a common ancestor. For this approach, an evolutionary timekeeper is necessary. Such a timekeeper must be able to use the components of the system, and be able to turn over in one’s mind differences and changes in the other regions appropriate to the assigned evolutionary distances. The 16S ribosomal RNAs meet these criteria as ribosomes are involved in protein synthesis in all living things. They are also highly interesting stay the same in many groups and some observed minor changes commensurate with expected evolutionary distances (Figure 3).

Figure 3. Evolutionary relationship between organisms in time

Living organisms are placed into three groups according to the accepted classification: Bacteria, Archaea and Eucarya. Archaea and Bacteria are prokaryotic while Eucarya are eukaryotic. The revolutionary relationships between these groups of living organisms are given in the diagram shown below (Figure 4).

Chapter 2. Objects of biotechnology and their application

13

2.3 Microorganisms in industrial microbiology and biotechnology The microorganisms commonly used in the industrial microbiology and biotechnology are mainly found in the bacteria and Eukarya; the Archaea are not used. However, the processes used in industrial microbiology and biotechnology are dynamic. Consequently, outdated procedures were discarded as new and more efficient were discovered. At the present time, organisms from Archaea are not used for industrial processes, but the situation may change in future (Figure 4). This idea is not so farfetched, as it may seem now. As shown below, one of the criteria, which supports microorganisms used for industrial purposes, is a possession of properties that will enable the organism to survive and be productive against contaminants (Table 1). Many organisms in Archaea, which are able to grow in extreme conditions such as temperature and salinity, and these conditions can be exploited in the industrial processes where extreme physiological properties may put a member of the Archaea at an advantageous position over contaminants. Plants, animals, and their cell cultures are widely used in biotechnology.

Figure 4. The three domains of living things based on Woese’s work

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Part I. Microbial biotechnology

Microorganisms have more advantages in biotechnology than plants or animals: 1. Microorganisms grow rapidly in comparison with plants and animals. The reproduction time (the time for organism to mature and reproduce) is about 12 years for humans; 24 months for cattle about, 18 months for pigs, 6 months for chicken, and only 15 minutes for E coli bacteria. The consequence is that biotechnological products, which can be obtained from microorganisms in a few days, can be obtained from animals or plants in many months. 2. The space requirement for growth of microorganisms is small. A 100,000-liter fermenter can be housed in about 100 square of space yards, whereas plants or animals needed to generate the equivalent of products in the 100,000 fermenter would require many acres of land. 3. Microorganisms are not subject to the problems of vicissitudes of weather, which may affect agricultural production, especially plants. 4. Microorganisms are not affected by plant and animal diseases, although they have their peculiar scourges in the form of phages and contaminants. Table 1. Summary of differences between three domains of living things (from Madigan and Martimko, 2006) S/ Characteristic No 1 2 Morphology and Genetics 1 Prokaryotic cell structure 2 DNA present in closed circular form 3 Histone proteins present 4 Muramic acid in cell wall 5 Nuclear membrane 6 Membrane lipids: fatty acids or Branched hydrocarbons 7 Ribosome size 8 Initiator 9 10 11 12

Introns in most genes Operons Plasmids Ribosome sensitive to Diphtheria toxin 13 Sensitivity to streptomycin 14 Transcription factors Physiological/Special Structure 15 Nitrification

Bacteria

Archaea

Eukarya

3

4

5

+ + + Fatty acids

+ + + Branched hydrocarbons 70S Methionine

+ + Fatty acids

+ + +

+ Rare +

+ -

+

+

+

-

-

70S Formylmethionine + + -

80S Methionine

15

Chapter 2. Objects of biotechnology and their application 1 16 17 18 19 20 21 22 23 24

2 Methanogenesis Nitrogen fixation Denitrification Chlorophyll based (plants) photosynthesis Gas vesicles Chemolitotroph Storage granules of poly-Bhydroxyalkanates Growth above 80oC Growth above 100oC

3

4

5

+ + + +

+ + -

+

+ + +

+ + +

-

+ -

+ +

-

Despite these advantages there are occasions when it is better to use either plants or animals; however, microorganisms are preferred for the reasons given above. 1) Histone proteins are present in eukaryotic chromosomes; histones and DNA give structure to chromosomes in eukaryotes. 2) Non-coding sequences in the genes; 3) Operons: present in prokaryotes, clusters of genes which are controlled by a single operator; 4) Transcription factor is a protein, which binds DNA in a specific promoter or enhancer region, or site, where it regulates transcription process. Control questions: 1. Which microorganisms are widely used in microbiology and biotechnology? 2. Advantages of microorganism as an object of microbiology. 3. What are the differences between three domains of living organisms?

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Part I. Microbial biotechnology

CHAPTER

3

MICROBIAL BIOTECHNOLOGY 3.1 Microorganisms – objects of biotechnology, their demands The main object of biotechnological process is a cell where the end product is synthesized. The cell is a small chemical factory where thousands of difficult compounds are synthesized. The base of modern biotechnological industry is synthesis of different substances with the help of microorganisms’ cells. Most microorganisms are unicellular. Microbial cell is separated from the external environment by the cell wall, sometimes only by the cell membrane, and contains different subcellular structures. A lot of attention in biotechnology is paid to bacteria. Nowadays more than 100 thousand species of microorganisms are known. Bacteria reproduce very fast through binary division. There is no clear border between the nucleus and cytoplasm. There is no nucleus membrane. The DNA in these cells does not create a structure like an eukaryote chromosome. Most prokaryotes do not create intercellular organelles surrounded by membrane; there are no mitochondria and chloroplasts. Selection of necessary forms of microorganisms for cultivation with predetermined features includes: 1. Receiving of microorganisms. The samples are selected from the inhabit places of microorganisms (soil, plant residue, etc.). For hydrocarbon oxidative microorganisms the soil can be taken near gas stations; wine yeasts plentifully live on grapes, anaerobic cellulose debasing and methane forming microorganisms in large numbers live in a hem of ruminants. 2. Formation of accumulative cultures. Samples are placed in liquid nutrient medium with a special structure; favorable conditions for producer development (temperature, pH, power sources, carbon, nitrogen, etc.) are created. 3. Production of pure cultures. On the nutrient mediums, sow samples from accumulative cultures. Separate cells of microorganisms on the dense nutrient mediums create isolated colonies or clones, at their resowing obtain pure cultures consisting cells of one producer species. 16

Chapter 3. Microbial biotechnology

17

4. Determination of ability to synthesize a target product – the main criteria in selection of producers. Microorganisms have to: –– correspond the following requirements; –– have high growth rate; –– use cheap substrates for life; –– be steady against infection with foreign microflora. 3.2 Stages and kinetics of microbial growth Microorganisms, having got into fresh full-fledged nutrient medium, begin to reproduce not at once. This period is called a lag phase (1-st phase). During this period, the culture gets used to new conditions of dwelling. Fermentable systems are activated if it is necessary, new fermentable systems are synthesized, cells prepare to the synthesis of nucleic acids and other compounds. Log phase is a phase of the accelerated growth, it is characterized by the beginning of cell division, increasing of general mass population and constant increase in the growth rate of the culture: usually it is. During this period the maximum growth rate of the culture is noticed, the intervals between the appearance of the previous and following generation are constant. Owing to the intensive growth and reproduction of the culture, the reserved necessary nutrients in the medium decrease. It is the main reason for reduction of the growth rate of the culture. Metabolism products collect in the medium; also, a certain concentration of them can disturb normal biochemical processes of metabolism. Sometimes in the nutrient medium a large number of cells is formed, then they take a lot of place and surface, which badly influences the new cell generation. The growth rate decreases, the number of cell divisions decreases, there comes the phase of growth rate reduction. The third phase is called the stationary phase or the phase of linear growth. The weight and number of all living cells reaches a maximum. The number of newly formed cells at this stage is equal to the number of the cells that have died and were destroyed by cellular enzymes. The balance is broken and the number of died cells is higher again. There comes the VI phase – a death phase. The growth cycle and development of population in the closed volume is characterized by dying microorganisms. At this stage, biomass of the cells considerably decreases as spare substances of cells are exhausted. Factors necessary for cultivation of any culture: 1) Viable sowing material; 2) Sources of energy and carbon; 3) Nutritional substances for biomass synthesis;

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Part I. Microbial biotechnology

4) Absence of growth inhibitors; 5) Corresponding physical and chemical conditions (temperature, environment pH, existence or lack of oxygen, etc.). 3.3 Products of microbial fermentation and metabolism Products of microbial fermentation and metabolism include primary metabolites, secondary metabolites, enzymes and cellular biomass (so-called proteins of unicellular microorganisms). Primary metabolites are low-molecular compounds (molecular weight is less than 1500 Dalton), necessary for growth of microbes: some of them are construction blocks of macromolecules, others participate in synthesis of coenzymes. It is possible to distinguish most important metabolites for the industry: amino acids, organic acids, purine and pyrimidine nucleotides, vitamins, etc. Initial strains for industrial processes presented by natural organisms and cultures with violations of regulation of synthesis of these metabolites as simple microbial cells do not make surplus of primary metabolites. Secondary metabolites are the low-molecular compounds formed at later stages of cultural development, they are not required for the growth of microorganisms. By the chemical structure, secondary metabolites belong to various groups of compounds. They include antibiotics, alkaloids, hormones of plants growth, toxins and pigments. Raw materials and composition of nutrient mediums for biotechnological production. Nutrient medium provides activity, growth, development of bioobject, effective synthesis of a target product. An integral part of the nutrient medium is water, nutrients, which form true solutions (mineral salts, amino acids, carbon acids, alcohols, aldehydes, etc.), and colloidal solutions (proteins, lipids, inorganic compounds – iron hydroxide). Separate components can be in strong aggregate state, can emerge, evenly distributed throughout the volume in the form of a suspension or a benthonic layer. The raw materials used for receiving a target product have to be not scarce, inexpensive, whenever possible easily available: molasses – a by-product of production of sugar, components of oil and natural gas, waste of agriculture, the woodworking and paper industries, etc. Most often as components of nutrient mediums waste of food productions is used. The beet molasses – withdrawal of production of sugar from beet, is rich in organic and mineral substances necessary for the development of microorganisms. It contains 45-60% of sucrose, 0.25-2.0% of inert sugar, 0.2-3.0% raffinose. Besides, the molasses contains amino acids, organic acids and their salts, betaine, mineral substances and some vitamins. It is used for industrial production of lemon acid, ethanol and other products.

Chapter 3. Microbial biotechnology

19

Molasses bard – withdrawal of molasses- alcohol production. Chemical composition of bards depends on the structure of the initial molasses and fluctuates over a wide range. By the chemical composition, the molasses bard is the full-fledged raw material for production of fodder yeast, which does not demand additives of growth substances because, it contains enough vitamins. The content of solids in the natural bard is 8-12%, in the reduced bard – 53%. The bard’s grain-potato is withdrawal of spirit production. The content of soluble solids usually makes 2.5-3.0%, including 0.2-0.5% of the reducing substances, it is an available source of nitrogen and minerals. It is applied to production of microbial protein. Brewing waste (beer pellet and malt sprouts) and waste of barley by-product are suitable. However, it is a small source of assimilable carbohydrates for receiving microbial white. For the production of fodder yeast these raw materials are well hydrolyzed and entered into nutrient medium in the ratio of S: 0.2: 0.05 (pellet: sprouts: waste of barley). Wheat bran – withdrawal of flour-grinding production, is used for preparations of nutrient mediums at a solid-phase cultivation. They have a rich chemical composition and are used as the only component of the nutrient medium. As wheat bran is an expensive product, it is mixed with cheaper components: sawdust, malt sprouts, fruit residue, etc. Whey is a by-product of production of cheeses, cottage cheese and casein. In connection with it, to distinguish subcheese, cottage cheese and casein serum. According to the chemical composition and energy value this product considered as “semi-milk”. Whey is very rich in various biologically active compounds, its dry residue contains on average 70-80% of lactose, 7-15% of albumens, 2-8% of fat, 8-10% of mineral salts. Besides, whey incorporates significant amount of hormones, organic acids, vitamins and microelements. The presence of digestible by many types of microorganisms carbon sources in milk whey and various growth factors promote it into a range of more valuable nutritional media for obtaining of microbial synthesis products. For example, production of protein preparations in industrial scale. In addition, it is to note important that application of milk whey does not require special difficult preparation and cultural liquid after cultivation of microorganisms can be used in the food and fodder purposes without processing. Nutrient media may have uncertain content, i.e. it may including biogenous (vegetable, animal, microbial) additives like meat extract, cornmeal, seaweed, etc. Also synthetic media prepared from clean chemical compounds in known ratios could be applied. The content of any nutritional medium is: water, carbon compounds, nitrogen, phosphorus and other mineral substances, vitamins. The water must be clean, colorless, without smack, smell and precipitate.

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Part I. Microbial biotechnology

Sugars glucose, sucrose, lactose, polyatomic alcohols: glycerin, mannitol, etc. belong to easily available sources of sugar. Then, polysaccharides such as cellulose, hemicellulose, starch can be sources of carbon after transformation by microbial enzymes hydrolyzing these substances into easily available low molecular oligosacharides. Such microorganisms are molds Aspergillus, Penicillium, Bacillus bacteria and others. In practice we meet a large number of microorganisms that successfully utilize organic acids, especially in anaerobic conditions. Low-molecular alcohols: methanol and ethanol are perspective types of raw materials. Much yeast of the Candida, Hansenula, etc. is capable to assimilate ethanol. Yeast of the Pichia, Candida and other Flavobacterium species use methanol as the only source of carbon – methanol. Some species of microorganisms (insignificant part) use hydrocarbons as a source of carbon and energy: n-alkanes and some fractions of oil. Nitrogen can be in a form of inorganic salts and acids. The majority of yeast well acquires ammoniac salts and ammonia from water solution. Only several species of yeasts require nitrates in their nutrition. In addition, organic compounds can be a source of nitrogen: amino acids, urea etc. that are easily available for microorganisms. It known that bacteria are more selectable to nitrogen sources than other microorganisms (fungi, actinomycetes and yeast). Phosphorus is the most important component of the cell. It is a part of ATP (adenosine triphosphate), ADP, and AMP, there by phosphorus provides the normal course of cell energy metabolism, synthesis of proteins, nucleic acids and other processes of biosynthesis. Phosphorus entered the nutrient medium in the form of salts of phosphoric acid. The demand of microorganisms in these compounds is various; nevertheless, practically all microorganisms grow in the presence of vitamins. Corn extract thanks to availability of vitamins in it, amino acids and mineral elements in easily assimilable forms were effective addition in the nutrient mediums. Other products also include yeast autolysate, yeast extract, potatoes juice, whey, extract of malt sprouts and other products. Minerals enter into the composition of nutrient mediums in microdoses; otherwise, they have the inhibiting effect on the microbial cells. At drawing up nutrient medium for a concrete species of microorganism, the most suitable sources of carbon, nitrogen, phosphorus and other substances should be selected. 3.4 Asepsis, antiseptics and disinfection. Sterilization methods in biotechnology Asepsis – prevention of microorganisms from getting from the environment into a human body at medical and diagnostic manipulations and in research ma-

Chapter 3. Microbial biotechnology

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terial and the nutrient mediums in biotechnological productions. Sterilization of tools, materials, hands of personnel and observance of sanitary and hygienic rules in work is required (for example, sterilization tools, use of dressing gowns, gloves, etc.). Antiseptics – destruction of microorganisms, which are capable to cause infection of skin and mucous, covers. Chemicals-antiseptics are used for achieving this purpose. Examples of antimicrobial effect: 70% ethyl alcohol, spirit 5% iodine solution, chloramine solutions (0.5-2%), formalin (0,5-1%), potassium permanganate (0.1%), 1-2% solutions of methylene blue or diamond green and others (for example, local debride by iodine in surgical therapy). Disinfection – disinfecting the objects of the environment with the help of chemicals: chloric lime (0.1-10%), chloramine (0.5-5%), phenol or carbolic acid (3-5%), lysol (3-5%), etc. (for example, processing of objects: floors, walls, etc., including bactericidal lamps). Sterilization is one of the major and necessary method in biotechnological practice. Cultivation of organisms is carried out in sterile conditions. Elimination of living microorganisms and their forms through the sterilization or disinfecting (dispute) in nutrient mediums, ware, dry materials, tools and other pieces of the laboratory equipment. Sterilization methods in biotechnology are divided by the action into physical, mechanical and chemical. Physical methods of sterilization: calcination in a flame; sterilization by dry heat (hot air in a drying cabinet); sterilization by saturated steam under pressure (autoclave): fractional sterilization (tyndallization); sterilization by ultra-violet radiation. Chemical methods of sterilization: disinfection by antiseptics. Mechanical method of sterilization: filtration with membrane filters and Zeits’ filters. Physical and chemical properties of object which will be the subject of sterilization, define the opportunity and expediency of application of these methods. In biotechnology and microbiological practice thermal sterilization by roasting in a torch flame is widely applied. Small glass and metal objects (a needle, a loop, tweezers, a scalpel, sticks, and the pallet) will sterilize by calcination in a flame before use. Sterilization could be reached by carbonization of the microorganisms that are on their surfaces. Sterilization in the autoclave under pressure is the most reliable and universal method of sterilization of nutrient mediums and materials – their sterilization by saturated steam under pressure higher than atmospheric. Elevated pressure of steam is created in special tight thick-walled devices (autoclaves). Objects that are subject of sterilization in the autoclave have to be wrapped in paper. Full sterilization of many nutrient mediums is provided by heating within 15-20 minutes at 120 °C and the excessive pressure of 1 atm., mediums with

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carbohydrates – 15 minutes at 0.5 atm. Disinfecting of the infected material is carried out during 20-25 minutes at 1.5-2 atm. Sterilization by boiling. Sterilization of metal tools, subject glasses, rubber tubes and others is carried out by boiling. Spores of some bacteria keep viability when boiling. It is recommended to carry out boiling sterilization in 2% sodium carbonate solution (for softening of water and increase in temperature of boiling) for not less than 30 min. Sterilization by dry heat (Pasteur’s furnace). Dry heat are usually applied to sterilize glassware. To avoid infections, objects before sterilization are wrapped in brown paper and they are taken out from it only before work. The method provides bactericidal effect of heated air up to 165-180 °C, at a higher temperature the paper will be burn. Sterilization by fluid steam (autoclave or Koch’s device). Nutrient mediums with carbohydrates and vitamins, milk, malt, gelatin and other objects, which spoil from the influence of dry heat, are sterilized by the current steam. Sterilizations by the current steam are made in Koch’s boiler or in the autoclave with the open gate. Water in them is boiling, and the formed steam flows round objects. Temperature of nutrient mediums reaches 100 °C (80-90 °C). Heating for 2045 minutes kills vegetative cells of bacteria, but not their spores. Heating should be repeated next day. In these conditions vegetative cells well be inactivated. For ensuring full sterility of liquid, it is left for several days and heating is repeated again. Tyndallization. Fractional sterilization at 56-5S°C within 1 hour for 5-6 days in a row. It is applied to substances which are easily blasted – blood serum, solutions of vitamins, etc. Pasteurization. Heating of liquids at a temperature less than 100 °C is the cornerstone of pasteurization. The purpose of pasteurization is a destruction of bacteria in liquids that lack nutritional properties during boiling (milk, beer, wine, etc.). Pasteurization is carried out by heating of liquids at 50-65 °C within 15 – 30 minutes, or at 70-80 °C within 5-15 minutes with subsequent fast cooling. Tools made from thermo-labile plastic. For example, centrifugal test tubes will be sterilized by ultraviolet rays (length of waves of 260-300 microns in special boxes). Time of radiation is set experimentally. It depends on the power of the bactericidal lamp and the distance between the lamp and the object. Mechanical filtering. Organic liquids do not bear heating, to removing bacteria they are passed via sterile close-meshed filters. These filters detaining microorganisms are called bacterial filters. Asbestos and membrane filters with various diameter of pores are widely used. Porcelain (from quartz sand and a kaolin) and glass filters from kieselguhr (from diatomite or diatomaceous earth) are also applied. Filters have their own numbers and brands. Asbestos filters are made with a diameter 35 and 140 mm. It is necessary to distinguish filtering and sterilizing brands. Membrane filters (from nitrocellulose or cellulose acetate) have number

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1-5, respectively, sizes 350-1200 nanometers. Before using, membrane filters must be sterilized by boiling or in the autoclave. Filters are placed in the warm distilled water and boiled for 30 minutes, changing it 2-3 times. Chemical sterilization. Chemical disinfecting of substances are applied. Usu­­ally, this method does not provide a predictable result for producers, and final pro­ducts. It does not have broad application in biotechnological productions. 3.5 Organization of biotechnological production Clean zones. In the production of biotechnological medicines any of three polluting factors could play crucial role. They are foreign microorganisms, aerosol particles, chemical pollution. The basic principle ensuring purity at such enterprises is creation of “clean” rooms. The clean room is a room where concentration of aerosol particles is controlled and which is constructed and used to minimize entry, generation and accumulation of particles indoors. If necessary, other parameters such as temperature, humidity, pressure should be controlled. During designing of clean rooms for biotechnology, all requirements of the state standard specifications ISO 14644 have to be applied. Lighting, temperature conditions, humidity and air balance of a supply and exhaust ventilation have to correspond to the specifications of room, not to exert direct or indirect negative impact on products. In biotechnology, the systems of clean rooms include the rooms of various classes of purity intended for various stages of production. The class of purity of the room is the accurately regulated requirements for the level of different impurities and particles in the air. Classes of cleanness are divided according to the quantity of particles with a certain size of volume. This parameter is the one of the major in classification of clean rooms. So, class A is a local zone for operations which have a high risk to the quality of production (zone of filling); Class B is the zone which surrounds zone A and is destined for preparation of solutions; Classes C and D are zones for carrying less liable stages of production of sterile production. Planning of rooms must correspond the logical sequence of production operations and demands on purity, to minimize a possibility of mixing of various medicines or their components, and cross contamination. The most important indicator of technological level of execution of the clean room is a level of intellect of control system. The problem of ensuring purity indoors is most effectively solved based on the comprehensive approach considering peculiar features of each concrete room (space-planning characteristics, technological appointment, qualifying standards on purity and climatic parameters) and the features characterizing the room as an element of set of rooms. This situation finds reflection in creation of complexes of clean rooms stated above whose basic design principles are:

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Part I. Microbial biotechnology

1) zoning on the functional modules of rooms; 2) creation of a physical barrier between modules: 3) creation of a physical barrier between modules and building constructions of the building; 4) ensuring the required settlement of air exchange; 5) preparation of stitched air with the required parameters on humidity, temperature and microbiological purity; 6) the rational organization of overflows of air from cleaner modules to less clean; 7) distribution of air in modules with the organization of the set direction of its movement, features of rooms and technological process; 8) highly effective purification of internal air of modules. Design of a complex is defined by a concrete purpose of clean rooms, their configuration and sizes, the existing standard requirements to the air environment. In a general view, the delivered complexes are made by the modular principle and include the following functional systems and elements: –– System of preparation, disinfection and distribution of air; –– Control system of microclimate of rooms; –– Complex design (structural elements for sealing the rooms); –– Biotechnological equipment, which is built in a complex design. In microbiological productions, the personnel often fulfill tasks of carrying out sterile works with a certain product (a powder, a suspension, etc.). At the same time, it is necessary that this substance does not get into the surrounding space and does not make any harm to the operator and the environment. For this case, laminar boxes that help to organize sterile workplace for personnel to work with loose fine substances in pharmaceutical and microbiological productions are used. Clean rooms in the biotechnological industry lead to the strict hygienic and microbiological control. Nowadays microbiological monitoring of air of clean rooms is carried out, using the following main methods of sampling: passive – sedimentation and active – impaction and filtering. The method of sedimentation consists in determination of the microbial particles settling on the surface of Petri dish with an agar. It does not give a quantitative characteristic of air semination, and serves as addition to other methods of sampling. Particles with big size settle on the Petri dish, small particles stay in the air and do not settle on the surface. This method should be combined with any active method of sampling. The method of impaction is used in many samplings. It is necessary to consider that the high speed of collision of particles with the agar medium can lead to a damage of microorganisms on them, to loss of their vi-

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ability and distortion of microbiological impurity results. The filtration method is usually is followed (in comparison with impaction) by death of microorganisms during sedimentation on the surface of the filter. For cleaning the industrial rooms, we should have the concept of the potential source of contamination and the methods of its prevention: 1. Enterprise personnel. Enterprise workers must wear special clothes that fully close the body (brat, pants, and robe), head (cap/hat, mask on the face), feet (boots) and hands (gloves); 2. Air. The room is equipped with devices of filtration ensuring vertical movement of air streams. In addition, sensors of pressure control, microclimate parameters, etc., are installed; 3. Surfaces. Surfaces of walls and a floor are laid down using special chemically resistant material, which provides absence of roughness and seams. Cleaning of rooms also has the features worth talking about in details. There are two approaches to cleaning of sterile rooms: 1. Application of disposable expendables. Cleaning of rooms made with the use of materials not for long service life that are thrown out after cleaning; 2. Use of reusable microfiber materials. After cleaning rooms for ensuring their sterility, waste must be send to the laundry and processed by specialized organizations. Today in the European countries the process of cleaning of sterile rooms and materials has to conform to the standards ISO14644-5. The document indicates that usual materials are not suitable for cleaning of sterile rooms because they do not meet the following conditions: do not remove surface qualitatively, do not delete microbes, do not prevent further distribution of bacteria. Therefore, the main materials of which the stock is made is polypropylene, polyester to which a small amount of cellulose is sometimes added. All products will be sterilized and packed in tight packings. The main stock used in the cleaning of sterile rooms includes carts, the holders washing nozzles and various napkins of one-time use and multiple use. Cleaning of sterile rooms is carried out by a damp method of preliminary preparation because it collects dirt well and at the same time the surface remains almost dry. In sterile rooms, cleaning begins with the farthest corner and is finished with the exit. At first, wipe the ceiling and walls, then surfaces of furniture and the equipment, at the end – the floor. When cleaning of rooms is over, all materials are disinfected. Nozzles and napkins are washed and pass sterilization by the method of autoclaving or gamma irradiation. Each part is checked for quality of processing and readiness for the following cleaning of rooms. At the end, materials are carefully packed into tight packages and delivered to the venue of works. Thus, aseptic production of medicines demands absence of foreign microorganisms and particles in the rooms. Observance of these requirements is one of

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the guarantees of efficiency of the biotechnological production connected with microorganisms and their metabolites. 3.6 Methods of microorganism cultivation Fermentation (cultivation) is a set of consecutive operations from inoculation of sowing material into temperature-controlled nutrient medium (inoculate) till completion of growth and biosynthesis processes. The set of processes of cultivation of microorganisms is known. They are divided: –– by the content of oxygen – aerobic and anaerobic; –– by quantity of fermenters – one – two-and multistage; –– by existence or lack of mixing – dynamic and static; –– by condition of nutrient medium – superficial and deep. At superficial cultivation, the sowing material sowed on the nutrient medium surface is distributed by a thin layer (about 10 cm) in metal ditches. At deep cultivation, immersion of microorganism cells is carried out due to continuous hashing during all fermentation process. Deep cultivation is more favorable in the industry in comparison with a superficial cultivation because it allows us to carry out full mechanization and automation of the process, and to avoid infection of technological process with foreign microflora. At a periodic cultivation, sterile nutrient medium is sowed with the initial culture of a producer, and further in the same capacity microorganisms under certain conditions pass through all stages of growth and development of population. When the cultivation process comes to the end, the capacity for cultivation is released and the cycle renews, beginning from sowing of nutrient medium with the initial culture of the producer. In such way of cultivation (it is possible to call it a “close” system when one of components can’t enter it or be removed from it) the biomass growth rate always has to tend to zero either because of lack of nutrients, or because of accumulation of toxic metabolites in the medium. The cultivation is carried out on the nutrient medium surface in test tubes, flasks, bottles, etc. The culture grown in these conditions was heterogenic in physiological range because the cells in the different parts of the medium were in the different layers, conditions and developed differently. Sometimes this method of cultivation is applied to the biomass growing. Nowadays, in the industry liquid nutrient mediums are used, the application of which allowed us to avoid shortcomings of dense nutrient mediums and increased the process yield due to use of big capacities for cultivation (fermenters). The use of liquid nutrient mediums has demanded hashing of culture for the purpose of alignment of microbe growth conditions in different parts of working ves-

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sel and aeration (saturation by oxygen). For this purpose mixers, rocking chairs, large bottles with gas bubbling are used. 3.6.1 The aerated stirred batch fermenter A typical fermenter (Figure 5) is an upright closed cylindrical tank fitted with four or more baffles attached to the side of the wall, a water jacket that heats and/ or cools, a device for forcible aeration (sparger), a mechanical agitator usually carrying a pair or more impellers introducing organisms and nutrients and taking samples, and outlets for exhaust gases. Modern fermenters are highly automated and usually have continuous monitoring, control and record of pH, oxidationreduction potential, dissolved oxygen, effluent O2 and CO2, and chemical components of the fermentation broth (or fermentation beer as the broth is called before it is extracted). Nevertheless, the fermenter may not have all these gadgets and many automated activities can be prosecuted manually. It is important to that required fermentation is to be clearly understood when a fermenter is planned; a fermenter is expensive and once installed, it is expensive to remodify it.

Figure 5. Structure of a Typical Fermenter (Stirred Tank Batch Bioreactor)

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Part I. Microbial biotechnology

Batch cultivation of microorganisms is used for production of sowing material at some stages, for microbiological production of amino acids, in production of vaccines etc. Continuous cultivation, as well as periodic, provides one-time loading and unloading of the fermenter. However, the cycle of microorganisms development in the prolonged periodic process is extended either due to feeding (periodic or continuous addition of nutrient medium), or due to long deduction of cells in the system (dialysis culture). In this case, the exponential phase and a phase of linear growth are longer. The essence of dialysis consists in development of the culture in the space limited by a semipermeable membrane, products of metabolism are diffused into external solution. The simplest dialysis method is cultivation in cellophane bags, shipped in nutrient medium. Multicyclic process of cultivation is a cycle of culture cultivation repeated several times without repeated sterilization of the capacity. Multicyclic cultivation can be different. It can be carried out in one fermenter, repeated in full stroke of cultural development without interruption in sterilization. The methods, realized in one fermenter, are called single-stage. Multistage is multicyclic processes based on the principle of repeated and serial periodic cultivation proceeding in several fermenters connected in the battery on purpose, which make possible a long use of culture. One of options of such a way consists in the following: the culture is grown up in one bioreactor and in the time, when it passes an exponential phase, an inoculate (sowing material) for sowing in the next reactor is taken from it. In the first reactor, the culture is grown to a necessary growth phase. When the culture in the second reactor reaches an exponential phase, its sowing material is also taken to the third reactor, etc. As the culture is oversown in the exponential phase all the time, there is no aging and degeneration. Besides, there is a gain in time because several fermenters work at the same time. Multicyclic processes of cultivation of microorganisms are applied to receiving a biomass and production of microbial synthesis products – antibiotics, extracellular enzymes, and amino acids. The use of this method allows us to reduce several times work expenses on production of the product in comparison with a periodic way. In semi-continuous systems, the full load and unloading of the fermenter are carried out once. Thus, the system functions drain-additive. Various options of semi-continuous systems are used in production of yeast, alga, antibiotics and citric acid. In continuous cultivation, microorganisms constantly receive inflow of fresh sterile nutrient medium, and biomass together with the formed metabolites is continuously selected from the device (this cultivation is called “open” system). At continuous cultivation, microorganisms should not lack a nutritious substratum because the speed of his inflow is balanced with a biomass exit speed. Besides, the culture is not poisoned with metabolism products; it is a big advantage

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of the continuous way of cultivation in comparison with periodic, the advantage of an “open” system in comparison with a “closed” one. Continuous fermentation can take place in the homogeneous system of ideal mixture, the system of full replacement and the system of solid-liquid type. Homogeneous systems of ideal mixture. In the system of ideal mixture, microorganisms grow in the cultural medium; therefore, a constant structure, in each timepoint is in the same physiological state, which is in the condition of the established dynamic balance. By the quantity of fermenters, homogeneous systems can be single-stage, two-phasic and multistage. For obtaining high concentrations of biomass, single-stage systems with return of cells in which the cells are separated from cultural liquid by means of the pump, return in fermenter, are used. Return of cells (recirculation) is important in those processes in which during stay in a fermenter cells do not manage to realize the potential opportunities concerning synthesis of a target product. Multistage systems consist of a row of sequentially connected fermenters – batteries. The use of multistage systems allows us to get a culture in case of any growth rate – from a log phase to exponential and stationary. Multistage cultivation is applied in the lactic acid, and ethyl alcohol production. The main device for cultivation of a continuous homogeneous system is the fermenter of ideal mixture with the device for a medium stream and discharge of culture supporting the constant level of the nutrient medium. This process called continuous-flowing, provides identical concentration of all products in the fermenter and in the flowing liquid. Continuous-flowing cultivation gives a chance to support constant conditions of microorganism growth due to limitation (restriction) of some factors of the medium. In case the factor limiting growth is chemical composition of the nutrient medium, the process is called chemostate cultivation. In the chemostate (a fermenter where chemostate cultivation proceeds), the speed of dilution of the nutrient medium is constant according to the set population density. During change of dilution speed, it is possible to receive the modes providing various growth rates. The other principle of process management is turbidostate. In it, the nutrient medium is carried out at the command of the photoelectric element registering the optical density of the culture in the fermenter. The speed of dilution is established automatically according to the set population density. Though, theoretically, the interrelation between the concentration of biomass and dilution speed follows the same regularities in the chemostatе and the turbidostatе, methods of management of processes are different. 3.6.2 Systems of cultivation with full replacement The main difference between the open system of full replacement and the system of ideal mixture is that that culture in it does not mix up, and represents a liquid stream

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Part I. Microbial biotechnology

through a pipe. The most widespread device for cultivation in this case is the tubular fermenter. It can have various forms (direct, S-shaped, spiral) and be mounted horizontally or vertically. The system of full replacement represents a spatial, flowing option of the periodic culture. Such culture goes through all stages of batch culture during time from inoculation till unloading. Phases of growth are distributed not in time, but in space, and each part of the fermenter in the set mode corresponds to a certain piece in the curve of growth. This cultivation is used for anaerobic processes. Inoculation is carried out continuously at the entrance of the fermenter along with medium supply. Systems of solid-liquid type. To the multiphase systems belongs systems wich culture grows on the border of different phases to the solid-liquid systems type: liquid – a solid phase – gas. In these systems, cells keep by sticking to the solid basis – filler and breed, forming a biomass film. A typical example is production of vinegar in the devices. In this system the limiting factor for aerobic microbes is oxygen and substrate (nutrients). In thin films, each cell attached to the surface is completely provided with these substances and is capable to grow and breed with the maximum exponential speed. The cells form a thicker film of biomass, their growth is limited (the top layers lacks oxygen, the lower – nutrients). Cultivation of microorganisms forming a film of biomass is carried out in a fermenter like a column with a filler. As the filler the macrocarrier (coke, rods, shaving, glass balls, etc.) and the microcarrier can be used (pitches, parts sefadex, etc.). The cells thus cultivated are called immobilized. The use of immobilized cells has several advantages. Firstly, the possibility of long operation of cells in case continuous fermen­ tation appears. Secondly, examples of increase in the resistance of cells under the influen­ce of various adverse external factors (temperatures, acidities, concentration of to­xic substances and others) because of immobilization. Thirdly, procedures of obtaining used cells and the cultural liquid containing a significant amount of the target product become simpler. Fourthly, thanks to application of immobilization energy consumption in the process decreases: due to reduction (reduction in cost) in the sizes of applied fermenters; and due to simplifications in procedures of allocation and cleaning of the final product. In industrial microbiology the systems of solid-liquid type have found application in sewage treatment, in production of organic solvents and acids, etc. 3.7 Obtaining of microbial synthesis products Production of a target product of microbiological biosynthesis is the closing stage of the biotechnological process. At the same time, the products of biosyn-

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thesis can collect in the cells of producers, allocated in cultural liquid. Stages of obtaining a target product of microbial biosynthesis are given below. At the first cleaning stage of the target product, first, it is necessary to separate biomass of microorganisms from the cultural liquid. Because the maintenance of microorganisms in the liquid medium is very low (1 liter contains 5-10 g of dry biomass), for this purpose, various methods are used. Flotation means capture of microorganisms’ biomass by bubbles of foam and its obtaining from foamy fraction. This method can be used if cells of producers collect in the top layers of the fermenter because of low wettability. At the same time, liquid is previously made foam, and then the top blanket containing vials of gas with the cells stuck to them are separated. Flotation is used at the first stage for clarification of cultural liquid. Filtration is transmission of suspension through the filtering material on which biomass of microorganisms lingers. Use filters of single and repeated use: drum, disk, tenial, dish-shaped, rotary, vacuum filters, filter presses various designs, membranous filters. This method can be used during antibiotic production, especially when the producer has a filamentous character. Centrifugation is sedimentation of the particles suspended in liquid with the use of centrifugal force. This method is used for yeast or bacteria in production of fodder biomass. It demands more expensive equipment, than filtering. Therefore, it`s application is reasonable when: a) Suspension pass filtration slowly; b) It is necessary to achieve maximum separation of biomass from cultural liquid; c) Filters are suitable only on periodic action and it is necessary to adjust continuous process of separation. Coagulation is addition of the reagents promoting creation in suspension and sedimentation of larger cellular agglomerates with their subsequent separation from liquid by upholding. The second stage of production of the products accumulated in cells is destruction of cells is selves cages. The process of destruction of a cellular membrane could be carried out by physical methods (unwinding, action of ultrasound, sharp freezing and thawing, excessive pressure and other) and chemical enzymatic (biotechnological) methods, which include various manipulations. Hydrolysis is destruction of cellular membrane under the influence of chemical reagents and temperature. Enzymatic method is destruction of cellular membrane under the influence of enzymes at increased temperature. Autolysis is a kind of enzymatic method when cell’s own enzymes are used for cell destruction. A lack of destruction processes, especially physical, is nonselectiveness for disintegrative influence.

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Part I. Microbial biotechnology

Extraction is transition of a target product from a water form in organic liquid, immiscible with water. Mainly fatty like substances extended by liquid hydrocarbons (like gasoline) but also many druty types of extragent are applied (chloroform, air, butyl acetate). Extraction directly from solid phase (including biomass of microorganisms) is called extraction. A special case is cold extraction (cryoextraction) when allocation of products from the frozen samples is supposed, in this case solvents with a low boiling temperature, which at the room temperature turn into gaseous state, are used. Cryoextraction is often used in a combination with cryopreservation of cells. Sedimentation is obtaining of a target product by addition to liquid of the reagent interacting with the dissolved product and transferring it to a solid phase. Sedimentation is carried out by physical (heating, cooling, dilution or concoction of solution) or chemical methods. Penicillinum is transferred to a crystalline precipitate in the presence of potassium or sodium compounds. Proteins precipitate by addition of ammonium sulfate, organic dissolvents (ethanol, acetone). Adsorption is transfer of a dissolved product in the liquid phase by its deposition (sorption) on special solid carriers (sorbents). A good adsorbent is charcoal, clays with the developed porous surface. Antibiotics and vitamins are obtained by adsorption. Ionic exchange is the same as adsorption but in this case ions (cations or anions) pass ed into a solid phase but not a whole molecule of a target product. Rectification is the methods used for receiving easily boiling products dissolved in cultural liquid. An example is ethyl alcohol. Ultrafiltration, nanofiltration and the return osmosis are applied to receive high-molecular compounds (proteins, polypeptides, polynucleotides). The return osmosis and nanofiltration allow us to separate small molecules. Ultracentrifugation is used for receiving viruses, cellular organelles, and high-molecular compounds. 3.8 Purification of microbial synthesis products Purification of products of microbial biosynthesis is carried out when production with a large amount of impurity is received. Chromatography is the process reminding adsorption. The dissolved substances with a similar structure collect on a solid sorbent: for example, mixtures of proteins, nucleotides, sugars, antibiotics. At adsorption, they separate from sorbent together, in chromatography they leave a sorbent in turn, which allows us to separate them and to clean from each other. Dialysis means the process where low-molecular substances can pass through a semipermeable septum, but high-molecular remain. This method is used to purify vaccines and enzymes from salts and other solutions.

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Crystallization is the process based on various solubility of substances at different temperatures. Slow cooling allows forming crystals of solutions of target products and their purity is usually very high. Thus, for example, it is possible to receive crystals of penicillin. It is possible to receive cleaner product if to dissolve crystals in water or solvent, and then again to crystallize (i.e. to carry out recrystallization). Concoction, dehydration, modification and stabilization of microbiological synthesis products. Concoction of a product is carried out by the methods of return osmosis, ultrafiltration, and evaporation. If the membrane passes water, detaining the substances dissolved in it, it is about osmosis. Direct osmosis is diffusion of a substance through the membrane dividing solution and solvent. Solution is placed in a bag from semipermeable material; the bag is immersed in a flask with solvent. Similar process represents the reverse osmosis used as a method of concoction of a product. Ultrafiltration – separation of substances by membranous filters. Some brands of filters are intended for units of coarse particles: immunoglobulins, colloid units, viruses. Membranes with small pores detain molecules of organic acids. The ultrafiltration proceeds with a high pressure (20-400 kPa) attached to liquid. It is applied for concoction of such low-stable products, as milk and glutamic acids, some antibiotics and enzymes. The most ancient method is evaporation. Its disadvantage is the heating at low pressure. Vacuum and evaporating devices are used. The heating agent, most often water vapor, is warmed with the liquid heat carrier or an electrical heating. Steam is characterized by big warmth of condensation and facilitates regulation of the evaporation process. Product dehydration – drying on trays, on the tape conveyor with heating, giving of the gaseous heating agent (air, CO2, combustion or furnace gases, etc.) in the drying device, in a vacuum – drying cabinets, in drum and the spray dryers. When drying in the boiling layer, the gaseous heating agent with a powerful stream rushes into the drying device from below and particles of the dehydrated product soar in the gas stream. Materials especially sensitive to heating, are dried in vacuum-drying cabinets with the lowered pressure and temperature. Drum dryers for dehydration of microbial suspensions consist of the rotating warmed-up drums are shipped in a flask with suspension. Adjoining a drum, suspension is dehydrated and dries in the form of a film to its walls. The dried biomass is scraped off from walls with a knife. Modification of a product – reorganization of the received compounds of animal, vegetable or microbial origin for the purpose of giving specific properties to them, necessary for people. Chemical modification is necessary when biotechnological process gives only “preparation” to a target product. For example, penicillin G formed from

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Part I. Microbial biotechnology

Penicillium notatum were modified for receiving ampicillin, methicillin and other semi-synthetic penicillin. Necessary substituents attach to the synthesized structure in the chemical way. An important point is to give optical asymmetry to the product. An object (for example, yeast) selectively consumes one of isomers (for example. L-amino acids) its optical antipode is left in the medium. The similar technology known since Louis Pasteur. Modification – necessary stage in receiving a number of enzymes, hormones, medicines of medical appointment. For example, in bull insulin “remove” the amino-acid residues then it becomes identical to human hormone. The consumer (addition of fillers, modification, etc) directs stabilization of a product to maintaining properties of a product during its storage and use. Drying increases resistance of a product to external influences. Dehydration of enzymes causes their resistance to heating. Stabilization of products, including fodder microbial white is made by addition of fillers from a fungi mycelium, wheat bran, cornmeal that have nutritional value. Stabilization of enzymes is reached by addition of glycerin, carbohydrates or inorganic ions – SO2. Mg2; Na + for a pectinase, formalin of 0.2% of solution for glucoamylase, antibiotics – a lysine for glucoamylase. Sometimes stabilization of a product represents a problem of special biotechnological process. The melange received from egg whites – valuable foodstuff, in several months of storage – darkens, it organoleptic properties worsen. Damage of melange can be prevented if carbohydrates are removed from it. For this purpose, it is recommended to grow up the propionic bacteria “eating” carbohydrates on melange. It leads to prolongation of storage of melange, raises nutritional value since propionic bacteria enrich it with organic acids and B12 vitamin. 3.9 Modern methods of substances separation Modern methods of separation of substances include chromatography, electrophoresis, isotachophoresis, electrofocusing based on the principles of extraction and adsorption. The separation of substances by chromatography is associated with their unequal distribution between two immiscible phases. Distinguish chromatography on paper, plates, columns. Chromatography on paper or on plates of one of the immiscible phases is a moving solvent, for example, butanol, and the other, a stationary phase, of a paper fiber or a particle of a plate-covering silica gel or other material. In column chromatography, the mobile phase is a solvent flowing through the column, a fixed adsorbent column, most often a granular gel. Column chromatography allows for scaling – increasing the size and throughput. It is used in industrial conditions and has several varieties.

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Chapter 3. Microbial biotechnology

1) Ion exchange chromatography – adsorbent pellets, for example carboxymethylcellulose, carry charged groups, cationic (-NH3) or anionic (-SO3), capable of capturing ions of the opposite sign. It is used to separate ionized compounds from a liquid, as well as to purify neutral compounds from impurities of ionic nature. One of the first ion-exchange columns used on an industrial scale was a column with a natural ion exchanger zeolite to soften the water, i.e., removing Ca2+ and Mg2+ from it. Ion exchange columns with amberlite are widely used for the separation of vitamin B12. 2) Chromatography is based on the separation of substances with different molecular weight and diameter (the method of “molecular sieves”, gel chromatography, gel filtration). Adsorbent particles capture and retain only low-molecular compounds, passing high-molecular. 3) Сhromatography аffinity is a component that forms a strong complex with a ligand fixed on carrier particles. Agents specifically binding one individual substance are used. For example, the enzyme is purified by binding on a column bearing a substrate or a specific inhibitor. The interaction of antigens and antibodies plays an important role in the separation and purification of protein and non-protein substances. Based on these interactions, chromatography was called immuneaffinity chromatography. New horizons opened before immune affinity chromatography is the use of monoclonal antibodies, allowing a 500-fold purification of human interferon. In affinity chromatography, group ligands used linking, for example, a whole group of enzymes similar in structure. These ligands include cofactors of enzymes and their analogues. In this case, the separation and purification of individual substances is based on their different affinity to the ligand, which allows them to be collected separately as separate fractions. Even less specific ligands can be used that bind whole classes of substances, for example, alkyl and arnyl groups or ligands, which are textile dyes (Table 2).



Ligands widely used in various variants of affinity chromatography (according to J.S. Jansen, 1984) The ligands Detachable product Enzyme Specific binding of individual substances Enzyme (Apoenzyme) Enzyme Lectin Substrate Glycoproteid Co-factor Receptor (coenzyme) Antigen Inhibitor Carbohydrate Lectin Hormone Antibody

Table 2

The ligands Hapten

Detachable product Antibody

Polynucleotide

Complementary nucleotidesequence

Group Alkyl or arylmoieties Functional groups of organic dyes Activated SH-groups

Various proteins Various proteins Proteins with SH-groups

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Affinity chromatography can provide complete purification of the product from a complex multicomponent mixture – the culture liquid, the extract of the cells – in a single step, while the more traditional methods of precipitation and ion exchange chromatography require multiple-stage purification, which involves a lot of labor and time. A certain inconvenience is caused by the relatively high cost of materials for affinity chromatography, in particular, substances used as ligands. The problem is also the rapid failure of the column when mixtures pass through it, the components of which block the gaps between the gel particles. Therefore, in production conditions, the columns are used in a batch, rather than continuous, mode. After passing a portion of the culture liquid from the column, from which the product is isolated, the gel particles are subjected to purification. Purifying methods are based on: а) the use of gel particles that exceed the density of the conglomerates of substances clogging the column: a difference in density makes it possible to purify the gel by selectively precipitating it or by flushing it to remove only the contaminants; b) giving the particles of the gel magnetic properties, which allows them to clean the gradient magnetic field; c) packing of gel particles in the form of a ribbon coated with a fine-mesh shell: the tape rotates and passes alternately through the liquid with the crude product and through a buffer solution into which contaminants pass. Scaling of the affinity chromatography process is limited by the destruction of the gel structure and the entrainment of its particles by the fluid current. This, in particular, is due to the fact that, in wide columns for large-scale cleaning of products, the walls of the column no longer serve as a support for particles of the gel dragged by the liquid. Increasing the height of the column leads to a proportional increase in the forces that destroy the lower layers of the gel. In recent years, they have been seeking means to strengthen the gels for large-scale affinity chromatography. Particles of agarose – the most promising material for gels – are supposed to be strengthened by cross-links. Along with affinity chromatography also called affinity adsorption in a gel, affinity precipitation and affinity separation are contemplated for large-scale separation and purification of products of biotechnological processes. In affinity precipitation, the ligand is attached to a soluble carrier. When a mixture containing the corresponding protein is added, its complex is formed with a ligand, which precipitates immediately after its formation or after the addition of the solution with electrolyte. Affinity separation is based on the use of a system containing two watersoluble polymers. One of the polymers, for example polyethylene glycol, carries specific ligands. Another polymer, for example high molecular weight dextran, has an affinity for the remaining impurity components. Thus, proteins, nucleic acids, fragments of cellular structures contained in the separated mixture prefer

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more polar dextran, while the target product, say, the enzyme, accumulates “in the networks” of polyethylene glycol carrying the molecules of the ligand (substrate, cofactor, inhibitor). Affine separation is the most highly effective of affinity purification methods. With the use of polyethylene glycol-6000 with the dye Cibacron blue F3G-A as a ligand, a successful purification of phosphofructokinase isolated from 1 kg of yeast was carried out. The volume of the system of two polymers was only 250 ml, and the purification lasted less than 1 h. Along with chromatography, promising for biotechnology methods of separation of substances are electrophoresis and its modifications, for example, isoelectric focusing.The separated mixture is placed in a powerful electric field, which drives its ionized components. The difference in electrophoretic mobility makes it possible to separate the components of the mixture. In modern versions of electrophoresis, as in chromatography, plates or columns with filler (agarose, polyacrylamide, sepharose, oxiapatite) forming a gel are used. In some cases, a gradient gel is used in which the density of its constituent particles increases or decreases linearly or exponentially in the direction of the electric field. The use of gradient gels makes it possible to synchronize the motion of ions, to reduce the spread between their velocities, so that ions form a compact front when moving in the gel, rather than a diffuse wide band. Widely used is a two-dimensional electrophoresis, in which two electric fields simultaneously perpendicular to each other act simultaneously on the mixture to be separated. In this variant of electrophoresis, non-linearly arranged bands are obtained, and spots are distributed along the gel plane corresponding to the separated components of the mixture. The problem arising when scaling the electrophoretic methods of separation and purification is the heating of the gel under the influence of the electric field. For gel layers 2 mm thick or thicker, cooling becomes a complicated procedure: not only thermal inactivation of the sample, for example, denaturation of the protein, but also the appearance of temperature differences in the thickness of the gel, which distort the picture of the distribution of components, is an electrophoregram. At present, electrophoresis systems with thermostatic gel plates are being developed. To more evenly distribute the bands corresponding to the components being separated, electrophoresis was developed in an inhomogeneous electric field. The field strength decreases in the direction of ion motion. This is achieved by the use of gels of wedge shape. Another way is to direct inside the gel of an electric field superimposed on the external applied to the gel. The gel is impregnated with a mixture of pH buffers giving a non-uniform distribution of charged particles over the volume of the gel (buffer gradient). Modification of the electrophoresis method is isoelectric focusing, electrofocusing. The solution to which the gel is drowned contains a compound

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with acid-base groups. The gel is placed between the two electrodes, and under the influence of the electric field, the acid-base groups of the buffer compound change the degree of ionization. A pH gradient is formed in the direction of the electric field. A sample of the mixture to be separated is applied to the gel, and its electrically charged components migrate towards the electrode of the opposite sign. Since the charged components move along the pH gradient, they gradually lose their charge and in the zone where the pH corresponds to the point of electro neutrality of the components, their movement ceases. Each component is concentrated in a certain area of the gel. In modern systems, electrofocusing in gels with immobilized acid-base groups is often used. Such a gel is designated immobilin. With the help of elec­tro­focusing in immobilin gel, the microheterogeneity of monoclonal antibodies is shown, that is, the presence of slightly different components in electrophoretic mobility. An important modification of electrophoresis is immunoelectrophoresis. In one variant of the method, called rocket immunoelectrophoresis, the antibody is added to the gel, and the material containing the antigen is added to the well at the edge of the gel plate. When the electric field is induced in the gel, the antigen is electrophoretically moved along the gel, and its propagation zone is delineated by a clearly visible contour line-the zone of formation of the antigen-antibody complex. The contour line resembles a missile in shape, hence the name rocket immunoelectrophoresis. A complicated variant of immunoelectrophoresis can be presented by cross, or two-dimensional immunoelectrophoresis, which allows us to separate multicomponent mixtures. The sample is introduced into the well at the edge of the gel plate and electrically propagated through the entire length of the gel, until the most mobile component approaches the opposite edge of the gel. A longitudinal track is obtained on the gel, which is cut out and transferred to another gel plate, and this path is oriented transversely to the direction of the electric field. The second gel contains antibodies to one or more components of the mixture, and after electrophoresis, “rockets” are formed on it, each of which corresponds to a certain antigen-component of the mixture. The other modifications of electrophoresis, we note an isotachophoresis based on the separation of ionic components of a mixture with different electrophoretic mobility. Two electrolytes are used, for example tris-C1 and tris-glycine. Anions C1- have a greater electrophoretic mobility than glycine anions. Accordingly, Tris-Cl is called the leading one, and Tris-glycine is the terminating electrolyte. Anionic components that can be separated in such an electrolyte system must have an electrophoretic mobility intermediate between the electrophoretic mobility of the anions Cl- and glycine. The mixture to be separated is introduced into the gel at the interface between the leading and terminating electrolyte. When an electric field of high intensity is applied, the

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anionic components of the mixture form clearly delineated zones in the gel. The speed of their movement coincides with the speed of movement of ions of the leading and terminating electrolyte, so the method was called isotachophoresis – movement (fores) with the same (iso) speed (tacho). Of the methods of separation and purification of substances considered, only a few, apart from ion exchange and affinity chromatography, have been introduced into large-scale production. Other methods are limited to laboratory use. However, among biotechnological products many such ones are produced so far in scales that have not exceeded the level of laboratory bioreactors. In such designs, thin preparative separation methods are used. Note developments on the preparation of E. coli polynucleotide phosphorylase (S.N. Zagrebelny and others, 1985), DNA and RNA ligases (Z.A. Baronaite et al., 1985), cellulase and glycoamylases (M.V. Gernet, 1985). Some products (drugs, enzymes for analytical purposes) are inexpedient to scale, since the demand can be satisfied with production in bioreactors of laboratory scale. Subtle preparative methods have prospects for their scaling by increasing the dimensions of the corresponding installations. There is also improvement of the tested methods of purification of substances. Thus, simple heating can provide a significant degree of purification of enzymes. Yu. I. Venyuzhins-kene, et al. (1985) achieved a 9-fold purification of a-aminoe-caprolactamhydrolase by selecting the optimum conditions for heat treatment of the Cryptococcus sp. homogenate cell: heating to 68°C at pH 6.3 during 15 minutes. The medium contained a pyridoxalphosphate, which protected the target enzyme from thermal denaturation. 3.10 Methods of microbial culture storage Preservation of microogranisms is based on slowing down of cells me­ta­ bolism by changing of environmental conditions. Consequently, during the cooling process, the viability of the cells quickly slows down, аnd when it freezes and dehydrated the cells fall into anabiosis. However, this cell retains its ability to survive, and when it is normal, it rebuilds its essential properties. The following storage methods are widely used. Store in the refrigerator.This is the simplest way to store culture in agar vial in the refrigerator for 1-2 months at a temperature of +4 ° -4 ° C. To preserve maintain the culture for a long time, it is necessary to re-cultivate it to maintain cells physiological and biochemical properties. Repeated cultivation should not be prolonged, as it uses medium nutrients during storage of microorganisms and the metabolism of products begins to accumulate, and such a property is harmful. In addition, dehydration of the environment in the process of preserving

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microorganisms leads not only to changes in the properties of the culture, but also to its destruction and loss. Storage of culture under a layer of mineral oil. The culture can be stored for several months in a solid sterilized medium under petroleum jelly or paraffin. The thickness of the fat layer on the surface of the agar-agar should be at least 1 cm to prevent moderate drying. If the fat layer is thick, the oxygen deficiency causes the death of the aerobic culture. After the physiological state of the culture is completely cured, it is poured into disinfected oil. Storage in sliding and crumbly material. This method is widely used due to its simplicity and reliability. Here, the suspensions or spores of microorganisms are impregnated with a non-fermented sprinkled material (sand, soil, seeds), dried at room temperature and stored in glass containers tightly closed with a cotton stopper. The microorganisms in the fibrous loose material should be washed in a Petri dish for cleaning and re-sown in an agar nutrient medium. Depending on the type of microorganism, the culture can be stored for several months. Some culture can be stored in frozen ice at a temperature of 165-196 °C (liquid nitrogen temperature).The culture is frozen as ice in an aqueous solution of 10% glycerol and welded into ampoules. In this case, some microorganisms can maintain all their physiological and biochemical properties for many years. Sublimation drying.Currently, sublimation drying is the best way to maintain microorganisms for a long time. This method is complex and proceeds in several stages. The protective environment (sucrose, broth, etc.) is first added to the culture of microorganisms, due to the interaction of cells and water, the state of lyophilization decreases, prevents cell inactivation, and then cells are placed in sterilized ampoules and sealed with a dampened cotton stopper. And then quickly frozen at temperature of -35-78 °C. Ampoules with ice culture are replaced by vacuum dryers, at room temperature and at a pressure of 1-10 kPa, for sublimation drying for 25-30 hours. After sublimation drying for 5-6 years, the culture can be maintained without loss of physiological properties. This method is also called culture preservation in conditions of lyophilization. In factories it is often used to store the culture under a refrigerator or Vaseline fat layer. Practical use of microorganisms is comprehensive. Microorganisms in medicine, light and heavy industry, agriculture, etc. are able to synthesize valuable biologically active substances. Microorganisms, products of biologically active substances, are the main area of microbiological technology.In fact, the producer of a microorganism is determined by many important indicators, the main ones are as follows: 1) Harmlessness (in consumption and production); 2) Biosynthesis activity (the rate of growth, the rate of accumulation of biomass, the rate of synthesis of biologically active substances); 3) Application of a carbon source (raw material application, etc.);

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4) Application of a nitrogen source; 5) Sensitivity to the cultivation condition (aeration, temperature, pH, growth factors, etc.); 6) Stability and phagage strength. The activity of the product, that is, the formation of a large number of me­ta­ bolic products, is a special characteristic of microorganism producers. Control questions: 1. Which products of microbial fermentation and metabolism do you know? 2. What are the primary and secondary metabolites? Give examples. 3. Which materials and products are used in the preparation of culture medium? 4. What is asepsis, antiseptics, disinfection? 5. The main principles in creation of clean zones 6. What does ISO standard mean? 7. What is a fermenter? Types of fermenters 8. What are multicyclic processes of cultivation? 9. What is continuous cultivation, periodic and continuous-flowing cultivation? 10. Which methods of product purifying do you know? 11. What qualities should be taken into account in the fermentation process to obtain the final product wich good quality? 12. Stages of final product concentration

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Part I. Microbial biotechnology

CHAPTER

4

SELECTION OF MICROORGANISMS – PRODUCERS OF IMPORTANT SUBSTANCES Industrially important waste products of microorganisms by their nature and value divide into three main groups: 1) large molecules (enzymes, polysaccharides with molecular mass from 10 thousands to several millions); 2) primary metabolites (bonds, necessary microorganisms for body growth: amino acids, purine and pyrimidine nucleotides, vitamins, etc.); 3) secondary metabolites (the compounds which is not necessary for microorganism development: antibiotics, toxins, alkaloids). Primary and secondary metabolites of a microbial parentage usually have molecular mass, quite low in comparison with enzymes – less than 1.5 thousands.These substances show the biological activity variously: satisfy the needs of the people and animals, interact with microorganisms, insects, and plants, and participate in decomposing of various organic substrates. Besides, some amino acids can serve as raw materials for further transformations based on chemical synthesis. Products of microbial synthesis become objects of profitable industrial production, obtained with a microbial cell in a medium and collected in a medium in quantities that would justify a raw and metabolic cost of microorganism cultivation and obtaining of a product in a form, necessary for further use. In most cases, the choice of a microbiological way of substance production is caused by the total absence or a very limited possibility of receiving it in other ways, firstly, by chemical synthesis. Nowadays it is much simpler to obtain many antibiotics, enzymes, biologically active isomers of amino acids, purine nucleotides, toxins from available and cheap raw materials, than to carry out difficult, multi-stage chemical synthesis, or even one-two stages of enzymatic synthesis, but on the basis of complex and often inaccessible raw materials. However, natural strains of microorganisms, as a rule, have no ability to form and accumulate in a medium, i.e. to produce many necessary products that would provide their low cost and the output demanded to the national economy or medicine. Natural strains of some groups of microorganisms (fungi, actinomycetes, bacilli) are capable to produce antibiotics, toxins or hydrolytic enzymes 42

Chapter 4. Selection of microorganisms – producers of important ...

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in a small amount. Primary metabolites, as a rule, cannot be obtained from microorganisms in a great amount (the synthesizable amount of these substances is strictly limited by properties of the cell). The exception of this rule – release of glutamic acid natural strains (the so-called group of glutamate-producing Corynebacterium) doesn’t extend to the vast majority of other amino acids. In this regard the main objective of the selector – creation of a “anew” producer from the strain of the wild type capable to synthesize the substance (for example, amino acid) not only intensifying the natural ability of a microorganism but also to confer a new property to produce a certain substance (an antibiotic, enzyme, toxin, etc.) but not capable to produce it. These tasks are fulfilled by receiving natural strains of hereditary changes – the mutations leading to intensification of the natural ability of microorganisms to synthesize and produce a certain substance and also emergence of a new ability – to synthesize a substance in the more amount than cells needs. Further increasing production of this or that substance of microorganism is a constant purpose of selectors as the most effective way of intensification of microbiological production, which does not demand additional capital investments, consists in the use of a more productive strain. Synthesis of primary or secondary metabolites by microorganisms can be imagined as the process beginning with absorption of a substratum by the cell (sources of carbon and nitrogen, minerals, etc.) followed by a number of stages catalyzed by various enzymes, which participate in regulation of synthesis of the necessary substance or its predecessors. At separate stages, the intermediate substances can serve as predecessors of other metabolites and spend for their synthesis. Predecessors of a certain substance can be intermediate or final products of other ways of synthesis, having their own regulation and spend for other cell requirements. Besides, the produced substance breaks the barrier of permeability and collects in the nutrient medium, without degradation under the influence of enzymes which the microbial cell can synthesize. Theoretically, the mutations promoting supersynthesis of the product can involve a large number of the structural genes coding enzymes at all stages of synthesis, transport and catabolism of this product and regulatory genes. The result of such mutations can be shown in various changes of cell metabolism: 1) increasing of absorption speed and utilization of a substratum by the cell; 2) increasing of synthesis of biosynthetic enzymes or their activity; due to violation of negative control of synthesis and activity of regulatory enzymes in the synthesis of the product or its predecessors; 3) blocking of collateral reactions of synthesis, for decreasing the expense of general predecessors on synthesis of other metabolites; 4) blocking of further intracellular transformation of the product if it occurs; 5) providing an effective excretion of the product from the cell;

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Part I. Microbial biotechnology

6) blocking of product degradation; 7) strengthening of positive forms of regulation of the product synthesis. If a desirable product is the enzyme (mostly hydrolytic enzymes, although now much attention is paid to oxireductases participating in the amino acid catabolism) then, the mutations which promote creation and activation, collecting in the nutrient medium can affect: 1) a structural gene, leading to the synthesis of the mutant enzyme which is not sensitive to inhibition of the final product of response and (or) increasing its activity (speed, i.e. the number of moles of the turned substratum in a minute); the mutation in the pro-motor part of a gene must intensify the frequency of transcription of initialization or cause a constitutive synthesis of enzyme; 2) genes, the encoding squirrels participating in regulation of synthesis of this enzyme (in particular, as the catabolite repression having the various forms of manifestation and in a general view expressing in inverse relation of synthesis of catabolite-sensitive enzyme from the growth rate of cells), mutations in these genes remove or weaken the factors restricting enzyme synthesis; 3) the genes encoding enzymes which can hydrolyze and inactivate the necessary enzyme mutations reduce or remove such opportunity; 4) the genes responsible for synthesis of diaphragms cellular components which participate in “assembly” (at eukaryotes) and an excretion of enzymes, a mutation in these genes can increase efficiency of the specified processes. That list of theoretically possible, “participating” in supersynthesis mutations, obviously, isn’t full as our data on regulation of biosynthesis of this or that metabolite and its interrelations with other processes in the cell aren’t exhaustive. Not any of the listed mutations can cause supersynthesis. The last is necessary for manifestation or the greatest expression of the first. At the same time, also the lack of considerable level of production is even possible in case the majority of mutations theoretically necessary for this purpose is obtained at a microorganism and, on the contrary, the selector can be saved from the need to obtain a set of different mutations if he has successfully chosen the initial natural type of a microorganism. 4.1 The choice of initial microorganism for selection

A variety of natural forms allows us to choose a microorganism, which has a smaller number of restrictions for supersynthesis of some substance even if they doesn’t produce it. It was very difficult to obtain an industrially significant level of production of L-lysine from colibacillus or pseudo-monads, but very easy – representatives have a glutamate producing Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum, etc. Proceeding from the available data it is possible to explain it by less difficult regulation of lysine synthesis at

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Corynebacterium (in the synthesis of a lysine only one enzyme is controlled as polyvalent inhibition of activity by a lysine and threonine, and this control is eliminated with the mutation blocking synthesis of homoserine – the predecessor of threonine and methionine, at the same time the general predecessors goes only for synthesis of a lysine), lack of degradation of a lysine in comparison with regulation at colibacillus (three controlled enzymes and more irregular shapes of regulation) and the expressed ability to degradation of a lysine at pseudomonads. In some cases natural strains with less difficult systems of supersynthesis limitation, receive on the medium some quantity of initial metabolite, for example, the strain of Egetothecium ashbyii fungi capable to produce vitamin B 2, and Rgopionibacterium shermanii – vitamin B 12. Such microorganisms become objects of selection on increasing the level of production of the produced substance. Suitability of the microorganism (which does not emit the necessary substance, usually a primary metabolite, but attracts the researcher by some properties) for use as an object of selection can be checked by obtaining a producer of this substance, by introducing one or several, easily tested mutations which theoretically have to cause supersynthesis of this substance and, may have been already “approved” on the other microorganism. It is the most reliable way of choice of an initial strain even if for this species of microorganisms there are no data on regulation of synthesis of the desirable substance. For producers of secondary metabolites and enzymes or polysaccharides the choice of the initial strain is predetermined by the ability of a natural microorganism to produce some amount of the necessary substance. When the same substance is emitted by the natural strains relating to different taxonomical groups (for example, fungi and bacilli), it is able to afford to choose more “technological” method for future production or selection of strain. Thus, the selector most often is not free in the choice of the strain, initial for strain selection, and cannot consider criteria of choice of microorganisms` genetic study and a possibility of various genetic methods application. The natural properties of strains defining this choice facilitate and accelerate selection work. However, the absence of many industrial microorganisms of exchange systems of genetic information does not allow us to study genetic control synthesis of the produced substance to facilitate saturation of producer genome mutations, necessary for supersynthesis. 4.2 Preparation of the initial strain for selection It is necessary to study natural variability of morphological features of the microorganism producent that taken for selection as well as the level of production of the desirable substances. After putting the initial strain on the Petri dish among not less than 100 (and it is better than several hundred) colonies it is

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Part I. Microbial biotechnology

necessary to reveal morphological, typical for this culture form and deviations from it. The isolated colony (clones) from the agar of a particular form (not less than 100 clones), so the available number of its morphological options (previously it was shown that clones keep the features) is estimated after the corresponding way of cultivation on the level of production of substance, applying a reliable analytical method. Such an assessment allows us to reveal a possible correlation between the ability to produce this substance and morphology of colonies. Several clones with the highest level of production in relation to the level of control, which is an initial culture, are selected and tested on production in several repeated experiences, and then one clone is selected that is characterized by high-reproduced level. Such a procedure, sometimes called “cleaning” of initial culture, leads to selection of a clone with a high production, and in certain cases – with a deviation from typical morphology. The clone, selected from a sieving of initial culture dissipates again, note morphological variability if it is, and then estimate not less than 100 typical for these clone colonies isolated on jambs on production level. It is convenient to distribute the values of levels of production of such subclones expressed as a percentage in relation to the production (parental) clone, initial for them, in a variation row and to calculate statistics: an arithmetic average of X, a quadratic deviation and coefficient of variability of cv = a*100/X. The subclones, which have an extreme right part of this row are selected, repeatedly estimated on the level of production. This subclone is dissipated and, as well as in the previous case, having checked not less than 100 colonies, build a variation row and calculate its indicators. Having received two variation rows, compare cv values of these ranks. If these values authentically don’t differ, it is possible to finish preparation of an initial strain for further selection with selection of a subclone from the first variation row, and to consider the second row (the number of this subclone) control for the following stage of selection with application of mutagen factors. At detection at cv of the second row of an obvious tendency to reduction it is expedient to carry out one more stage of cloning, having chosen the “best” clone from the right part of the second row, to construct the third variation row on the basis of its sieving and to determine cv. After comparison of cv values of the second and third ranks the further cloning is stopped if cv of both rows does not differ, and continued if cv of the third row decreases. The purpose of the cloning step is “to stabilize” the initial culture on quantitative sign, obtained on the basis of action of the stabilizing selection of the population, most uniform in this sign, as a secure control for assessment of the variability induced by mutagens and the subsequent selection of mutants. It must be kept in mind that the uniformity of the selected culture decreases at repeated browning and long storage. Therefore the initial culture, which has to serve as control at selection of mutants needs to be maintained

Chapter 4. Selection of microorganisms – producers of important ...

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by the periodic cloning carried out at the periods established for this culture. To increase cloning production level, i.e. to select a mutant, it isn’t possible. The average level of production of clones selected from the right part of the variation row constructed according to the natural variability of culture is usually equal to the average level of production of parental culture. In the case, when the initial culture does not produce the necessary substance, it is necessary to choose a colony, which according to the taxonomical characteristic completely corresponds to this species of microorganism from its sieving, and to use this clone in further work with application of mutagens. 4.3 Production of mutants Various types of mutations are produced by various physical and chemical mutagen factors. The biological material subjected to the influence of mutagen factors has to be discrete and contain the minimum number of kernels that allows us to eliminate or reduce a segregation stage (Figure 6). Usually it is spores, vegetative cells or even scraps of a mycelium at the non-spore of organisms. The suspension containing cells or spores possibly deprived of lumps – conglomerates as the mutation in one of the cells of conglomerate at its germination on the agarized environment will be lost or at best will be shown in the form of the sector. Lumps break on a rocking chair, filter suspension, but completely it isn’t possible to get rid of their presence at the suspension processed by a mutagen. Physical factors (UF-radiation and different types of ionizing radiation) process water suspension of spores or cells. During the processing by chemical factors (most often it is alkylating agents: alkyl methanesulfonates, alkyl – sulfates, an alkyl nitrosourea, methylnitrosoguanidine, etc.) it is necessary to meet the conditions promoting the maximum manifestation of mutagen activity of this substance. The large role plays pH of solution, in this regard processing is carried out in buffer solutions at the most effective pH values for this mutagen. At the same time it must be kept in mind that the same mutagen for different microorganisms can be active at different pH values. It is shown, on different types of mutations the methylnitrosoguanidine of colibacillus and yeast the greatest number of mutants induces at pH 6.0 – 6.5, and at actinomycetes – at 9.0. Sometimes the chemical mutagen is more effective in a gas phase, but not in a liquid phase. Actinomycetes and corynebacteria processed by diethylsulphate vapors (for this purpose it is enough to apply a substance drop on a test tube wall with the grown culture and to mature several hours in the thermostat) have several times more morphological mutations, than after processing in water solution of a mutagen.

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Part I. Microbial biotechnology

Figure 6. Scheme of selection due to biologically active agents

The dose of influence on mutagen is expressed in terms of radiation, corresponding to the radiation type. For chemical mutagens, the dose characterized by concentration of a mutagen in the processed suspension and an exposition at a certain temperature, is determined. After the expired exposition, processing by a chemical mutagen is interrupted with material washing (applying sedimentation by cells centrifugation), placing in buffer mix with pH value non-optimal for mutagen effect, and (or) a series of cultivations in physiological solution preceding seeding on the agarized environment. If by search of certain types of mutations, suspension has to be sowed on an agar without cultivation, then washing from the mutagen is necessary. In the choice of mutagen dose guided by survival of processed microorganism, the last is determined by the relation of number of colonies, which have grown on an agar after the mutagen influence, to the number of the colonies,

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which have grown after seeding of the same, but not processed mutagen (control) suspension of cells, expressed in percentage. The survival depends both on the mutagen dose, and on the sensitivity of this microorganism to the lethal effect of the mutagen, and the sensitivity can differ considerably at several strains of one species of microorganism. In selection, the doses providing survival of cells are used in the range from 0.1 to 50 – 80%. 4.4 Methods of selection of mutants with high productive level After processing by a mutagen, among survivors of initial strain cells in microorganism, it is necessary to select mutants with desirable properties. For this purpose there are two alternative ways: 1) selection of random mutations after assessment of this quantitative sign, i.e. level of production of desirable substance at big number of the clones received from the survived cells; 2) selection of the quantitative sign among mutants with a certain phenotype, for example the auxotrophic, resistant to an anti-metabolite, etc., which in each case are obtained from the survived clones by applying its own method of selection. 4.5 Selection of random (unpredictable) mutations Selection of the random mutations controlling this quantitative sign applied when the selector has no data, way of synthesis in desirable product, its regulation and interrelations with synthesis of other metabolites. 40 years ago when Penicillin and then other antibiotics opened, this method was unique. With large effect, it was applied for many years in selection of producers of these most valuable drugs and, in a sense, framed industrial production of the antibiotics available to broad masses of the population. A classic example of such selection is the well-known Wisconsin series of Penicillin producers that carried out in USA based on a stock strain of Penicillium chrysogenum NRRL 1951 with use of various physical and chemical agents. The selection of strains of this series continuing more than 30 years allowed increasing the level of production of Penicillin of several thousands of times in comparison with the level of a stock strain. The method of random selection of mutations always include several stages; therefore, it is called multi-stage selection with the use of mutagens. The initial culture, prepared for selection processed by mutagen taken in several doses provides different survival. The use of several different mutants from the colonies grown after the influence of mutagen separate dose sift not less than 100 colonies for further assessment of desirable substance production.

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4.6 Receiving practically important strains through genetic engineering In the first half of the 1970s the development of molecular biology in the USSR led to emergence of new experimental equipment called “genetic engineering”, and in the western literature called “work with recombinant molecules of DNA”. The essence of this technology consists in fragmentation of DNA molecules in strictly defined sites, reunion of such fragments, i.e. creation of new recombinant DNA molecules in vitro. It is important that all matrix processes are based on recognition of cell enzymes on the sites at the beginning and end of process. For the enzymes, copying a matrix or working at a matrix, the sequence of nucleotides between signals of the beginning and end of process is indifferent. The situation when alien DNA capable to be replicated in structural gene, transcribed and broadcast in the form of new hybrid (“chimeric”) protein is introduced (Figure 7).

Figure 7. Scheme of matrix processes in the prokaryotic cell in case of introduction of alien genetic information into the genome

Transfer of genes of any organism to the other organism is possible today. Considering specifics of this book, we will consider only transfer of genes in microorganisms and generally those aspects of transfer, which can be important

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51

for creation of industrially useful strains-producers. We will not consider in detail the equipment of genetic engineering that is well described in literature. Nevertheless, it is necessary to say in several words about those basic elements without which genetically engineered manipulations are impossible. First, it necessary to have a well-defined system: the owner – a vector. The vector is a small molecule of DNA capable to autonomous replication in a certain microorganism. It can be a bacteriophage or a plasmid. Naturally, it is necessary to have an effective way of introduction of a vector and a recombinant molecule in the microorganism. Vector molecules have to have a number of properties allowing convenient introduction of alien DNA and its subsequent expression. Now vector systems are well developed in such bacterium as Escherichia coli. A problem of applied microbiology is to develop systems: the owner – a vector for industrial microorganisms: bacilli, pseudomonads, actinomycetes, yeast, etc. Achievements in this area are summarized in the review “Achievements of microbiology” in 1983. Methods of designing strains by genetic engineering methods in many cases depend on the industrial purposes. The clearest and best of the developed tasks is production of microbiological synthesis of human proteins important for medicine, or proteins of farm animals for veterinary science. From the genetic engineering point of view, this task comes to introducing of alien gene in the microorganism and optimization of its expression.

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CHAPTER

5

APPLICATION OF BIOTECHNOLOGICAL OBJECTS IN DIFFERENT INDUSTRIES 5.1 Food biotechnology 5.1.1 Microorganisms used in fermentation and production of yeast In fermentation industry, the production of yeast, bacteria and mold are applied widely. Yeasts are unicellular immovable fungi, having a size from 8 to 10 microns. The shape of these organisms is oval or elongated. They are widely distributed in soil, on substrates of plants that have a lot of glucose. They do not have moving organelles. The cell has an outer membrane. In the cytoplasm of the cell there is a nucleus, vacuoles and other substances (fats, glycogen, volutin). There are wild yeasts in nature. They damage agricultural products causing harm. The useful yeasts are called cultural yeasts. Yeasts are extensively used in industry. They cause fermentation of sugar with the release of carbon dioxide and alcohol. These qualities of yeasts are used in bakery and dairy industry, as well as in the production of alcohol, different types of wine and beer. Yeasts contain a lot of protein and vitamins (B, D, E), and therefore are used in the food and feed industry. Yeast multiply by monopolar, bipolar and lateral budding (Figure 8).

Figure 8. Method of reproduction of yeasts: unipolar budding (а); bipolar budding (b); lateral budding (c)

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Reproduction is characterized by sporulation and simple division. They have species reproduced sexually. During budding, a tubercle is formed first on the mother cell, then it grows and turns into a bud. After this, the young cell is separated from the mother’s body. With a favorable condition, the process of budding takes two hours. In the nutrient medium rich with carbohydrate and nitrogen, the budding process of the yeasts can be seen under a simple biological microscope. Reproduction by sporulation proceeds sexually and asexually. The number of spores varies from two to twelve in the cells of yeasts. During reproduction of nonsexual spores, vegetative cells are divided into small particles, each of these particles forms outer membrane. And with the sexual pathway of sporulation, two cells are combined and covered with a common membrane. Spores can be round and oval. The systematization of yeasts is based on the methods of reproduction and physiological properties. They are divided into two families: Saccharomycetes and not saccharomyces. Saccharomycetes. Saccharomycete includes cultural yeasts. They reproduce by budding and sporulation. Therefore, they are called real yeasts. Cultural yeasts include bakery, wine and brewer’s yeast. The species of Sacch������������������ а����������������� romyces cerevisiae and Saccharomuces elipsoideus are most important in the industry (Figure 9).

Figure 9. Yeast cells of Sacchаromyces cerevisiae

On April 24, 1996 it was announced that Saccharomyces cerevisiae had become the first eukaryotic organism. The cell of Saccharomyces cerevisiae has a spherical and ovoid shape (2-picture). They are used for brewing, bakery and for production of alcohol. There are groups – races that live in certain conditions and temperatures. The kind of wine yeast Saccharomuces elipsoideus causes alcoholic fermentation, as a result, glucose breaks down into wine alcohol and carbon dioxide. Therefore, it is used for wine fermentation. Cell shape of Saccharomyces ellipsoideus is ellipsoid. Some races take part in the formation of the aroma of wine. Yeast of Thallum is unicellular. Vegetative reproduction is carried out through bud formation. Under favorable conditions, reproduction is so rapid that the cells do not have time to separate from each other.

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Yeasts are not Saccharomycetes – false yeasts. The yeasts are not able to form spores, they reproduce by budding. Many of them damage the products in different industries. However, among of them there are two economically important genera for industry: Torula and Mycoderma. The yeasts of Torula genus are spherical and form fermentation in small amounts during the fermentation process. Torula kefir is used to prepare such dairy products as kefir and kumys, and Torula utility plays an important role in production of food and fodder yeasts. The cell of the yeasts of Mycoderma genus has an oblong form. They are not able to form alcohol but can oxidize alcohol and organic acid to water and carbon dioxide. If Mycoderma settles on the surface of an alcohol-containing beverage, it can form a pleated film and break the smell and taste of the drink. Along with this, Mycoderma destroys dairy products, salted vegetables, causing significant harm in the production of vinegar and berry yeast. Rodotorula belongs to the yeasts. It contains a reddish pigment and destroys some food products. A group of Candida genus has the properties that cause diseases.

Figure 10. Lactic Acid Bacteria: а – Lactobacillus plantarum; b – Lactobacilus bulgaricus.

Most bacteria belonging to the lactobacilli family are found in milk and dairy products. They cause the disintegration of mono and disaccharides to lactic acid. They are called lactobacilli. These bacteria have a great importance in the industry and agriculture. There are spherical and rod-shaped bacteria in this family. Bacteria of Propionibacteriaceae family, close to this family, are of great importance in the production of cheese. They are Gram-positive, rod-shaped, immobile bacteria, do not form spores. In fermented production, yeast types can be

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improved through the genes transformed into the Aspergillus cell amylase-coding bacteria (Figure 11).

Figure 11. Aspergillus niger and its dried conidiospores

These methods were used to improve strains of S.cerevisiae in the production of cheese. Increased demand for light beer, that is, for beer containing carbohydrates in a smaller volume led to the search for new strains of dextrin fermentation. But beer obtained with the help of new strains had an unpleasant smell and taste. Then scientists came up with the introduction of genes coding for dextrin fermentation. The introduction of the amylase gene and glucoamylase into the S.cerevisiae cell provided the production of alcohol from starch. 5.1.2 Yeast production In Russian the word “yeast” means “jitter, shudder, tremble, fret”, but in English “yeast” means “foaming, boiling, gassing”. Yeasts are probably one of the most ancient “domestic organisms”. Thousands of years people used it for fermentation and baking. In the ruins of ancient Egyptian cities archaeologists found millstone and bakery, as well as drawings of bakers and brewers. It is assumed that the Egyptians began to brew beer 6000 years before Christ, and by 1200 BC they mastered the technology of baking yeast bread along with baked fresh. To start fermenting a new substrate, people used the remains of the old one. As a result, in different economies, yeast breeding has occurred for centuries and new physiological races have been formed that are not found in nature, many of them were described as separate species originally. In 1680, the Dutch naturalist Anton van Leeuwenhoek first saw the yeast in an optical microscope; because of lack of movement, he did not recognize living organisms. The tour was proved in 1857 by the French microbiologist Louis Pasteur in the work “Mémoire sur la fermentation alcoholique” . Pasteur proved that

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alcoholic fermentation is not just a chemical reaction, as was previously thought, but also a biological process produced by yeasts. In 1861, Louis Pasteur proposed a Pasteur flask for the pure storage of yeast culture. This flask consists of a long tube and the neck of the flask has an S shape that excludes the ingress of microorganisms (Figure 12).

Figure 12. Pasteur`s test of spontaneous generation

In the 1850-1860 in Vienna, compressed yeast was first produced. In 1870 yeast was used for growth in the test. In 1881, Emil Christian Hansen, a worker at the laboratory of the Danish company Carlsberg, singled out a pure culture of yeast, and, firstly, in 1883 used it to produce beer instead of unstable starter cultures. At the end of the XIX century with the participation of Hansen, the first classification of yeast was created. The science of yeast (zymology), in addition to practical issues, began to pay attention to the ecology of yeast in nature, cytology, genetics in the second half of the XX century. 5.1.3 Beer fermentation. Conditions for beer making Beer is a kind of drink obtained by special treatment of germinating barley, by fermentation, carbon dioxide-enriched frothy beverage. About 5-7% of ethyl alcohol is in the beer composition. In the production of beer, yeast strains of Saccharomyces cerevisiae, S. Сarlsbergensis and others are used. The right choice of strains affects color, smell, and the strength of the beer. The beer contains maltose – 53, glucose – 12, maltotriose-13, dextrin – 22 and maltotetraose in a small volume. At present, the genetics have a goal to produce beer in which yeast is capable to assimilate dextrin, known as the light beer (low carbohydrate content). In Kazakhstan the brewing process is intensively developing.

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5.1.4 Manufacture of dairy products. Microorganisms used in the production of dairy products. Sour-milk drinks and technological processes of their production The process of lactic acid fermentation occurs in milk. The process of lactic acid fermentation is widespread in nature and everyday life. In the industry, the production of pure lactic acid, the preparation of various products (ayran, sour cream, cottage cheese, etc.), ensilage of feed, fermentation of vegetables are based on the property of a sour-milk bacterium. In 1847, S.Blondo first showed that lactic acid is a product of the fermentation process, and Louis Pasteur proved the role of bacterium in this fermentation process. The production of lactic acid by microbiological synthesis is known to technologists since 1881 and used constantly. Lactic acid is widely used in the food industry, in medicine, as raw materials in chemical synthesis and other industries. In the production of lactic acid as a raw material, it is possible to use molasses, mineral salts, malt and extract of maize, the remains of fruit drink production, whey, ultrafiltrate of milk and whey. The fermentation process is carried out in an acidic environment for 2-7 days at 49-50°C; under seasonal cultivation conditions, the separation and purification of lactic acid causes some difficulties. Lactic acid is poorly crystallized in the form of colorless syrup (mainly in the form of 65% solution). Many lactic products, butter and cheese are made through fermentation of lactic acid, and it also plays a major role in cabbage salting and canning of berries, ensilage of forages. Intermediate products of lactic fermentation suppress the growth of the other microflora, give good organoleptic properties to fermented mixtures, have a beneficial effect on the human and animal organism. Milk is considered to be the best nutritional product. Its composition includes valuable substances. Dairy products play an important role in human life, they account for 16% of the used food. The composition of natural milk is very complex. On average, it comprises 3.7% fat, 3.5% protein, 4.9% lactose (lactic sugar), 0.7% mineral substances and 87.2% water. 5.1.5 Ayran and technology of its production At present, many foodstuffs are made from milk. One of them is ayran. In the production of ayran, add cream to the required level fat content. Then the grains of fat are homogenized, pasteurized (90-96°C), cooled at 30-45°C temperature, and special yeast is added for fermentation. Ayran refers to lactic acid products. To prepare boiled milk add yeasts and lactic acid bacteria. Special microorganisms for ayran – Streptococcus lactis, Lactobacillus etc (Figure 13).

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Figure 13. Microflora of ayran: Lactobacillus bulgaricum. (28.5 %). Streptococcus Lactis. (35.7 %). Streptococcus diacetilactis.(21.4 %). Saccharomyces Kefiri. (14.2 %).

Set of proteins The Ayran preparation goes in two processes simultaneously, lactic acid and alcohol fermentation. The temperature regime plays an important role because at +20°C temperature the process of lactic fermentation occurs, and at +15°C temperature and below favorable condition for alcohol fermentation (Table 3). Ayran is very tasty, slightly sour, thick drink. It contains about 1% of alcohol and 1% of lactic acid. Before addition of leaven, ayran pasterilized at +85 – 90°C temperature. Then, the pasterilized milk cooled 10-15 minutes, so the temperature of milk decreases to +30°C temperature. To the prepared milk, add leaven, then in 6-8 hours ayran will be ready. Ayran stored in cool place. Bulgarian bacillus widely used in the milk acid production. Characteristics of microbiological composition of some lactic acid products: №

Dairy

Culture

Table 3

Temperature, duration of incubation 22°С, 18 hours.

1

Sour cream

Streptococcus lactis, S.cremoris, Leuconostoc cremoris, S.diacetilactis

2

Yogurt

43 – 45°С, 2,5–3 hours

3

Ayran

Streptococcus termophilis, Lactobacillus bulgaricus Streptococcus lactis, Lactobacillus, Yeast

4

Kumys

Lactobacillus bulgaricus, and L.casei, Saccharomyces lactis

15 – 25°С, 24-36 hours

5

Cheese

Streptococcus lactis S.cremoris, Leuconostoc cremoris,

22°С, 18 hours or 35°С, 5 hours

15 -20°С, 15-20 hours

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5.1.6 Technology of Kumys and Shubat production One of the most frequently used lactic acid products is kumys. Researchers have long wanted to turn the manufacture of kumys into a production base. Therefore, manufacturers of kumys tried to obtain it through pure cultures of Lactobacillus bulgaricus, L.casei and yeast Saccharomyces lactis. Kumys is a drink prepared by fermentation of horse milk with the addition of lactic acid bacteria and yeast. Making of kumys has two processes at the same time. During the lactic fermentation process, lactic acid forms; in the alcoholic fermentation process, ethyl alcohol and carbon dioxide are formed. In kumys, due to simultaneous processes of lactic acid and alcohol fermentation, the composition contains 2.5% of alcohol. According to the new technology kumys is made in the manufactory. In special farms and complexes, the manufactory consists of a reception room, a laboratory, a cleaning and fermentation room, a kumys making room, a cooling room and a storage room for milking machines. For the correct course of the process of kumys making, the following tools should be used: a jar, a sieve, a milk meter, enameled buckets, bottles, boxes. After milk enters the manufactory, it is measured, re-filtered, an average sample is taken to check the quality. Preparation of kumys begins with the addition of yeast, then at 26-280C temperature it is pasteurized for a long time. Kazakh people say that the quality of kumys depends on leaven and saba. Every 10-15 days, the saba is freed from kumys, dried out, re-treated with smoked and smeared with fat. Now it is known that the microflora of kumys is well preserved in dry form. In summer, when the kumys season begins, it is first prepared by yeast and ferment. To prepare the starter in the fall, the kumys is kept at home for a long time to settle. Then the serum of kumys is separated to the surface, a thick sediment remains at the bottom. Sediment (casein) is put in a gauze bag, filted and dried under the light, stored in a container in a cool place till the next stage. To «rejuvenate» again the leaven powder of 3-4 tablespoons is mixed in 5 liters of mare milk. Then this leaven is used in kumys fermentation. Then, 5 liters of mare milk are added in a liter of fermented kumys. It is cooked into the sabu, in the evening 25-40% of the daily mare’s milk is poured over and mixed. At the next day, pour fresh milk shaked well. Thus, the addition of milk every evening increases the supply of kumys. In 1969, technical conditions for preparation of natural kumys were developed.Kumys is a drink easily assimilated by the organism. Lactic acid in kumys affects the digestive glands, improves digestion, small amounts of alcohol and carbon dioxide enhance metabolic processes. Currently, all the medicinal properties of kumys are known. Yeasts causing breakdown of milk sugar suppress the growth of tuberculosis bacteria in human

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body. In recent years, kumys has been prepared from a cow milk with pure cultures of lactic acid bacteria and yeasts. Lactic acid bacteria use acidophilus and a Bulgarian bacillus for the alcohol fermentation process, champagne and bread yeasts are also used. The following technology is used: 20% water is added to the skim milk, diluted and 5% sugar beet is added. This mix is pasterilized at +90°C temperature for 15 minutes. The purpose is destruction of microorganisms contaminating milk. Then, adjusted at +40 -45°C, about 3-5% of previously prepared yeast from clean microbes is added. The process of fermentation proceeds at +35-37°C temperature. Then inoculated milk is mixed carefully, barmy fungi at 30°C temperature is added and mixed repeatedly. The termination of kumys is spilled in bottles at +10-12°C temperature in the special room. Thus kumys will be ready in 12-16 hours. The disadvantage of kumys from cow’s milk is the strengthened gas generation in the summer. Therefore in summertime kumys preparation adds sugar in small (3%) amount. To apply kumys in the medical purposes instead of water sour curdled milk is added. Microflora of Kumys: In 1934 A.F. Voytkevich and E. Runov obtained lactic sticks from the kumys. According to the morphological and biochemical structure they belong to Bulgarian bacillus. Bacterium was rod-shaped (length 1.8-7.7 and width 0.450.6 μm), gram-positive. It did not grow in a simple peptone nutrient medium because it was a very strong (260 – 326°C) oxidant. The optimum growth temperature of bacteria was 40-50°C, the maximum temperature was 50°C, and the minimum temperature was 20°C. In kumys preparation, the composition of yeast included Lactobacillus bulgaricus. Along with this, Lасtobacillus casei occured in kumys. Feature – the optimal temperature was higher and the acidity was lower. In the Mongolian kumys, Streptobacterium group was present with Lасtobacillus casei. In 1949 M.G. Kuramshin examined the coccal form of lactic acid bacteria. In Kazakh kumys, there are about 160 lactic acid bacteria, 35 are lactis streptococci and diplococci. When researching of streptococci growth in meat-peptone nutrient medium they formed smooth, glossy columns. They developed at 20°C temperature. The diplococci bacterium belongs to Streptococcus lactis. In 1961 K.Ch. Mahanta found in Bashkir kumys Streptococcus lactis and Streptococcus termophilis. In 1964, Gritsenko T.T. isolated a thermophilic bacterium, but in comparison with a rod-shaped bacterium it was found in small amounts. In Bashkir kumys Lactobacillus bulgaricus, Lасtobacillus casei, Streptococcus lactis were rare. M.H. Shigaeva and M.Sh. Ospanov during the research of Kazakh kumys met rod-shaped and coccoid lactic acid bacteria. According to their morphological and physiological properties, bacteria were divided into 3 groups:

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Group I – lactobacillus, 3 x 10 – 0.7 x 0.8 μm long, gram-positive, immobile, does not form spores, facultatively anaerobic, poorly grow in agar medium with hydrolyzed milk and other dense media. They grow well in liquid nutrient medium (hydrolyzed and skim milk). The growth temperature of the isolated culture is at minimum 15-18°C, the optimum 37-45°C, the maximum 50°C. For bacterium, the growth to be saved the optimal pH is 5.8 – 6.5, acidity of milk – 350ºT. The duration of milk fermentation is 4-6 hours. Isolation of strains according to the classification of M. Rogoso, M.E. Sharpe (1959) refers to Lactobacilus bulgaricus. Group II – short rod-shaped, 0.5 to 0.7 μm in size. Gram-positive, motionless, does not form spores. One or two are cellular, form a short chain. They form a dotted, spherical colony in the nutrient medium. Deep colonies are boat-shaped. The culture develops well in a liquid nutrient medium. The optimum cultivation temperature is 37°C, the minimum temperature is 10°C, and the maximum temperature is 45°C. The optimal pH is 6.6-6.9. Fermentation of milk occurs at 30°C for 10-20 hours. The limiting acidity is 250ºT. According to morphological and biochemical properties these cultures belong to Lactobacilus casei. Group ІІІ – sour-milk streptococci. They are isolated from various samples of kumys. They are spherical, fixed, Gram-positive, do not form spores. Mostly diplococci, sometimes unicellular cocci. The average length of the cells is 0.8 – 1.1 μm. The cultivation in a dense nutrient medium forms a chest-shaped form with an edge. In agar medium with hydrolyzed milk, after 2 days, it forms S-shaped colony, rod-shaped, glossy, white, with edge. The colony size is 1-2 mm. At the edges of the colony, in the culture medium of the sugar and limestone forms colorless zones. In agar medium with hydrolyzed milk all strains grow well. All strains actively produce acid. Fermentation takes 4-8 hours with the formation of a dense, homogeneous yeast. In sterilized milk, the limiting acidity is 110ºT. As they do not grow at 45°C, they refer to Streptococcus lactis. The culture grows well in a nutrient medium with 2% and 4% NaCl. The isolated culture develops well in a medium with pH-7.2, the cells transferred at 60ºC temperature for 30 minutes, are killed at 60ºC. The minimum growth temperature is 10-15ºC, the optimal temperature is 2530ºC, and the maximum temperature is 38-40ºC. The culture causes fermentation of glucose, galactose, arabinose, psilozane, lactosan, sorbitol, does not digest raffinose and mannitol. The isolated strains of lactate streptococci belong to Streptococcus lactis. The samples of Kazakh kumys are similar to Mongolian kumys because of lactic acid bacteria, there are Streptococcus lactis, Lactobacilus casei. In addition, species of Lactobacilus bulgaricus, Streptococcus lactis, Lactobacilus casei have been identified. Since the ancient times the drink – shubat is known from lactiferous products of inhabitants of Central Asia and Africa, especially Kazakh people. Shubat is

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made from camel milk. It is a very tasty, high-caloric drink having medical properties. Nowadays, the medical propertis of shubat are known very well. It is used to treat diseases of the stomach, kidneys, intestines. The composition of camel’s milk is very different from the composition of cow’s milk. There are also the processes of lactic acid and alcohol fermentation. For preparation of shubat from camel milk, add 10-40% of previously prepared yeast. For fermentation, milk is poured into wooden utensils or a jug of burnt clay. Shubat with a specific smell and taste will be ready in 8 hours at a temperature of +25-30°C. The method of shubat making has been studied by many scientists: In 1954 N.T. Kiselev proposed a method of shubat making from pure culture. According to his technique, the composition of the shubat composed of 3 types of microorganisms: Lactobacillus casei acidic sticks, small amount of Streptococcus termophilis and the Torula yeast. The 10% yeast was added to camel milk, kept at 25°C for 8 hours, then at 20°C for 16 hours. In 1958 K. Kuliyev made some changes in the method of shubat making. The composition of yeast included the above-listed microorganisms, but their ratios were different: 6:2 parts of sour-milk bacteria, 6:4 parts of yeasts. 10% of yeast added to camel milk, kept at 30°C for one day. Finished shubat had acidity 134.2 ° T and 1.14% alcohol. The microbiological processes in shubat continue after the shubat is ready. It depends on the storage temperature. The taste of shubat is slightly sour, liquid, creamy in consistency and has a lot of foam. The storage at high temperature (18 – 20°C) one day, on the second day the taste becomes acidic, highly liquid, not foaming. The drink stored for a long time at 4-6°C. At 18-20°C the biochemical process proceeds rapidly. 5.2 Agricultural biotechnology Biotechnology is used to address the problems in all branches of agricultural production and processing. It includes plant breeding to raise and stabilize yields; to improve resistance to pests, diseases and abiotic stresses like drought and cold; to increase the nutritional content of foods. Biotechnology is used to develop low-cost disease-free planting materials for crops, for example, cassava, banana and potato and creates new tools for the diagnosis and treatment of diseases of plants and animals and for measurement and conservation of genetic resources. Biotechnology is used to speed up breeding programs for plants, livestock and fish; to extend the range of traits, which it can be addressed. Animal feeds and feeding practices changed by biotechnology to improve animal nutrition and to reduce environmental waste. Biotechnology is used in disease diagnostics and for the production of vaccines against animal diseases.

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5.2.1 Biopesticides Biopesticides are pesticides obtained from animals, plants, bacteria and some types of minerals. They are less harmful to the environment, and their action is directed only to a certain group of pests and doesn’t exert impact on other insects, birds and mammals. The American agency EPA (Environmental Protection Agency) distinguishes three main types of biopesticides. Biopesticides based on the microorganisms (bioagents: bacteria, viruses, fungi, etc.) are characterized by the area of application. Some species of fungi can be used in the control of plant growth and others – for elimination of certain groups of insects. The most effective and widely applied (about 90-95% of the market of biopesticides) in fight against insects are medicines based on a grampositive bacteria of Bacillus thuringuesis. The incorporated protectants of plants (plant-incorporated protectants). These are substances, which make the plant independently from the activity of the genetic material entered into it. Use of genetic material in Europe is strictly regulated by various regulatory bodies on protection of the environment. 5.2.2 Biochemical Pesticides Biochemical pesticides represent the natural substances influencing insects by non-toxic methods. Biochemical pesticides include, for example, pheromones or plant extracts. Biopesticides have been used in agriculture recently, the history of the industry takes 10–20 years. At the present time the market grows promptly while the production of chemical pesticides increases annually by 1–2%, the segment of organic substances shows stable growth at the level of 10–15%, and its volume is estimated in 300 million dollars. 5.2.3 Microbial Pesticides The phenomenon of antagonism is widely spread among microbes. One of them suppresses the other’s growth. It explains that microorganisms can produce antibiotics – substances that suppress the growth or destroy some groups of microbes. Each antibiotic has its own spectrum of action, i.e. it suppresses only certain microorganisms. Antibiotics differ from each other by their impact on microorganisms. Some of them stop microbes growth or have bacteriostatic action, others kill microbial cells, act bactericidal. The third cause not only death, but also lysis (disintegra-

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tion) of microorganisms’ cells. The influence of antibiotic action depends on its dosage. Nowadays, the antibiotics are used to protect plants against pests successfully. Antibiotics in small amounts enter into forages for acceleration of the growth of young animals. The practical value of antibiotics began to extend in the 40s of the XX century. However, the antagonism phenomenon of microorganisms was known before. L. Pasteur noted oppression of an anthracic bacillus by culture of a pyocyanic rod. I.I. Mechnikov studied the antagonism phenomenon of intestinal microflora. In the 70s of the XIX century, the Russian doctors V.A. Manassein and A.G. Polotebnov found in the culture of fungi Penicillium antibacterial substance and tried to use it in the medical purposes. The interest in antibiotics increased sharply lately. Fungi (Penicillium, Aspargillus and others), many actinomycetes, and bacteria can produce antibiotics. Among them, the medicals influence better than chemicals. However, not all antibiotics might be used in practice. According to the chemical nature of antibiotics, they belong to different groups of chemical compounds. Here are the acyclic and aromatic compounds, quinones, heterocyclic substances containing nitrogen, lipeptides and others. Microbes-antagonists and their antibiotics play an important role in fighting with phytopests. For production of microbial insecticides, viruses, fungi, protozoa, protozoa – spore-forming bacteria are used. Microbial insecticides are highly specific and act only on certain harmful insects, and are non-harmful for useful insects. Pathogenicity of microorganisms is caused by the action of certain toxins and developing of resistance to biological products in insects does not occur. Microbial pesticides (entomopathogenic preparations based on bacteria, fungi, or viruses) are biodegradable. Microorganisms can regulate the growth of plants and animals to suppress disease. Some bacteria alter the acidity and salinity of the soil, produce other compounds that bind iron, and others – produce growth regulators. As a rule, microorganisms inoculate seeds or plants before planting. In animal biotechnology, the technique of monoclonal antibodies, genetic improvement of animal breeds is used for diagnosis, prevention, treatment of diseases. Widely used in biotechnology techniques is artificial insemination. Biotechnology is used to ensile fodder, allowing increasing the absorption of plant biomass, for the disposal of cattle farms and ecologically cleaning of organic fertilizers based on recycling crop and livestock production. Some substances produced by microorganisms can be used as food supplements, others inhibit harmful microflora in the gastrointestinal tract or stimulate the formation of specific microbial metabolites.

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5.2.4 The nitrogen cycle and nitrogen fixation Biological nitrification – is the process of transformation of nitrogen, contained in the atmosphere in the form of chemical inert N2 into the available form of nitrates and ammonia for plants. Nitrogen makes 78% from the total volume of the atmosphere and is non-available for plants in this form. That is why people have to introduce nitrogen fertilizers for increasing productivity of agricultural plants. Fixation of atmospheric nitrogen is provided by bacteria living in symbiosis with representatives of legume crops (tubercle bacteria from Rhizobium genera) or free-living nitrogen fixators like Azotobacter. Biological nitrifixation is the process of transformation of nitrogen contained in the atmosphere in a form of chemically inert N2 into forms of nitrates and ammonia accessible for plants. Nitrogen makes 78% from the total volume of the atmospheric air and is non-available for plants. That is why people have to introduce nitrogen fertilizers for increasing the productivity of agricultural plants. The fixation of atmospheric nitrogen occurs by bacteria living in the symbiosis with representatives of legume crop family (tubercular bacteria from Rhizobium genera) or free-living nitrogen fixators like Azotobacter. For settling acceleration of rhizosphere, we usually use bacterial fertilizers containing cultures of nitrogen fixation microorganisms. Bacterial fertilizers based on the tubercular bacteria enter under the legume crops cultures by symbionts. Mutants of tubercular bacteria with increased ability to nitrogen fixation are obtained. Nowadays the creation of nitrogen fixation plants capable to symbiosis with cereal plants occurs. There are bacterial medicals improving phosphorus nutrition of plants (Table 4). Data from various sources, compiled by DF Bezdicek & AC Kennedy, in Microorganisms in Action. Type of fixation

N2 fixed (1012 g per year, or 106 metric tons per year)

Non-biological Industrial Combustion Lightning Total Biological Agricultural land Forest and non-agricultural land Sea Total

Table 4

about 50 about 20 about 10 about 80 about 90 about 50 about 35 about 175

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Nitrogen cycle is a biogeochemical cycle of nitrogen. The most part depends on the microorganisms provided by the action of living organisms. The main role in the nitrogen cycle is played by soil microorganisms. It is expressed in the soil nitrogen change – cycle of nitrogen in soil which present in the form of simple substance (gas – N2) and ions: nitrites (NO2-), nitrates (NO3-) and ammonia (NH4+). Concentration of these ions reflects the status of soil compounds. The condition of biota (plants, microflora), atmosphere status, and substances soil washing. They can decrease the concentration of nitrogen containing substances, which badly influences living organisms. They can prevent toxic ammonia to less toxic nitrates and to biological inert atmospheric nitrogen. Therefore, the microflora of soil supports stability of its chemical indicators. 5.2.5 Mechanism of biological nitrogen fixation After Burris and Wilson’s discovery, which showed that nitrogen fixing activity is specifically inhibited by hydrogen and carbon monoxide at Rhizobium, Azotobacter and at Nostoc muscorum, numerous confirmations of identity of enzymatic systems operating by nitrogen fixation at alga and bacteria have been received. Studying of the mechanism of biological nitrogen fixation has a particular interest. Chemically inert molecule N2 enters fast reactions of synthesis at usual temperatures and pressure, which is not reached in the chemical industry yet. The mechanism of nitrogen fixation by alga and other microorganisms has made blue-green alga perspective objects for studying of the general regularities of biological fixation of nitrogen. At the same time the combination of nitrogen fixation and photosynthesis attracts specific interest in these organisms as they show the most direct way of use of solar energy for providing plants with available nitrogen of blue-green alga. Studying the ratio of photosynthesis and nitrogen fixation has been undertaken by Fogg. He noted that blue-green alga, as photosynthesizing bacteria have no features in photosynthesis that can be specifically connected with ability to nitrogen fixation; the way of carbon in photosynthesis and products of photosynthesis are the same in green alga and higher plants. Processes of photosynthesis and nitrogen fixation can be independent; however, they proceed at the same time in one cell, which forces us to assume a close interlacing of these two processes. Fogg noted that nitrogen fixation on light goes more intensively. It turned out that the hydrogen donors necessary for restoration of molecular nitrogen and formed by many organisms irrespective of light, are connected with photochemical reactions. Such photochemical reduction is confirmed for an obligate phototrophic Anabaena cylindrica; the intracellular reducers, which are formed during water

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photolysis, were quite suitable for nitrogen restoration. It is supposed that the photochemical reduction of nitrogen under certain conditions excluded, then restoration goes at the expense of hydrogen which is released in Krebs’s cycle. The biological nitrogen fixation may be written in this equation, where 2 moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and supply of electrons and protons (hydrogen ions): N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi This equation is performed only by prokaryotes (the bacteria and related organisms), using enzyme complex called nitrogenase. This enzyme includes two proteins: iron protein and molybdenum-iron protein (Figure 14). The process occurs when N2 is bound to nitrogenase enzyme complex. Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP molecules and reduces molybdenum-iron protein that donates electrons to N2, producing HN=NH. In both cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and then this in turn is reduced to 2NH3. According to the type of microorganism, the reduced ferredoxin that supplies electrons for this process is generated by photosynthesis, fermentation or respiration.

Figure 14. Mechanism of biological nitrogen fixation

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The nitrogen-fixing organisms. Nitrogen fixing bacteria are subdivided into three groups: symbiotic, free living and associative. Symbiotic nitrogen fixators acquire molecular nitrogen being in symbiosis with a plant. Symbiosis between tubercle bacteria of Rhizobium genera and legume plants is especially important. Bradyrhizobium genera bacteria belong to symbiotic nitrogen fixators (symbiosis with a lupine, soy, a cowpea, peanut, etc.), Azorhizobium bacteria (symbiosis with legume plants). Bacteria of Rhizobium, Bradyrhizobium and Azorhizobium generas enter α – subgroup of proteobacteria, form root, and stem (Azorhizobium) tuber in legume plants. Actinomycetes of Frankia genera also live as endosimbiont in the tubers, which are formed on roots of not legume plants, wood, shrubby and grassy among them are alder, a sea-buckthorn, a beef-wood, a waxberry, an oleaster, etc. Some symbiotic nitrogen fixators related to Klebsiella form tuber on leaves of Pavetta and Psychotria. Cyanobacteria Anabaena azollae forms symbiotic association with a water fern Azolla, making a big contribution to nitrogen fixation on rice plantations where this fern grows on a water surface. Cyanobacteria of Nostoc genera enter symbiosis with mosses – liverworts and a tropical plant Gunnera macrophylla. Symbiotic cyanobacteria are present at the lichens representing association of pokaryotes with fungi. Thanks to cyanobacteria that carry out nitrogen fixation and have pho­ toautotrophic type of metabolism, required for growth only CO2, N2 and mineral salts, lichens the first occupy inorganic environments, creating conditions for development of other organisms. Some species of bacteria Clostridium (C. pas­ teu­rianum, C. butyricum, C. acetobutyricum, C. felsineum, C. pectovorum, etc.), bac­teria Azotobacter, Azomonas, Beijerinckia, Derxia, the majority of phototroph bac­teria, many cyanobacteria, facultative anaerobe bacterias (Klebsiella pneu­ mo­niae, Bacillus polymyxa), bacteria Xanthobacter autotrophicus, Alcaligenes latus, methylotroph bacteria (Methylomonas, Methylobacterium and Methy­lo­ coc­cus), sulfatereducing bacteria (Desulfotomaculum and Desulfovibrio) and methanogenic bacteria belong to free-living nitrogen fixators (Table 5). Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium) Free living Anaerobic (see Winogradsky column for details) Azotobacter Clostridium (some) Beijerinckia Desulfovibrio Klebsiella (some) Purple sulphur bacteria* Cyanobacteria (some)* Purple non-sulphur bacteria* Green sulphur bacteria* Aerobic

Table 5

Symbiotic with plants Legumes Other plants Rhizobium

Frankia Azospirillum

Data from various sources, compiled by DF Bezdicek & AC Kennedy, in Microorganisms in Action

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5.2.6 Products obtained from nitrogen fixation Agrophil. Is obtained from Agrobacterium (A.radiobacter, strain 10). This medical is enriched with vitamins, carbon and microelements. It is enriched with 50-55% of fertilizers. Powder. The 1g of medical contains 10 billion activated bacterial cells. Increases the resistance of vegetables to infectious diseases. Agrophor. Is obtained from Agrobacterium spp. (A.radiobacter, strain 57/136). Stimulates the growth of plants, is used in the production of cabbage, tomatoes and others. Azorizin (diazobacterin). This medical received from Azospirillum spp. Azospirillas increases the productivity of cereal cultures like rice, corn, etc. Bioplant-K. Received from Klebsiella (K.planticola, strain ТСХА-91) in A.K.Timiryazev Moscow agricultural academy, microbiology department. It is used as bacterial fertilizer for vegetables against nitrogen fixation bacteria and phytopathogenic fungi like Penicillum, Aspergillus, Alternaria, Mucor and other. The medical increases productivity of cucumber by 21-23%, tomato-31%, potato-21%. Mizorin. Received from Arthrobacter spp.(A.mysorens, strain 7). In 1g of medicine there are 8-10 billion bacterial cells. This medicine is enriched with nutrient medium, contains 50-55% of fertilizers. Powder. It increases productivity of cereal cultures and vegetables. Mycolin. Obtained from Bacillus spp. (B.cereus var.mycoides). Bacteria stimulate the growth of potato and cabbage through the activity in their rhyzospheres. Rhyzogarin. Obtained from Agrobacterium spp.(A.radiobacter, strain 204). There are 8-12 billion bacterial cells in 1g of medical. Bacteria increase the productivity of rice, corn and other agricultural products through the activity in the rhizosphere, and contain their proteins 0.5-1%. Rhyzoenterin. Obtained from Enterobacter spp. (E.aerogenes, штамм 30). There are 6 billion bacterial cells in 1 g of medical. Rhyzoenterin is enriched with nutrient medium and contains 45-50% of fertilizers. Powder. It is used to increase rice productivity. Flavobacterin. Obtained from Flavobacterium sp., strain 130. There are 5-10 billion bacterial cells in 1g of medical. Powder. It is used to get additional 3-5c/ga of cereal cultures, 20-60 c/ga of vegetables, 60-70 c/ga of sugar beet. 5.3 Environmental biotechnology 5.3.1. Biodegradation Biodegradation (Greek bio(s) – life and lat. degradatio – decrease, movement back, deterioration) – destruction of the pollutants with a help of living

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microorganisms in the environment. For biodegradation of xenobiotics, associations of microorganisms or artificially obtained strains are usually used as they are more effective, than separately taken natural species. At the same time types of communications in association can be various. One species of microorganisms can participate in decomposition of xenobiotics, and another – to deliver missing nutrients. It can be metabolic “attack” to a substratum when different components of enzymatic complex, or a chain of enzymatic reactions (multiple substrate conversion), etc. are synthesized. The most capable to biodegradation of pollutants of various types are representatives of Pseudomonas – they are almost “omnivorous”. Advantage of bacterial cleaning in comparison with chemical is that it doesn’t cause appearance of new polluting agents in the environment. At the same time, the density of phytoplankton after bacterial cleaning increases. Some microorganisms are capable to change a molecule of xenobiotic and to make it available and attractive to other microorganisms (cometabolism), for example, decomposition of paration insecticide under the influence of two strains Pseudomonas – P. aeruginosa and P. stutzeri. In certain cases, there is only modification of a molecule of a xenobiotic (phosphorylation, methylation, acetylation, etc.). Biodegradation is one of the main mechanisms of waste destruction in nature: waste of human activity and industrial wastes. In greater or lesser extent biodegradation acts on almost all organic and many inorganic pollutants, except radioactive materials. Biodegradation is the main mechanism of selfrepair/resistance of ecosystems to the anthropogenic influences. 5.3.2 Anaerobic biodegradation of pollutants In anaerobic conditions, in the absence of oxygen oxidizer, destruction of aromatic substances proceeds more difficulty, in multi-stage process with the participation of various enzymes. Microorganisms are capable to use a broad set of aromatic substrates in nitrate – sulfate – iron and carbonate – restoring conditions. The main stages of the process of biodegradation are activation of a benzene ring, its rupture and formation of S1-and C2 bonds. Activation of the ring can be a result of carboxylation reactions, anaerobic hydroxylation and creation of CoA-thyoether aromatic acids. In the last reaction, soluble, non-specific, inducible CoA-ligases or CoA-transferases participate. The central intermediate of aromatic bonds of biodegradation is a benzoil-CoA, which is exposed to a series of reductions under benzoil-CoA-reductase action and hydrolytic cleavage of derived cyclohexane. The first non-aromatic product is pimelil-CoA. Further there is a number of oxidations and decarboxylation of Glutaconyl-CoA with creation of Acetyl-CoA (Heider, 1997; Kleerebezem, 1999; Lochmeyer, 1992).

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Only a few microorganisms capable to biodestruction of aromatic compounds were received in the form of pure cultures. They are Pseudomonas sp., Thauera aromatica, T. chlorobenzoica, Desulfobacterium anilini, Azoarcus evansii, Magnetospirillum sp., Delftia acidovorans, Rhodopseudomonas palustris, Syntrophus gentinae and S. buswellii. In the methanogenic conditions the final stages of biodestruction are presented by archeae Methanobacterium, Methanospirillum, Methanosarcina, Methanosaeta generas. In the lack of oxygen a rupture of C – S – bonds of sulfoaromatic compounds bacteria can proceed with fermentative type of metabolism. Processes of anaerobic degradation of alkylbenzolsulfonates occur in the community containing microorganisms Clostridium, Desulfovibrio, Methanobacterium, Methanosarcina. 5.3.3 Role of microorganisms in biodegradation of pollutants Biodegradation is associated with environmental bioremediation. So, biodegradation is a way of nature to recycle wastes, or break down organic compounds into nutrients that can be used and reused by other organisms. In the microbiological sense, “biodegradation” means – the process of decay of all organic materials carried out by a huge assortment of life forms comprising mainly bacteria, yeast and fungi, and possibly other organisms. Methods of bioremediation and biotransformation try to harness the naturally occurring, astonishing, microbial catabolic diversity to degrade, transform/ accumulate huge range of compounds involving hydrocarbons (for example oil), polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), radionuclides and metals. 5.3.4 Biodegradable pollutants Hydrocarbons are the organic compounds consisting of carbon atoms and hydrogen. Hydrocarbons are considered as basic compounds of organic chemistry – all other organic compounds are considered to be their derivatives. As carbon has four valent electrons, and hydrogen – one, the simplest hydrocarbon is methane (CH4). In systematization of hydrocarbons it is necessary to take into account a carbon structure skeleton and type of bonds in carbon atoms. According to the structure of carbon skeleton, hydrocarbons subdivide into acyclic and carbocyclic. Depending on frequency rate of carbon – carbon bonds, hydrocarbons subdivide into saturated (alkanes) and unsaturated (alkenes, alkynes, dienes). Cyclic hydrocarbons divide into alicyclic and aromatic.

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Polyaromatic hydrocarbons (PAH) are organic compounds characterized by the presence of two and more condensed benzene rings in chemical structure. In the nature polyaromatic hydrocarbons are formed in the pyrolysis of cellulose and are met in layers of stone, brown coal and anthracite and as an uncomplete combustion product at wildfires. The main sources of emission of technogenic PAH to the surrounding environment are the enterprises of power complex, the motor transport, chemical and oil-processing industry. The thermal processes connected with burning and processing of organic raw materials are the cornerstone of all technogenic sources of PAH: oil products, coal, wood, garbage, food, tobacco, etc. The polychlorinated diphenyls (PCD) or polychlorinated biphenyls (PCB) are the group of organic compounds including all chlorsubstituted diphenyl derivatives (1-10 atoms of chlorine connected with any atom of diphenyl carbon, which molecule is made of two benzene rings) responding to the general formula C12H10-nCln. They were firstly synthesized in 1929. The feature of these substances is heat resistance and possibility to use as insulator in electrical engineering. They are colourless and flavourless, PCB are also chemically stable. For these reasons, PCB began to add in many materials. The polychlorinated biphenyls (PCB) belong to the group of resistant organic pollutants whose monitoring in air, water and the soil is mandatory in the developed industrial countries owing to their high danger to the environment and health of the population. Pesticides (Latin pestis “infection” + caedo “kill”), agricultural toxic chemicals, are chemical agents used for pest control and plants illnesses, against various parasites, weeds, wreckers of grain and grain products, wood, products from cotton, wool, a skin, against ectozoons of pets, carriers of dangerous diseases of people and animals. Pesticides unite the groups of substances: herbicides destroying the weeds, insecticides destroying insects, wreckers, the fungicides destroying the pathogenic fungi, zoocides destroying hematothermal animals, etc. The most part of pesticides is poisons, it targets poisoning organisms and also provide sterilizers (the substance causing sterility) and growth inhibitors. Dyes – the chemical compounds that have ability to intensively absorb and transform energy of electromagnetic radiation in visible and to ultra-violet and infrared areas and are applied to giving this ability to other things. The term “dye” was introduced in 1908 into scientific terminology by A.E. PorayKoshitsu. A distinctive feature of a dye is ability to impregnate the painted material (for example, textiles, paper, fur, hair, skin, wood, food – food colorings) – processes of diffusion give color to all its volume, being fixed on the active centers – sorption processes. The terms “dye” and “pigment” frequently used as equivalent, designate accurately differing concepts. The dyes soluble in the tinctorial medium (solvent), pigments

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are insoluble. In the coloring, dyes get in material and form more or less strong communication with fibers; the using of pigments provides communication with the painted material binding, but not pigments. Dyes in paint are binding (drying oil, nitrocellulose, etc.), and properties of paint depend more from binding, than on a pigment. Dyes usually – organic substances; pigments mostly – small dispersion of minerals. Radionuclides, radioactive nuclides (radioactive isotopes, radioisotopes) – nuclides, which nuclei are unstable and experience radioactive decay. The majority of known nuclides are radioactive (only about 300 from 3000 nuclides known to science are stable). All nuclides having the charging number equal to 43 or 61 or bigger than 82 are radioactive; the corresponding elements are called radioactive elements. There are radionuclides (mainly beta-unstable) with other charging numbers (from 1 to 42, from 44 to 60 and from 62 to 82). Heavy metals – a group of chemical elements with properties of metals (including semi-metals) and the atomic weight or density of environment pollutants. Many heavy metals, such as iron, copper, zinc, molybdenum, participate in biological processes and in certain quantities are necessary for functioning of plants, animals and human minerals. On the other hand, heavy metals and their compounds can make harmful effects on a human body, capable to collect in the tissue, causing many diseases. The metals, which do not have a useful role in biological processes, such as lead and mercury, are defined as toxic metals. Some elements, such as vanadium or cadmium that usually have toxic influence on live organisms, can be useful to some types. The major microbial processes influencing bioremediation of metals are given in Figure 15.

Figure 15. Microbial processes used in the bioremediation technologies modified from Lloyd and Lovley.

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5.3.5 Types of bioremediation Bioremediation – a complex of treatment methods of water, soil and the atmosphere with the use of metabolic potential of biological objects – plants, fungi, insects, worms and other organisms. The first simplest methods of sewage treatment – the field of irrigation and the field of filtration – have been based on the use of plants. The plant influences the environment in different ways. The main ways: – Rhizofiltration – roots soak up water and chemical elements necessary for activity of plants; – Phytoextraction – accumulation of dangerous pollutions (for example, heavy metals) by plant; – Phytovolatilization – evaporation of water and emission of chemical elements (As, Se) by leaves of plants; – Phytotransformation: – Phytostabilization – transfer of chemical compounds to less mobile and active form (reduces risk of distribution of pollution) – Phytodegradation – degradation by plants and symbiotic microorganisms of organic part of pollution – phytostimulation – stimulation of development of the symbiotic microorganisms which are taking part in cleaning process The plant is a biofilter, creating for them a habitat (plants provide oxygen, soil loosening). In this regard, the process of cleaning also proceeds out of the vegetation period with a little reduced activity. Rhizofiltration is a technology of metal extraction from water by roots of plants by the flowing systems in which the polluted water is pumped repeatedly by periodically deleted plants. The system of treatment can be in situ – rafts on ponds and ex situ – the designed tanks. After the procedure of water purification, roots of plants could be buried or used for metal recovery. Use of unicellular alga, for example Chlamydomonas reinhardtii is one type of rhizofiltration. These algae are capable to absorb significant amounts of heavy metals. Some algae transfer surplus heavy metals thanks to obtaining of phytohelatines. The ability of plants to create the microenvironment promoting concentration and penetration of substances into plants around root system is used. The advantage of rhizofiltration technology consists in its low cost and opportunity to use widespread plants, including wood. Plants for application in this technology must have the following properties: a rapid growth, intensively accumulate biomass, possess powerful root system. Generally, it is the broad-leaved, monocotyledonous perennial plants, which are well growing in conditions of both warm and mild climate. Many water and marsh plants meet these requirements.

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Phytoextraction. Use of the natural plants-accumulators capable to accumulate metals in elevated organs of specially removed plant varieties, and certain processing of soil for transfer of element-pollutants to elevated parts of plant which are then utilized. It is known that metals do not decay, and clarification of soil demands their removal. This way demands big expenses and is connected with application of physical and chemical agents that have negative effect on the ecosystem. At the same time, phytoextraction is a cheap and safe alternative method. Many experiences have shown that plants have the potential of removal many toxic metals from the soil. Application of processing of soil by agents increases the bioavailability of heavy metals and radionuclides, which increases the efficiency of phytoextraction (citric acid, EDTA, etc.). Recently there were developments allowing us to combine phytoremediation (phytoextraction) with production of bioenergy. Phytostabilization is based on the ability of plants to immobilization of metals in the soil by absorption, sedimentation and complex formation. Phytodegradation is based on the possibility of plants together with soil microorganisms to carry out enzymatic splitting of organic pollutants. The phytodegradational technology is effective in pollution of soils by phenols, pesticides, oil products, etc. Phytovolatilization or evaporation. In the basis of this technology is the ability of some species of plants (Lucerne, Poplar, some species of Acacia, etc.) to emit from the surface of leaves metals and metalloids (generally, it is mercury, arsenic and selenium) in the form of gaseous compounds. However, the serious restriction of phytovolatilization is possible pollution of atmospheric air. The greatest effect in treatment of polluted territories can be reached by combination of different strategies of remediation, for example, phytoremediation and use of microbial communities (Figure 16).

Figure 16. Cycle of waste remediation

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5.3.6 Bioremediation technology 1. Bioremediation of polluted soil and ground is a set of techniques based on application of biological agents for treatment of soils by pollutants. Most often the microorganisms, usually bacteria and fungi are used in bioremediation of soil; rarer – plants. The choice of a certain technology of bioremediation is based on the criteria such as environment of the place, property of soil, concentration and level of toxicity by pollutants, etc. The technologies, which are applied in bioremediation of soil, can be united into two groups: in-situ methods and ex-situ methods. In-situ bioremediation. It is based on treatment of the environment without removing polluted soil from the area of pollution. Technologies of this type do not demand carrying out digging works, they are cheaper, less dust goes to air and less flying pollutants, than in the ex-situ technology. One of approaches of in-situ bioremediation consists of introducing oxygen in the polluted soil by special equipment to stimulate the growth of microorganisms and aerobic biodegradation of pollutants. This equipment is most often used to treat various oil products. Besides oxygen, stimulation of biodegradation can be carried out by introducing of nutrients into soil for stimulation microorganisms’ growth and metabolism, which results in degradation of pollutants. Most often for these purposes nitrogen – and phosphorus-containing fertilizers are used. The other widespread approach is introduction of microorganisms into the soil (including genetically modified) or enzymes to accelerate degradation of organic pollutants in the soil. Ex-situ bioremediation. It is based on removing of polluted layer of soil and its treatment from pollutants outside the place of pollution, it makes this approach more expensive than in-situ bioremediation. Nevertheless, this technology has a number of advantages: it demands less time and provides complete control of cleaning process. One type of in-situ technologies applied in bioremediation uses bioreactors. Before putting into bioreactor, large stones are removed from the soil, soil is exposed to hashing that makes it more uniform; after water addition clay suspension is formed. The microorganisms which provide pollutant’s treatment in the soil are introduced in the reactor with optimum conditions. After completion of treatment process the soil is dried up and comes back to the environment. The other approach of in-situ bioremediation is that the removed soil is placed on the certain territory, provided with aeration, nutrients and water for stimulation of microbial growth and metabolism carrying out bioremediation. In comparison with treatment with bioreactors, this technology demands a lot of place and takes long time. It is possible to obtain several various methods of such approach.

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In one variant, the polluted soil is removed from the place of pollution and distributed by a thin layer on the place, which is fenced in perimeter for prevention of pollution distribution out of its limits. The soil is plowed to provide oxygen to soil microorganisms, and stimulating substances are added for their growth. Also over the soil, water is sprayed that allows maintaining optimum humidity and decrease dust in the air. Contaminated soil can be put in a thick layer of 1-3 meters height, where aeration by ploughing is replaced by aeration through a system of pipes delivering air into the soil to stimulate biodegradation. In this case, the soil is usually mixed with some loose substances (e.g., straw) to facilitate aeration. In the process of remediation, air blowing is used for evaporation of various substances including pollutants from the soil, so the system must be provided with a sensor composition of soil evaporation. Also, fertilizer is added to the ground and a certain level of humidity is maintained. 5.3.7 Natural attenuation Natural attenuation or bioatteuation is a decrease in contaminant concentrations in the environment (soil, water, air) through biological (aerobic, anaerobic biodegradation, also plant, animal uptake), physical (dispersion, advection, diffusion, dilution, volatilization and sorption/desorption), and chemical processes (ion exchange, abiotic transformation, complexation). Terms like intrinsic remediation or biotransformation are included in the definition of natural attenuation. One of the most significant components in natural attenuation is biodegradation, compounds from change that carried out by microorganisms. In good conditions, microorganisms can cause or assist chemical reactions, which change the form of contaminants so little or no health risk remains. Natural attenuation occurs at most polluted sites. However, good conditions must exist underground to purify sites properly. If not, cleanup will not proceed quickly or completely. Scientists studied these conditions to make sure natural attenuation works. It was called monitored natural attenuation or (MNA). Therefore, Monitored Natural Attenuation is a special technique used in monitoring or testing the progress of natural attenuation processes, which can degrade contaminants in the soil and groundwater. It might be used with other remediation processes like a finishing option or remediation process if the rate of contaminant degradation is fast to protect human health and the environment. Natural processes can mitigate the remaining big amount of pollution; regular monitoring of soil and groundwater can verify these reductions. When the environment is polluted with various chemicals, the nature works in four ways to clean it up: 1) Tiny bugs or microbes, which live in the soil and groundwater use some chemicals for food. When they completely digest the

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chemicals, they convert them into water and harmless gases. 2) Chemical substances can stick or sorb in soil that holds them in the place. This does not purify the chemicals, but it can keep and hold them from polluting groundwater and leaving site. 3) Pollutants move through the soil and groundwater, they can mix with clean water. This decreases or dilutes pollutant’s concentration. 4) Several chemical compounds, like oil and solvents, can evaporate that means they transform from liquids to gases within the soil. If these gases diffuse in the air at the ground surface, sunlight may destroy them.If the natural attenuation is not fast or complete enough, bioremediation could be increased by biostimulation or bioaugmentation. 5.3.8 Biostimulation Biostimulation includes the addition of nutrients from soil, trace minerals, electron acceptors, or electron donors enhancing biotransformation of soil contaminants. There are a lot of examples of biostimulation of pollutant biodegradation with the help of indigenous microorganisms. These two chemical compounds: trichloroethene and perchloroethene can be fully converted to ethane by microorganisms in a short time with the addition of lactate during biostimulation. Electron shuttles like humic substances (HS), can play a major stimulation role in the anaerobic biotransformation of organic pollutants via enhancing the electron transfer speed. The anthraquinone-2,6-disulfonate (AQDS) from the HS category may serve as an electron shuttle to promote the decrease of iron oxides and transformation of chlorinated organic pollutants.

5.3.9 Bioaugmentation Bioaugmentation, (bio-augmentation) [gr. bio(s) – life and Latin augmentare – to increase] is biomass increasing; introduction of natural microbial strains or genetically engineered variants into the environment to achieve bioremediation. For example, it is used in the treatment of wastewater (Figure 17). Bioaugmentation is the method used for increasing the biological efficiency of systems of waste treatment by programmed addition with special microorganic formulas. Selectively adapted microorganisms are introduced into the system of waste processing in a large amount to cope with simple and/or complex chemicals. Application of microorganisms worked well for many septic processing of waste. Since the 1950s the use of chemical detergents sharply increased. These

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detergents effectively destroy populations of microorganisms, seriously limiting functioning of waste systems.

Figure 17. Bioremediation of pollution utilizing biodegradation features of microorganisms involves bioattenuation, although it may be changed by engineered techniques, by addition of special microorganisms (bioaugmentation) or biostimulation, where nutrients are added. In addition, to develop biodegradation, genetic engineering uses abilities of microorganisms.

5.3.10 Microbial metabolism Metabolism is the sum of reactions proceeding under the action of enzymatic systems of cells governed by various external and internal factors, which provides the ability to exchange substances and energy between the environment and the cell. Reactions leading to the cleavage and oxidation of substances to obtain energy are called catabolism; the way leading to the synthesis of basic compounds is called anabolism. Catabolism and anabolism are two independent pathways in the metabolism, although some areas can be the same. Such common areas, characteristic of catabolism and anabolism, are called amphibolites. Catabolic and anabolic transformations performed in sequence, as the reaction product of the preceding stage, are a substrate for the next stage (Figure 18).

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Figure 18. Microbial metabolism

Regulation of metabolism in microbial cells is a complex interdependent system, which “turns on” and “off” certain enzymes by using various factors: pH, concentration of substrates, some intermediate and end metabolites, etc. Studying the regulation ways of certain products of metabolism in the cell opens unlimited possibilities for determination of optimal conditions for biosynthesis of target products with the help of microorganisms. Coordination of chemical transformations, providing thrifty metabolism, means that the microorganisms have three basic mechanisms: –– regulation of enzyme activity, including retroengineering; –– regulation of enzyme synthesis volume (induction and repression of enzyme biosynthesis); –– catabolic repression. In the process of retroengineering (inhibition by feedback principle) enzyme activity (allosteric protein), standing at the top of a multi-stage transformation of substrate, is inhibited by the end-metabolite. Low molecular weight metabolites transmit information about the level of their concentration and the metabolic state to key enzymes of metabolism. Key enzymes are the regulators of the frequency of product formation. Using the described mechanism, the final products self-regulate biosynthesis. Inhibition is a method of precise and fast regulation of the created product. The analogues of metabolism affect the metabolism. Practical importance of microorganisms is associated with the peculiarities of metabolism. Substances of microbial origin include primary and secondary metabolites, enzymes, capsular polysaccharide and cell’s biomass. The primary metabolites are low molecular weight compounds required for microorganisms’ growth. Some of them are involved in the construction of biopolymers, in the synthesis of coenzymes.

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Enzymes catalyze all processes in the cell, so the regulation of microbial metabolism is performed on the enzymatic level through changes in the synthesis and activity of enzymes. Enzymes synthesized regardless on the environmental conditions, are called constitutive, while the enzymes, produced only in a particular environment, – inducible, typically these enzymes are used in the intermediate metabolism. The rate of microbial cell’s metabolism usually depends on the frequency of transcription (formation of messenger RNA-copies of DNA using the enzyme RNA-polymerase). DNA structural genes are responsible for the synthesis of enzymatic proteins. The DNA molecule responsible for transcription of enzymatic proteins consists of several sections (regulatory gene (R) that encode the synthesis of protein-repressor; promoter (R) – gene-activator, which is “recognized” by RNA polymerase, responsible for starting transcription; operator (O), encoding the functioning of structural gene and the structural gene (S) where the formation of iRNA occurs). These areas are connected functionally and when one of them is locked, the transcription is impossible. Promotor, operator and structural gene form the structural unit of the gene-operon. Secondary metabolites are high molecular weight compounds that are not required for growth of pure cultures. Usually they are a mixture of related compounds within the same chemical group (e.g., antibiotics, alkaloids, hormones, toxins, etc.). Secondary metabolites at the early stages of growth and in the period of intensive biomass accumulation have a negative impact on the body-producer, which eventually acquires resistance to them. As a rule, these substances are not a material for the formation of cellular structures and are synthesized at later stages of growth. During lack of one of the sources in nutrient medium, synthesis of secondary metabolites increases dramatically. Therefore, to protect organisms from self-destruction, it is necessary, after reaching rapid growth phase, to maintain a certain level by dosed introduction of nutrients. Based on artificial regulation of the synthesis the secondary metabolites are created by the technology of producing antibiotics, the largest class of pharmaceutical compounds. Control questions: 1. The use of microorganisms in fermentation to obtain a valuable product and their biochemical effects. 2. Use of yeast, molds and bacteria in food production. 3. The main stages of yeast production technological processes and its importance. 4. What are dairy products? 5. Ayran (kefir) and its production technology. 6. Microflora of ayran. 7. Stages of cheese production. 8. What is the role of biopesticides in agriculture? 9. What is the difference between microbial and chemical pesticides?

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Part I. Microbial biotechnology 10. What microorganisms participate in the nitrogen conversion? 11. What is nitrogen fixation? 12. Explain the reaction of nitrogen fixation. What is a nitrogenase? 13. Which types of bacteria participate in degradation? 14. Which factors affect the microbial degradation? 15. What are in-situ and ex-situ technologies? 16. What is the role of microbial metabolism in bioremediation?

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PART IІ ANIMAL BIOTECHNOLOGY

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THE SUBJECT, OBJECTS, TOOLS AND METHODOLOGY OF ANIMAL BIOTECHNOLOGY The developing world is grossly unprepared for the new technological and economic opportunities, challenges and risks that lie on the horizon. In most developing countries, biotechnological applications related to livestock must be made suitable for animal owners who are resource-poor small-scale operators owning little or no land and few animals. Animal biotechnology is quite a new science, which aroused from such basic sciences as Developmental Biology, Cell Biology, Genetics and Molecular Biology, and Gene Engineering. In this regard, we would like to mark the amazing scientific diversity of animal biotechnology: its development and achievements are closely related and depend on a complex of knowledge not only in biological sciences, but also in many other areas (see Figure 1).

Figure 1. Interdisciplinary nature of Animal Biotechnology

Animal Biotechnology is an innovative discipline. Animal Biotechnology provides new technologies for getting basic knowledge in: –– Biology –– Agriculture –– Medicine –– Pharmacology –– Ecology etc. 84

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Animal biotechnology is the use of science and engineering to modify living organisms. Its goal is to make products, to improve animals and to develop organisms for specific agricultural and medicine uses. Animals are playing a growing role in the advancement of biotechnology, as well as increasingly benefiting from biotechnology. Combining animals and biotechnology results in advances in four primary areas: –– Advances in human health –– Improved animal health and welfare –– Enhancements in animal products –– Environmental and conservation benefits Examples of animal biotechnology include creation of transgenic animals (animals with one or more genes introduced by human intervention or animals with a specific inactivated gene using gene knock out technology), chimeric (or alophaenic) animals) and production of nearly identical animals by somatic cell nuclear transfer (or cloning) (see Figure 2).

Figure 2. Three types of animals created by the tools of Animal Biotechnology

Animal biotechnology is a huge field of study including the following topics: use of animals in research, clones, transgenic animals, gene pharming, and animal health. Along with the scientific study, researchers must also deal with many tough scientific and ethical challenges. Animal biotechnology provides new tools for improving human health and animal health and welfare and increasing livestock productivity. Biotechnology improves the food we eat – meat, milk and eggs (see Figure 3).

Figure 3. Animal Biotechnology provides a number of products we use in everyday life

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Biotechnology can improve animal’s impact on the environment. And biotechnology enhances ability to detect, treat and prevent diseases. Just like other assisted reproduction techniques such as artificial insemination, embryo transfer and in vitro fertilization, livestock cloning improves animal breeding programs allowing farmers and ranchers to produce healthier offspring, and therefore to produce healthier, safer and higher quality foods more consistently. Livestock is becoming increasingly important to economic growth in developing countries and application of biotechnology is largely dictated by commercial considerations and socio-economic goals. Using technology to support livestock production is an integral part of viable agriculture in multienterprise systems. Livestock is part of a fragile ecosystem and a rich source of animal biodiversity, as local species and breeds possess genes and traits of excellence. Molecular markers are increasingly used to identify and select the particular genes that lead to these desirable traits, and it is now possible to select a superior germ plasm and disseminate it using artificial insemination, embryo transfer and other assisted reproductive technologies. These technologies have been used in the genetic improvement of livestock, particularly in cattle and buffaloes, and the economic returns are significant. However, morbidity and mortality among animals produced using assisted reproductive technologies lead to high economic losses, so the principal application of animal biotechnology at present is in the production of cheap and dependable diagnostic kits and vaccines. Animal Biotechnology provides new methods of reproduction and saving Animals (see Figure 4): –– Artificial insemination –– In vitro fertilization –– Cloning –– Cryobanking of sperm, eggs and embryos

Figure 4. The basic methods of reproduction and saving animals

Animal Biotechnology to Improve Animal Health and Advance Human Health For decades, farmers have been improving livestock herds through enhanced animal husbandry practices and more modern technologies, such as artificial insemination, embryo transfer, in vitro fertilization, genetic mapping and cloning.

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Through biotechnology, farmers can enhance breeding, resulting in healthier herds. Additionally, the animal health industry has developed treatments that can prevent and treat diseases. New vaccines, diagnostic tests and practices can help farmers treat animal diseases, while reducing food borne pathogens at the farm level. Animals have been used for years to produce medicines for humans. Animal made pharmaceuticals transform biotech animals into “factories” to produce therapeutic proteins in their milk, eggs, and blood, which can be used in the development of biopharmaceuticals. In addition, biotechnology can be used to produce human – compatible transplant organs, tissues and cells in pigs that can be vital to enhancing human health. So, Animal Biotechnology provides new technologies (see Figure 5) for diagnosis and therapy of Animal and Human diseases.

Figure 5. New technologies for diagnosis and therapy of Animal and Human diseases

Biotechnology to Develop More Nutritious Food Improved animal health conditions from vaccines, medicines and diagnostic tests result in safer foods for consumers. In addition, food quality may be improved by introducing desirable traits through introduction of new genes into farm livestock and poultry. In the future, meat, milk and egg products from animals can be nutritionally enriched with the usage of biotechnology. Conservation of Environment and Animals Biotechnology can help produce environmentally friendly animals, as well as conserve endangered species. Farm animals and their feeds have been improved through biotechnology to reduce animal wastes, minimizing the impact on the environment. Today’s reproductive and cloning techniques offer the possibility of preserving the genetics of endangered species. Genetic studies of endangered animals can also result in increased genetic diversity which can result in healthier populations of species. Objects All living animals and their cells can be an object of Animal Biotechnology (see Figure 6). There are laboratory animals, farm animals, pets, rare and endangered

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species. Tissue and cultures are also the objects of Animal Biotechnology. Most applicable tissue and cell cultures are stored in the big scientific centers. For example, National Cancer Institute has more than 60 Human cancer cell lines ((NCI60); ESTDAB database etc.). Among them there are HeLa (cervical cancer), DU145; Lncap, PC3 (prostate cancer), MCF-7, MDA-MB-438 (breast cancer), THP-1 (acute myeloid leukemia), U87 (glioblastoma), SHSY5Y (Human neuroblastoma cells, cloned from a myeloma, Saos-2 cells (bone cancer). In big biobanks there are Primate cell lines: Vero (African green monkey Chlorocebus, kidney epithelial cell line initiated in 1962), Rat tumor cell lines, GH3 (pituitary tumor), PC12 (pheochromocytoma), Mouse cell lines, MC3T3 (embryonic calyarium). Biotechnologists also use other species cell lines (Zebra fish ZF4 and AB9 cells, Madin-Darby canine kidney MDCK, epithelial cell line Xenopus A6, kidney epithelial cells, etc.).

Figure 6. Objects of Animal Biotechnology

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Animal models There are 4 types of animal models, which we use for animal biotechnology aims: –– Living animals These animals are living and usually no threat to their wellbeing. Such animals may be known as laboratory scientific animals (mouse, drosophila, Chinese hamster, sea urchin, xenopus, rat, rabbit etc.). Agriculture research often uses experimental groups of animals. –– Living animal tissues (cell cultures) or systems Animal tissues can be cultured in the lab. This saves use of animals as well as expenses on feeding, housing and cleaning up after animals. –– Non-Living systems Non-living systems include using non=-living mechanical models which reflect animal activity. Very often these relate to skeletal movement and lo­co­ mo­tion. Artificial replacement parts, such as hip joints can be studied using nonliving systems. –– Computer and Mathematical approaches Computer simulation with virtual reality and other applications help in biotechnology. Computer modeling may be done with a proposed biotechnology practice before it is tested with animals. Methodology The methodology of Animal Biotechnology has a long history (see Table 1). We can believe that some of the first biotechnology methods in use include traditional breeding techniques that date back to 5000 B.C. Such techniques include crossing diverse strains of animals (well known as the hybridizing method) to produce a greater genetic variety of animals. The offspring from these crosses are then bred selectively to produce the greatest number of desirable traits. For an example, female horses have been bred with male donkeys to produce mules, and male horses have been bred with female donkeys to produce hinnies, for use as work animals, for the past 3,000 years. This method is still used today. The modern era of biotechnology began in 1953, when James Watson, American biochemist, and Francis Crick, British biophysicist, presented their double-helix DNA model to the world. That was followed by the discovery of the Swiss microbiologist Werner Arber in the 1960s of special enzymes, called restriction enzymes (or restrictases), in bacteria. These enzymes cut the DNA strands of any organism at precise sites. In 1973, American geneticist Stanley Cohen and American biochemist Herbert Boyer removed a specific gene from one bacterium and inserted it into another bacterium strain using restrictases. That event marked the beginning of recombinant DNA technology, or genetic engineering. In 1977, genes from other organisms were transferred to bacteria, the achievement that eventually led to the first transfer of a human gene.

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Part II. Animal biotechnology Some Milestones in the History of Animal Biotechnology

Date 1 3-9000 B.C. 8-9000 B.C. 1400 B.C. 300 B.C. 1651 1665 1674 1760 1780 1856 1859 1891 1900 1919 1935 1944 1947 1949 1950's 1953 1957 1960 1966 1973 1975 1977 1978

Event 2 Domestication of cattle and horses / livestock. Orchiectomy of young bulls. Artificial incubation of eggs. Embryo development systematized. Circulation of blood (Harvey). Plant compartments called "cells" (Hooke). Simple lenses used to see microscopic organisms (Leeuwenhoek). Genetic selection to improve livestock.

Implication(s) 3 Birth of animal agriculture.

Successful artificial insemination (dogs). Existence of microbes demonstrated (Pasteur). On the Origin of Species published (Darwin). First successful embryo transfer (Heape). Application of artificial insemination in food animal breeding (Ivanov). Term "biotechnology" coined (Ereky). First virus discovered. DNA identified as the genetic material. Elements of DNA are transposable (McClintock). Cryoprotectants/ / cyropreservation of sperm. Mammalian tissue culture technology. DNA described as 'double-helix' of nucleotides (Watson and Crick). Liquid nitrogen cyropreservation Radioimmunoassay (RIA) of hormones (Yalow). Microinjection technology developed. DNA from one organism 'recombined' with that of another. Monensin approved as feed additive. Human gene cloned (Itakura). Commercial estrous synchronization (cattle).

Birth of "AI".

Table 1

Growth/behavior modification. Birth of poultry 'industry'. Birth of embryology. Modern Physiological principles. Concept of "cells" born. Birth of microscopy. "Engineering" of livestock begins.

Germ theory described. Theory of evolution. Embryo manipulation technology established. Increased pace of genetic improvement. 'Biotechnology' in the lexicon. Vectors for genetic mutations. Molecular basis of heredity. Concept of natural genetic engineering. Freezing/shipping gametes & cells. Tissues/cells grown in Lab. Gene structure described. Long-term storage of cells/gametes. Assay of hormones at physiological levels. Physical manipulation of genes. "Recombinant DNA" technology. Improved metabolic efficiency (cattle). Genes can be copied. Timed 'AI' and embryo transfer.

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2002 2003

1984-2003

2007 2009

2 First transgenic mice (mice bearing foreign genes). Transfer of murine embryonic stem (ES) cells. Polymerase chain reaction (PCR) described (Mullis). First transgenic domestic animals produced (pig). Targeted gene disruption (gene 'knockout'). Targeted DNA integration and germ-line chimeras (mice). Recombinantly produced growth hormone (rbGH / 'POSILAC') approved for dairy cows. Functional nucleic acid vaccines introduced. Sheep cloned by somatic (body) cell transfer.  Human embryonic stem cells derived. A diagnostic test allows quick identification of Bovine Spongicorm Encephalopathy (BSE, also known as “mad cow” disease) and CreutzfeldtJakob Disease (CJD) FDA approves Gleevec® (imatinib), a gene-targeted drug for patients with chronic myeloid leukemia. Gleevec is the first gene-targeted drug to receive FDA approval. The banteng, an endangered species, is cloned for the first time. China grants the world’s first regulatory approval of a gene therapy product, Gendicine (Shenzhen SiBiono GenTech), which delivers the p53 gene as a therapy for squamous cell head and neck cancer. The Human Genome Project (HGP) completes sequencing of the human genome. FDA approves the H5N1 vaccine, the first vaccine approved for avian flu. FDA approves the first genetically engineered animal for production of a recombinant form of human antithrombin.

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3 Mammalian genetic engineering. Totipotent ES cells aid transgenics. Rapid amplification, detection and cloning of genes. Genetic engineering of livestock. Loss of gene function studies/ therapies. Potential for tissue engineering and gametic transmission of transgenes. Pharmacologically enhanced milk production. Engineering medicines. True mammalian cloning possible. Multiple therapies for genetic and immunological disorders. Quick development of diagnostic tests for farm animal diseases

Pharmacogenetics and targeted therapy are fast developing.

Endangered species cloning researches. Gene therapy products in clinics.

The HGP can help us understand human and animal diseases. Commercial development of genomics research related to DNA based products. Developing of animal disease protecting vaccines. Animals modeling in pharming

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Animal biotechnology in use today is based on the science of genetic engineering. Under the umbrella of genetic engineering there exist other technologies, such as allophaens, transgenics and cloning, which are also used in animal biotechnology. Nowadays 3 basic methodological approaches of Animal Biotechnology (xenotransplantation, cloning, and genetic engineering) do not exist independently. Pointed methodological approaches complement and enrich each other. Figure 7 demonstrates the different genetic background of chimeras, clones, and transgenic organisms.

Figure 7. The genetic background of 3 types of animals produced by biotechnology tools

Chimeras The main methodological approach to get chimeras is transplantation of cells, tissues or organs from one individual to another. The special term – xenotransplantation means the transplantation of living cells, tissues or organs from one species to another. Transplanted cells, tissues or organs are called xenografts or xenotransplants. It is contrasted with allotransplantation when cells, tissues or organs are transplanted from the other individual of same species. If two genetically identical individuals of the same species exchange cell tissue or organs it will be termed as syngeneic transplantation. And Autotransplantation defines the transfer of one part of the body to another in the same individual. Clones Research uses reproductive cloning techniques to produce multiple copies of mammals that are nearly identical copies of other animals, including transgenic animals, genetically superior animals and animals that produce high quantities of milk or have some other desirable trait. To date, cattle, sheep, pigs, goats, horses, mules, cats, rats and mice have been cloned, beginning with the first cloned animal, a sheep named Dolly, in 1996.

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The somatic cell nuclear transfer (SCNT) technology led to the development of Reproductive cloning. Using SCNT scientists remove the nucleus from an egg cell (oocyte) and replace it by the nucleus from a donor adult somatic cell, which is any cell in the body except for an oocyte or sperm. For reproductive cloning, the embryo carrying the nuclei of somatic cell is implanted into the uterus of a surrogate female, where it can develop into a live being. Transgenics Transgenics is the transfer of a specific gene from one organism to another. The approach is also known as recombinant DNA technology. Gene splicing is used to introduce one or more genes of an organism into another organism. A transgenic animal is created once the second organism incorporates the new DNA into its own genetic material. In gene splicing, DNA cannot be transferred directly from its original organism, the donor, to the recipient organism, or the host. Previously the donor DNA must be cut and pasted, or recombined, into a compatible fragment of DNA from a vector – an organism that can carry the donor DNA into the host organism. Often the host organism is a rapidly multiplying microorganism such as a harmless bacterium (E. coli, B. subtilis), which serves as a factory where the recombined DNA can be copied in large quantities. Then, the produced protein can be removed from the host and used as a genetically engineered product in humans, other animals, plants, bacteria or viruses. The donor DNA can be introduced directly into an organism by such techniques as injection through the cell walls of somatic cell or into the fertilized egg of an animal. This transfer of genes alters the characteristics of the organism by changing its protein composition. Proteins, including enzymes and hormones, perform many vital functions in organisms. Individual genes control animal’s characteristics through the production of proteins. Other Technologies In addition to the use of transgenics and cloning, scientists can use gene knock out technology to inactivate, or “knock out,” a specific gene. This technology provides a possible source of replacement of organs for humans. As we mentioned before the process of transplanting cells, tissues or organs from one species to another is referred to as xenotransplantation. Currently, the pig is the major animal considered as a viable organ donor for humans. Unfortunately, pig cells and human cells are not immunologically compatible. Pigs, like almost all mammals, have markers on their cells that enable the human immune system to recognize them as foreign and reject them. Genetic engineering is used to knock out the pig gene responsible for the protein that forms the marker to the pig cells. Milestones of applications –– Improving animals

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Transgenic animals with increased growth rates, enhanced lean muscle mass, enhanced resistance to disease or improved use of dietary phosphorous to lessen the environmental impacts of animal manure. –– Farming Transgenic poultry, swine, goats and cattle that generate large quantities of human proteins in eggs, milk, blood or urine, using these products (enzymes, clotting factors, albumin and antibodies) as human pharmaceuticals. –– The use of animal organs in humans Pigs are currently used to supply heart valves for insertion into humans, but they are also considered as a potential solution to the severe shortage in human organs available for transplant procedures. The Future of Animal Biotechnology –– The government agencies will more actively participate in the regulation of animal biotechnology Likely they will rule on pending policies and establish processes for com­ mercial uses of products created through the transgenic and cloning technologies –– The use of animal organs in human transplant operations. –– Inventions of new methods of in-vitro fertilization (IVF) and preim­ plantation diagnostic (PGD) –– The widespread use of stem cells technologies in medicine Related Issues Positive: –– enhanced nutritional content of food for human consumption; –– a more abundant, cheaper and varied food supply; –– agricultural land-use savings; –– a decrease in the number of animals needed for the food supply; –– improved health of animals and humans; –– development of new, low-cost disease treatments for humans; –– and increased understanding of human diseases. Negative: –– potential effects of transgenic food products on Human; –– potential effects on the environment and the effects on animal welfare; –– the majority of the public is uncomfortable with genetic modifications to animals, cloning of animals and potentially Human, and also IVF technology. Control questions: 1. What are the subject and methods of Animal Biotechnology? 2. Indicate the place of animal biotechnology in the system of Biological Sciences. 3. What do you know about the main stages in the development of Animal Biotechnology? 4. How do you understand the embryological basis of Animal Biotechnology? 5. Can you explain the genetic basis of Animal Biotechnology?

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BIOSAFETY AND BIOETHICS ISSUES IN ANIMAL BIOTECHNOLOGY Before considering ethical points of Animal Biotechnology we need to present some definitions. Biosafety deals with prevention of large scale loss of biological integrity focusing both on ecology and human health. It is related to several fields such as ecology, agriculture, medicine, chemistry and ecobiology. Bioethics is the philosophical study of the ethical controversies brought about by advances in biology and medicine. It is concerned with the ethical questions that arise in the relationships among life sciences, biotechnology, medicine, politics, law, philosophy and theology. It is concerned with the nature of life and death, the kind of life to be considered worth living, what constitutes murder, how people in very painful circumstances should be treated, what are the responsibilities of one human being to the others, and other such living organisms. What do we expect from Animal Biotechnology? There are some answers to this question. First of all, we should expect growing use and demand for agricultural animal biotechnology. Secondly, it is the development of animal biotechnology for biomedical purpose, for needs of Humans and companion animals. This includes the genetic selection, gene therapy, disease management. The government agencies will be more involved in the regulation of animal biotechnology, will likely rule on pending policies and establish processes for commercial uses of products created through the animal transgenic and cloning technologies, the use of animal organs in human transplant operations, inventions of new methods of in-vitro fertilization (IVF) and preimplantation diagnostic (PGD), a widespread use of stem cells technologies in medicine, the new gene therapy protocols for inherited monogenic diseases, neuro-generative and cancer states, the invention of new gene therapy methods. Politicians expect potentially negative effects of transgenic food products on Human, potential effects on the environment and effects on animal welfare, the majority of the public is uncomfortable with genetic modifications of animals, cloning of animals and potentially Human, and also IVF technology. 95

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There are some areas of ethical concern about Animal Biotechnology. First of all, we should mention a possible safety/risk, long/short–term effects, environmental impact, distributive justice. Is really a biotechnologically produced animal harmful? And who benefits from it? In this regard we can note the most discussed problems (see Figure 8): –– Designer babies –– Perfecting humans –– Human cloning –– Genetically modified (GM) food –– Reproductive tourism

Figure 8. Most discussed bioethical problems

Genetic engineering of animals has increased significantly in recent years. In connection with the widespread use of this technology, many ethical issues arise, some of which are related to animal welfare defined by the World Organization for Animal Health as “the state of the animal…how an animal is coping with the conditions in which it lives”. These issues need to be considered by all stakeholders, including veterinarians, to ensure that all parties are aware of the ethical issues at stake and can make a valid contribution to the current debate regarding the creation and use of genetically engineered animals. In addition, it is important to try to reflect societal values within scientific practice and emerging technology, especially publicly funded efforts that aim to provide societal benefits, but that may be deemed ethically contentious. Genetically engineered animals bring extra challenges, and as a result of that, governing organizations have started to develop relevant policies, often calling for increased vigilance and monitoring of potential animal welfare impacts. Veterinarians can play an important role in carrying out such monitoring, especially in the research setting when new genetically engineered animal strains are being developed.

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Several terms are used to describe genetically engineered animals: genetically modified, genetically altered, genetically manipulated, transgenic, and biotechnology-derived, amongst others. In the early stages of genetic engineering, the primary technology used was genetical transformation, transgenesis. These terms mean the transfer of genetic material from one organism to another. However, with advances in the field, new technology emerged that did not necessarily require transgenesis: recent applications allow for the creation of genetically engineered animals via the deletion of genes, or the manipulation of genes already present. Human Genome projects achievements are broadly discussed in the world. We should point it. Benefits of HGP: –– The genes for many diseases (various cancers, Alzheimer’s disease, and polycystic kidney disease) have been already identified. –– Genomic sequencing allows rapid and accurate diagnosis for individuals. –– Earlier detection of genetic predispositions to disease can be used for late onset of genetically-inherited diseases. –– Many gene therapy protocols are being tested –– Other advantages of the sequencing include genetic testing and screening, and its use in reproductive technologies for preimplantation diagnosis. –– We can make rational drug design, so that drugs can be designed to target the cause of the disease. The drugs can be designed for specific individuals, pharmacogenics, “custom drugs”, which will change the prescription of drugs. –– The risk assessment of health damage and risks caused by radiation exposure, including low-dose exposure, and assessment of health damage and risks caused by exposure to mutagenic chemicals and cancer causing toxins, to reduce the likelihood of heritable mutations. –– DNA fingerprinting methods was invented and databases were designed. Since the beginning of the Human Genome Project, many ethical questions were raised. Recognizing that, the U.S. Department of Energy (DOE) and the National Institutes of Health (NIH) allocated 3–5% of their total expenditure on HGP for the Ethical, Legal, Social Implications (ELSI) arising out of the Genome Project. This represented the world’s largest bioethics program. The European Commission only started funding the HGP when it had set up an ELSI program. Since then there have been contemporary efforts going on to answer some of the bioethical challenges of the Human Genome Project. Bioethics can be called as love of life. It is the concept. Bioethics could be viewed in descriptive, prescriptive and interactive ways. Interactive bioethics is discussion and debate between people and groups within society. Different sectors of society have been involved in the HGP, from ordinary people, patients, scientists to industry, governments, legal system, regional and international organizations, and the United Nations.

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To control the bioethics issues many countries developed special documents. The safety of GM foods in the European Commission is controlled by Regulation EC No. 258/97, which had introduced a mandatory premarket safety assessment for all novel foods produced after May 1997. In USA: Food and Drug Administration (FDA) controls GM food safety and distribution. In GB: UK Consumers’ Association. In Kazakhstan the law about genetic engineering activity and GM products is still discussed. –– Due to multiple regulation documents and rules of GM food controlling we can mark the common traits: –– Checking the source of any food is one among the primary duties of individual consumers. –– You should buy GM food only for yourself. –– The first use of GM food labeling arose from the collaboration of two government agencies, Health Canada and the Canadian Food Inspection Agency (CFIA). –– Different countries use different systems of labeling and control for GM food. Reproductive techniques also induce bioethical concern. Development of public attitudes in this field: –– Unnatural/Irreverent – genetic engineering, cloning, Frankenstein, Playing God, hubris –– Valuing of human life –– Speed of scientific developments – black run –– Further breaking link between sex and reproduction –– Perceived threat to “traditional family” and relationships –– Extension of practices for social reasons/convenience –– Addition of desirable characteristics –– Reproductive tourism In this regard I would like to mention the reasonable flexible system controlling reproductive techniques in the UK (Committee of inquiry into human fertilization and embryology, Warnock Reports). Development of stem cells technologies is also regulated by this Committee. Stem cells: what is permitted in the UK: –– Adult stem cells (bone marrow) –– Fetal cells (cord blood) –– Embryonic stem cells (pre-implantation embryos) –– “spare” IVF embryos –– embryos created from donated gametes –– somatic cell nucleus transfer from donated oocytes Techniques not currently permitted in the UK: –– Use of spermatids –– Fragment removal

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–– Ooplasmic transfer –– Selection of embryos for non-medical genetic traits –– Human reproductive cloning It is very important that therapeutic cloning is permitted – cloning of modified (healthy) cells and then transfer to a sick body. But reproductive cloning is not permitted. Creation of the regulating documents in the field of animal biotechnology is not a fast procedure and many points should be taken into account. There are: human rights, animal rights, authority, autonomy, ownership, justice, confidentiality and privacy, responsibility, scientists and social duty, etc. A very good example of regulation rules gives Good Laboratory Practice (GLP). GOOD LABORATORY PRACTICE (GLP) PRINCIPLES 1. Test Facility Organization and Personnel Management’s responsibilities Test facility management should ensure that the principles of Good Laboratory Practice are complied with in the test facility. 2. At minimum it should (a) ensure that qualified personnel, appropriate facilities, equipment, and materials are available; (b) maintain a record of the qualifications, training, experience and job description for each professional and technical individual; (c) ensure that personnel clearly understand the functions they are to perform and, where necessary, provide training for these functions; (d) ensure that health and safety precautions are applied according to national and/or international regulations; (e) ensure that appropriate standard operating procedures are established and followed; (f) ensure that there is a Quality Assurance Program with designated personnel; (g) where appropriate, agree to the study plan in conjunction with the sponsor; (h) ensure that amendments to the study plan are agreed upon and documented; (i) maintain copies of all study plans; (j) maintain a historical file of all Standard Operating Procedures; (k) for each study ensure that a sufficient number of personnel is available for its timely and proper conduct; (l) for each study designate an individual with the appropriate qualifications, training, and experience as the Study Director before the study is initiated. If it is necessary to replace a Study Director during a study, this should be documented; (m) ensure that an individual is identified as responsible for the management of the archives.

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Another ethical question is intellectual property rights. For discussion I’ll give you some examples of special patent application in the field of biotechnology (examples from the USA): –– The Harvard mouse was the first patented transgenic animal –– The polymerase chain reaction (PCR) –– Hepatitis B –– HIV protease inhibitors –– Primate embryonic stem cells Patenting of living organisms USA: Since 1984. Harvard University and the US Patent Office. Harvard applied for a patent on a genetically modified mouse. This was the first time when it was officially decreed that an animal could indeed be classed as an invention. Moreover, it was a mouse specifically engineered to have an increased probability of suffering malignant tumors – for use as a “model” for studying human cancers and carcinogens. Europe: The question of patenting “animate matter” had given long term headaches to the European Patent Office in Munich, and in March 1995, it led to the first ever rejection by a European Commission Directive of the European Parliament. A new draft EC Directive on patenting is currently being discussed in the early committee stages of the European Parliament, and is again the subject of deep seated controversy between industry proponents and many diverse groups which include church groups, NGO’s, environmental and animal welfare organizations, as well as many doctors, farmers and ethicists. You cannot patent a mere discovery. It must have a non-obvious “inventive step”, and some specified practical application. Patent law was framed in an industrial context, and typically applied to objects, chemicals, designs and processes. Agriculture was seen as lying outside this realm. You could patent a mouse trap, but not a mouse. But, with the rise of biotechnology, a shift has occurred. Once it became possible to alter the genetic makeup of living things, researchers could genuinely claim an “inventive step” in the organism itself. Oncomice, transgenic sheep, or whatever: should we be patenting our fellow creatures at all? If this is true, is this a shift in perception we should be counteracting? Patenting the Human Genome–Losing Investments or Losing our Humanity? Sections of the human genome are being identified by the thousand. Should these be patentable, if you could prove they weren’t just “discoveries”? Many US researchers with an eye to the main chance thought “yes”? –– If animals or our own genes are products of nature then we cannot claim an invention. –– If they are nothing more than products – it can be patented.

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–– If they are both, – we need a new system of intellectual property involving living material. Much depends on what we need to patent. We can patent only discovery not invention. –– Parts of the human genome –– Sequence itself –– Identification of human genes function etc. The patent for such research also has a moral dimension. To patent parts of the human genome as such, even in the form of “copy genes”, would be ethically unacceptable to many in Europe. In response it is argued that patenting is the legal assessment of patent claims, and should not be confused with ethics. But now we have brought cancerous mice and human genetic material in the potential frame of intellectual property, ethics has moved to a much more central position. Control questions: 1. What is bioethics? 2. What is biosafety? 3. What do we expect from Animal Biotechnology? 4. Indicate the ethical concerns about Animal Biotechnology. 5. How do bioethics and biosafety control?

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BASIC TECHNIQUES IN ANIMAL CELL CULTURE Cell culture is the process by which prokaryotic, eukaryotic, plant or animal cells are grown under controlled conditions. But due to complexity of animals, as highly specialized multicellular organisms, culturing of cells derived from animal cells requires special conditions. Animal cells are more difficult to culture than microorganisms and plant cells because they require many more nutrients and typically grow only when attached to specially coated surfaces. Rich media are required for culturing of an animal cell. Animal cell culturing requires knowledge of molecular contents of natural medium for cells in the body and factors stimulating their growth. So, it was a long history of scientific events before animal cell culturing became a real methodological approach of animal biotechnology (see table 2). Animal cell culture was first successfully undertaken by Ross Harrison in 1907. But in 1885 Roux first maintained embryonic chick cells in a cell culture. The first development was the use of antibiotics which inhibits the growth of contaminants. The second was the use of trypsin to remove adherent cells to subculture further from the culture vessel. The third was the use of chemically defined culture medium. Milestones in the history of animal cell culturing Date

Who

Table 2

Event

1878

Claude Bernard

Proposed that physiological systems of an organism can be maintained in a living system after the death of an organism. maintained embryonic chick cells in a saline culture.

1885

Roux

1897

Loeb

demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma.

1903

Jolly

observed cell division of salamander leucocytes in vitro.

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1907

Harrison

Frog embryo nerve fiber outgrowth in vitro. (cultivated frog nerve cells in a lymph clot held by the 'hanging drop' method and observed the growth of nerve fibers in vitro)

1912

Alexis Сarrel

Explants of chick connective tissue; heart muscle contractile for 2–3 months

1916 Rous & Jones 1920s/30s Carrel & Ebeling, 1923 1925– Strangeways & 1926 Fell 1940s Keilova, 1948; Cruikshank & Lowbury, 1952 1943 Earle

Trypsinization and subculture of explants Subculture of fibroblastic cell lines Differentiation in vitro in organ culture Introduction of the use of antibiotics in tissue culture (penicillin and streptomycin) Establishment of the L-cell mouse fibroblast cell line; first continuous cell line Growth of virus in cell culture

1949

Enders

1952

Dulbecco

Use of trypsin for generation of replicate subcultures Virus plaque assay

1952

Gey

1955 1959 1961

Eagle Puck & Marcus Sorieul & Ephrussi Macpherson & Stoker

Establishment the first human cell line, HeLa, from a cervical carcinoma Development of defined media Cloning of HeLa on a homologous feeder layer Cell fusion–somatic cell hybridization

1962

Establishment and transformation of BHK21

104 1964 1965 1967 1968 1969 1975

1976 1976 1977

Part II. Animal biotechnology Klein Smith & Pierce Ham Hoober & Cohen Stoker et al. Metcalf Kohler & Milstein

Pluripotency of embryonal stem cells

Illmensee & Mintz Hayashi & Sato Nelson-Rees & Flandermeyer

Totipotency of embryonal stem cells

Serum-free cloning of Chinese hamster cells Epidermal growth factor Anchorage-independent cell proliferation Colony formation in hematopoietic cells Hybridomas-monoclonal antibodies

Growth factor-supplemented serum-free media Confirmation of HeLa cell cross-contamination of many cell lines

Areas where cell culture technology is currently playing a major role. Model systems for: –– Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies –– Toxicity testing –– Study the effects of new drugs –– Cancer research –– Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells Cultivation of virus for vaccine production, also used to study the infectious cycle. Production of commercial proteins, large scale production of viruses for use in vaccine production, e.g. polio, rabies, chicken pox, hepatitis B & measles. During the development the gene therapy method cells having a functional gene can be replaced to cells which have a non-functional gene.

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Types of animal cells which can be cultured Basing on morphology (shape and appearance) or on their functional characteristics, animal cells, able to exist in culture medium, are divided into three types: –– Epithelial like-cells attach to a substrate and appear flattened and polygonal in shape –– Lymphoblast like-cells do not attach, they remain in suspension with a spherical shape –– Fibroblast like-cells attach to a substrate, appear as elongated and bipolar Figure 9 demonstrates the different morphology of animal cells. Media Nine amino acids, referred to as the essential amino acids, cannot be synthesized by adult vertebrate animals and thus must be obtained from their diet. Animal cells grown in culture also must be supplied with these nine amino acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In addition, most cultured animal cells require cysteine, glutamine, and tyrosine. In the intact animal, these three amino acids are synthesized by specialized cells. For example, liver cells produce tyrosine from phenylalanine, and both liver and kidney cells can produce glutamine. Animal cells within the organism and also in culture can synthesize the 8 remaining amino acids; thus, these amino acids need not be present in the diet or culture medium. The other essential components of a medium for culturing animal cells are vitamins, which the cells cannot make at all or in adequate amounts; various salts, glucose, and serum, the noncellular part of the blood.

Figure 9. Cell morphologies strongly depend on the cell type.

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Serum is a mixture of hundreds of proteins. Serum contains various factors needed for proliferation of cells in culture. For example, serum contains insulin, a hormone required for growth of many cultured cells of vertebrates, and transferrin, an iron-transporting protein, essential for incorporation of iron by cells in culture. Although many animal cells can grow in a serum-containing medium, such as Eagle’s medium, certain cell types require specific protein growth factors that are not present in serum. For instance, precursors of red blood cells require the hormone erythropoietin, and T lymphocytes of the immune system require interleukin 2 (IL-2). These factors bind to receptor proteins that span the plasma membrane, signaling the cells to increase in size and mass and undergo cell division. A few mammalian cell types can be grown in a completely defined, serumfree medium supplemented with trace minerals, specific protein growth factors, and other components. The three basic classes of media, which differ in their requirement for supplementation with serum, are: basal media, reduced serum media, and serum free media. Basal (Basic) Media: –– Basal Medium is a defined medium that contains essential and nonessential amino acids, vitamins, inorganic salts, organic compounds, and trace elements, but does not contain the Growth Supplements necessary for cell growth. –– Balanced salt solutions (BSS) e.g. phosphate-buffered saline (PBS) –– DMEM and RPMI 1640 (with or without glutamine) Reduced-Serum Media: –– Reduced-serum media are basal media formulations enriched with nutrients and animal-derived factors, which reduce the amount of serum that is needed. Serum-Free Media: –– Serum-free media (SFM) circumvents issues with using animal sera by replacing serum with appropriate nutritional and hormonal formulations. –– Serum-free media formulations exist for many primary cultures and cell lines, including Chinese Hamster Ovary (CHO), hybridoma cell lines, VERO, MDCK, MDBK cell lines etc. –– One of the major advantages of using serum-free media is the ability to make the medium selective for specific cell types by choosing the appropriate combination of growth factors. The choice of media depends on the type of cell being cultured. The components or reagents of suitable culture media include balanced salt solution, buffers and chemicals, cell dissociation reagents, and supplements. Balanced salt solutions can provide an environment that maintains structural and physiological integrity, pH and osmotic pressure of cells in vitro. It maintains osmolarity, regu-

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lates membrane potential (Na+, K+, Ca2+) and provides ions for cell attachment and enzyme cofactors. Cell dissociation reagents are the cell detachment solutions of proteolytic and collagenolytic enzymes (Papain, powder; Trypsin 0.25% (1X), liquid; Trypsin 2.5% (10X), liquid; Dispase (Neutral Protease), powder; Elastase, powder; Hyaluronidase; Pepsin A, powder; Collagenase type 1,2,3,4; Trypsin Inhibitors) Supplements include L-glutamine; Glutamax™; Non-essential amino acids (NEAA); Growth Factors and Hormones (e.g.: insulin); Vitamines (MEM Vitamin Solution); 2-Mercaptoethanol; Lipid Supplement; CHO Supplement. To avoid microbial contamination the medium is supplemented with antibiotics viz. penicillin, streptomycin etc. Prepared medium is filtered and incubated at 4 °C. On the market there are many animal cell culture media: Minimum Essential Medium (MEM); GMEMm (Glasgow Minimum Essential Medium); EMEM (Eagle’s MEM); DMEM (Dulbecco’s Modified Eagle Medium); Medium 199; BME (Basal Medium Eagle); Ham’s F-10 Medium; Ham’s F-12 Medium; RPMI 1640 medium; Leibovitz L-15 medium; CMRL 1066; Dulbecco’s Modified Eagle’s Medium (DMEM-001); MCDB 131; McCoy’s 5A. But commonly used Medium are GMEM, EMEM, DMEM etc. Conditions Within the tissues of intact animals, most cells of organism tightly contact and interact specifically with other cells via various cellular junctions. The cells also contact with the extracellular matrix, a complex network of secreted proteins and carbohydrates that fills the spaces between cells. The extracellular matrix constituents are secreted by cells themselves. The matrix helps form the tissue binding the cells in tissues together. It also provides a lattice through which cells can move, particularly during the early stages of animal differentiation. In various animal tissues the extracellular matrix consists of several common components: fibrous collagen proteins, hyaluronan (or hyaluronic acid), a large mucopolysaccharide, and covalently linked polysaccharides and proteins in the form of proteoglycans (mostly carbohydrate) and glycoproteins (mostly protein). However, the exact composition of the matrix in different tissues depends on the specialized function of the tissue. For example, in the connective tissue the major protein of the extracellular matrix is a type of collagen that forms insoluble fibers with a very high tensile strength. Fibroblasts, the basic cell type in the connective tissue, secrete this type of collagen as well as the other matrix components. Receptor proteins in the plasma membrane of cells bind various matrix elements, imparting strength and rigidity to tissues. The ability of animal cells in vivo interact with one another and with the surrounding extracellular matrix is mimicked in their growth in culture. In contrast to bacterial and yeast cells, which can be grown in suspension, most cultured

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animal cells require a surface (often agarizied) to grow on (see Figure 10). Many types of cells can adhere to and grow on glass, or on specially treated plastics with negatively charged groups on the surface (e.g., SO32-). The cultured cells secrete collagens and other matrix components. These bind to the culture surface and function as a bridge between it and the cells. Cells cultured from single cells on a glass or a plastic dish form visible colonies in 10–14 days. Some tumor cells can be grown in suspension because of the tumor transformation of adhesion and motility behavior. This is a considerable experimental advantage because equivalent samples are easier to obtain from suspension cultures than from colonies grown in a dish.

Figure 10. Monolayer of cells, adherent cells

Once the available substrate surface is covered by cells (a confluent culture) the growth slows and ceases. Cells to be kept in a healthy and growing state have to be sub-cultured or passaged. It is the passage of cells when they reach 80-90% confluence in flask/dishes/plates. Explantation techniques (see Figure 11) depend on the cell type and supporting culture cell equipment. Adherent cells: –– Cells which are anchorage dependent –– Cells washed with PBS (free of Ca & Mg) solution. –– Add enough trypsin/EDTA to cover the monolayer –– Incubate the plate at 37 C for 1-2 mins –– Tap the vessel from the sides to dislodge the cells –– Add complete medium to dissociate and dislodge the cells with the help of pipette which remain to be adherent –– Add complete medium depends on the subculture requirement either to 75 cm or 175 cm flask Suspension cells: –– Easier to passage as no need to detach them –– As the suspension cells reach confluence –– Asceptically remove 1/3rd of medium –– Replaced with the same amount of pre-warmed medium

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Figure 11. Explantation techniques.

Cells are cultured as anchorage dependent or independent. Derived from normal tissues cell lines are considered as anchorage-dependent, they grow only on a suitable substrate, for example, the tissue derived cells. Suspension cells are anchorage-independent, for example, the blood cells. Transformed cell lines either grow as a monolayer or as a suspension. Such enzymes as trypsin, dipase, collagenase in combination with EDTA break the cellular glue that attached the cells to the surface. Basic environmental requirements for “happy” cells are controlled temperature, good substrate for cell attachment, appropriate culture medium and CO 2-incubator that maintains the correct pH and osmolality. The control of cell toxicity is very important for culturing of animal cells. Cytotoxicity causes inhibition of cell growth. Then morphological alteration in the cell layer or cell shape is observed. Characteristics of abnormal morphology are giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleus. Cytotoxicity is determined by substituting materials such as medium, serum, supplement flasks, etc. Cell culture contaminants of two types: –– Chemical contaminants are difficult to detect, they are caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible. –– Biologically-caused visible effects on the culture are caused by mycoplasma, yeast, bacteria or fungus or cross-contamination of cells from other cell lines. Biological contaminants compete for nutrients with host cells. Secreted acidic or alkaline by-products cease the growth of the host cells. Degraded arginine & purine inhibits synthesis of histone and nucleic acid. It also produces H2O2 which is directly toxic to cells. In general, indicators of contamination are turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, grain-

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ing cellular appearance, vacuolization, inclusion bodies and cell lysis. Yeast, bacteria & fungi usually show visible effect on the culture (changes in medium turbidity or pH). Mycoplasma is detected by direct DNA staining with intercalating fluorescent substances, e.g. Hoechst 33258. Mycoplasma is also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA. The best and the oldest way to eliminate contamination is to discard the infected cell lines directly. Basic aseptic conditions: –– If working on the bench, use a Bunsen flame to heat the air surrounding the Bunsen. –– Swab all bottle tops and necks with 70% ethanol. –– Flame all bottle necks and pipettes by passing very quickly through the hottest part of the flame. –– Avoid placing caps and pipettes down on the bench; practice holding bottle tops with the little finger. –– Work either left to right or vice versa, so that all material goes to one side, once finished. –– Clean up spills immediately and always leave the work place neat and tidy. Safety aspect in cell culture: –– Possibly keep cultures free of antibiotics in order to be able to recognize the contamination. –– Never use the same media bottle for different cell lines. If caps are dropped or bottles touched unconditionally, replace them with new ones. –– Necks of glass bottles prefer heating at least for 60 s at a temperature of 2000C. –– Switch on the laminar flow cabinet 20 mins prior to start working. –– Cell cultures, which are frequently used, should be subcultered and stored as duplicate strains. Rules for working with cell culture: –– Never use contaminated material within a sterile area. –– Use the correct sequence when working with more than one cell lines. Figure 12 demonstrates how the animal cell culturing should be controlled.

Figure 12. Control for culture contamination

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Basic equipment used in cell culture: –– Laminar cabinet: Vertical are preferable. –– Incubation facilities: Temperature of 25-300C for insect and 370C for mammalian cells, CO2 2-5% and 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approximately 38.50C. –– Refrigerators: Liquid media kept at 40C, enzymes (e.g. trypsin) and media components (e.g. glutamine & serum) at -200C. –– Microscope: An inverted microscope with 10x to 100x magnification. –– Tissue culture ware: Culture plastic ware treated by polystyrene. How to do tissue culture? Tissue culture means in vitro cultivation of organs, tissues and cells at defined temperature using an incubator supplemented by a medium containing cell nutrients and growth factors. Different types of cells grown in vitro culture include connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (from liver, breast, skin, kidney) and many different types of tumor cells. Tissue culture is used as a generic definition to include the in vitro cultivation of organs, tissues or cells. The ability to survive and grow tissues outside the body in an artificial environment is very important for preparing the tissue culture. Tissue culture can be subdivided into three major categories: cell culture, organ culture, explant culture (see Figure 13).

Figure 13. Types of tissue culture

Figures 14 demonstrates the main steps for preparing the tissue culture.

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Figure 14. A typical scheme of the tissue culture

Organ culture The entire embryos or organs are excised from the body and culture. Organ culture refers to a three-dimensional culture of tissue retaining some or all of the histological features of the tissue in vivo. (Histological structure maintained) The tissue is cultured at the liquid–gas interface (on a grid or gel), which favors the retention of a spherical or three-dimensional shape. Advantages: –– Normal physiological functions are maintained. –– Cells remain fully differentiated. Disadvantages: –– The particular cells of interest may be very small in number in a piece of tissue so the response produced may be difficult to detect and quantify. Interactions between epithelial and mesenchymal tissues constitute a central mechanism regulating the development of most embryonic organs. Studies on the nature of such interactions require the separation of the interacting tissues from each other and the follow-up of their advancing development in various types of recombined explants. The tissues can be either transplanted and their development followed in vivo, or they can be cultured as explants in vitro. Although the transplantation methods offer certain advantages, including physiological environment and the possibility for long-term follow-up, organ culture techniques are superior in many other aspects. The cultured tissues can be manipulated in multiple ways, and their development can be continuously monitored. The culture conditions are reproducible, and the composition of the medium is known exactly and can be modified. Furthermore, the in vitro culture conditions allow analyses of the nature of the inductive signals.

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Many types of organ culture systems have been used over the years for studies on embryonic organ development. The Trowell method (see Figure 15) has been widely applied, and it has proven to be suitable for the analysis of the morphogenesis of many different organs. In this system, the explants are cultured in vitro at the medium/gas interface on thin membrane filters supported by a metal grid. We have used the Trowell technique as modified by Saxen.

Figure 15. Schematic representation of the Trowell method (Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ)

The most popular methods include the use of a grid combined with the growth on filter or agar media (see Figure 16).

Figure 16. Organ culture: using the agar (A, B, C) and filter (D) on the grid.

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Organ culture techniques include: 1. Plasma clot method or Watch glass method 2. Organ Culture on Agar (Agar gel method) –– Raft method –– Grid method 3. Organ Culture in Liquid Media (using filter) –– Trowell method Primary cell culture The primary culture is a term for the culture derived from cells surgically or enzymatically removed from an organism and placed in a suitable culture environment, which attached and grow together. A cell strain is a lineage of cells originating from the primary culture. Normal animal tissues (skin, kidney, liver etc.) or whole intact embryos are commonly used to establish the primary cell cultures. To prepare tissue cells for culture (also to remove adherent cells from a culture dish for biochemical or molecular-genetic studies), trypsin or other protease is used to destroy the proteins in the junctions, which normally interconnect cells. Most cell types were difficult, if not impossible, to culture for many years because they need stimulating growth factors. The identification and preparation of various protein growth factors which can stimulate the replication of specific cell types, as well as other recent modifications in culture protocols, now permit scientists to grow various types of highly specialized cells. However, many studies with vertebrate cells are still performed with those few cell types that grow most readily in culture. These are not cells of a defined type, rather, they represent whatever grows when a tissue or an embryo is placed in culture. The cell type that usually predominates in such kind of cultures is called a fibroblast because it secretes the types of proteins associated with fibroblasts in fibrous connective tissue of animals. Cultured fibroblasts have the morphology of tissue fibroblasts, but they retain the ability to differentiate into other cell types; thus, they are not as differentiated as tissue fibroblasts. Some studies are performed with primary cultures of epithelial cells. Basically, external and internal surfaces of tissues and organs are covered by a layer of epithelial cells known as an epithelium. These highly differentiated cells are polarized because the plasma membrane is organized into at least two discrete regions. Certain cells cultured from blood, spleen, or bone marrow adhere poorly, if at all, to a culture dish but nonetheless grow well. In the body, such non-adherent cells are held in suspension (for example, in blood), or they are weakly adherent (for example, in the bone marrow or spleen). As these cells often come from immature stages of the development of differentiated blood cells, they are very

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useful for studying normal blood cell differentiation and the abnormal development of leukemia. In cases when cells are removed from an embryo or an adult animal, most of the adherent ones grow continuously in culture for only a limited time period before they spontaneously cease growing. Such a culture eventually dies out after many cell divisions, even if it is provided with fresh supplies of all known nutrients that cells need to grow, including serum. For instance, when human fetal cells are explanted into cell culture, the majority of cells die within a relatively short time. “Fibroblasts,” although also destined to die, proliferate for a while and soon become the predominant cell type. They divide about 50 times before they cease growth. Starting with 106 cells, 50 cell divisions can produce 106×50, or more than 1000 cells, which is equivalent to the weight of more than 100 people. So, even though its lifetime is limited, a single primary culture, if carefully maintained, can be studied through many generations. Such a lineage of cells originating from one initial primary culture is called a cell strain. The traits of primary cell culture: –– Primary cells have a restricted life span –– Primary culture contains a very heterogeneous population of cells –– Sub culturing of primary cells leads to the generation of cell lines –– Cell lines have a limited life span. They pass several times before they become senescent. –– Cells such as macrophages and neurons do not divide in vitro conditions, so cannot be used as primary cultures. Biologists often want to maintain cell cultures for many generations, more than 100 cell divisions because they want to clone an individual cell type, or a specially transformed single cell, modify cell behavior or select mutants. This opportunity is possible with cells derived from some tumors and with rare cells that arise spontaneously because they have undergone genetic changes that endowed them with the ability to grow indefinitely. The genetic changes that allow these cells to grow indefinitely are due to oncogenic transformation. And such cells are oncogenically transformed and become immortalized. A culture of cells with an indefinite life span is called immortal, and such culture is called a cell line to distinguish it from an impermanent cell strain. The ability of cultured cells to grow indefinitely or their possibility to be transformed varies depending on the animal species from which the cells were extracted. For instance, normal chicken cells are rarely transformed and die out after only a few cell divisions, even tumor cells from chickens almost never exhibit immortality. Among human cells, only tumor cells are immortalized and grow indefinitely. The HeLa cell, the first human cell type to be grown in culture, was originally obtained in 1952 from a malignant tumor (cervical carcinoma). This cell line exists for more than 60 years and has been invaluable for research on human cells.

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In contrast to described human and chicken cells, cultures of embryonic adherent cells from rodents routinely give rise to cell lines. When adherent rodent cells are first explanted, they grow well, but after a number of serial replantings they lose potential to growth, and the culture goes into crisis (see Figure 17). During this period most of the cells in the culture die, but often a rapidly dividing variant (mutant) cell arises spontaneously and takes over the culture. A cell line derived from such a mutant variant will grow forever if it is provided with the necessary nutrients. Cells in spontaneously established rodent cell lines and in cell lines derived from tumors often have abnormal chromosomes or chromosomal rearrangements. In addition, their chromosome number is usually greater than in the normal cell from which they arose, and it continually expands and contracts in culture. Cells having an inappropriate number of chromosomes are said to be aneuploid and are obviously mutants.

Figure 17. Stages in the establishment of a cell culture (Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W.H. Freeman; 2000)

Although most cell lines are undifferentiated, some of these can have many functional characteristics of the normal differentiated cells from which they are derived. One example is provided by some hepatoma cell lines (HepG2) that synthesize most of the serum proteins produced by normal hepatocytes (the major cell type in the liver) from which they are derived. The HepG2 represented by highly differentiated hepatoma cells are commonly studied as models of normal

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hepatocytes. Cultured myoblasts, muscle precursor cells, are another example of transformed cells that continue to exhibit many functions of a specialized, differentiated cell. Transformed myoblasts grown in the culture can be induced to fuse and form myotubes. These resemble differentiated multinucleated muscle cells and synthesize many of specialized proteins associated with contraction. Certain lines of epithelial cells from different sources have also been cultured successfully. One such line, Madin-Darby canine kidney (MDCK) cells, can form a continuous sheet of polarized epithelial cells one cell thick that exhibits many of the properties of the normal canine kidney epithelium from which the cell line was derived. The MDCK line has proved valuable as a model for studying the functions of epithelial cells. Continuous cell lines Most cell lines grow for a limited number of generations after which they cease. Cell lines either occur spontaneously or are induced virally or chemically transformed into Continuous cell lines. Characteristics of continuous cell lines: –– Cells are smaller, more rounded, less adherent with a higher nucleus/ cytoplasm ratio. –– Cells are able to fast growth and have aneuploid chromosome number. –– Cells have reduced serum and anchorage dependence and grow more in suspension conditions. –– Culture is able to grow up to a higher cell density. –– Cells are different in phenotypes from donor tissue. –– Cells stop expressing tissue specific genes. Figure 18 demonstrates the ability to growth of animal cells.

Figure 18. Growth of animal cells in culture

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Figure 19 was created to summarize knowledge about types of cell lines in animal cell culturing.

Figure 19. Isolation of cell lines in vitro culture.

Hybridoma or Interspecific Hybrids Cultured animal cells infrequently undergo cell fusion spontaneously. However, the fusion rate increases greatly in the presence of certain viruses that have a lipoprotein envelope similar to the plasma membrane of animal cells. A mutant viral glycoprotein in the envelope promotes cell fusion. Cell fusion can also be promoted by chemicals – polyethylene glycol, which causes the plasma membranes of adjacent cells to adhere to each other and to fuse. As most fused animal cells undergo cell division, the nuclei eventually fuse, producing viable cells with a single nucleus (instead of two) that contains chromosomes from both parents. Fusion of two cells that have genetically different origins leads to formation of a hybrid cell called a heterokaryon. Cultured cells from different mammals can be fused to produce interspecific hybrids, which have been widely used in genetics of the somatic-cell. For instance, hybrids can be obtained from human cells and mutant mouse cells that lack an enzyme required for synthesis of a particular essential metabolite. As the human-mouse hybrid cells grow and divide, they gradually lose human chromosomes in a random order, but retain the mouse chromosomes. In a medium supporting growth of both human cells and mutant mouse cells, the hybrids eventually lose all human chromosomes. However, it is interesting that in a medium lacking the essential metabolite that the mouse cells cannot produce, the one human chromosome which contains the gene encoding the needed enzyme will be

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retained, because any hybrid cells that lose it following mitosis will die. All other human chromosomes are eventually lost. Researchers have prepared various panels of hybrid cell lines using different mutant mouse cells and media in which they cannot grow. Each cell line in such panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes. As each chromosome can be identified visually under a light microscope, such hybrid cells provide a means for assigning or “mapping” individual genes in specific chromosomal sites. For instance, suppose a hybrid cell line is shown microscopically to contain a particular human chromosome. That hybrid cell line can then be tested biochemically or molecular genetically for the presence of various human enzymes or genes, exposed to specific antibodies to detect human surface antigens, or subjected to DNA hybridization and cloning techniques to locate particular human DNA sequences. The genes encoding a human protein or containing a human DNA sequence detected in such tests must be located on a particular human chromosome carried by the cell line being tested. Panels of hybrids between normal mouse and mutant hamster cells have also been established; in these hybrid cells, the majority of mouse chromosomes is lost, allowing mouse genes to be mapped to specific mouse chromosomes. The hybridomas are often used for monoclonal antibodies production (see Figure 20). For this purpose there are some specialized media, which allow us to select mutant and wild type variants (for example, HAT medium). But this question will be described further in regard with animal cell markers.

Figure 20. The usage of hybridomas for monoclonal antibodies production (https://en.wikipedia.org/wiki/Hybridoma_technology)

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Part II. Animal biotechnology Control questions: 1. Describe main applications of cell cultures. 2. Indicate the significance of in vitro cultivation technique. 3. What discoveries are the milestones in the history of in vitro cultivation? 4. Indicate the areas where the cell culture technology is currently playing a major role. 5. Describe the most important principles of protocols for in vitro cultivation of animal cells

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Chapter 4. Stem cells technology

CHAPTER

4

STEM CELLS TECHNOLOGY Diversity of Human Cells Adult humans consist of more than 200 kinds of cells. They are nerve cells (neurons), muscle cells (myocytes), skin (epithelial) cells, blood cells (erythrocytes, monocytes, lymphocytes, etc.), bone cells (osteocytes), and cartilage cells (chondrocytes). There are cells essential for embryonic development but not incorporated into the body of the embryo, they are included into the extra-embryonic tissues, placenta, and umbilical cord. All these cells are generated from a single, totipotent cell, the zygote, or fertilized egg (see Figure 21).

Figure 21. The origin of Stem Cells (https://www.researchgate.net/figure/8551939)

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Stem cells are undifferentiated cells that can differentiate into specialized types of cells and can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. Mammals represent two broad types of stem cells: embryonic stem cells, which can be isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. The main function of stem cells and progenitor cells in adult organisms is reparation of the body, replenishing of adult tissues. Stem cells of the developing embryo can differentiate into all the specialized cells – ectoderm, endoderm and mesoderm (induced pluripotent stem cells), but can also maintain normal homeostasis of regenerative organs, such as blood, skin, or intestinal tissues. A stem cell is a “blank” cell/precursor cell that can give rise to multiple tissue types such as a skin, muscle, or nerve cell. Essentially a stem cells acts as a building block of the body. Features of Stem Cells: –– Stem Cells are very unique cells. –– Stem Cells are able to develop into multiple cell types in the body. –– Stem Cells act as a repair system for the body. –– Stem Cells can theoretically divide unlimitedly in a living organism in order to replenish various types of cells. –– When a stem cell undergoes the division, each new (daughter) cell has a potential to either remain a stem cell or become another (differentiated) type of cell with a more specialized function (for example, a muscle cell, a red blood cell, a brain cell, etc.). Stem cells have three unique properties which allow them to differ from other cells in the body: –– Stem cells are capable to divide and renew themselves for long periods. –– Stem cells are “unspecialized” and can differentiate and give rise to specialized cell types. –– A stem cell fate is “uncommitted”, until cell receives a signal to develop into a specialized cell. Stem cells have the ability to divide asymmetrically. It means that one portion of the cell division becomes a differentiated cell while the other becomes another stem cell. Stem cells are unspecialized. A stem cell does not have any tissue-specific structures that allow it to perform specialized functions of body organs. A precursor stem cell in the tissue cannot function as a neighboring cell in the tissue, for example, to pump blood through the body like a heart muscle cell. It also cannot carry molecules of oxygen through the bloodstream like a red blood cell and it cannot fire electrochemical signals to other cells that allow the body to move like a nerve cell. Stem cells are capable to divide and renew themselves for long periods. Stem cells may undergo mitosis divisions and replicate many times. The following di-

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visions of cell called proliferation. In the laboratory the stem cells can proliferate for many months yielding millions of cells. So, stem cells are capable of longterm self-renewal. Stem cells can differentiate and give rise to specialized cell types. The term differentiation means the process when unspecialized stem cells become specialized. Stem cell differentiation is triggered by signals inside and outside cells. The internal signals are controlled by cell’s own genes. The external signals are chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. Stem cells exist in both embryos and adults. Stem cells function in embryos to generate new organs and tissues. In adults, their function is to replace cells during the natural course of cell turnover. You should know the distinguishing features of progenitor/precursor cells and stem cells (see Figure 22). The figure demonstrates that the stem cell is an undifferentiated cell which develops into a variety of specialized cell types. The results of stem cell division give one additional stem cell and a specialized cell. For instance, a hematopoietic stem cell produces a second generation stem cell and a neuron. A progenitor cell (a precursor cell) is capable of undergoing cell division and can yield two specialized cells. For example, a myeloid progenitor/ precursor cell undergoing cell division yields two specialized cells – a neutrophil and a red blood cell.

Figure 22. Distinguishing features of progenitor/precursor cells and stem cells (https://www.researchgate.net/figure/8551939)

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Two main properties characterize the stem cells: 1) Self-renewal – The ability to go through numerous cycles of cell division maintaining the undifferentiated state. 2) Potency – The capacity to differentiate into specialized cell types. This requires stem cells to be either totipotent or pluripotent, that means have a possibility to give rise to any mature cell type. Sometimes multipotent or unipotent progenitor (precoursor) cells are referred to as stem cells. But it is a narrow application of this term as a result of the clinical usage a tissue progenitor cells for restoration of one or a limited number of cell types in this tissue. Real stem cell function should be regulated in a feedback mechanism. Self-renewal Two approaches exist to ensure that a stem cell population is maintained: 1) The mechanism of obligatory asymmetric replication: a parent stem cell divides into one mother cell which is identical to the original stem cell, and another daughter cell which is differentiated. 2) The mechanism of stochastic differentiation: when one parent stem cell develops into two differentiated daughter cells, another stem cell undergoes mitosis giving two stem cells identical to the original parent cell. Potency definition The term potency specifies the potential of differentiation of the stem cell, which means the potential to differentiate into different cell types. Totipotent stem cells can differentiate into embryonic and extraembryonic cell types. Totipotent stem cells can create a complete, viable organism. This kind of cells is produced from the fusion of an egg and a sperm cell. In animals, cells of early embryo produced by the first few divisions of the fertilized egg are also totipotent. Pluripotent stem cells are the descendants of totipotent cells of early embryo and can differentiate into nearly all types of cells, i.e. cells derived from any of the three germ layers. Multipotent stem cells can differentiate into a limited number of cell types, and only those of a closely related family of cells. Oligopotent stem cells differentiate into only a few cell types, such as lymphoid or myeloid stem cells. Unipotent cells can differentiate into only one cell type, their own, but have the property of self-renewal, that distinguishes them from non-stem cells (for example, progenitor cells, muscle stem cells). Figure 23 demonstrates types of stem cells taking into account the potency of cells.

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Figure 23. Potency characterized types of stem cells

In Mammalians totipotent stem cells are the products of only first divisions of zygote. The fertilized egg is totipotent. The origin of word came from the Latin totus, meaning “entire”. It has the potential to generate all cells and tissues that make up an embryo. It supports embryonic development in utero. Between different kinds of Mammalians there is some restriction of totipotency, which can differ. For example, the stage of 8-celled embryo – in Human and Mouse, and for Cattle – the 16-celled embryo stage. “Pluri” is derived from the Latin plures and means several or many. Thus, pluripotent cells have the potential to differentiate to any type of cell. Pluripotent stem cells are descendants of the totipotent stem cells of the embryo. These cells develop for about four days after fertilization. They can give rise into any cell type, except for totipotent stem cells and the placenta cells. Pluripotent, embryonic stem cells originate as inner cell mass (ICM) cells within a blastocyst (see Figure 24). These cells cannot re-create a complete organism but differentiate to a large number of mature tissue types, for example, brain and muscle. These stem cells can give rise to any tissue in the body, excluding a placenta. Only cells from an earlier stage of the embryo (before morula stage) are totipotent, able to give rise to all tissues in the body and the extraembryonic placenta. As mentioned before, the multipotent stem cells (see Figure 25) are descendants of pluripotent stem cells and antecedents of differentiated cells in particular tissues. For instance, hematopoietic stem cells, which were found primarily in the bone marrow, give rise to all cell types found in the blood, including red blood cells, white blood cells, and platelets.

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Figure 24. The origin of pluripotent stem cells (https://www.researchgate.net/figure/8551939)

Figure 25. The differentiation ways for multipotent stem cells (https://www.researchgate.net/figure/8551939)

Figure 26 demonstrates the origin of Human stem cells taking into account the potency feature.

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Figure 26. Developmental stages and potency of Human stem cells (http://www.celldiagram.net/frequently-asked-questions-on-stem-cells.html)

Figure 27 demonstrates the differentiation potential of the bone marrow stem cells.

Figure 27. Bone marrow stem cells differentiation potential. (https://stemcells.nih.gov/info/basics/4.htm)

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Unipotent stem cells are the cells of adult organisms. These cells are capable of differentiating only along one cell lineage. The word “Uni” is derived from the Latin word unus, which means one. Thus, Progenitor cells are unipotent stem cells, which can produce only one specialized cell type. For instance, erythroid precoursour cells differentiate into only red blood cells. Terminally differentiated cells represent the end of the long chain of cell divisions. These cells are permanently committed to specific functions, such as a liver cell or lung cell. Stem cell сlasses The class of animal stem cells depends on their potency. In animals, there are 2 main stem cell types (see Figure 28) that come from different places in the body or were formed at different times in the live cycle: 1) embryonic stem cells (ESC) that exist only at the earliest stages of development, which are different for different animals due to their ontogenesis peculiarities; 2) various types of tissue-specific basal cells (or adult stem cells – ASC), which include the progenitor cells that appear during fetal development and remain in animal and human bodies throughout life. The new class of stem cells was recently developed by biotechnologists using reprogramming of potency of adult cells – induced pluripotent stem cells (iPSCs).

Figure 28. Different types of stem cells

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Embryonic Stem Cells (ESC) Embryonic stem cells class includes 2 subtypes – embryonic stem cells and embryonic germ cells. ESC type presents stem cell colonies that are not differentiated. Properties of Embryonic Stem Cells: –– Derived from the inner cell mass of the blastocyst. –– Capable to undergo an unlimited number of symmetrical divisions without differentiating. This property is called as long-term self-renewal. –– Exhibit and maintain a stable set of diploid number of chromosomes (karyotype). –– Pluripotent ES cells can give rise to differentiated cell types that are derived from all three primary germ layers of the embryo (endoderm, mesoderm, and ectoderm). ESC was first extracted in 1981from a Mouse embryo by Cf. G.R. Martin et al. Isolation of Human ESC was first done by James Thomson’s team in 1998. hESCs were isolated by transferring the inner cell mass of a 3-5-day old embryo onto a mouse fibroblast feeder layer. If the embryonic stem cells are grown in culture under appropriate conditions, ESC can remain undifferentiated or unspecialized. But if cells are clump together, they can form embryoid bodies and begin differentiate spontaneously growing up into all types of differentiated cells such as muscle cells, nerve cells, and many other cell types. Although spontaneous differentiation is a good sign that a culture of embryonic stem cells is healthy, the differentiation process is uncontrolled and therefore represents an inefficient strategy to produce cells of certain specific cell types. Thus, to produce cultures of specific types of differentiated cells (for example heart muscle cells, blood cells, or nerve cells) the scientists try to control the differentiation of embryonic stem cells. The approach is to change the chemical composition of the culture medium, alter the surface of the culture dish, or modify the cells by inserting specific genes. The results of numerous experiments are some basic protocols for the directed differentiation of embryonic stem cells into some specific cell types (see Figure 29). If scientists can reliably direct the differentiation of embryonic stem cells into specific cell types, they may be able to use the resulting, differentiated cells to treat certain diseases in the future. Diseases that might be treated by transplanting cells generated from human embryonic stem cells include diabetes, traumatic spinal cord injury, Duchenne’s muscular dystrophy, heart disease, and vision and hearing loss.

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Figure 29. Directed differentiation of mouse ESC (https://stemcells.nih.gov/info/basics/4.htm)

Regarding Human there are two main sources of embryonic type stem cells: embryos and fetus. ESCs are obtained by harvesting living embryos which are generally 5-7 days old. The removal of embryonic stem cells invariably results in the destruction of the embryo. Another kind of stem cell, called an embryonic germ cell, can be obtained from either miscarriages or aborted fetuses. Potential sources of stem cells are: –– fetal tissue that becomes available after an abortion –– excess embryos from assisted reproductive technologies such as commonly used in fertility clinics –– embryos created through in vitro fertilization specifically for research purpose, and –– embryos created asexually as a result of transfer of a human somatic cell nucleus to an egg with its own nucleus removed. –– Other sources of stem cells are those from umbilical cord blood, and bone marrow.

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–– In addition, neural stem cells, haematopoetic stem cells and mesenchymal stem cells can be obtained from fetal blood and fetal tissue. Advantages of Embryonic Stem Cell:    –– Flexible – have the potential to make any cell. –– Immortal – one embryonic stem cell line can potentially provide an endless supply of cells with defined characteristics. –– Availability – embryos from in vitro fertilization clinics. Disadvantages of Embryonic Stem Cell: –– Difficult to differentiate uniformly and homogeneously into a target tissue. –– Immunogenic – embryonic stem cells from a random embryo donor are likely to be rejected after transplantation –– Tumorigenic – capable of forming tumors or promoting tumor formation. –– Destruction of developing human life. Embryonic stem cells are easier to identify, isolate and harvest than adult stem cells. There are more of them. They grow more quickly and easily in the lab than adult stem cells. They can be more easily manipulated (they are more plastic). Adult Stem Cells (ASC) Animal or human adult stem cells are undifferentiated cells or multipotent, which are found among differentiated cells in a basal layer of a tissue or an organ. The adult stem cell can renew itself and differentiate to yield some or all of the major specialized cell types of the tissue or organ. The main function of adult stem cells in the adult organism is to maintain and repair the tissue in which they are found. Biologists also use the term somatic stem cell instead of adult stem cell, where somatic refers to the cells of the body as opposite to the germ cells (sperm or eggs). Unlike embryonic stem cells which present in early embryo, the origin of adult stem cells is some mature tissues. But initially they arose from embryo and contain their multipotency in the adult body. Studying of ASC led to surprising results. Biologists have found adult stem cells in many more tissues than they once thought possible. These findings made researchers and clinicians think whether adult stem cells could be used for transplantations. In fact, adult hematopoietic, or blood-forming, stem cells from bone marrow have been used in transplants for more than 40 years. Scientists now have evidence that stem cells exist in the brain and heart, two locations where adult stem cells were not at first expected to be found. The development of protocols to control the adult stem cells differentiation in vitro conditions has become the basis of transplantation-based therapies.

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The history of research on ASC began more than 60 years ago. In the early 1950s, it was revealed that the bone marrow contains at least two kinds of stem cells. The population, called hematopoietic stem cells, forms all the types of blood cells in the body. Another population, called bone marrow stromal stem cells, was discovered a few years later. This population is also called mesenchymal stem cells or skeletal stem cells. These non-hematopoietic stem cells make up a small proportion of the stromal cell population in the bone marrow and can generate bone, cartilage, and fat cells which differentiate to blood and fibrous connective tissue. In the 1960s researches studying rats discovered 2 regions in brain that contained dividing cells that ultimately become nerve cells. Before this discovery most scientists believed that the adult brain could not divide and generate new nerve cells. Only in the 1990s researchers agreed that the adult brain does contain stem cells capable to generate the three major cell types in brain: astrocytes and oligodendrocytes, which are non-neuronal cells, and neurons, or nerve cells. Thus, ASCs have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. They are thought to locate in a specific area of each tissue, which researchers termed as a “stem cell niche”. In many tissues, current evidence suggests that some types of ASCs are pericytes, cells that compose the outermost layer of small blood vessels. Stem cells may remain quiescent, non-dividing, for long time periods until they are activated by a normal need for more cells to maintain tissues, or by a disease or tissue induction. As a rule, a very small number of stem cells is present in any tissue. Once removed from the body, the capacity of ASC to divide is limited, making generation of large quantities of stem cells quite difficult. In many laboratories, researchers are trying to find better ways to grow large quantities of ASC in vitro cell culture and to manipulate them for generation of specific cell types and for disease treatment. Some instances of potential treatments include regeneration of bones using cells derived from bone marrow stroma, development of insulin-producing cells for type 1 diabetes, and repair of damaged heart muscles following a heart attack with cardiac muscle cells. In a living animal, ASC are able to divide for a long period until they are needed, and can give rise to mature cell types that have characteristic shapes and specialized structures and functions of a particular tissue. Figure 30 shows examples of differentiation pathways of ASC in vitro or in vivo (see Figure 30).

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Figure 30. Adult type of Stem cells (ASC)

All types of blood cells give rise to hematopoietic stem cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, and macrophages. Mesenchymal stem cells are present in many tissues of the body. Mesenchymal stem cells from bone marrow, specifically bone marrow stromal stem cells and skeletal stem cells, give rise to a variety of cell types: bone cells (osteoblasts and osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and stromal cells supporting blood formation. But it is not yet clear how similar or dissimilar mesenchymal cells derive from non-bone marrow sources. Brain neural stem cells give rise to three major cell types: nerve cells (neurons) and two categories of non-neuronal cells–astrocytes and oligodendrocytes. The epithelial stem cells lining the digestive tract occur in deep crypts. They give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells. The epidermal stem cells represented by skin stem cells are located in the basal layer of the epidermis and at the base of hair follicles. They give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis. Certain ASC types can differentiate into cell types seen in organs or tissues other than those expected from the cells’ predicted lineage, for example, brain stem cells that differentiate into blood cells or blood-forming cells that differentiate into cardiac muscle cells, and so forth. This phenomenon was called transdifferentiation. The examples of transdifferentiation have been observed in some vertebrate species, whether this phenomenon actually occurs in humans is still discussed. The observed instances in human may involve fusion of a donor cell with a recipient cell.

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Another possibility is that transplanted stem cells are secreting factors that encourage the recipient’s own stem cells to begin the repair process. Even when transdifferentiation has been detected, only a very small percentage of cells undergo this process. In transdifferentiation experiments, scientists have recently demonstrated that certain adult cell types can be “reprogrammed” into other cell types in vivo using a well-controlled process of genetic modification. This strategy is used as a way to reprogram available cells into other cell types that have been lost or damaged due to disease. For instance, one recent experiment in mice shows how pancreatic β-cells, the insulin-producing cells lost or damaged in diabetes, could possibly be created by reprogramming of other pancreatic cells. By “re-starting” expression of three critical β-cell genes in differentiated adult pancreatic exocrine cells, researchers were able to create β cell-like cells that can secrete insulin. The reprogrammed cells were similar to β-cells in appearance, size, and shape. They expressed genes which characterize β-beta cells, and they were able to partially restore blood sugar regulation whose own β-cells had been chemically destroyed. While the gene modification is not a real transdifferentiation process, this method for reprogramming adult cells may be used as a model for direct reprogramming of other adult cell types. Sources of Human adult type stem cells (see Figure 31): –– Umbilical Cords, Placentas and Amniotic Fluid – Adult type stem cells can be derived from various pregnancy-related tissues. –– Adult Tissues – In adults, stem cells are present within the bone marrow, liver, epidermis, retina, skeletal muscle, intestine, brain, dental pulp and elsewhere. –– Cadavers – Neural stem cells have been removed from specific areas in post-mortem human brains as late as 20 hours following death.

Figure 31. Potential sources of ASCs

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Differentiation pathways of ASCs: –– Neural stem cells in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal cells – astrocytes and oligodendrocytes. –– Epithelial stem cells in the lining of the digestive tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells. –– Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. –– The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. –– The follicular stem cells can give rise to both the hair follicle and to the epidermis Despite a big variety of differentiation ways of embryonic stem cells due to their totipotency or multipotency, the adult stem cells are preferable to embryonic stem cells because of the following reasons: 1) adult stem cells naturally exist in animal and human bodies, and they provide a natural repair mechanism for many specialized tissues; 2) they belong in the microenvironment of the adult body, while embryonic stem cells belong in the microenvironment of the early embryo, that’s why they tend to cause tumors and immune system reactions. Advantages of Adult Stem Cells: –– Adult stem cells from bone marrow and umbilical cords appear to be as flexible as the embryonic stem cells; –– Specialized inducement may be simpler. –– Not immunogenic – recipients who receive the products of their own stem cells will not experience immune rejection. –– Relative ease of procurement – some adult stem cells are easy to harvest (skin, muscle, marrow, fat) –– Non-tumorigenic – tend not to form tumors. –– No harm done to the donor. Disadvantages of Adult stem cells: –– Limited quantity – it is sometimes difficult to obtain them in large numbers. –– Finite – may not live as long as embryonic stem cells in culture. –– Less flexible – may be more difficult to reprogram to form other tissue types Induced pluripotent Stem Cells (iPSC) In addition to reprogramming cells to become a specific cell type, it is now possible to reprogram adult somatic cells to become like embryonic stem cells

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through the introduction of embryonic genes. The scientists term this kind of stem cells – induced pluripotent stem cells (iPSCs). Thus, a source of cells can be generated that are specific to the donor, thereby increasing the chance of compatibility if such cells to be used for tissue regeneration. However, like embryonic stem cells, determination of the methods by which iPSCs can be completely and reproducibly committed to appropriate cell lineages is still under investigation. iPSCs are adult cells that have undergone genetic modification and have been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. In 2006 the mouse iPSCs were first reported, and human iPSCs were first announced in late 2007. Mouse iPSCs demonstrate important peculiarities of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage of development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. Although additional research is still needed, iPSCs are already useful tools for new drug development and modeling of diseases to understand the mechanisms. Scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatment for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. In any case, this breakthrough discovery has created a powerful new way to “de-differentiate” cells whose developmental fates were previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body. The iPSCs resemble ESCs in their properties and potential to differentiate into a range of adult cell types. Transgenic expression of only four transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram these cells to a pluripotent state. The schematic image of the iPSC method is represented in Figure 32.

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Figure 32. The schematic image of the iPSC method

iPSC is a potential for studying human disease mechanisms, for getting basic biology knowledge, and drug screening. iPSCs were generated from patients with a variety of genetic diseases (Park et al., 2008; Dimos et al., 2008): amyotrophic lateral sclerosis, Parkinson disease, Huntington disease, Alzheimer disease, Juvenile-onset, type 1 diabetes mellitus. Besides iPSCs the Somatic Cell Nuclear Transfer (SCNT) method provides the potential alternative to stem cells. SCNT technology allows us to transfer the nucleus from the adult cell to the enucleated egg and reprogram it. Table 3 shows a comparison of these two alternatives to ESC. Potential alternatives to ESC Somatic cell nuclear transfer (SCNT) 1 The nucleus from adult cell is transferred to enucleated egg (the nucleus was removed)

Table 3

Induced pluripotent stem cells (iPSC) 2 Human cells are injected with genes that made them behave like human embryonic cells

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1 Advantages over ESC: • No embryo derived cells • Well characterized system in mice • No ethical restrictions • Potential for generating stem cells from any individual

2 Advantages over ESC: • No embryo derived cells • Adult cells only • No ethical restrictions • Potential for generating stem cells from any individual

Challenges: • Limited success in primates • Human egg donation • Labor intensive

Challenges: • New technology (2007) • The infection process makes the gene integrate randomly into the DNA. (Potential cancer in clinic applications)

Applications of stem cells Figure 33 demonstrates potential applications of stem cells in medicine.

Figure 33. Potential uses of Stem cells (https://en.wikipedia.org/wiki/Stem_cell)

SCNT is applicable for therapeutic cloning. Biotechnologists first remove the nucleus from a normal egg cell of a healthy woman. Then they extract a nucleus from a somatic cell – that is, any cell other than an egg or sperm – from a patient who needs an infusion of stem cells to treat a disease or injury, and insert the nucleus into the egg. The egg, which now contains the patient’s genetic material, is allowed to divide and soon forms a blastocyst. Cells from the inner cell mass were isolated and used to develop new embryonic stem cell (ESC) lines. Figure 34 represents the possibility of using SCNT for therapeutic cloning.

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Figure 34. Therapeutic cloning strategies

Therapeutic cloning was used for tissue repair. There are many examples with using the repair of human skin. Skin is readily cultured to provide replacement tissue for burn victims. Healthy skin cells from the patient can be grown rapidly in vitro to provide self-compatible skin grafts. The human skin cell divides once in two days. Stem cell transplantation (SCT) is the term now used in preference to bone marrow transplantation (BMT). When a patient’s bone marrow fails to produce new blood cells, for whatever reason, he or she will develop anemia, be prone to frequent, persistent infections and may develop serious bleeding problems. In order to restore blood cell production a patient may be given healthy stem cells. Cell therapy: –– Treatment of neural diseases such as Parkinson’s disease, Huntington’s disease and Alzheimer’s disease. –– Stem cells could be used to repair or replace damaged neurons. –– Repair of damaged organs such as the liver and pancreas. –– Treatments for AIDS. Stem cells are very promising for cancer treatment: –– Intense chemotherapy damages person’s bone marrow, where the stem cells for blood reside. –– Depleted of a fresh supply of blood cells, the patient is left vulnerable to infection, anemia and bleeding. –– These side effects of chemotherapy are often treated with a bone marrow transplant.

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–– “Transplanting bone marrow tissue into a chemo-cancer patient may involve hundreds of thousands or millions of cells – of which only two or three may be actual stem cells. –– It would be much more efficient if you could inject a thousand of purified stem cells. Is Stem Cell Research Ethical? –– Embryonic Stem Cells – are always morally objectionable, because the human embryo must be destroyed in order to harvest its stem cells. –– Embryonic Germ Cells – are morally objectionable when utilizing fetal tissue derived from elective abortions, but morally acceptable when utilizing material from spontaneous abortions (miscarriages) if the parents give informed consent. –– Umbilical Cord Stem Cells – are morally acceptable, since the umbilical cord is no longer required once the delivery has been completed. –– Placentally-Derived Stem Cells – are morally acceptable, since the afterbirth is no longer required after the delivery has been completed. –– Adult Stem Cells – are morally acceptable. Control questions: 1. What do you know about the origin and characteristics of animal cells? 2. Describe the properties of totipotency, multipotent, pluripotent and unipotent animal cells. 3. What do you know about sources and types of embryonic stem cells? 4. What do you know about sources and types of adult stem cells? 5. What are the induced pluripotent stem cells (iPS cells)?

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REPRODUCTIVE TECHNOLOGIES IN ANIMAL BIOTECHNOLOGY Reproductive manipulations, including superovulation, semen collection, artificial insemination (AI), embryo collection, In vitro fertilization (IVF) and embryo transfer (ET), are used in the production of both transgenic animals and animals produced by nuclear transfer (NT). Commercial livestock breeders also use many of these manipulations routinely. However, in spite of many experiments (Matthews, 1992; Moore and Mepham, 1995; Seamark, 1993), these procedures do raise animal welfare concerns, these generally are not specific to the production of genetically engineered animals. As recently shown by Van der Lende et al., 2000, only few of these procedures have received systematic study from the perspective of animal welfare. Grandin (1993) demonstrated that handling and restraint can be distressful to farm animals, but are essential for almost all husbandry procedures, that include those involving reproductive manipulations. Certain reproductive manipulations, for example, the administration of injections to induce ovulation, can cause additional transient distress, as can electroejaculation. Artificial insemination and embryo collection with fallowing embryo transfer present a range of animal welfare issues depending on the species used. These procedures in cattle can be accomplished with minimally invasive non-surgical procedures – the latter under epidural anesthesia. However, in sheep, goats, and pigs these manipulations involve surgical or invasive procedures, like a laparotomy or laparoscopy, and hence the potential for operative and postoperative pain. In poultry species the hen is killed in order to obtain early-stage embryos. In fish, eggs and milt might be hand-stripped in some species (causing handling discomfort), while in others the males or females must be killed to obtain eggs or sperm. Farm animals might be subjected to these reproductive manipulations repeatedly during their lifetime because breeding livestock was valuable. As noticed by Eyestone (1994) this was due to the problems involved in screening microinjected embryos prior to implantation to ensure that they are actually carrying the transgene of interest. Recipient cows might be a subject of transvaginal amniocentesis 141

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for genotyping. Nontransgenic fetuses or male fetuses are then aborted and the cows reused as recipients (Brink et al., 2000). While this limits the number of recipient animals used, it also raises welfare concerns over the repeated exposure of individual animals to procedures likely to cause pain and distress. However, many scientists (Moore and Mepham, 1995; Seamark, 1993) believe that some reproductive manipulations are available for replacements or alternatives to known techniques. For instance, a method has been devised for non-surgical embryo transfer in pigs, and ova for some purposes can be obtained from slaughterhouses, which eliminates the need for manipulation of live donor livestock females. The use of nuclear transfer to produce transgenic animals (Eyestone and Campbell, 1999) could eliminate the problem of repeated elective abortion and reuse of recipient animals, since cell populations with specific genotypes or phenotypes could be selected before embryo reconstruction. Endocrine control of reproductive function in animals Achievements in endocrinology and biology of animal reproduction significantly expanded the possibilities of regulating Animal reproductive function by biotechnological methods. The development of the Mammalian reproductive system and its functioning, especially in later stages of ontogenesis, largely depends on the gonadostimulating hormones of the anterior lobe of the pituitary gland. In Mammalian, adenohypophysis secretes 3 hormones, which have stimulating and regulating action on reproductive system. There are follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL). FSH, a glycoprotein operating in conjunction with LH, stimulates development of the graafian follicle, a small, egg-containing vesicle in the ovary of the female mammal; in the male, it promotes the development of the tubules of the testes and the differentiation of sperm. Though in the male the presence of FSH is necessary for the maturation of spermatozoa, additional FSH may not be required for months because testosterone can maintain this activity. In the female, however, there is a rhythmic, or cyclical, increase and decrease of FSH, which is essential for monthly ovulation. LH is a glycoprotein and operates in conjunction with FSH. Following the release of the egg (ovulation) in the female, LH promotes transformation of the graafian follicle into the corpus luteum, an endocrine gland that secretes progesterone. In the male, LH stimulates the development of the interstitial cells of the testes, which secrete testosterone, a male sex hormone. The production of LH is cyclical in nature (especially in the female). The major action of PRL, which is a protein hormone, is to initiate and sustain lactation. In breast-feeding mammal females, tactile stimulation of the nipples and the breast by the suckling infant blocks the secretion of hypothalamic dopamine (which normally inhibits prolactin) into the hypophyseal-portal circu-

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lation of the pituitary gland. This results in a sharp rise in serum prolactin concentrations, followed by a prompt fall when feeding stops. High serum prolactin concentrations inhibit secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, thereby decreasing the secretion of gonadotropins (LH and FSH), and may also inhibit the action of gonadotropins on the gonads. Thus, high serum prolactin concentrations during lactation reduce fertility, protecting lactating females from a premature pregnancy. Figure 35 demonstrates regulation of the estrous cycle in Mammalian by hormones produced by adenohypophysis.

Figure 35. The estrous cycle in Mammalian (https://en.wikibooks.org/wiki/Anatomy_and_Physiology_of_Animals/Reproductive_System)

Estrous cycle: –– Reoccurs and repeats itself as long as female is not pregnant. –– Is controlled by hormones preparing the reproductive tract for ovulation and pregnancy –– Includes two phases: 1. Follicular phase – a short phase, a period from regression of the corpus luteum (CL) to ovulation 2. Luteal phase – a long phase, a period from ovulation to CL regression –– Reoccurs and repeats itself as long as female is not pregnant. –– Is controlled by hormones preparing the reproductive tract for ovulation and pregnancy –– Is categorized by frequency of occurrence throughout the year.

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Phases of Estrous: –– Proestrus: ovary is about to release an egg –– Estrus: female receptivity –– Metestrus: uterus prepares for pregnancy, a fertilized egg attaches to uterus –– Diestrus: the longest period of cycle inactive –– Estrous Cycles stop after conception, and begin soon after Parturition (birth) Ovulation is a realization of the egg cell from ovary. It occurs near the end of the estrous period. Before ovulation the egg cell is contained in the follicle. During ovulation the follicle breaks, releases the egg into the oviduct. If sperm is present, the egg may become fertilized. After ovulation CL forms on ovary and releases progesterone. If the egg is not fertilized, CL does not grow, allowing another follicle to grow and another estrous period to occur. Functions of progesterone: –– Prepares uterus for implantation of the embryo. –– Stops other eggs from forming –– Maintains pregnant condition –– Develops mammary glands which produce milk to feed young after they are born. Human, Horse, Cow are monoestral animals. Mice, Rat, Dog, Cat, Pig – are polyestral animals. Ship, Goat have 1-2 estrous cycles. Seasonably polyestrous cycles occur only during certain times of the year. Long-day breeders (for example, horses) cycle when the day length increases. Short-day breeders (for an example, ships) cycle when the day length decreases. Figure 36 presents the scheme of hormone-controlling estrous cycle at horses.

Figure 36. Estrous cycle at horses (https://www.pinterest.com/pin)

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The cow is a non-seasonal polyestrous species. Which means a cow can have multiple estrous cycles throughout the year. The ovarian changes during a typical 21-deay estrous cycle in which pregnancy does not occur. The development and regression of the corpus luteum and the follicles are continuous processes (see Figure 37).

Figure 37. Estrous cycle at cow (http://www.cahe.nmsu.edu/pubs/_b/b-212.pdf) Table 3

presents the longevity of the estrous cycle at agricultural animals Animal Mare Sow Eve Cow Gyp

Age of puberty

Age of first service

18 months 24 months 6 months 8-10 months 6-12 months (first fall) First or second fall 6-18 months 14 months 6-12 months 12 months

Length of estrous cycle 21 days 21 days 16 days

Length of estrous period 5 days 18 days 2 days

Gestation period in days 336 114 150

21 days 6-12 months

2 days 9 days

283 63

Efficient and profitable reproductive performance of a dairy herd requires routine but conscientious heat detection and proper timing of artificial insemination. Failure to detect estrus (heat) is a major factor contributing to low fertility. Methods of Heat Detection:

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–– Visual Appraisal –– Gomer Bulls –– Electronic device –– Ultrasound machine –– Marking harness (sheep and goats) –– Teaser Stallion Common symptoms of Estrous for agricultural animals: –– Standing heat is the best indicator –– Restlessness –– Discharge from vulva –– Red, swollen vulva –– Mares with raise tail, and “winking” Each kind of agricultural animal has its own peculiarities which reflect in physiological signs and behavioral symptoms of estrous. Control questions: 1. What kind of Animal biotechnology tools can you include to reproductive technologies? 2. Indicate the milestones in the history of reproductive technology. 3. How are reproductive functions controlled in animals? 4. What is the estrous cycle? 5. Indicate the differences in estrous cycle of the main farm animals.

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CHAPTER

6

INDUCTION OF SUPEROVULATION

Superovulation plays an important role in the reproductive technologies programs: artificial insemination (AI), in vitro fertilization (IVF), embryo transfer (ET). It aims at inducing a high number of ovulations and a high yield of embryos of good quality. One of the most problematic aspects of the reproductive procedure is the variable response by the donor to superovulatory treatment and the percentage of embryos available for transfer from each donor. Despite much attention, little progress has been made during the last years, and the injection of exogenous gonadotrophins is still the only practical means of producing multiple ovulations from donor. The earliest descriptions of superovulation date back to Smith and Engle in 1927, who used anterior pituitary preparations to induce a fourfold increase in the ovulation rates of rats and mice. A few years later, Cole and Hart in the USA demonstrated that the blood serum of pregnant mares would induce multiple ovulations in rats, establishing the basis for what was to become the most widely used gonadotrophin in the treatment of farm animals. Pregnant mare’s serum gonadotrophin (PMSG) is a glycoprotein found in the blood of the mare between days 40 and 130 of gestation and is unique among gonadotrophins in possessing both FSH and luteinizing hormone (LH) biological properties within the one molecule. It is now known that PMSG is secreted by specialized trophoblastic cells that invade the mare’s endometrium between days 3 and 40 of gestation; for such reasons, the term equine chorionic gonadotrophin (eCG) rather than PMSG is preferred by many researchers. The name notwithstanding, early research on superovulation in cattle and other farm animals invariably involved the use of PMSG. In the first decade of human in vitro fertilization (IVF), on the other hand, superovulation was usually by way of human menopausal gonadotrophin (hMG), countless thousands of healthy babies being born as testimony to the safety and efficacy of this urinary gonadotrophin. Although, initially, relatively undefined preparations such as PMSG were used for superovulation in cattle and other farm ruminants, these were subse147

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quently replaced by purified pituitary extracts from pigs, horses and sheep. One practical consequence of using pituitary preparations, because of their much shorter half-life, was the need to administer them by multiple injections rather than a single administration. Such variability in superovulatory response in cattle, which is reflected in buffaloes, sheep and goats, is known to be related to differences in the gonadotrophin preparation, the total dose of FSH administered and duration and timing of treatment and the use of additional hormones in the superovulation regimen. There is evidence that pretreatment with recombinant bovine growth hormone (recombinant bovine somatotrophin (rBST)) or increased dietary intakes, which induce an increase in the population of small follicles, can significantly improve the response to standard superovulatory protocols; it is evident that ovarian status can be manipulated in various ways to improve superovulatory response (see Figure 38).

Figure 38. Induced ovarian superovulation of ewes after gonadrotipin containing drug Folligon (Netherlands) (kindly presented by Toishibekov E.M.)

Superovulation in cattle The principle of superovulation in cattle is basically simple: to induce more ovulations than the normal rate by giving a gonadotrophin stimulus (at critical moments of follicular development), followed by control of luteolysis, synchronous ovulation, high fertilization and early embryonic development rates. The majority of donor cows will give the best superovulatory response if superovulation treatment is established between days 8 and 14 of the cycle. Therefore, the donor cow must always be examined prior to treatment to detect any abnormality and to establish the presence of a normal, functional corpus luteum. Although widely used in standardised protocols, superovulation is still not a well-controlled technique. The superovulation treatments usually yield an aver-

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age of 6 transferable embryos, but this technique continues to be associated with variable and unpredictable responses in ovulation rates and recovery of transferable embryos. Normally, no transferable embryos are recovered from about 20 percent of donors and only 1 to 3 transferable embryos are obtained from another 20 percent. An ideal response of 5 to 12 good quality embryos is obtained from about one third of the donors. The preparations to induce superovulation include: equine chorionic gonadotrophin (eCG) derived from the serum of pregnant mares (usually called pregnant mare’s serum gonadotrophin, PMSG); extracts of domestic animal pituitaries, particularly those of the pig, of various degrees of purity and FSH to LH ratios; recombinant FSH; and gonadotrophins of pituitary origin extracted from human post-menopausal urine (human menopausal gonadotrophin, hMG). The pregnant mare’s serum gonadotrophin (PMSG) is a glycoprotein that produces both FSH and LH biological effects. Due to high content of carbohydrate side chains and sialic acid, PMSG has a very long half-life of about 5 days. Traditionally, a single dose of 1500 to 3000 IU of PMSG during the mid-luteal phase of the estrous cycle has been used to superovulated cows. A luteolytic dose of prostaglandin F2 or an analogue is administered 2 to 3 days later. The donor is expected to show heat signs 2 days after prostaglandin injection. The result is an interval of about 4 days between starting PMSG treatment and the onset of estrous. The advantage of using PMSG for superovulation is its availability in large quantities at a low cost and possibility to use it in the countries where the import of certain porcine products is banned. PMSG can be also administered as a single dose compared with the multiple injections normally required when using pituitary preparations. Control questions: 1. Indicate the functions of the main hormones in the reproductive control. 2. What is the biological and genetic sense of ovulation? 3. What is the difference between normal ovulation and superovulation? 4. What type of animal treatment is needed for superovulation? 5. Describe the main principal steps in the superovulation procedure.

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CHAPTER

7

COLLECTION OF SPERM, OOCYTES AND EARLY EMBRYOS Collection of sperm, oocytes and early embryos is an important step in: –– Artificial insemination –– In Vitro Fertilization –– Cloning –– Embryo-engineering (Chimera getting) –– Transgenic technologies –– Biobanking Collection of sperm Semen collection refers to the process of obtaining semen from animals or male humans with the use of various methods, for the purposes of artificial insemination, or medical study (usually in fertility clinics). Semen can be collected via masturbation (e. g., from stallions and canids), prostate massage, artificial vagina, penile vibratory stimulation (vibroejaculation) and electroejaculation. Semen can be collected from endangered species for cryopreservation of genetic resources. In animals, for example, bull semen can be collected by using an artificial vagina (AV), electro-ejaculator, or by massaging the ampule of the bull by hand. AV is the most desired use. The AV is made up of a piece of heavy rubber hose about 5 cm (2.0 in) in diameter and up a 46 cm or more (in only some cases less) in length. A small screw cap attachment is fitted or drilled preferably in the middle of the housing. A piece of latex or silicone rubber (usually rough inside, but in some cases smooth) is then put through the hose and pulled up at both ends. Another latex or silicone cone shaped attachment is placed on the one end of the AV held by a rubber band. On the end of the cone a glass or plastic centrifuge tube is attached, usually 15ml in size). Through the cap attachment water usually around 54°C is filled until the AV is 1/2 full, temperature may vary from bull to bull. This water is between the inside rubber and the inside hose wall of the AV. A small amount of K-Y jelly or Vaseline is placed just inside on the unconed end and then it is smeared with a pipette or greasing stick. Finally, in most cases and AV jacket is tied or attached onto the cone end to cover the cone and tube. This 150

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will help prevent cold shock to the semen by keeping the cone and tube warm. The collector then goes cautiously to the side of the bull when he is in mount, directs the bull’s penis inside the AV by grabbing the sheath directly behind the extended penis never touching the penis itself. The bull then ejaculates after his penis slides through the AV. The bull should be ejaculating through the AV into the cone of the AV so there is very little chance of temperature shock to the semen. After the bull has dismounted, the AV should be held in the upright position so the semen runs into the tube onto the end of the cone. Then the tube is detached and placed upright in a water bath of about 29-32°C. A bull is generally brought to a collection area where gates or other protection areas are set up. In most cases of Dairy Bulls, the bull has a halter or rope inserted into his nose ring. In this case he can be led and his handler can be a safe distance away, yet have some control over the animal. In the collection area there usually is a stall or stanchion where a mount animal is tied in. A mount animal can be a steer, a cow, or even another bull. Some bulls can be rather aggressive and have problems with mounting another animal. Others are rather passive and may show little or no interest in mounting. Different scenarios are used to try to entice him to mount. Such scenarios may include a different breed of steer, a different bull, or even a cow. Sometimes a bull may be wary of the individual who is approaching him to collect. In which cases a blindfold may have to be put over his eye in that side. The bull mounts and semen is able to be collected as the bull doesn’t worry about what is going on. In human: Shortly before or after the oocyte collection the male partner will be asked to give a sperm sample. Sexual abstinence of 3-4 days should be exercised. Sperm is collected for 60-90 minutes prior to fertilization. Sperm is liquefied, centrifuged, suspended in the culture medium, and incubated for 30-60 mins at 37OC. The most active sperms are located on the surface of the medium. Sperm may be obtained from the testicle, epididymis, or vas deferens from men whose semen is void of sperm either due to obstruction or lack of production. Figure 39 represents a semen collection example.

Figure 39. Procedure of collection (A), centrifugation (B) and suspension (C) of sperms in culture medium

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Sperm can also be collected from testicles with assisted surgical methods: percutaneous epididymal sperm aspiration (PESA) and microsurgical epididymal sperm aspiration (MESA) (see Figure 40).

A

B A – PESA;

B – MESA

Figure 40. Surgical sperm retrieval

Sperm quality control is a required procedure. It includes analysis of sperm count, sperm morphology and motility (see Figure 41). The types of morphological abnormalities of sperm are presented in Figure 42. Many computer programs were developed for morphological analysis and motility assessment (see Figure 43).

Figure 41. Sperm quality control

Figure 42. Morphological sperm abnormalities

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A

153

B А-Computer morphological analysis; B- Individual sperm movement tracks Figure 43. Computer analysis of sperm quality (Kindly presented by Toishibekov E.M.)

Collection of oocytes and embryos Initial IVP (in vitro embryo production) used oocytes from slaughterhouse ovaries. During early experimentation this approach worked well when large numbers of immature eggs (oocytes) were needed to develop procedures. In the 1980s, it was proposed that the IVP application in animals would likely be used in rare exotic animals and in genetically valuable seedstock. Early attempts to retrieve oocytes from potential donor cattle included the surgical and less invasive laparoscopic procedures/ But there was a limit to how many procedures could be performed without causing injury. At this stage, there was essentially no safe, repeatable method of harvesting oocytes from live farm animals. Then in the late 1980s, a method was developed in humans for retrieving oocytes using ultrasonography to visualize the ovary while a needle was guided transvaginally into the follicle. The oocyte could be aspirated from the follicle and subjected to in vitro maturation, fertilization and then to culture procedures. These efforts paved the way for the new reproductive technology now available for farm animals. Currently, TUGA, the transvaginal ultrasound-guided oocyte aspiration, also known as ovum pick-up (OPU) in human medicine, is now used in cows, goats, mares and more recently in pigs and exotic hoof stock species. The problem with the larger farm animals is that their gestation periods are considered to be long in comparison with those of dogs (62 days) and cats (63 days) and that the animals are out of embryo production during their gestation. Cows have 9.5-month and mares have 11-month pregnancies. But both are known to continue follicle wave development during early to mid-gestation. Another goal has been to take advantage of these developing ovarian follicles and attempt to produce IVF-derived offspring from oocytes harvested from females during pregnancy.

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The ongoing pregnancies affecting from oocyte aspiration procedure were the main concern of biotechnologists. But soon this oocyte aspiration approach proved not to be a problem, and pregnant donors were found to consistently produce more oocytes per collection than similar nonpregnant, cyclic females. The first offspring produced from oocytes collected by transvaginal ultrasound-guided aspiration from pregnant donor animals resulted from cows and horses. Ultrasound-guided follicle aspiration has also been used successfully in other kinds of animals, with modifications made primarily to account for anatomical differences of the donor animals. The aspiration technique has been used successfully to harvest oocytes from adult pigs, as long as rectal manipulation of the ovaries was possible within the donor females. Also transvaginal ultrasound-guided oocyte aspiration has also been used successfully to harvest oocytes in llamas. However, there is still much to be studied in the use of assisted reproductive technologies to maximize reproductive potential in genetically valuable animals. Repeatable oocyte retrieval methods are being fine-tuned, it is likely that these procedures will become routinely used to obtain oocytes for further gamete and embryo research and also by seedstock producers for in vitro embryo production from farm animals in the commercial way. Transvaginal ultrasound-guided oocyte aspiration is now used to harvest valuable oocytes from minor farm animal breeds, from domestic females representing rare bloodlines, clinically infertile females and cows too old to become pregnant. Research continues to find applications for this technology, including harvesting oocytes from injured females, young prepubertal heifers and early postpartum beef cows for in vitro embryo production. There are some plans to use ultrasound-guided oocyte aspiration to obtain oocytes for in vitro embryo production to help preserve germplasm of endangered exotic species. Another way to obtain female germ line cells is use of abattoir materials for collecting oocytes and embryos, which is also popular among farmers and researchers. Although superovulation remains the chosen method of producing highquality bovine embryos for most commercial embryo transfer (ET) purposes, its cost may well make it prohibitive for many research programs. On the other hand, abattoir materials form an inexpensive and readily available source of oocytes for research in embryo production and for use in cloning and production of transgenic animals. In the production of cattle embryos from slaughterhouse ovaries, it was necessary to develop methods that permit the recovery of several goodquality oocytes per ovary; the number actually recovered will vary with different collection procedures (see Figure 44).

Chapter 7. Collection of sperm, oocytes and early embryos

A

155

B A – abbatoir material uses for embryo collection; B – Ovum pickup in the cow Figure 44. Different methods of embryo and oocyte collections in cow

An alternative approach to oocyte collection from slaughterhouse ovaries is ovum pick-up (OPU) technique that allows us to collect eggs from live donor females (Figure 43B). Biotechnologists have developed OPU devices that are practical and economical for routine use in oocyte retrieval, without causing negative effects on ovarian structure and subsequent ovarian function. As it was shown by Lopes et al. (2003), although donor cows used for OPU vary considerably in their yield of oocytes, there is evidence that a certain oocyte number may be characteristic of each donor animal and determined by its genetic constitution. In using OPU, a scanner with an intravaginal sector probe and a guided needle is employed; the needle is connected to a test-tube and to a vacuum pump to aspirate the follicular fluid and the oocyte contained within it. Using an ultrasound scanner with good resolution, it is possible to envisage ovarian follicles down to 2-3 mm in size; the recovery procedure can be carried out on the farm with the donor sedated and confined in a crush. It is possible to collect oocytes not only from cyclic heifers and cows but even from animals in the early months of pregnancy, from those in the early post-partum period and from prepubertal heifers. After collection of oocytes, they should be maintained in media such as Dulbecco’s phosphate buffered saline (PBS) or tissue culture medium (TCM)-199 (HEPES-buffered). It is clear that oocytes are extremely sensitive to temperature shock, making it important to monitor temperature carefully during the collection procedure. As proposed by Merton et al. (2003), the follicle-stimulating hormone (FSH) pre-stimulation of donors prior to OPU is available to improve cattle embryo production efficiency, much better than other methods. In the Czech Republic, Cech et al. (2003) also demonstrated that pre-stimulation of dairy cows with FSH increased the number of oocytes recovered by OPU in the first trimester of pregnancy.

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Equipment, methods and practical applications of OPU are described by Christie et al. (2002), who also drew attention to the benefits and associated problems of this new technology. OPU/IVF can produce more embryos over a given period of time than conventional superovulation and embryo recovery, but the in vitro-derived embryos have proved to be significantly less viable than in vivo counterparts. This also results in a higher incidence of abortion, perinatal mortality and fetal and placental abnormalities and a tendency to increased birth weights among IVP embryos. Nonetheless, a large majority of IVF pregnancies do result in normal healthy calves and the technique provides a valuable new means of producing additional offspring from donors unable to produce calves by any other means In human oocytes collected during IVF program: –– Collected from females desirable of having baby. –– Cannot be collected from females with non-functional ovaries. –– Can be collected during a natural or induced menstrual cycle. –– Time for this is determined by monitoring rise in the level of Luteinizing Hormone (LH) in urine or blood. Human ovulation may be stimulated by administration of Clomiphene or Human menopausal Gonadotropin (hMG). Follicle development may be arrested at the optimum stage by administering Human chorionic gonadotropin (hCG) so that ova are not released (see Figure 45). Recovery of oocytes can be done most conveniently by a laparoscopic instrument that allows the visualization of ovary through a monitor, aspiration (suction) of the follicular fluid containing the oocytes and the necessary surgical manipulation of ovary using sensors, laparoscopic scissors and an aspirating apparatus inserted into the abdomen of the female via a suitable tube (see Figure 46).

Figure 45. Ovarian follicles, stimulated by ovulation medications, visible on ultrasound. The dark, circular areas are the follicles.

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Figure 46. The eggs are aspirated (removed) from the follicles through the needle connected to a suction device.

From practice it is known, that usually around 10-15 oocytes are aspirated. The eggs are prepared and stripped from the surrounding cells. After the eggs are retrieved, they are examined in the laboratory for maturity and quality. Mature eggs are placed in an IVF culture medium and transferred to an incubator to await fertilization by the sperms. Oocytes are identified by microscopic examination of the follicular fluid aspirated during laparoscopy, and are incubated for 10-15 hours depending upon the expected time of maturation. The following mediums may be used for serving the purpose: modification of Ham’s F10 medium; Earl’s solution; modified Whitten’s medium; Whittingham’s T6 medium. Evaluation and maturation of the oocyte, selection criteria The methods of selection of the cumulus–oocyte complexes (COCs) are usually based on parameters such as the morphology of the cumulus, the combined morphology of the cumulus and the ooplasm, the size of the follicle and the oocyte and the level of follicular atresia. COCs with a compact and complete cumulus mass and a uniform appearance seem to present a higher developmental ability. Many reports have proposed classification schemes based on the compactness and number of layers of cumulus cells surrounding the oocyte and on the appearance of the oocyte itself. Oocytes with the highest developmental competence are expected to possess an even, smooth, finely granulated cytoplasm, surrounded by fewer than three compact layers of cumulus cells.

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There are those suggesting the need to revise the criteria employed for selecting oocytes. The data reported by Vassena et al. (2003) suggest that oocytes collected during the static or regressing phases of the follicular wave are preferable to those collected from follicles in the growing phase; it appears that the effects of early follicular atresia are beneficial to oocyte competence, although the reasons for this phenomenon are still unclear. The results reported by Alm and Torner (2003) showed that the staining of bovine COCs with brilliant cresyl blue before IVM could be used to increase the number of developmentally competent oocytes and to act as a marker of oocyte quality for such techniques as cloning. Brilliant cresyl blue stain determines the intracellular activity of glucose-6-phosphate dehydrogenase (G6PD), which is known to play a critical role in cell growth. The quality control and evaluation are necessary steps in collecting oocytes and embryos. Criteria used in evaluating cattle embryos (Lonergan, 1992). Developmental stage Morula Compact morula Early blastocyst

Blastocyst or midblastocyst

Expanded blastocyst Hatched blastocyst Hatched expanded blastocyst

Table 4

Identifying features Individual blastomeres are difficult to discern from one another. The cellular mass of the embryos occupies most of the perivitelline space Individual blastomeres have coalesced, forming a compact mass. The embryo mass occupies 60-70% of the perivitelline space This is an embryo that has formed a fluid cavity or blastocoel and has the general appearance of a signed ring. The embryo occupies 70-80% of the perivitelline space. Visual differentiation between trophoblast and inner cell mass may be possible at this stage of development. Pronounced differentiation of the outer trophoblast layer and the darker, more compact inner cell mass is evident. The blastocoel is highly prominent, with the embryo occupying most of the perivitelline space Overall diameter of the embryo dramatically increases (1.2-1.5 times), with a concurrent thinning of the zona pellucida to approximately one-third of its original thickness Embryos can be undergoing the process of hatching or may have completely shed the zona pellucida A re-expanded embryo with a large blastocoel and a round, very fragile appearance or, in later stages, an elongated shape

Figure 47 presents eve oocytes and embryos, which were successful in quality control.

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A – an eve oocyte surrounded by cumulus cells; B – an oocyte after cultivation in vitro; C – a sheep zygote; D – 2-celled embryos; E -2- and 4-celled embryos; F – 8-celled embryos and early morulas; G – embryos from one ewes-donor (No. 01424) of Kazakh Arkharomerinos; Figure 47. Quality control selection of sheep oocytes and embryos (kindly presented by Toishibekov E.M.) Control questions: 1. What for is the collection of sperm, oocytes and early embryos needed? 2. How can we collect sperm of farm animals? 3. How can we collect oocytes from farm animals? 4. How can we collect the early embryos of farm animals? 5. Describe the application of germ line cells and early embryos collection for Human.

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CHAPTER

8

ARTIFICIAL INSEMINATION Artificial insemination (AI) is used in animals ranging from the honeybee to the elephant. Some of the milestone events in the development of AI technology are detailed in Table 5. Milestones of AI technology (I.R. Gordon 2004. Reproductive Technologies in Farm Animals) Date 1677 1780

Event Discovery of sperm by the use of magnitying lens Artificial insemination of a dog bitch and the subsequent birth of pups 62 days later 1803 Freezing of stallion sperm in the sbow and motility recovered after warming 1890 AI in horses first attempted in France 1899 Started work on horse AI at Moscow State University 1912 Demonstrated AI in horses, achieved results comparable to those obtained by natural service. Achieved success in cattle and sheep AI and trained hundreds of inseminators 1914 Start of work in Italy which led to artificial vagina for semen collection in the dog 1920s and Development in Russia of artificial vaginas for use in 1930s bulls, stallions and rams, development a simple dilluents 1936 Shipment of ram semen from cambridge in the UK to Poland, birth a lamb after AI 1937 Development in Denmark of the rectovaginal method of AI in cattle 1941 Development of egg-yolk citrate semen diluent for cattle 1946 Antibiotics (penicillin and streptomycin) used to control pathogenic microorhganisms in semen used for AI 1949 Method of freezing sperm of several species discovered 1952 First calf born (Frostly I) after use of frozen-thawed bull semen in Cambridge 1960 Liquid nitrogen became the refrigerant of choice for preserving bull semen. Most countries used 100% frozen bull semen

160

Table 5

Researcher(s) Anton van Leeuwenhoek Spallanzani Spallanzani Repiquet Ivanov Ivanov Amantea Milovanov Arthur Walton Varios Danish workers Glenn Salisbury Almquist Chris Polge Chris Polge and Tim Rowson Many researchers in various countries

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The earliest reference to any form of AI is in the 13th-century Arabic scriptures featuring the horse. The first systematic exploitation of this technology was also in horses, with the work of the Russian physiologist Ivanov at a government stud farm more than a century ago. Without doubt, AI was the most important reproductive technology applied during the 20th century to cattle; unlike technologies such as embryo transfer, which in the cow calls for considerable expertise on the farm and in the laboratory to be successful, AI is relatively cheap and simple to apply. Some reliable authorities estimate that the contribution made by AI to improved dairy production worldwide since the Second World War was equal to the combined contributions of better health, husbandry and nutrition; the technique was to greatly accelerate genetic selection, most notably with dairy cattle. Current world statistics for AI in cattle stand at 232 million doses of semen produced as a frozen product and 11.6 million as liquid (Vishwanath, 2003). Fresh rather than frozen semen is primarily restricted to New Zealand, with limited amounts used in Africa, Australia, France, Germany and Eastern Europe. Insemination procedure In the 1940s, vaginal or shallow cervical insemination in cattle, performed with the aid of a vaginal speculum, was replaced by deep cervical or intrauterine insemination, involving the technique of cervical fixation per rectum; this method proved more efficient and was rapidly adopted as standard by the cattle AI industry (see Figure 48). Based on the assumption that the deposition of semen nearer to the site of fertilization (ampulla of the oviduct), the next logical step in cattle AI was to attempt deep AI; however, when this was done, it was found to have little effect on the success of AI.

Figure 48. Rectovaginal method of cow insemination (Hammond et al., 1983)

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Deep intracornual deposition of semen in cattle was first proposed in the late 1950s to increase the efficiency of AI, to reduce the required number of sperm per insemination dose and to enhance the use of sperm from genetically superior bulls that were in demand. More recent times have seen a resurgence in interest in deep inseminations. A study in Estonia reported by Kurykin et al. (2003), for example, showed that with deep intracornual insemination of PG-synchronized heifers at a fixed time (80–82 h) after the second of two PG injections, a sperm dose of 2 million sperm was as efficient as a dose of 40 million. It should be noted that intracornual AI requires greater care on the part of the inseminator because of the risk of perforating the uterine wall due to the tonicity of the uterus at oestrus and the danger of rupturing the ovulatory follicle while palpating the ovaries per rectum to determine the probable site of ovulation. Although there is ample evidence showing that a single laparoscopic insemination in sheep with frozen–thawed semen can result in acceptable conception rates, its use in commercial practice is limited by its cost and even more so by animal welfare considerations. Among the alternative insemination procedures studied is that of transcervical AI, which involves the passage of an instrument through the cervix and into the uterus; unfortunately, this technique, quite apart from not being possible with all sheep, has serious welfare implications because of the manipulations of ewe and cervix involved. Despite the long history of AI and its successful application to cattle breeding programmes, there are still areas of semen technology in which improvements can be made. Rejection of ejaculates, due to low motility, before or after freezing, is still common in AI centres; some bulls that apparently present a normal sperm picture and have an acceptable NRR of 60–70% may still yield ejaculates of which more than 50% are unsuitable for use. Factors influencing the sex ratio Sex is determined in farm mammals by the sex chromosome content of the sperm produced; females are produced by gametes containing an X-chromosome and males by sperm carrying the Y-chromosome. The sex ratio in a litter of piglets at birth (the so called secondary sex ratio) is determined by the primary sex ratio in the litter (the sex ratio immediately after fertilization) and eventual selective prenatal mortality. In several farms and other mammals, variation has occasionally been reported in the sex ratio of offspring, which is apparently influenced by genetic and environmental factors. The way in which chromosomes segregate at meiosis in the testes ensures that sperm carrying X- and Y-chromosomes are produced in equal numbers. There are, however, several interesting aspects to the actual ratio of males to females that are born under some conditions; it may not always be a matter of equal

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numbers of each sex. It now seems probable that, in species living in socially structured herds or flocks, females in good body condition or of high social rank produce more male than female offspring; increased dietary energy appears to be the factor that skews the sex ratio in favour of males. In deer, the suggestion is that male fetuses may be more vulnerable than females to the mother’s nutritional stress (arising from high population densities or other causes of food deprivation) because of faster male growth rates in utero; male fetuses require more nutrients and so may well be more adversely affected by food restrictions. Others who have speculated on the cause of sex ratio skewing suggest that one sex may signal its presence to the mother more strongly; alternatively, the uterine tract environment may favour embryos of one sex more than the other (Roberts et al., 2002). It is known that expanded female cattle blastocysts produce about twice asmuch interferon (IFN) as male blastocysts. It is also evident from various studies that male embryos have a greater ability to survive in a glucoserich medium; the uterine glucose environment may be greater in well-fed cows, thereby providing male embryos with a survival advantage. The higher production of IFN by female blastocysts may be a factor in their survival in a glucose-rich uterine environment. Data presented by Roberts et al. (2003) emphasize the high sensitivity of embryos, particularly females, to glucose; one possible explanation is that female embryos are compromised because of the presence of two transcriptionally active X-chromosomes, which causes imbalance in glucose metabolism. The semen-sorting technology currently applied commercially by Cogent in the UK is based on work at the American Beltsville Agricultural Research Center. It has been recognized for some time that the X-chromosome is larger and carries more DNA than the smaller Y-chromosome. In domestic livestock the DNA difference between X- and Y-bearing sperm varies from 3.5% to 4.2%. The first calves born from semen sexed by the Beltsville technique were produced by IVF. Clearly, far fewer sperm is required for fertilization when IVF is employed, although the techniques require modification to take account of the reduced motility and viability of sorted sperm (Zhang et al., 2003). According to Galli et al. (2003c), the use of sexed semen for IVF would increase if an efficient intracytoplasmic sperm injection (ICSI) procedure could be employed. Although there is a genuine interest among many cattle farmers in semen sexing, not all researchers and commercial concerns have expressed enthusiasm about sexing bull sperm by flow-cytometry; there are those taking the view that the high cost and lower pregnancy rates associated with flow-cytometry sorting make the approach impracticable for widespread use. For such reasons, some commercial concerns have supported work which seeks to detect sex-specific differences in sperm surface antigenicity in cattle. One approach by a Canadian biotechnology company was based on the assumption that bull and boar sperm

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have sex-specific proteins on their surface and that these can be separated using appropriate antibodies. Under this scenario, addition of male antibody would permit X-chromosome sperm to be filtered out, without causing cell membrane damage, enabling the sperm to be used fresh (pigs) or frozen (bull) in the normal way. Although some Canadian researchers expressed optimism about developing a viable immunological sperm-sexing procedure, elsewhere researchers have shown reservations on the possibility of identifying membrane proteins specific to X- or Y-bearing sperm. In Brazil, Matta et al. (2001) reported testing a monoclonal antibody against a malespecific protein for sexing semen, claiming that there was a cytolytic effect on male gametes and no effect on female cells; they reported almost 80% of female embryos after IVF. It is probably wise never to say that the seemingly impossible cannot happen. Control questions: 1. What do you know about artificial insemination applications in Animal Biotechnology? 2. What do you know about insemination procedure? 3. What do you know about factors influencing the sex ratio? 4. Describe the technical tools used for AI. 5. How is AI used at farm animals?

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CHAPTER

9

IN VITRO FERTILIZATION There are many reasons for interest in embryos that can be produced in the laboratory, rather than recovered from the living animal. Progress in cellular and molecular embryology in farm animals was difficult in the past due to the limited availability of suitable experimental material at an acceptable cost. Although oocytes and embryos can come from superovulated donor animals, this is likely to be expensive and not always free from animal welfare concerns. For such reasons, in vitro production (IVP) techniques, particularly those based on ovaries recovered after the donor’s demise, have received much attention in the past 10– 15 years (Galli and Lazzari, 2003). Europe has been at the forefront of applying such technologies. In the commercial cattle embryo transfer (ET) industry, in vitro embryo production is now an alternative to conventional means of obtaining embryos for transfer, using immature oocytes collected from the ovaries by ovum pick-up (OPU) of donor cattle of differing ages and physiological states. Reliable methods are now available for the maturation and fertilization of bovine oocytes in vitro; culture methods, although still imperfect, enable embryos to be grown to the stage at which they are suitable for transfer or cryopreservation. In vitro embryo production involves three steps, which have been developed to the greatest extent in cattle: oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC (in vitro culture)). During the past decade, IVP of bovine embryos has become a routine research tool in many laboratories; on the farm, the method is employed in commercial cattle breeding programmes in several countries. Statistics gathered for 25 European countries in the year 2003 showed more than 8000 in vitro-produced (IVP) embryos for commercial use, oocytes being collected either by aspiration from abattoir ovaries or by OPU from live donors. Although commercial uptake of such embryo-based biotechnology in cattle remains limited in the context of overall cattle ET activity, on the research front the ability of laboratories to generate large numbers of embryos is likely to have a considerable impact on the accumulation of biological knowledge on factors 165

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influencing early embryonic development in this species. In due course, marked improvements in embryo production technology can be expected, which will help to improve the efficiency of cattle production. OPU is a very flexible procedure which does not interfere with the productive and reproductive career of donors, yet allow the production of more embryos than conventional superovulation. Nonetheless, IVP of cattle embryos remains technically demanding and requires specific laboratory expertise and equipment to ensure the production of high-quality embryos. The term in vitro means in glass or in artificial conditions, and IVF refers to the fact that fertilization of the egg by sperm had occurred not in uterus but outside the uterus at an artificially maintained optimum condition. In recent years the IVF technology has revolutionized the field of animal biotechnology because of production of more and more animals as compared to animal production through normal course. For example, an animal produces about 4-5 offsprings in her life through normal reproduction, whereas through IVF technology the same can produce 50-80 offsprings in her life. Therefore, the IVF technology holds a great promise because a large number of animals may be produced and gene pool of animal population can also be improved. The IVF technology is very useful. It involves the following procedure: taking out the eggs from ovaries of female donor; in vitro maturation of egg cultures kept in an incubator; fertilization of the eggs in test tubes by semen obtained from the superior male; and implantation of seven-day-old embryos in reproductive tract of the other recipient female which acts as a foster mother or surrogate mothers. These are used only to serve as an animal incubator and to deliver offspring after normal gestation period. The surrogate mothers do not contribute anything in terms of genetic makeup as it comes from the egg of the donor mother and semen from artificial insemination. Steps in IVF: –– Initial evaluation –– Suppression of natural hormonal cycle –– Ovarian stimulation –– Collection of oocytes –– Collection of sperms –– In vitro fertilization of oocytes –– Embryo transfer The maturation of eggs is a very important point of the IVF technology and depends on many physiological and technical factors. Table 6 presents milestones in the development of the optimum conditions for animal egg maturation.

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Chapter 9. In vitro fertilization History of understanding the oocyte maturation (I.R. Gordon 2004. Reproductive Technologies in Farm Animals) Date 1935

Event Observation of spontaneous resumption of meiosis in rabbit oocytes 1939 Observation of resumption of meiosis in human follicular oocytes 1955 Detailed study of maturation of rabbit oocytes 1962-1965 Nuclear maturation in vitro achieved in several mammalian species 1968 Chronology of nuclear maturation in cattle oocytes 1977 Appreciation of importance of cytoplasmic and nuclear maturation 1978 Less than 1% of ruminant oocytes attained developmental competence after artificial maturation 1984 Crucial support of follicle cells in maturation of sheep oocytes

Table 6

Researcher(s) Pincus & Enzmann Pincus & Saunders Chang Edwards Sreenan Thibault et al. Moor & Warnes Stiugmiller & Moor

The immature oocytes are incubated in vitro so that they can mature. However, immature oocytes should be taken out from follicles because they cannot mature in it but degenerate. Thus, full potential of superovulation and all the oocytes can be utilized by IVF technology. Moreover, metabolic and hormonal requirement for oocytes during IVM should be found out so that the present rate of maturation (20%) could be improved. In majority of cases ovarian follicles never reach maturity and degenerate due to unknown causes. There is a possibility that they have genetic defects. Certain preliminaries must occur before sperm is in a position to effect fertilization. In embryo production, one of the first steps is the selection of sperm for use in IVF. A common practice is to select frozen–thawed sperm on the basis of the Percoll separation method. Mammalian sperm must undergo epididymal maturation, capacitation and the AR to be able to fertilize the oocyte. Studying epididymal sperm maturation in pigs, Burkin and Miller (2000) concluded that porcine sperm develops zona pellucida binding sites on the acrosomal ridge while they reside in the corpus region of the epididymis, thereby gaining the ability to fertilize oocytes. Studies reported by workers in Japan indicate that an increasing percentage of goat sperm acquires the potential to undergo the AR and fuse with the oocyte plasma membrane during transit through the caput epididymidis. During capacitation, several biochemical modifications occur in the sperm’s surface membrane; such changes are essential in permitting sperm–oocyte binding and the AR. During the AR, hydrolytic enzymes are released by exocytosis to enable the sperm to penetrate the zona pellucida.

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In Hungary, Somfai et al. (2002) showed that sperm with a higher viability and acrosome integrity could be obtained by Percoll separation than by a ‘swim-up’ method. Sperm capacitation is the biochemical modification of sperm that must undergo within the female tract before the cell can bind to the zona pellucida and undergo the acrosome reaction (AR). Capacitation is possible in vitro in the absence of reproductive-tract fluids and several compounds are known to induce in vitro capacitation (Table 7); the most common of these is the glycosaminoglycan (GAG) heparin. Although there are anecdotal reports from commercial clinics suggesting that heparin may not be needed for capacitation of frozen–thawed bull sperm prepared for IVF by centrifugation through Percoll, this is not supported by Mendes et al. (2003); they showed that heparin improved cleavage rates and embryo production in vitro, even when sperm were centrifuged through Percoll. The same workers found the commercial preparation Puresperm to be a useful alternative to Percoll when separating cryopreserved bull sperm for IVF. IVF of unfertilized eggs is carried out in small droplets (or microdroplets) of culture medium. Each microdroplet comprises of about 10 oocytes. The culture medium should be supplemented with penicillamine, hypotaurin, and epinephrine because they facilitate penetration of sperms into oocytes. Moreover, one dose of sperm is given that consists of about one million sperms per one ml of medium. Thus, in vitro fertilized embryo should be kept in in vitro conditions for a few days before it develops into blastocyst stage. For sheep and goats, it takes about seven days, for cattle it needs eight days. Many farm biotechnology laboratories are known where about 60 per cent IVF embryos of cattle were successfully cultured to blastocyst stage. Artificial capacitation of bovine sperm in vitro (I.R. Gordon 2004. Reproductive Technologies in Farm Animals) Date 1982 1983 1984 1984 1985 1986 1988 1988 1989 1989

Method High-ionic-strenth (HIS) medium Bovine follicle fluid Standard-ionic-strength medium Heparin Ionophore A23187 Liposomes Percoll gradient/hypotaurine Caffeine TEST yolk Oviductal cell monolayer

Researcher(s) Brackett et al. Fukui et al. Iritani et al. Parrish et al. Hanada Graham et al. Utsumi et al. Niwa et al. Ijaz & Hunter Guyader et al.

Table 7

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The term delivery from cultured embryo is very low due to occurrence of high loss during the first two months of pregnancy. This may be due to abortion of fetuses arising from the presence of genetic defects. It should be noted that before birth about 80 per cent genes play a key role in differentiation and development of fetuses. The oocytes which are forced to mature in vitro occasionally get some defects. Sometimes environmental mutagenesis occurs in eggs, sperms or embryos. Artificial culture media should be improved because oxygen may have a toxic effect. Therefore, gas atmosphere should be carefully controlled. Read and Smith proved this in 1996. The other most successful method of IVM is to place the fertilized zygotes into agar (so that it may wrap around it) and implant them in oviduct of synchronized sheep or rabbit where the environment for early development of embryo is perfect. For early bovine embryo the oviduct of rabbit and sheep has been used as in vitro culture system. Hundreds of cattle eggs can be put into the oviduct of a sheep and many of these are recovered after a week. A good quality of embryo at the late morula or blastocyst stage of development with a yield of about 40 per cent or more was recorded by Lu et al. (1987). Brackett et al. (1982) reported the birth of the first IVF calf after getting success in fertilizing the eggs recovered from ovulated cow. Thereafter, hundreds of IVF calves have been born in Japan, India, U.K., etc. The general standard scheme of IVF in cattle is presented in Figure 49.

Figure 49. IVF in cattle

ICSI Intracytoplasmatic injection of spermatozoids (ICSI) is a quite new method, invented for human in the case of male infertility, but now also used for animals. For various reasons, there may be a need to explore novel means of achieving fertilization. Some of the possible approaches are illustrated in Figure 50.

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Figure 50. Four approaches for micro-assisted fertilization. SUZI – subzonal spern injection. (I.R. Gordon 2004. Reproductive Technologies in Farm Animals)

As a result of poor IVF results, often with only 15–30% of oocytes being fertilized, ICSI has been employed in dealing with horse oocytes. Various workers have shown that ICSI is capable of increasing fertilization rates compared with IVF, and several foals have been born from IVM horse oocytes fertilized by sperm injection. In Italy, studies have demonstrated that a blastocyst rate of about 30% can be achieved after ICSI of IVM equine oocytes. According to Squires et al. (2003), the ICSI technique provides the possibility of obtaining pregnancies from stallions that may have low sperm numbers or poor semen quality. Although ICSI is the method of choice for fertilizing horse oocytes in vitro, the reasons for this are not well understood and embryo development rates have remained low. For such reasons, a study undertaken by Tremoleda et al. (2003) in The Netherlands sought to characterize the nuclear and cellular events occurring in horse oocytes during fertilization after sperm injection; it was concluded that, until conventional IVF becomes more reliable, ICSI probably remains the best way to produce equine embryos, to perform fundamental research into the cellular and molecular events of fertilization, to investigate infertility and to understand the cellular basis of early pregnancy failure in horses. ISCI has proved to be a valuable addition to the technology of assisted reproduction, especially in humans and, to a more limited extent, in horses. The technique also provides an opportunity for research into cell-cycle control and mechanisms involved in sperm-induced oocyte activation. The same sperm injection technique also has relevance as a sperm vector system for transgenic animal production and with freeze-dried sperm for which the maintenance of motility is not required.

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Other fertilization approaches in animal artificial reproduction Gamete intra-Fallopian tube transfer (GIFT) has been used in horses to deal with stallions having low sperm numbers or in situations in which frozen semen is in limited supply or sexed semen is being employed. In the GIFT technique, oocytes and sperm are transferred to the mare’s oviduct. Results from studies by Squires et al. (2003), who inseminated fresh semen into the oviduct, showed an 82% pregnancy rate compared with an 8% rate with the use of frozen–thawed sperm; the authors note that further work is required to determine why pregnancy rates are depressed when using cooled or frozen semen compared with fresh semen for GIFT transfer. When techniques such as GIFT are used in the horse, the culture and subsequent transfer of donor oocytes may pose additional risks for disease transmission; although the zona pellucida provides pathogen protection, the cumulus cells surrounding the oocyte, which are inevitably transferred with the gamete, could harbour intracellular viral pathogens. IVF in human Assisted reproduction technologies are treatments or procedures that include handling of human eggs and sperm or embryos for the purpose of establishing pregnancy. To understand the techniques, you will need some special definitions. Infertility is inability to conceive after 1 year of properly timed unprotected intercourse. Fecundity rate, a likelihood of achieving pregnancy in a given month in a couple with normal fertility is approximately 20% per month. Cumulative pregnancy rate after 12 months is 93%. In order to get pregnant under normal conditions: –– Healthy sperms аSperm ascent аAn ovum аFertilization аCell division. –– Implantation аWomen with blocked fallopian tubes (Tubal Factor) –– Ovulation problems (Diminished ovarian reserve; Endometriosis; Advancing age; Third party reproduction) –– Seminal problems for the man: sperm transport, motility… (Severe male factor) –– Unexplained infertility cases. –– Presence of seminal antibodies in the woman’s body Current methods of IVF in human include: –– Precycle Work-up –– Ovarian Stimulation –– Egg Retrieval –– Embryo Transfer –– PGD –– Laboratory Handling

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Ovarian stimulation is the first step controlled by vaginal ultrasound which allows specialists to monitor the number and size of the follicles and adjust the dose of the injections accordingly. HCG Hormone injection is given for final maturation of the eggs. Oocytes (eggs) retrieval is done 34-37 hours after taking the HCG injection. The fluid collected from the follicles in special tubes is examined by the embryologist and the eggs are isolated. The semen sample is taken in the same day and the eggs are fertilized either using the conventional IVF or ICSI. Suppression drugs preventing spontaneous ovulation are also an important step in the IVF cycle because that natural ovulation should not occur – if the eggs leave the ovary, the doctor will not be able to retrieve them. Ovarian stimulation is used to produce multiple mature follicles, rather than a single egg normally developed each month. It produces many good follicles to be Fertilized. Multiple eggs are stimulated because some eggs will not fertilize or develop normally after fertilization. Regular monitoring by ultrasound scan is done. Generally, eight to 14 days of stimulation is required. Used medications are presented in Figure 52.

Figure 52. Medications used to superovulation treatment in human

Possible side effects of ovarian stimulation: –– Discomfort, bruising or swelling at the injection site –– Rash –– Allergic sensitivity –– Headache –– Mood swings –– Abdominal discomfort and bloating –– Chance of multiple pregnancies –– Ovarian Hyperstimulation Syndrome (OHSS) Ovarian hyperstimulation can result in enlargement of the ovaries with leakage of fluid into the abdomen and rarely into the lungs. Most cases are mild, but a

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small proportion is severe and fatal. Treatment of OHSS depends on the severity of the hyperstimulation. Mild OHSS can be treated conservatively. Oocyte retrieval is done by using vaginal ultrasound, Transabdominal ultrasound or laparascope is used under general or local anaesthetic. Eggs are mixed with thousands of sperms in a special dish. Special incubators simulating natural conditions with 5% of CO2 are used. Fertilization is observed in the laboratory microscope. After fertilization is assured and cell division is observed, the embryos (usually 3) are returned to the woman’s uterus through the cervix using a special catheter (done in 2-5 days). Luteal phase support is also needed. Before implantation of embryos the genetic screening procedure is required (see Figure 53).

Figure 53. IVF genetic screening

It is recommended to transfer embryos at the blastocyst stage. Culture fertilized ova and early embryos with cells that normally surround the oocyte, so they can provide growth factors. Screen early embryos for chromosome abnormalities and implant only those with normal karyotypes. ICSI indications: –– Moderate to severe male factor:

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–– Severe problems in the seminal fluid such as a severe deficiency in the number or motility of the sperms or both. –– Epididymal or testicular sperm: In cases when there are no sperms in the seminal fluid but there are in testicles (absent duct). In these cases the sperms are taken from either the epididymis or the testes (PESA, TESA). –– History of failed fertilization with IVF –– Antisperm antibodies –– Low egg number Percutaneous Epididymal Sperm Aspiration (PESA) is recommended in the following cases: absence of the vas deferens; past infections which resulted in obstruction; cases who had vasectomy and surgical reversal failed. Testicular Sperm Aspiration (TESA) can be done by aspiration using a needle or surgical extraction (���������������������������������������������������� е��������������������������������������������������� taking a very small piece from the testes). The sbsence of sperms in the epididymis and absent or obstructed epididymis are indications for TESA. Applying ICSI at human the egg is injected with a single sperm, taken from the husband’s sperm (after preparation in the lab). A special needle is used to go through the wall of the egg and the sperm is introduced into the cytoplasm of the egg (see Figure 54).

Figure 54. ICSI at human Damage to the oocyte (meiotic spindle)

ICSI concerns with override natural safeguards that serve to prevent fertilization by abnormal sperm; transmission of paternal genetic abnormalities (Sex chromosomal abnormalities, Y chromosome microdeletions); karyotyping and Y chromosome deletion analysis should be offered to all men with severe male factor infertility, who are undergoing ICSI. The fertilization is observed later and after the division occurs, the embryos are transferred into the woman’s uterus. If repeated implantation fails, in special cases when the wall of the embryo is thick, the assisted hatching is recommended (see Figure 55).

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Figure 55. Assisted hatching in human

Various protocols have been described. They are partial zona dissection; acid tyrode’s assisted hatching; laser-assisted hatching; zona pellucida thinning. A small hole is made in the wall of the Embryo using either a needle, and a special chemical or a laser beam. Conventional methods of GIFT and ZIFT are still actual in assisted reproductive technologies in human. GIFT involves laparoscopical placing of mature eggs into the healthy fallopian tube along with washed sperm. In the case of ZlFT, the zygotes are placed directly into the fallopian tubes via laparoscopy or transcervical fallopian tube catherization. Since the day in 1978 when Louise Brown was born in England (Henig, 2003), human IVF has led to the birth of an estimated 1 million and more babies worldwide. At that time, media attention verged on the hysterical, with reports suggesting that testtube babies were “the biggest threat since the atom bomb”. Although such predictions turned out to be quite wrong, there are those, some with the benefit of hindsight, who have introduced a strong note of caution in applying human-assisted reproduction techniques without a solid body of research evidence in support. It is now recognized that the unregulated nature of human IVF failed to put appropriate emphasis on follow-up studies on the children born after IVF. There are those who have commented that techniques with such an impact on human welfare should have been under government-sponsored regulation from the start so that appropriate follow-up information would be guaranteed. In the USA, for example, the National Institute of Child Health and Human Development never funded human IVF research in any form. There are those, such as Winston (2003) in the UK, who draw attention to worrying aspects of more recent advances, such as intracytoplasmic sperm in-

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jection (ICSI). In this context, there is an obvious need for those engaged in the animal field to share their experiences with those in the human field. Control questions: 1. Indicate the milestones in the IVF technology development. 2. Describe the main steps in the IVF protocol. 3. Indicate where human oocytes can be collected during IVF program. 4. Describe the technical tools used for IVF. 5. What do you know about selection criteria of sperm, oocytes and embryos?

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Chapter 10. Embryo transfer

CHAPTER

10

EMBRYO TRANSFER The first embryo transfer (ET) dates back to Walter Heape more than a century ago in England. Many years were to pass before this novel form of reproductive technology was to reach the farm; when it did, it was predominantly in the breeding of cattle. Tim Rowson and associates in Cambridge, working with sheep in the mid1950s, showed usefulness of ET as a research tool. The technique provided the means for testing in sheep the relative importance of genetic and environmental factors for the developing sheep embryo. Although the birth of the first calf by ET occurred in the USA (Betteridge et al., 2000) in the early months of 1950, it was to be a further two decades before Rowson’s work in Cambridge led to commercial application of ET technology. The ET is a technique that increases both the commercial production and the genetic potential in livestock. The embryo transfer technique provides a rapid rate of improvement of animal genetic quality offering access to the highest-quality genetics at a lower cost than purchasing a live animal. Techniques inducing superovulation are used in conjunction with the ET to expedite the propagation of animals with genetic selection for desirable traits. A considerable variation in superovulatory response as well as the fact that the percentage of the superovulated donors does not produce transferable embryos, are the major limiting factors to the routine application of ET techniques. The main purpose of embryo transfer (ET) in domestic animals is to spread the genetic quality of livestock production for desirable traits. Although the basic procedures employed in the ET are now well established, there is a considerable scope for improvement of the ET technology in various areas. The main point in the ET is the donor-recipient synchrony. Over the years, much evidence has been accumulated on the importance of synchrony between donor and recipient in terms of their cycle stage. Exact synchrony should be the aim, but recipients out of phase by ± 1 day are generally regarded as acceptable, although some reduction in pregnancy rate is to be expected; cattle that are out of synchrony by as much as 2 days would not normally 177

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be used because of the reduced pregnancy rates. Some workers have searched for the ways of making synchrony as exact as possible. In Arkansas, for example, the use of an electronic estrous detection system to continuously monitor cattle permitted more precise timing of ET and resulted in improved pregnancy rates; Rorie et al. (2002) reported the data suggesting that continuous monitoring of embryo donors and recipients and selection of recipients with synchrony of ±12 h could improve pregnancy rates (see Table 8). Work in Ireland in the mid-1970s and elsewhere showed that it is possible to establish pregnancies by a non-surgical procedure involving the use of the standard Cassou inseminating instrument. The cow embryo is loaded, held in a small volume of medium (for example, phosphate-buffered saline (PBS) supplemented with 15% bovine serum), into a plastic straw (usually 0.25 ml capacity). At transfer, the straw is inserted into the inseminating instrument (“gun”) in the usual way and the same procedure as for AI is followed, the main difference being that the embryo is deposited around the mid-horn position (ipsilateral horn); before carrying out the transfer, the recipient animal is given an epidural anaesthetic and tranquillizer. Embryo-recipient synchrony and pregnancy rates (Rorie et al., 2002) Estrous synchrony category (h) -12 to -24 0 to -12 0 0 to +12 +12 to +24 0 to ±12 ±12 to ±24

Number of embryo transfers 37 67 9 78 37 126 102

Table 8

Pregnancy rate (Mean±sem) 51.4±8.2 58.2±6.1 66.7±16.6 61.5±5.6 48.6±8.2 62.7±4.4 50.0±4.9

During the past quarter-century, several variants of the standard transfer instrument have been marketed, with appropriate modifications to ensure that the embryo is deposited safely in the uterus. Pregnancy rates in mares after non-surgical ET are usually lower than for surgical transfer and are highly dependent upon operator experience and expertise to pass the sterile transfer pipette through the closed diestrous cervix. Cambridge workers have reported a new transfer method which obviates the need for such operator skill; they reported an 85% pregnancy rate, a marked improvement on rates previously achieved (50–55%) using the conventional transfer method (Wilsher and Allen, 2003). It was concluded that the new technique is manipulatively uncomplicated, simple to perform and remarkably successful. Maiden

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heifers are usually preferred as recipients in conventional cattle ET operations; quite apart from being free of problems arising from previous pregnancies, such animals are likely to cost less and be easier to acquire than cows. However, in terms of ease of transfer, the parous cow has a definite advantage; various studies have shown that 10% or so of heifer recipients may be difficult, if not impossible, to use for cervical transfers. Certain categories of maiden heifers as recipients may also pose very real welfare problems. In the UK, for example, the use of beef-type heifers as recipients for embryos from large dairy breeds (for example, Holstein–Friesian) and doublemuscled beef breeds has occasionally resulted in a proportion requiring surgery to deliver the fetus. Clearly, it is undesirable for unsuitable embryos to be transferred to recipients; legislation has been enacted in several countries to prohibit transfer of embryos likely to produce large calves. The importance of minimizing stress in recipient animals is rightly emphasized in various reports. Any routine treatment (e.g. antiparasitic) should take place at least 3 weeks prior to transfer; changes in the feeding regimen should be prohibited for 3-4 weeks before and after transfer. Recipients should be located where they can be easily and quietly handled on the day of transfer. Several authors stress the need for ET teams to examine their procedures to reduce stress and improve the welfare of all animals involved in their activities. Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) which has a number of beneficial actions in addition to its analgesic and antipyretic effects. There is evidence that substances that inhibit cyclo-oxygenase enzyme isoforms may improve IVF outcome in humans; treatment may take the form of a daily dose of 100 mg aspirin. It is also known that ibuprofen improves pregnancy rates in recipient cattle after ET and may be a useful, effective and safe adjunct to assisted reproduction in cattle (Elli et al., 2001). Practical applications of embryo transfer: –– Gender preselection –– Genetic preservation of endangered breeds –– Interspecific embryo transfer –– As a research tool Control questions: 1. What is the embryo transfer? 2. What do you know about embryo transfer applications in Animal Biotechnology? 3. What is the significance of the donor-recipient synchrony in ET? 4. What do you know about farm animal pregnancy rate after ET? 5. What are the main technical tools for ET?

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CHAPTER

11

CRYOPRESERVATION OF ANIMAL CELLS AND TISSUES Cryo-preservation or cryo-conservation is a process where organelles, cells, tissues, extracellular matrix, organs or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures (typically −80°C using solid carbon dioxide or −196 °C using liquid nitrogen). One of the most important early theoreticians of cryopreservation was James Lovelock (born 1919) known for Gaia theory. He suggested that damage to red blood cells during freezing was due to osmotic stress. During the early 1950s, Lovelock also suggested that increasing salt concentrations in a cell as it dehydrates to lose water to the external ice might cause damage to the cell. Cryopreservation of tissue during recent times began with the freezing of fowl sperm, which during 1957 was cryopreserved by a team of scientists in the UK directed by Christopher Polge. The process was applied to humans during the 1950s with pregnancies obtained after insemination of frozen sperm. However, the rapid immersion of the samples in liquid nitrogen did not, for certain of these samples – such as types of embryos, bone marrow and stem cells – produce the necessary viability to make them usable after thawing. Increased understanding of the mechanism of freezing injury to cells emphasized the importance of controlled or slow cooling to obtain maximum survival on thawing of the living cells. A controlled-rate cooling process, allowing biological samples to equilibrate to optimal physical parameters osmotically in a cryoprotectant (a form of anti-freeze) before cooling in a predetermined, controlled way proved necessary. The ability of cryoprotectants, in the early cases glycerol, to protect cells from freezing injury was discovered accidentally. Freezing injury has two aspects: direct damage from the ice crystals and secondary damage caused by the increase in concentration of solutes as progressively more ice is formed. During 1963, Peter Mazur, at Oak Ridge National Laboratory in the USA, demonstrated that lethal intracellular freezing could be avoided if cooling was slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. That rate differs between cells of differing size and water permeability: 180

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181

a typical cooling rate around 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulphoxide, but the rate is not a universal optimum. Cryopreservation is based on the ability of certain small molecules to enter cells and prevent dehydration and formation of intracellular ice crystals, which can cause cell death and destruction of cell organelles during the freezing process. At low enough temperatures, any enzymatic or chemical activity, which might cause damage to the biological material in question, is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing additional damage caused by the formation of ice during freezing. Traditional cryopreservation has relied on coating the material to be frozen with a class of molecules termed cryoprotectants. Two common cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol. Glycerol is used primarily for cryoprotection of red blood cells, and DMSO is used for protection of most other cells and tissues. A sugar called trehalose, which occurs in organisms capable of surviving extreme dehydration, is used for freeze-drying methods of cryopreservation. Trehalose stabilizes cell membranes, and it is particularly useful for the preservation of sperm, stem cells, and blood cells. The side effects of cryoprotectants: –– True chemical toxicity –– (Baxter and Lathe, 1971; Fahy, 1986). –– In vitrification methods very high concentrations of these compounds are necessary to achieve and maintain a vitreous state. –– The precise nature of the toxic effects of CPA remains, to a large degree, uncertain. –– Cryoprotectants have been shown to alter cytoskeletal components in mammalian oocytes, particularly the filamentous acting network and meiotic spindle (Johnson and Pickering, 1987;Vincent et al., 1990;Vincent and Johnson, 1992). New methods are constantly investigated due to the inherent toxicity of many cryoprotectants. By default it should be considered that cryopreservation alters or compromises the structure and function of cells unless it is proven otherwise for a particular cell population. Most systems of cellular cryopreservation use a controlled-rate freezer. This freezing system delivers liquid nitrogen into a closed chamber into which the cell suspension is placed. Careful monitoring of the rate of freezing helps to prevent rapid cellular dehydration and ice-crystal formation. In general, the cells are taken from room temperature to approximately −90 °C (−130 °F) in a controlledrate freezer. The frozen cell suspension is then transferred into a liquid-nitrogen freezer maintained at extremely cold temperatures with nitrogen in either the vapour or the liquid phase. Cryopreservation based on freeze-drying does not require use of liquid-nitrogen freezers.

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Cryoconservation of animal genetic resources is the process in which animal genetic material is collected and stored with the intention of conservation of the breed. Vitrification For successful commercial application, a simple process that permits direct embryo transfer and gives high pregnancy rates is what is required. The most significant steps in the cryopreservation of cattle embryos in recent years include the ability to freeze and transfer embryos in straws without dilution and the development of the open pulled straw (OPS) method for efficient vitrification of embryos and oocytes. In vitrification, ice-crystal formation is prevented by using high concentrations of cryoprotectants and high cooling and warming rates. Although vitrification as a method of cryopreserving embryos appeared on the scene in the mid-1980s as an alternative to the traditional slow freezing of cattle embryos, its suggested advantages (simplicity, cost, speed) had little impact on commercial ET operations and its application remained largely confined to research studies. Vitrification is an ultra-rapid cooling technique based on direct contact between the vitrification solution, containing cryoprotectant agents, and liquid nitrogen. The protocols for vitrification are simple, allowing cells and tissue to be placed directly into the cryoprotectants and then plunged directly into liquid nitrogen. It may be noted that the literature of cryopreservation technology makes a distinction between ‘thawing’ as applied to embryos and oocytes preserved by conventional freezing, and ‘warming’, which is the term used in bringing embryos back to ambient temperature after vitrification. In human-assisted reproduction, vitrification protocols are starting to be employed, with several births reported using protocols that have been successfully applied to bovine oocytes and embryos (Liebermann et al., 2002). Milestones in the application of vitrification to various mammalian species are detailed in Table 9. Milestones in vitrification of embryos Date 1985 1986 1986 1988 1989 1990 1994 1998

Species Mouse Cow Hamster Rat Rabbit Sheep/goat Horse Pig

Researcher(s) Rall & Fahy Massip et al. Critser et al. Kono et al. Smorag et al. Scieve et al. Hochi et al. Kobayashi et al.

Table 9

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Vitrification has the attraction of avoiding the need for expensive equipment, as required in a conventional cryopreservation program. In physical terms, vitrification is a process of solidification in which crystalline ice does not separate and there is no concentration of solutes, as in conventional freezing; there is an abrupt increase in the viscosity of the holding medium, producing a glasslike solid. Very high cooling rates are employed but initial exposure to the vitrifying solution is at refrigerator temperature and very brief or avoid adverse effects from cryoprotectant toxicity. Warming rate is also rapid to avoid crystal formation as the temperature returns to normal. Using the standard French ministraw as an embryo container, vitrification enabled a maximum cooling rate of about 2000°C/min. Vajta’s OPS method, on the other hand, permits much higher cooling and warming rates (> 20,000°C/min); the method involves loading the cattle embryos into a straw previously heat-pulled to half the diameter and thickness of the wall. In the freezing of embryos, the methods originally used by Whittingham and others with mice were found to be entirely unsuccessful with pigs; the three approaches taken towards the cryopreservation of the pig embryo are shown in Fig. 3.6. It early became evident that pig morulae are extremely sensitive to cooling below 15°C and it eventually became clear that this sensitivity to cooling and freezing was the result of their high lipid content; it also became clear that pig embryos at the expanded and hatched blastocyst stages have a higher tolerance to cooling than early blastocysts or morulae. Cryopreservation and freeze-drying of sperm Cryopreserved sperm is successfully used for AI and IVF. Although millions of sperms are normally used to inseminate different farm animal species, in all cases, only a minute fraction of these sperms reach the site of fertilization. There are those who believe that the difference among species in the ability of their sperm to survive freeze–thawing is related to their tolerance of osmotic stress. It is critically important that the osmotic behavior of sperm be determined and that cryopreservation protocols are adjusted to make it possible for sperm with appropriate motility and survival ability to be inseminated. It is likely that future research will continue to be concerned with semen storage. Although it has been known for many years that mouse sperm does not freeze well, it now appears that an alternative approach to long-term storage in this species may lie in freeze-drying. The production of mouse pups from freeze-dried sperm has recently been reported by Japanese researchers; they used low temperature and pressures to remove water from the sperms and stored them at 4°C for periods of up to 3 months. In reconstituting the gametes, it was simply a matter of adding water, removing the sperm heads and

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injecting them into mouse oocytes; of 57 oocytes injected, 54 survived and 49 began development in vitro. The transfer of 46 embryos to the uterus of recipient mice resulted in the birth of 14 normal young. Freeze-dried mouse sperm is motionless and is not fertile in the conventional sense; however, ICSI can enable it to achieve fertilization. In farm animals, a study by Keskintepe et al. (2002) was the first to report the production of apparently normal cattle blastocysts after the injection of frozen–thawed bull sperm that was selected, freeze-dried and stored at 4°C until use. Other work in the USA working with pigs, has demonstrated that cytologically dead boar sperm, which had been freeze-dried, were capable of fertilizing oocytes by way of ICSI and of delivering exogenous genetic material for the production of transgenic pig embryos. Although freeze-thawing does produce damage to the cells with loss of up to 50% of pre-freeze motility, since large numbers of cells are available, successful fertilization can be achieved even with low cryosurvival rates. Effects of cryopreservation of sperm: –– Sperm membranes have an unusual lipid composition –– Reduction in temperature alters the membrane lipid organization and modifies the kinetics or intra-membrane proteins, leading to lowered permeability and loss of fluidity. –– Loss of fluidity led to poor sperm survival –– Frozen/thawed sperm behaves similarly to capacitated sperm, which may lead to a shortened lifespan within the female tract; therefore the timing of insemination is important when using frozen sperm samples. –– Difference in sperm cryosurvival rates between normal semen and semen with abnormal parameters Methods to improve: –– Sperm preparation in order to remove immotile and damaged sperm prior to freezing may help to select a population of sperm with a better chance of survival. –– Use of stimulants such as pentoxifylline may also improve survival after thawing Freezing embryos Freezing of mammalian embryos was first shown to be possible in 1971, when David Whittingham and colleagues in London obtained live mice pups after the transfer of frozen–thawed embryos that had been frozen using either glycerol or dimethyl sulphoxide (DMSO). Some milestones in the freezing of mammalian embryos are detailed in Table 10.

Chapter 11. Cryopreservation of animal cells and tissues First young born after transfer of frozen-thawed embryos Date 1971 1973 1974 1974 1975 1976 1982 1984 1985 1988 1989 1989

Species Mouse Cow Rabbit Sheep Rat Goat Horse Human Hamster Cat Pig Rhesus monkey

185 Table 10

Researcher(s) Whittingham et al. Wilmut &Rowson Bank & Maurer Wiladsen Whittingham Bilton & More Yamomoto et al. Zeilmaker et al. Ridha & Dukelow Dresser et al. Hayashi et al. Wolf et al.

It is believed that sperm and embryos are capable of remaining viable at a temperature of −196°C (liquid nitrogen) for perhaps 1000 years or more, the only source of damage at such a temperature being direct ionization from background radiation. For normal purposes, however, there is little need to think in terms of storage for other than a few months or years. Many studies have shown that the usual length of cryopreservation of cattle embryos in liquid nitrogen does not affect their viability after thawing. Cambridge workers were at the forefront in developing effective techniques for the freezing of cattle embryos, the work of Chris Polge, Ian Wilmut and Steen Willadsen being particularly valuable. It was found that slow freezing (0.3°C/min) of cattle embryos to low subzero temperatures (−80°C) required slow thawing; slow freezing of embryos to relatively high subzero temperatures (−25°C to −35°C), on the other hand, required rapid thawing (360°C/min). Such findings, initially described by Willadsen in the freezing of sheep embryos, subsequently formed the basis of the cryopreservation technique that was to be widely adopted in commercial practice; since the late 1970s, the method has been the standard technique in freezing the embryos of many species, including human embryos. The first calf born after transfer of a frozen embryo in the early 1970s at Cambridge (Frosty II) did much to stimulate such research. In the course of the next three decades, countless thousands of cattle embryos were frozen and thawed for transfer in countries around the world. In cattle, as in other domestic species and humans, the study of cryobiology as it relates to embryo preservation is one of the most intensively researched areas of embryo biotechnology.

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Numerous protocols of cryopreservation were to be proposed (conventional slow freezing, ultra-rapid freezing and vitrification), each methodology with its advantages and disadvantages. Disease control is an important consideration in the export and import of embryos and recent years have seen moves to minimize or abandon altogether the use of animal proteins (serum or bovine serum albumin (BSA)) in the medium employed in bovine embryo freezing. A study reported by George et al. (2002) substituted plant protein (wheat peptones) for BSA, the workers finding that the substitution did not affect blastocyst survival and quality. During recent years, ethylene glycol has been effectively employed as a cryoprotectant for bovine embryo preservation. The molecular weight of this agent (62.1) is lower than that of glycerol (92.1), propylene glycol (76.1) and DMSO (78.1) and it seems possible that its beneficial effect is partly due to its high permeability; the fact that it permeates the embryo rapidly also eliminates the need for the stepwise dilution of the cryoprotectant at the time of thawing. In a survey of the cattle ET industry in North America, workers reported the growing popularity of ethylene glycol and direct embryo transfers, recording that, in 1997, 55.4% of frozen–thawed embryos transferred in the USA and 87.6% of those in Canada were frozen using the agent. In France, workers have recently reported studies with 2134 transfers in which ethylene glycol was compared with a glycerol–sucrose combination; they recorded an improved success rate with ethylene glycol (55.4% vs. 47.2%). Further information on the use of ethylene glycol in the freezing of cattle embryos in the USA is provided by Hasler (2002). Embryos can be frozen successfully by slow and ultrarapid freezing. Timing of pronucleate freezing is crucial, and the process must be initiated while the pronuclei are still distinctly apparent, no later than 20–22 hours after insemination. 2 to 8 cell embryos should be of good quality, grade 1 or 2, with less than 20% cytoplasmic fragments. Uneven blastomeres and a high degree of fragmentation jeopardize survival potential; embryos with damage after thawing may still be viable and result in pregnancies, but their prognosis for implantation is reduced. Cryopreservation of oocytes The first report on successful freezing of a mammalian oocyte was the work of David Whittingham in London in the late 1970s with mice; it was shown to be possible to obtain live offspring after IVF of mouse oocytes frozen with dimethyl sulphoxide (DMSO) and stored in liquid nitrogen. Despite such success, the history of oocyte freezing during the past quarter-century has revealed difficult problems; certainly, in terms of applications in human assisted reproduction, current cryopreservation protocols are regarded as far from optimal (Van der Elst, 2003). Several workers have reported on their efforts to develop effective vitrification techniques for the cryopreservation of bovine oocytes. In Nottingham, for

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example, Mavrides and Morroll (2002) suggest that the cryo-loop vitrification technique followed by ICSI could be effective. The cryo-loop is a technique where a thin nylon loop is used to suspend a film of cryoprotectant containing the oocytes before they are immersed in liquid nitrogen. Using bovine oocytes, the Nottingham workers reported a survival rate of 90.5% in comparison with a slow-freezing technique (54.4%). Mammalian oocytes are particularly susceptible to freeze-thaw damage due to their size and complexity. They must not only survive thawing, but also preserve their potential for fertilization and development. Cryopreserving of tissues Some have attempted to preserve oocytes by vitrifying ovarian tissues. A study by Al-Aghbari and Menino (2002), for example, reported that sheep oocytes could be successfully cryopreserved by vitrification of ovarian tissues; they subsequently exhibited IVM rates similar to those of vitrified and non-vitrified oocytes. An important application of cryopreservation is in the freezing and storage of hematopoietic stem cells, which are found in the bone marrow and peripheral blood. In autologous bone-marrow rescue, hematopoietic stem cells are collected from a patient’s bone marrow prior to treatment with high-dose chemotherapy. Following treatment, the patient’s cryopreserved cells are thawed and infused back into the body. This procedure is necessary, since high-dose chemotherapy is extremely toxic to the bone marrow. The ability to cryopreserve hematopoietic stem cells has greatly enhanced the outcome for the treatment of certain lymphomas and solid tumour malignancies. In the case of patients with leukemia, their blood cells are cancerous and cannot be used for autologous bone-marrow rescue. As a result, these patients rely on cryopreserved blood collected from the umbilical cords of newborn infants or on cryopreserved hematopoietic stem cells obtained from donors. Since the late 1990s it has been recognized that hematopoietic stem cells and mesenchymal stem cells (derived from embryonic connective tissue) are capable of differentiating into skeletal and cardiac muscle tissues, nerve tissue, and bone. Today there is an intense interest in the growth of these cells in tissue culture systems, as well as in the cryopreservation of these cells for future therapy for a wide variety of disorders, including disorders of the nervous and muscle systems and diseases of the liver and heart. Control questions: 1. What is cryopreservation? 2. Why do researchers need cryopreservation of germ line cells and embryos? 3. What kinds of cryoprotectants are used for animal cells? 4. Describe the main steps in cryopreservation of germ line cells and embryos. 5. What are the differences between cryopreservation protocols of sperm and oocytes/embryos?

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CHAPTER

12

CHIMERAS. XENOTRANSPLANTATION A chimera or an allophenic animal is the animal consisting of different genotype cells which originated from more than one zygote (see Figure 56). Different cell populations can be combined in one organism during any stage of development.

Figure 56. Chimeric mouse born from 3 “parents” (S. F. Gilbert et al., Developmental Biology, 11th edition, 2016)

In biological research, chimeras are artificially produced by selectively transplanting embryonic cells from one organism onto the embryo of another, and allowing the resultant blastocyst to develop. Chimeras are not hybrids, which form from the fusion of gametes from two species that form a single zygote with a combined genetic makeup. An animal chimera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic. Chimeras are formed from at least four parent cells (two fertilized eggs or early embryos fused together). Each 188

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population of cells keeps its own character and the resulting organism is a mixture of tissues. As with cloning, the process of creating and implanting a chimera is imprecise, with the majority of embryos spontaneously terminating. Successes, however, have led to major advancements in the field of embryology, as creating chimeras of one species with different physical traits, such as color, has allowed researchers to trace the differentiation of embryonic cells through the formation of organ systems in the adult individual. The first known primate chimeras are the rhesus monkey twins, Roku and Hex, with each having six genomes. They were created by mixing cells from totipotent four cell blastocysts; although the cells never fused they worked together to form organs. It was discovered that one of these primates, Roku, was a sexual chimera; as four percent of Roku’s blood cells contained two x chromosomes. A major milestone in chimera experimentation occurred in 1984, when a chimeric geep was produced by combining embryos from a goat and a sheep, and survived to adulthood. The creation of the “geep” revealed several complexities to chimera development. In implanting a goat embryo for gestation in a sheep, the sheep’s immune system would reject the developing goat embryo, whereas a “geep” embryo, sharing markers of immunity with both sheep and goats, was able to survive implantation in either of its parent species. In 2003, researchers at the Shanghai Second Medical University in China reported that they had successfully fused human skin cells and rabbit ova to create the first human chimeric embryos. The embryos were allowed to develop for several days in a laboratory setting, then destroyed to harvest the resulting stem cells. In 2007, scientists at the University of Nevada School of Medicine created a sheep whose blood contained 15% human cells and 85% sheep cells. Chimeric mice are important animals in biological research, as they allow the investigation of a variety of biological questions in an animal that has two distinct genetic pools within it. These include insights into such problems as the tissue specific requirements of a gene, cell lineage, and cell potential. The general methods for creating chimeric mice can be summarized either by injection or aggregation of embryonic cells from different origins. The first chimeric mouse was made in the 1960s by Beatrice Mintz, he used the aggregation of eight-cell-stage embryos. Richard Gardner and Ralph Brinster developed the method of injection cells into blastocysts to create chimeric mice with germ lines fully derived from injected embryonic stem cells (ES cells). Chimeras can be derived from mouse embryos that have not yet implanted in the uterus as well as from implanted embryos. ES cells from the inner cell mass of an implanted blastocyst can contribute to all cell lineages of a mouse including the germ line. ES cells are a useful tool in chimeras because genes can be mutated in them through the use of homologous recombination, thus allowing gene targeting. Since this discovery occurred

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in 1999, ES cells have become a key tool in the generation of specific chimeric mice for many purposes. The ability to make mouse chimeras comes from an understanding of early mouse development. Different parts of the mouse embryo retain the ability to give rise to a variety of cell lineages between the stages of fertilization of the egg and the implantation of a blastocyst into the uterus. Once the embryo has reached the blastocyst stage, it is composed of several parts, mainly the trophectoderm, the inner cell mass, and the primitive endoderm. Each of these parts of the blastocyst gives rise to different parts of the embryo. The inner cell mass gives rise to the embryo proper, while the trophectoderm and primitive endoderm give rise to extra embryonic structures that support growth of the embryo. From two- to eight-cell-stage embryos are competent for making chimeras, since at these stages of development, the cells in the embryos are totipotent, and not yet committed to give rise to any particular cell lineage. They could give rise to the inner cell mass or the trophectoderm. In the case where two diploid eight-cell-stage embryos are used to make a chimera, chimerism can be later found in the epiblast, primitive, endoderm and trophectoderm of the mouse blastocyst. It is possible to dissect the embryo at other stages so as to accordingly give rise to one lineage of cells from an embryo selectively and not to the other. For example, subsets of blastomeres can be used to give rise to a chimera with specified cell lineage from one embryo. The Inner Cell Mass of a diploid blastocyst, for example, can be used to make a chimera with another blastocyst of eight-cell diploid embryo. The cells taken from the inner cell mass will give rise to the primitive endoderm and to the epiblast in the chimera mouse. ES cell contributions to chimeras have been developed from this knowledge. ES cells can be used in combination with eight-cell-and twocell-stage embryos to make chimeras and exclusively give rise to the embryo proper. Embryos that are to be used in chimeras can further be genetically altered in order to specifically contribute to only one part of chimera. An example is the chimera built off of ES cells and tetraploid embryos, tetraploid embryos which are artificially made by electrofusion of two two-cell diploid embryos. The tetraploid embryo will exclusively give rise to the trophectoderm and primitive endoderm in the chimera. Methods of creating chimeras (see Figure 57): –– Embryonic aggregation chimeras –– Injection of embryonic cells to blastocyst –– Therato-carcinomas (for example, the introducton of embryonic cells to the adult) –– Xenotransplantation (transplantations of cells, tissue, organs from one adult to another)

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Figure 57. Injection method of creating chimeras

Steps in creating a chimerical mouse: –– Isolation of eggs from donors with different genotypes; –– Embryos should be cultivated on standard culture media (base-buffered saline, STC and lactate, glucose, albumin) to stage of 8 blastomeres; –– 8-cells morula aggregation (aggregation method), and cultivation in vitro to the blastocyst stage; –– chimeric embryo should be implanted into the uterus of female recipient Animal chimeras are produced by the merger of multiple fertilized eggs or fusing of stem cells. There is a variety of combinations that can give rise to a successful chimera mouse and – according to the goal of the experiment – an appropriate cell and embryo combination can be picked. They are common but not limited to diploid embryo and ES cells, diploid embryo and diploid embryo, ES cell and tetraploid embryo, diploid embryo and tetraploid embryo, ES cells and ES cells. The combination of embryonic stem cell and diploid embryo is a common technique used for making of chimeric mice, since gene targeting can be done in the embryonic stem cell. These kinds of chimeras can be made through either aggregation of stem cells and the diploid embryo or injection of the stem cells into the diploid embryo. For gene targeting to make a chimera the embryonic stem cells are used by the following procedure: 1) a construct for homologous recombination for the gene targeted will be introduced into cultured mouse embryonic stem cells from the donor mouse, by electroporation; 2) cells positive for the recombination event

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will have antibiotic resistance, provided by the insertion cassette used in the gene targeting; and be able to be positively selected for. ES cells with the correct targeted gene are then injected into a diploid host mouse blastocyst. These injected blastocysts are then implanted into a pseudo pregnant female surrogate mouse which will bring the embryos to term and give birth to a mouse whose germline is derived from the donor mouse’s ES cells. The same procedure can be achieved through aggregation of ES cells and diploid embryos, diploid embryos are cultured in aggregation plates in wells where single embryos can fit, to these wells ES cells are added, the aggregates are cultured until a single embryo is formed and has progressed to the blastocyst stage, and can then be transferred to the surrogate mouse. Xenotransplantation is the transplantation of living cells, tissues or organs from one species to another. Such cells, tissues or organs are called xenografts or xenotransplants. It is contrasted with allotransplantation (from other individual of the same species), Syngeneic transplantation (Grafts transplanted between two genetically identical individuals of the same species) and Autotransplantation (from one part of the body to another in the same person). The first serious attempts at xenotransplantation (then called heterotransplantation) appeared in the scientific literature in 1905, when slices of rabbit kidney were transplanted into a child with renal insufficiency. In the first two decades of the 20th century, several subsequent attempts to use organs from lambs, pigs and primates were published. Scientific interest in xenotransplantation declined when the immunological basis of the organ rejection process was described. The next waves of studies on the topic came with the discovery of immunosuppressive drugs. Even more studies followed Dr. Joseph Murray’s first successful kidney transplantation in 1954 and scientists, facing the ethical questions of organ donation for the first time, accelerated their effort in looking for alternatives to human organs. In 1963, doctors at Tulane University attempted chimpanzee-to-human kidney transplantations in six people who were near death; after this and several subsequent unsuccessful attempts to use primates as organ donors and the development of a working cadaver organ procuring program, interest in xenotransplantation for kidney failure dissipated. An American infant girl known as “Baby Fae” with hypoplastic left heart syndrome was the first infant recipient of a xenotransplantation, when she received a baboon heart in 1984. The procedure was performed by Leonard L. Bailey at Loma Linda University Medical Center in Loma Linda, California. Fae died 21 days later due to a humoral-based graft rejection thought to be caused mainly by an ABO blood type mismatch, considered unavoidable due to the rarity of type O baboons. The graft was meant to be temporary, but unfortunately a suitable allograft replacement could not be found in time.

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Xenotransplantation of human tumor cells into immunocompromised mice is a research technique frequently used in oncology research. It is used to predict the sensitivity of the transplanted tumor to various cancer treatments. Human organs have been transplanted into animals as a powerful research technique for studying human biology without harming human patients. This technique has also been proposed as an alternative source of human organs for future transplantation into human patients. For example, researchers from the Ganogen Research Institute transplanted human fetal kidneys into rats which demonstrated life supporting function and growth. Xenotransplantation differs from other uses of genetically engineered animals in that it has the potential to create something entirely new–permanent human–animal chimeras–in which cells of distantly-related species survive and function for long periods of time in the most intimate contact possible. Given its potential for alleviating human diseases due to irreversible tissue or organ failure (see Table 11), and given the acute shortage of human organs for transplant, there are very active research programs underway, in both commercial and academic laboratories, to overcome the significant immunologic and physiologic barriers, and thereby to bring xenotransplantation into standard medical practice. Table 11 Application of xenotransplantation in medical practice (https://www.ncbi.nlm.nih.gov/books/NBK207578/table/ttt00004/?report=objectonly) Indication Organ Failure Acute liver failure Diabetes Parkinson's disease, Huntington's Disease, Focal epilepsy, Stroke Burn, Skin injury

Transplant Status Pig heart, kidney, liver, etc. No successful experience Extracorporeal perfusion Some trials have been performed. Pancreatic islets (or cells) Some trials have been performed. Neural tissue Some trials have been performed. Skin autograft Successful trials have been (co-cultured with mouse performed. cells)

At present, the only animal under serious consideration as a xenotransplant donor is the pig. For regulatory purposes, human cells cultured ex vivo with the cells of any other animal, such as mouse cell lines, are also considered to be xenotransplants (DHHS, 2001); co-cultivation with mouse cell lines has been used in the preparation of some cultured skin grafts as well as human stem cell lines; Thomson et al., 1998). While nonhuman primates, such as the baboon, would seem to have physiologic and immunogenetic advantages such as the lack of a hyper-acute immune response, their scarcity as well as difficulty of clearing

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them of adventitious infectious agents (as well as ethical concerns) makes them impractical for further consideration. The field of xenotransplantation covers a great spectrum of procedures, ranging from implantation of single cells to treat Parkinson’s disease and tissues, such as pancreatic islets, to treat diabetes; extracorporeal use of intact organs, such as perfusion of patient blood through pig livers to provide short-term support in cases of liver failure; to transplantation of whole organs–heart, kidney, liver, and so on. While whole-organ xenotransplantation remains far in the future, development of the simpler modalities is underway, and hundreds of human subjects have received porcine cells or tissues as part of clinical trials in the United States, Russia, Israel, and many European countries (Paradis et al., 1999). Given the nature of infectious disease issues, regulatory concerns are not limited to the United States alone, but extend to the international health community as well. The development of xenotransplantation as a part of clinical practice promises great benefits in terms of making possible essentially infinite supplies of replacement tissues and organs where severe shortages exist today. This development will naturally entail both great potential benefit as well as considerable risk to the study participant, but such risk is not qualitatively different from that entailed in the development of any other new medical procedure and will not be considered further. The principal concern is that the uniquely close relationship created between recipient and host will allow novel opportunities for transmission of infectious disease, and possible creation of new disease agents in the process. While the history of close contact between humans and pigs is a very long one, and one would imagine that all possible transmission of infectious agents between the two species had already been seen and thoroughly studied, it is possible that the “co-culture” environment of a transplant would be qualitatively different in ways that would allow different outcomes. Control questions: 1. Give the definition of xenotransplantation. 2. What do you know about xenotransplantation applications in Animal Biotechnology? 3. What is a chimeric organism? 4. Describe the methods of creating chimeras. 5. What do you know about application of xenotransplantation in medical practice?

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CHAPTER

13

CLONING TECHNOLOGY TO PRODUCE ANIMAL CLONES Cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms reproduce asexually. Cloning is a natural form of reproduction that has allowed life forms to spread for more than 50 thousand years. So, a clone is an organism that descended from and is genetically identical to a single common ancestor. Clones are abundant in nature – when a zygote splits in two, identical twins are formed. Cloning technology involves techniques making genetically identical copies of an organism, using asexual reproduction. Animal cloning history spans more than 100 years. The milestones of animal cloning history are represented in Table 12. Table 12

Milestones in animal cloning history Date

Species

1 1885

2 Sea urchin

1902

Salamander

1928

Salamander

1952

Frog

Researcher(s)

Event

3 4 Hans Adolf Ed- First-ever demward Dreisch onstration of artificial embryo twinning Hans Spemann Artificial embryo twinning in a vertebrate Hans Spemann First instance of nuclear transfer Robert Briggs First successful & Thomas nuclear transfer King

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Implication 5 The sea urchin is a relatively simple organism that is useful for studying development. Embryos from a more-complex animal can also be “twinned” to form multiple identical organisms– but only up to a certain stage in development. The cell nucleus controls embryonic development Nuclear transfer was a viable cloning technique. Embryonic cells early in development are better for cloning than cells at later stages.

196 1 1958

1975 1984 1987

1996

1996

1997

1997

19981999 2001

2007

2013

Part II. Animal biotechnology 2 1.Frog

3 John Gurdon

4 Nuclear transfer from a differentiated cell

5 Nuclei from somatic cells in a fully developed animal could be used for cloning. Cells retain all of their genetic material even as they divide and differentiate. Rabbit J. Derek Brom- First mammalian Mammalian embryos could be crehall embryo created by ated by nuclear transfer. nuclear transfer Sheep Steen WilFirst mammal cre- It is possible to clone a mammal ladsen ated by nuclear by nuclear transfer–and that the transfer clone could fully develop. Cow Neal First, Nuclear transfer Cows added to the list of mammals Randal Prather, from embryonic that could be cloned by nuclear & Willard Eye- cell transfer. stone Sheep Ian Wilmut and Nuclear transfer Cultured cells can supply donor nuKeith Campfrom laboratory clei for cloning by nuclear transfer. bell cells It is possible to use such modified cells to create transgenic animals– such as cows that could make insulin for diabetics in their milk. Sheep Ian Wilmut & Dolly: First mam- Arrival of Dolly started conversaKeith Campmal created by so- tions about the implications of bell matic cell nuclear cloning, bringing controversies transfer over human cloning and stem cell research into the public eye. Rhesus mon- Li Meng, John First primate cre- Primates and humans,’ closest relakey ated by embrytives, can be cloned. Ely, Richard Stouffer, & Don onic cell nuclear transfer Wolf Sheep Angelika Nuclear transfer Sheep could be engineered to Schnieke, from genetically make therapeutic and other useful Keith Campengineered labora- proteins in their milk, highlighting bell, Ian tory cells the potential medical and commerWilmut cial uses for cloning. Mice, cows, Multiple More mammals Several more animals had been and goats groups cloned by somatic successfully cloned including cell nuclear transfer transgenic animals. Gaur and Multiple Endangered aniScientists began to explore clonMouflon groups mals cloned by so- ing as a way to create animals matic cell nuclear belonging to endangered or extinct transfer species. Rhesus mon- Shoukhrat Primate embryonic Possibility of human therapeutic key Mitalipov and stem cells created cloning: creating individual-specifcolleagues by somatic cell ic stem cells that could be used to nuclear transfer treat or study diseases. Human Shoukhrat Human embryonic First successful usage of somatic Mitalipov and stem cells created cell nuclear transfer to create a hucolleagues by somatic cell man embryo that could be used as nuclear transfer a source of embryonic stem cells.

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A short description of the key experiments in cloning will give an input to getting knowledge to understanding the basic questions in developmental biology, area of the scientific opportunities and applications. Dreisch’s experiment revealed that it was possible to separate cells by merely shaking two-celled sea urchin embryos. After separation each cell grew into a full sea urchin. This experiment showed that each cell in the early embryo has its own complete set of genetic instructions and can grow into a full organism. Spemann’s experiment was to figure out how to split the two cells of an embryo much stickier than sea urchin cells. He fashioned a tiny noose from a strand of baby hair and tightened it between two cells of a salamander embryo until they separated. Each cell grew into a normal adult salamander. Also, Spemann tried to divide more advanced salamander embryos using this method, but he revealed that cells from these embryos did not develop successfully into adult salamanders. This experiment showed that embryos from a more-complex animal can also be “twinned” to form multiple identical organisms, but only up to a certain stage in development. Again using a strand of the baby hair tied into a noose, Spemann temporarily squeezed a fertilized salamander egg to push the nucleus to one side of the cytoplasm. The egg divided into cells, but only on the side with the nucleus. After four cell divisions, which made 16 cells, Spemann loosened the noose, letting the nucleus from one of the cells slide back into the non-dividing side of the egg. He used the noose to separate this “new” cell from the rest of the embryo. The single cell grew into a new salamander embryo, as did the remaining cells that were separated. Essentially the first instance of nuclear transfer, this experiment showed that the nucleus from an early embryonic cell directs the complete growth of a salamander, effectively substituting for the nucleus in a fertilized egg. Robert Briggs and Thomas King first transferred the nucleus from an early tadpole embryo into an enucleated frog egg, a frog egg from which the nucleus had been removed. The resulting cell developed into a tadpole. The scientists created many normal tadpole clones using nuclei from early embryos. However, just like Spemann’s salamander experiments, the cloning procedure was less successful with donor nuclei from more advanced embryos: only few tadpole clones that did survive grew abnormally. It is important, that this experiment showed that nuclear transfer was a viable cloning technique. It also reinforced two earlier observations: first, the nucleus directs cell growth and, ultimately, an organism’s development; second, cells of early stage of embryo development are better for cloning than cells from embryo at later stages. John Gurdon transplanted the nucleus of a tadpole intestinal cell into an enucleated frog egg. By this way, Gurdon created tadpoles that were genetically identical to the one from which the intestinal cell was taken. This experiment showed that, despite previous failures, nuclei from somatic cells in a fully devel-

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oped animal could be used for cloning. Importantly, it suggested that cells retain all of their genetic material even as they divide and differentiate, although some wondered if the donor DNA came from a stem cell, which can differentiate into multiple types of cells. The sizes of mammalian egg cells are much smaller than those of frogs or salamanders, so mammalian eggs are harder to manipulate. Bromhall first transferred the nucleus from a rabbit embryo cell into an enucleated rabbit egg cell using a glass pipette as a tiny straw. Bromhall considered the procedure a success when a morula, or advanced embryo, developed not more than couple of days after fertilization. This experiment showed that Mammal’s embryos could be created by nuclear transfer. To demonstrate that the embryos could continue developing, Bromhall would have had to place them into a mother rabbit’s utery. Steen Willadsen used a chemical process to separate one cell from an 8-cell lamb embryo. He used a not powerful and short-timed electrical shock to fuse it to an enucleated egg cell. And the new reconstructed cell started to divide successfully. To that time, in vitro fertilization techniques had been developed, and they had been used successfully to help human couples have babies. Using the IVF approach, a few days later after reconstruction, Willadsen placed the lamb embryos into the womb of the surrogate mother sheep. And three live lambs were born from the surrogate mother. The experiment showed that it was possible to clone a mammal by nuclear transfer–and that the clone could fully develop. But it is important to use cells of early embryo for donor nuclei. The experiment was considered a great success. Prather and Eyestone repeated the Willadsen’s experiment using cows instead of sheep, and successfully produced two cloned calves, which were called Fusion and Copy. This experiment added cows to the list of mammals that could be cloned by nuclear transfer. But by that time, mammalian cloning was still limited to using embryonic cells as nuclear donors. Using nuclei from differentiated adult somatic cells for cloning still wasn’t thought possible. In all previous successful cloning experiments the scientists used donor nuclei from cells of early embryos. Ian Wilmut and Keith Campbell set up the experiments where the donor nuclei came from a slightly different source: cultured cells of adult sheep, which were kept alive in the laboratory. Wilmut and Campbell made a nuclear transfer into enucleated sheep egg cells using nuclear from cultured somatic cells. The lambs named Megan and Morag were born from this procedure. So, it was revealed that cultured cells can supply donor nuclei for cloning by nuclear transfer. Soon the nuclei transfer technique was started to use for creation of transgenic animals because biotechnologists had already known how to do gene transfer into cultured cells. It was possible to use such modified cells to create transgenic animals, for example, cows that could produce milk containing insulin for diabetics.

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Cloning Dolly sheep was a landmark experiment because never before had a mammal been cloned from an adult somatic cell. Wilmut and Campbell created a clone lamb using transfer of nucleus from an adult sheep’s udder cell into an enucleated egg of another sheep. Despite the fact that every cell of the adult organism contains a nucleus with a complete set of genetic information, differentiated adult cells shut down the genes that they don’t need for their specific functions while the embryonic cells are ready to activate any gene. When scientists use an adult cell nucleus as a donor, the genetic information of the “adult” nucleus must be reset to an embryonic state. Very often the reprogramming process is incomplete, and the reconstructed embryos fail to develop. In Wilmut and Campbell’s experiment, of total 277 attempts, only one produced a normal embryo that was carried to term in a surrogate mother’s body. The name of this famous lamb was Dolly. This successful experiment brought cloning into the public attention. Appearance of Dolly started conversations about the implications of cloning, bringing controversies over human cloning and stem cell research into the public eye. The new usage of nuclear transfer and cloning techniques started intensively. Due to evolutionary relations between humans and primates, scientists started to use primates as a good model for studying human disorders. Li Meng et al. first received identical cloning primates with purpose to decrease the genetic variation of research animals, and therefore the number of animals needed in research studies. Like in the previous cloning experiments, Wolf with collaborators fused the early-stage embryonic cells with the enucleated monkey egg using an electrical shock. Then the reconstructed embryos were implanted into surrogate mothers. Two monkeys were born out of 29 cloned embryos. One of these was a female named Neti, and the other was a male called Ditto. So, this experiment showed that primates, the closest relatives of human, can be cloned. The experiment of A. Schnieke et al. regarding nuclear transfer from genetically engineered laboratory cells was an exciting combination of previous discoveries. As described before, by that time, Campbell and Wilmut had already created a clone using a cultured cell as a donor of nucleus. Schnitke’s team introduced the human gene encoding Factor IX (“factor nine”) into the genome of sheep skin cells grown in a laboratory dish. Factor IX, a protein that helps blood clot, is intensively used to treat hemophilia, a genetic disease where blood doesn’t form proper clots. To make a transgenic sheep, the biotechnologists carried out the nuclear transfer using donor DNA from the cultured transgenic cells. A sheep Polly, that produced Factor IX protein in her milk, was the result of that experiment. Scientists realized that sheep could be engineered to make therapeutic and other useful proteins in their milk, highlighting the potential medical and commercial uses for cloning. The approach was termed “farming”. After the successes with Polly and Dolly, other researchers wanted to use similar techniques to clone other mammalian species. And soon several more

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animals were successfully cloned. Among them were transgenic animals, clones made from fetal and adult cells, and a male mouse. It should be noted that all previous clones had been females. The list of successfully cloned animals grew, and biotechnologists began to explore cloning procedure as a way to create animals belonging to endangered or extinct species. To clone the endangered and extinct species scientists need to find closely related animals to use their cells as egg donors and surrogates. The mouflon and gaur were chosen in part because they are close relatives of domestic cattle and sheep, respectively. The first extinct animal, a Spanish mountain goat called the bucardo, was cloned in 2009 by another group of researchers. They used goats as egg donors and surrogate mothers. Unfortunately, the one kid that survived gestation died soon after birth because of lung defect. A very exciting experiment at Rhesus monkey was done by S. Mitalipov with collaborators. Researchers took a nucleus from the adult monkey’s cells and fused it with an enucleated egg cell. The reconstructed embryo was allowed to develop for a short time. Then its cells were placed in the cell culture and grown in a culture dish. These cells were called embryonic stem cells, because they can differentiate to form any cell type. This experiment confirmed that nuclear transfer in a primate was possible, in spite of the fact that researchers had tried to use this technique for years unsuccessfully. This opened the door to the possibility of human therapeutic cloning which means the usage of genetic modifications and cloning techniques to improve the patient’s cells and disease treatment. But this approach demands creation of individual-specific stem cell culture, that could be used to treat or study diseases. In 2004-2005 South Korean researchers claimed that they used SNT technique to create human embryonic stem cell lines, but it was false. After that cloning controversy, biotechnologists community required much stronger evidences from scientists reporting about experiments with human embryonic stem cells lines. Mitalipov with his team had to overcome a lot of technical problems and colloquies suspicions to present the results of his exciting experiment. Researchers used a skin cell from the baby with a rare genetic disorder, improved the mutated gene by genetic engineering tools, and then made a nuclear transfer into the enucleated donated egg. The resulting embryo could be used as a source of embryonic stem cells to cure the patient because stem cells are specific to the patient they came from. The improvements in the technique used in the experiment: 1) modifications of the cell culture medium and liquid for nuclear transfer procedure; 2) series of electrical pulses were used for stimulation of the development of the reconstructed egg. Scientists use two terms for cloning technology: reproductive and non-reproductive. Reproductive cloning is the creation of genetically identical offspring to the mother or donor of the nuclei. Two main approaches to getting genetically

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identical clones: 1) nuclear transfer, and 2) splitting of embryos. Non-reproductive cloning means the stem cells usage to generate replacement cells, tissues or organs in medical purposes, for an example to treat particular human diseases or conditions. Animals can be cloned by two different methodical approaches: –– mechanical embryo splitting (figure 58) –– nuclear transfer (figure 59).

A – Cutting the 2-celled embryo; B – Aspiration of blastomeres at 6-8-celled embryo Figure 58. Cloning by embryo splitting method (http://169.237.28.91/animalbiotech/Biotechnology/Cloning/index.htm)

Currently, embryo splitting of Mammals has successfully been established in livestock species. In several livestock species, the embryo splitting was efficient and safe for animals used in assisted reproduction. Experimentally all techniques including embryo splitting and single blastomer cloning were developed in mice system. As we mentioned before, experiments with humans were promoted by success with embryo splitting of nonhuman primate embryos which resulted in several pregnancies. Human embryo splitting has been started only recently. The experiments have shown that embryo splitting at the 6-8 cell stage provided a much higher developmental potential compared to splitting of 2-5 celled embryo. In spite the fact that embryo splitting is intensively used at farm animals, the efficiency of the procedure and stages of embryo development are still under discussion. The experiments with sheep revealed that it is more efficiently to split

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2- and 4-cell embryos because in this stage about 36% of embryos are developed to term the following transfer to recipient females. In cattle, the 4-celled embryos separated into blastomeres could further develop to term and give rise to multiple monozygotic (identical) healthy calves. The efficiency of embryo splitting in cattle after bisection of early bovine embryos was comparable with the control results because it gave pregnancy rates similar to the rates of pregnancy obtained from intact control embryos. That’s why embryo twinning in cattle was proposed for suitable applications in field conditions. Moreover, the success was proved by the experiments with cryopreserved bovine embryos, which were split after their time-separated thawing, then transferred into uterine of surrogate mothers and finished by live-born monozygotic calves of different ages. Monozygotic twin kids of goat were produced from bisected 2-celled early embryos. Split embryos of the pig were capable of full-term development giving rise to healthy genetically identical twin piglets. In the horse, split embryos created using blastomer biopsy at 2- or 8-celled stage resulted in term pregnancy and appearance of healthy monozygotic foals. The genetic identity of split embryos to these foster females were they been transferred after splitting and developed in term in allogeneic pregnancies supports the success of embryo splitting at farm animals.

Figure 59. Cloning by Somatic cell nuclear transfer (SCNT) method (http://169.237.28.91/animalbiotech/Biotechnology/Cloning/index.htm)

Another methodological approach to cloning is based on somatic cell nuclei transfer. Nuclear transfer is the process when the nucleus of a differentiated adult cell is placed in an enucleated egg cell. Later this technique was refined and became known as somatic cell nuclear transfer (SCNT). SCNT represented an extraordinary advance in the biotechnology, particularly in the cloning area, because genetically identical clones of already grown animals are easy to produce

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using this technology. It was highly appreciated by farmers. The scientific impact is in understanding the fact that it was possible for the DNA of adult differentiated somatic cells to revert to an undifferentiated embryonic stage by reestablishing pluripotency and create a new normal embryo. The possibility to reprogram the nuclei of somatic cells to a pluripotent state significantly influenced research into therapeutic cloning and stem cell therapies. To understand this, we need to remind the preliminary experiments with nuclear transfer. The experiments of Briggs and Kings with cloning a frog (Rana pipiens) embryo in November of 1951 gave a base for development of SCNT. They did not simply break off a cell from an embryo, they carefully took the nucleus out of a frog embryo cell and then placed the nucleus into unfertilized enucleated frog egg cell (see Figure 60).

Figure 60. R. Briggs and T.J. King experiment: cloning the Rana pipiens by SCNT

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Gurdon’s experiments done in 1962 at Xenopus laevis revealed that the nuclei from differentiated cells could be reprogrammed step by step if he used the serial nuclear transfer of specialized nuclei into enucleated eggs. By nuclear transplantation he introduced the nuclei from intestinal epithelial cells into eggs that had been enucleated by ultra violet irradiation. After serial transplantation these nuclei gave rise to a complete animal (Figure 61). Serial nuclear transfer is a way to return differentiated cells to nondifferentiated states. Sir John Gordon got a Nobel Prize in Physiology or Medicine in 2012 for his research in the field of developmental biology and for the discovery that mature cells can be converted to stem cells. The famous cloned mammalian – Dolly was part of a series of experiments done in Roslin Institute (Scotland) in attempts to develop a better method for producing genetically modified animals. Scientists believed that if the experiment with cloning sheep by nuclear transfer of differentiated nucleus was successful, it would lead to a decrease in the number of animals needed to be used in future experiments. Another task of the experiment with Dolly sheep was to learn more about differentiation capability of cells during animal development and to answer the question whether a specialized cell, such as a cell of skin or brain, could be used to make a complete healthy animal.

Figure 61. J. Gordon experiment: cloning the Xenopus laevis by nuclear transfer using serial nuclear transplantation

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The team of Professor Sir Ian Wilmut carried out experiments on SCNT resulted in cloning Dolly. Ian Wilmut’s team was interdisciplinary and included biotechnologists, geneticists, embryologists, surgeons, vets and, of course, farm staff. The reason was the nature of the research. The project started in 1986.  The claimed goal was to create a sheep that produced a certain chemical in its milk.  Ian Wilmut decided to genetically alter adult cells, which were successfully cultivated in laboratory in vitro conditions, then clone them and produce animals with the altered gene in all cells of the organism.  The paperwork began in 1987, and research started in 1990. Lots of experimental work was done, and a lamb was born from a genetically different surrogate mother on July 5, 1996, as a result of cloning from a frozen mammary cell taken from another adult sheep. Cell-donor of the nucleus was taken from the mammary gland of a six-year-old Finn Dorset sheep. The egg cell was taken from a Scottish Blackface sheep (see Figure 62). It should be mentioned, that when scientists were working at Dolly creation, she was the only lamb born from 277 attempts. From 277 cell nuclear transfer procedures, only 29 early embryos developed and were implanted into 13 surrogate mothers. But only one pregnancy went to full term (148 days) and finished successfully by appearance of 6.6 kg Finn Dorset lamb named Dolly. Dolly was born by Scottish Blackface surrogate mother. The phenotype of Dolly’s white face was one of the first signs that clone was produced because her surrogate mother was black faced, but donor of nucleus had a white face. So, Dolly’s DNA came from a mammary gland cell of Finn Dorset sheep. Interestingly, that the cloned sheep was named Dolly after the country singer Dolly Parton, whose phenotypic characteristics were very expressive (Figure 62).

Figure 62. Ian Wilmut experiment: cloning the sheep Dolly

World got acquainted with Dolly after sensational publication in Nature on 22nd February, 1997. This publication immediately attracted attention of mass

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media, and Dolly captured the public’s imagination. Possible benefits and dangers of cloning were the most discussed topics in public debates. Sheep Dolly attracted attention because she was the first mammal cloned from an adult cell. Dolly’s birth proved that specialized cells of adult mammals can be used for creation of a genetically identical copy of the animal they came from. This knowledge changed the existing scientific developmental theories and opened up lots of possibilities for using the SCNT in biology and medicine. One of the most important possibilities is the development of personalized stem cells which can be used for therapy of serious and inherited diseases. The possibility to reprogram specialized cells of adult mammals developed in induced pluripotent stem cells (iPS) technology. But cloning technology has some implications. When Dolly was at the age of one year, the analysis of her DNA revealed that, in Dolly’s body cells the telomeres length was shorter than it would be expected for a normal sheep of the same age. The telomeres are the repeated DNA sequences on the ends of chromosomes that protect the DNA from damage. Each cell division following after DNA replication makes telomeres shorter, and this is a reason that with ageing of an animal or a human their telomeres become progressively shorter, exposing the DNA to more damages. Dolly had shorter telomeres because her DNA came from an adult sheep which underwent multiple cell divisions, and Dolly’s telomeres had not been fully restored during her new development. Due to genetic age Dolly was “older” than her actual age. But at the time, the extensive screening of Dolly’s health status did not find any conditions which could be directly related to premature or accelerated ageing. All her life Dolly spent at The Roslin Institute with the other sheep, quite apart from the occasional media appearance. From her normal crossing with a Welsh Mountain ram called David Over a total of six lambs were born. The first lamb named Bonnie was born in April 1998. The twins (Sally and Rosie) were born in 1999, and triplets (Lucy, Darcy and Cotton) were born the year after. After burning the triplets, the health screening of Dolly (September, 2000) revealed that she had become infected by a JSRV virus (Jaagsiekte sheep retrovirus), which causes lung cancer in sheep. It was a common infection because other sheep at The Roslin Institute had also been infected with JSRV in the same outbreak. Soon, in 2001, the farm staff noticed her walking stiffly, and Dolly was diagnosed with arthritis. Dolly was successfully treated by anti-inflammatory medication, and she continued to have a life of normal quality until February, 2003, when she developed a cough. A computer tomography scan revealed tumors growing in lungs. Scientists decided to euthanize Dolly rather than risk of her suffering. So, Dolly was put to sleep at the age of six on February 14, 2003.

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As we mentioned before, the main goal of the experiment with cloning Dolly was to learn to clone sheep and other large animals in hopes of producing medicines in the milk of such animals. Biotechnologists from Roslin Institute have managed to transfer human genes that produce useful proteins into big farm animals as sheep and cows. Currently farm animals can produce, for instance, the factor IX, blood clotting agent to treat hemophilia, or alpha-1-antitrypsin to treat cystic fibrosis and other lung conditions. Among “farming” products produced by cloned farm animals there are human antibodies against infectious diseases and even cancers. “Foreign” genes have also been transferred into zebra fish, which are widely used in laboratories as a model object. Cloned embryos from these fish successfully express the foreign protein. So, SCNT development allows us to imagine the flocks of genetically engineered animals all producing medicines in their milk. The research interest in cloning is also based on other medical and scientific reasons. The SCNT is already being used in combination with genetic techniques in xenotransplantation, which means the development of animal organs for transplant into humans. For example, alongside with genetic techniques the cloning of pigs would lead to a reliable source of suitable donor organs for transplantation, for the first time achieved in March 2000. The usage of pig organs for xenotransplantation was hampered by the presence of sugar and alpha gal on the surface of pig cells, but the “knockout” technology developed in 2002 helped scientists to turn off the genes encoding these undesirable proteins. Knockouted pigs could be bred naturally. However, virus transmission, used for genetic transformation, still worries scientists. Implications of cloning are still expected. For example, cloned mice become obese, and related symptoms manifest, such as raised plasma insulin and leptin levels. But the offspring of cloned mice does not suffer from obesity and are normal. The achievements in animal cloning and study of clones and cloned cells could lead to better understanding of the embryo development and problem of ageing and development of age-related diseases. The SCNT cloning technology could be used for creation of better animal models of human diseases. This approach could in turn lead to further progress in understanding the disease mechanisms and cure protocols to treat persons suffering from a disease. The SCNT cloning technology could even enhance the biodiversity by supporting the continuation of rare breeds and endangered species. Control questions: 1. What is a clone in biological and genetic sense? What are the differences between cloning genes and cloning organisms? 2. Indicate the milestones in the history of cloning. 3. What do you know about cloning applications in Animal Biotechnology? 4. Describe the main steps in animal cloning techniques 5. How can we use specialized cells from an adult animal to clone it to organism?

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CHAPTER

14

TRANSGENIC TECHNOLOGIES IN ANIMAL BIOTECHNOLOGY The term transgenic animal refers to an animal which was genetically modified by introducing one or more foreign genes in its genome. The foreign DNA construction should be made by recombinant DNA technology, then the foreign DNA is introduced into the animal, and then it must be transmitted through the germ line. Thus, every cell of the transgenic organism should contain the same modified genetic material and pass it through generations. The term transgenic was first used by Gordon and Ruddle (1981). The combining contributions of genetic engineering and developmental biology allowed rapid development of the technology for creation of transgenic animals. Gordon and Ruddle (1981) firstly used the DNA microinjection technique in mice. It was the first evidence of transgenic technology successful in mammals, and soon this technology was applied to various other species such as rats, rabbits, sheep, pigs, birds, and fish. The retrovirus-mediated transgenesis was proposed by Jaenisch (1976), and embryonic stem cell-mediated gene transfer – by Gossler et al. (1986). These three main techniques significantly promote the success in producing transgenic animals. Rapid development of usage of genetically engineered animals opened up an increasing number of applications for the technology. The milestones in transgenic research are presented in Table 13. Milestones in transgenic animal research Date 1 1971 1977 1980 1980-1981 1981

Event 2 First demonstration of sperm-mediated gene transfer mRNA transferred into Xenopus eggs mRNA transferred into mammalian embryos Transgenic mice was first recorded Transgenic mice was first documented

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Researcher(s) 3 Brackett et al. Gordon Brinster et al. Palmiter et al. Six research groups

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2 Transgenic mice demonstrate an enhanced growth phenotype (Super-mouse) First transgenic pigs and sheeps were produced Production of transgenic sheep by nuclear transfer of nuclei from transfected fetal fibroblasts

3 Palmiter et al. Hammer et al. Schnieke et al.

In the past few years, the techniques and science of producing genetically engineered animals have advanced very rapidly. Currently it is possible to create animals carrying useful novel properties for dairy, meat, or fiber production, for environmental control of waste production, for biomedical purposes or other human consumption, or generate a plenty of copies of farm animals, which represent the valuable traits, such as good quality milk or meat production, high fertility. There is a number of methods which are currently used for genetic engineering of various animal species. Most of techniques were developed using fully genetically investigated animal model systems, such as mouse and Drosophila. Application of these to domesticated animals has only been extended more recently. Access to the germline of mammals can be obtained by: – direct manipulation with the fertilized egg, followed by implantation of reconstructed egg into the uterus; – manipulation with sperm used to generate the zygote; – manipulation with early embryo tissues and cells; – the usage of ES cell lines which, after genetic manipulation and selection ex vivo, can be introduced into early embryos, some of whose germlines will develop from the ES cells (Smith, 2001); – manipulation with cultured somatic cells, whose nuclei can then be transferred into enucleated oocytes and thereby provide the genetic information required to produce a complete animal. The manipulation with ES cells allows researchers to do preliminary selection of desirable genetic modifications before undertaking the expensive and lengthy process of generating animals. ES cells are not available for all animal species of interest. But, usage of nuclear transfer from modified somatic cells for generation of transgenic embryos is a suitable approach for genetic engineering and cloning of nearly genetically identical animals. This method was proposed by Westhusin et al. (2001). For example, manipulation with the avian germline is difficult because ES lines are not available and the early embryo is difficult to access from birds. Many scientists focus on the usage of blastodermal cells or primordial germ cells. These embryonic cells can be cultured in vitro briefly and allow us to manipulate with germline cells for genetic modifications prior to introducing into fresh embryos to create chimeras from which modified lines can eventually be developed. This

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approach was proposed by Aritomi and Fujihara (2000). However, the efficiency of getting transgenic avian is still low. Steps of genetic transformation: – Cloning of needed genes – Construction of genetic vector – Introduction of genetic vector to the host cell or early embryo – Selection of genetically transformed cells or organisms – Checking the foreign gene expression in needed cells or organs – Creation of homozygous genetically transformed strain The most important question in creation of transgenic animals is ensuring that the desirable foreign gene product is expressed in the correct place (specialized cells or tissues), at the appropriate level and time. Many specifics are known how this goal can be achieved, and the approaches vary considerably depending on the used animal system. But, some general principles can be elucidated. First of all, there is a big range of tissue-specific promoters, which can be chosen for transgenic construction. But most of such promoters are complex, large, and hard to regulate in animal bodies. Therefore, scientists use a small range of wellcharacterized promoters to fuse them with genes of interest. Another approach is explained by the fact that transgene expression depends on the place of transgene insertion into the host genome. This question is especially important for the transfection method of foreign DNA introducing, because the transgene insertion will be occasional, and the presence of control elements around the integration site of host DNA can influence the transgene expression (Wolf et al., 2000). These consequences were called positional effect. The positional effects often lead to gene silencing or unregulated expression, not only for the transgene, but also for the surrounding host genes (Bonifer et al., 1996; Henikoff, 1998). To elucidate the problem Wolf et al. (2000) proposed to use some special DNA sequences, such as insulators, or locus-control regions, but the efficiency of these techniques is still not predictable. While these effects do not directly affect the safety or utility of transgenic animals, they do introduce considerable inefficiency into production of transgenic animal. Gene knockout technology A special goal of the transgenic technology is to create genetically engineered animals that lack specific genes (knockout), or where the undesirable gene are replaced by another gene specially engineered for this purpose. To study the genes or protein interactions scientists can directly turn off some gene functions. Producing a homozygous strain on disabling gene (contains two copies of disabled gene alleles) in the model animal system, it is possible to obtain information about the development of descendants in the absence of a particular protein and elucidate protein function or gene interactions. This knockout

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technology was developed in 2001 (University of Guelph) and was widely used at mouse models. The methodological approach to creation of a “knockout” animal includes: usage of ESCs for genetic transformation; introducing of modified ESC into the developing embryo; embryo transfer into the womb of a host animal to get a chimeric organism; selection of the host offspring for genetically transformed individuals, which result from a portion of transformed ESC carrying the knocked-out gene in germ line of chimeric organism. In many cases it is not necessary to delete the undesirable gene, it can also be modified in an expression or function. A “targeted mutation” effect refers to the situation when a gene is altered but not shut down. The differences between two described methodologies are reflected in the terms (MGD, 2002): “knockin” gene instead of “knocked” gene. Impossibility to use xenotransplantation of organs or tissues from non-primates (i.e. pigs) to humans is an example of application of the gene knockout technology. It is well known that galactose-1,3-galactose is present on the surface of pig cells, but it not expressed by primate cells (Galili, 2001). This carbohydrate induces a dramatic “hyperacute” immune response of human recipients. To elucidate the xenotransplantation problem scientists applied the knockout technology for gene encoding the galactosyl transferase (GT) enzyme in donor pigs (Lai et al., 2002). GT-deficient pigs are used for growing tissues or organs to replace human organs. Another practical requirement related to elimination of the cattle genome is the gene encoding prion-related protein (PrP). PrP protein induces the scrapie in sheep and bovine spongiform encephalopathy (BSE), which is called mad cow disease. Using the mouse model scientists did a knockout of this gene (Bueler et al., 1992). Removal of Prp gene makes mice completely resistant to this disease. Thus, if the mouse model is proved at cattle, homozygous knockout of bovine PrP could lead to the elimination of BSE. The variety of mouse ES cell lines supports the gene knockout or knockin technology applications. The protocols for gene knockout/knockin include the mechanisms of homologous recombination between identical sequences in the genome and the transfecting DNA vector. The technology was developed by Bronson and Smithies in 1994. The most common protocol describes the usage of neomycin resistance gene as a selective marker, which is inserted in vector construction into the homological to targeted gene region of host DNA. After vector transfection, due to homological recombination the marker gene is inserted into the recipient targeted gene destroying its structure and functioning. The successful insertion is established by selection of the medium containing the neomycin-related antibiotic G418. Because the insertion of the transgene is an occasional event, the progeny animals will be heterozygous for the knockout/knockin gene. To make a homozygote the researcher must set up ta system of crossing between transgenic mice and

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breed the homozygous animals. Each result should be proved by molecular genetic analysis. The galactosyl transferase-knockout pigs were obtained in this way using genetic modification of cultured fetal fibroblasts. Nuclei from modified cells were then transferred into an enucleated egg as described before. Nuclear transfer method in producing transgenic animals Very fast development of SCNT technology and its success in operating with animals have become an efficient tool of propagation of farm animals with desired traits. There is no difference, whether animals are naturally selected from the population or genetically modified (Betthauser et al., 2000; Lanza et al., 2001; Westhusin et al., 2001). Mouse models have become the main objects used in the field of transgenesis due to their good learned genetics, their short generation time, small size and low cost of housing in comparison to that for larger vertebrates. As for big animals, the ability to reprogram the specialized donor cell nuclei for successful nuclear transfer will depend on many factors, such as possibility of culturing cells which will serve as a donor of nuclei; nuclear transfer procedure; used animals (donor of egg, recipient, surrogate mothers) cell cycle synchrony (Stice et al., 1998; Wilmut et al., 1998); the developmental peculiarities of used animal species etc. The actively dividing animal cells (epithelial cells, fibroblasts etc.) have been used successfully for this purpose (Cibelli et al., 1998; Kasinathan et al., 2001; Kuhholzer et al., 2001). The hypothesis that differences in timing of embryonic genome activation contributes to the differences in cloning efficiency among animal species (Stice et al., 1998) is actively discussed now. Many experimental results showed that only oocytes can be used successfully as a recipient for a foreign nucleus because only the egg can convert the differentiated nuclei into undifferentiated stages and can stimulate the development of reconstructed embryo (Campbell, 1999; Fulka et al., 2001). But molecular mechanisms of this reprogramming are currently unknown. These complications explain the fact that currently the propagation of farm animals by SCNT technology is inefficient. Statistical data show that in average about 10 percent of reconstructed embryos result in live offspring (Cibelli et al., 2002). It was shown that most of abnormalities occur during embryo development. For example, lethality of cattle and sheep embryos most often occurs in the first third of pregnancy, and the frequency of perinatal deaths of reconstructed embryos is much higher than that of normally obtained embryos. Scientists believe, that, in cattle, problems with in vitro culturing provide a higher influence on this situation than nuclear transfer technology itself. But, even with this existing of SCNT low efficiency at farm animals, there are many potential applications of this technology to reproduce highly valuable genotypes, such as rare or endangered species, household pets, elite sires or dams, breeds with desirable production traits but low fertility, sterile animals such as castrates and

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mules, or transgenic animals that have high value and for which rapid propagation is desirable. Application of SCNT technology for dissemination of germplasm as embryos and consequent reduction in the risk of associated disease spread is also very important. In this way the researcher should take into account the significant differences in fertilizing capacity of farm animal sperm. For example, in cattle, the single bull ejaculate can fertilize 400-500 eggs in AI programs. As for pigs, the single boar ejaculate can be used to breed only 10-20 females. That’s why nuclear transfer technology for propagation of pigs is more preferable that artificial insemination. Genetic transformation tools In genetic transformation, if desirable gene has been cloned, for transfer it into another cell vectors are used. Vector is a specific DNA sequences used as a vehicle to artificially transfer foreign genetic material into the target cell, where it can be replicated and then expressed. The recombinant DNA technology provides all tools for vector construction. The four major types of vectors known in genetic engineering: – plasmids, – viral vectors, – cosmids, – artificial chromosomes. Selectable gene markers allow us to select and identify the vector in transformed cells or organisms. There are the following types of gene markers: – visible (coding phenotypic trait) – markers of cell surface – biochemical (antibiotic resistance, alcohol sensitive, enzymes of ad­di­ tional way of nucleotide biosynthesis) To be recognizable the marker gene must code the negative trait or the trait that distinguishes it from the wild type. As a rule, the marker is represented by the mutated gene, and the recipient cell or organism must by normal. Most common selectable markers are antibiotic resistant. Constructing the vector scientists do not need to use the promoter sequence of transgene in nature because sometimes they are not strong working in occasional place of foreign chromatin. There are commercial proposals of very strong promoters, most of them have viral origin (HSV1, CMV, HPV etc.). Sometimes scientists use the inducible promoter that can turn on or turn of the transcription of transgene in some environmental conditions (promoter of human metalothioneine gene). Common to all engineered vectors are the origin of replication, a good promoter, cloning foreign gene (transgene), a selectable marker, and regulatory sequences. System of bi-directional selection One metabolic pathway – the additional way for nucleotide biosynthesis – has been particularly useful in cell-fusion and genetic transformation experiments

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with animal cells, and especially development of the system of bi-directional selection, which allows us to select mutant and normal cells by little modification of cultural medium HAT (contains hypoxanthine, aminopterin, thymidine). Most animal cells can synthesize the purine and pyrimidine nucleotides de novo from simpler carbon and nitrogen compounds, rather than from already formed purines and pyrimidines (Figure 62, top). The folic acid antagonists amethopterin and aminopterin interfere with the donation of methyl and formyl groups by tetrahydrofolic acid in the early stages of de novo synthesis of glycine, purine nucleoside monophosphates, and thymidine monophosphate. These drugs are called antifolates, since they block reactions involving tetrahydrofolate, an active form of folic acid. Many cells, however, contain enzymes that can synthesize the necessary nucleotides from purine bases and thymidine if they are provided in the medium; these salvage pathways bypass the metabolic blocks imposed by antifolates (Figure 63, bottom). In a normal medium, cultured animal cells synthesize purine nucleotides (AMP, GMP, IMP) and thymidylate (TMP) by de novo pathways (blue). These require the transfer of a methyl or formyl group from an activated form of tetrahydrofolate (e.g., N5, N10-methylenetetrahydrofolate), as shown in the upper part of the diagram. Antifolates, such as aminopterin and amethopterin, block the reactivation of tetrahydrofolate, preventing purine and thymidylate synthesis. Normal cells can also use salvage pathways (red) to incorporate purine bases or nucleosides and thymidine added to the medium. Cultured cells lacking one of the enzymes of the salvage pathways – dehydrofolate reductase (HGPRT), adenofolate reductase (APRT), or thymidine kinase (TK) – will not survive in the media containing antifolates.

Figure 63. De novo and salvage pathways for nucleotide synthesis (Lodish H. et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman. 2000)

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A number of mutant cell lines lacking the enzyme needed to catalyze one of the steps in a salvage pathway have been isolated. For example, cell lines lacking TK can be selected on the medium containing the thymidine analog 5-bromodeoxyuridine because TK- cells (with a TK mutation) are resistant to this otherwise toxic substance. TK+ cells can convert 5-bromodeoxyuridine into 5-bromodeoxyuridine monophosphate. This nucleoside mono- phosphate is then converted into a nucleoside triphosphate by other enzymes and is incorporated by DNA polymerase into DNA, where it exerts its toxic effects. This pathway is blocked in TK- cells because they lost normal TK enzyme function. However, TK− mutants are resistant to the 5-bromodeoxyuridine toxic effects. Cells lacking the HGPRT enzyme have also been selected in the same manner because they are resistant to the otherwise toxic guanine analog 6-thioguanine. HGPRT− cells and TK− cells are useful partners in cell fusions with one another or with cells that have salvage-pathway enzymes but that are differentiated and cannot grow in culture by themselves. The special culture medium, which is most often used to select mutant and normal cells, was called HAT medium, because it contains hypoxanthine (a purine), aminopterin, and thymidine. Normal cells can grow in HAT medium because even though aminopterin blocks de novo synthesis of purines and TMP, the thymidine in the media is transported into the cell and converted to TMP by TK and the hypoxanthine is transported and converted into usable purines by HGPRT. On the other hand, neither TK− nor HGPRT− cells can grow in HAT medium because each lacks a functional enzyme of the salvage pathway. However, hybrids formed by fusion of these two mutants will carry a normal TK gene from the HGPRT− parent and a normal HGPRT gene from the TK− parent. The hybrids will thus produce both functional salvage-pathway enzymes and grow on HAT medium. Likewise, hybrids formed by fusion of mutant cells and normal cells can grow in HAT medium. The system of bidirectional selection of mutated and normal gene variants is presented in Figure 64.

tk – thimidine kinase; gprt – hypoxanthine phosphoribosyl transferase; HAT – HAT medium Figure 64. Bidirectional selection of cells on HAT medium

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Introduction of foreign DNA into recipient cells and organisms Several methods were developed for genetic transformations of animal cells growing in culture: – Hypertonic salt method – It is based on the osmotic pressure shift. In presence of 0.5-1.0 M NaCl water solution of transgene easily goes into the recipient cell. – DEAE-dextran method – Polycatione diethyl-aminoethyl-dextran, in which transgene is packed, attracts to negatively charged cell membrane. – Calcium-phosphate method – CaCl2 in presence of phosphate buffer precipitates on cell surface and cell phagocytes transgene. – Method of fusion of cultured cells with bacterium protoplasts in the presence of PEG – In the presence of polyethileneglicol the bacterium protoplasts containing transgene easily fuse. – Use of liposomes – Specialized to definite cell type liposome can do the targeted transformation. – The use of transducing retroviral vectors – Retroviral vectors infect animal cells. – Method of microinjection – Direct injection of transgene into cell and its nuclei using a glass needle (see Figure 65). – Electroporation – Short-timed and high-powerful electric pulse makes cell membrane permeable for transgene (see Figure 66). – Gene gun – Inertial movement of small particles covered by transgene after the bullet stopped. It is not used for animal cell culture because the method is not targeted and shot can damage the cell.

1 – recipient cell; vector DNA carrying transgene; 3 – buffer solution A-Injection; B-Piercing Figure 65. Modifications of the microinjections method

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Figure 66. Electroporation

There are two basic methodologies currently used for inserting transgene into vertebrate animal germline cells: transfection and infection with retrovirus vectors. The third approach includes the usage of mobile genetic elements. This kind of vectors has been commonly used for insects and is being explored for germline modification of vertebrate animals (Izsvak et al., 2000). The three principal methods used for the creation of transgenic animals are (see Figure 67): – DNA microinjection – embryonic stem cell-mediated gene transfer – retrovirus-mediated gene transfer.

1 – Egg; 2 – Microinjection of transgene to the fertilized egg; 3 – Transgene transfection of ES cells; 4 – nuclei transfer; 5 – transfer of transformed ES cells into the blastocyst Figure 67. The scheme of approaches to obtaining a transgenic animal

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Transfection The usage of viral vectors for transfer of foreign DNA into the animal cells or embryos is based on the animal-specific transfection properties of retroviruses and adenoviruses. Another preferable property of retroviruses and adenoviruses is their ability to integrate into chromosomes. Technically transfection methods include the following procedures: – direct microinjection of DNA into the cell nucleus; – electroporation – introduction of DNA through transient pores opened by controlled electrical pulses; – usage of polycations to neutralize charges on DNA and the cell surface that prevent efficient uptake of DNA; – lipofection, or enclosure of DNA in lipid vesicles that enter a cell by membrane fusion much in the manner of a virus; – sperm-mediated transfection, possibly in conjunction with intracyto­plas­ mic sperm injection (ICSI) or electroporation. Each researcher decides how to introduce the transgene and what method to choose basing on the used animal system. And the manner of transgene transfection is determined empirically for each kind of animal. Excepting the possibility of homologous recombination, the structure of carrying transgene DNA vector does not determine the integration site in the recipient DNA. Very often only a fragment of the transfected DNA is integrated into the chromosome, frequently in multiple copies. In case of retroviral transfection, the vector is often integrated in the region of long tandem repeats (LTR) in the result of homological recombination between viral and host LTRs. In case of transfecting cultured somatic cells or ESC, a selectable marker (for example, gene encoding phosphotransferase), is often included as part of the DNA allowing selection for its presence either in eukaryotic cell lines or in the bacteria in which the DNA was massively produced. Retrovirus Vectors Retroviruses are viruses containing RNA. Retroviruses are known as viruses that specifically infect the animal cells. The replication of retroviruses is a unique mechanism involving copying of the viral RNA genome that converts it to DNA (cDNA). This process is called reverse transcription. Reverse transcription is followed by its specific and stable integration into host cell DNA using the mechanism of homological recombination between viral and host LTRs. The inserted viral DNA can then be expressed using the normal transcriptional machinery of the cell. Retroviruses are commonly used to introduce foreign genes into cells in the culture or into somatic tissue of the experimental animals, such as mice (Soriano et al., 1986; Miller, 1997). Retroviruses have also been used for modification of fish fertilized eggs (Amsterdam et al., 1997), for mollusks (Lu et al., 1996), chickens (Thoraval et al., 1995), mice (Soriano et al., 1986). They have

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been used for genetic transformation of cattle (Chan et al., 1998). In addition to the gene of interest, selectable marker and regulatory elements, the retroviral vector construction contributes to the presence of transcriptional viral promoter and flanked sequences (LTRs), which are necessary for replication and integration into the host genome. Sequences necessary for packaging of the transcript in virions and gene encoding reverse transcriptase must also be included. So, retroviral vectors are quite big. The capacity of retroviral vector contributes not more than 8,000 base-pair-long transgene. Introduction of the retroviral vector into the cells leads to the creation of infectious virions containing an RNA copy of the transgene. After the transfection procedure, the vector’s RNA is copied into DNA and integrated at random sites in the recipient genome. Transposons Transposons or mobile elements are the DNA sequences that can change their location in genome. The retrotransposons contain a gene encoding transposase, the enzyme with the same function as a reverse transcriptase of retroviruses. Retrotransposons (or transposons) are the most used type of mobile elements for genetic transformation of animal cells. A variety of transposons has been found in insects (Handler, 2001), and in fish (Ivics et al., 1997). Usage of transposons as a vector for genetic transformation of insects was very effective (Braig and Yan, 2002). No active transposons of these types which have been observed in mammals, although the human genome contains thousands of copies of a DNA sequence homological to Drosophila transposons (Lander et al., 2001). Several reports suggest that natural transposons found in insects might provide useful and efficient tools of transgenes integration in animal germlines. Sherman (1998) showed the activity of Mariner transposon in chick zygotes, which was introduced by microinjected plasmid into the germline. Interestingly, that modified copy of Sleeping Beauty transposon originated from fish has been successfully used for animal cells and embryo transformation (Ivics et al., 1997; P. Hackett, University of Minnesota). However, the efficiency of transposon usage in animal transformation is still under discussion. DNA microinjection The microinjection method involves the direct microinjection of a vector containing a single foreign gene or a combination of genes into the pronucleus of a fertilized ovum or the nucleus of a cell in culture. As shown by Gordon and Ruddle (1981), microinjection is the first method that proved to be effective in mammals. The transgene integration may result in the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. It is known that insertion of foreign DNA is a random not site-specific process.

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There is a high probability that the introduced transgene will insert into the right site of the host DNA that will permit its expression and will not disrupt the important genes. The microinjection method proved the efficiency for many types of animal cells and early embryos. An important advantage of this method is its applicability to a wide variety of species. Embryonic stem cell-mediated gene transfer. As mentioned before, this technology involves the usage of in vitro culture of embryonic stem cells, the retroviral vector and its insertion to the recipient genome by the mechanism of homologous recombination. Because ESC are undifferentiated, they have the potential to differentiate into any type of cell and therefore to give rise to a complete organism. After genetic modification these cells might be incorporated into an embryo at the blastocyst stage of development, and then placed into the foster mother uteri. A chimeric animal grows up from this modified blastocyst. ESC-mediated gene transfer is the method of choice for gene inactivation by knockout/knockin technology. This technology is of particular importance for studying mechanisms of genetic control of the developmental processes. The development of this technique was carried out on the mouse model. The advantages of this method include: precise targeting of defined mutations in the gene via homologous recombination; preliminary selection of transgene copies and integrated sites; availability of selection for transgene homozygous state. Retrovirus-mediated gene transfer. As mentioned before, retroviruses are most commonly used vectors for genetic transformation of animals. Offspring derived from retrovirus-mediated gene transfer is chimeric. Transgene transmission is possible only if the retrovirus integrates into some place of the germ cell genome. In spite of many advantages and a variety of techniques for animal transgenesis, the success rate in terms of pregnancies and live birth of animals containing the transgene is extremely low. The frequent result of genetic manipulation with vertebrate animals is a chimeric organism called a founder of transgenic strain. If the genetic manipulation does not lead to abortion of a modified embryo, the first generation of animals should be tested for the transgene expression. After birth, the so-called germ line chimeras or animal-founders should be selected for transgenic offspring and then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell, and the transgene will be inherited in a clear manner. After this, embryos carrying the transgene in the homozygous state can be isolated, frozen and stored for subsequent implantation or storage.

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Usage of this way for animal transformation depends on cell specialization, physiological characteristics, transgene size and function. The researcher can select a desirable method to introduce the transgene. By using the mouse as a model all genetically transformation tools were developed, the mouse demonstrates success in different methods of transformation (see Table 14). State of the art of transgenic technology for selected organisms (https://www.ncbi.nlm.nih.gov/books/NBK207572/) Organism

Transfection Viral vectors

Transposon

Mouse 4 2 1 Cow 3 1 0 Sheep 3 0 0 Goat 3 0 0 Pig 3 0 0 Rabbit 3 0 0 Chicken 1 2 1 Altlantic salmon 3 0 0 Channel catfish 2 0 0 3 0 0 Tilapia 1 0 0 Zebrafish Crustaceans 1 1 0 Mollusks 1 1 0 2 2 2 Drosophila Mosquito 1 0 2 0 – no significant progress 1 – has been accomplished experimentally (proof of concept) 2 – Routine experimental use 3 – Commercialization sought 4 – Widespread production

Table 14

ES cells

Nuclear transfer

4 0 0 0 0 1 0 0 0 0 1 0 0 2 0

2 2 2 2 2 0 0 0 0 0 1 0 0 0 0

Positive – negative selection A very important question for transgene expression are the chromosomal localization of the DNA vector and its orientation. After transfection of embryonic stem cells in culture with a DNA vector designed to insert within a specific chromosomal site, some cells will have a transgene integrated at nontarget (wrong) sites, whereas in the other cells, integration will occur at the target (correct) site. The target site should be located in a region of genomic DNA that does not contain the important genes encoding essential products. So that after transgene integration, there is no interference with any developmental or cellular functions. Moreover, it is important that the transgene can be integrated into a part of the genome that does not prevent

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it from being transcribed, for example, in euchromatin regions rather than heterochromatin. In most of the embryonic stem cells, the foreign DNA will not be integrated at all. To achieve the correct transgene integration to the target site, a technique called positive-negative selection is implemented. This strategy uses positive selection for cells that have the DNA vector integrated somewhere in their genomes and negative selection against the DNA vector sequence integrated at wrong occasional sites. A targeting DNA vector for the positive-negative selection procedure usually contains: – two blocks of DNA sequences (HB1 and HB2) that are homologous to separate regions of the target site; – the transgene, which will confer a new function on the recipient; – a DNA sequence that codes for resistance to the compound G-418 (Neor); – two different genes for thymidine kinase (tk1 and tk2) from herpes simplex virus types 1 and 2 (HSV-tk1 and HSV-tk2). The arrangement of these sequences is a key to the positive-negative selection procedure. Between the two blocks of DNA that are homologous to the target site are the genes for the transgene and G-418 resistance (Neor gene). Outside of each of the homologous blocks are the genes HSV-tk1 and HSV-tk2. If integration occurs at a spurious site, i.e., not at HB1 and HB2, either one or both of the HSV-tk genes have a high probability of being integrated along with the other sequences (Fig. 68). A more direct way to detect embryonic stem cells carrying a transgene at a targeted chromosomal site is to use PCR. The targeting DNA vector contains two blocks of DNA that are homologous to the target site, with one on either side of both the transgene and a cloned bacterial or synthetic (unique) DNA sequence that is not present in the mouse genome (Figure 69). After the transfection of embryonic stem cells, the cells are pooled and samples are screened by PCR. One of the primers (P1) for PCR is complementary to a sequence within the cloned bacterial or synthetic (unique) DNA sequence of the integrating vector. The other primer (P2) is complementary to the DNA sequence that is part of the chromosome adjacent to the region of one of the homologous blocks of DNA. If integration is at a random site, the predicted amplified DNA product is not synthesized. However, if site-specific integration occurs, the PCR amplifies a DNA fragment of known size. In this way, pools of cells with embryonic stem cells containing the desired gene at the targeted site can be identified. By subculturing from these pools, cell lines carrying the site-specific integration can be established.

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A-Nonspecific integration: Both genes for thymidine kinase (tk1 and tk2), the two DNA sequences that are homologous to a specific chromosomal region in the recipient cells (HB1 and HB2), a gene (Neor) that confers resistance to the cytotoxic compound G-418, and the transgene (TG) are incorporated into the chromosome. After transfection, cells are selected for resistance to both G-418 and the compound ganciclovir, which becomes cytotoxic to cells that synthesize thymidine kinase. Other nonhomologous integrations may occur and produce inserts with one or the other of the thymidine kinase genes. After treatment with G-418 and ganciclovir, all the cells with nonspecific integration of the input DNA that includes at least one of the thymidine kinase genes are killed. B-Specific integration – result of homologous recombination. The product of a double crossover between homologous blocks (HB1 and HB2) of DNA on the vector DNA and on chromosomal DNA does not contain either of the two thymidine kinase genes (tk1and tk2). After treatment with G-418 and ganciclovir, only cells that have undergone homologous recombination survive. Figure 68. Positive – negative selection (B.R. Glick & J.J. Pasternak. Molecular Biotechnology – Principles and Applications of Recombinant DNA. 3rd Edition. 2003)

Not only can a transgene be inserted into a specific chromosome site by homologous recombination in embryonic stem cells to provide a new function, but a specific mouse gene can also be targeted for disruption by the incorporation of a DNA sequence, usually a selectable marker gene, into its coding region (Fig. 70). One of the aims of targeted gene disruption (gene knockout) is to determine the developmental and physiological consequences of inactivating a particular gene. In addition, a transgenic line with a specific disabled gene can be used as a model system to study the molecular pathology of a human disease.

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A-After nonspecific integration of the vector DNA, one of the primers (P2) is not able to hybridize to a chromosomal site that is a predetermined distance from the site of hybridization of P1, so a DNA fragment with a specific size is not amplified. P1 hybridizes to a unique segment (US) of the input DNA that does not occur in the chromosomal DNA of the recipient cells. TG, transgene; HB1 and HB2, homologous blocks. B-Homologous recombination between DNA sequences (HB1 and HB2) of the input DNA that are complementary to chromosomal sites (CS1 and CS2) creates hybridization regions for both P1 and P2 that are a predetermined distance apart. Amplification by PCR generates a DNA fragment of a specific size that can be visualized by gel electrophoresis. In this case, the transgene (TG), which lies between the homologous blocks (HB1 and HB2), is integrated at a specific chromosomal location. Figure 69. Testing for nonspecific integration and homologous recombination in transfected cells by PCR (B.R. Glick & J.J. Pasternak. Molecular Biotechnology – Principles and Applications of Recombinant DNA. 3rd Edition. 2003)

The target vector carries a selectable marker gene (SMG) with flanking DNA sequences that are homologous to regions of the targeted gene. In this example, the targeted gene has five exons (1 to 5). Homologous recombination disrupts (i.e., knocks out) the targeted gene. p, promoter; pa, polyadenylation signal. Figure 70. Gene disruption by targeted homologous recombination (B.R. Glick & J.J. Pasternak. Molecular Biotechnology – Principles and Applications of Recombinant DNA. 3rd Edition. 2003)

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Necessity of transgene identification and selection of transgenic animals The first selection criterium is a manifestation of a selectable gene marker. In case of applying a microinjection to a fertilized egg this selection will be informative. But in case of usage of the ES cell transformation in culture and the following return of the transformed cells into the blastocyst, the result of genetic transformation is a mosaic (or chimeric) animal-founder. And the researcher needs to check the transgene presence using molecular-genetic tools. The polymerase chain reaction (PCR) using specific primers to transgene DNA sequence is the most popular method for it. It helps to quickly select the transformed and non- transformed animals among the offspring of the animal-founder checking their DNA samples. The Southern blot hybridization using labeled probes which complement to the transgene also shows the transgene presence. But this method is more complicated. The in situ hybridization on the cytogenetical slide helps to establish the sites of transgene localization in chromosomes. To control the transgene expression scientists need to check transgene RNA or coding protein presence doing RT-PCR, Northern blot hybridization or Western blotting. Immunostaining of histological slides by specific antibodies to the transgene product can help recognize the tissue or cell localization. Animal germline modification remains a technology of “hit-or-miss”. Usage of most techniques results in the fact that only a very small fraction of obtained progeny has the desired properties of transgene insertion, expression, copy number, and absence of genetic damages. So, large numbers of animals must be screened for the presence and copy number of the inserted transgene sequence, for its properly regulated expression, for the ability of this expression to survive transmission through the germline and, finally, for the desired phenotypic characteristics and absence of unintended genetic side effects. Such selection and testing could require several generations of breeding before one can be confident of the absence of recessive genetic damages, and the failure rate of the overall process is very high. As nuclear transfer technology improves, techniques requiring direct introduction of DNA into the animal germline followed by extensive screening of progeny are likely to be replaced by much simpler manipulation and prior selection of cells in culture, followed by recreation of animals with the desired properties directly from the nuclei of the manipulated cells. Applications of transgenic animals Combination of the successful transfer of genes into mammalian cells and the possibility of creating genetically identical animals by transplanting nuclei from somatic cells into enucleated eggs (nuclear transfer, or nuclear cloning) led researchers to consider putting single functional genes or gene clusters into the chromosomal DNA of higher organisms. The strategy used to achieve this:

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–– A cloned gene is injected into the nucleus of a fertilized egg. –– The inoculated fertilized eggs are implanted into a receptive female because a successful completion of mammalian embryonic development is not possible outside of a female. –– Some of the offspring derived from the implanted eggs carry the cloned gene in all of their cells. –– Animals with the cloned gene integrated in their germ line cells are bred to establish new genetic lines. This approach has many practical applications. If, for example, the product of the injected gene stimulates growth, animals that acquire this gene should grow faster and require less feed. An enhancement of feed efficiency by a few percent would have a profound impact on lowering the cost of production of either beef or pork. Transgenesis has become a powerful technique for studying fundamental problems of mammalian gene expression and development, for establishing animal model systems for studying human diseases, for producing foreign proteins in bird eggs, and for using the mammary gland to produce pharmaceutically important proteins in milk. With this last application in mind, the term “pharming” was coined to convey the idea that milk from transgenic farm (“pharm”) animals can be a source of authentic human protein drugs or pharmaceuticals. There is a number of reasons why the mammary gland should be used in this way. Milk is a renewable, secreted body fluid that is produced in substantial quantities and can be collected frequently without harm to the animal. A novel drug protein that is confined to the mammary gland and secreted into milk should have no side effects on the normal physiological processes of the transgenic animal and should undergo posttranslational modifications that at least closely match those in humans. Finally, purification of a protein from milk, which contains only a small number of different proteins (Table), should be relatively straightforward. Transgenic mice: Applications Transgenic mice (see Figure 71) can be used as model systems for determining the biological basis of human diseases and devising treatments for various conditions. In addition, transgenesis of mice is an exemplary system for proving whether the production of a potential therapeutic agent is feasible. Whole animal models simulate both the onset and progression of a human disease. However, a mouse is not a human, even though it is a mammal, and so the information gathered from some transgenic models may not always be medically relevant. In other instances, however, critical insights into the etiology of a complex disease can be gained.

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Figure 71. The scheme for obtaining transgenic mice (http://11e.devbio.com/wt031002.html)

With this in mind, mouse models for human genetic diseases, such as Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, arthritis, muscular dystrophy, tumorigenesis, hypertension, neurodegenerative disorders, endocrinological dysfunction, and coronary disease, as well as many others, have been developed. Transgenic cattle: Applications Conceptually, the methods used to generate transgenic cattle are similar to those used for transgenic mice. The essential steps in a modified mouse transgenesis DNA microinjection protocol entail: –– Collecting oocytes, for example, from slaughterhouse-killed animals; –– In vitro maturation of oocytes; –– In vitro fertilization with bull semen; –– Centrifugation of the fertilized eggs to concentrate the yolk, which in normal eggs prevents the male pronuclei from being readily seen under a dissecting microscope; –– Microinjection of input DNA into male pronuclei; –– In vitro development of embryos before blastocyst stage; –– Nonsurgical implantation of one embryo into one recipient foster mother in natural estrus; –– DNA screening of the offspring for the presence of the transgene. Firstly, although mice have proven to be useful in biomedical research as models of human diseases and for testing of disease treatments, the physiology,

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anatomy, and life span of a mouse are different from those of humans. Thus, livestock animals are often better for modeling disease processes, gene regulation, and immune system development. Secondly, many livestock animals produce large amounts of milk and therefore can be used to produce and secrete large amounts of recombinant proteins and other molecules of pharmaceutical importance. Thirdly, genetic engineering can be used to rapidly and specifically improve livestock traits, such as growth rate, disease resistance, and milk quality. There are some main applications of transgenic livestock: –– Production of Pharmaceuticals –– Production of Donor Organs –– Disease-Resistant Livestock –– Improving Milk Quality –– Improving Animal Production Traits Some human proteins that have been expressed in the mammary glands of transgenic animals: Antithrombin III; α1-Antitrypsin; Calcitonin; Erythropoietin; Factor IX; Factor VIII; Fibrinogen; Glucagon-like peptide; α-Glucosidase; Growth hormone; Hemoglobin; Serum albumin; Insulin; Insulin-like growth factor 1; Interleukin 2; α-Lactalbumin; Lactoferrin; Lysozyme; Monclonal antibodies; Nerve growth factor; Protein C; Granulocyte colony-stimulating factor; Superoxide dismutase; Tissue plasminogen activator. Transgenic poultry: Applications Several features that are unique to avian reproduction and development make the production of transgenic strains by microinjection of DNA into fertilized eggs extremely inefficient. For example, during fertilization in birds, several sperms penetrate the ovum instead of one, as usually occurs in mammals. As a result, it is impossible to identify the male pronucleus that will fuse with the female pronucleus. Also, the DNA injected into the cytoplasm of the fertilized egg does not integrate into genomic DNA. Finally, even if nuclear DNA microinjection were practicable, the technique would be difficult to implement because the avian ovum after fertilization becomes, in rapid succession, enveloped in a tough membrane, surrounded by large quantities of albumin, and enclosed in inner and outer shell membranes. Despite these disadvantages, it is possible to inject a transgene into the region (germinal disc) on the yolk that contains the female and male pronuclei. The germinal disc is present before the eggshell is formed. After the administration of DNA to a germinal disc, each egg is cultured in vitro, and when an embryo forms, it is placed in a surrogate egg to produce a hatchling. Despite the technical difficulties, some transgenic lines of chickens have been established by this method (see Figure 72). By the time an avian egg outer shell membrane has hardened, the developing embryo (blastoderm stage) has two layers consisting of 30,000 to 60,000 cells. In trial experiments, inoculation of the blastoderm stage with replication-defective retrovirus vectors containing bacterial marker genes resulted in a few chickens and quail carrying these DNA sequences in their germ lines.

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Transgenesis could be used to improve the genetic makeup of existing chickens with respect to built-in (in vivo) resistance to viral, bacterial, and coccidial diseases; better feed efficiency; lower fat and cholesterol levels in eggs; and better meat quality. Avian researchers have also suggested that the egg, with its high protein content, could be used as a source for pharmaceutical proteins. The expression of a transgene in the cells of the reproductive tract of a hen that normally secretes large amounts of ovalbumin could lead to the accumulation of a transgene-derived protein that becomes encased in the eggshell. Ovalbumin constitutes more than 50% of the protein of egg white; therefore, expression of a transgene under the control of the ovalbumin promoter and regulatory elements can yield high levels of recombinant protein. Yields of up to 1 g of recombinant protein have been achieved per egg, and considering that a single hen lays more than 300 eggs per year, the productivity of these animal bioreactors could be substantial. The recombinant protein could either be fractionated from the sterile egg packages or consumed as a nutraceutical. Currently, as “proof-of-principle” experiments, transgenic chickens that synthesize monoclonal antibodies, growth hormone, insulin, human serum albumin, and alpha interferon have been created. Regulatory approval of therapeutic proteins produced in eggs may be more straightforward, as chicken eggs are already used to produce vaccines for injection into humans. Isolabe blastoderm cells

Blastoderm

Transgene

Irradiated blastoderm

Transfect Inject transfected blastoderm cells into the subgerminal apace

Subgerminal space Chimera

Transgenic founder

Figure 72. The scheme of obtaining transgenic chickens (B.R. Glick & J.J. Pasternak. Molecular Biotechnology – Principles and Applications of Recombinant DNA. 3rd Edition. 2003)

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Transgenic fish: Applications As natural fisheries become depleted, production of this worldwide food resource has come to depend more heavily on aquaculture. In this context, enhanced growth rates, tolerance to environmental stress, and resistance to diseases are some of the features that may be created by transgenesis. To date, transgenes have been introduced by microinjection or electroporation of DNA into the fertilized eggs of a number of fish species, including carp, catfish, trout, salmon, Arctic char, and tilapia. The pronuclei of fish are not readily seen under a microscope after fertilization; therefore, linearized transgene DNA is microinjected into the cytoplasm of either fertilized eggs or embryos that have reached the four-cell stage of development. Unlike mammalian embryogenesis, fish egg development is external; hence, there is no need for an implantation procedure. Development of transgenic fish occurs in temperature-regulated holding tanks. The survival of fish embryos after DNA microinjection is high (35 to 80%), and the production of transgenic fish ranges from 10 to 70%. The presence of a transgene is scored by PCR analysis of either nucleated erythrocytes or scale DNA. Founder-fish are mated to establish true-breeding transgenic lines. Concerns related to germline transgenic technology Introduction of foreign DNA into an animal cell (somatic or germline) is not a well-controlled process and can lead to a number of undesired genetic consequences. Here we should discuss a number of safety issues that arise as a consequence of manipulation of the animal germlines. All safety problems can be divided into several levels of concern: 1) from the animal or group of animals; 2) to the human handler, 3) recipient, or user of the transgenic animal or its products; 4) to the human population as a whole; 5) to the environment. –– Unintended genetic side effects Insertion of a transgene into random or wrong sites in the germline is a mutagenic event that will affect any gene that happens to be at or near the site of integration. The most obvious effect is disruption of the gene integrity at the location where the insertion occurs. It is well known that a large part of the mammalian genome is noncoding DNA derived from various kinds of silenced transposable elements. Not all integration events will lead to gene inactivation. However, a fraction of animals selected for the presence of a transgene/transgene product has been found to carry associated genetic damages. For example, in mice, as it was recorded by Boeke and Stoye (1997), about 5% of MLV proviruses integrated into the germline had led to mutations of this kind. Direct transgene introduction can lead to numbers of integrated copies at multiple sites, which leads to a risk of creating animals with a variety of genetic defects. Thus, the transgenic animals should be carefully screened for in the course of subsequent breeding. For instance, one of

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the first transgenic mouse strains generated, intended to contain an inserted active oncogene, also suffered a lesion that caused a severe recessive developmental limb defects (Woychik et al., 1985). A number of other examples of insertional inactivation of transgenes introduced into mice are known, and this approach has been proposed as a useful technique for mutagenesis (Woychik and Alagramam, 1998). Another concern is related to the fact that vector DNA contains not only transgene sequences, but also the regulatory elements. So, regulatory sequences can influence the expression of transgene and other host genes. And the damage can be at a location different from the active transgene. This kind of damage is often recessive because it is not selected by the experimenter. Thus, it can only be detected by inbreeding of animal-founder offspring at the homozygous state. As a rule, researchers use strong promoters for vector construct, and this promoter can induce the gene expression in the vicinity of the transgene. This kind of unfavorable activity is the mechanism of cancer induction in animals infected by a variety of retroviruses, and it has been well-studied as a model for oncogenesis. Rosenberg and Jolicoeur (1997) revealed that promoter and enhancer insertion, as well as gene fusion and introduction of elements that stabilize messenger RNA, can prevent this negative effect of adjacent activation. The altered methylation can stimulate unfavorable activity of genes at genome sites far removed from the inserted transgene location (Muller et al., 2001). In transgenic animals, the negative activation effects are likely to reveal themselves as dominant mutations that can have a variety of phenotypic consequences, from derailing normal development to causing a high rate of cancer later in life. –– Unexpected Effects of Modification Except for the expected effects of transgene expression, genetic modifications can stimulate unexpected effects on the physiology of the transgenic organism. For example, knockout of galactosyl transferase gene in pigs, which leads to rejection of human hyperacute immune reaction at xenotransplantation of organs and tissues, also provides protection against zoonotic infection by enveloped viruses (Weiss, 1998). The similarity of pigs and viral galactosyl-galactose structure is the reason for the expected immune reaction at human. This response can result in rapid elimination of viruses transmitted from animals before infection could occur. But, pigs with the knockouted gene would have a potential to transmit viruses, such as influenza, much more readily to human handlers. And human genes are encoding cell-surface proteins and introduced into pig genome as transgenes could render pigs susceptible to human viruses, which will increase their risk of disease and provide alternative hosts for the spread of human disease. Racaniello and Ren (1994) showed that the human poliovirus receptor (CD155) renders mice susceptible to poliovirus infection when introduced as a transgene. Weiss’s experiments (1998) revealed that the human complement-response modifying proteins CD46 and CD55, introduced into pigs to protect xenografts from

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rejection, also serve as receptors for measles and Coxsackie human viruses. This could not only render these animals susceptible to infection by the human viruses, but could also provide a new evolutionary pathway for adaptation of pig viruses to human cells. And because receptors for many other viruses have not yet been identified, the potential for this kind of undesirable effects exists whenever a human cell-surface protein is introduced into another animal species. –– Marker Genes When the researcher constructs a vector for genetic transformation he should select the marker gene whose function is different from that of the desired transgene. Commonly used marker genes are typically genes encoding drug-resistance or a phenotypic trait. The main properties of the marker gene are its fast selectivity and its harmlessness or neutrality regarding the organism being modified. For example, the neo gene encoding neomycin phosphotransferase has been widely used for selecting animal transgenic cells in the culture containing neomycin. At genetic transformation the marker genes remain in vectors sequences. There is a potential for marker genes to cause undesired side effects to the recipient animals, for example, provide a novel antibiotic resistant pathogens or novel allergens. This potential may be far from a real danger, but it is hard to prove that marker genes are harmless in consumer products. The presence of marker genes in the transgenic construction raises concerns about food and environmental safety of genetically engineered animal products. –– Undesired Inserts Except the transgene, the vector design includes the marker gene and regulatory sequences which often originate from prokaryotes (microbes and viruses). Cloning of extracted DNA fragments includes their propagation in bacterial systems. Despite intensive purification of the DNA fragments, some amounts of contaminating material derived from the host bacterium can remain. And it is difficult to detect them in DNA samples by standard methods like gel electrophoresis because such fragments can be heterogeneous in size and sequence. Chakraborty (1994) and Scadden (1990) paid attention to the problem arising from the usage of retroviral vectors, because host cells, often mice cells, could contain large amounts of endogenous viruses and virus-like sequences. In some cases, they can be present in vector DNA samples, accidental introduction of such sequences into the transgenic animal germlines not only provides a potential for creating unintended genetic damages, but can also contribute by recombination to the generation of novel infectious viruses. Purcell with the team (1996) obtained evidences of generation of replication-competent MLVs containing such multiple recombinants during the growth of the vector containing a globin gene. MLVs were highly pathogenic in rhesus monkeys, causing a fatal lymphoma, which is symptomatically similar to the disease induced by MLV in mice. –– Potential for Mobilization

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Usage of viral vectors stimulates other particular concern regarding the mobility of this kind of vectors. In case of viral vectors, usage of genes for introduction into the germline of animals has a potential for inadvertent transmission of the gene to other individuals, not necessarily of the same species. If genetically modified animals were to be infected with a vector sufficiently similar to the virus, it could be packaged into virions as a virus. Usage of avian retrovirus vector for creation of a transgenic chicken may result in infection of the transgenic chicken by any related virus. Such viruses are quite commonly found in commercial poultry operations. This could lead to the production and release of a virus that could transmit the gene to other animals where its presence and expression might be highly undesirable, for example, in wild bird populations. Since many species contain endogenous retroviruses in their genomes, the generation of a replicating virus could occur in the absence of exogenous infection. For example, usage of the murine leukemia virus-based vector constructs in cats leads to mobilization of introduced genes and transfection of other cats or their hosts. Researchers use a HIV based vectors in hope to improve the efficiency of introduction of new genes into the germline of many animal species. However, HIV-vectors in germline could also be mobilized and transfect recipient’s close relatives. It should be mentioned, that viruses closely related to HIV were found only in African primates. However, phylogenetically related to HIV viruses were described at other animals. For example, Lentiviruses are fairly common in cats (feline immunodeficiency virus or FIV), cattle (bovine immunodeficiency virus or BIV), and sheep (visna-maedi virus or VMV). Despite a quite distant relationship, the Lentiviruses can induce the mobility of HIV-based sequences. Some studies (Berkowitz et al., 2001; Browning et al., 2001) present the evidence that FIV was able to transfer HIV-based vector constructs from one cell to another. A related concern arises from the usage of mobile elements as vectors for genetic transformation of animals. For example, Mariner and related transposons, such as Sleeping beauty, have been found in multiple copies (about 14000 copies) in planaria, nematodes, centipedes, mites, insects (Robertson, 1997), and in human genome (Robertson and Zumpano, 1997; Lander et al., 2001). Scientists suggest the possibility of horizontal gene flow via transposition among highly diverse hosts (Robertson and Lampe, 1995; Hartl et al., 1997; Hamada et al., 1997; Kordis and Gubensek, 1998; 1999; Jordan et al., 1999; Sundararajan et al., 1999). A risk of transfer of mobile elements in the animal germline could result in unexpected genetic damages. The most compelling argument for horizontal gene flow in eukaryotes is the ubiquity of transposable elements and endogenous retroviruses in genomic DNA. Transposase or hopase are the enzymes encoding viral genes, they cause mobility of viruses and transposons. Vectors containing these genes in the trans configuration could express the transposase or hopase, and after integration to

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chromosome, these genes were deleted from the transgene constructs. Thus, once inserted into the host’s chromosome, the element should be immobilized. The hazards of their presence in vectors could be minimized or eliminated if this requirement was strictly observed in transposable element vector systems for genetic engineering of animals. –– Potential for Creation of New Pathogens Vector transgene sequences can also contribute elements to infecting agents that might modify their ability to cause disease. The example of this theoretical risk is represented by the usage of drug-resistance genes to bacteria. Their widespread presence in transgenic livestock is a potential risk. Possible generation of new retroviruses following recombination between endogenous or exogenous viruses, which are used as vectors for animal transgenes, is another concern. The recombination event could result in appearance of new genes or regulatory elements (such as LTRs), that could adversely modify the pathogenic potential of the infecting virus. An example from a natural event is generation of a highly virulent virus HPRS-103 (or subgroup J avian leukemia virus (ALV)) through recombination between an infectious avian retrovirus and a distantly related endogenous element (Payne et al., 1992; Benson et al., 1998). The natural possibility of the HPRS-103 appearance has a very small probability, but, nevertheless, this virus quickly spread worldwide and had become a source of considerable economic loss to poultry breeders (Venugopal, 1999). The recombinant DNA technology for genetical modifications of domestic animals is being advanced at a very rapid rate. And we should expect some problems from the use of unnecessary genes and regulatory sequences in vector constructions used for generation of engineered animals, and undesirable effects of the technology on the welfare of the engineered animals themselves. Control questions: 1. What is a transgenic animal in biological and genetic sense? 2. Indicate the milestones in the history of transgenic technologies development. 3. Describe the main approaches to obtaining a transgenic animal. 4. What are the marker genes? 5. Describe the main techniques for introduction of foreign genes in the recipient genome. 6. What do you know about special types of selection used for vector constructs of right insertion? 7. What do you know about cloning applications in Animal Biotechnology? 8. What do you know about concerns of transgenic technology?

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PART III PLANT BIOTECHNOLOGY

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CHAPTER

1

THE SUBJECT, OBJECTS, HISTORY AND METHODOLOGY OF PLANT BIOTECHNOLOGY Plant biotechnology is the area of biotechnology involving applications to agriculture, industry, medicine, food industry. The main object of plant biotechnology is plant tissue or cell culture. In general, culture in vitro is plant cells, tissues, plant organs, or whole plants growing in nutrient medium in vitro, under aseptic conditions, e.g. cell culture, tissue culture, embryo culture, shoot-tip culture, microspore/anther culture. Plant tissue culture has become popular among plant breeders, horticulturists and industrialists because of its varied practical applications. The latest advances in plant biotechnology provide a potential for making improvements much more quickly than by conventional plant breeding. It is also being applied to study basic aspects of plant growth and development. Studies on all aspects of plant development and multiplication in whole plants are often complicated by interactions between various processes that underlie growth and development. It is, therefore, desirable to simplify matters so that controlling influences can be easily identified and studied. This can be done by isolating and culturing parts of the plants in vitro. An important contribution made through tissue culture is the revelation of the unique capacity of plant cells, called “cellular totipotency”. It means that all living plant cells are capable of regenerating whole plants irrespective of their nature of differentiation and ploidy level. Tissue culture also provides the best means to elicit the cellular totipotency of plant cells and, therefore, it forms the backbone of the modern approach to crop improvement by bioengineering methods. Totipotency is the potentiality or property of a plant cell to produce a whole organism or whole parent plant in the presence of correct physical and chemical stimulus. Cell division can be induced from almost all plant tissues. When plant cells and tissues are cultured in vitro, they generally exhibit a very high degree of plasticity, which allows one type of tissues or organs to be initiated from another type under the influence of chemical stimuli. In this way, whole plants can be subsequently regenerated. For example, embryos may be developed in vitro from somatic cells and haploid cells, as well as from normal zygote and all these, in 236

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turn, could develop into whole plants. Therefore regeneration is a process in tissue culture, a morphogenetic response that results in the formation of new organs, embryos or whole plants from cultured explants. A piece of tissue or a plant organ used to initiate a culture is called explant. Regeneration of plants from cultured cells has also found many applications. The earliest application of plant tissue culture was to rescue hybrid embryos and the technique became a routine aid with plant breeders to raise rare hybrids, which normally failed due to post-zygotic sexual incompatibility. Hybrid embryos, formed by the fusion of gametes from distant relatives, frequently abort naturally because the endosperm is not compatible with embryo. Under in vitro conditions, in the presence of correct nutritional medium, these embryos can be developed and form a plant. Even the triploid cells of endosperm are totipotent, which provides a direct and easy approach to regeneration of triploid plants difficult to raise in vivo. The plant protoplasts (plant protoplasts are naked cells from which the cell wall has been removed) can be fused to form somatic hybrids. Such fusion products are the result of uniting of two or more protoplasts from similar or dissimilar parents (asexual hybridization). Plant regeneration from cultured somatic cells is proving to be a rich source of genetic variability, called “somaclonal variation”. Several somaclones have been processed into new cultivars. Also, induced mutagens produce more frequency of mutants than spontaneous ones and screening them at cellular level also inhibits chimeric formation, which is a drawback in mutation breeding. Methods of modern biotechnology allow the process of breeding to be accelerated, and haploid production is one of the most widely used biotechnological methods in the breeding of self-pollinating plants. Regeneration of plants from microspore/pollen culture in vitro provides the most reliable and rapid method to produce haploids, which are extremely valuable in plant breeding and genetics. These plants contain a single set of chromosomes, as those of gametes, and are haploid in nature. The chromosome set of these haploids can be diploidized by mutagenic treatment like, colchicine, to develop homozygous diploid lines or pure breeding lines. With doubled haploids (DH), homozygosity can be achieved in a single step; the method accelerates breeding by 3-5 years. This is particularly important for highly heterozygous, long-generation plant species. DH production is aimed at gene transfer into the homozygotic state in the first generation. Recessive mutations, important recombinations, and other genomic changes can be found in doubled haploids more easily. DH can be used for genetic analysis, gene mapping, and gene engineering. Haploid plants provide a unique opportunity to screen gametic variation at sporophytic level. This approach has enabled selection of several gametoclones (gametoclonal breeding), which could be developed into new cultivars. The gametoclonal and somaclonal variations induced in in vitro cultures can be utilized to raise new improved varieties which may have commercial value such as, new flower color, big canopy plants, large sized grains etc.

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The most popular commercial application of plant tissue culture is clonal propagation in vitro and propagation of disease-free plants. In vitro propagation of plants vegetatively by tissue culture to produce genetically similar copies of a cultivar is referred to as micropropagation or clonal propagation. Sexually propagated plants (through generation of seeds) demonstrate a high amount of heterogeneity since their seed progenies are not true to type whereas asexual reproduction (by multiplication of vegetative parts) gives rise to genetically identical copies of the parent plant. Thus, it permits perpetuation of the parental characters of the cultivars among the plants resulting from micropropagation. Actively-dividing young cells (meristem) are placed in a special medium and treated with plant hormones to produce many similar sister plantlets. Since the meristem divides faster than the disease-causing virus, clean materials are propagated and hundreds of uniform plantlets are produced in a short time. Thus, over a million plants can be produced in a year starting from a small piece of tissue. The enhanced rate of multiplication can considerably reduce the period between the selection of plants and raising enough planting material for field trials. In tissue culture, propagation occurs under disease-free and pest-free conditions. It is important in those plants which are otherwise difficult to propagate, have a high value or where speed of propagation is important. It can also help to raise disease-free stock of a crop by apical meristem culture where the entire plant is infected with pathogenic bacteria or viruses. Therefore in vitro clonal propagation, popularly called micropropagation, offers many advantages over the conventional methods of vegetative (asexual) propagation: 1) many species (e.g. palms, papaya) which are not amenable to in vivo vegetative propagation are being multiplied in tissue cultures; 2) the rate of multiplication in vitro is extremely high and can continue round the year, independent of the season and climate conditions; 3) the reduced growth cycle and rapid multiplication as shoot multiplication has a short cycle and each cycle results in exponential increase in the number of shoots; 4) virus free plants can be raised and maintained through meristem culture which is the only method available for this. Characteristics like, herbicide resistance, resistance to abiotic and biotic factors that could not be achieved by conventional plant breeding methods, can be transferred from one plant species to another by Agrobacterium-mediated gene transfer or by gene transfer methods like, particle gun method (biolistics), electroporation, microinjection and polyethylene glycol-mediated gene transfer. The plants obtained by gene transfer technique and cell culture methods will be transgenic plants (this organism is called “genetically modified organism” or “GMO”). Genetic engineering techniques are utilized to produce transgenic plants with desirable genes like disease resistance, insecticide resistance, herbicide resistance, increased shelf life of fruits, etc. Also, molecular breeding has hastened the process of crop improvement for e.g. molecular markers like RFLP, SSRs provide

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powerful tools for indirect selection of both qualitative and quantitative traits and also for studying genotypic diversity. Plant tissue culture is a technique of in vitro cultivation of plant cells and organs, which divide and regenerate into callus or particular plant organs and whole plants. Tissue culture is a straightforward technique and many developing countries have already mastered it. Its application only requires a sterile workplace, nursery, green house, and trained manpower. 1.1 History of Plant Biotechnology In 1902, a German physiologist, Gottlieb Haberlandt developed the concept of in vitro cell culture. He isolated single fully differentiated individual plant cells from different plant species like palisade cells from leaves of Laminum purpureum, glandular hair of Pulmonaria and pith cells from petioles of Eichornia crassiples, etc., and was the first to culture them in Knop’s salt solution enriched with glucose. In his cultures, cells increased in size, accumulated starch but failed to divide. Therefore, Haberlandt’s prediction failed that the cultured plant cells could grow, divide and develop into an embryo and then into a whole plant. The idea that cell division may be initiated by a diffusible factor originated with the plant physiologist G. Haberlandt. He demonstrated that vascular tissue contains a water-soluble substance or substances that could stimulate the division of wounded potato tuber tissue. This potential of a cell is known as totipotency, a term coined by Steward in 1968. Despite lack of success, G.Haberlandt made several predictions in media about the requirements to the experimental conditions which could possibly induce cell division, proliferation and embryo induction. G. Haberlandt is thus regarded the father of tissue culture. Taking experience from Haberlandt’s failure, Hannig (1904) chose embryogenic tissue to culture. He excised nearly mature embryos from seeds of several species of crucifers and successfully grew them to maturity on mineral salts and sugar solution. In 1908, Simon regenerated callus, buds and roots from Poplar stem segments and established the basis for callus culture. For about next 30 years (up to 1934), there was very little further progress in cell culture research. Within that period, an innovative approach to the tissue culture using meristematic cells like root and stem tips was reported by Kolte (1922) and Robbins (1922) working independently. All these research attempts involving culture of isolated cells, root tips or stem tips ended in the development of calluses. There were two objectives to be achieved before putting Haberlandt’s prediction to fruition. First, to make the callus obtained from the explants to proliferate endlessly and, second, to induce these regenerated calluses to undergo organogenesis and form whole plants. It

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was in the 1930s, when the progress in the plant tissue culture accelerated rapidly owing to an important discovery that vitamin B and natural auxins were necessary for the growth of isolated tissues containing meristems. In 1934, Philip White demonstrated that tomato roots could be grown indefinitely in a simple nutrient medium containing only sucrose, mineral salts, and a few vitamins, with no added hormones but could be repeatedly subcultured to fresh medium of inorganic salts supplemented with yeast extract. Later (1937) he replaced the YE by vitamin B, namely pyridoxine, thiamine and proved their growth promoting effect. In 1926, Fritz Went discovered the first plant growth regulator (PGR), indoleacetic acid (IAA). IAA is a naturally occurring member of the class of PGRs termed ‘auxins’. Roger J Gautheret (1934) reported the successful culture of cambium cells of several tree species to produce callus and 2 addition of auxin enhanced the proliferation of his cambial cultures. Callus tissue sometimes forms naturally in response to wounding, or in graft unions where stems of two different plants are joined. Further research was done by Nobecourt (1937), who could successfully grow continuous callus cultures of carrot slices, and White (1939), who obtained similar results from tumour tissues of hybrid Nicotiana glauca x N. langsdorffi. Thus, the possibility of cultivating plant tissues for an unlimited period was independently endorsed by Gautheret, White and Nobecourt in 1939. In contrast to roots, isolated stem tissues exhibit very little growth in culture without added hormones in the medium. Even if auxin is added, only a limited growth may occur, and usually this growth is not sustained. Frequently, this auxin-induced growth is due to cell enlargement only. The shoots of most plants cannot grow on simple medium lacking hormones, even if the cultured stem tissue contains apical or lateral meristems, until adventitious roots form. Once the stem tissue has rooted, shoot growth resumes, but now as an integrated, whole plant. These observations indicate that there is a difference in the regulation of cell division in root and shoot meristems. They also suggest that some root-derived factor(s) may regulate growth in the shoot. Adding to the ongoing improvements in the culture media, Johannes Van Overbeek (1941) reported growth of seedlings from the heart-shaped embryos by enriching the culture media with coconut milk besides the usual salts, vitamins and other nutrients. This provided a tremendous impetus for further work in embryo culture. Stem tip cultures yielded success when Ernest Ball (1946) devised a method to identify the exact part of shoot meristem that gives rise to the whole plant. After 1950, there was an immense advancement in knowledge of the effect of PGRs on plant development. The fact that coconut milk (embryo sac fluid) is nutritional requirement for tobacco callus besides auxin, indicated the non-aux-

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inic nature of milk. This prompted further research and so other classes of PGRs were recognized. A great number of substances was tested in an effort to initiate and sustain the proliferation of normal tissues in culture. Materials ranging from yeast extract to tomato juice were found to have a positive effect, at least with some tissues. However, the culture growth was stimulated most dramatically when the liquid endosperm of coconut, also known as coconut milk, was added to the culture medium. Philip White’s nutrient medium, supplemented with an auxin and 10 to 20% coconut milk, will support the continued cell division of mature, differentiated cells from a wide variety of tissues and species, leading to the formation of callus tissue. This finding indicated that coconut milk contains a substance or substances that stimulate mature cells to enter and remain in the cell division cycle. Eventually coconut milk was shown to contain the cytokinin zeatin, but this finding was not obtained until several years after the discovery of the cytokinins. The first cytokinin to be discovered was the synthetic analog of kinetin. Skoog and Tsui (1957) demonstrated induction of cell division and bud formation in tobacco by adenine. This led to further investigations by Skoog and Miller (1955) who isolated ‘kinetin’- a derivative of adenine (or aminopurine) 6- furfuryl aminopurine. Kinetin and many other compounds which show bud promoting activities are collectively called cytokinins, a cell division promoter in cells of highly mature and differentiated tissues. In the presence of an auxin, kinetin would stimulate tobacco pith parenchyma tissue to proliferate in culture. No kinetin-induced cell division occurs without auxin in the culture medium. Several years after the discovery of kinetin, extracts of the immature endosperm of corn (Zea mays) were found to contain a substance that has the same biological effect as kinetin. This substance stimulates mature plant cells to divide when added to a culture medium along with auxin. Letham (1973) isolated the molecule responsible for this activity and identified it as trans-6-(4-hydroxy-3-methylbut2-enylamino)-purine, which he called zeatin. The molecular structure of zeatin is similar to that of kinetin. Both molecules are adenine or aminopurine derivatives. Although they have different side chains, in both cases the side chain is attached to the 6 nitrogen of the aminopurine. Because the side chain of zeatin has a double bond, it can exist in either the cis or the trans configuration. Skoog and Miller worked further to propose the concept of hormonal control of organ formation (1957). Their experiment on tobacco pith cultures showed that high concentration of auxin promoted rooting and high kinetin induces bud formation or shooting. However, now the concept is altered to multiple factors like source of plant tissue, environmental factors, composition of media, polarity, growth substances being responsible for determination of organogenesis. Besides PGRs, scientists tried to improve the culture media by changing essentially the mineral content. In this direction, Murashige T. and Skoog F.(1962) prepared a

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medium by increasing the concentration of salts twenty-five times higher than Knops. This media enhanced the growth of tobacco tissues by five times. Even today MS medium has an immense commercial application in tissue culture. Having achieved success and expertise in the growth of callus cultures from explants under in vitro conditions, the focus is now shifted to preparation of single cell cultures. Muir (1953-1954) demonstrated that when callus tissues were transferred to the liquid medium and subjected to shaking, callus tissues broke into single cells. Bergmann (1960) developed a technique for cloning of these single cells by filtering suspension cultures. This technique called Plating technique is widely used for cloning isolated single protoplasts. The next step for realization of Haberlandt’s objectives was the development of a whole plant from the proliferated tissue of these cells. Vasil and Hilderbrandt were the first to regenerate plantlets from colonies of isolated cells of hybrid Nicotiana glutinosa x N. tabacum. In 1966, the classical work of Steward on induction of somatic embryos from free cells in carrot suspension cultures brought an important breakthrough by final demonstration of totipotency of somatic cells, thereby validating the ideas of Haberlandt. This ability of regenerating plants from single somatic cells through normal developmental process had great applications in both plant propagation and genetic engineering. For example, micropropagation where small amounts of tissue can be used to continuously raise thousand more plants. Morel utilized this application for rapid propagation of orchids and Dahlias. He was also the first scientist to free the orchid and Dahlia plants from viruses by cultivating shoot meristem of infected plants. The role of tissue culture in plant genetic engineering was first exemplified by Kanta and Maheshwari (1962). They developed a technique of test tube fertilization which involved growing of excised ovules and pollen grains in the same medium thus overcoming the incompatibility barriers at sexual level. In 1966, Guha and Maheshwari, for the first time, showed the possibility of producing haploid plants from anther culture (androgenesis method) of Datura inoxia that strongly stimulated research in this direction. Haploid production through the use of unfertilized ovaries and ovules had been practiced since the 1950s. A considerable contribution to the development of this method was made by Indian scientists Sachar and Kapoor (1959), Guha and Johri (1966), Rangan (1982). They gave a theoretical basis to the possibility of growing female gametophyte in vitro. San Noeum (1976) obtained the first haploid plants from unfertilized ovary culture of Hordeum vulgare. This discovery received significant attention since plants recovered from doubled haploid cells are homozygous and express all recessive genes thus making them ideal for pure breeding lines. The results of these investigations demonstrated that haploid production is the result of abnormal development of the female gametophyte and subsequent embryo- or callus formation.

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Next breakthrough in application of the tissue culture came with isolation and regeneration of protoplasts first demonstrated by Prof. Edward Cocking in 1960. Cocking produced large quantities of protoplasts by using cell wall degrading enzymes. After success in regeneration of protoplasts, Carlson (1972) isolated protoplasts from Nicotiana glauca x N. langsdorfii and fused them to produce first somatic hybrid. Since then many divergent somatic hybrids have been produced. With the advent of restriction enzymes in the early 1970s, the tissue culture headed towards a new research area. The totipotent plant cells could now be altered by insertion of specific foreign genes giving rise to genetically modified crops. In 1970, Smith and Nathans isolated the first restriction enzyme from Haemophillus influenzae which was later purified and named Hind III. The same year witnessed the other Nobel Prize winning discovery by Baltimore who isolated Reverse transcriptase from RNA tumor viruses. This is a useful enzyme in genetic engineering which functions to convert RNA to DNA and hence useful in construction of complementary DNA from messenger RNA. Another pathbreaking discovery establishing potential of genetic engineering came in 1972 when Paul Berg working at Stanford University produced the first recombinant DNA in vitro by combining DNA from SV40 virus with that of lambda virus. This led to construction of the first recombinant organism by Cohen and Boyer in 1973. Genetic engineering’s potential was first exploited when a man-made insulin gene was used to manufacture a human protein in bacteria. Agrobacterium tumefaciens plays a crucial role in plant genetic engineering. The involvement of this bacterium in the crown gall disease in plants was recognized as early as 1907 by Smith and Townsend. However, it was only in 1974, that Zaenen et al. discovered that Ti- plasmid is a tumor-inducing principle of Agrobacterium. This was followed by its successful integration in plants by Chilton et al. in 1977. Zambryski et al. in 1980 isolated and studied the detailed structure of T-DNA and its border sequences. Soon thereafter in 1984, transformation of tobacco with Agrobacterium was accomplished to develop transgenic plants. Simultaneously, there was an upsurge in the development of techniques of genetic engineering in the mid-1970s. Sanger et al. (1977) and Maxam and Gilbert (1977) reported techniques for large scale DNA sequencing. This was followed by complete genome sequencing projects on many prokaryotes and eukaryotes like Haemophilus influenzae in 1995, E. coli in 1997. 1.2 The development of biotechnology research in Kazakhstan In the last 70-80 years of the twentieth century, scientific research institutes were established in Kazakhstan. They were engaged in fundamental and applied

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problems in the fields of biotechnology, molecular biology, genetics, biochemistry and plant physiology. In Kazakhstan, a great contribution to the development of plant biotechnology and the creation of the national school of Biotechnology was made by such scientists as Academician M.A. Aytkhozhin, Academician I.R. Rakhimbaev, Professor G.Zh. Valikhanova and others (Figure1).

М.А.Aytkhozhin

I.R.Rakhimbaev

G.Zh.Valikhanova

Figure 1. Scientists and professors – the founders of Plant Biotechnology in Kazakhstan

Murat Abenovich Aytkhozhin (1939-1987) – Doctor, Professor, a specialist in the field of molecular biology and biochemistry, Member of the Academy of Sciences (AS) of the Kazakh SSR (1983), winner of the Lenin Prize (1976), a disciple of Academician A. Spirin. Murat Aytkhozhin was the founder of molecular biology and biotechnology in Kazakhstan. M. Aytkhozhin was one of the first in the world of science to conduct a comparative study of the protein synthesizing system in higher organisms, and studied physical and chemical properties of informosomes in plant cells. Informosomes are free cytoplasmic, polysomeassociated and nuclear, including RNA-binding, proteins. He studied physical and chemical properties of influence of informosomes on biosynthesis at the level of mRNA translation and its biogenesis. In 1978-1983, M. Aytkhozhin headed the Institute of Botany, AS of the Kazakh SSR. In 1983, M.A. Aytkhozhin organized the Institute of Molecular Biology and Biochemistry in Alma-Aty, then for four years he headed the Institute. Murat Aytkhozhin was the first scientist who introduced a course in Molecular Biology and several special courses for students at the Kazakh State University at the Biology faculty. In 1987, he founded the Kazakh Agricultural Biotechnology Center and organized research of plant cell and genetic engineering. In 1986-1987, he was the President of the Academy of Sciences of the Kazakh SSR. Under the leadership of M. Aytkhozhin a set of instruments for automation of experiments in Molecular Biology was designed (it was protected by 15 copyright certificates and 16 patents in the leading countries). For the research series “Discovery of informosome – a new class of intra-

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cellular particles” and “Molecular mechanisms of plant protein biosynthesis” he was awarded the Lenin Prize and the Gold Medal of the Peace Fund (1987). Izbasar Rakhimbaevich Rakhimbaev (1936) – Doctor, Professor, Member of the National Academy of Sciences (NAS) of the Republic of Kazakhstan, Honorable member of the Presidium of the Russian Plant Physiology Society and VicePresident of the Botany Society in Kazakhstan. I. Rakhimbaev is the founder of plant biotechnology in Kazakhstan, and a well-known specialist in the field of Plant physiology and biotechnology. He is the author of about 400 scientific works and publications, including 7 monographs (“Vegetative micropropagation of plants” (1985), “Cell culture and Plant Biotechnology” (1991), “Biotechnology of crops” (1992), “Cell culture and cell engineering of plants” (1994), “Plant Embryology” (1996) etc.), 9 textbooks, 12 methodological manuals and 5 patents. Professor I. Rakhimbaev and his disciples at the Institute of Plant Biology and Biotechnology created 4 new wheat cultivars and 2 rice cultivars. Under his supervision, 30 PhD and 8 Doctor’s theses were defended in Kazakhstan and foreign countries. In 1993-2007, he was the editor-in-chief of the Scientific Journal “Biotechnology. Theory and Practice”, “Proceedings of the National Academy of Sciences” and Chairman of the Dissertation Council on the Defense of Theses to Confer PhD and Doctorate Degrees in Biology. From 1981 to 1987, he was Head of the Department of Plant Physiology and Biochemistry at the Kazakh State University. In 1988-1992, Professor I. Rakhimbaev headed the Main Botanical Garden in Almaty. In 1993 he founded the Institute of Plant Physiology, Genetics and Bioengineering (from 2005 to present time known as the Institute of Plant Biology and Biotechnology). He was awarded the medals “For the development of virgin lands” (1968), “For Valiant Labor” (1973), “Gold Medal of the National Academy of Science” and “Respect” (2007). Since 2007, Dr. Rakhimbaev I. has been a main researcher of the Institute of Plant Biology and Biotechnology at the National Biotechnology Center of the Republic Kazakhstan. At the present time, many students, alumni of Professor I. Rakhimbaev, work in the leading research companies, Research and Academic Institutes, National Centres, Universities in Kazakhstan and abroad. Gulzhannet Zhansultanovna Valikhanova (1939) – Biologist, Professor of the Department of Plant Biotechnology, Biochemistry and Physiology (Department of Biotechnology) at Al-Farabi Kazakh National University. In 1961, she graduated from the Kazakh State University in Alma-Ata and gained her Candidate Diploma (PhD) at the State University named after A.A. Zhdanov in Leningrad (St. Petersburg). From 1966 to 1971, G. Valikhanova worked in the Laboratory of Biochemistry at the Institute of Botany of the Kazakh SSR Academy of Sciences. In 1971-1981, G. Valikhanova was a senior lecturer at the Department of Plant Physiology and Biochemistry. In 1981, she was an Associate Professor and

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in 1998 she was awarded the academic rank of Professor of the Kazakh National University. In 1989-1990, Professor G. Valikhanova headed the Department of Plant Physiology and Biochemistry at the Biology faculty of the Kazakh National University. She was a co-author of the National mandatory standards of education in biotechnology for undergraduate and graduate programs, published in 2002 and 2004. Over a number of years, she was an author of the working curricula for the specialization in biotechnology at the faculty, was the head of the Methodological Bureau of the Faculty and head of the Educational and Methodological Section on Biotechnology in Kazakhstan. G.Zh. Valikhanova makes an essential contribution to the development of teaching Biotechnology in Kazakhstan by publishing the original textbooks “Plant Biotechnology” (1st edition in 1996, 2nd edition in 2001, 3d edition in 2009) in Russian and in Kazakh, methodological manuals on Biotechnology, a Test-set in the disciplines of Plant Physiology and Plant Biotechnology, education programs for obligatory basic biotechnological disciplines (Cell Biotechnology, Plant Biotechnology, Plant Physiology) and special courses/pacticums. Professor G. Valikhanova is the author of about 90 scientific publications. Her main publications are given below: G.Zh. Valikhanova. Plant Biotechnology. Textbook (in Russian). Almaty: Konzhyk, 1996 – 272 p.; G.Zh. Valikhanova. Plant Biotechnology. Textbook (in Kazakh). Almaty: Kazakh University, 2001(1st edition), 2009 (2nd edition); G.Zh. Valikhanova, B.A. Sarsembayev. Tests for control of knowledge on physiology of plants. Almaty: Kazakh University, 2001-210 p.; S.K. Mukhambetzhanov, G.Zh. Valikhanova, A.E. Yerezhepov. Methodical guidelines on laboratory works on plant biotechnology. Shymkent: Academic material. 2007. – 110 p. In Kazakhstan, research on plant biotechnology started in 1970. Extensive research on tissues, cells and protoplast culture of cereal crops was conducted in M.A. Aytkhozhin Institute of Molecular Biology and Biochemisrty and the Institute of Plant Phyziology, Genetics and Bioengineering (now Institute of Plant Biology and Biotechnology). This led to further investigations by I.R. Rakhimbaev, B.B. Anapiyaev, S.V. Kushnarenko (from the Institute of Plant Biology and Biotechnology, IPBB), S.S. Bekkuzhina (S.Seifullin Kazakh Agro Technical University) who clarified the regularities of proliferation, differentiation, morphogenesis and regeneration in reproductive and somatic cell culture. The implementation of haploid technology based on culture generative cells, in combination with convenient breeding methods allows the selection of varieties and hybrids with valuable traits. Associate Professor B.A. Zhumabaeva (1995) obtained heterozygous somaclonal variants of wheat in somatic cell culture based on established theoretical concepts. Research of Professor N.K. Bishimbaeva et al.(2006) identi-

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fied the way of regulation of cytodifferentiation and morphogenesis in long-term cultured embryogenic tissues of barley and wheat, growing in normal conditions and under abiotic stress. Genotype-independent cell technology of plant regeneration during longterm subcultivation was improved, which allows us to overcome the differences in plant regeneration capacity in vitro for wheat varieties and lines. The technology of regeneration from repeatedly subcultured callus tissue was created. In 1996-2004, O.I. Smolyakova, A.B. Iskakova, N.V. Terletckaya and B.R. Kudarov cultured anthers of potato and barley, and raised embryos which developed into haploid plants initiating androgenesis for potato and chromosome elimination for barley hybrids. Regulations for the regeneration of homozygous doubled haploid plants using anther culture and the “bulbosum”-method were developed. A.E. Erezhepov, S.K. Mukhambetzhanov, L.N. Tuypina (Al-Farabi Kazakh National University and IPBB) have improved the methods of embryo culture developed to increase the viability of wheat hybrid embryos with its wild relatives. Professor S. Svanbaev and E.D. Dzhangalina (from the Institute of Plant Physiology, Genetics and Bioengineering) obtained “artificial seeds” of Medicago sp. (Fabaceae) from embryogenic suspension culture through induction somatic embryogenesis in vitro. Biotechnological protocols of micropropagation in tissue culture and isolated organs of rare and endangered species, as well as medicinal, fodder, technical and ornamental plants were developed under the supervision of Dr. I. Rakhimbaev and Professor V.K. Mursalieva and senior researcher S.V. Nam (1993, 2008). The concentration of growth regulators in the nutrion medium was optimized in accordance with the content of endogenous plant hormones in the tissue of intact plants. Professor K.K. Boguspaev and Associate Professor S.K. Turasheva from Al-Farabi Kazakh National University (2015) proposed a methodological approach that led to an improvement in the efficiency of in vitro clonal propagation of rare and endangered endemic plants. At the Institute of Plant Biology and Biotechnology the cyto-physiological patterns of influence of ultra-low temperatures on plant cells for improving the technique of cryopreservation of the germplasm of fruit and berry cultivars were studied by Professor S.V. Kushnarenko et al. (2013). The influence of space factors (such as a high dose of irradiation, high temperature, zero-gravity conditions) on the growth processes and morphogenesis in wheat callus tissue, as well as on the development of generative organs and cells (anthers, ovary, micro/ megaspores) of wheat was studied in 19992002 by Z.R. Mukhitdinova, M.A. Berdina, S.K. Turasheva and N.K. Ismagulova. They showed that the formation of gametes and embryo development in a culture of isolated ears of wheat can be realized in the zero-gravity of the outer space. A significant damage and decrease in the rate of cell division, callus growth and

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cell secretory activity was detected. After the extended space flight and further cultivation under Earth conditions, somaclonal variantions in a culture of somatic tissue of potato were obtained by Professor M.K. Karabaev, Dr. L. Ligay, R.M. Turpanova, Zh.T. Lesova from M.A.Aytkhozhin Institute of Molecular Biology and Biochemisrty. In 2002, the scientists first reported about the cultivar “Tokhtar” obtained from somaclones. It should be mentioned that the cultivar was named after the first Kazakh astronaut Tokhtar Aubakirov. Research in applications of the tissue culture led to isolation and regeneration of wheat and maize protoplasts demonstrated in 1995 by Professors M. Karabaev and Zh. Dzhardemaliev from M.A. Aytkhozhin Institute of Molecular Biology and Biochemisrty. In 1993 the National Center for Biotechnology – the leading biological center in Kazakhstan, implementing the State policy of support and development of biotechnological industry, was founded. The mission of the NСB is generation of high-level scientific knowledge and its application to solving problems of human and animal healthcare, environmental, and agricultural challenges, collaborating with industries and ensuring the transfer of technology. Nowadays, the center implements and coordinates the governmentfunded scientific-technical programs in biotechnology, biosafety and ecology. A priority area in the development of biotechnology in Kazakhstan is genetic engineering. Scientists from the National Centre for Biotechnology (NCB) and M.A. Aytkhozhin Institute of Molecular Biology and Biochemistry as well as from the Institute of Plant Biology and Biotechnology and L.N.Gumilyov Eurasian National University – R.T. Omarov, Sh.A. Manabaeva, A.A. Kakimzhanova, O.N. Khapilina, B.K. Iskakov, N.K. Bishimbaeva and N.N. Galiakparov – had previously demonstrated methods of stable genetic transformation and regeneration of transgenic crops, potato, cotton and grapes (2010-2013). In that period the transgenic potato with genetically fixed Y-resistant virus was obtained. A viral genome fragment was introduced into the potato genome in the antisense orientation. Genetic transformations of potato and tobacco were implemented by transferring genes to protoplasts that code for the synthesis of antifreeze proteins involved in resistance to low temperatures. As a result of the research, scientists demonstrated that tobacco is the best host for transient production of heterologous proteins compared to other plants from Solanaceae (2014) GMO cotton was also created in the laboratory. In the traditional Kazakh cotton variety “Turkestan” was introduced a bacterial gene for resistance to the herbicide widely used in agriculture. The conditions for in planta transformation of domestic cotton variety “Maktaaral-4005” using agrobacterial plasmid with the reporter GUS gene and marker nptII gene were also determined. The cotton flowers were pollinated with the transformed pollen and also transgenic plants were obtained from transformed shoot apexes. Thus, the variety was significantly improved, and as a result, productivity of the important agricultural crop was increased and hence the competitiveness of domestic cotton producers.

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Professor K.Zh. Zhambakin and colleagues have recently developed the protocol for the production of transgenic canola (rapeseed plants) expressing the gene transcription factor HvNHX2 by using Agrobacterium-mediated transformation (2015). Under the supervision of Professor Ye.M. Ramankulov – General Director of NCB – the National Centre for Biotechnology is actively involved in the state programs and projects promoting industrial and agricultural biotechnology. More than 2,000 new genetic lines of wheat, new varieties of spring-planted soft wheat resistant to fungal infections, new varieties of potato, cotton, grapes are just part of the developments made in the scientific institutions. The genomes of many plants were sequenced successfully in the 20th – 21st century, thus laying the foundations of genomics, which is the main focus of the present day biotechnology. In the first decade of the 21st century, developments in plant genomics and genetics have led to a much greater understanding of the plant genome, leading to new tools and approaches. Now molecular genetic analysis allows the use of molecular markers for identifying specific plant traits. The use of molecular markers can reduce the labor and resources needed to analyze the sample. Academic K.R. Urazaliev and co-workers at the Kazakh Research Institute of Agriculture and Crop Production, Dr. A.S. Rsaliev at the Research Institute for Biological Safety Problems, as well as Professor A.K. Bisenbaev at Al-Farabi Kazakh National University, Dr. S.I. Abugalieva, Dr. E.B. Turuspekov and Dr. A.M. Kokhmetova at IPBB use molecular genetic analysis, quantitative trait loci (QTL), marker assisted selection (MAS) to improve and increase the efficiency of breeding of local cultivars (2012-2014). The results obtained by our scientists have been actively used for the identification of new genes associated with stress abiotic resistance, search for new resistance genotypes and studies for genotype x environment or genotype x pathogen interaction patterns. Methods for DNA-genotyping and breeding using molecular markers can accelerate the transfer of valuable genes and QTL (that are associated with yield, plant resistance and seed/grain quality) in the selection process and provide for the development of new varieties with a set of useful properties. Control questions: 1. What is biotechnology, and how is it used in agriculture, industry, medicine? 2. What is Plant Biotechnology? 3. What do you understand by the terms “Culture in vitro”, “Totipotency”, “Regeneration”? 4. Define the terms “explant”, “in vitro”, “in vivo”. 5. What do you know about applications of Plant Tissue Culture? 6. What basic historical periods were in the development of Biotechnology? 7. What do you know about biotechnology research in Kazakhstan?

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CHAPTER

2

TECHNIQUES AND METHODS OF CULTIVATION PLANT TISSUE IN VITRO Tissue culture has been applied to diverse research techniques such as viral elimination, clonal propagation, gene conservation, in vitro fertilization, mutation, induction for genetic diversity, genetic transformation, protoplast isolation and somatic hybridization, secondary metabolite production and other related techniques. During in vitro condition, plantlets are grown under fixed and controlled environment in sterile formulated medium which contained macronutrients, micronutrients, vitamins and plant growth regulators. After the plantlets reached optimum growth in the culture containers after a certain growth period, they can be transferred to ex vitro condition to allow continuous growth of the plantlets. 2.1 Aseptic technique. Tissue culture technique Tissue culture is a technique of growing plant cells by culturing explant aseptically on a suitable nutrient medium. This technique relies on the following conditions: –– Explant; –– Aseptic environment; –– Nutrient media. Explant. A small tissue excised from any part of the plant is called explant which is the starting point. It can be initiated from any part of the plant-root, stem, petiole, leaf or flower, choice of the explant varies with species. Meristems are more responsive and give better success as they are actively dividing. The physiological state of the plant also has an influence on its response to initiation of the tissue culture. Therefore, the parent plant must be healthy and free from obvious signs of disease or decay. 250

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Aseptic Technique Aseptic Technique is the procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures. The aseptic technique is absolutely necessary for the successful establishment and maintenance of the plant cell, tissue and organ cultures. The in vitro environment in which the plant material is grown is also ideal for the proliferation of microorganisms. Since the plant cell division is slower than the growth of bacteria, fungi and even minor contaminants can easily overgrow the plant tissue culture. In most cases the microorganisms outgrow the plant tissues, resulting in their death. Contamination can also spread from culture to culture. The environmental control of air is also of concern because room air may be highly contaminated. Large transfer rooms are best sterilized by exposure to ultraviolet (UV) light. Most contamination is introduced with the explant because of inadequate sterilization or just very dirty material. It can be fungal or bacterial. This kind of contamination can be a very difficult problem when the plant explant material is harvested from the field or greenhouse. Bacteria are the most frequent contaminants. They are usually introduced with the explant and may survive surface sterilization of the explant because they are in the interior tissues. Fungi may enter cultures on explants or spores may be airborne. Yeast is also a common contaminant of plant cultures. Yeasts live on the external surfaces of plants and are often present in the air. Initial contamination is obvious within a few days after cultures are initiated. Bacteria produce “ooze” on solid medium and turbidity in liquid cultures. Fungi look “furry” on solid medium and often accumulate in little balls in liquid medium. Latent contamination is particularly dangerous because it can easily be transferred among cultures. This kind of contamination is usually bacterial and is often observed long after cultures are initiated. Apparently, bacteria are present endogenously in the initial plant material and are not obviously pathogenic in situ. Contamination can also occur as a result of poor sterile technique or dirty lab conditions. The purpose of aseptic technique is to minimize the possibility that mic­ro­organisms remain in or enter the cultures. Aseptic environment during culture work is required to avoid contamination from microorganisms. Therefore, all the materials like glassware, instruments, medium, explant, etc., to be used in culture work must be freed of microbes using several techniques. Laminar flow is a mandatory prerequisite for any tissue culture laboratory for contamination free work (Figure 2). Laminar airflow hoods are used in com­ mer­cial and research tissue culture settings.

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Figure 2. Laminar flow with HEPA filter (“High Energy Particle Air”)

A horizontal laminar flow unit is designed to remove particles from the air. Room air is pulled into the unit and pushed through a HEPA (High Energy Particle Air) filter with a uniform velocity of 90 ft/min across the work surface. The air is filtered by a HEPA (high efficiency particulate air) filter so nothing larger than 0.3 micrometer, which includes bacterial and fungal spores, can pass through. This renders the air sterile. The positive pressure of the air flow from the unit also discourages any fungal spores or bacteria from entering. It is possible to use germicidal UV lamps to sterilize items in the transfer hood when no one is working there. Laminar flow hoods are easily sterilized by turning on the hood and wiping down all surfaces with 95% ethyl alcohol 15 min before initiating any operation under the hood.

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Sterilization and Use of Supplies and Equipment Sterilization procedure involves: 1. Sterilizing tools, media, vessels etc. 2. Surface-sterilizing Plant Material. Autoclaving is a method of sterilizing with water vapor under pressure. Cotton plugs, gauze, labware, plastic caps, glassware, filters, pipettes, water, and nutrient media can all be sterilized by autoclaving. Autoclaving is the method most often used for sterilizing heat-resistant items. In order to be sterilized, the item must be held at 121° C, 15 psi, for at least 15 minutes. It is important that items reach this temperature before timing begins. Therefore time in the autoclave will vary, depending on the volume in the individual vessels and the number of vessels in the autoclave. Most autoclaves automatically adjust time when temperature and psi are set, and include time in the cycle for a slow decrease in pressure (Figure 3). Empty vessels, beakers, graduated cylinders, etc., should be closed with a cap or aluminum foil. Tools, glassware, aluminum foil, etc., can also be sterilized by exposure to hot dry air (130°-170°C) for 2-4 hr in a hot-air oven. All items should be sealed before sterilization but not in paper, as it decomposes at 170°C. Autoclaving is not advisable for metal instruments because they may rust and become blunt under these conditions. Metal Instruments such as scalpels and forceps are best sterilized using a glass bead sterilizer. These sterilizers heat to approximately 275-350° C and will destroy bacterial and fungal spores that may be found on the instruments. The instruments simply need to be inserted into the heated glass beads for a period of 10 to 60 sec. The instruments should then be placed on a rack under the hood to cool until needed. Plastic containers that cannot be heated are sterilized commercially by ethylene oxide gas. These items are sold already sterile and cannot be resterilized. Examples of such items are plastic petri dishes, plastic centrifuge tubes, etc. Plant tissue culture media is normally rich in sucrose and other organic nutrients that can support organogenesis in plants but also the growth of many microorganisms (like bacteria and fungi). To overcome and prevent contamination in media preparation, sterilization should be done thoroughly. Sterilization of nutrient media can be done in an autoclave (a large pressure cooker), less often by filtration and seldom by irradiation. The container with the medium should be properly closed and autoclaved at 121ºC, 105 kPa, for 20 minutes. It is also identified that good sterilization relies on time, pressure, temperature and volume of the object to be sterilized. Two methods (autoclaving and membrane filtration under positive pressure) are commonly used to sterilize culture media. Culture media, distilled water, and other heat stable mixtures can be autoclaved in glass containers that are

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sealed with cotton plugs, aluminum foil, or plastic closures. However, solutions that contain heat-labile components must be filter-sterilized. For small volumes of liquids (100 ml or less), the time required for autoclaving is 15-20 min, but for larger quantities (2-4 liter), 30-40 min are required to complete the cycle. The pressure should not exceed 20 psi, as higher pressures may lead to the decomposition of carbohydrates and other components of the medium. Too high temperatures or too long cycles can also result in changes in the properties of the medium.

А

В

Figure 3. Glass bead sterilizer for sterilization tools (A) and autoclave (B)

Organic compounds such as some growth regulators, amino acids, and vitamins may be degraded during autoclaving. These compounds require filter sterilization through a 0.22 µm membrane. Several manufacturers make nitrocellulose membranes that can be sterilized by autoclaving. They are placed between sections of the filter unit and sterilized as one piece. Other filters come pre-sterilized. Larger ones can be set over a sterile flask and a vacuum is applied to pull the compound dissolved in liquid through the membrane and into the sterile flask. Smaller membranes fit on the end of a sterile syringe and liquid are pushed through by depressing the top of the syringe. The size of the filter selected depends on the volume of the solution to be sterilized and the components of the solution. Nutrient media that contain thermo labile components are typically prepared in several steps. A solution of the heat-stable components is sterilized in the usual way by autoclaving and then cooled to 35°-50° C under sterile conditions. Solutions of the thermo labile components are filter-sterilized (Table 1). The sterilized solutions are then combined under aseptic conditions to give the complete medium.

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In spite of possible degradation, however, some compounds that are thought to be heat labile are generally autoclaved if results are found to be reliable and reproducible. These compounds include ABA, IAA, IBA, kinetin, pyridoxine, 2-ip and thiamine. Surface-sterilizing Plant Material Treatment of stock plants with fungicides and/or bacteriocides is sometimes helpful. It is sometimes possible to harvest shoots and force buds from them in clean conditions. The forced shoots may then be free of contaminants when surface-sterilized in a normal manner. Seeds may be sterilized and germinated in vitro to provide clean material. Covering growing shoots for several days or weeks prior to harvesting tissue for culture may supply cleaner material. Explants or material from which material will be cut can be washed in soapy water and then placed under running water for 1 to 2 hours. Ethanol, sodium or calcium hypochloride, Hg2Cl2, AgNO3, diacid or mercuric chloride, hydrogen peroxide are the powerful sterilizing agents but also extremely phytotoxic. Therefore, plant material is typically exposed to them for only seconds or minutes. The tender the tissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, or unopened flower buds can be treated for longer periods of time since the tissue that will be explanted or that will develop is actually within the structure that is being surface-sterilized. Usually, 70% ethanol is used prior to treatment with other compounds. Sodium hypochloride, usually purchased as laundry bleach, is the most frequent choice for surface sterilization (Table 1). It is readily available and can be diluted to proper concentrations. Commercial laundry bleach is 5.25% sodium hypochloride. It is usually diluted to 10% – 20% of the original concentration, resulting in a final concentration of 0.5 – 1.0% sodium hypochloride. Plant material is usually immersed in this solution for 10 – 20 minutes. A balance bet­ ween concentration and time must be determined empirically for each type of explant, because of phytotoxicity. Surfactant (e.g., Tween 20) is frequently ad­ded to the sodium hypochloride. Calcium hypochloride is used for surface ste­ri­li­ zation of plant material. It is obtained as a powder and must be dissolved in water. The concentration that is generally used is 3.25 %. The solution must be filtered prior to use since not all the compound goes into solution. Calcium hypochloride may be less injurious to plant tissues than sodium hypochloride. Mercuric chloride is extremely toxic to both plants and humans and must be disposed of with care. Since mercury is so phytotoxic, it is critical that many rinses be used to remove all traces of the mineral from the plant material. The concentration of hydrogen peroxide used for surface sterilization of plant material is 30%, ten times stronger than that obtained in a pharmacy. Some researchers have found that hydrogen peroxide is useful for surface-sterilizing material while in the field.

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The use of antibiotics and fungicides in vitro is not very effective in eliminating microorganisms and these compounds are often quite phytotoxic. But there are compounds that could be further evaluated. After plant material is sterilized with one of the above compounds, it must be rinsed thoroughly with sterile water. Typically three to four separate rinses are done. Sterilization techniques used in Plant Tissue Culture Technique Steam sterilization/Autoclaving (121°C at 15 psi for 20-40 min) Dry heat (160-180°C for 3h) Flame sterilization Filter sterilization (membrane filter made of cellulose nitrate or cellulose acetate of 0.450.22 µm pore size) Alcohol sterilization Sodium hypochlorite, hydrogen peroxide, mercuric chloride etc

Table 1

Materials sterilized Nutrient media, culture vessels, glasswares and plasticwares Instruments (scalpel, forceps, needles etc.), glassware, pipettes, tips and other plasticwares Instruments (scalpel, forceps, needles etc.), mouth of culture vessel Thermolabile substances like growth factors, amino acids, vitamins and enzymes. Worker’s hands, laminar flow cabinet Surface sterilization Explants

Nutrient media The type and composition of culture media very strongly govern the growth and morphogenesis of plant tissues. The choice of tissue culture medium largely depends upon the species to be cultured. For example, some species are sensitive to high salts or have different requirements for PGRs (Plant Growth Regulators). Some tissues show better response to a solid medium while others prefer a liquid medium. Therefore, the development of culture medium formulations is the result of systematic trial and experimentation considering specific requirements of a particular culture system. White’s medium is one of the earliest plant tissue culture media originally formulated for root culture. Murashige and Skoog (MS) medium is the most suitable and commonly used medium for plant regeneration from tissues and callus (Appendix, Table 1). B5 medium has been defined for the growth of cell suspensions of soybean root cells in the presence of 2,4 D. This is a high salt medium due to the content of potassium and nitrogen salts. B5 medium works well for protoplast culture. It has smaller amounts of nitrate and particularly ammonium salts than MS medium. Nitsch’s medium developed for anther culture contains salt concentration intermediate between MS and White. Chu (N6) medium is defined to improve the

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formation, growth and differentiation of pollen callus in rice. The concentration of ammonium proved to be crucial for the development of callus. Schenk and Hildebrandt medium has been developed for growth of both monocotyle and dicotyle cell suspensions. Components of Tissue Culture Medium: 1. Inorganic Nutrients/elements (macro- and microelements); 2. Carbon Source; 3. Organic Supplements; 4. Plant growth regulators (PGRs); 5. Solidifying agents (only for solid or semi-solid medium) Inorganic Nutrients In vitro growth of plants also requires combination of macro and mic­ ronutrients like in vivo growth. Macronutrients are classified as those elements which are required in concentration greater than 0.5 mM/l. They include nitrogen, potassium, phosphorus, calcium, magnesium and sulphur in the form of salts in the media. Nitrogen is usually supplied in the form of ammonium (NH4+) and nitrate (NO3-) ions. Nitrate is superior to ammonium as the sole N source but the use of NH4+ checks the increase of pH towards alkalinity. Nitrogen is an essential macronutrient in plant life. It is an important component of proteins and nucleic acids. Nitrate is the main source of nitrogen. NO3- is reduced to ammonium NH4+ after uptake. Plants have ability to use the reduced form of nitrogen for their metabolism. Nitrate uptake happens effectively in an acidic pH. But after nitrate uptake, the medium becomes less acid. Ammonium uptake makes the medium more acidic. The pH of the plant culture media is important because in a buffered media, existence of both ions affects efficient nitrogen uptake. The form and amount of nitrogen in the media have significant effects on the cell growth and differentiation. pH controlling in the media is not the only reason for using both ions, excessive ammonium ions are toxic to the plants. Even when the pH of the medium is kept neutral, most plants grow better if they have access to both NH4+ and NO3– because absorption and assimilation of the two nitrogen forms promotes cation– anion balance within the plant. The medium containing high levels of NH4+ also inhibits chlorophyll synthesis. The culture media should contain at least 25 mM/l nitrogen and potassium. Other major elements are adequate in concentration range of 1-3 mM/l. Sulfur is found in two amino acids and is a constituent of several coenzymes and vitamins essential for metabolism. Phosphorus (as phosphate, PO43–) is an integral component of important compounds of plant cells, including the sugar–phosphate intermediates of respiration and photosynthesis, and the phospholipids that make up plant membranes. It is also a component of nucleotides used in plant energy

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metabolism (such as ATP) and in DNA and RNA. Potassium, present within plants as the cation K+, plays an important role in regulation of the osmotic potential of plant cells. It also activates many enzymes involved in respiration and photosynthesis. Calcium ions (Ca2+) are used in the synthesis of new cell walls, particularly, the middle lamellae that separate newly divided cells. Calcium is also used in the mitotic spindle during cell division. It is required for the normal functioning of plant membranes and has been implicated as a second messenger for various plant responses to both environmental and hormonal signals. In plant cells, magnesium ions (Mg2+) have a specific role in the activation of enzymes involved in respiration, photosynthesis, and the synthesis of DNA and RNA. Magnesium is also a part of the ring structure of the chlorophyll molecule. Most species utilizing the C4 and CAM pathways of carbon fixation require sodium ions (Na+). In these plants, sodium appears vital for regenerating phosphoenolpyruvate, the substrate for the first carboxylation in the C4 and CAM pathways. Sodium stimulates growth through enhanced cell expansion, and it can partly substitute for potassium as an osmotically active solute. For example, in B5 medium nitrate was required in a concentration of 2030 mM. An addition of 2 mM ammoniumsulphate led to an increase in the cell growth. NH4+ when added as the sole source of nitrogen, did not support growth. Similar results were obtained when NH4NO3 was substituted for (NH4)2SO4. However, ammonium ions depressed growth when the concentration exceeded 2 mM. Variations in the concentrations of phosphate, calcium and magnesium resulted in relatively minor changes in the growth rate. In Chu (N6) medium the optimum concentration of NH4+ is 7.0 mM (equal to 3.5 mM (NH4)2SO4). Higher concentrations of ammonium drastically inhibited the growth and differentiation of the rice pollen. The concentration of KNO3 and the other medium components did not affect the development of the callus. Micronutrients are those elements which are required at a concentration less than 0.05 mM/l. These include iron, manganese, zinc, boron, copper and molybdenum. These inorganic elements although required in small quantities are essential for plant growth. Iron has an important role as a component of enzymes involved in the transfer of electrons (redox reactions), such as cytochromes. In this role, it is reversibly oxidized from Fe2+ to Fe3+ during electron transfer. Iron is not available at low pH. Therefore, it is provided as Fe-EDTA complex to make it available in the wide range of pH. When iron is supplied as an inorganic salt such as FeSO4 or Fe(NO3)2, iron can precipitate out of solution as iron hydroxide. If phosphate salts are present, insoluble iron phosphate will also form. Precipitation of the iron out of solution makes it physically unavailable to the plant, unless iron salts are added at frequent intervals. Earlier researchers tried to solve this problem by adding iron together with citric acid or tartaric acid. Compounds such

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as these are called chelators because they form soluble complexes with cations such as iron and calcium in which the cation is held by ionic forces, rather than by covalent bonds. Chelated cations are thus physically more available to a plant. More modern nutrient solutions use the chemicals ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA, or pentetic acid) as chelating agents. Boron in plant metabolism plays roles in cell elongation, nucleic acid synthesis, hormone responses, and membrane function. Like iron, copper is associated with enzymes involved in redox reactions being reversibly oxidized from Cu+ to Cu2+. An example of such an enzyme is plastocyanin, which is involved in electron transfer during the light reactions of photosynthesis. Manganese ions (Mn2+) activate several enzymes in plant cells. In particular, decarboxylases and dehydrogenases involved in the tricarboxylic acid (Krebs) cycle are specifically activated by manganese. The best defined function of manganese is in the photosynthetic reaction through which oxygen is produced from water. Many enzymes require zinc ions (Zn2+) for their activity, and zinc may be required for chlorophyll biosynthesis in some plants. Molybdenum ions (Mo4+ through Mo6+) are components of several enzymes, including nitrate reductase and nitrogenase. Nitrate reductase catalyzes the reduction of nitrate to nitrite during its assimilation by the plant cell, nitrogenase converts nitrogen gas to ammonia. Carbon Source Sugar is a very important part of nutrient medium as energy source, since most plant cultures are unable to photosynthesize effectively owing to inadequately developed cellular and tissue development, lack of chlorophyll, limited gas exchange and carbon dioxide in tissue culture vessels, etc. Hence they lack auxotrophic ability and need external carbon for energy. The most preferred carbon or energy source is sucrose at a concentration of 20-60 g/l. While autoclaving the medium, sucrose is hydrolysed to glucose and fructose which are then used up for growth. Fructose, if autoclaved, is toxic. Other mono- or disacharide and sugar alcohols like glucose, sorbitol, raffinose, etc., may be used depending upon plant species. Carbohydrates also provide osmoticum and hence in anther culture higher concentration of sucrose (6-12%) is used. Organic Supplements: –– Vitamins; –– Complex organics; –– Activated charcoal; –– Plant hormones/plant growth regulators. Vitamins are organic substances required for metabolic processes as cofactors or parts of enzymes. Hence for optimum growth, medium should be

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supplemented with vitamins. Thiamine (B1), nicotinic acid (B3), pyridoxine (B6), pantothenic acid (B5) are commonly used vitamins of which thiamine (0.1 mg/l to 5 mg/l) is essentially added to the medium as it is involved in carbohydrate metabolism. The other vitamins are promontory. Linsmaier and Skoog studied the organic requirements of cultures Nicotiana tabaccum. It was found that of all MS vitamins only thiamine and inositol are essential. The optimum concentration for thiamine HCl was 0.4 mg/l (MS 0.1 mg/l). At a lower concentration the growth decreased and the cells became necrotic after 4 weeks. Inositol also had a very stimulatory effect on the cell growth but was not as essential as thiamine. All other Murashige & Skoog vitamins were not required for cell growth and could be omitted without any disadvantageous effect. Folic acid, p-Aminobenzoic acid, l-Glutamic acid and Ascorbic acid also had a positive influence on the cell growth of Nicotiana tabaccum, however the effect was much less than that of thiamine and inositol Addition of amino acids to the media is important for stimulating cell growth in protoplast cultures and also in inducing and maintaining somatic embryogenesis. This reduced organic nitrogen is more readily taken up by plants than the inorganic nitrogen. L-glutamine, L-asparagine, L-cystein, L-glycine are commonly used aminoacids which are added to the culture medium in the form of mixtures as individually they inhibit cell growth. Complex organics are the group of undefined supplements such as casein hydrolysate, coconut milk, yeast extract, orange juice, tomato juice, etc. These compounds are often used when no other combination of known defined components produce the desired growth. Casein hydrolysate has given significant success in the tissue culture and potato extract also has been found useful for anther culture. However, these natural extracts are avoided as their composition is unknown and vary from lot to lot and also vary with age affecting reproducibility of results. Activated charcoal acts both in promotion and inhibition of culture growth depending upon plant species being cultured. It is reported to stimulate growth and differentiation in orchids, carrot, ivy and tomato, whereas it inhibits tobacco, soybean, etc. It absorbs brown-black pigments and oxidized phenolics produced during culture and thus reduce toxicity. It also absorbs other organic compounds like PGRs, vitamins, etc., which may cause inhibition of growth. Another feature of activated charcoal is that it causes darkening of medium and so helps root formation and growth. In vitro plant tissue culture needs the formulation of a complete nutritional medium and to explore plant physiological processes, it needs the addition of effective plant growth regulators. These two aspects can be considered for plant tissue culture as the wings to take off. Usage of common or specific media and

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selection of appropriate plant tissue culture, enable induction of cell division, callus growth, differentiation of shoots, roots and embryos. Plant growth regulators (PGRs) PGRs stimulate cell division and hence regulate the growth and differentiation of shoot and roots on explants and embryos in semisolid or in liquid medium cultures. The four major PGRs used are auxins, cytokinin, gibberellins and abscissic acid and their addition to the culture medium Auxin was the first hormone to be discovered in plants and is one in the expanding list of chemical signaling agents that regulate plant development. The most common naturally occurring form of auxin is indole-3-acetic acid (IAA). One of the most important roles of auxin in higher plants is the regulation of elongation growth in young stems. Low levels of auxin are also required for root elongation, although at higher concentrations auxin acts as a root growth inhibitor. Auxins induce cell division, cell elongation, apical dominance, adventitious root formation, somatic embryogenesis. When used in low concentrations, auxins induce root initiation and in high, callus formation occurs. Commonly used synthetic auxins are 1-naphthaleneacetic acid (NAA), 2.4- dichlorophenoxyacetic acid (2.4-D), indole-3-acetic acid (IAA), indo­ lebutyric acid (IBA), etc. Both IBA and IAA are photosensitive, so the stock solutions must be stored in the dark. 2.4-D is used to induce and regulate somatic embryogenesis. Cytokinins are N6-substituted aminopurines that will initiate cell proliferation in many plant cells when they are cultured on a medium that also contains an auxin. The principal cytokinin of higher plants – zeatin, or trans-6-(4-hydroxy3-methylbut-2-enylamino) purine – is also present in plants as a riboside or ribotide and as glycosides. These forms are generally also active as cytokinins in bioassays through their enzymatic conversion to the free zeatin base by plant tissue. Cytokinins (CK) promote cell division and stimulate initiation and growth of shoots in vitro. Zeatin, 6-benzylaminopurine (BAP), kinetin, 2-iP are frequently used cytokinins. They modify apical dominance by promoting axillary shoot formation. When used in high concentration, CK inhibits root formation and induces adventitious shoot formation. The ratio of auxin and cytokinin in the culture determines morphogenesis. If this ratio is high, it leads to embryogenesis, callus initiation and root initiation whereas if ck/auxin is high, it gives rise to axillary and shoot proliferation (Figure 4). Gibbrellins and abscissic acid are rarely used PGRs. Gibberellins are a family of compounds defined by their structure. They now number over 125, some of which are found only in the fungus Gibberella fujikuroi. Gibberellins induce dramatic internode elongation in certain types of plants, such as

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dwarf and rosette species and grasses. Gibberellin induces transcription of the gene for α-amylase biosynthesis in cereal grain aleurone cells. Gibbrellic acid (GA3) is mostly used for internode elongation and meristem growth. Abscissic acid (ABA) is used only for somatic embryogenesis and for culturing woody species. ABA can control water and ion uptake by roots, promote adventitious shoots, absorb and prevent the phenolic production. As ethylene is a gaseous plant hormone, it is moved by diffusion around the plant rather than translocation. It stimulates the final stage of fruit development and flower fall. The main function of ethylene in plant tissue culture is that it can stimulate respiration, seed germination, peroxidase enzymes and regulate the level of auxins. The low concentration of ethylene induces the proper resistance to the developed plant.

Figure 4. The influence ratio of auxin (NAA or IAA) and cytokinins on morphogenesis in the plant culture

For example, in Schenk and Hildebrandt medium a high level of auxin-type growth regulators, 2,4-D (0.5 mg/l) and 4-CPA (2.0 mg/l), generally favoured monocoty-ledonous cell cultures, while low levels of cytokin, kinetin (0.1 mg/l), were essential for most dicotyledonous cell cultures. Solidifying agents Solidifying agents are used for preparing semi-solid tissue culture media to enable the explant to be placed in right contact with the nutrient media (not submerged but on the surface or slightly embedded) to provide aeration. Agar is high molecular weight polysaccharide obtained from sea algae Gelidium amansii, which can bind water. It is added to the medium in concentration ranging from 0.5% to 1 %( w/v). To make a semi-solid medium, a gelling agent is added to the liquid medium before autoclaving. Gelling

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agents are usually polymers that set on cooling after autoclaving. Agar is preferred over other gelling agents because it is inert, neither does it react with media constituents nor digested by plant enzymes. Agarose, a purified extract of agar is used for protoplast culture. Alternative gelling compounds like gelrite, etc., form clear gels (unlike agar which is translucent) and hence can easier detect contamination which might develop during culture growth. Gelrite is produced by bacterium Pseudomonas elodea . It can be readily prepared in cold solution at room temperature. Unlike agar, the gel strength of gelrite is unaffected over a wide range of pH. However, few plants show hyperhydricity on gelrite due to freely available water. Gelatin is used at a high concentration (10%) with a limited success. This is mainly because gelatin melts at low temperature (25°C) and as a result the gelling property is lost. The pH affects absorption of ions and also solidification of gelling agent. The pH of a solution is a measure of the concentration of hydrogen ions in the solution. The pH of most culture media is adjusted to 5.7 ± 0.1 before autoclaving. Values of pH lower than 4.5 or higher than 7.0 greatly inhibit growth and development in vitro. The pH of culture media generally drops by 0.3 to 0.5 units after autoclaving and keeps changing through the period of culture due to oxidation and also differential uptake and secretion of substances by growing tissue. The pH can influence the solubility of ions in nutrient media, the ability of agar to gel, and the subsequent growth of cells. Thus accurate determination and control of the pH of tissue culture media are necessary. Generally, pH is determined with a pH meter. Preparation of Media This is a very crucial step for the experiment to be successful. While making the media taking individual constituents, each ingredient is separately weighed and dissolved before putting them together. After making up the volume by water, pH is adjusted and then the medium is autoclaved. Preferably, following four stock solutions are prepared: –– Major salts (20X concentration) –– Minor salts (200X concentration) –– Iron (200X concentration) –– Organic nutrients (200X concentration) Separate stock solution for each growth regulator is prepared. Commonly used plant growth regulators are listed in Table 2. Appropriate quantities are taken from stocks and mixed to constitute a basal medium. Make up the final volume of the medium with distilled water (according to formula (1)):

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Dilutions: required concentration x medium volume = volume of stock required (1) concentration of stock solution Table 2

Stock solutions of growth regulators Compound

Abbreviations

mg/50 ml (1mМ or 10-3 Molar)*

1 CYTOKININS**

2

3

6-Benzyladenine

BA, C12H11N5

11.25

2-iP, C10H13N5 Kinetin, C10H9N5O ZEA, C10H13N5O TDZ, C9H8N4OS

10.15 10.75 10.95 11.00

IAA, C10H9NO2 IBA, C12H13NO2 NAA, C12H10O2 2.4-D, C8H6ClO3

8.76 10.16 9.31 11.05

МСРА, C9H9ClO3

10.03

2.4.5-T

12.78

Dicamba, C8H6C12O3 4-CPA, C8H6ClO3 PIC, C6H3C13N2O2

9.33 12.06

GA3, C19H22O6

17.32

ABA

13.20

N -(2-isopentenyl) adenine 6-Furfurylaminopurine Zeatin Thidiazuron AUXINS*** β-Indole-3-acetic acid Indole-3-butyric acid α-Naphthaleneacetic acid 2,4-Dichlorophenoxyacetic acid 2-������������������������ Metyl������������������� -4-���������������� clorophenoxyacetic acid 2,4,5-Trichlorophenoxyacetic acid 3,6-Dichloro-2-methoxybenzoy acid 4-Chlorophenoxyacetic acid 4-Amino-3,5,6-Trichloropicolen acid (Picloram) OTHER PGRs**** Gibberellic acid 6

Abscisic acid

Note: *1М=1 molar = the molecular weight in g/l; 1 mM = the molecular weight in mg/l; ppm = parts per million = mg/l; **Dissolve cytokinins in a few drops of 1N NaOH; stir, heat gently and make to volume. TDZ is dissolved in 95% ethanol ***Dissolve auxins in 95% ethanol or 1N NaOH; stir, heat gently, gradually add water to the volume. Dissolve picloram in DMSO. **** Dissolve in 95% ethanol or 1N NaOH; stir, heat gently, gradually add water to the volume

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Required quantities of agar, sucrose and organic supplements if needed are added separately. A suitable medium may be devised by trying many combinations of different concentrations of major components like PGRs, salt, sucrose. Also, various combinations of low, medium and high concentration of the following four categories of components can be evaluated i.e. minerals, auxins, cytokinin, organic nutrients to get a suitable medium. The exact conditions required to initiate and sustain plant cells in culture or to regenerate intact plants from cultured cells, are different for each plant species. Each variety of species will have a particular set of cultural requirements. Despite all knowledge that has been obtained about plant tissue culture during the twentieth century, these conditions have to be identified for each variety through experimentation. Culture room. The room for maintaining cultures should be maintained at temperature 25±2°C, controlled by air conditioners and heaters attached to a temperature controller are used. For higher or lower temperature treatments, special incubators with built-in fluorescent light can be used outside the culture room. Cultures are generally grown in diffuse light from cool, white, fluorescent tubes. Lights can be controlled with automatic time clocks. Generally, a 16-hour day and 8-hour nights are used. The culture room requires specially designed shelving to store cultures. Insulation between the shelf lights and the shelf above will ensure a uniform temperature around the cultures. Plant Tissue Culture Techniques have the following characteristics: 1. Environmental condition optimized (nutrition, light, temperature). 2. Ability to give rise to callus, embryos, adventitious roots and shoots. 3. Ability to grow as single cells (protoplasts, microspores, suspension cultures). Plant tissue culture has value in studies such as cell biology, genetics, biochemistry, and many other research areas such as crop improvement, seed production (plant propagation technique), genetic material conservation, etc. Types of in vitro culture (explant based) There are several types of in vitro culture: 1. Culture of intact plants (seed and seedling culture); 2. Embryo culture (immature and mature embryo culture); 3. Organ culture; 4. Protoplast culture; 5. Callus culture; 6. Cell suspension culture (Figure 5) Seed culture is growing seeds aseptically in vitro on artificial media. The plant seed us cultivated in vitro for the following aims:

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–– Increasing efficiency of germination of seeds that are difficult to germinate in vivo; –– Precocious germination by application of plant growth regulators; –– Production of clean seedlings for explants or meristem culture. Embryo culture is growing an embryo aseptically in vitro on artificial nutrient media. It is developed from the need to rescue embryos (embryo rescue) from wide crosses where fertilization occurred, but embryo development did not occur. Isolated embryos (usually immature embryo) cultivate in vitro on nutrient media: for their further development for production of plants from embryos developed by non-sexual methods; overcoming embryo abortion due to incompatibility barriers; overcoming seed dormancy and self-sterility of seeds; shortening of breeding cycle.

Figure 5. Modes of culture in vitro

Organ culture – any plant organ can serve as an explant to initiate cultures, for example, shoot tip culture, root culture, leaf culture, anther/ovary culture. Shoot apical meristem cultivate for production of virus free germplasm and mass production of desirable genotypes, also, for in vitro conservation or cold storage (cryopreservation) of germplasm. Root organ cultivate to obtain secondary methabolites accumulated in root cells. Some plants can produce methabolites by hairy root cultures in liquid-phase bioreactors. Reproductive

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organ culture (ovary or ovule culture and anther/microspore culture) can be used to obtain haploid plants. It is possible to use in vitro fertilization to produce distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth. Also, it may enable to overcome abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers. Generative organs of plants are used for production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines and also, to uncover mutations or recessive phenotypes. Protoplast culture is the living material of a plant cell, including the protoplasm and plasma membrane after removal of the cell wall. Protoplast culture is used as a recipient model for gene transformation in genetic engineering, for obtaining somatic hybrids in cell engineering, etc.

2.2. Methods of cultivation of plant tissue in vitro The plant cells if cultured on a solid surface will grow as friable, pale brown, unorganized mass of cells called callus. In nature, in vivo callus is a tissue that develops in response to injury caused by physical or chemical means but in vitro there are cells, which are differentiated and are often highly unorganized within the tissue. Tissues and cells of plant cultured in a liquid medium aerated by agitation grow as suspension of single cells and cell clumps. For growth, the cells need to divide, whereas, the cells breaking up from explant are mature and nondividing. Therefore, the differentiated tissue undergoes modifications to become meris­tematic, which means having the characteristics of a meristem, especially high mitotic activity. Meristem is a localized group of actively dividing cells, from which permanent tissue system i.e. root, shoot, leaf and flower are derived. This phenomenon of a mature cell reverting back to the meristematic state to form undifferentiated callus tissue is called dedifferentiation. Callus culture The culture of undifferentiated mass of cells on agar media produced from an explant of a seedling or other plant part is called callus culture (Figure 6).

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G

H Note: А-C-morphogenic callus (х10), D-F-non-morphogenic callus (х20),G-H-embyogenic callus Figure 6. Callus cultures with different capacity to morphogenesis

For callus formation, auxin and cytokinins, both are required. Callus can be subcultured indefinitely by transferring the same small piece to fresh agar medium. Subculturing needs to be done every 3-5 weeks in view of cell growth, nutrient depletion and medium drying. The rate of growth of callus grown on solid agar medium is relatively slow. The new cells are formed on the periphery of existing callus mass. Consequently, callus consists of cells which vary considerably in age. Since nutrients are gradually depleted from the agar, a vertical nutrient gradient is formed. Because of low degree of uniformity among cells in callus, slower growth rate and development of nutrient gradients, usefulness of callus in the experimental system is limited. The main use of the callus culture is for purposes of maintaining cell lines and for morphogenesis. Morphogenesis is anatomical and physiological events involved in the growth and development of an organism resulting in the formation of its characteristic organ and structures, or in regeneration. For the initiation of a callus culture, the following factors are important: the origin of explants used for the establishment of the callus culture, the cellular/tissue; differentiation status, external plant growth regulators, culture media and culture conditions. Cellular competence to plant

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hormones is understood as the status in which a cell must possess the ability to perceive a transducer and respond to a signal. The callus may remain in a differentiated condition regardless of the hormones and nutrients to which it is exposed the secondary metabolites. These metabolites have biological activity [2]. Cell suspension culture The culture of tissues and cells cultured in a liquid nutrient medium produce a suspension of single cells and cell clumps, this is called suspension culture. A callus mass friable in texture is transferred to the liquid medium and the vessel is incubated on the shaker (Figure 7) to facilitate aeration and dissociation of cell clumps into smaller pieces. Gradually, over several weeks by subculturing, cells of callus dissociate and a liquid suspension culture is obtained (Figure 8). Cell suspensions are also maintained by subculturing of cells in the early stationary phase to a fresh medium. Their growth is much faster than that of callus cultures and hence they need to be subcultured more frequently (3-14days). Cell suspension cultures when fully established consist of a nearly homogeneous population. This system has an advantage that the nutrients can be continually adjusted and hence it is the only system that can be scaled up for large scale production of cells and even somatic embryos. The suspension cultures are broadly classified as: 1) batch culture; 2) continuous culture; 3) single cell culture.

A

B

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C Figure 7. Shakers for obtaining a suspension culture (A, C), industrial bioreacters (C)

Batch culture The culture medium and the cells produced is retained in the culture flask. These cultures are maintained continuously by subculturing i.e. by transferring a small aliquot of inoculum from the grown culture to a fresh medium at regular intervals. The biomass or the cell number of a batch culture follows a typical sigmoidal curve, where at first the culture passes through the lag phase during which the cell number is constant, followed by a brief exponential or log phase where there is a rapid increase in the cell number because of culture cell division (Figure 9). Finally, the growth decreases after 3-4 generations which is the doubling time (time taken for doubling of the cell number) and culture enters the stationary phase during which the cell number again becomes static. The cells stop dividing due to depletion of nutrients and accumulation of cellular wastes. Batch cultures undergo a constant change in cell density and metabolism and, hence, are not used for studies related to aspects of cell behaviour. But batch cultures are convenient to subcultivation, hence they are used for initiation of cell suspension and scaling up for continuous cultures.

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Figure 8. Cell suspension culture

Continuous culture In this type of culture the steady state of the cell density is maintained by regular replacing of a portion of the used up medium with a fresh medium. Continuous culture is further classified into two types: 1) Closed; 2) Open. In the closed type, the used medium is replaced with a fresh medium, hence, the cells from the used medium are mechanically retrieved and added back to the culture and thus, the cell biomass keeps increasing. In the open type, both cells and used medium are replaced with a fresh medium thus maintaining the culture at a constant and submaximal growth rate. There are further two types of open continuous suspension culture: turbidostat and chemostat. In turbidostat, cells are allowed to grow up to a certain turbidity (decided on the basis of optical density) when a predetermined volume of the culture is replaced by a fresh culture. On the other hand, in chemostat, the fresh culture medium to be added has one nutrient kept at such a concentration that it is depleted rapidly and becomes growth limiting while other nutrients are still in the concentration higher than required. An increase or a decrease in the concentration of the growth limiting factor is correspondingly expressed by an increase or a decrease in the growth rate of cells or density. The density of the suspension culture has been calculated by the formula (2): X=

where Х is the number of cells, М is the average number of (2) cells in the chamber, n is a dilution

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Figure 9. The growth curve for plant cell suspension grown in the closed system. The four different growth phases are labeled: Lag phase, Exponential phase, Linear phase, Stationary phase

Thus, the desired rate of cell growth can be maintained by adjusting the level of concentration of the growth limiting factor with respect to that of the other constituents. Chemostats are useful for the determination of effects of individual nutrients on cell growth and metabolism. Single cell culture Free cells isolated from plant organs or cell suspensions, when grown as a single cell under in vitro conditions thus producing a clone of identical cells, are called a single cell culture. –– Isolation of a single cell from plant organs. Leaf tissue in particular is utilized as it has homogeneous population of cells using either of the following two methods: mechanical and enzimatic. 1. Mechanical. Small pieces of leaves are cut and macerated in mortar and pestle in a grinding buffer. This homogenate is filtered through muslin cloth followed by centrifugation to finally pellet down the cells. 2. Enzymatic. Leaves are cut into moderate pieces after peeling the lower epidermis off. Cut pieces are then incubated with macerozyme or pectinase which degrade the middlelamella and cell wall of parenchymatous tissue. A suitable osmoticum like 0.3M mannitol is added to the culture which provides protection to the cell wall from any damage to cells by enzymatic action. –– Isolation of Single Cells from Cell Suspension. Suspension cultures are prepared from friable calli as described earlier from which isolation is carried out by filtering and harvesting the cells by centrifugation. Culture of single cells. Isolated single cells are unable to divide in normal tissue media, therefore, they are cultured on nurse tissue where well grown callus

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cultures are made to diffuse their exudates through filter paper placed on them. The single cells placed on the filter paper derive their nutrition from these exudates, thus called nurse tissue. This technique of culturing single cells is known as Filter paper-Raft nurse tissue technique. Besides this, there are other techniques for single cell culture like microchamber, microdrop, Bergmann’s plating technique, thin layer liquid medium, etc., out of which Bergmann’s plating technique is widely used. In this technique, free cells are suspended in a liquid medium. Culture medium with agar (1%) is cooled and maintained at 35°C in a water bath. Equal volumes of liquid and agar medium are mixed and rapidly spread in a petri dish. The cells remain embedded in the soft agar medium. These embedded cells in the soft agar are observed under inverted microscope. When cell colonies develop, they are isolated and cultured separately. The other point to be taken care of here is that since light has a detrimental effect on cell proliferation, single cells should be cultured in dark. Synchronisation of suspension cultures. Cell suspension is mostly asynchronous, it contains different cells of different size, shape, DNA, nuclear content and also in different stages of the cell cycle (G1, S, G2, M). This is not desirable in cell metabolism studies. Hence, it is essential to obtain synchrony in suspension cultures and it can be achieved by the following methods: starvation, inhibition, and mitotic arrest. A) Starvation: Cells are starved of a nutrient like a growth regulator which is necessary for cell division resulting in arrest of cell growth during G1 or G2. After some time, when the nutrient is supplied, all arrested cells enter divisions synchronously. B) Inhibition: Using a biochemical inhibitor of DNA synthesis like 5 aminouracil, cells are arrested at G1 so that removal of inhibitor leads to synchronous division of cells. C) Mitotic arrest: Colchicine is widely used to arrest cells at metaphase but only for short duration as longer colchicine treatment may induce abnormal mitosis and chromosome stickiness. Cell viability test The objective of cell suspension culture is to achieve rapid growth rates and uniform cells with all cells being viable. The viability of cells can be determined by the following approaches: phase contrast microscopy, reduction of tetrazolium salts, fluorescent diacetate (FDA). Live cells having a well-defined healthy nucleus and streaming cytoplasm are easily observed under phase contrast microscope. When cell masses are stained with 1-2 % solution of 2,3,5- triphenyl teterazolium chloride (TTC) the living cells reduce TTC to red coloured formazon which can be extracted and measured spectrophotometrically for quantification of viability. This approach is not used for single cells. Enzyme Esterase present in live cells cleaves FDA to produce fluorescein which fluoresces under UV so that live cells appear green under UV (Figure 10). Evan’s Blue staining is the only dye which

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is taken up by dead cells. Therefore, Evan’s blue is usually used to complement FDA. All plant tissue culture techniques can be mainly divided into two categories based on establishing a particular objective in the plant species: I. Quantitative Improvement –– Adventitious shoot proliferation (leaves, roots, bulbs, corm, seedlingexplants etc.) –– Nodal segment culture –– Meristem/Shoot-tip culture –– Somatic embryogenesis –– Callus culture II. Qualitative Improvement –– Anther/ Microspore culture –– Ovary/ Ovule culture –– Endosperm culture –– Cell culture –– Protoplast culture The above techniques are discussed in detail in subsequent chapters.

60 rpm

120 rpm

Note: A)  cellular clump at 60 rpm, showing unstained dead cells in the centre of the cell aggregate and live cells fluorescent green at the periphery; B) the cultures maintained at 120 rpm in the cell suspension, showing aggregates of live and healthy, fluorescent green stained cells; C) the same at 240 rpm, showing dead clumps (dark bodies) and sheared cells. Figure 10. Cells stained with 1% fluorescein diacetate solution  Control questions: 1. Why is tissue culture important? 2. What are some of the plants that we might use for tissue culture? 3. Why is a sterile environment important in tissue culture? 4. What is a sterile environment?

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5. What happens if you open your sterile plant container when it is not inside a sterile environment? 6. What are some possible substitutes of agar as a medium matrix in plant tissue culture? 7. How is batch culture different from continuous or semi-continuous culture of plant cell suspension?

2.3. Biology of cultured plant cells The ability of mature cell to dedifferentiate into callus tissue and the technique of maintaining by subculturing isolated single cell in vitro discussed earlier in this chapter have demonstrated that the somatic cells can differentiate to a whole plant under particular conditions. Plant cells growing in vitro have three fundamental abilities: a) the potential or inherent capacity of a plant cell to develop into a whole plant if suitably stimulated. Plant cells are totipotent, able to regenerate a whole plant. It implies that all the information necessary for growth and reproduction of the organism is contained in the cell; b) capacity of mature cells to return to meristematic condition and development of a new growing point followed by redifferentiation, which is the ability to reorganize into a new organ (dedifferentiation); c) Competency is the endogenous potential of a given cell or tissue to develop in a particular way. Dedifferentition is possible because the non-dividing quiescent cells of the explant, when grown in a suitable culture medium revert to meristematic state. The potential of somatic cell to divide and develop into a multicellular plant is termed as cellular totipotency. To express totipotency, after dedifferentiation (when mature cells revert to meristematic state to produce callus), the cell has to undergo redifferentiation or regeneration which is the ability of the dedifferentiated cell to form a plant or plant organs. The ability of the callus cells to differentiate into a plant organ or a whole plant is regarded as redifferentiation. Plant Morphogenesis/Regeneration Pathways Redifferentiation, morphogenesis and regeneration may occur through either of these processes: –– Organogenesis (relies on the production of organs either directly from an explant or indirectly from callus structure) –– Somatic Embryogenesis (embryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells –– Existing Meristems (microcutting), uses meristematic cells to regenerate a whole plant. Organogenesis is a process involving redifferentiation of meristematic cells present in callus into shoot buds and roots. These organs may arise out of pre-existing meristems or out of differentiated cells (Figure 11). Also, organogenesis is 275

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the ability of non-meristematic plant tissues to form various organs (adventitious organs) de novo. Organogenesis refers to the formation of shoots/roots. Developing from unusual points of origin, such as shoots or roots arising from a leaf or stem tissues other than the axils or apex; and embryos from any cell other than the zygote are called adventitious organs. In the case of indirect organogenesis the morphogenetic processes develops according to this scheme: Explant → Callus (undifferentiated mass of cells) → Meristemoid (a localized group of meristematic cells that arise in the callus and may give rise to roots and/or shoots)→ Primordium. Cells become organogenically competent and fully determined for primordia production after induction by different chemical and physical factors. Organ formation generally follows cessation of unlimited proliferation of callus. Individual cells or group of cells of smaller dimensions may form small nets of cells scattered throughout the callus tissue, the so-called meristemoids. These meristemoids become transformed into cyclic nodules from which shoot bud or root primordia may grow as shoots/roots. Shoot bud formation may decrease with age and subculture duration of the callus tissue but the capacity of rooting may persist for longer period. In some calli, rooting occurs more often than in other forms of organogenesis. During organogenesis, if the roots are first formed, then it is very difficult to induce adventitious shoot bud formation from the same callus tissue. If the shoots are first formed, it may form roots later on or may remain in rootless condition unless and until the shoots are transformed to another medium or hormone less medium or conditions that induce root formation.

Figure 11. Pathways of morphogenesis and plant regeneration in vitro

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In certain cases root and shoot formation may occur simultaneously, but organ connection (vascular connection) between root and shoot primordial is essential for the regeneration of complete plantlet from the culture. Shoot formation followed by rooting is the general feature of organogenesis. The shoot buds are monopolar structures which in turn give rise to leaf primordial and apical meristem. Shoot Apical Meristem is an undifferentiated tissue, located within the shoot tip, generally appearing as a shiny dome-like structure, distal to the youngest leaf primordium and measuring less that 0.1 mm in length when excised. The buds have procambial strands connected with preexisting vascular tissue present in the explant or callus (Figure 12). Adventitious shoot proliferation in plant cell and tissue culture, in response to hormonal manipulation of the culture medium, require de novo differentiation of meristematic region, randomly, all over the tissue other than the pre-existing meristem. It is a series of intracellular events, collectively called induction that occurs before the appearance of morphologically recognizable organs. Propagation via adventitious shoot regeneration may occur directly or indirectly via the intervening callus phase (Figure 13). Indirect regeneration often results in somaclonal variations, making this strategy less desirable for large-scale clonal multiplication. Therefore, regeneration of shoots directly from the explants is regarded as the most reliable method for clonal propagation. Various explants like leaf, cotyledon, embryo and root have been tried with different media combinations by the scientists to obtain adventitious shoot proliferation.

Note: A, B – Development of vascular nodules randomly in the callus, note a small shoot-bud originated from vascular tissue in figure B. C, D – Shoot-buds establish a connection with the preexisting vascular tissue developed from the callus Figure 12. Shoot differentiation from the callus tissue (from website http://nptel.ac)

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Under optimal conditions, meristems formed from the callus are random and scattered. Transferring to the medium supporting organized growth first promotes appearance of localized clusters of cambium-like cells (Figure 12 A). These meristemoids (or nodules or growing centers), which may become vascularized due to the appearance of tracheidal cells in the centre, are the site for organ formation in the callus, as seen in above Figure 12 B-C. Initially, the meristemoids exhibits plasticity and can form shoots and roots. Regeneration in the plant tissue culture will be successful by maintaining various factors involved, including media factors and environmental factors. Stimulation of shoot bud differentiation in plants depends on many factors, which differ for different plant species. The following factors affect organogenesis and shoot-bud differentiation: –– growth media (minerals, growth factors/PGH, carbon source); –– explant source (usually, the younger, less differentiated explant, the better for tissue culture); –– environmental factors (light, temperature, photoperiod, sterility, culture media); –– different species show differences in ability to tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture.

Figure 13. Organogenesis from leaf explants indirectly via callusing A – Shoot differentiation; B – Root differentiation (from website http://nptel.ac)

The media factors include media constituents, macronutrients, micronutrients, vitamins, amino acids, carbon source, complex nutritive mixtures, gelling agents, activated charcoal, plant growth regulators and pH of the medium. Environmental factors on the other hand are the culture conditions under which

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explants are maintained. The environmental factors include the temperature and illumination of the culture room, agitation process and incubation period of the cultures [3]. The form and the amount of nitrogen in the media have significant effects on cell growth and differentiation. It has been known that, for example, the root growth is induced by NO3- and reduced by NH4+. But morphogenesis is being controlled by the total amount of nitrogen in the medium and it needs both NO3- and NH4+. Because of using optimum NH4+: NO3- has a key role in morphogenesis, therefore the balance between NO3- and NH4+ differs for different plants and different kinds of cultures. This situation implies that this ratio should be specifically adjusted for each plant species and for different purposes. Changing the NO3- to NH4+ ratio by small alterations affects differentiation and growth. The classical studies of Skoog and Miller (1957) demonstrated that the relative ratio of CK (cytokinin) and auxin is important in determining the nature of organogenesis in tobacco pith tissue. After the discovery of kinetin, it was observed that the differentiation of cultured callus tissue derived from tobacco pith segments into either roots or shoots depends on the ratio of auxin to cytokinin in the culture medium. Whereas high auxin:cytokinin ratios stimulated the formation of roots, low auxin:cytokinin ratios led to the formation of shoots. At intermediate levels the tissue grew as an undifferentiated callus. One of the primary determinants of the plant form is the degree of apical dominance. Although apical dominance may be determined primarily by auxin, physiological studies indicate that cytokinins play a certain role in initiating the growth of lateral buds. For example, direct applications of cytokinins to the axillary buds of many species stimulate cell division activity and growth of the buds. The phenotypes of cytokininoverproducing mutants are consistent with this result. Wild-type tobacco shows strong apical dominance during vegetative development, and the lateral buds of cytokinin overproducers grow vigorously, developing into shoots that compete with the main shoot. Consequently, cytokinin-overproducing plants tend to be bushy. But there have been studies in other plant species which don’t follow this concept of auxin/CK ratio. In most cereals, callus tissue exhibits organogenesis when it is subcultured from a medium containing 2,4-D to a medium where 2,4-D is replaced by IAA or NAA. Gibberellin GA3, in general, has an inhibitory effect on shoot buds whereas many species show enhanced shoot regeneration due to abscissic acid. Different responses of different plant species to the growth regulators are explained by the fact that the requirement of exogenous GRs depends on their

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endogenous levels, which might differ in different plant species and also in different plant materials. Under the optimal growth regulator combinations, the cells induced to form a specific organ will continue to develop into that organ even if the inductive growth regulators are removed. Hence, induction favours the irreversible commitment of cells to follow a particular developmental pathway. For example, Brassica juncea, undergoes the induction of organogenic differentiation where a cytokinin, BAP induces shoot-bud differentiation at the cut end of the cotyledon petiole. In the absence of BAP (basal medium) only roots are formed at the same site. The cotyledons transferred to basal medium after 11 days of incubation on BAP leads to the development of only shoots and no roots. Similarly, the cotyledons lose the potential to form shoots on BAP medium if they are pre-cultured on BAP free medium for more than 7 days. Other factors affecting organogenesis are size and source of the explant. Regenerability of an explant is influenced by several factors such as, the organ from which it is derived, the physiological state of the explant like age of the explant, young versus. mature, position of the explant on the plant and the explant size. The larger the explant (containing parenchyma, cambium and vascular tissue), the more is the likelihood of shoot bud formation. Orientation of the explant in the medium and the inoculation density may also affect shoot bud differentiation. Generally, the explants inoculated horizontally in the medium produced three times more shoots than those planted vertically. There may be a decline in the number of shoots per culture and the percentage of cultures showing regeneration with increasing age of the seedlings. Also, the genotype of the explant affects shoot regeneration as the explant taken from different plant varieties of the same species show different frequencies of shoot bud differentiation. The genotype and plant growth regulators are well known to affect regeneration frequency. Plant growth regulators play a major role in the regeneration, which mainly depends upon the concentration and type of growth regulators used. For in vitro differentiation the genotype plays an equal, if not more critical, role as the growth regulator. Besides, the other factors that play a critical role in regeneration are physical factors. Light has been shown to have inhibitory effect. The quality of light also influences organogenic differentiation. Even the quality of light has a certain effect as blue light has been shown to induce shoot formation and red light induced root formation in tobacco. Alternating light and dark periods (diffused light,

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15-16 h) proved to be the best. Callus maintained under continuous light remained whitish and may not exhibit organogenesis. Calli of Brassica oleracea grown in dark for 20 days formed shoot-buds 12 days after transfer to light, whereas those shifted to light after 12 days of growth in dark differentiated shoots within 9 days. Light, especially red light, is required for bud formation in the moss Funaria. In the dark, buds fail to develop, but cytokinin added to the medium can substitute for the light requirement. Cytokinin not only stimulates normal bud development, but it also increases the total number of buds. Even very low levels of cytokinin (picomolar, or 10 -12 M) can stimulate the first step in bud formation: swelling at the apical end of the specific protonemal cell. The optimum temperature is 25±2 0 C but it depends on plant species. Growth of callus increased with rise in temperature up to 33°C, but for shoot-bud differentiation 18°C was optimum. Explants grown on liquid or semisolid medium give different degrees of organogenesis. In a few species, like tobacco, the medium with 1% agar showed only flower formation. With lowering the agar concentration the frequency of flower formation dropped and vegetative bud differentiation occurred. In liquid medium, the tissue exhibited callusing and vegetative bud formation. Regeneration of plant from the cultured explant may occur either through differentiation of shoot-buds or somatic embryogenesis (Figure 14). Plant regeneration from isolated cells, protoplasts or unorganized mass of cells (callus) is generally more difficult than that obtained from the intact explants such as, cotyledons, hypocotyl segments and immature embryos. The regeneration obtained through de novo differentiation of shoot buds or somatic embryogenesis directly from explants may also exhibit genetic variability. The shoot-bud and embryo formation can be distinguished by the distinct morphological features.

Figure 14. Direct differentiation of somatic embryos from hypocotyl explants

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Somatic embryogenesis is a process involving redifferentiation of meristematic cells into nonzygotic somatic embryo which are capable of germinating to form complete plants. Also, somatic embryogenesis is the formation of adventitious embryos in vitro. It usually involves a callus intermediate stage which can result in variation among seedlings. F���������������������������� ����������������������������� or non-zygotic embryos various terms are used. For example, adventious embryos are somatic embryos arising directly from other organs or embryos; parthenogenetic embryos (apomixis) are somatic embryos formed by the unfertilized egg; androgenetic embryos are somatic embryos formed in the male gametophyte culture. Somatic embryos are bipolar structures with radicular and plumular ends in contrast to monopolar shoot bud with only plumular end in organogenesis. The gradation or change in the character occurs along the axis from one end to the other and the condition is referred as “polarity”. In plants, like in animals, the axes appear very early in the development and are mostly polar in nature. It is visible as morphological differentiation during the development of shoots and roots or is invisible, physiological effect, which is expressed during reactivity of cells, tissues and organs in determining cell division and cell growth, and to geotropic or phototropic stimuli. The entire plant is bipolar in nature consisting of two ends – plumular end (where the shoots develop) and radicular (where the roots develop). Besides, there are two other terms, “distal” and “proximal”. Distal refers to the part of the plant which is the farthest from the original point of attachment, i.e. the tip of the leaf or shoot or root, while proximal means the nearest to the point of attachment. While developing into a somatic embryo, the mersitematic cell breaks any cytoplasmic or vascular connections with other cells around it and becomes isolated. Therefore, unlike the shoot bud, the somatic embryos are easily separable from explants. There are two routes to somatic embryogenesis: direct embryogenesis, when embryos initiate directly from explant in the absence of callus formation and indirect embryogenesis – callus from explant takes place from which embryos are developed. Regeneration of plant from the cultured explant may occur either through embryogenic callus induction, development of embryogenic callus, maturation and germination process and relies on the scheme of indirect somatic embryogenesis: Explant → Callus Embryogenic → Maturation → Germination. Regeneration in plant tissue culture will be successful by maintaining various factors involved, including medium factors and environmental factors. Plant growth hormons such as auxines (2.4-D, NAA, dicamba) are required for induction from proembryogenic masses and embryogenic callus. However, at the next step, auxin must be removed for embryo development because of con-

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tinued use of auxin inhibits embryogenesis. The following stages are similar to those of zygotic embryogenesis: Globular stage, Heart and Torpedo stages, cotyledonary (Figure 15). The most important stages of embryogenesis in many angiosperms, are these: 1. The globular stage embryo. The first division is asymmetric and occurs at right angles to the long axis. This division creates two cells – an apical and a basal cell, which have very different fates. After the first division, the apical cell undergoes a series of highly ordered divisions, generating an eightcell (octant) globular embryo. Additional precise cell divisions increase the number of cells in the sphere. The basal cell also divides, but all its divisions are horizontal, at right angles to the long axis. The result is a filament of six to nine cells known as the suspensor that attaches the embryo to the vascular system of the plant. Only one of the basal cell derivatives contributes to the embryo. 2. The heart stage embryo. This stage forms through rapid cell divisions in two regions on either side of the future shoot apex. These two regions produce outgrowths that later will give rise to the cotyledons and give the embryo bilateral symmetry. 3. The torpedo stage embryo. This stage forms as a result of cell elongation throughout the embryo axis and further development of the cotyledons.

1

2

3

4

5

Note: 1 – early globular stage, which has developed a distinct protoderm (surface layer); 2 -early heart stage; 3 – late heart stage; 4 – torpedo stage; 5 – mature embryo. (From West, M. A. L., and Harada, J. J. 1993. Embryogenesis in higher plants: An overview. Plant Cell. 5: 1361–1369.) Figure 15. Stage of embryogenesis Arabidodsis thaliana

4. The maturation stage embryo. Toward the end of embryogenesis. Axial polarity is established very early in embryogenesis. Maturation requires complete development with apical meristem, radicle, and cotyledons. In this stage,

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sometimes repetitive embryons (polyembryoids) are obtained. Complete maturation often requires ABA and necessary production of storage protein. Plant hormone such as ABA is often required for normal embryo morphology, fasciation and precocious germination. At the last stage in culture medium add 10% sucrose, 4% mannitol. The final moisture content must be 10-40%. In general, somatic embryogenesis involves three distinct steps which are absent in organogenesis: –– Induction is the initiative phase where cells of callus are induced to divide and differentiate into groups of meristematic cells called embryogenic clumps (ECs). These ECs develop into initial stages of somatic embryo, i.e. globular stage (Figure 16); –– Maturation: In this phase somatic embryos develop into mature embryos by differentiating from globular to heart shaped, torpedo to cotyle­ do­nary stages. The mature embryo here undergoes biochemical changes to acquire hardiness; –– Conversion: Embryos germinate to produce seedlings. The studies of Brazilian scientists demonstrated histological events during somatic embryogenesis of Heliconia stricta [4]. The globular embryogenic structures covered most of the surface of the callus (Figure 16 A). Embryogenic callus of Heliconia stricta can be obtained from floral shoots after incubation in MS medium supplemented with 22.62 μM 2,4-D and 14.27 μM IAA for four to six months. The callus in most treatments with IAA+2,4-D demonstrated cells with reduced sizes, nucleolus not clearly defined, dense cytoplasmic content and intense metabolic activity (Figure 16 B). Cells that demonstrated these characteristics were considered embryogenic cells. Transverse sections showed that these structures consisted of meristematic cells surrounded by a protoderm. Some globular embryogenic structures became larger, while others seemed to fuse and acquire an elongated shape. The inner tissue of the callus mostly consisted of parenchyma cells and vascularized zones (Figure 16 C). After the callus-induction phase, the yellowish friable callus demonstrated cellular acquisition of morphogenetic competence during which proembryogenic sectors were formed. The images obtained by scanning electron microscopy show embryogenic tissue with several pro-embryos (Figure 16 D). A close-up of this embryogenic tissue shows two spherical cells at the time of cytokinesis (Figure 16 E) and demonstrates how the daughter cells of the embryogenic tissue remain as a coherent tissue (Figure16 F). The expression of embryogenic program could subsequently be observed when the proembryogenic clusters developed into the first visible globular embryos.

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Somatic embryogenesisis is influenced by following factors: –– Growth regulators. The presence of auxin (generally 2,4-D) in the medium is essential for the induction phase. 2,4-D induces dedifferentiation of explant cells to form ECs. When auxin is removed or its concentration is reduced, ECs convert to somatic embryos (Figure 17). Once induced, cells don’t need PGRs. Still some doses of CK at maturation and conversion make better plants. Maturation is achieved by culturing somatic embryos on high sucrose medium. Also, ABA is added as it gives hardening due to water loss which is important for embryo maturation. Ethylene inhibits both somatic embryogenesis and organogenesis. Therefore, silver nitrate is added to the medium as inhibitor of ethylene for plant regeneration;

Note: 1A-Somatic embryos on the surface of the callus (arrows) (Bar = 1.25 mm); 1B- Proembryogenic cells with densely stained cytoplasm indicating high metabolic activity (Bar = 1 mm); 1C- Cross-section of the callus showing meristematic nodules (MN) and vascularized zones (VZ) (Bar = 25 mm); 1D-General view of the grouping of pro-embryos in calluses as viewed using scanning electron microscopy (SEM); 1E-Close-up of embryogenic tissue shows two spherical cells at the time of cytokinesis (arrow) under SEM; 1F-Close-up (SEM) of a region with a cylindrical shaped somatic embryo (SE) demonstrating how the daughter cells of the embryogenic tissue remain as a coherent tissue. Figure 16. General aspects of the callus with embryogenic cells, and histological sections of the callus derived from transversal ovary sections of cv. Sexy Pink after 90 days of treatment with 14.27 μM IAA plus 22.62 μM 2,4-D in vitro culture (Ulisses C. et al. [4])

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–– Nitrogen source. NH4+ form of nitrogen is essential for induction of somatic embryogenesis while NO3- form is required during the maturation phase; –– Other factors. Like shoot bud differentiation, explant genotype also has influence on somatic embryogenesis. In cereals, the use of maltose as a carbohydrate source promotes both somatic embryo induction and maturation. Histological analyses of callus material Cleome rosea revealed heavily stained cells located on the surface of the callus (Figure 17). Callus induced by 2,4-D presented a nodular appearance (Figure 17 a), which is considered a typical physical feature of embryogenic calli. Embryos at the globular stage were observed 7-9 days after transfer to the media with a reduced 2,4-D concentration and some of them presented a proliferation of cells near the callus tissue, suggesting the presence of a suspensor-like structure (Figure 17 b). The globular embryos presented a protoderm formed by cells dividing in the anticlinal plane (Figure 17 c).

Note: a) Embryogenic callus produced on stem explant cultivated on medium with 9.0 μM 2,4-D (Bar = 1.6 cm); b) Longitudinal section of a globular embryo exhibiting a suspensor-like structure (arrow). (Bar = 50 μm); c) A detail of a globular embryo showing the protoderm (pt) formed by cells in anticlinal division (arrow), the ground meristem (gm) and the procambium (pc). (Bar = 30 μm); d) Longitudinal section of an embryo with an elliptical shape showing the procambium (arrow). (Bar = 60 μm); e) A mature embryo detached from the callus. (Bar = 0.06 cm); f) Longitudinal section of a mature embryo showing procambial strands connecting the root and the shoot apex (arrow). (Bar = 640 μm); g) Plants regenerated from somatic embryos after 3 months of planting. (Bar = 2.6 cm). Figure 17. Aspects of the somatic embryogenesis in Cleome rosea (Simões, C. et al. [5])

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The protoderm is considered one of the unique features of somatic embryo development and could regulate the embryogenic process by applying physical and cell division limitations. Embryos with an elliptical shape, which could be recognized as an intermediary stage between the heart and torpedo stages, were visualized (Figure 17 d). Mature embryos loosely attached to the callus surface were observed 15 days after transfer to the medium with reduced 2,4-D concentration (Figure 17 e). These embryos showed a bipolar structure with the presence of procambial strands connecting the root and shoot apices and with no vascular connection to the callus tissue (Figure 17 f) [5]. Somatic embryogenesis and organogenesis, both of these technologies can be used as methods of micropropagation. It is not always desirable because they may not always result in populations of identical plants. The most beneficial use of somatic embryogenesis and organogenesis is in the production of the whole plants from a single cell (or a few cells). Microcutting propagation is a specialized form of organogenesis, it requires breaking apical dominance. Microcutting propagation involves the production of shoots from pre-existing meristems only. Control questions: 1. What fundamental abilities do plant cells growing in vitro have? 2. What plant morphogenesis pathways do you know? 3. Give a brief note on induction of organogenic differentiation 4. What is organogenesis? 5. What factors affect organogenesis and shoot-bud differentiation? 6. What is somatic embryogenesis? 7. What are the differences between direct embryogenesis and indirect embryogenesis? 8. What factors influence somatic embryogenesis?

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CHAPTER

3

INDUSTRIAL AND AGRICULTURAL APPLICATIONS OF PLANT IN VITRO CULTURE 3.1. Production of secondary metabolites in plant cell culture Plant cells produce a large, diverse array of organic compounds that appear to have no direct function in growth and development. These substances are known as secondary metabolites. Plants being an important source of a variety of chemicals are used in pharmacy, medicine and industry. The past two decades plant cell biotechnology has evolved as a promising new area within biotechnology, focusing on the production of plant secondary metabolites. Plant cell cultures are an attractive alternative source to whole plants for the production of high-value secondary metabolites. Plant secondary metabolites can be divided into three chemically distinct groups: terpenes, phenolics, and nitrogen-containing compounds. The terpenes, or terpenoids, constitute the largest class of secondary products. The diverse substances of this class are generally insoluble in water. They are biosynthesized from acetyl-CoA or glycolytic intermediates. Terpenes are composed of five-carbon isoprene units. Plant cells produce a large variety of secondary products that contain a phenol group – a hydroxyl functional group on an aromatic ring. These substances are classified as phenolic compounds. Phenolics, which are synthesized primarily from the products of the shikimic acid pathway, have several important roles in plants. Plant phenolics are a chemically heterogeneous group of nearly 10,000 individual compounds: some are soluble only in organic solvents, some are water-soluble carboxylic acids and glycosides, and others are large, insoluble polymers. A large variety of plant secondary metabolites have nitrogen in their structure. Included in this category are such well-known anti herbivore defenses as alkaloids and cyanogenic glycosides, glucosinolates, nonprotein amino acids, and proteinase inhibitors, which are of considerable interest because of their toxicity to humans and their medicinal properties. Most nitrogenous secondary metabolites are biosynthesized from common amino acids. Alkaloids are usually synthesized from one of a few common amino acids, in particular, lysine, tyrosine, and tryptophan. However, the carbon skeleton of some alkaloids contains a component derived from the terpene pathway. For most compounds of interest, e.g. morphine, 288

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quinine, vinblastine, atropine, scopolamine and digoxin, one has so far not been able to come to a commercially feasible process. In contrast to the production of antibiotics by microorganisms, the plant is an already existing, although not always a reliable, source of these compounds. The study of plant secondary metabolites has many practical applications. By virtue of their biological activities against herbivorous animals and microbes, many of these substances are employed commercially as insecticides, fungicides, and pharmaceuticals, while others find uses as fragrances, flavorings, medicinal drugs, and industrial materials. Breeding of increased levels of secondary metabolites into crop plants has made it possible to reduce the need for certain costly and potentially harmful pesticides. The present research particularly focuses on the possibilities of applying metabolic engineering to improve yields to commercially interesting levels. Cell cultures are effectively utilized for production of these chemicals on a commercial scale for enhanced yield and better production control. Plant cell cultures provide an excellent system for studying biosynthesis of secondary metabolites. Plants are the source of a large variety of biochemicals, which are produced as both primary and secondary metabolites. Primary metabolites include nucleic acids, proteins, carbohydrates and fats which along with their intermediates function for survival of cell and organism. Compounds like alkaloids, non-protein aminoacids, terpenoids and phenolics are grouped under secondary metabolites which don’t participate in vital metabolic function of cell. Sugars, amino acids and nucleotides synthesized by plants are used to produce essential polymers. Primary metabolites essentially provide the basis for growth and reproduction, while secondary metabolites for adaptation and interaction with the environment. The formation of bioactive compounds in plants in response to the stress caused by physical factors (drought, flood, salinity, alkalinity, radiation, etc.) or wounding caused by insects, pests and microbes is a natural process. The primary role of accumulated products is the protection of plants from the natural and induced stress, and they play a vital role in ailment healing. Although the presence of secondary metabolites in small quantity is sufficient to protect the plant body, because of their pharmaceutical importance for treating human and animal diseases, they warrant a huge availability; hence, the ruthless collection of plant parts for the extraction leads to the depletion of plant population, even in some cases where the secondary metabolites are obtained from root or fruit parts endangering their population more severely. Thus, to potentially protect the valuable germplasm, the plant cell culture could be used as an alternative measure. Primary metabolites are typically found in all species within broad phylogenetic groupings, and are produced using the same metabolic pathway (Figure 18). As secondary metabolites provide industrially important natural products like colour, insecticides, antimicrobials, fragrances, therapeutics, etc., they are of great economic importance (Table 3). Therefore, plant tissue culture is being potentially used as an alternative to plants for production of secondary metabolites. The first large scale production was successfully done for shikonin produced from Lithospermum erythrorhizon. It is used as antiseptic and as dye for

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cosmetics. Artabotrys hexapetalus, a climbing herb, secretes oil used in perfume industry. Stem barks and roots of Tinospora cordifolia, a woody climber, are used in dysentery and diarrhoea. Toddalia asiatica, an evergreen climber, produced nitidine secondary metabolite which contains anti-HIV and antimalarial and anticancerous properties. Since then many valuable secondary metabolites like taxol, berberine, etc. have been obtained using tissue culture [6, 7]. Tissue culture technology has emerged as a supplementary branch to fulfill the demands for this valuable secondary metabolite. Various in vitro methods for enhancement of secondary metabolites are available such as hairy root culture, treatment of elicitors, and use of precursors and introduction of any foreign gene via bacterial transformation. Once interesting bioactive compounds were identified from plant extracts, the first part of the work consisted in collecting the largest genetic pool of plant individuals that produce the corresponding bioactive substances. However, a major characteristic of secondary compounds is that their synthesis is highly inducible, therefore, it is not certain, if a given extract is a good indicator of the plant potential for producing the compounds. For a long time, it was believed that undifferentiated cells, such as callus or cell suspension cultures were not able to produce secondary compounds, unlike differentiated cells or specialized organs.

Figure 18. A simplified view of the major pathways of secondary-metabolite biosynthesis and their interrelationships with primary metabolism (such as glycolysis, shikimate and production of aliphatic amino acids)

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It is well known that callus can undergo somaclonal variations, usually during several subculture cycles. This is a critical period where, due to in vitro variations, production of secondary metabolite often varies from one subculture cycle to another. When genetic stability is reached, it is necessary to screen the different cell (callus) lines according to their aptitudes to provide an efficient secondary metabolite production. Hence, each callus must be assessed separately for its growth rate as well as intracellular and extracellular metabolite concentrations. This allows an evaluation of the productivity of each cell line so that only the best ones will be taken for further studies, for example, for production of the desired compound in suspension cultures. Table 3

Classification of secondary metabolites Terpenes

Phenols

Type Monoterpenes Sesqui-terpenes Diterpe-nes

Example Farnessol

Type Lignan

Example Lignan

Limonene Taxol

Tannins Flavo-noids

Gallotan-nin Anthocya-nin

Triterpe-nes

Digitogenin Carotene Spina-sterol

Couma-rins

Umbellife- rone

Tetra-terpenoids Sterols

Nitrogen and/or sulphur containing compounds Type Example Alkaloids Nicotine Atropine Glucosinolates

Sinigrin

Cell suspension cultures represent a good biological material for studying biosynthetic pathways. They allow the recovery of a large amount of cells from which enzymes can be easily separated. Compared to cell growth kinetics, which is usually an exponential curve, most secondary metabolites are often produced during the stationary phase. This lack of production of compounds during the early stages can be explained by carbon allocation mainly distributed for primary metabolism when the growth is very active. On the other hand, when the growth stops, carbon is no longer required in large quantities for primary metabolism and secondary compounds are more actively synthesized. However, some of the secondary plant products are known to be growth-associated with undifferentiated cells, such as betalains and carotenoids. Plant organs are alternative to cell cultures for the production of plant secondary metabolites. Two types of organs are generally considered for this objective: hairy roots and shoot cultures. Shoots exhibit some properties comparable to hairy roots, genetic stability and good capacities for secondary metabolite production. They also provide the possibility of gaining a link between growth and

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production of secondary compounds. All plants produce secondary metabolites, which are specific to an individual species, genus and are produced during specific environmental conditions, which makes their extraction and purification difficult. As a result, commercially available secondary metabolites, for example, pharmaceuticals, flavours, fragrances and pesticides, etc. are generally considered high value products as compared to primary metabolites and they are considered to be fine chemicals. At the International Research and Production Holding “Phytochemistry” (in the city Karaganda of Central Kazakhstan) complex development of the original domestic phytodrugs is carried out: search for biologically active substances, cultivation of medicinal raw material, processing and production of pilot lots of medicinal forms of new phytodrugs [8]. One of the basic scientific directions of the Holding ”Phytochemistry” is the study of natural raw materials for production of new materials and drugs as well as carrying out of research works on the development of high technologies for production of medical drugs corresponding requirements of international standards GMP and their introduction into industry. The Head of the Holding “Phytochemistry” is Academician of the National Academy of Science of the Republic of Kazakhstan, Doctor, Professor S.A. Ade­ kenov. The main scientific directions of the Holding are: – search for new biologically active compounds from natural sources and development of methods for their extraction, determination of molecular structure and investigation of the properties of the obtained compounds; – development of methods for the synthesis of organic compounds and production of new derivatives based on the molecules of natural compounds; – creation of the effective system for biological and pharmacological screening of natural compounds and their derivatives; preclinical and clinical research of original phytopreparations; – development of methods for deriving substances and standard samples of medicinal drugs, phytopreparation biotechnology and introduction of quality control in pharmaceutical production. An analysis of species diversity and perspectives of using Kazakhstan medicinal flora in official and folk medicine allowed us to establish that to date at least 1406 species of medicinal plants of 612 orders pertaining to 134 families presenting one fourth of the whole species of higher plants of Kazakhstan flora grow within Kazakhstani areas. Among them are species that traditionally were used in Kazakh folk medicine such as aconite, ferula, sagebrush, knotweed, pigweed, fritillary, eminium, angelica, roseroot, maral root, etc. In the collection in vitro holding Phytochemistry has 16 endemic plant species of Kazakhstan. The methods of introduction to the culture of plants represented in the collection in vitro are: Berberis karkaralensis Korn. et Potap., Centaurea bipinnatifida (Trautv.) Tzvel., Scorzonera tau-saghyz Lipsch. et Bosse, Atraphaxis decipiens

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Jaub. et Spach., Tanacetum ulutavicum Tzvel., Tanacetum scopulorum (Krasch.) Tzvel., Artemisia leucodes Schrenk., Artemisia hippolytii Butk., Artemisia glabella Kar. et Kir., Artemisia kasakorum (Krasch.) Pavl., Pyrethrum kelleri (Kryl. et Plotn.) Krasch., Artemisia filatovae Kupr., Rhaponticum karatavicum Regel et Schmalh., Serratula kirgisorum Iljin, Galatella bectauatense Kupr. et Korolyuk, Hieracium bectauatensis Kupr. in order to preserve and restore endemic, rare and endangered plant species [8, 9]. Commercial demand for secondary metabolites is increasing every day, necessitating the exploitation of many endangered medicinal plants. In vitro propagation has the potential to quickly provide very high multiplication rate leading to conservation of endangered medicinal plants. It also enhances the production of desirable secondary metabolites from callus and suspension cultures throughout the years without any hindrance of external factors. The most important advantage of in vitro grown plants is that it is independent of geographical variations, seasonal variations and environmental factors. It offers a defined production system, continuous supply of products with uniform quality and yield. Novel compounds, which are not generally found in the parent plants, can be produced in the in vitro grown plants through plant tissue culture. In addition, stereo- and region- specific biotransformation of the plant cells can be performed for the production of bioactive compounds from economical precursors. It is also independent of any political interference. Efficient downstream recovery of products and rate of production are its additional advantages. Among the plant secondary metabolites that exhibit evident toxicity to humans and animals, alkaloids, terpenes, steroids, and phenolic compounds have led to drugs or templates for drug design. Many of these molecules affect neural transmission or cell division processes, which have given rise to drugs for treating central nervous disorders and cancer. In addition to secondary metabolites, toxic plant proteins such as lectins have emerged as tools for disease diagnosis and as candidates to develop new anticancer drugs. The International Research and Production Holding Phytochemistry makes a certain contribution to the preparation of promising new drugs from medicinal plants. Today, medicinal plants, and the extracts from them, are an indispensable raw material for creation of some herbal remedies. The most important and promising species of introduced medicinal plants include: Artemisia glabella Kar.et Kir. Extract, which is used to create phytopreparation “Arglabin”, which can stimulate the immune system (Figure 19), Salsola collina Pall., the extract of which is used to manufacture phytopreparation “Salsocollin” possessing anti-inflammatory, anti-oxidant effect (Figure 20), Serratula coronata L. based on the extract used to obtain phytodrug “Ecdyphyt” possessing anabolic and tonic properties (Figure 21) and Ajania fruticulosa Ledeb, used to obtain phytopreparation “Aefrol” having wound-healing properties. Arglabin, a new herbal anti-tumor medicine, is developed and used in Kazakhstan. The method of production of an antitumor preparation Arglabin was

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patented in 11 countries of the world: the USA, the Great Britain, Germany, Switzerland, France, etc. Most of the medicinal plants growing on the site are for herbal tea, which has both preventive and curative properties [9]. Artemisia is a large, diverse genus belonging to the family Asteraceae. The constituents of Artemisia species can be used as medicines and cosmetics. The plants of Artemisia genus have been studied most intensively by chemical means. Most studies have been devoted to flavonoids, sesquiterpene lactones, and essential oil components from different geographic regions. These compounds are responsible for some sorts of biological activity.

A

B

C

D

Figure 19. Phytodrug “Arglabin” (B). Chemical structure of sesquiterpene lactone Arglabin (C, D), extracted from endemic plant Artemisia glabella Kar.et Kir. (A) /from website www.phyto.kz/

Lipids extracted from the aerial part of Artemisia species contain polyprenols composed of 12 up to 22 isoprene units with PP-15-18 as the dominating prenologues. Polyprenols can be identified by the application of HPLC. Comparing the chromatographic profiles of samples of lipophilic extracts, 20 paraffin hydrocarbons with normal and branched structure and chain length С16-С36 were identified, aliphatic and triterpenic alcohols were found. Some species contain polymethoxylated flavonoids [10].

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1. Artemisia annua L. is a plant which has been successfully introduced into the culture in many countries. This is a major source of artemisinin, which is recommended by WHO for the treatment of malaria. Rupestonic acid, first isolated from the dichloromethane extract of leaves of Decachaeta scabrella and named pechueloic acid, was isolated from dry Artemisia rupestris L., a traditional Chinese medicine used in China. It is a multifunctional sesquiterpenoid compound. In order to find high activity and low toxicity anti-influenza virus compounds, more than 200 rupestonic acid derivatives have been synthesized and in vitro activities against influenza viruses A and B were assayed.

A

C

B

D

E

Figure 20. Endemic plants Saussurea salsa Pall (A) and Salsola collina Pall (C) – a source of sesquiterpene lactone Cinaropicrin (B) and rutin (E); phytodrug “Salsocollin” /from website www.phyto.kz/

Sesquiterpene lactones (SLs) are a varied group of secondary plant metabolites, occurring widely within the families Asteraceae and Apiaceae. SLs, including recognized agents such as artemisin, parthenolide, costunolide and helenalin, are known as inhibitors of NF-kappaB activation. This effect explains their suppressive effects on immune-stimulated cytokine and NO production [11]. Essential oils of Artemisia contain a large amount of chamazulene. Oils may find application as a source of new medicines and dietary supplements and have

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antiseptic, anti-inflammatory and antimicrobial properties. Components of essential oils are very interesting to the synthetic and theoretical chemistry because of their high reactivity and availability as reagents in the preparation of practically useful compounds. All components of essential oils have elopathic activity. Chemical compounds of essential oils of Artemisia halophila Krasch (white wormwood) and Artemisia arenaria Dc. (desert wormwood) were observed by researchers from the International Research and Production Holding Phytochemistry. Essential oils were extracted by hydrodistillation of leaves and flowers of the plant. The major compounds of essential oils of these plants are 2,6-octadien1-ol, 3,7- dimethylacetate, trans-geraniol, chrysanthenone, camphor, chrysanthenyl acetate and sabinyl acetate [12].

Figure 21. Chemical structure of polyhidroxisteroid – the basic compound of phytodrug “Ecdyphyt”, extracted from endemic plant Serratula coronata L. /from website www.phyto.kz/

Artemisia cina Berg. is a taxon of Artemisia herba, which is endemic in Southern Kazakhstan region in the valleys of the Syr Darya and Arys. The herb, a mixture of leaves, inflorescence axes and unopened baskets, are used as raw materials. Leaves of the young stems and especially the flower baskets contain Artemisia cina santonine (sesquiterpene compound), essential oil (1.53%), which consists of cineole (70-80%), pinene, terpinene and 1-terpineol, terpinenol, 1-camphor, carvacrol, a sesquiterpene alcohol – sesqi-artemizol, betaine, choline, bitter and colorants, malic acid and acetic acid. The formation and accumulation of santonine is characteristic of many species of Artemisia. It should be noted that the tight buds of Artemisia cina can contain up to 7% santonine, green leaves and tops of the stems – to 5.41%, none found in the santonine seeds and roots [9]. Research of Kazakh scientists at the Institute of Plant Biology and Biotechnology and International Research and Production Holding Phytochemistry showed significant differences in the frequency of callus formation, total weight

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and callus growth during cultivation cycle between the species. Artemisia kasakorum showed the maximum intensity of callusgenesis with the growth index RI ranged from 2.37 to 4.98 depending on the medium hormonal composition. The callus cultures had a similar S-shaped growth curve with the latent (first 10 days), a logariphmic stage (from 11 to 20-25 days), slow growth and stationary growth (from 26 to 40 days) phases. The obtained callus cultures differed by callus biomass accumulation [13]. For isolation of several phenolic acids, phenolic acid glycosides and esthers, triterpenic acids, a cardenolide glycoside and a cardenolide aglycone the following scheme was used: plant material was extracted with 70% MeOH, once at room temperature and two times at 45°C. The collected alcohol-aqueous extract can be concentrated and successively partitioned between CHCl3-MeOH (1:2; 2:3), ethylacetate and n-BuOH. The obtained fractions were subjected to the column chromatography on Sephadex LH-20, elute with methanol. The sub-fractions were further separated by RP-HPLC (Waters XTerra Prep MSC18 column, 300x7.8 mm i.d.) at flow rate 2.0 ml/min using different mixtures of MeOH:H2O in isocratic conditions. Dracocephalum nutans (Labiatae) has been used for treatments of bronchial asthma, hepatitis and urethritis in Kazakh traditional medicine. Biologically active constituents including essential oils, diterpeniods, triterpenoids, alkaloids, flavanoids and phenylpropanoids from other species of Dracocephalum L. (D. kotschyi, D.rupestre, D. polychaetum and D. surmandinum) have been reported with some pharmacological actions such as immunoinhibitory, protection against doxorubicin-induced toxicity in cardiomyocytes, antimicrobial, antivirus, cytotoxicity and antioxidant activities. In the search of Kazakh and Chinese scientists at Al-Farabi National University and Xinjiang Technical Institute of Physics and Chemistry for structurally and biologically interesting natural products of the aerial parts of D.nutans, the methanol extract was partitioned with n-hexane, CHCl3, and n-BuOH. The CHCI3 layer was separated using a silica gel column, a sephadex LH-20 column, and preparative HPLC to obtain 7 known compounds in which four flavonoids together with Oleanolic acid, Ursolic acid, and Stigmasterol were isolated for the first time from the plants which grow in Kazakhstan [14]. Lentil (Lens culinaris Medikus) is an annual plant of the family Fabaceae, which is cultivated mainly for food. Lentils are common in Europe, Asia, Africa and in some areas of the United States. This plant is widely used in traditional medicine: to treat metabolic disorders, colitis, ulcers of the stomach and duodenal ulcers, diseases of the genitourinary and nervous systems, diabetes. Lentils contain various biologically active substances, but in phytochemical aspect phenolic compounds are of greatest interest that causes pharmacological activity of the

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raw material. For detection of phenolic compounds, alcohol-water extracts and ethyl acetate butane fraction were used. Qualitative reactions were performed: reaction with iron (III) chloride – red-brown coloring; cyanidin sample by Bryant – pink coloring; reaction with alkali – a bright yellow color. To determine aglycones belonging to the flavonoid glycosides authentic samples in solvent systems were used: chloroform-acetic acid-water (13:6:1) and benzene-ethyl acetate-acetic acid-water (50:50:1:1) (after pre-acid hydrolysis). Total flavonoid content can be measured spectrophotometrically in terms of reaction using rutin flavonoids complexation with aluminum chloride. Many plant extracts, their fractions and isolated compounds present in them possess various pest control properties against many pests such as antifeedants, larvicides, ovicidal and oviposition deterrent activities and insect growth regulators. Different flavonoids in the plants have diverse functions that include providing much of the colour to flowers and fruits, pollinator attraction mechanism, symbiotic relationship with N2-fixing rhizobia, protection from UV, pathogens and insects, allelopathy and inhibition of auxin transport. The presence of various groups of flavonoids such as flavones, flavonols, flavanones, anthocyanins and chalcones in the plants plays a significant role in insect-plant interactions. Plantbased pesticides are not accumulated in the food chain as the synthetic chemicals which are the major concern for the environmental pollution. Further, plant based secondary chemicals are non-toxic, targeted for a wide range of pests and could be potent alternatives to synthetic pesticides. Juice of leaves, containing flavonoids is alterative and given in neglected syphilitic complaints. Bitter tonic from the root is used as antidote, analgesic, anti-asthmatic tonic and for treating inflammatory and rheumatic diseases [15]. Leaves and fruit of sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae) are sources of valuable biologically active substances. They are used in folk medicine for diseases of skin and gastrointestinal tract. More than 200 constituents such as carotenoids, flavonoids, carbohydrates, aliphatic and triterpenoic acids, vitamins, glycosides, sterols, aldehydes, alcohols, hydrocarbons, polyprenols, dolichols were identified in these parts of the plant. Lipids from sea buckthorn leaves and fruit have been investigated previously and recommended as antiburn and woundhealing agents. For exhaustive extraction of biologically active hydrophilic constituents it is recommended to use some polar solvents as extragents (for example hexane, it is an analog of commercial solvent and guarantees exhaustive extraction of lipophilic constituents). The composition of the extract can be investigated by HPLC (High Performance Liquid Chromatography). The high content of active triterpenic acids (up to 5%) and alcohols (5.8%), polyprenols and dolichols (up to 4.2%) makes tree green of sea buckthorn the prospective raw material for producing medicines, food additives and cosmetics [10].

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Alkaloids are one of the largest groups of plant secondary metabolites, being present in several economically relevant plant families. 537 plant species from 228 genera and 61 families growing in Kazakhstan contain alkaloids. Among them, 213 species of plants have not previously been studied in the alkaloid content, 32 are endemic [16]. Alkaloids encompass neuroactive molecules, such as caffeine and nicotine, as well as life-saving medicines including emetine used to fight oral intoxication and antitumoral vincristine and vinblastine. Alkaloids can act as defense compounds in plants, being efficient against pathogens and predators due to their toxicity. Toxic effects, in general, depend on specific dosage, exposure time, and individual characteristics, such as sensitivity, site of action, and developmental stage. At times, toxicity effects can be both harmful and beneficial depending on the ecological or pharmacological context. Different strategies are used to study alkaloid metabolism and accumulation. An efficient approach is to monitor gene expression, enzyme activities, and concentration of precursors and the alkaloid itself during controlled attacks of pathogens and herbivores upon simulation of their presence through physical or chemical stimulation. Detailed understanding of alkaloid biosynthesis and mechanisms of action is essential to improve production of alkaloids of interest, to discover new bioactive molecules, and to sustainably exploit them against targets of interest, such as herbivores, pathogens, cancer cells, or unwanted physiological conditions. Primary biological tests of natural alkaloids and their derivatives on the physiological activity showed that many of them exhibit antiviral, antibacterial, anti-inflammatory, cytotoxic and phagocytosis stum action, allowing us to set them on the basis of new drugs that would increase the range of domestic pharmaceutical products. Thus, the analysis of the data on chemical studies of alkaloids from plants of Kazakhstan, including selection, determination of the molecular structure of alkaloids, chemical modification, determination of the biological activity of the compounds obtained, indicates the prospects of studying the flora of Kazakhstan as a source of new and effective drugs [17]. Table 4 presents the major alkaloid types and their amino acid precursors. Nearly all alkaloids are toxic to humans when taken in sufficient amount. For example, strychnine, atropine, and coniine (from poison hemlock) are classic alkaloid poisoning agents. At lower doses, many are useful pharmacologically. Morphine, codeine, and scopolamine are just a few of the plant alkaloids currently used in medicine. Other alkaloids, including cocaine, nicotine, and caffeine, enjoy widespread nonmedical use as stimulants or sedatives. Many alkaloids interfere with components of the nervous system, especially chemical transmitters, others affect membrane transport, protein synthesis, or miscellaneous enzyme activities.

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Part II. Animal biotechnology Major types of alkoloids, their amino acid precursors and well-known examples of each type

Alkaloid class Pyrrolidine Tropane

Biosynthetic precursor Ornithine (aspartate) Ornithine

Examples

Human uses

Nicotine Atropine

Stimulant, depressant, tranquilizer Prevention of intestinal spasms, antidote to other poisons, dilation of pupils for examination Stimulant of the central nervous system, local anesthetic Poison (paralyzes motor neurons) Restoration of heart rhythm

Cocaine Piperidine Quinolizidine

Lysine (or acetate) Lysine

Coniine Lupinine

Isoquinoline

Tyrosine

Codeine

Indole

Tryptophan

Table 4

Morphine

Analgesic (pain relief), treatment of coughs Analgesic

Psilocybin

Halucinogen

Reserpine

Treatment of hypertension, treatment of psychoses Strychnine Rat poison, treatment of eye disorders

The biosynthetic capacity of the hairy root cultures is equivalent or sometimes higher than that of the corresponding plant roots. Therefore, hairy root cultures have been developed as an alternate source for the production of root biomass and root derived compounds. For example, the total dry weight of 2-hydroxy-4-methoxy benzaldehyde was enhanced in hairy root cultures of Decalepis hamiltonii [18]. Another advantage of plant hairy roots is the large-scale plant micro-propagation. This technique is an effective way of propagating massive amounts of disease-free plants and is genetically uniformed by “artificial” seeds, which can be achieved through direct organogenesis from hairy roots. Transformed root cultures provide a promising alternative to biotechnological exploitation and a method for constant and standard production of valuable metabolites of plant cells. Hairy roots are obtained after a successful transformation of a plant with Agrobacterium rhizogenes. They have received considerable attention of plant biotechnologists for the production of secondary compounds. They can be subcultured and indefinitely propagated on a synthetic medium without phytohormones and usually display interesting growth capacities owing

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to the profusion of lateral roots (Figure 22). This growth can be assimilated to an exponential model, when the number of generations of lateral roots becomes large. Hairy roots have the following properties: 1) high degree of lateral branching; 2) profusion of root hairs; 3) absence of geotropism; 4) high growth rates in culture, due to their extensive branching, resulting in the presence of many meristems; 5) they do not require conditioning of the medium. Hairy roots are genetically stable, hence, they exhibit biochemical stability that leads to stable and high-level production of secondary metabolites. Hairy root cultures apparently retain diploidy in all species so far studied. The stable production of hairy root cultures is dependent on the maintenance of organized states. The factors which promote disorganization and callus formation depress secondary metabolite production. Productivity of hairy root cultures is stable over many generations in contrast to disorganized cell cultures. This stability is reflected in both the growth rate and the level pattern of secondary metabolite production. For the production of hairy root cultures, the explant material is inoculated with a suspension of A. rhizogenes. The bacterial suspension is generated by growing bacteria in Yeast Mannitol Broth (YMB) medium for 2 days at 25°C under shaking conditions. Thereafter, pelleting by centrifugation (5 x 10 rpm; 20 min) and resuspending the bacteria in YMB medium to form a thick suspension (approx. 1010 viable bacteria/ml). Transformation may be induced in aseptic seedlings or surface sterilized detached leaves, leaf-discs, petioles, stem segments, from greenhouse grown plants by scratching the leaf midrib or the stem of a plantlet with the needle of a hypodermic syringe containing a small (about 5-10 μl) droplet of thick bacterial suspension of A. rhizogenes. For example, an effective biotechnological protocol for large-scale artemisinin production was established by cultivation of Artemisia annua  hairy roots in the nutrient mist bioreactor (NMB). Artemisinin is used for the treatment of cerebral malaria. It was extracted from hairy root culture induced by Agrobacterium rhizogenes-mediated genetic transformation of explants. The high (23.02 g/l) final dry weight of hair roots along with the artemisinin accumulation (1.12 mg/g, equivalent to 25.78 mg/l artemisinin) was obtained in this bioreactor, which is the highest reported artemisinin yield in the gas-phase NMB cultivation [19]. Many medicinal plants have been transformed successfully by A. rhizogenes and the hairy roots induced show a relatively high productivity of secondary metabolites, which are important pharmaceutical products (for example, alkaloids produced by hairy roots, including Atropa belladonna L., Catharanthus trichophyllus L., and Datura candida L.).

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Native roots of Scutellaria baicalensis Georgi.

Hairy roots culture of Rubia tinctorum

Hairy roots culture of S. baicalensis Georgi

Freeze-dried hairy roots of Rubia tinctorum

Figure 22. Hairy root cultures as an alternate source for obtaining root- derived bioactive compounds

Among tropane alkaloids, scopolamine is a more valuable secondary metabolite due to its fewer side-effects, usefulness in the treatment of Parkinson’s disease, and relaxing and hallucinogenic properties. Many efforts have been made to extract scopolamine from the plants but because of the low endogenicity of scopolamine content, the commercial production of scopolamine is limited. However the highest amount of scopolamine production (1.59 mg/g-1 dry wt) occurred in the bioreactor with aeration and agitation 1.25 vvm (volume per minute) and 70 rpm, respectively (Figure 23). The production of tropane alkaloids, especially scopolamine, in bioreactors instead of native plants, is an efficient way, because the process is performed under clean and controlled conditions, which consequently controls or decreases the diversity in the component quality and the alkaloid yield [20]. The hairy root system is stable and highly productive under hormone-free culture conditions. The fast growth, low doubling time, easy maintenance and

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ability to synthesize a range of chemical compounds of hairy root cultures gives additional advantages as continuous sources for the production of plant secondary metabolites. Usually root cultures require an exogenous phytohormone supply and grow very gradually, resulting in a poor or insignificant synthesis of secondary metabolites. Hairy roots are also a valuable source of photochemicals, which are useful as pharmaceuticals, cosmetics and food additives. These roots synthesize more than a single metabolite, prove economical for commercial production purposes.

1

2

Note: 1 – Obtaining hairy root culture (A-aseptical seedling of Atropa belladonna); B – Different seedling parts including roots, stems and leaves; C – Hairy root induction in vitro culture of Panax ginseng; D – The cultivated hairy root in hormone – free MS/2 medium; 2 – Hairy root culture obtained in bioreactors. Bioreactor with agitation 70 rpm and aeration 1.75 vvm (А) and Bioreactor with agitation 110 rpm and 1.75 vvm (В) [20]. Figure 23. Hairy root culure

The scale-up of hairy root cultures becomes complicated because of the need to simultaneously provide nutrients from both liquid- and gas-phases. To design bioreactors for hairy root cultures many factors should be considered, such as the growth characteristics, nutrient requirements and utilization rates, mass transfer, mechanical properties, requirement of a support matrix, and the possibility of flow restriction by the root mass in certain parts of the bioreactor. Moreover, in order to obtain an optimal biomass yield, roots should be evenly distributed in the bioreactor. While designing a suitable bioreactor for hairy root cultures the physiology and morphology of the hairy roots should be taken into consideration. The major problem in bioreactor cultivation of hairy roots is their tendency to form clumps resulting from the bridging of primary and secondary roots. This results in dense-

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ly packed root beds and reduces mass transfer (both oxygen and nutrients). Root thickness, root length, the number of root hairs and root branching frequency are some of the factors which should be taken into consideration for hairy root cultures in bioreactors (Figure 23). Immobilization of hairy roots by horizontal or vertical meshes as well as by cages or polyurethane foam promotes their growth in submerged stirred bioreactors, bubble columns, air lift reactors and drum reactors where the roots are immersed in the culture medium. Isolation of the roots from the impeller also rules out the possibility of root damage even at low tip speeds in stirred bioreactors. The oxygen transfer limitation in hairy root cultures in bioreactors can be reduced by growing them in gas phase bioreactors, spray or droplet reactors and mist reactors. Here the roots are exposed to humidified air or a gas mixture and nutrients are delivered as droplets by spray nozzles. Spray and mist reactors also provide the additional advantage of low hydrodynamic stress. A bioreactor must have a unique configuration to compensate for the heterogeneous, cohesive, structured, and entangled nature of fibrous roots. In flask and bioreactor cultures, the oxygen transferred to hairy roots and submerged in the medium exhibits three external mass transfer resistances: gas-liquid, liquid-liquid, and liquid-solid resistances. In shake-flask cultures, the important parameters affecting flask properties are flask size, shaking speed, closure type, and medium volume. Large-scale plant production through cell tissues in bioreactors is a key way for industrial plant propagation. In a biochemical context, bioreactors are usually explained as self-contained, sterile environments, which capitalize on liquid nutrient or liquid/air inflow and outflow systems. They are designed for intensive culture and providing maximal opportunity for monitoring and controlling micro-environmental conditions (e.g., agitation, aeration, temperature, dissolved oxygen, pH, etc.). Plant cell cultures are increasingly utilized for the production of valuable natural products such as pharmaceuticals, flavors, fragrances, and fine chemicals. On the other hand, various problems associated with low cell productivity, slow growth, genetic instability, and an inability to maintain photoautotrophic growth have limited the application of plant cell cultures. Increasing productivity of secondary metabolites by cell cultures In plants, most of the secondary metabolites are produced in differentiated cells or organized tissues. However, callus and cell suspension culture lack organ differentiation and hence produce low yields of these biochemicals. The ability of plants to produce certain bioactive substances is largely influenced by physical and chemical environments in which they grow. Plants also produce certain chemicals to overcome abiotic stresses. In this aspect plant tissue culture developed callus influenced by medium, explants, plant growth regulators, color lights, temperature, photoperiod and carbon sources are helpful to produce valuable secondary metabolite compounds in many studies.

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Growing a plant outside its natural environment under ideal conditions may, therefore, result in being unable to produce the desired bioactive substances, hence, there arises the need for prior evaluation. Chemical substances are synthesized in particular cells and are transferred to other cells, which in extremely small quantities influence the development process. Several strategies are being followed to improve yields of secondary metabolites in plant cell cultures. First of all, the screening and selection of high producing cell lines and the optimization of growth and production media can be mentioned as common approaches (Figure 24). In the past years new approaches have been developed: culturing of differentiated cells (e.g. shoots, roots and hairy roots), induction by elicitors and metabolic engineering [7]. With the culture of differentiated cells one has in most cases been able to get production of the desired compounds in levels comparable to that of the plant, however the culture of such differentiated tissues on a large-scale in bioreactors is a major constraint. For studies of the biosynthesis, such systems are very useful. The yield of secondary metabolite by undifferentiated tissue or cell cultures can be increased by the following techniques: –– Select proper cell line. The heterogeneity within the cell population can be screened to select lines capable of accumulating higher level of metabolite. –– Medium manipulation. The constituents of culture medium like nutrients, phytohormones as well as the culture condition like temperature, light, etc. influence the production of metabolites. For example, if sucrose concentration is increased from 3% to 5%, the production of rosamarinic acid is increased by five times. In case of shikonin production, IAA enhances the yield whereas 2,4-D and NAA are inhibitory. The second approach mentioned, the use of elicitors has been successful in several cases. However, it remains limited to a certain type of compounds for each plant, compounds which most likely act as phytoalexins in these plants. –– Elicitors. Compounds that induce the production and accumulation of secondary metabolite in plants are known as elicitors. Elicitors produced within the plant cells include cell wall derived polysaccharides like pectin, pectic acid, cellulose, etc. Product accumulation also occurs under stress caused by physical or chemical agents like UV, low or high temperature, antibiotics, salts of heavy metals, high salt concentration grouped under abiotic elicitors. These elicitors when added to medium in low concentration (50-250 ng/l) enhance metabolite production. Elicitation is one of the biotechnological strategies which holds the ability of enhancing secondary metabolite accumulation in plant cells and their quality production in cell suspension cultures.

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–– Permeabilisation. Secondary metabolites produced in the cell are often blocked in the vacuole. By manipulating the permeability of cell membrane, they can be elicited out (secreted out) to the media. Permeabilisation can be achieved by electric pulse, UV, pressure, sonication, heat. Even charcoal is added to the medium to absorb secondary metabolites.

Figure 24. Steps involved in the production of secondary metabolites from the plant cell

–– Immobilisation. Cell cultures encapsulated in agarose and calcium alginate gels or entrapped in membranes are called immobilised plant cell culture (Figure 25). Immobilization of plant cells allows better cell to cell contact and the cells are also protected from high shear stresses. These immobilized systems effectively increase the productivity of secondary metabolites in a number of species. Elicitors can also be added to these systems to stimulate secondary metabolism.

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–– Addition of precursors. Precursors are the compounds, whether exo­ genous or endogenous, that can be converted by a living system into useful compounds or secondary metabolites. It has been possible to enhance the bio­ synthesis of specific secondary metabolites by feeding precursors to cell cultures. For example, amino acids have been added to suspension culture media for production of tropane alkaloids, and indole alkaloids. The amount of precursors is usually lower in callus and cell cultures than in differentiated tissues. Phenylalanine acts as a precursor of rosmarinic acid. Addition of phenylalanine to Salvia officinalis suspension cultures stimulated the production of rosmarinic acid and decreased the production time as well. Phenylalanine also acts as precursor of the N-benzoylphenylisoserine side chain of taxol. Addition of Taxus cuspidata cultures with phenylalanine resulted in increased yields of taxol. Timing of precursor addition is critical for an optimum effect. The effects of feedback inhibition must surely be considered when adding products of a metabolic pathway to cultured cells.

A

B

D

C

E

Note: Cell cultures encapsulated in calcium alginate gels (A) and in agarose (B); cells entrapped in membranes or adsorbed in substrate (C); cells immobilised by biomacromolecules / lectins/ (D); cells connected by covalent bonds (E) Figure 25. Immobilised plant cells (Valikhanova G.Zh. 2009, [21] )

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Plant cells, which in general are shear-stress tolerant, can be cultured on a large scale in stirred bioreactors. To increase yields, metabolic engineering seems to be a promising approach, but requires understanding of the regulation of secondary metabolism at all its levels: genes, enzymes, products, transport and compartmentation. The attention is more and more focused on metabolic engineering, which can be used to increase yields. Several possibilities can be envisaged: –– increase activity of enzymes which are limiting in a pathway; –– induce expression of regulatory genes; –– block competitive pathways; –– block catabolism. The first two possibilities require the expression of genes yielding active enzymes, the latter two approaches blocking genes by antisense genes. In all cases the respective biosynthetic pathway has to be known on the level of products, enzymes and genes, as well as the regulation on all these levels, including aspects as compartmentation and transport. For example, in Tabernae montana species mevalonate is used for the mterpene biosynthesis after elicitation, channeling away this precursor from the alkaloid pathway [7]. Blocking such pathways by means of antisense genes might be of interest to increase the availability of these precursors for the alkaloid biosynthesis. Catabolism is another important factor in the accumulation of alkaloids. Identification of the enzymes involved and cloning of the encoding genes is thus of interest to eventually use antisense gene technology to block catabolism and thus increase the production. Current progress has been made in the field of molecular biology through alteration in the metabolic skeleton of the plant secondary metabolism. With the use of various genes (involved in the synthesis of enzymes and their regulatory proteins), diverse pathways have been traced and are transformed. Antisense technology has emerged as an additional alternative for enhancement of secondary metabolites. In Tylophora indica enhancement in kaempferol, an antioxidant compound was observed by using precursors such as salicylic acid, ornithine, cinnamic acid, tyrosine and phenylalanine [18]. Metabolic engineering offers new perspectives for improving production of secondary metabolites by overexpression of single genes. This approach may lead to an increase of some enzymes involved in metabolism and, consequently, results in the accumulation of the target products. This method utilizes the foreign genes that encode enzyme activities not normally present in a plant. This may cause modification of plant metabolic pathways. At present, analytical tools based on genomics, proteomics, metabolomics, bioinformatics, and other twenty-first-century technologies are accelerating identification and characterization of natural products. On the other hand, many new bioactive compounds fail due to a lack of efficacy in the clinic, which demands

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new strategies for pharmacological research. The fusion of ‘omics technologies’ with ethnomedicine, systems biology, and studies of plant endophytes are exciting approaches to search for new drugs from natural sources. Control questions: 1. How are secondary metabolites in the plant cells produced? 2. What is the importance of plant cell culture in production of secondary metabolites? 3. What steps are involved in the production of secondary metabolites from plant cell? 4. How is production of secondary metabolites associated with primary metabolites? 5. What are the strategies for the enhancement of production of secondary metabolites? 6. What is hairy root culture? 7. Describe the establishment of hairy root culture. 8. What are the factors which influence the transformation of hairy root culture? 9. What are the properties of hairy roots? 10. Give some example of pharmaceutical products from hairy root culture.

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CHAPTER

4

MICROPROPAGATION IN VITRO Micropropagation as an alternative method to conventional propagation is a suitable way to produce a large number of progeny plants, which are genetically identical to the stock (donor) plant in a short time. The important property of the plant cells is totipotency which is a capacity to produce the whole plant from different plant parts. Micropopagation is a tissue culture method developed for production of disease-free, high quality planting material and for rapid production of many uniform plants. Micropropagation has some features to be chosen in commercial production such as multiplicative capacity in a relatively short time, healthy and disease-free production capacity and ability to generate population during a year. More than 600 million micropropagated plants are produced every year in the world [22], therefore micropropagation is one of the few areas of plant tissue culture in which the techniques have been applied commercially. To circumvent these impediments, clonal or vegetative propagation has been deployed for recovering dominant, additive and epistatic genetic effects to select superior genotypes. Plant tissue culture methods offer an important option for effective multiplication and improvement of plants within a limited time frame. The genetic pattern of the plant is a key element in the selection of the propagation method. Micropropagation techniques in plant biotechnology applications are more expensive than conventional propagation methods. Propagation by using in vitro techniques instead of conventional methods offers some advantages such as utilizing small pieces of plants (explants) to maintain the whole plant and to increase their number. The main point is to evolve new strategies to reduce the time and cost consumed per plant. In tissue culture applications, selection of the initial material is important in the beginning of the culture. Therefore it is easy to provide virus-free clones in a short time. Production of plants during all year long independent of seasonal changes, and long storage periods make micropropagation preferable to propagate plants in a short time. There are also some disadvantages of micropropagation. Adaptation of cultured plants to the environmental 310

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conditions need transitional period to allow the plants to produce organic matter by photosynthesis. Summarizing the above we can state that the advantages of micropropagation over conventional propagation methods are: –– Genotype constitution maintained as there is lesser variation in somatic embryo; –– Easier transport and storage is facilitated by small size propagules and their ability to grow in soil-free medium; –– Control over growing conditions as the production of planting material is completely under artificial control in vitro; –– Reduced growth cycle and rapid multiplication as shoot multiplication has a short cycle and each cycle results in the exponential increase in the number of shoots; –– Selective multiplication can be done for e.g. auxotrophs, aneuploids, selected sex in dioecious species; –– Virus free plants can be raised and maintained through meristem culture, which is the only method available for this. Micropropagation proves useful for propagation of: 1) sexually sterile species like triploids, aneuploids, which cannot be perpetuated by seeds; 2) seedless plants like banana; 3) cross bred perennials where heterozygosity is to be maintained; 4) mutant lines like auxotrophs which cannot be propagated in vivo; 5) disease free planting material of fruit trees and ornamentals. Disadvantages of micropropagation: –– High cost; –– Somaclonal variation- any variation if occurs during multiplication may go unnoticed; –– Recalcitrancy of species/genotype – many tree species like mango, etc. do not respond to in vitro growth. Application of micropropagation 1. Commercial production of secondary metabolites. The compounds/biochemicals which are not directly involved in primary metabolic processes like respiration, photosynthesis, etc. are secondary metabolites. These include a variety of compounds like alkaloids, terpenoids, phenyl propanoids, etc. with various biological activities like antimicrobial, antibiotic, insecticidal, valuable pharmacological and pharmaceutical activities. Therefore, micropropagation allows their commercial scale production from cell cultures viz. shikonin derivatives used in dyes, pharmaceuticals are produced from cell cultures of Lithospermum erythrorhizon. Also, cultured cells of many plant species produce novel biochemical, which have otherwise not been detected in whole plants. 2. Production of synthetic seeds. Synthetic seed is a bead of gel containing a somatic embryo or a shoot bud with growth regulator, nutrients, fungicides, pesticides,

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etc. needed for the development of a complete plantlet. These are better propagules as do not need hardening and can be sown directly in the field (Figure 26).

А

В

С

Note: A, C – the bead(s) of gel containing somatic embryo; B – a plantlet, growing from the gel-capsule Figure 26. Synthetic seeds

3. Raising somaclonal variants. The genetic variability occurring in somatic cells, plants produced in vitro by tissue culture are referred to as somaclonal. When these variations involve traits of economic importance, these are raised and maintained by micropropagation. 4. Production of disease-free plants. Most of the horticultural fruit and ornamental crops are infected by fungal, viral, bacterial diseases. Micropropagation provides a rapid method for production of pathogen free plants. In case of viral diseases, especially, the apical meristems of infected plants are free or carry very low concentrations of viruses. Thus culturing meristem tips provides disease free plants. Actively-dividing young cells (meristem) are placed in a special medium and treated with plant hormones to produce many similar sister plantlets. Since the meristem divides faster than disease-causing virus, clean materials are propagated and hundreds of uniform plantlets are produced in a short time. Methods of in vitro propagation The main methods of in vitro propagation can be classified into two groups: 1. Propagation from axillary or terminal (apical) buds/meristems (Fig. 27, 28) 2. Propagation by the formation of adventitious shoots or adventitious somatic embryos The shoot apical meristem is located at the extreme tip of the shoot, but it is surrounded and covered by immature leaves. These are the youngest leaves produced by the activity of the meristem. It is useful to distinguish the shoot apex from the meristem proper. The meristem and shoot tip cultures (apical meristems

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cultures) are used to establish virus-free plant culture. Many important horticulture crops were propagated by meristem culture for rapid growth and virus elimination. Axillary meristems are formed in the axils of leaves and are derived from the shoot apical meristem. The growth and development of axillary meristems produces branches from the main axis of the plant. Propagation from axillary or terminal buds is the most reliable method to have the highest genetic stability during in vitro propagation of plants. Apical meristem

Figure 27. Shoot apex and apical meristem, axillary bud with meristem

Adventitious shoots or adventitious somatic embryos are established directly or indirectly. Somatic cell cultures are directly started with the excised explants from the mother plant tissues for organogenesis or embryogenesis. If shoots or embryos regenerate on the previously formed callus or in cell culture, they are called indirect organogenesis or embryogenesis. When propagation occurs via an indirect callus phase, the genetic identity of the progenies decreases. An important problem in commercial propagation is to affect the uniformity of progenies. Callus formation also increases the somaclonal variation. Increasing of somaclonal variation incidence is a crucial result of long term period of callus growth. The origin of the callus also causes somaclonal variation.

Figure 28. Micropropagation from axillary and apical buds/meristems

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Micropropagation involves the following major stages: Stage 1. Selection and maintenance of stock plants for culture initiation (about 3 months) – the stage of selection and preparation of mother plants Stage II. Preparation and establishment of explant on suitable culture medium (usually shoot tips and axillary buds used) – stage of in vitro culture establishment (3-24 months); Stage III. Regeneration: multiplication of shoots or somatic embryos on defined (10-36 months) culture medium – stage of shoot multiplication; Stage IV. Rooting of regenerated shoots/ somatic embryo in vitro (16 weeks) – stage of rooting of microshoots; Stage V. Transfer of plantlets to sterilized soil for hardening under greenhouse environment – stage of acclimatization (Figure 29). These stages are necessary for successful micropropagation. Establishing aseptic culture conditions can be classified as Stage 1-2 which contains presurface sterilization applications of explants to reduce contamination of stock plants. The success of Stage 2-3 depends on different factors such as plant species, cultivar or genotype, plant growth regulators, the ingredients of the medium and physical culture conditions. Stage 4 is responsible for rooting of microshoots. It depends on the factors given in Stage 3 (PGR, chemical and physical culture conditions). Transplantation of rooted shoots to the environment is the main step of Stage 5. This is also the important part of micropropagation. Acclimatization needs to be well controlled to avoid loss of propagated plants. Factors affecting micropropagation In vitro propagation methods have several advantages over conventional propagation like flexible adjustment of factors affecting regeneration such as genotype, explant type, nutrient and plant growth regulator levels and conditions of the environment, production of clones in desired rate, continued production during seasonal changes using tissue culture methods also increase the multiplication rate of plants. Genotype and explant type. The genotype of the mother plant plays an important role in in vitro propagation. Different genotypes had different responses to the same culture conditions. For this reason, it is necessary to establish a suitable procedure for each variety of plants that can be adapted to commercial production.

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introduction in vitro

in vitro culture establishment

rooting of microshoots

shoot multiplication

stage of acclimatization

Figure 29. The stages of plant micropropagation

The success of tissue culture depends on the correct choice of explants. Shoot or shoot tips and node cultures are the most commonly used culture types in micropropagation of plants (Figure 30). Explants from shoot tips and nodal stem segments are suitable for enhanced axillary branching. Selection of explant type to induce callusogenesis and organogenesis is important for plants. In direct and indirect organogenesis studies, use of young leaf explants is important for the success of the culture. Jahan M.T. et al. observed higher number of shoots in the brown young lamina explants than young green lamina as well as indicated the importance of using new brown leaves for callus induction [23]. Culture medium. Culture medium influences the propagation efficiency in plant tissue culture applications. Organic compounds, vitamins and plant growth regulators are used to stimulate healthy growth. The rate of tissue growth and morphogenetic responses are highly affected by the features of nutrients. There are several basal media such as Murashige and Skoog (MS), Gamborg’s B5 (B5), Nitsch and Nitsch (NN), Murashige and Tucker (MT). These media are successfully used for establishing tissue cultures of different explants of various plants. In plant tissue culture studies, different combinations of every medium based on different concentrations of macro and micronutrients have been used to develop efficient protocols [24]. The rapid and efficient tissue culture protocols are important for micropropagation of plants. The success of plant tissue culture depends on the composition of the medium used. Each

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plant species has its own medium composition or it should be improved for better results. The modifications can be made up in macro- and micronutrients, sugar content, plant growth regulators, vitamins and other nitrogen supplements.

Figure 30. Clonal micropropagation of Guava (Psidium guajava L.)

Plant growth regulator levels. In culture conditions, the use of synthetic chemicals with similar physiological activities as plant hormones can induce plant growth as desired. Auxin and cytokinins are the most important hormones regulating growth and morphogenesis in plant tissue culture. Their combinative usage promotes growth of callus, cell suspensions, root and shoot development and has capability to regulate the morphogenesis. Different combinations and concentrations of plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA), benzylaminopurine (BAP) and kinetin were used to indicate callus formation from different kinds of explants of Scorzonera tau-saghyz Lipsch et Bosse. In preliminary studies, induction and regeneration of callus followed by shoot and root regeneration are the main steps of tissue culture of whole plants. As an important commercial rubber plant, to develop a rapid and more effective tissue culture protocol to reduce the time for breeding processes is the main objective of endemic rare plant Tau-saghyz, growing in the Karatau mountains of the Southern Kazakhstan [25]. Endemic

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species are defined as “which grow only in limited area and often rare species”. Rubber plant Tau-saghyz (Scorzonera tau-saghyz Lipsch. et Bosse) belongs to the family Asteraceae, a rare, endemic species with a reduced amount. This is a perennial plant 25–40 cm high, with the powerful branching caudexes and deep rod root. The content of rubber in roots is about 20–40 % of the dry weight of roots, it depend on the age and a cultivar. This compound of the medium influences the efficiency of in vitro cultivation. A rapid multiplication rate could be obtained from leaf explants by combining the phytohormones in MS medium (Figure 31). Addition of 0.5 mg/l GA to MS medium containing 1 mg/l BA and 2 mg/l NAA resulted in an increase in the mean numbers as well as the mean length of the shoot. It implies that cytokinin in combination with GA and auxin plays vital role in organogensis and further regeneration from leaf explants of S. tau-saghyz. This is the first protocol which has a great potential for rapid multiplication, propagation and conservation of rare species S. tau-saghyz Lipsch. et Bosse and also for creation a collection of Tau-saghyz, representing scientific and commercial interest. This reliable technique is currently available for largescale micropropagation of rubber plants on a commercial scale. Under the supervision of Professor K.K. Boguspaev and Associated Professor S.K. Turasheva at Al-Farabi Kazakh National University the extensive experiments on enhancement of the frequency of somatic embryo induction and plant regeneration in rubber plants were carried out. Studies were conducted to optimize culture conditions, nutritional and hormonal requirements during somatic embryogenesis. Hormonal balance of the medium and explant, mineral and carbohydrate nutrition, interaction of growth regulators, sucrose and calcium on callus friability, the role of sucrose and abscisic acid in embryo induction and carbohydrate types have also been evaluated. In this research the explants were isolated (leaf, root, apical meristems of shoots) from 1-3 year-old plants and were placed on the Gamborg and MS media supplemented with kinetin (0.5-5 mg/l), 6-benzilaminopurine (0.1-1 mg/l), NAA (0.1-2 mg/l). MS medium with 5 mg/l was the optimum for the development of axillary meristems from the plantlets and MS medium with ratio 1:2 of 6-benzilaminopurine to NAA was the optimum for induction of adventious shoots from the apical buds. The physiological stage of the explants was found to play a significant role in micropropagation. In this study it was shown that usage of young leaf explants is important for the success of culture rubber plants. Optimum callus induction was obtained in modified MS medium supplemented with 2.0 mg/l 2,4-D and 0.5 mg/l kinetin. Somatic embryo induction was found to be better with 1 mg/l kinetin and 0.2 mg/l NAA. Further development of the embryos into plantlets was achieved on a hormone free medium. Cytological analysis revealed that all the plantlets tested were diploid [26].

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A

D

B

F

C

G

Note: Scorzonera tau-saghyz Lipsch. et Bosse (rubber plant) at flowering stage (A); the roots of rubber plants (B); callusogenesis (C) and organogenesis (D) in tissue culture of Scorzonera tausaghyz; plant-regenerant of Scorzonera tau-saghyz (F); chromosoms at metaphase in the cells of root meristem (G) Figure 31. Micropropagation of rare rubber endemic plant Scorzonera tau-saghyz

In rubber plant breeding, the current propagation method of grafting onto unselected seedlings, maintains intraclonal heterogeneity for vigour and productivity and hence a great improvement may be expected by using micropropagation in vitro. Micropropagation with nodal and shoot tip explants derived from seedlings is possible as with mature clonal explants. The major problem in using clonal material from mature rubber trees is the failure to produce an adequate tap root system necessary for tree stability, and the poor response to culture conditions. The latter problem has been overcome to a significant level by in vitro micrografting. Recently, there has been an increasing interest in the induction of somatic embryogenesis in rubber plants using different explants, media compositions and conditions, especially for use in genetic transformation studies. Genetic transformation offers a viable approach to overcoming different problems and to introducing specific agronomically important traits without disrupting their otherwise desirable genetic constitu-

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tion. A reproducible plant regeneration system for each genotype of rubber plants through tissue culture is essential for crop improvement programs. An efficient plant regeneration pathway through somatic embryogenesis is essential for crop improvement through transgenic approaches besides using this as a micropropagation system. An increase in the auxin concentration with the higher concentration of cytokinins resulted in the production of callus at cut ends of nodal segments, possibly due to the accumulation of auxins, which stimulates cell proliferation in the presence of cytokinins. Similar findings were reported in medicinal plants like Tylophora indica, Holostemma annulare, Holostemma adakodien, Spilanthes acmella (Figure 32). Micropropagation is an efficient tool for the conservation and mass multiplication of endangered important medicinal plants. An efficient protocol of axillary bud proliferation and direct organogenesis has been developed for Crocus alatavicus Regel et Semen, a highly important medicinal plant. Crocus alatavicus is a source of phenols, flavonoids, xanthones, fatty acids, carotenoids which have a high biological activity. Overexploitation for its antidiabetic, anti-inflammatory, antiviral, antioxidant properties concentrated in roots and stem has made it endangered, causing the need for its conservation. Propagation of Crocus alatavicus Regel et Semen in vitro is a promising way for its conservation. To develop the micropropagation protocol, the germplasm was screened for selection of a suitable ecotype with high content of mangiferin estimated with High Performance Liquid Chromatography technique. Nodal segments were cultured on MS supplemented with different growth regulators. The most efficient shoot multiplication was obtained with the supplementation of 3.5 mg/l BA and 0.5 mg/l IAA. Elongation of the micro-shoots was achieved by subculture every 20 days. The elongated micro-shoots were efficiently rooted in vitro on half strength MS supplemented with IBA. Plantlets were successfully established in the soil in 6-8 weeks and were morphologically similar to those of the source plant. The protocols developed presently for direct shoot regeneration and root induction could be successfully applied for the development of high quality planting stocks [13].Sometimes the complex of biologically active substances affected root formation as well as plant growth regulators. For example, the research of Kazakh scientists showed that in test systems in vitro and in vivo the total extracts and individual tannin fractions of Bergenia crassifolia (L.) Fritsch. and Sanguisorba officinalis have a synergetic effect with low concentration IBA on the root development.

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Figure 32. Scheme of nodal segment culture Spilanthes acmella for clonal  propagation by axillary   shoots proliferation (from http://nptel.ac)

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Under the supervision of Dr. V.K. Mursalieva at the Institute of Plant Biology and Biotechnology the growth-regulatory activity of Bergenia crassifolia (L.) extract, which consists of hydrolysable and condensed tannins (20.87%), flavonoids, phenolic acids, amino acids and carbohydrate compounds, was determined. In the samples of Sanguisorba officinalis the highest content of hyrolysable tannins (21.08 %), phenol acid, amine compounds, flavonoids and steroids were determined [27]. Treatment with IBA (148.0 μM), followed by ex vitro rooting and adaptation in the mixture of peat and sand, significantly increases the yield of a high-quality planting material. Conditions of the environment. For root induction, well developed shoots inoculate on different root initiation media comprising half strength MS with different concentrations of IBA or NAA. The micro-shoots with roots were transferred to MS for further growth and finally washed before transfer to small pots containing soil + vermicompost (2 : 1) for hardening and acclimation. The pots were covered with polythene bags to maintain high relative humidity for two weeks and transferred to the glasshouse after emergence of new leaves and later to the field [24]. At the fourth step of shoot rooting the hydroponic set was used. The technique of growing plants with their roots immersed in nutrient solution without soil is called solution culture or hydroponics. Successful hydroponic culture (Figure 33) requires a large volume of nutrient solution or frequent adjustment of the nutrient solution to prevent nutrient uptake by roots from producing radical changes in nutrient concentrations and pH of the medium. A sufficient supply of oxygen to the root system, which is also critical, may be achieved by vigorous bubbling of air through the medium.

Figure 33. Hydroponic system for growing plants in nutrient solutions in which composition and pH can be automatically controlled.

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Hydroponics is used in the commercial production of many greenhouse crops. In one form of commercial hydroponic culture, plants are grown in a supporting material such as sand, gravel, vermiculite, or expanded clay. Nutrient solutions are then flushed through the supporting material, and old solutions are removed by leaching. In another form of hydroponic culture, plant roots lie on the surface of a trough, and nutrient solutions flow in a thin layer along the trough over the roots. Each plant should be carefully removed from its tube of media and planted into a small pot containing a clean light potting mix. Gently wash off all the agar medium prior to planting. The plants will still need to be protected at this stage since they are not accustomed to the drier air of the room when compared to the moist environment of the tube of media. Acclimatization. Acclimatization process is to be carried out while the plants are still under in vitro conditions. A few days before the process was to be carried out, the cover of test tube is removed. With the relative humidity at 50-70% in the culture room, this will increase the epicuticular wax development on the upper leaf surfaces of the plantlets and their survival rate will rise from 70 to 90%. The plantlets exposed to the normal environment in stages will wilt due to rapid changes of relative humidity and light intensity. In vitro plantlets that reached 3-5 cm height are taken out from culture tubes and the excess media rinse to avoid contamination. They are put into plastic pots and planted out in soil at a ratio of 1:1:1 for garden soil, sand and loam. Plantlets are planted in a pot and covered with transparent plastic lid. The type of substrate or medium used for germination is important to seedling establishment. In general, a substrate should be light and porous to provide adequate oxygen, yet retain moisture and allow for proper drainage. Most commercial germination mixes contain a blend of peat moss, vermiculite, perlite, and sometimes sand (Figure 34). When propagation substrate components are combined, a lightweight porous moisture holding soilless mix is produced. After the seedling has developed its first true leaves, a mild fertilizer solution can be applied. Nitrogen concentrations of 50 to 75 ppm are recommended. For example, for acclimatization of Saintpaulia ionantha, various substrates were used, such as autoclaved mixed soil (compost, sand, and black soil in the ratio 1:1:2) and nonautoclaved mixed soil. The regenerated plants must reach 4-5 cm before transferring them into pots of mixed soil. After transplanting, the plantlets were watered regularly to prevent from drying. For the first 3 weeks the regenerated plants were maintained in the culture room at 25±2 °C. Successful micropropagation of plants, which can survive under the natural environmental conditions, depends on the acclimatization process. Most species grown in vitro required an acclimatization process in order to ensure that sufficient number of plants can survive and grow vigorously after being transferred to ex vivo soil. The excess media was cleaned from the roots and the plants were

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transplanted in an adequate substrate such as peat or soil. Plantlets were maintained in a confined environment temporarily before they can be adapted progressively in typical environment within drier air, high light intensity and temperature variations [24]. Light, in particular light level, has a tremendous effect on the success of rooting cuttings. Cuttings should be protected with shade cloth during parts of the year with high light levels and if light levels are too low in the winter, propagators should provide supplemental light. A general light level range for the propagation of floricultural crops is 1,000 to 2,000 footcandles.

Figure 34. Different substrates for plantlets

Generally, clonal multiplication of cultivars can be achieved by vegetative propagation. However, where the entire population of the clone is infected the only way to obtain pathogen-free stock is to eradicate the pathogen from vegetative parts of the plants and regenerate full plants from such tissues. Once pathogen free plants are obtained they can be multiplied indefinitely under conditions which would protect them from chance reinfection. Plant diseases Diseases thrive in environments which allow for easy survival and transmittance of fungi and bacteria. Propagators should establish strict sanitation measures to prevent diseases, thus production is optimized and profits always exceed losses. Plant virus diseases are critical problems in agriculture and their occurrence may be excluded if preventive strategies are established. Identification of the major viruses present in crops provides support for further studies to establish integrated management of viruses in producing regions and development of virus-resistant cultivars. Virus infection has a variety of symptoms in the field, perhaps, because of the particular virus present in the host and possible mixed infections. The symptoms of a sample with different viruses were analyzed on Cucurbita pepo L., Nicotiana benthamiana Domim. and Datura stramonium Thunb. The virus inoculum (Papaya ringspot W, PRSV-W), Zucchini yellow mosaic

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virus (ZYMV), Watermelon mosaic virus (WMV, potyvirus), Cucumber mosaic virus (CMV-Cucumovirus) and Zucchini lethal chlorosis virus (ZLCV, Tospovirus) sprinkle with abrasive silicon carbide by rubbing the virus suspension against the leaf tissue and keep in plants until the appearance of symptoms for further analysis and studying expression of symptoms. Normally, multiple infections cause severe symptoms such as leaf distortion, mosaic, bubbles, narrowing leaf, leaf rolling, necrosis, spur and underdevelopment, ruffled edges, blistering, leaf narrowing, shoestring, leaf rolling, necrosis, stunting (Figures 35-37) [28].

A. Mosaicwith chlorotic rings

B. Bubbles

C. Narrowing leaf

Figure 35. Symptoms of viral infections of watermelon leaves

Signs are indications of the presence of a disease-causing organism – e.g., fruiting body or mycelium of fungi. Symptoms are changes in the host – exudations, resinosis, necrosis (death of tissue or tree), hypotrophy (dwarfing), hypertrophy (overgrowths -galls, witches brooms). Organisms causing biological diseases: 1) Fungi and fungus-like organisms; 2) Viruses, viroids, and prions; 3) Bacteria; 4) Phytoplasmas; 5) Nematodes; 6) Parasitic plants; 7) Protozoans. Fungi cause the greatest problems on woody plants because they have enzymes to break down cellulose and lignin. Bacteria and viruses make more problems for soft tissued plants. Perhaps, there are a million species of fungi, but only 100,00 are known. 10,000 are plant pathogens. Viruses are organisms with protein coat and nucleic acid, Viroid–low MW RNA, Prion–infectious protein molecule. Viruses and viroids can move through plant in phloem or xylem, or stay localized in foliage and can also become part of plant genome. The symptoms of virus infection are the following: foliage streaking, spotting, mottling, brooms or rosettes, growth reduction. Bacteria (one-celled prokaryotes with a cell wall) easily exchange genetic material on plasmids. Bacteria cause disease by enzymes that digest cell walls, toxins, or tumors. Typical symptoms are: water soaking, wet

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wood, shoot blight, bleeding cankers, galls. In addition to the crown gall bacterium, Agrobacterium tumefaciens, and other pathogenic bacteria may stimulate plant cells to divide. For example, Corynebacterium fascians is a major cause of the growth abnormality known as witches’ broom. The shoots of plants infected by C. fascians resemble an old-fashioned straw broom because the lateral buds, which normally remain dormant, are stimulated by the bacterial cytokinin to grow. Phytoplasmas, also called MLO’s, or Mycoplasm-like organisms are like bacteria without cell walls. Phytoplasmas cannot be cultured apart from the host, infect phloem and cause a systemic, lethal disease (causes elm yellows, X disease of cherry, coconut lethal yellowing, and others). Symptoms of phytoplasmas infection: yellowing, epinasty, witches brooms, defoliation. Plant parasitic nematodes have styletto pierce plant cell walls. They cause injury by feeding, toxins. They are the vectors of other diseases, contributing factors in declines. Evolution towards plant parasitism has occurred at least 8 times in the flowering plants. Many are in the order Santalales: Loranthaceae (leafy mistletoes), Santalaceae (root and stem hemiparasites), Viscaceae (for example Phoradendron–leafy mistletoes, Arceuthobium– dwarf mistletoes).

A

B

C

D

Figure 36. Symptoms of plant diseases caused by viruses: Odondoglossum ringspot (А); Cymbidium mosaic virus (В); «Taiwan virus» Phalaenopsis (С); Orchid fleck virus (D) /http://forum.bestflowers.ru/

Parasitic plants are usually visible. Symptoms: brooming, galls, reduced growth. Protozoans such as phytoflagellates can parasitize milkweed, tomato, onion and chive plants. A vast number of plant pathogens from viroids of a few hundred nucleotides to higher plants cause diseases in our crops. Their effects range from mild symptoms to catastrophes in which large areas of crops planted for food are destroyed [29].

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Figure 37. Bacterial leaf spot, bacterial gall of Douglasifir caused by Pseudomonas pseudotsugae, bacterial Wetwood

Plant pathogens are difficult to control because their populations are variable in time, space, and genotype. Most insidiously, they evolve, often overcoming the resistance that may have been the hard-won achievement of the plant breeder. In order to combat the losses they cause, it is necessary to define the problem and seek remedies. At the biological level, the requirements are for the speedy and accurate identification of the causal organism, accurate estimates of the severity of disease, its effect on yield, and identification of its virulence mechanisms. Disease may then be minimized by the reduction of the pathogen’s inoculum, inhibition of its virulence mechanisms, and promotion of genetic diversity in the crop. Conventional plant breeding for resistance has to play an important role, which can now be facilitated by marker-assisted selection. There is also a role for transgenic modification with genes that confer resistance. Plant viruses cannot keep up with the rapid growth in the shoot tips of plants, therefore scientists grow new plants from these shoot tips on sterile media, then culture test them over several months to ensure clean stock plants. The technique is also used to eliminate bacteria or fungi from plants. Production of pathogen free plants The plants infected with bacteria and fungi can be treated by bactericidal and fungicidal compounds, but there is no commercially available treatment to cure virus-infected plants. A large number of viruses is not transmitted through seeds. Therefore, it would be possible to obtain virus free plants from infected individuals by using seeds as propagules. However, genetic variation often occurs from the sexually reproduced plants when propagated by seeds. Apical meristem culture for virus elimination. In a young plant, the most active meristems are called apical meristems. They are located at the tips of the stem and the root (Figure 38). At the nodes, axillary buds contain the apical meristems for branch shoots. The shoot apex consists of the apical meristem and the most recently formed leaf primordia. The shoot apical meristem is the undiffer-

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entiated cell population only and does not include any of the derivative organs. The shoot apical meristem consists of different functional regions that can be distinguished by the orientation of the cell division planes and by cell size and activity. The angiosperm vegetative shoot apical meristem usually has a highly stratified appearance, typically with three distinct layers of cells. Each layer has its own stem cells, and all three layers contribute to the formation of the stem and lateral organs. The shoot apical meristem is a flat or slightly mounded region, 100 to 300 μm in diameter, mostly composed of small, thin-walled cells, with a dense cytoplasm, and lacking large central vacuoles. apical meristem apical meristem intercalar meristem

leaf primordial leaf rudiments

lateral meristem

procambium

Figure 38. Longitudinal section of the shoot showing positions of meristems

It is well known that the distribution of viruses in plants is uneven. In infected plants the apical meristems are generally either free or carry a very low concentration of viruses. In older tissues the virus titer increases with increasing distance from the meristem-tips. The reasons proposed for the escape of meristem from virus invasion are: a) viruses readily move in the plant body through the vascular system which is absent in the meristem; b) the alternative method of cell-to-cell movement of the virus through plasmodesmata is too slow to keep pace with the actively growing tip; c) high metabolic activity in the actively dividing meristem cells does not allow virus replication; d) the ‘virus inactivating systems’ in the plant body, if any, have higher activity in the meristem than in any other region. Thus, the meristem is protected from infection; e) a high endogenous auxin level in shoot apices may inhibit virus multiplication. In vitro propagation through a meristem culture is the best possible means of virus elimination and produces a large number of plants in a short span of time. The term ‘meristem culture’ is a meristem with no leaf primordial or at most

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1-2 leaf primordial which are excised and cultured (Fig. 39). The pathway of regeneration undergoes several steps: starting with an isolated explant, with dedifferentiation followed by re-differentiation and organization into meristematic centers.

А

В

С

D

E

F

Figure 39. Scheme of obtaining virus free plants in vitro

Factors affecting eradication of viruses through meristem tip culture Culture medium, explant size and incubation conditions affecting plant regeneration from meristem-tip cultures have a pronounced effect on virus eradication. Besides, thermotherapy or chemotherapy and physiological stage of the explants also affect virus elimination by shoot-tip culture. Culture medium. The nutrients, growth regulators and nature of the medium highly influence the development of virus free plants from meristem tip cultures. Maximum success is achieved from MS medium which promoted a healthy, green shoot development in comparison with the other nutrient media. The main reason for the suitability of medium for meristem-tip culture could be the presence of high levels of K+ and NH4+ ions. There is no critical assessment of the role of various vitamins or amino acids but sucrose or glucose is the most commonly used carbon source in the medium, at the range of 2-4%, to raise virus free plants from meristem-tip cultures. Large meristem-tip explants, measuring 500 µm or more in length, may give rise to plants even in the basal medium but generally the presence of an auxin or a cytokinin or both plays a major role in the development of excised apical meristem. In angiosperms, the meristematic dome in the shoot-tip does not synthesize auxin on its own, but it is supplied by the second pair of the youngest leaf primordia. Therefore, for development of excised meristem in culture, without the leaf primordia, requires the supply of exogenous auxin. The plants requiring only auxin must have a high endogenous cytokinin level in their meristems. Among auxins, the use of 2,4-D should be avoided as it promotes only callusing. NAA and IAA are widely used auxins and NAA is preferred due to better stability. The role of GA3 is also emphasized by a few authors, who suggest that it promotes better growth and differentiation and suppresses callusing from meri-

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stem explants. Both liquid and semi-solid media have been tried for meristem–tip culture but, agar medium is generally preferred. Explant size. Survival of the meristem tips, under the controlled condition, is determined by the size of the explant. The larger is the explant, the greater are the chances for plant regeneration. However, the survival of the explants cannot be treated independent of the efficiency with which virus elimination is achieved that is inversely proportional to the size of the explant. Thus, explants should be small enough to eradicate viruses and large enough to be able to develop into a complete plant. Besides the size of the explant, the presence of leaf primordia influences the ability of the meristems to form plants. In some plants it is essential to excise shoot meristems with two to three leaf primordia. Leaf primordia supply auxin and cytokinin to the meristem necessary for its growth and differentiation. In a culture medium containing essential growth regulators, the excised meristem domes develop bipolar axes very quickly during reorganization. Once the root-shoot axis is established, further development follows the same pattern as that for seedlings. Physiological conditions of the explant. Meristem-tips should be collected from actively growing buds. In a few cases, the tips taken from terminal buds proved to be better than those taken from axillary buds. Seeing the higher number of axillary buds present per shoot, in majority of the reports axillary buds were utilized as explants to increase the overall production of virus-free plants (Figure 40).

А

В

С

D E Note: A, B – plant-regenerants in vitro; C-assay of contamination on Vissa-medium; D-virusfree plantlets; E-plantlets of potato in the soil Figure 40. Virus-free potato obtained in apical meristem culture

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The time excision of buds is also critical, especially for trees with periodic growth. For example, in temperate trees the growth of the plant is limited to only a very short period in the spring and afterwards dormancy starts. In such cases to increase the success rate, the meristem-tip cultures can be raised during spring only. Storage conditions. Generally, light incubation of meristem tip culture is found better than dark incubation. The light intensity could range from 100 lx to 4000 lx which should increase in succession as the differentiation of meristem explant progresses. There is no clear information on the effect of temperature on regeneration of plants from excised meristem tips. The cultures are normally stored under room temperature (25±2°C) conditions. Thermotherapy Apical meristems are not always free of virus and it cannot be considered as a universal occurrence. There are certain viruses like, Tobacco Mosaic Virus (TMV), Potato Virus X (PVX) and Cucumber Mosaic Virus (CMV), which invade the meristematic region of the growing tips and interrupt the growth of the meristematic tissue. In such cases it has also been possible to obtain virusfree plants by combining meristem-tip culture with thermotherapy. In this technique, first the mother plants are exposed to heat treatment before excising the meristem-tips or, alternatively, shoot-tip cultures are exposed to high temperature regimes (35°C-40°C) for certain duration (6 h to 6 weeks) to obtain virus free plants. In the latter case, continuous exposure to a very high temperature causes deterioration of the host tissues. The first procedure of treating the mother plant has added advantage where larger explants can be taken from the treated stock and thus, favors relatively higher chances of the tip survival. Chemotherapy Chemotherapy is the treatment of an ailment by chemicals especially by killing microorganisms. It will not eradicate the virus completely. However, a large number of antibiotics, growth regulators, amino acids, purines and pyrimidines can be tested for inactivation of viruses. A nucleotide analogue ribavirin has been found to be the most efficient viracide for plant viruses. This broad spectrum antiviral agent, effective against both plants and animals, was reported to eliminate PVY, CMV and TMV from tobacco explant cultures, Chlorotic Leaf Spot Virus (CLSV) in apple cultures when incorporated into the medium. Vidarabine (adenine arabinoside) and antiserum are also known to reduce the titre of viruses. The effectivity of the compound may vary with the virus and the host genotype. Virus identification: diagnostic methods Even after subjecting the meristem-tips to various treatments favoring virus eradication, only a fraction of the cultures yields virus free plants. Therefore, it is

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required to test all plants, regenerated through meristem-tip or callus cultures, for specific viruses before being used as a mother plant to produce virus-free stock. The individual plants consistently showing negative results for virus titre can be marked as ‘virus tested’ for specific virus/es and can be released for commercial purposes. The following tests can be performed for virus testing: 1. Microscopic examination. The simplest test for the presence or absence of viruses in plant tissues is to examine the leaves and stem for the visible symptoms characteristic of the virus. Direct microscopic examinations of ill tissues are especially suitable for biological agents. The lactophenol blue staining technique could be used and slides examined at X 400 magnification could be prepared. 2. Another test is the sap transmission test or ‘bioassay test’ or ‘infectivity test’. It is a very sensitive test and can be performed at a commercial scale. To perform this, ground the test leaves in equal volume (w/v) of 0.5 M phosphate buffer using a mortar and pestle. Leaves of the indicator plant (a plant very susceptible to specific viruses), dusted with 600-grade carborundum, are swabbed with the leaf sap from the test plant. After 5 min the incubated leaves are gently washed with water to remove the residual inoculum. The inoculated indicator plants are maintained in a glasshouse, separately from the other plants. It may take from several days to several weeks, depending on the nature of virus and the virus titre, for the symptoms to appear on the indicator plants. It is used to detect some viruses and viroids, but is a slow process requiring several days to months. 3. The method of enzyme-linked immunosorbant assay (ELISA) is a more rapid serological test which allows quick detection of important viruses. It relies on the use of antibodies prepared against the viral coat protein, requires only a small amount of antiserum and can be performed with simple equipment. However, it is not applicable to viroids and viruses which have lost their coat proteins. The identification of viruses can performed by Dot-ELISA with specific antisera for different virus infection present in samples collected from commercial crop fields and from plants cultivated in vitro (Figure 41). The samples macerate and prepare at a ratio of 1g leaf tissue to 10 mL PBS solution (0.08 M NaH2PO4xH2O; 0.02 M K2HPO4; 1.4 M NaCl; 0.02 M KCl; pH 7.4) [22]. Sample extract (4 μL) is placed on a nitrocellulose membrane. The membrane is dried at room temperature for 30 min and incubated with blocking solution of 0.5% PBS plus 3% non-fat Molico milk under gentle agitation for 3 h. Subsequently, the membranes are placed in PBS solution containing specific antibodies for each isolated virus at a dilution of 1 μg/ml. The second antibody of goat antirabbit IgG alkaline phosphatase conjugate is used at a dilution of 1:30 000 in 0.5%. For detection tests, BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) substrates are used according to the manufacturer’s protocol. Through micropropagation, it is now possible to provide clean and uniform planting materials in plantations – oil palm, plantain, pine, banana, abaca, date,

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rubber tree; field crops – eggplant, jojoba, pineapple, tomato; root crops – cassava, yam, sweet potato; and many ornamental plants such as orchids and anthuriums.

Figure 41. Dot-ELISA assay

Micropropagated plants were found to establish more quickly, grow more vigorously and taller, have a shorter and more uniform production cycle, and produce higher yields than conventional propagules. Control questions: 1. Tissue culture uses a small piece of tissue from a mother plant to grow many new copies of the original plant. What term is used to refer to this small piece of tissue? 2. Why is tissue culture used for propagation of some plants rather than just planting seeds? 3. Why is node culture considered the most reliable and rapid method for in vitro propagation of true-to-type plants? 4. Why is it important to have virus-free seed stocks? 5. What is the source of microbial contamination for in vitro plantlets? 6. Why is the rate of virus elimination higher in meristem cultures than in the nodal segment culture? 7. How does heat act as a therapeutic treatment?

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Chapter 5. Cell engineering

CHAPTER

5

CELL ENGINEERING Application of protoplast technology or cell engineering for improvement of plants offers fascinating option to complement conventional breeding programs. The ability of isolated protoplasts to undergo fusion and take up macromolecules and cell organelles (cell reconstruction) offers many possibilities in genetic engineering and crop improvement. A fundamental difference between plant and animal cells is that ‘plant cells are totipotent’, which formed the basis of plant tissue culture. Another important difference is that the ‘presence of the cell wall in plants’ paved the way for the most significant development in the field of plant tissue culture, which is isolation, culture and fusion of protoplasts. Plant cells, from which the cell wall has been removed, are termed protoplasts. Figure 42 demonstrates the microscopic view of protoplasts isolated from tobacco leaves. Cultured protoplasts, besides being used for genetic recombination through somatic cell fusion, can also be used for taking up foreign DNA, cell organelles, bacteria and virus particles. Therefore, protoplast culture has achieved an important status in plant biotechnology.

Figure 42. Microscopic view of isolated protoplasts from tobacco

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The experiments involving protoplasts (or protoplast culture) consist of three stages – protoplast isolation; protoplast fusion (leading to gene uptake); development of regenerated fertile plants from the fusion product (Hybrid). Depending upon the species and culture conditions, the protoplasts may have the potential to: –– regenerate a cell wall –– dedifferentiate to form callus –– divide mitotically and proliferate clonally –– redifferentiate into shoots, roots or embryos and produce a complete plantlet. Isolation of Protoplast. Plant protoplasts were first isolated by Klercker in 1892 from onion bulb scales plasmolysed in hypertonic solution. This mechanical procedure gave low yield of protoplasts and could be utilized for only highly vacuolated and non meristematic cells. It was in 1960, when Cocking demonstrated the isolation of intact protoplasts by the use of the cell wall degrading enzyme, cellulase prepared from the fungus, Myrothecium verrucaria. By 1968, commercial preparations of purified cell wall degrading enzymes such as macerozyme, cellulase and hemicellulose became available that gave further progress to enzymatic isolation of protoplasts. Hemicelluloses and pectins may be modified and broken down by a variety of enzymes that are found naturally in the cell wall. Glucanases and related enzymes may hydrolyze the backbone of hemicelluloses. Xylosidases and related enzymes may remove the side branches from the backbone of xyloglucan. Transglycosylases may cut and join hemicelluloses together. Such enzymatic changes may alter the physical properties of the wall, for example, by changing the viscosity of the matrix or by altering the tendency of the hemicelluloses to stick to cellulose. Conditions required for enzyme activity –– Enzymes are pH and temperature dependent, thus, for enzymatic release of protoplast an enzyme showing activity at pH range 5-6 and temperature range of 25-30°C is used; –– Duration of enzyme pretreatment and condition of light presence required for incubation may also be determined; –– The enzyme mixture should mainly consist of cellulose, hemicellulase and pectinase, which facilitate the degradation of cellulose, hemicelluloses and pectin, respectively; –– The concentration of sugar alcohols used as osmoticum (mannitol) must be empirically defined. Enzymatic method of protoplast isolation can be classified into two groups:

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1. Sequential enzymatic: This involves two steps where first macerated plant tissues are incubated with pectinase to get single cells followed by cellulase treatment to get protoplast. 2. Mixed enzymatic: This involves simultaneous separation of cells and degradation of their walls to convert protoplast by immersing plant tissues in the mixture of pectinases and cellulases. Protoplasts have been isolated from all plant parts like shoot tips, cotyledons, flower petals, however, leaf mesophyll tissue is widely preferred because of its high reproducible potential for regeneration. Generally, the youngest, fully expanded leaves from young plants or seedlings are used as a source of protoplast. Preconditioning plants in darkness or cold (4-10°C) for 24-72h before protoplast isolation improves protoplast yield. The leaves are surface sterilized and cut into small pieces and floated on an osmotically adjusted solution at 20-25°C for 1-24 h. During this step, plasmodesmatal complex is broken down, water moves out of cell which causes the cell contents to shrink and draw away from the cell wall. This allows cells to retain their integrity after the cell wall is removed otherwise they start fusing. This step is followed by incubation of leaf pieces with digestive enzymes in darkness on shaker (30-50rpm). Incubation time and temperature vary with species and time. The osmotic concentration of enzyme mixture and subsequent media is elevated by adding sorbitol or mannitol to stabilize protoplasts or they will burst. Addition of 50-100mM/l CaCl2 improves stability of plasma membrane. Acidic pH in the range of 5.0-6.0 is optimal. The buffer often contains phosphate at 3mM to minimize shifts in pH during digestion. The most commonly used compounds such as plasmolyticum are the sugar alcohols – mannitol and sorbitol. Of these, mannitol is the most preferred since it is not metabolized by the plant cells. Once the protoplast divides and regenerates the cell wall, no more osmoticum is required. It, therefore, should be removed gradually from the medium otherwise the cell division stops. To slowly remove the osmoticum from the medium, the protoplast can be isolated in a high osmoticum mixture consisting of both mannitol and sucrose, the sucrose will be metabolized by the dividing protoplasts and thus, will reduce the osmolarity of the medium. Normally, mannitol is used at concentration range of 11-13%. A solution into which the osmoticum is often, but not always, added is called CPW salts mix or CPW for short. This has been observed much more beneficial than using distilled water as a solvent in obtaining high yields of viable protoplasts (Table 5). Although CPW is the most widely used solution into which osmoticum or enzymes are added, sometimes the culture medium used to grow cells or plants can also be utilized for protoplast isolation at one tenth concentration. Low concentration of the culture medium is much more advantageous when compared with CPW.

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Table 5 Salt mix of protoplast washing media solution (Cocking, Peberdy and White – CPW) Components KH2PO4 KNO3 CaCl2 x2 H2O MgSO4x7 H2O KJ CuSO4x5 H2O

Concentration, mg/l 27.2 101 1480 246 0.16 0.025 pH=5.8

In case of monocots, where mesophyll cannot be the source, cell suspension cultures are used for isolation of protoplasts. The released protoplasts are separated by centrifugation at a low speed of about ten minutes followed by washing two to three times before transfer to culture medium to remove enzymes and debris. Protoplast viability and Plating Density Before culturing protoplasts, their viability is estimated by staining with Fluorescein diacetate (FDA). Viable protoplasts exhibit green fluorescence under UV. It should be examined within 5-15 min after the FDA treatment after FDA dissociates. Some protoplasts have leaky membranes through which metabolites are leached out. Like cell cultures, the initial plating density of protoplasts has a profound effect on plating efficiency. Protoplasts are cultured at a density of 1 x 104 to 1 x 105 protoplasts ml-1 of the medium. At high density the cell colonies arising from individual protoplasts tend to grow into each other resulting into chimera tissue if the protoplast population is genetically heterogeneous. Cloning of individuals cells, which is desirable in somatic hybridization and mutagenic studies, can be achieved if protoplasts or cells derived from them can be cultured at a low density. If sufficient density of protoplasts is there, metabolites can be absorbed back. Therefore, a minimum plating density is required for growth to begin and it is estimated by haemocytometer. For genetic engineering, single or fewer protoplast culture is required for which either a conditional medium or a feeder layer is used. Conditional medium is the one where the plant cells were already grown, and so it has metabolites leached into it. After filtering, this medium is used for growing isolated protoplasts. The feeder layer is prepared by plating solid medium with protoplasts followed by irradiation which inactivates the nucleus but protoplasts remain viable. Protoplasts at lower density can now be plated on this feeder layer. Protoplast Culture The following techniques have been adopted in order to maintain the number of protoplast population between minimum and maximum effective densities after plating up:

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Liquid method. This method is preferred at earlier stages of culture as it provides easier dilution and transfer, the osmotic pressure of liquid media can be effectively reduced after a few days of culture, the cell density can be reduced or cells of special interest can be isolated easily. In liquid medium, the protoplast suspension is plated as a thin layer in petri plates, incubated as a static culture in flasks or distributed in 50-100 μl drops in petriplates and stored in a humidifier chamber. Embedded in Agar/ Agarose. Agarose is a preferred choice in place of agar and this has improved the culture response. This method of agar culture keeps protoplast in fixed position, thus, prevents it from forming clumps. Immobilized protoplasts give rise to clones which can then be transferred to the other media. In practice, the protoplasts suspended in molten (40°C) agarose medium (1.2% w/v agarose) are dispensed (4 ml) into small (3.5-5 cm diameter) plates and allowed to solidify. The agarose layer is then cut into 4 equal sized blocks and transferred to larger dishes (9 cm diameter) containing liquid medium of otherwise the same composition. Alternatively, protoplasts in molten agarose medium are dispensed as droplets (50-100 μl) on the bottom of petri plates and after solidification the droplets are submerged in the same liquid medium. Feeder layer. In order to culture protoplast at a low density, the feeder layer technique is adopted. The feeder layer of X-ray irradiated non-dividing but metabolically active protoplasts after washing is plated in soft agar medium. Non-irradiated protoplasts of low density are plated over this feeder layer. The protoplasts of the same species or different species can be used as a feeder layer. Co-culturing. This method involves co-culture of protoplasts from two different species to promote their growth or that of the hybrid cells. Metabolically active and dividing protoplasts of two types – slow and fast growing are cultured together, the fast growing protoplast provides other species with diffusible chemicals and growth factors which helps in cell wall formation and cell division. The co-culture method is generally used where calli arising from two types of protoplasts can be morphologically distinguished. For example, protoplasts isolated from albino plants and green plants are easily distinguishable based on color where albino protoplast will develop non green colonies. Freshly isolated protoplasts are spherical because they are unbound by cell wall. Viable protoplasts regenerate a new cell wall within 48 to 96 h after isolation which can be determined by staining with calcafluor white. Protoplasts with new cell wall fluoresce bluish white under UV. Protoplasts that fail to regenerate a wall generally will not divide and die eventually. Also, all the healthy protoplasts may not divide and therefore, plating efficiency is calculated to estimate cell vigor. Plating efficiency is the number of dividing protoplasts/the total number of protoplasts plated. The protoplasts capable of dividing undergo first division within 2- 7 days after isolation (Figure 43).

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Figure 43. Protoplast culture techniques

The delicate nature of protoplasts demands modifications in MS and B5 media or any other culture medium used for organ regeneration from explants. Besides higher osmotica, the inorganic salt concentration is adjusted (Ammonium nitrate concentration is lowered and calcium level is increased), more of organic components, vitamins and PGRs are added to hasten and promote cell wall synthesis. Due to sensitivity to light, protoplasts are cultured in diffuse light for initial 4-7 days. After early culture, when protoplasts have regenerated new cell wall and divided, they are transferred back to normal medium where plants regenerate by shoot formation or somatic embryogenesis. Plant regeneration from protoplasts has been reported from alfalfa, tobacco, carrot, tomato, etc., but cereals still pose problems.

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Protoplast Fusion and Somatic Hybridization Sexual hybridization since time immemorial has been used as a method for crop improvement but it has its own limitations as it can only be used within the members of the same species or closely related wild species. Thus, this limits the use of sexual hybridization as a means of producing better varieties. Development of viable cell hybrids by somatic hybridization, therefore, has been considered as an alternative approach for the production of superior hybrids overcoming the species barrier. The technique can facilitate breeding and gene transfer, bypassing problems associated with conventional sexual crossing such as, interspecific, intergeneric incompatibility. Purified protoplasts once obtained from any two different sources (can be different tissues, different plants or species or different genera), they can be fused together to form somatic hybrids. This non-conventional method of genetic recombination involving protoplast fusion under in vitro conditions and subsequent development of their product to a hybrid plant is known as somatic hybridization. This technique of hybrid production via protoplast fusion allows combining somatic cells (whole or partial) from different cultivars, species or genera resulting in novel genetic combinations including symmetric somatic hybrids, asymmetric somatic hybrids or somatic cybrids (Figure 44). Protoplast fusion could be spontaneous during isolation of protoplast or it can be induced by mechanical, chemical and physical means. During spontaneous process, the adjacent protoplasts fuse together as a result of enzymatic degradation of cell walls forming homokaryons or homokaryocytes, each with two to several nuclei. The occurrence of multinucleate fusion bodies is more frequent when the protoplasts are prepared from actively dividing callus cells or suspension cultures. Since the somatic hybridization or cybridization requires fusion of protoplasts of different origin, the spontaneous fusion has no value. To achieve induced fusion, a suitable chemical agent (fusogen) like, NaNO3, high Ca2+, polyethylene glycol (PEG), or electric stimulus is needed. First, somatic hybrid plant of Nicotiana glauca (+) N. langsdorfii was reported by Carlson in 1972. Protoplasts can be induced to fuse by variety of chemical fusogens or electrical manipulations which induce membrane instability (Figure 45). Most commonly reported fusion inducing chemical agents is sodium nitrate (used by Carlson), high pH/Ca2+ concentration and Polyethylene glycol (PEG) treatment. Sodium nitrate treatment results at low frequency of heterokaryon formation, high pH and high Ca2+ concentration suit few plant species, whereas PEG is the most favoured fusogen for its reproducible high frequency of heterokaryon formation and low toxicity. However, treatment with PEG in the presence of high pH/ Ca2+ is reported to be the most effective in enhancing heterokaryon formation and their survivability.

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Figure 44. Scheme of technique of hybrid production via protoplasts fusion

Note: A – two separate protoplasts; B – agglutination of two protoplasts; C, D – membrane fusion at a localized site; E, F – development of spherical heterokaryon Figure 45. Sequential stages in protoplast fusion

The chemical fusion of plant protoplast has many disadvantages: 1) the fusogens are toxic to some cell systems; 2) it produces random, multiple cell aggregates; 3) must be removed before culture.

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A more selective, simpler, quick and nontoxic approach is electrofusion which utilizes an electric shock or a short pulse of high voltage to promote membrane fusion between two cells. Moreover, the somatic hybrids produced by this method show much higher fertility than those produced by PEG-induced fusion. Electrofusion protocol involves a two-step process. First, the protoplasts are introduced into a small fusion chamber containing parallel wires or plates which serve as electrodes. Second, a low-voltage and rapidly oscillating AC field are applied, which causes protoplasts to become aligned into chains of cells between electrodes. This creates a complete cell-to-cell contact within a few minutes. Once alignment is complete, the fusion is induced by application of a brief spell of high-voltage DC pulses (0.125-1 kVcm-1). A high voltage DC pulse induces a reversible breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent membrane reorganization. The entire process can be completed within 15 min. Many useful somatic hybrid plants produced by electrofusion have been reported like Nicotiana plumbaginifolia (+) N. tobacum, Solanum tuberosum (+) S. charcoense (resistant to Colorado potato beetle) [7]. The most common target using somatic hybridization is the gene of symmetric hybrids that contain the complete nuclear genomes along with cytoplasmic organelles of both parents. This is unlike sexual reproduction in which organelle genomes are generally contributed by the maternal parent. On the other hand, somatic cybridization is the process of combining the nuclear genome of one parent with the mitochondrial and/or chloroplast genome of a second parent. Cybrids can be produced by donor-recipient method or by cytoplast-protoplast fusion. Incomplete asymmetric somatic hybridization also provides opportunities for transfer of fragments of the nuclear genome, including one or more intact chromosomes from one parent (donor) into the intact genome of a second parent (recipient). Cybrids provide the transfer of plasmogenes of one species into the nuclear background of another species in a single generation, and even in sexually incompatible combinations, recovery of recombinants between the parental mitochondrial or chloroplast DNAs (genomes), and production of a wide variety of combinations of the parental and recombinant chloroplasts with the parental or recombinant mitochondria. Methods to produce cybrids They are produced in variable frequencies in normal protoplast fusion experiments due to one of the following methods: 1. Fusion of normal protoplast with an enucleated protoplast. The enucleated protoplast can be produced by high speed centrifugation (20,000-40,000xg) for 60 min with 5-50% percoll. 2. Fusion between a normal protoplast and another protoplast with a nonviable nucleus or suppressed nucleus.

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3. Elimination of one of the nuclei after heterokaryons formation. 4. Selective elimination of chromosomes at a later stage. 5. Irradiating (with X-rays or gamma rays) the protoplasts of one species prior to fusion in order to inactivate their nuclei. 6. By preparing enucleate protoplasts (cytoplasts) of one species and fusing them with normal protoplasts of the other species. These methods provide genetic manipulation of plants overcoming hurdle of sexual incompatibility, thereby, serving as a method of bringing together beneficial traits from taxonomically distinct species which cannot be achieved by sexual crosses. Several parameters such as, the source tissue, culture medium and environmental factors influence the ability of a protoplast derived hybrid cells to develop into a fertile plant. Verification and characterization of Somatic Hybrids Protoplast population following fusion treatment is a heterogeneous mixture of unfused parents, homokaryons, heterokaryons, fused protoplasts with independent two nuclei, etc., thus necessitating selection of stable hybrids. Therefore, some identification and selection system should be incorporated into each parental cell line before fusion. Usually, two parental cell lines with different requirements as selective screens make selection of hybrid convenient as only those fused cells that possess complementary traits of both parents will thrive. Somatic hybrids in most of the cases show characters intermediate between the two parents such as, shape of leaves, pigmentation of corolla, plant height, root morphology and other vegetative and floral characters. The method is not much accurate as tissue culture conditions may also alter some morphological characters or the hybrid may show entirely new traits not shown by any of the parents. Resistance to antibiotics, herbicides and ability to grow on specific amino acid analogues are a few of the selection methods used. Besides these, two different vital stains which do not affect viability of cells like fluorescein isothiocynate (FITC) and rhodomine isothiocyanate (RITC) have been successfully used. Under fluorescent microscope, FITC stained cells appear green and RITC stained protoplasts appear red whereas fused cells having both RITC and FITC fluoresce yellow. Chromosome counting of the hybrid is an easier and more reliable method to ensure hybridity as it also provides the information of ploidy level. Cytologically the chromosome count of the hybrid should be a sum of the number of chromosomes from both parents. Besides, the number of chromosomes, the size and structure of chromosomes can also be monitored. However, the approach is not applicable to all species, particularly, where fusion involves closely related species or where the chromosomes are very small. Moreover, sometimes the somaclonal variations may also give rise to different chromosome numbers.

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Multiple molecular forms of the same enzyme which catalyses similar or identical reactions are known as isozymes. Electrophoresis is performed to study the banding pattern as a check for hybridity. If the two parents exhibit different band patterns for a specific isozyme, the putative hybrid can be easily verified. The isozymes commonly used for hybrid identification include acid phosphatase, esterase, peroxidase. Specific restriction pattern of nuclear, mitochondrial and chloroplast DNA characterizes the plastomes of hybrids and cybrids. Molecular markers such as RFLP, RAPD, ISSR can be employed to detect variation and similarity in banding pattern of fused protoplasts to verify hybrid and cybrid. Application of Somatic Hybridization –– Genetic recombination in asexual or sterile plants: Protoplast fusion has overcome the impediment of reproduction in haploid, triploid and aneuploid plants. Somatic hybridization can be used as a method for the production of autotetraploids. Genomes of asexually reproducing plants can be recombined using this approach viz.protoplasts isolated from dihaploid potato clones have been fused with protoplasts of S. brevidens to produce hybrids of practical breeding value. –– Genetic recombination between sexually incompatible species: The incompatibility barriers in sexual recombination at interspecific or intergeneric levels are also overcome by somatic hybrisation. Generally, somatic hybrids are used for transfer of useful genes such as disease resistance, abiotic stress resistance or genes of industrial use for e.g. Datura hybrids (D. innoxia + D. discolor, D. innoxia + D. stramonium) show heterosis for scopolamine (alkaloid) content which is 20-25% higher than in parent species and therefore has industrial application [2]. –– Cytoplasm transfer. Somatic hybridization minimizes the time taken for cytoplasm transfer to one year from 6-7 years required in the back cross method. Also, this method allows cytoplasm transfer between sexually incompatible species. Cybrids have cytoplasm from both parents but nucleus of only one. Nucleus of the other parent is irradiated. This approach has been potentially used to transfer two desirable traits – cytoplasmic male sterility (CMS) and resistance to atrazine herbicide, both coded by cytoplasmic genes in Brassica to different crops like tobacco, rice, etc. –– Novel interspecific and intergeneric crosses, which are difficult to produce by conventional methods, can be easily obtained. –– Plants in juvenile stage can also be hybridized by means of somatic hybridization. Control questions: 1. Why can the protoplast be used as a starting material for genetic manipulation? 2. What is the role of cell wall degrading enzymes?

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Part II. Animal biotechnology 3. Name the best source used for protoplast isolation. 4. Which chemical is considered to be the most important media component for protoplast isolation and culture? 5. How do you count protoplasts and why it is essential? 6. List out different protoplast culture techniques. Why do you think a sequential reduction in osmoticum is necessary during culture? 7. What are the different methods of protoplast fusion? Explain the mechanism involved in PEG and High Ca2+ method of fusion. 8. Can you differentiate fusion partners during protoplast fusion?

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Chapter 6. Cell selection

Chapter

6

CELL SELECTION Development of modern experimental biology depends on new model systems which allow estimation of the potential of the plant cell in biotic and abiotic stress conditions (selective conditions). The conventional model system allows us to select at the level of the intact plant. Plant response to abiotic stress is a complex phenomenon, which could be approached efficiently through in vitro culture. Tolerant lines derived from conventional breeding programs or resulting from transgenic transformations could be screened via in vitro culture. This is particularly attractive for certain abiotic stresses where appropriate screening methods are unavailable or not efficient. Plant tissue culture techniques provide a promising and feasible approach to develop salt-, drought-, heavy metall-, heat- tolerant, pathogen-, insect tolerant plants. Cell selection is the select in culture means the select at the level of the single cell. Selection strategies are: –– Positive –– Negative –– Visual –– Analytical Screening. Positive selection allows us to select cells, which have resistance to stress factors (biotic and abiotic). In positive selection a toxic compound (e.g. hydroxy proline, kanamycin and other stress agents) is added into the nutrient medium, after 2-4 weeks cells resistant to toxic substance are selected. In this case only the cells able to grow in the presence of the selective agent give colonies. It is possible to select one colony from millions of plated cells in a day work (possible to plate up to 1,000,000 cells on a Petri-dish). In negative selection, one should add an agent that kills dividing cells (for example, chlorate / BUdR, or treatment by mutagenic agents) into medium, then plate out leave for a suitable time, wash out agent and put on growth medium. In this case, all cells growing on selective agent will die leaving only non-growing cells to grow. This selection strategy is useful for selecting auxotrophs. 345

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Visual selection is useful only for coloured or fluorescent compounds, e.g. shikonin/ Berberine/ some alkaloids. At this type of selection one can plate out at about 50,000 cells per plate and pick off coloured / fluorescent compounds. It is possible to screen about 1,000,000 cells in a day work. Salinity is one of the main abiotic stresses that has been addressed by in vitro selection. High temperature and drought have detrimental effects on plant growth and development, such as tassel initiation and time of flowering, pollen sterility, rate and duration of endosperm cell division. High temperature induced oxidative stress in plants, which caused lipid peroxidation and consequently membrane injury, protein degradation, enzyme inactivation, pigment bleaching and disruption of DNA strands. In addition, this is the major factor influencing the embryogenic response and plant regeneration [30]. Stress is usually defined as an external factor that exerts a disadvantageous influence on the plant. Under both natural and agricultural conditions, plants are exposed to unfavorable environments that results in some degree of stress. Water deficiency, heat stress and heat shock, chilling and freezing, salinity, and oxygen deficiency are major stress factors restricting plant growth such that biomass or agronomic yields at the end of the season express only a fraction of the plant’s genetic potential. The capacity of plants to cope with unfavorable environments is known as stress resistance. Stress caused by water deficiency leads to the expression of sets of genes involved in acclimation and adaptation to the stress. These genes mediate the cellular and whole-plant responses. Sensing and activation of signal transduction cascades mediating these changes in gene expression involve both an ABA-dependent pathway and an ABA-independent pathway. Heat stress and heat shock are caused by high temperatures. Some CAM species can tolerate temperatures of 60° to 65°C, but most leaves are damaged above 45°C. Heat stress inhibits photosynthesis and impairs membrane function and protein stability. Heat shock proteins synthesized at high temperatures act as molecular chaperones that promote stabilization and correct folding of cell proteins, and biochemical responses leading to pH and metabolic homeostasis are also associated with acclimation and adaptation to rapid rises in temperature. Chilling and freezing stress ensue from low temperatures. Chilling injury occurs at temperatures that are too low for normal growth but are above freezing, and it is typical of species of tropical or subtropical origin exposed to temperate climates. Membrane lipids of chilling-resistant plants often have a greater proportion of unsaturated fatty acids than those of chilling-sensitive plants. Transgenic plants overexpressing cold stress–activated signaling components demonstrate increased cold tolerance. Salinity stress results from salt accumulation in the soil. Some halophyte species are highly tolerant to salt, but salinity depresses growth and photosynthesis in sensitive species. Salt injury ensues

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from a decrease in the water potential of the soil that makes soil water less available and from toxicity of specific ions accumulated at injurious concentrations. Plants avoid salt injury by exclusion of excess ions from leaves or by compartmentation of ions in vacuoles. Some of the molecular determinants of Na+ exclusion and vacuolar partitioning have been determined, and no signaling pathway, the SOS pathway, regulating the expression of these genes involved in ion homeostasis has been established. Oxygen deficiency is typical of flooded or waterlogged soils. Oxygen deficiency depresses growth and survival of many species. On the other hand, plants of marshes and swamps, and crops such as rice, are well adapted to resist oxygen deficiency in the root environment. Most tissues of higher plants cannot survive anaerobically, but some tissues, such as the embryo and coleoptiles from rice, can survive for weeks under anoxic conditions. Cellular responses to stress include changes in the cell cycle and cell division, changes in the endomembrane system and vacuolization of cells, and changes in cell wall architecture, all leading to enhanced stress tolerance of cells. At the biochemical level, plants alter metabolism in various ways accommodate to environmental stresses, including producing osmoregulatory compounds such as proline and glycine betaine. The molecular events linking the perception of a stress signal with the genomic responses leading to tolerance have been intensively investigated in recent years [30, 31]. The major advantages of cell culture systems over the conventional cultivation of the whole plants are: –– Higher and quicker yields of product from very small amount of plant material needed to initiate the culture in contrast to large amounts of mature plant tissues processed to achieve low yields of final product, for example, the dry weight of shikonin produced from cell culture is 20% more than from plants. –– In case plant material is facing threat of extinction or is limited in supply like L-erythrorhizon, in vitro production of secondary metabolites is a saving option. –– Controlled environmental conditions in cell culture ensure continuous supply of metabolites. In the conventional system, the source plant may be seasonal, location specific and also subject to environmental degradation. Also, in vitro culture of cells is more economical for those plants which take long to achieve maturity (Figure 46).�������������������������������������������������������������������� Population can be stored for years and used for many different projects. It is an advantage of induced mutagenesis. Population can also be used in forward genetic screens.

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Figure 46. Conventional system and in vitro cell culture for producing stress-resistant plant via induced mutagenesis

–– Bioconversion: Low cost precursors are supplied as substrates to cell cultures for conversion to the high cost final product, thus minimizing labor, cost and time. Also, specific substrates can be biotransformed to a more valuable product by a single-step reaction in vitro. –– Production of novel compounds: Mutant cell lines can be utilized to produce novel compounds which were not previously found naturally in plants viz. cell suspension cultures of Rauwolfia serpentina produce novel glucosides of ajmaline (alkaloid) [2]. In vitro tissue culture could be an important means of improving crop tolerance and yield through genetic transformation as well as by induced somaclonal variation. Therefore, it is important to devise an efficient protocol of callus proliferation to start in vitro selection for biotic and abiotic stress tolerance, and to broaden opportunities for genetic manipulation of plants through tissue culture, including trying various explants and media. Plants generally exhibit cytogenetic and genetic variations which help the plant breeders in crop improvement. When such variants arise through the cell and tissue culture process using any plant portion as an explant material, variations arising are termed as somaclonal variations. Variants obtained using callus cultures are referred as “Calliclones” (Skirvin, 1978) while variants obtained using protoplast cultures are known as “Protoclones” (Shepard et al. 1980). Larkin and Scowcroft (1981) proposed a general term ‘Somaclonal variation’ to describe genetic variation in plants regenerated from any form of cell cultures. Accordingly, the plants derived from cell and tissue cultures are termed as ‘somaclones’,

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and the plants displaying variation as ‘somaclonal variants’. Another term suggested by Evans et al. (1984) as ‘gametoclonal variation’ for those variations arising in cell cultures of gametic origin like, in pollen and microspores cultures, to distinguish them from somatic cell derived regenerants. However, generally the term somaclonal variation is used for genetic variability present among all kinds of cell/plants obtained from cell cultures in vitro. Plants regenerated from tissue and cell cultures show heritable variation for both qualitative and quantitative traits. Several useful somaclonal variants have been obtained in a large number of plant species such as, potato, sugarcane, banana, tomato, etc. Chaleff (1981) labeled plants regenerated from tissue cultures as R0 generation and their successive sexual generations as R1, R2 and so on [3]. Somaclonal Variation The genetic variability present in somatic cells, plants or plant progenies derived from cells/tissue cultured in vitro is called somaclonal variation. Larkin and Scrowcroft (1981) coined this term for all plant variants derived from any form of cell or tissue culture. Some variants are obtained in homozygous condition in the plants regenerated from cells cultured in vitro (R0 generation) but mostly variants are recovered in the selfed progeny of tissue culture regenerated plants (R1 generation). This variation includes aneuploids, sterile plants and morphological variants, some of which may involve traits of economic importance for crop plants. Somaclonal variation may be genetic or epigenetic. Since only gametic variation follows the Mendelian inheritance pattern and is transmitted to the next generation, they are important for crop improvement. Therefore, in several crops, R0, R1 and R2 progenies are analysed for transmission of variant trait to sexual progeny (R1) and 3:1 segregation leading to isolation of true breeding variants (R2). The significance of somaclonal variation in crop improvement was first demonstrated in sugarcane and potato where a few somaclones with disease resistance against Fiji, downy mildew (sugarcane) and late and early blight in case of potato were recovered [3]. Major causes of Somaclonal Variation The basic cause of these variations may be attributed to changes in karyotype – genetical changes: chromosome rearrangements (chromosome number and structure), somatic crossing over, sister chromatid exchange, DNA amplification and deletion, transposable elements and DNA methylation. Somaclonal variation can be characterized based on morphological, biochemical (isozymes) and DNA markers such as, Random Amplified Polymorphic DNA (RAPDs), Restriction Fragment Length Polymorphism (RFLPs) and Inter-Simple Sequence Repeats (ISSR).

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The variations could also arise in tissue culture due to physiological changes induced by the culture conditions. Such variations are temporary and are caused by epigenetic changes. These are non-heritable variations and disappear when the culture conditions are removed. Variations induced by physiological factors in culture medium for e.g. prolonged exposure to PGRs (2.4-D; 2,4,5-T) results in variability among the regenerants. Often such variations are epigenetic and hence donot follow Mendelian inheritance. The somaclonal variations observed in plants regenerated from cultured cells are derived from two sources: 1) some of the variations could be revelation of the inherent cellular heterogeneity of the explant, and 2) culture conditions may bring about new genetic changes. All the alterations at chromosomal level are grouped under genetic cause of variation observed in regenerants. Chromosomal rearrangements such as deletion, duplication, translocation, inversion polyploidy, aneuploidy, have been reported to be the chief source of somaclonal variation (Figure 47). Meiotic crossing over involving symmetric and asymmetric recombination could also be responsible for variation observed among somaclones. Transposable elements like Ac-Ds in maize have been shown to get activated in in vitro culture. In maize (Zea mays L.) and broad beans (Vicia faba L), late replicating heterochromatin is the main cause of somaclonal variation. Single gene mutations in cultures also give rise to variations which are not detected in plants regenerated in vitro from any cell or tissue (R0 plants) but express in R1 plants (after selfing R0 plants). Biochemical: The most common kind of biochemical variation is change in carbon metabolism leading to failure of photosynthesis viz. albinos is rice. Any variation in other cell processes like starch biosynthesis, carotenoid pathway, nitrogen metabolism, antibiotic resistance, etc. also lead to somaclonal varaiation (Figure 48). Mechanisms of genetic changes (or spontaneous mutations)

Figure 47. Sources of somaclonal variation

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A

B

Figure 48. Mutagenesis creates heritable changes in DNA (A); The range of mutation types can have differing functional consequences (B) /from “Plant Breeding Unit” Activities Report. FAO/IAEA Programme. Nuclear Techniques in Food and Agriculture. 2008/

Application in Crop Improvement Somaclonal variation represents a useful source for introduction of valuable variations to plant breeders. Cell culture systems are well defined controlled environments, away from limitations of availability of space, time and variations due to the environmental effects which are the major bottlenecks in conventional mutation breeding [32]. In “mutation breeding” plants were exposed to γ-rays, X-rays, protons, neutrons, α-, and β- particles to see if these could induce useful mutations. Mutation-inducing chemicals such as sodium azide and ethyl- (or methyl)- methanesulphonate, were also used to cause mutations. Mutation breeding efforts continue around the world today. In the 75 years of mutation breeding (1939-2015), a total of 3,218 varieties obtained through mutation breeding have been registered in the IAEA database. Staple crops such as rice has registered 824 varieties, barley (312), wheat (274), maize (96), common bean (57), tomato (20), potato (16), sugarcane (13), soybean (2), as well as other important crops that were improved to possess agronomically-desirable characteristics [32; http://www-infocris.iaea.org]. Somaclonal variation occurs at much higher frequencies than induced mutants which are associated with undesirable features. Cell culture systems allow plant breeders to have greater control on selection process as here they have the option to select from a large amount of genetically uniform material. Therefore, this is the only approach to genetic improvement in perennial species limited by narrow germplasm and long regeneration cycle, asexually propagated plants like bananas, for isolation of biochemical mutants like auxotrophs. Somaclonal variants have been isolated for a variety of valuable traits like disease resistance, stress (salt, low temperature) resistance, improved yield and

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efficient nutrient uptake, etc. Bio-13 is a somaclonal variant of Citronella java, a medicinal plant which yields 37% more oil and 39% more citronellol than the control varieties [22]. Control Questions: 1. Why is a single cell culture advantageous over complete plant tissue culture? 2. What is the importance of stress-resistant plants for crop improvements? 3. Explain the factors, which influence the induction of somaclonal variants? 4. What are the main causes of somaclonal variation? 5. What are the differences between somaclonal variation in vitro and genetic variability in vivo?

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Chapter 7. Embryo and endosperm culture

Chapter

7

EMBRYO AND ENDOSPERM CULTURE Double fertilization is unique to the flowering plants. In plants, as in all other eukaryotes, the union of one sperm with the egg forms a single-celled zygote. In angiosperms, however, this event is accompanied by a second fertilization event, in which another sperm unites with two polar nuclei to form the triploid endosperm nucleus, from which the endosperm (the tissue that supplies food for the growing embryo) will develop. Embryogenesis occurs within the embryo sac of the ovule while the ovule and associated structures develop into the seed. Embryogenesis and seed development are highly ordered, integrated processes, both of which are initiated by double fertilization. When completed, both the seed and the embryo within it become dormant and are able to survive long periods unfavorable for growth. The ability to form seeds is one of the keys to the evolutionary success of angiosperms as well as gymnosperms. Embryogenesis and endosperm development typically occur in parallel with seed development, and the embryo is part of the seed. Endosperm may also be part of the mature seed, but in some species the endosperm disappears before seed development is completed. In monocots, the food reserves are stored mainly in the endosperm. In many dicots, the endosperm develops rapidly early in embryogenesis but then is reabsorbed, and the mature seed lacks endosperm tissue. Embryo culture in vitro under controlled conditions supports the development of embryos which use nutrient compounds from the culture media that is the same as the composition of the endosperm. Embryos isolated at the early stage of embryogenesis are used as explants. For embryo culture of ornamental trees the basal media 1/2 DCR and 1/2 LV supplemented with 300 mg/l ascorbic acid and 300 mg/l glutathione, 1 mg/l 2.4-D and 2 mg/l 6-BA were used for initiation somatic embryogenesis. At first 1-2 weeks embryo culture were incubated at 24±20 C in darkness. Morphological, onthogenetic and quantitative parametrs of proliferating embryos’ tissue were different and depend on the medium composition used for initiation and proliferation embryogenic suspensor mass (ESM). The addition of glutathione into the medium increased the frequency of 353

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ESM formation on the initiation stage, but inhibited the embryos development and even led to their degradation in the proliferation stage. Some factors contributed by the germinating embryo are required for the stimulation of mature and dried endosperms of a few plant species. In general, it has been found that mature endosperm requires the initial association of embryo to form callus but immature endosperm proliferates independent of the embryo. However, in neem the association of the embryo proved essential to induce callusing of immature endosperm; the best explant was immature seeds. However, the embryo factor can be overcome by the use of GA3 as was observed in Croton bonplandianum and Putranjiva roxburghii. It is reported that during germination, the embryo releases certain gibberellin like substances, which may promote the endosperm proliferation. However, the mature endosperms of Achras sapota, Santalum album, Emblica officinalis and Juglans regia could proliferate without the association of embryo or pre-soaking of them in GA3. Endosperm is a unique tissue in its origin, development and ploidy level. It is a product of double fertilization but unlike the embryo it is triploid and develops into a formless tissue. It is, therefore, an interesting tissue for morphogenesis. Any abnormality in the development of endosperm may cause the abortion of embryo resulting in sterile seeds. The endosperm may be totally consumed by developing embryo leading to the formation of exalbuminous (non-endospermous) seeds or when it persists, the seed is called albuminous (endospermous). In albuminous seeds, it is used as a food source which may contain proteins, starch or fats and the embryo can utilize this food during seed germination. Cellular totipotency of endosperm cells was first demonstrated by Johri and Bhojwani in 1965 [3]. To date, differentiation of shoots/embryos/plantlets from endosperm tissue has been reported for more than 64 species belonging to 24 families. In many of these reports the regenerants were shown to be triploid. A key factor in the induction of cell divisions in mature endosperm cultures is the association of embryo. The embryo factor is required only to trigger cell divisions; further growth occurs independent of the embryo. Triploid plants are usually seed-sterile. However, there are many examples where seedlessness caused by triploidy is of no serious concern or, at times, even advantageous. Some of the crops where triploids are already in commercial use include several varieties of apple, banana, mulberry, sugar beet and watermelon. Natural triploids of tomato produced larger and tastier fruits than their diploid counterparts. Traditionally, triploids are produced by crossing induced superior tetraploids and diploids. This approach is not only tedious and lengthy (especially for tree species) but in many cases it may not be possible due to high sterility of autotetraploids. The first step in the process is to produce tetraploids by colchicine treatment of germinating seeds, seedlings or vegetative buds. In most of these cases the rate of induction of tetraploids has been low (7-22%). Moreover, the

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treatment is lengthy and laborious. Once tetraploids have been produced, their cross with the diploid parent may not be successful in majority of the cases. In successful crosses the seed-set, seed germination and survival rate of the seedlings is low. Moreover, all sexually produced triploids do not behave uniformly, which may be due to segregation both at tetraploid level and subsequent population of crosses with putative diploid. In contrast, in vitro regeneration of plants from endosperm, a naturally occurring triploid tissue, offers a direct, single step approach to triploid production. The selected triploids, expected to be sexually sterile, can be bulked up by micropropagation. Organogenesis from endosperm tissue was first shown in Exocarpus cupressiformis (a member of the family Santalaceae) [3]. The pathway of plant regeneration includes shoot-bud differentiation or embryogenesis directly from the explants or indirectly from proliferating callus (Figure 49 A-C). In almost all the parasitic angiosperms, the endosperm shows a tendency of direct differentiation of organs without prior callusing, whereas in the autotrophic taxa the endosperm usually forms callus tissue followed by the differentiation of shoot buds, roots or embryos. Direct shoot regeneration from the cultured endosperm was observed in a number of semiparasitic angiosperms including Exocarpus, Taxillus, Leptomeria, Scurrula and Dendrophthoe .

Note: A- an immature seed in culture has split open after 2 weeks releasing the green embryo and callused endosperm; B-white fluffy endosperm callus can be seen from the fully opened seed after three weeks. Embryo is lying at one end of the explant; C- 6-week-old subculture of endosperm callus showing the differentiation of distinct shoots and nodules as well (from website http://nptel.ac) Figure 49. Shoot regeneration from endosperm callus of Azadirachta indica

In Exocarpus, an auxin (IAA) along with cytokinin (Kinetin) was required for direct shoot regeneration. Addition of zeatin in WM gave rise to green shoots from the intact seed (i.e. endosperm with embryo) culture of Scurrula pulverulenta which on subculture gave rise to characteristic haustoria. In Taxillus vestitus, shoot bud formation occurred on WM supplemented with IAA, kinetin

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and casein hydrolysate, after seven weeks. Replacement of IAA with IBA could induce shoot regeneration in 55% cultures and haustoria in 60% cultures. Here, the embryo had an adverse effect on bud differentiation from endosperm. Injury to the endosperm was found to be beneficial for shoot induction in some cases; shoot buds first develop along the injured region. The position of the explant on medium plays a significant role in regeneration of shoot in Taxillus spp. When half split T. vestitus endosperm without embryo was placed on medium with its cut surface in contact with the medium containing kinetin, 100 % of the cultures produced shoots. In Leptomeria acida, IBA proved more efficient than IAA in terms of rapid callus proliferation. However, on IAA supplemented medium the callus gave rise to shoots in 100% cultures [21]. The callus proliferation from endosperm and the subsequent shoot orga­ no­genesis was also reported in Jatropa panduraefolia, Putranjiva roxburghii, Codiaeum variegatum, Malus pumila, Oryza sativa, Annona squamosa, Actinidia chinensis, Mallotus philippensis, Actinidia deliciosa, Morus alba, Azadirachta indica and Actinidia deliciosa. In Actinidia species callus initiation occurred on MS medium supplemented with 2,4-D and kinetin. Transfer of these calli to MS medium containing IAA and 2ip resulted in shoot and root organogenesis. In apple, endosperm proliferated into callus on MS medium supplemented with kinetin + 2,4-D/BA + NAA and subsequent regeneration occurred on MS medium fortified with BA and casein hydrolysate. In Annona squamosa the callusing of endosperm occurred on WM supplemented with two cytokinins (kinetin and BA), an auxin (NAA) and Gibberellic acid (GA3). But organogenesis in the callus occurred on Nitsch’s medium supplemented with BA and NAA [3, 21]. In Mallotus philippensis, a continuously growing callus was obtained on MS medium supplemented with 2, 4-D and kinetin. These calli when subcultured on MS with BA and casein hydrolysate gave rise to various morphologically distinct cell lines, of these, only the green compact cell line was responsive for organogenic differentiation. Shoot regeneration occurred in this callus when subcultured on MS medium fortified with BA and NAA. Immature endosperms of neem (Azadirachta indica) showed best callusing on MS supplemented with NAA, BA and casein hydrolyzate. When the callus was transferred to a medium containing BA or kinetin, shoot buds differentiated from all over the callus. Maximum regeneration in terms of the number of cultures showing shoot-buds and the number of buds per callus cultures occurred in the presence of BA. The percentage of response was the highest on BA and NAA containing medium. However, the number of shoots per explants was maximum when TDZ alone was used. Physical factors include effect of temperature, light and pH on endosperm proliferation. Straus and La Rue (1954) observed that corn endosperms develop better in dark than in light conditions. But in Ricinus reverse is the case where a

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continuous light period was found optimum for endosperm proliferation (Srivastava 1971). In some cases, the endosperms were cultured along with the embryo and kept in the diffuse light with 16 h photoperiod. Light conditions facilitate the early germination of embryo and the embryos can be removed easily due to their characteristic green colour. In coffee, the endosperm callus grows better under 12 h light/dark conditions (Keller et al. 1972). In Lolium the light doesn’t have any significant role on endosperm proliferation. Not much research has been carried out up to date with regard to the effect of temperature and pH on endosperm proliferation. In available literature the optimum temperature for endosperm growth was reported to be 25°C [31]. The pH varies from 4.0 for Asimina to 5.0 for Ricinus, 5.6 for Jatropa and Putranjiva and 6.1 for Zea mays. In general, 5.5 to 5.8 pH seems to support the best growth of endosperm tissues in cultures [21]. In Rice, there was a striking difference in the growth response of immature and mature endosperm. Immature endosperm underwent two modes of differentiation i.e. direct regeneration from the explant or indirectly via intervening callus phase. In mature endosperm, shoot organogenesis was always preceded by callusing. Callus from mature endosperm was initiated and maintained on MS with 2,4-D; shoot differentiation from callus occurred on MS supplemented with IAA and kinetin. The proliferation of immature endosperm and occasional shoot formation occurred on YE supplemented medium; addition of IAA and kinetin improved the response further [33]. The development of a new rice plant type for West Africa (NERICA – New Rice for Africa) was a result of wide crosses between the Asian Oryza sativa and the African rice Oryza glaberrima. It employs embryo rescue in the initial breeding and in the successive back crossing work followed by anther culture to stabilize the breeding lines. The new plants combined yield traits of the sativa parent with local adaptation traits from glaberrima. Embryo rescue involves the culture of immature embryos of plants in a special medium to prevent abortion of the young embryo and to support its germination (Figure 50). This is used routinely in breeding parental lines having different or incompatible genome such as in introducing important traits of wild relatives into cultivated crops.

A. Emasculation

B.Pollination

C.Excision of the embryo

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D. Embryo culture in 1/4 MS medium

F.Germination

G.Rooting

Figure 50. Embryo Rescue (from http://www.warda.cgiar.org website of West Africa Rice Development Association /WARDA/)

Wild rice is a rich source of traits for resistance to pests and abiotic stresses. At the International Rice Research Institute (IRRI), embryo rescue is utilized and facilitated the transfer of bacterial blight resistance genes from wild rice Oryza longistaminata to variety IR24 resulting to a bacterial blight resistant line (IRBB21). Oryza rufipogon is a source of tungro resistance to a number of rice varieties. At IRRI, a new super salt tolerant rice was developed by saving the embryo produced in the cross between highly salt tolerant wild rice Oryza coarctata with cultivated rice variety IR56. The research team headed by Dr. Kshirod Jena has been attempting to cross the two rices since the mid 1990s and has been successful only recently. Selected salt tolerant lines will be tested further by farmers in salt affected locations for a possible release within 4 to 5 years (http://www.researchgate.net/publication /223276104). Control questions: 1. What are the most defining benefits of endosperm culture in vitro? 2. Name the plants where endosperm culture has been extensively used for practical applications. 3. How can you differentiate between triploid and diploid plants during the growth until maturity? 4. What are advantages of embryo culture?

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Chapter 8. Haploid technology

Chapter

8

HAPLOID TECHNOLOGY Plants usually reproduce through sexual means – they have flowers and seeds to create the next generation (Figure 51). Egg cells in the flowers are fertilized by pollen from the stamens (male part) of the flower of the same plant (self-pollination) or another plant (cross). Each of these sexual cells contains genetic material in the form of DNA. During sexual reproduction, DNA from both parents is combined creating off springs similar to the parents (in self-pollinated crops), or in new and unpredictable ways, creating unique organisms (in cross-pollinated crops). Some plants and trees on the other hand need several years before they flower and set seeds, making plant improvement difficult. Plant scientists have developed the science and art of tissue culture to assist breeders in this task. In vitro culture of reproductive organs – anthers and microspores, ovaries and ovules can be used as experimental models for the study of embryology and genetics. At the same time, the culture of male and female gametophytes increases the potential for practical application in breeding programs for obtaining haploid and then homozygous plants. The production of homozygous plants is a difficult and long procedure. Conventional methods of plant breeding need many cycles of selection to isolate a promising genotype and genetically constant lines. When the conventional method is used, this procedure takes approximately twelve to fifteen years. Haploid plant obtained with haploid technique (anther or ovary culture in vitro) accelerates the breeding cycle by shortening the time required to attain homozygosity (Figure 52) [35]. Haploid plant is defined as a sporophyte with gametophytic chromosome number (n). Double haploid techniques provide plant breeders with pure lines in a single generation. The use of elite lines as crossing parents combines the opportunity to select more efficient agronomic traits in homozygous plants. Therefore, double haploid breeding strategies have competitive advantages compared with conventional methods. Anther culture of F1 plants, which are 359

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progenies in a specific breeding objective, would allow many more different types of regenerants. This is because the genetic constitution of the pollen will be more varied than those from the inbreds, thus breeders will have a wider range of traits to choose from. This technology has been employed in the successful development of doubled haploid lines of rice, wheat, sorghum, barley, and other field crops. When the F1 is used, this technique has the advantage that genetic uniformity is achieved in a short time after the initial hybridization. A gameto-phyte makes gametes which fuse to make sporophytes

A sporo-phyte makes spores which germinate to form gameto-phytes

Alternation of generations Figure 51. Life Cycle of Higher Plants

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Figure 52. Pure line (inbred line) development with conventional breeding (A) and anther culture derived haploid plants for hybrid sorting (B)

The in vitro production of haploid plants can be achieved by many techniques such as: –– Delayed pollination which may not result in fertilization and hence only female genome grows up to form a haploid plant. –– Temperature shock – extremes of temperature (both high and low) are used to suppress syngamy or make pollen inactive, thus leading to induction of haploidy. –– Irradiation effect – X rays, UV rays induce chromosomal breakage in pollen cells thus making them sterile which in turn results in haploid production. –– Chemical treatment – treatment with colchicines, maleic hydrazide and toluene blue, etc., also induces chromosomal elimination. –– Genome elimination by distant hybridization – in case of distant crosses like inter-generic and inter-specific crosses where during the developmental process, one of the parental genomes is selectively eliminated subsequently leading to formation of haploid plants. Production of a haploid plant where the egg cell is inactivated and only a male genome is present is called androgenesis. Similarly, production of a haploid by the development of an unfertilized egg cell due to inactivation of pollen is called gynogenesis. Among all the methods illustrated above, anther culture is the most popular and successful for haploid production (Figure 53). Anther culture is a tissue culture method used to develop improved varieties in a short time. Pollen within an anther contains half dose of the genome (haploid) which spontaneously doubles (diploid) during culture. In some species, however,

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colchicine treatment is necessary to induce doubling. Doubling of the genome will allow the expression of recessive traits which were suppressed, masked or undetected in routine plant breeding. Anther culture allows a rapid production of appropriate genotypes for breeding purposes in an effort to identify promising pure lines [36]. Pure line development involves firstly, the selection of lines in the existing germplasm, which expresses the desired characteristics such as resistance to pest and diseases, early maturity, yield, and others. These traits may not be present in only one line, thus selected lines are bred together by hand.

Figure 53. Pathways of plant haploid production (from Forster B.P., Heberle-Bors E., Kasha K.J., Touraev A. The resurgence of haploids in higher plants. Trends Plant Sci., 2007, N 12 (8), pp.368-375)

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In self-pollinated plants, flowers are emasculated by removing the anthers or the male part of the flower by hand, and are pollinated by pollen from another line. The female parent is usually the line that possesses the desired agronomic trait while the male parent is the donor of the new trait. F1 (first filial generation) offsprings are planted and selfed, as well as the F2 generation. Breeders then select in the F3 and F4 generation the lines which exhibit their desired agronomic characteristics and the added trait. Tests on resistance to pests and abiotic stresses are also conducted at this time. Lines with desired traits rated intermediate to resistance/tolerance to the pests and abiotic stresses are selected and selfed in two to three more generations. Lines which do not lose the new traits and are stable are termed pure lines. In hybrid seed technology, two pure lines with complementing traits derived from diversely related parents are bred together by hand. F1 hybrids are tested for hybrid vigor in all agronomic and yield parameters and compared to both parents. The resulting offsprings will usually perform more vigorously than either parent. With the proven impact of hybrid seed technology, new tools for hybrid breeding were discovered and utilized for self-pollinating crops including cytoplasmic male sterility (cms). Cytoplasmic male sterility is a condition where the plant is unable to produce a functional pollen and would rely on the other pollen source to produce seeds. This greatly facilitates large-scale hybrid seed production, by-passing hand pollination. The technique of interspecific and intergeneric hybridization can be combined with anther culture techniques for obtaining new genotypes with alien chromosomes. Thus, new genotypes with various reconstructed chromosome complements can be obtained after their successful chromosome doubling. The production of double haploids through androgenesis represents a modern tool for the improvement of cultivated species. Anther culture procedure: anthers are placed in a special medium, and immature pollen within the anther divide and produce a mass of dividing cells termed callus. Healthy calli (plural of callus) are picked and placed in another medium to produce shoots and roots (regeneration). Stable plantlets are allowed to grow and mature in the greenhouse. Plant breeders can then select the desired plants from among the regenerated plants. Developmental stage of harvested spikes/flower buds, culture medium, duration of cold pretreatment and genotype may affect haploid induction in anther culture whereas among them genotype plays a major role in embryoid and green plant production. Anther Culture Culturing anther on a suitable media to regenerate into haploid plants is called anther culture. Haploid plants were first discovered in Datura stramonium by A.D. Bergner in 1921. Guha and Maheshwari (1964) pioneered the formation of embryos from anthers of Datura innoxia grown in vitro. In 1989-2004, the

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research team (K.Zh. Zhambakin, B.B. Anapiyaev, K.K. Boguspaev, S.K. Tu­ra­ sheva) headed by Professor I.R. Rakhimbaev obtained haploid plants of wheat, barley, rise, potato which are used in the breeding program for creation of new cultivars in Kazakhstan. After this, haploid plants have been produced via anther culture in more than 170 species. Strategies for doubled haploid production Young healthy plants grown under controlled conditions are used as experimental material from which the flower bud of right stage (varies with species) is excised. Antiseptic treatment. Disinfestation, excision and culture of anther. –– Surface sterilization of buds or inflorescences –– Surface sterilization of anthers Flower buds are surface sterilized in the laminar flow chamber followed by excision of anther from the bud. Stage of pollen development is determined by squashing an anther in acetocarmine and observing it under microscope. Isolation of anthers. Isolation of microspores –– Manual –– Blender –– Magnet stirring While excising anthers from flower buds, care is taken that anthers are not injured as injury leads to callusing, hence, giving a mixture of diploids, haploids and aneuploids. Stress treatment for reprogramming –– Starvation –– Heat shock –– Cold treatment (inflorescence, bud, microspores) –– pH –– sugars, hormones Culture medium conditions: The anthers are generally cultured on a solid agar medium where they develop into embryoids for anther culture under alternate light and dark period (Figure 54). Medium should have sucrose for induction of embryogenesis. For microspore culture use only liquid medium. Transfer to “germination” medium In species following direct androgenesis i.e. which develop through embryoid formation, small plants emerge in 3-8 weeks after culturing, which are then transferred onto a rooting medium with low salt and small amount of auxin. Those species undergoing indirect androgenesis involving callus formation, callus is removed from the anther and placed onto a regeneration medium with suitable ratio of cytokinin to auxin. The haploid plants thus produced in both cases are transplanted to soil in small pots and maintained under controlled conditions in greenhouse.

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Diploidisation of haploid plants Haploid plants produced from anther culture maintained in vitro can grow till the flowering stage but cannot be perpetuated. Since these plants are haploid and have only one set of homologous chromosomes of the diploid species, they cannot form viable gametes and hence no seed setting takes place for further perpetuation. Therefore, it is necessary to double the chromosome number of haploids by treatment with colchicine or colchicine derivatives (microtubule-disrupting agents) to obtain homozygous diploids or dihaploid plants followed by their transfer to culture medium for further growth (Figure 55). Cochicine is a microtubule-depolymerizing drug. The chemical modification of natural colchicine (an analogue of colchicine, colchicine derivatives) used for induced diploidization of haploids, is more efficient as chromosome-doubling agents and less toxic.

A

B

C

D

Note: Embryoid (A) and morphogenic callus induction, x40 (B), green plant regeneration in N6 medium (C), haploid plant-regenerant (D) Figure 54. The different steps from androgenic structure, embryoid induction to maturity

The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies of the factors such

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as induction medium composition, induction medium support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C17 induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants was produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F1 crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plants were obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, was sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain [36].

Colchicine

N-deacetil-N-(β,γ-thioepoxipropil) aminocolchi-cine (N2)

N-Cysteinocolchicine

N-aminoethylthiouronium colchicine chloralhydrate (L5)

N-deacetil-N-(β, γ-epoxi-propil) N-deacetil-N-(carboxy-amino) aminocolchicine (N19) dioxiethylamino-colchicine (N5)

Figure 55. Colchicine and colchicine derivatives

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Gynogenesis Gynogenic development of plants from unfertilized cells of female gametophyte (embryo-sac) in ovary/ovule/young flower cultures is one of the available alternatives for haploid production. It was first reported in barley San Noeum (1976). Cultivation of female gametophytes has been less studied than the anther culture approach. Nonfertilized ovaries and ovules are of interest because cultivation of nonfertilized ovaries is a unique possibility for obtaining haploids from male sterile plants. Female gametophytes can be alternative sources to recovering haploids from anthers of plants which do not respond to culture conditions or produce calli with low morphogenetic potential [37]. The frequency of regeneration of green plants from female gametophytes is higher than from male gametophytes. As a consequence, the frequency of production haploids from ovaries culture is greater than from anther culture. A comparative study of posterity androgenetic and parthenogenetic plants demonstrated superiority of the latter approach. The recovery of haploid plants from ovary culture is difficult and there are many upsets on unsuccessful attempts. During cultivation, ovaries and ovules often only increase their size by cell proliferation of somatic tissue around the female gametophyte but the embryo sac elements did not show morphogenetic activity. The first haploid calli were obtained from unfertilized ovaries of Ginkgo biloba (Gymnosperms) by Tulecke in 1964. He stimulated haploid calli formation the White’s medium supplemented with 6 mg/l 2.4-D and coconut milk but organogenesis was not induced. Callus induction from in vitro culture of Angiosperms was first demonstrated by Uchimiya et al. in 1971. They observed division of haploid cells from unfertilized ovary culture of Zea mays and ovules of Solanum melongena. But in this case, regeneration of plants was not achieved [36, 37]. A simple anther culture protocol for Australian spring wheat cultivars was developed using ovary co-culture. The inclusion of ovaries in the induction medium significantly increased the production of embryo-like structures (ELS), green and albino plants in two spring wheat cultivars tested. When five ovaries were added to the induction medium, the mean number of ELS per spike increased from 7.6 to 50.1 and green plants per spike increased from 0.6 to 8.9. The addition of 10 ovaries, however, did not further increase the production of ELS or green plants. The growth regulator combination of 2,4-D and kinetin was compared with IAA and BA. There were no significant differences in the numbers of ELS or green plants although significantly fewer albino plants were produced with IAA and BA. Eight additional cultivars were screened using the protocol with either 5 or 10 ovaries in the induction medium. Green plants were obtained from nine varieties at frequencies ranging from 0.3 to 33.0 green plants per spike. Regenerant plants at maturity exhibited chromosome fertility rates in different cultivars ranging from 15% to 100%.

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The influence of the genotype, the developmental stage of micro-, megaspores on the formation of haploid plants, nutrient medium composition and cultivation conditions have been shown to be very important. Factors affecting haploid androgenesis and ginogenesis There are numerous endogeneous and exogeneous factors that affect in vitro haploid production. These factors can be genetic, physiological, physical and chemical, which may also interact amongst each other to divert the microspores/ egg cell/megaspores to enter into a new developmental pathway. Induction of haploid development depends on genotype, stage of male/female gametophyte development, composition of nutrient media and conditions of cultivation. Genotype of the donor plant. The genotype of the donor plants, i.e. genetic factor, has a great influence on the anther, ovary and ovule culture response. In earlier studies, significant differences in callus formation using varieties or crosses were observed. In San Noeum (1979) experiments, the frequency of haploid plant formation varied from 0.2 to 1.1% depending on the genotype. In some species only a few genotypes have responded of many tested. We have experimental evidence that the formation of haploid plants depends on the genotype for in vitro ovary culture of Oryza sativa, Zea mays, Beta vulgaris, Allium cepa [37]. During cultivation, callus formation from nonfertilized ovaries of Oryza sativa was observed in only two varieties; one of var. japonica, and two varieties of var.indica. In some cases, it has been observed that hybrid materials give better results than standard varieties. In experiments on in vitro culture of unfertilized ovaries of wheat, it was also discovered that success of cultivation is determined by the genotype. In Triticum aestivum, 15 cultivars were tested: 7 interspecific hybrids and 8 lines of intergenus hybrid (Triticum aestivum x Aegilops squarrossa). A high degree of callus formation in two varieties: Grecum-476 (29.3 %) and Kazakhstanskaya-126 (19.6 %) was observed on MS medium, whereas in other cultivars it was 0.9-4.3%. Among interspecific hybrids, the number of nonfertilized ovaries forming callus varied from 4.3 % (K36446 x Gostianum 88, F2) to 15.5% (K 437x Gostianum 88, F2) on the same medium [38]. Empirically selected conditions for obtaining haploids for one type of cultivar or species usually prove to be unsuitable for another and require considerable modification. Most of the families, which produced embryoids, morphogenetic callus and regenerants, originated from high yielding genotypes. The differences between high yielding and middle yielding genotypes in embryoid production and green plant grown on the same medium may be due to genotypes because both androgenic response and regeneration capacity were greatly genotype dependent. A major problem with anther culture in cereals is the low numbers of plants that are regenerated. Often this problem is deteriorated by a high frequency of albinos. In the anther culture

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of wheat the high yielding donor plants produced more embryoids and green fertile plants than the middle yielding plants. In fact, genetic factors contribute in a major way to the differences in the number of haploid plants produced. Physiological status of the donor plant. The physiological conditions of the donor plant, i.e the environmental conditions and age of the donor plant, directly affect both in vitro androgenesis and in vitro gynogenesis in almost all plant species. A correlation between plant age and anther response has also been demonstrated. Similar is the case with ovary culture. The frequency of androgenesis is usually higher in anthers harvested at the beginning of the flowering period and showed a gradual decline in relation to plant age. Varying temperature and light conditions during the growth of donor plants also affect anther response. In the anther culture of grape, the induction frequency of embryoids derived from spring flowers was higher than that derived from summer flowers. The microscopical observations showed that some varieties of rubber tree often have a lot of degenerated and sterile microspores in their anthers in early spring or hot summer due to the influence of unfavourable climatic conditions. As a result no pollen embryoids were obtained from such anthers but only the somatic calli. The developmental stage of micro-, megaspores. An important factor for successful cultivation of female/male gametophytes is the stage of development of embryo sac/microspores. The stage of microspores at the time of inoculation is one of the most critical factors in the induction of androgenesis. Detailed cytological studies conducted on Angiosperms have shown that androgenic callus and embryos were mainly induced through a deviation of the first pollen mitosis to produce two undifferentiated nuclei. Besides affecting the overall response, the microspore stage at culture also has a direct bearing on the nature of plants produced in anther culture. About 80% of the embryos obtained from binucleate microspores of Datura innoxia, a highly androgenic species, were non-haploids (Sunderland et al, 1974) [36]. In a vast majority of species, where success has been achieved, anthers were cultured when microspores were at the uninucleate stage of microsporogenesis (Figure 56). The early divisions in responding pollen grains may occur in any one of the following four pathways: 1) Pathway I – The uninucleate pollen grain may divide symmetrically to yield two equal daughter cells both of which undergo further divisions e.g. Datura innoxia. 2) Pathway II – In some other cases e.g. N.tabacum, barley, wheat etc., the uninucleate pollen divides unequally. The generative cell degenerates, callus/embryo originates due to successive divisions of the vegetative cell. 3) Pathway III – But in a few species, the pollen embryos originate from the generative cell alone; the vegetative cell either does not divide or divides only to a limited extent forming a suspensor like structure.

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4) Pathway IV – Finally in a few other species e.g. Datura innoxia, the uninucleate pollen grains divide unequally, producing generative and vegetative cells, but both these cells divide repeatedly to contribute to the developing embryo/callus (Figure 57).

A

B

C Figure 56. Uninucleate (A) and binucleate microspores, х640 (B) of wheat; pathways of division of pollen grains (C)

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maturation

bi-cellular pollen

stress

mature pollen male gametophyte

sugar and nitrogen starvation, heat shock

uni-cellular microspore:

cell with restricted developmental potential

embryogenesis

embryogenic microspore

embryo, sporophyte

totipotent cell

Figure 57. Pathways of microspores development in vitro

The degree of development of the embryo sac is indirectly defined during the stage of development of pollen grain or with the help of histological preparations of ovules, which are at an identical stage with cultivated ovules. In different species of Angiosperms plants, the culture of different developmental stages of embryo sac may be optimal; however, induction of haploid plants is apparently

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possible for the same genotype from ovules that have embryo sacs in different stages of development. For example, induction of haploid plants of Hordeum vulgare is possible from ovaries in which embryo sacs were at different stages, from uninuclear to mature. The mature embryo sac stage is optimal for cultivation of nonfertilied ovaries of Oryza sativa. In Nicotiana tabacum, induction of haploid plants was observed on mature ovaries and on ovaries containing early stages of embryo sacs megaspore mother cells and tetrads up to the stage of the uninuclear embryo sac. The optimal stage development of the female gametophyte of wheat corresponded to the late stage of development of microspores in anthers. However, in cultivation of nonfertilized ovaries of wheat, using ovaries with a mature embryo sac was optimal for cultivars, while for interspecific hybrids – the eighth nuclear stage of female gametophyte development was optimal [37, 38]. Culture medium. Culture medium is a principal factor controlling the induction and development of intact plants. But, it is still difficult to draw a conclusion as to the most suitable composition for different plant species. As species or even genotypes may demand different nutritional conditions, no general recommendations can be given. Moreover, the conditions needed for sporophytic macrospore induction, for callus, embryo and plantlet formation normally differ. The important ingredients in the nutrient medium are inorganic salts, organic additives, carbohydrates, growth regulators. For most donor hybrid plants influence of the genotype on the induction of morphogenesis and regeneration in anther culture is higher (43.75%) than the impact of the culture medium factor (20.24%). There is a positive correlation between the yielding capacity of the lines and the anther response. The optimal culture medium for production of wheat doubled haploids by anther culture is N6 culture medium (Appendix 1, Table 3) including 100 mg/l myo-inositol, 9% (w/v) sucrose and 1 mg/l 2.4-D [35]. Sucrose is generally used at a concentration of 2-3%. For corn anthers and ovaries culture, 9-12 % sucrose was initially used. For wheat, it was found that 6 % maltose promoted embryos formation and inhibited the proliferation of somatic tissues. Several amino acids, vitamins, and inositol stimulate parthenogenesis. Quite often, nutrient media containing yeast extract, casein hydrolysate, and coconut milk were effective for the development of nonfertilized ovaries. The presence of ammonium ion is usually sufficient for induction of embryogenesis in callus or suspension cultures containing NO3-, but on media where NH4+ is absent, casein hydrolysate, or an amino acid such as alanine, or glutamine, is often promotory [39]. For embryogenesis in wheat anther cultures, it was shown that in a medium containing potassium nitrate, reduced nitrogen in the form of ammonium chloride matched the effectiveness of an equivalent concentration of nitrogen from casein hydrolysate. Casein hydrolysate could be replaced by glutamine, glutamic acid or alanine. Suspensions of haploid cells (microspores)

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grew and produced embryos on a medium containing either glutamine or casein hydrolysate as the sole nitrogen source. In many cases, embryogenic callus and/ or embryo formation did not occur without the presence of the amino acid source, suggesting that without amino acid, the medium was deficient in NH4+ or total nitrogen. It was found that the frequency of friable callus and embryo formation from anther culture of wheat increased almost linearly with the addition of up to 40 mM proline to N6 medium [total N, 34.99 mM; NO3/NH4 ratio, 3.99], but there was no benefit from adding proline to Potato medium, containing 100 mg/l glutamine) [total N, 60.01 mM; NO3/NH4 ratio, 1.91]. The N6 medium proved superior for the production of green plantlets in 17 genotypes. This basic medium (N6) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture than in the anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher. However, the regenerant plantlets from the anther culture were mainly albinos, whereas predominantly green plantlets were regenerated from isolated microspore culture. The production of green plantlets from isolated microspore culture was 2.9 times higher than from anther culture. The male gametophyte culture protocol is an efficient tool for the production of microspore-derived green plantlets in wheat [35, 39]. The embryoid formation and plant regeneration in anther cultures of three barley (Hordeum vulgareL.) cultivars (Niki, Karina, Thermi), one F1 hybrid (Niki × Thermi), two F2 populations (Niki × Thermi, Niki × Karina), and two F3 populations (Niki × Thermi, Niki × Karina) were investigated in two solid induction media after cold pretreatment for 14 and 28 days at 4°C . The media used (N6 and FHG) differed in their composition and source of energy (maltose in FHG vs. sucrose in N6). Embryoid frequency and green plant regeneration depended on both the induction medium composition and cold pretreatment. The combination of the FHG induction medium with 28-day-long cold pretreatment was the most efficient in haploid embryoid formation and green plant production. In addition, the green plant production was genotype-dependent. Cv. Thermi and F1 hybrid Niki × Thermi exhibited the highest frequency of green plant production. The parent with high or even moderate frequency of embryoid formation in anther culture could lead to the effective production of green plants from the F1 hybrid or the F2 generation for breeding purposes [36]. Addition of growth regulations to the basal medium is necessary. But recent investigations indicate that growth regulations are not essential in some monocotyledonous species. However, it should be noted that the high concentration of growth regulations stimulates callus formation from somatic tissue and inhibits embryo formation from haploid elements of the embryo sac or anther.

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The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11–39 nmol g−1 DW), with IBA constituting only 1 % of the total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121–424 pmol g−1 DW) and zeatin ryboside (cZR, 146–432 pmol g−1DW). Surprisingly, a relatively high level (10–64 pmol g−1 DW) of kinetin was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of ‘responsive’ DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and kinetin in comparison with ‘recalcitrant’ genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGR homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential [36]. Methods and conditions of cultivation Methods for obtaining haploid plants from male and female gametophytes usually use anthers with one-, binuclear microspores, nonfertilized ovaries and ovules as explants. Sometimes buds or segments of young flowers and even floating culture of flowers, as in case with rice, have been used. Moreover, haploid protoplasts have been isolated from male and female gametophytes. Isolated anthers, ovaries and ovules are usually cultivated at +22°...+26° C, but sometimes lower or higher temperatures for incubation were optimal (+20...+38° C). The process of callus formation on rice and barley was observed to occur in light or dark conditions. But for regeneration of plants from pea and sugarbeet it was observed that an optimal light regime was necessary [28]. The ability for in vitro morphogenesis has the individual cells of the embryo sac (egg dell, synergids, antipodals egg cell-synergids, egg cell-antipodals, synergids-antipodals). Synergids fromed only callus but antipodals gave rise to embryo. During embryogenesis, cells first differentiated into two proembryo cells, which developed into a pollycellular globular embryo, which was morphologically similar to the zygotic embryo. Regeneration of haploid plants by direct embryogenesis was observed in Triticum aestivum, Hordeum vulgare, Morus in-

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dica [35-39]. Wheat embryos formed from haploid cells (egg cells, microspores), antipodal cells and from diploid cells of somatic tissues of the anthers, nucellus and integuments. Direct embryogenesis is the most desirable pattern because, in this case, plants are photosynthetized, while plants from callus may be albino or demonstrate different levels of ploidy. In general, majority of induced mutations are recessive and, therefore, are not expressed in diploid cells due to the presence of dominant allele. Since, haploid plants have only one set of chromosomes, their dominant and recessive characters can be seen simultaneously on individual plants. It is extremely advantageous to provide a convenient system for the induction of mutations and selection of mutants with desirable traits in the absence of their dominant counterparts. The ability to fix mutations via doubled haploidy is therefore a key, especially, as induced mutations are predominantly recessive and cannot be detected until the second generation of selfing of putative mutants (M2 generation) at the earliest. The anther culture for producing double haploid has been recognized as an important tool, which can be used to capture the mutations leading to desirable traits in a homozygous, pure line rapidly [40]. Homozygous line is also important for the identification of molecular markers linked to traits of interest. Application of haploid production Plant breeding is focused on continuously increasing crop production to meet the needs of an ever-growing world population, improving food quality to ensure a long and healthy life and address the problems of global warming and environment pollution, together with the challenges of developing novel sources of biofuels. The breeders’ search for novel genetic combinations, which can be used to select plants with improved traits to satisfy both farmers and consumers, is endless. About half of the dramatic increase in crop yield obtained in the second half of the last century was achieved thanks to the results of genetic improvement, while the residual advance was due to the enhanced management techniques (pest and disease control, fertilization, and irrigation). Biotechnologies provide powerful tools for plant breeding, and among these ones, tissue culture, particularly haploid and doubled haploid technology, can effectively help to select superior plants. In fact, haploids (Hs), which are plants with gametophytic chromosome number, and doubled haploids (DHs), which are haploids that have undergone chromosome duplication, represent a particularly attractive biotechnological method to accelerate plant breeding. Currently, haploid technology, making possible through gametic embryogenesis the single-step development of complete homozygous lines from heterozygous parents, has already had a huge impact on agricultural systems of many agronomically important crops, representing an integral part in their improvement programmes. Diploidisation of haploid plants

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results in rapid achievement of homozygous traits in doubled haplouds, hence, these anther derived haploid plants have been used in breeding and improvement of crop species. 1. Production of homozygous lines: The most important use of haploids is the production of homozygous lines which may be used directly as cultivars or in the breeding program. For example. doubled haploids have been used for rapid development of inbred lines in the hybrid maize program. The anthers from F1 hybrids of selected or desirable cross are excellent breeding material for raising anther derived homozygous plants or doubled haploids in which complementary parental characteristics are combined in one generation. The doubled haploid plants are subjected to selection for superior plants (see Figure 52). This approach is described as hybrid sorting where recombinant superior gametes are virtually being selected since the heterozygous gene combination in the F1 hybrid is transformed into homozygous combinations. Hybrid sorting reduces the time required for haploid breeding by 4-5 years as in conventional breeding by the pedigree/ bulk method, the same requires ten years. Also, selection among DH lines reduces the size of breeding population. 2. Gametoclonal variation: The variation observed among haploid plants having gametic chromosome number developing from anther culture is called gametoclonal variation. Such variations resulting in desirable traits are subjected to selection at haploid level followed by diploidisation to get homozygous plants which can be released as new varieties. The concept of gametoclonal variation evolved from that of somaclonal variation. Both somaclonal and gametoclonal variations were detected in cultured cells and regenerated plants for morphological, biochemical characteristics, and chromosome number and structure. The life cycle of higher plants comprises a sporophytic (2n) and a gametophytic (n) generation. For genetic reasons, it is necessary to distinguish between plants regenerated from somatic (2n, somaclones) and gametic tissues (n, gametoclones), and also between somaclonal and gametoclonal variation. The concept of somaclonal variation is built on this definition. Variation is induced either during plant development or in vitro cell cultures, in the mitotic process. On the other hand, the term ‘gametoclonal variation’ can be described as the variation among derivatives of gametic cells in culture, or sexual progeny of plants regenerated from gametic cells in culture. These variants are obtained from both meiotic and mitotic divisions. There are four distinct sources of variations when referring to gametoclonal variation: a) New genetic variations induced by the cell culture procedures; b) Variations resulting from segregation and independent assortment; c) New variation at the haploid level induced by the chromosome doubling; d) New variation induced at the diploid level, resulting in heterozygosity.

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3. Selection of desirable mutants: Haploids offer a system where even recessive mutations are expressed unlike diploids where they express only in segregrating single plant progeny in M2 generation. Therefore, in several crops desirable mutants including traits like resistance to diseases, antibiotics, salts, etc. have been isolated from haploids derived from anther culture. For example, tobacco mutants resistant to black shank disease and wheat lines resistant to scab (Fusarium graminearum) have been selected and used as improved cultivars. Problems associated with haploid plants: –– Many species are not yet amenable for haploid production –– Deleterious mutations may be induced during in vitro phase. –– Plants having more or lesser than gametic chromosome number are also obtained, which necessitates cytological analysis first. –– Occurrence of gametoclonal variation limits the use of anther derived embryos for genetic transformation As microspore culture is a single cell system, it makes selection at the single cell level possible and, furthermore, offers new prospects for genetic manipulation like mutagenesis and transformation. Direct gene transfer by microinjection offers the possibility of transgenic plant formation by using isolated pollen culture having high regeneration efficiency. Moreover, if transgenes can be incorporated into the haploid microspore genome, prior to DNA synthesis and chromosome doubling, the doubled haploids may also be homozygous for the transgenes. Thus, isolated microspores not only provide a good target for bombardment but, also are readily amenable to transgene in vitro selection. Control questions:  1. Why is an isolated microspore culture advantageous over complete anther culture? 2. What is the importance of haploid plants for crop improvements? 3. With does the flow chart briefly describe the early patterns of cleavage in cultured pollen grains and the different modes of subsequent development of the proembryogenic mass thus obtained? 4. Explain briefly, the three crucial factors, which influence the induction of androgenesis. 5. Explain the factors, which influence the induction of ginogenesis.

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Chapter

9

GENETIC ENGINEERING Since the 1970s, there have been considerable developments in the methods and techniques used to study biological processes at the molecular level. The discovery of restriction enzymes, transformation technique within this period were breakthrough developments which allowed scientists to cut, splice and alter DNA, the molecule that carries the blueprints for life. This further led to isolation of a single DNA sequence from one organism by breakage of the DNA molecule at two desirable places and then inserting it at a desired position in another DNA molecule from a completely different organism to form a recombinant DNA and the technique involved is called the recombinant DNA technique. This has also been termed as genetic engineering because of the potential for creating novel genetic combinations. Using this technique, a single copy of a gene or DNA can be isolated and cloned into indefinite number of identical copies and this is known as gene cloning. Recombinant DNA technique. The basic steps involved in the recombinant DNA technique can be outlined as follows: 1. Isolation of a desired DNA fragment or gene of interest 2. Insertion of the isolated gene in a Figure 58. Recombinant DNA technique suitable vector. (Genomes. Garland Science, 2007) 3. Introduction of this recombinant molecule into the host cell by transformation 4. Selection of transformed host cells 5. Multiplication and expression of the introduced gene in the host (Figure 58) 378

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Although there are many diverse and complex techniques involved in genetic engineering, its basic principles are reasonably simple. It is however, very important to know the biochemical and physiological mechanisms of action, regulation of gene expression and safety of gene and gene product to be utilized. The process of genetic engineering requires the successful completion of a series of five steps: Step 1. Nucleic acid (DNA/RNA) Extraction Step 2. Gene cloning Step 3. Gene Design and Packaging Step 4. Transformation Step 5. Detection of Inserted Genes To explain these steps of gene cloning, understanding of a few basic techniques is essential which are discussed below. Several methods of plant DNA extraction have been developed over the last few years. Older methods for DNA isolation from plants require large amounts of tissue due to low yields and are also time consuming. This is an obvious problem when DNA must be isolated from numerous small individuals. Recently, easier and more efficient methods have been developed, mostly with the use of hexacetyltrimethylammonium bromide (CTAB). The method is used for DNA isolation from fresh, frozen or dried leaves of barley and wheat seedlings. With this method the usual yield is approx. 100 μg of DNA of quality suitable for PCR based molecular markers. Gel electrophoresis is a technique used to separate molecules based on physical characteristics such as size, shape, or isoelectric point. A gel is composed of agarose or polyacrylamide. Agarose is used for separating DNA fragments in size range of 100bp to 20kb. Polyacrylamide is preferred for smaller DNA fragments. Separation is achieved by moving the negatively charged nucleic acid molecules through agarose matrix with an electric field. The rate of migration of DNA molecules is inversely proportional to their molecular weight. Smaller molecules move faster than larger ones (Figure 59).

Figure 59. Agarose gel electrophoresis of DNA (ethidium bromide stained gel showing migration of DNA molecules)

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Conformation of the DNA molecule is also an important factor. Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds such as: supercoiled plasmid DNA will move fastest followed by linearised DNA, while open circular DNA will move at the slowest pace. To visualise DNA, gels are stained with ethidium bromide which intercalates in DNA and fluorescse under UV. Restriction endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases, so that restriction fragments generated from different genes can be joined together (Figure 60). These enzymes cleave DNA at specific sites and their recognition sequences or have two fold axis of rotational symmetry or are palindrmic (i.e. the sequence on one strand reads the same in the same direction on the complementary strand).

Restriction nucleases produce DNA fragments that can be easily joined together

Figure 60. Mechanism of restriction with EcoRI, EcoRV endonuclease (A, B) and ligases(at http://www.ncbi.nlm.nih.gov /bookshelf/ br.fcgi.book)

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Figure 60 explains the action of EcoRI restriction enzyme. These enzymes are often used in genetic engineering to make recombinant DNA for introduction into bacterial, plant, or animal cells. Inserting a particular fragment of DNA into the purified DNA genome of a self-replicating genetic element, typically into a plasmid, a new recombinant DNA molecule can be introduced into a bacterial cell. The normal replication mechanisms of the bacteria can produce a lot of identical copies of DNA molecules. The plasmid used in this way is known as a cloning vector, and the DNA propagated by insertion into it is said to have been cloned. Polymerase chain reaction (PCR). Polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied to millions of times in predetermined ways. It usually consists of a series of 20 to 35 cycles. Commonly, PCR is carried out in three steps such as denaturation, primer annealing followed by primer extension (Figure 61).

Figure 61. Typical PCR reaction (from http://bitesizebio.com/2008/01/23/ the-essential-PCR-troubleshooting-checklist)

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Basic PCR components: –– DNA template; –– Primers flanking the region to be amplified; –– Deoxyribonucleotides (dNTPs); –– DNA polymerase; –– Buffer with MgCl2; 1. Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96°C to ensure that most of the DNA template and primers are denatured. Also, some PCR polymerases require this step for activation. Following this hold, cycling begins, with denaturation step at 94-96°C for 20-30 seconds. 2. The denaturation is followed by the annealing step. In this step the reaction temperature is lowered so that the primers can attach to the single-stranded DNA template. The temperature at this step depends on the Tm of the primers and is usually between 50-64°C for 20-40 seconds. 3. The annealing step is followed by an extension/elongation step during which the DNA polymerase copies the DNA template, starting at the primers annealed to both of its strands. The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases, during the extension a temperature of 72°C is used. PCR can be used for a lot of molecular biology experiments such as introduction of restriction enzyme sites, or mutation (change) of particular bases of DNA. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (qPCR) which allows for quantitative measurement of DNA or RNA molecules Southern blotting. It is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with the methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the Edwin Southern. In this method, DNA samples are separated by gel electrophoresis and then transferred to a membrane by blotting via capilliary action. The membrane can then be probed using a labelled DNA complementary to the sequence of interest. Most original protocols used radioactive labels, however now non-radioactive alternatives are available. Southern blotting is less commonly used in laboratory science due to the capacity of using PCR to detect specific DNA sequences from DNA samples. However, these blots are still used for some applications, such as measuring transgene copy number in transgenic plants. Similarly, northern blotting is a technique used in research to study gene expression. It takes its name from the similarity of the procedure to the Southern blot procedure but the key difference is that instead of DNA,

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RNA is used for blotting and the analysis by electrophoresis and detection with a hybridization probe. Northern blot analysis determines whether the transcript or the messenger RNA (mRNA) of the introduced DNA is present and is correctly transcribed in the transgenic plant. The messenger RNA of the transgenic plants are isolated and processed to bind to the nitrocellulose membrane. Labeled DNA is used to bind to the mRNA and can be visualized through autoradiography. Western blotting is a method used to detect protein in a given sample of tissue homogenate or extract. The proteins transferred to membranes are “probed” using antibodies specific to the protein. Western blot analysis or protein immuno blotting is a technique used to detect whether the transgenic plants produce the specific protein product of the introduced gene. Protein samples are extracted from the transgenic plants, processed into denatured proteins and transferred to a nitrocellulose membrane. DNA Sequencing The term DNA Sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Currently there are two types of DNA sequencing methods: 1. Maxam-Gilbert sequencing. In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases. The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first ‘cut’ site in each molecule. The fragments are then size-separated by gel electrophoresis, with the four reactions arranged side by side. To visualize the fragments generated in each reaction, the gel is exposed to X-ray film for autoradiography, yielding an image of a series of dark ‘bands’ corresponding to the radiolabelled DNA fragments, from which the sequence is inferred. 2. Sanger and Coulson method. Chain-termination method������������� – Sanger method - �������������������������������������������������������������������� use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators. The chain-terminator method developed by Sanger was more efficient and rapidly became the method of choice. The Maxam-Gilbert technique requires the use of highly toxic chemicals, and large amounts of radiolabeled DNA, whereas the chain-terminator method uses fewer toxic chemicals and lower amounts of radioactivity. The key principle of the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators. The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elon-

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gation. The DNA sample is divided into four separate sequencing reactions, containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). These dideoxynucleotides are the chain-terminating nucleotides, lacking a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides during DNA strand elongation. Incorporation of a dideoxynucleotide into the nascent (elongating) DNA strand therefore terminates DNA strand extension, resulting in various DNA fragments of varying length. The dideoxynucleotides are added at lower concentration than the standard deoxynucleotides to allow strand elongation sufficient for sequence analysis. The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide), by gel electrophoresis on a denaturing polyacrylamideurea gel. Each of the four DNA synthesis reactions is run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. The terminal nucleotide base can be identified according to which dideoxynucleotide was added in the reaction giving that band. The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence. Gene cloning Isolation of the desired gene. The DNA fragment of interest can be obtained from any of the following: a) Genomic library is a large collection of recombinants in plasmid or phage vector, so that the total sum of DNA inserts in this collection represents the entire genome of the concerned organism. The genomic library is prepared by shotgun approach where the total genomic DNA of the organism is extracted and subjected to partial digestion by any restriction endonuclease or by sonication. These partial digests are then separated on agarose gel by electrophoresis or by sucrose density gradient centrifugation for selection of appropriate size fragments. The selected fragments are then inserted into phage λ vector or cosmid vector since these vectors can take up DNA inserts of up to 23-25 kilobase (kb) pairs. The recombinant molecules are then cloned in a suitable bacterial host, thus constructing a genomic library. The clone having the desired DNA insert is identified by screening the library using a suitable probe. The probe may be mRNA of the gene, complementary DNA (cDNA) of its mRNA, homologous gene from another organism or synthetic oligonucleotide representing the sequence of a part of the desired gene. Once identified, the desired clones are picked up from the library.

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b) cDNA library is a population of bacterial transformants or phage lysates in which mRNA isolated from tissue or organism is represented as its cDNA inserts. cDNA is complementary DNA produced by reverse transcriptase using mRNA as template. Isolation and identification of the desired clone from the cDNA library is done by screening in a similar manner to that of genomic library. However, cloned eukaryotic cDNAs have their own special uses since they lack intron sequences that are usually present in the corresponding genomic DNA. Introns are noncoding sequences that often occur within eukaryotic coding sequences (exons) and are sliced out while post transcriptional processing to produce mRNA. Since, bacteria do not possess the enzymes necessary for splicing of intron, eukaryotic cDNA clones become essential when the expression of eukaryotic gene is required in a prokaryote. Also, if the sequence of genomic DNA is known, the intron/ exon boundaries can be assigned by comparison with the cDNA sequence (Figure 62). c) Chemical synthesis of a gene. The basic sequence of any gene can be deduced from the nucleotide sequence of mRNA or amino acid sequence of the protein coded by it. The polynucleotide of the deduced base sequence can be synthesized chemically using automated DNA synthesizers. d) Gene amplification through PCR. The polymerase chain reaction technique amplifies a single copy of the desired DNA to billion copies in few hours. This PCR based approach is quicker and simpler than library construction and screening and hence preferred to all the aforesaid approaches Insertion of the isolated gene in a suitable vector. Vector is a DNA molecule into which exogenous DNA is integrated for cloning and which has the ability to replicate in a suitable host cell. Vectors are used to assist in the transfer, replication and sometimes expression of a specific DNA sequences in a target cell. Therefore, vector must have the following properties: –– Vector must have the origin of replication to replicate autonomously in the cell population as the host organism grows and divides. Their maintenance should not necessarily require integration into host genome. –– Vector must have unique sites for many restriction enzymes called multicloning site (MCS) into which DNA insert can be cloned without disrupting as essential function. –– Vector must be fairly small, low molecular weight DNA molecules to facilitate their isolation and handling. –– Vector must have some selectable marker that will enable the recombinant vector to be selected from a large population of cells that have not taken up foreign DNA.

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Figure 62. DNA cloning (Genomes. Garland Science, 2007)

A variety of vectors have been developed to function as cloning vehicles: 1) Plasmids are self-replicating circular duplex DNA molecules which are stably inherited in an extrachromosomal state. Plasmid vectors are used for cloning DNA of small size (up to 12 kb). The circular plasmid DNA which is to be used as a vector is first cleaved by restriction endonuclease (RE) to give linear DNA molecule. The foreign DNA to be inserted is also cut by the same endonuclease followed by ligation (joining) of the linearised vector and insertion of DNA resulting in a bigger circular DNA that can now be separated by gel electrophoresis on the basis of its size. Selection of the chimeric DNA can also be done if insertion of a foreign DNA at the endonuclease site inactivates a gene whose phenotype is readily scorable. Usually, the selectable marker is resistance to different antibiotics (Fig.65). The naturally occurring plasmids of Escherichia coli have been modified, shortened, reconstructed and recombined to create many different plasmids of enhanced utility as vectors for e.g. pBR322, pUC series(pUC18, pUC19, pUC8, etc.)

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Figure 63. Cloning Vectors

2) Bacteriophage vectors are viruses of bacteria that either infect the cells and lyse them (lytic cycle) or integrate into bacterial chromosome and multiply with it as prophage (lysogenic cycle). They act as cloning vehicles for larger pieces of DNA (23-25kb) as compared to plasmid vectors. Also, screening of recombinants is easier as phages form plaques (a clear zone where lysis has occurred in the bacterial lawn) in contrast to plasmids where bacterial colonies are screened. Most commonly used phage vectors are lambda (λ) and M13 phages. 3) Cosmid vectors are plasmids which contain a fragment of λ������������ ������������� DNA including the cos site since the cos site enables the DNA to get packed in λ particle in vitro. Recombinant cosmid DNA is injected and circularized like phage DNA which infects host cells, which is more efficient than plasmid transformation. But it replicates as a plasmid without expression of any phage functions. With a cosmid vector of 5 kb, large DNA inserts (32-47 kb) selected as the distance between two cos sites must be between 38 and 52 kb for packaging. Therefore, cosmids can accommodate up to 45 kb long DNA inserts, which is much more than a phage vector. Because of their capacity of taking up large fragments of DNA, cosmids are particularly used as vectors for constructing libraries of eukaryotic genome fragments. 4) Phagemid vector. Those vectors that have origin of replication derived from both a plasmid and a phage are known as phagemids. Under normal circumstances, the plasmid ori is used for replication but following a phage infection

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the other ori is used and single stranded DNA is produced which is useful for sequencing. One such plasmid is pBluescript which has its MCS flanked by T3 and T7 promoters, enabling expression of the cloned insert to be obtained regardless of its orientation. 5) Phasmid vectors are plasmid vectors containing λ attachment site (λatt) and functional origins of replication of both plasmid and λ. λatt allows plasmid to insert into a phage λ genome by means of site specific recombination, responsible for lysogen formation. This is a reversible recombinational insertion which generates phasmids. Phasmid vectors are preferred for the advantage that DNA may be cloned in plasmid vector and recombinant plasmid can be converted to phage which is easier to store, have long shelf life and screening by plaque hybridization gives cleaner results than colony hybridization. M13 is a highly developed phasmid vector being used widely. With the help of enzyme ligase, the isolated gene is inserted into linearized vector DNA, thus producing recombinant DNA as schematically shown in Figure 50. Introduction of the recombinant into suitable host. The recombinant vector is now introduced into a host cell for its multiplication and expression. The host cells are made competent or permeable by either Calcium chloride treatment for transformation or electric shock for electroporation. Alternatively, cosmids, phasmids and λ particles are used which then infect E. coli cells. Selection of recombinant clones. After transformation, there is a mixture of host cells which are not transformed or cells transformed with self-ligated vector and transformed cells carrying recombinant vectors. So, identification of recombinat transformed cells employs the reporter genes of the vector. A reporter gene produces a phenotype which permits either easy selection or quick identification of cells in which it is present. For example, genes conferring drug resistance or nutritional deficiencies are selectable markers which allow only cells which possess it to survive under selective conditions. On the other hand, for easy identification of recombinants, scorable markers are used which produce phenotypes different from those that do not have them such as LUX-gene codes for luciferase which produces phosphorescence, GUS-gene codes for β- galactourinidase which produces blue colour in the presence of substrate X-gluc. Reporter genes are cloned into the vector in close proximity to the gene of interest, to facilitate the identification of transformed cells as well as to determine the correct expression of the inserted gene. In general, reporter genes that have been used include: the beta glucuronidase gene (GUS A-gene) which acts on a particular substrate producing a blue product, hence making the transformed cells blue; the green fluorescent protein (GFP) which allows transformed cells to glow under the green light; and luciferase gene that allows cells to glow in the dark, among others. Several genetic sequences can also be cloned in front of the promoter sequences (enhancers) or within the genetic sequence itself (introns, or non-coding

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sequences) to promote gene expression. The next step is to identify the clone having DNA of interest from a population of transformed cells with recombinants. This is achieved by nucleic acid hybridization methods by colony or plaque hybridization if a specific probe for the DNA insert is available. In cases where the inserted gene is expressed, clones synthesizing a foreign protein can be detected immunochemically using specific antibody. Methods for Gene Transfer The uptake of foreign DNA or the recombinant DNA by cells is called gene transfer or transformation. Conventionally, the gene transfer necessary for crop improvement is obtained through sexual and vegetative propagation. However, biotechnological approaches like somaclonal variation, protoplast fusion, etc., have successfully speeded up the process of generating genetic variation and introgression of foreign genes. The most potential biotechnological approach for transferring recombinant DNA is based on genetic engineering which involves various techniques for gene transfer discussed ahead. Various gene transfer techniques used are grouped into two broad categories: –– Direct gene transfer –– Agrobacterium mediated 1. Direct Gene Transfer is a process where no vector is involved and can be applied to any species or genotype. The methods for direct gene transfer are further classified into two classes: –– Physical methods where usually naked DNA is directly transferred. Therefore, it is also referred to as DNA mediated gene transfer. –– Particle bombardment or Gene gun technique; The various physical methods for gene transfer are electroporation and microinjection technique. Electroporation is a process where the cells are exposed to electrical impulses of high voltage to reversibly make cell membranes permeable for uptake of DNA. Electroporation has been used extensively for transformation of protoplasts. Recently, transformation of intact plant cells of sugarbeet and rice has been successfully reported [41]. This method is convenient, simple and quick. However, electroporation cannot be applied to all the tissues, cell viability drops due to electric shock. Also, regeneration of plants from protoplasts is still difficult. The microinjection technique is a direct physical approach to inject DNA directly into the plant protoplasts or cells (specifically into the nucleus or cytoplasm) using a fine tipped (0.5-1.0 µm diameter) capillary glass needle or micropipettes. Through microinjection technique, the desired gene is introduced into large cells, such as oocytes, eggs, and the cells of early embryo (Figure 64).

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Figure 64. Microinjection technique

Particle bombardment. Instead of relying on a microbial vehicle, researchers can use a gene gun to literally blast tiny metal beads coated with DNA into a plant cell. The process is rather hit or miss – and more than a little messy – but some of the plant cells will adopt the new DNA. Gene guns are typically used to shoot DNA into the nucleus of a plant cell, but they can also be aimed at the chloroplast, the part of the cell that contains chlorophyll. Plants have between 10 and 100 chloroplasts per cell, and each chloroplast contains its own bundle of DNA. Whether they target the nucleus or the chloroplast, researchers must be able to identify the cell that has incorporated the new DNA. In one common approach, they combine the gene of interest with the gene that makes the cell resistant to certain antibiotics. This gene is called a marker gene or reporter gene. After firing the gene gun the researchers collect the cells and try to grow them in a medium that contains a specific antibiotic. Only the genetically transformed cells will survive. The antibiotic-resistant gene can then be removed before the cells grow into mature plants, if the researcher so desires. This is a relatively recent development but is widely used and is effective in introduction of DNA into plant cells (Figure 65). The technique involves coating 1µm diameter particles of tungsten or gold known as microprojectiles with DNA, which are then accelerated to high speed using a pulse of high pressure Helium gas inside a vacuum-filled chamber (into an evacuated chamber containing the target tissues). These DNA coated particles penetrate through the cell wall releasing DNA from particles which can express transiently or get integrated into nuclear genome of that cell. With appropriate tissue culture and selection, transgenic plants can be regenerated. Particle bombardment has been used for transformation of monocotyledonous crop plants such as maize, rice, wheat etc. 2. Agrobacterium mediated Gene Transfer is a form of the most successful plant transformation system. Wounding of plant tissues induces cell divisions at the wound

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site. Even highly specialized cells, such as phloem fibers and guard cells, may be stimulated by wounding to divide at least once. Wound-induced mitotic activity is typically self-limiting; after a few divisions the derivative cells stop dividing and redifferentiate. However, when the soildwelling bacterium Agrobacterium tumefaciens invades a wound, it can cause the neoplastic (tumor-forming) disease known as crown gall. This phenomenon is dramatic natural evidence of the mitotic potential of mature plant cells.

Figure 65. Biolistic Gun for gene transformation. Diagrammatic illustration of gene transfer using Gene Gun method

Without Agrobacterium infection, the wound-induced cell division would subside after a few days and some of the new cells would differentiate as a protective layer of cork cells or vascular tissue. However, Agrobacterium changes the character of the cells that divide in response to the wound, making them tumorlike. They do not stop dividing; rather they continue to divide throughout the life of the plant to produce an unorganized mass of tumorlike tissue called a gall (Figure 66).

A

B

Figure 66. Tumor (A) that formed on a plant stem infected with the crown gall bacterium Agrobacterium tumefaciens (B)

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After a gall has formed on a plant, heating the plant to 42°C will kill the bacterium that induced gall formation. The plant will survive the heat treatment, and its gall tissue will continue to grow as a bacteria-free tumor. Tissues removed from these bacteria-free tumors grow on simple, chemically defined culture media that would not support the proliferation of normal stem tissue of the same species. However, these stem-derived tissues are not organized. Instead, they grow as a mass of disorganized, relatively undifferentiated cells called callus tissue. Crown gall tumors are a specific type of callus, whether they are growing attached to the plant or in culture. Infection with a close relative of the crown gall organism, Agrobacterium rhizogenes, causes masses of roots instead of callus tissue to develop from the site of infection. A. rhizogenes is able to modify cytokinin metabolism in infected plant tissues. Crown gall tissues contain substantial amounts of both auxin and free cytokinins. Furthermore, when radioactively labeled adenine is fed to periwinkle (Vinca rosea) crown gall tissues, it is incorporated into both zeatin and zeatin riboside, demonstrating that gall tissues contain the cytokinin biosynthetic pathway. During infection by Agrobacterium tumefaciens, plant cells incorporate bacterial DNA into their chromosomes. The virulent strains of Agrobacterium contain a large plasmid known as the Ti-plasmid. So, the agent responsible for crown gall is Ti-plasmid and not the bacterium itself. Plasmids are circular pieces of extrachromosomal DNA that are not essential for the life of the bacterium. However, plasmids frequently contain genes that enhance the ability of the bacterium to survive in special environments. A small portion of the Ti-plasmid, known as the T-DNA, is incorporated into the nuclear DNA of the host plant cell (Figure 67). T-DNA carries genes responsible for tumor formation and necessary for the biosynthesis of cytokinin (trans-zeatin) and auxin, as well as a member of a class of unusual nitrogencontaining compounds (unusual aminoacid derivatives) called opines (most common are octopine and nopaline). Opines are not synthesized by plants except after crown gall transformation (Figure 68). These opines are used as sole carbon/nitrogen source for inducing Agrobacterium strain. The ipt gene (isopentenyl transferase) and the two auxin biosynthetic genes of T-DNA are phyto-oncogenes, since they can induce tumors in plants. Because their promoters are plant eukaryotic promoters, none of the T-DNAgenes are expressed in the bacterium; rather they are transcribed after they are inserted into the plant chromosomes. Transcription of the genes leads to synthesis of the enzymes they encode, resulting in the production of zeatin, auxin, and opine. The bacterium can utilize the opine as a nitrogen source, but cells of higher plants cannot. Thus, by transforming the plant cells, the bacterium provides itself with an expanding environment (the gall tissue) in which the host cells are directed to produce a substance (the opine) that only the bacterium can utilize for its nutrition. Plant tissues transformed by Agrobacterium carrying a wild-type Ti plasmid proliferate

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as tumors as a result of the overproduction of both auxin and cytokinin. And as mentioned already, if all of the other genes in the T-DNA are deleted and plant tissues are transformed with T-DNA containing only a selective antibiotic resistance marker gene and the ipt gene, shoots proliferate instead of callus.

Figure 67. Tumor induction by Agrobacterium tumefaciens. (After Chilton (1983) / Chilton, M.-D. A vector for introducing new genes into plants. Sci. Am. 248(00): 50–59.1983.)

The genes responsible for transfer of T-DNA called virulence genes (vir genes) are also contained on the Ti-plasmid. Agrobacterium infection requires wounded plant tissue because vir genes are induced by phenolic compounds released by the injured plant cells. The regions of T-DNA absolutely required for its transfer and integration into the plant genome are border regions which are short repeat sequences of 25 bp. Any DNA sequence inserted between the border repeats will be transferred to and integrated into the plant genome. Therefore, Ti-based plasmids are excellent vectors for introducing foreign genes into plants.

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In order to use them as vectors, the genes responsible for tumor formation must be removed. Ti-based plasmids lacking tumorigenic functions are known as disarmed vectors. These disarmed vectors are still too large to be conveniently used as vectors. Thus, smaller vectors described below have been constructed that are suitable for manipulation in vitro.

Figure 68. The two major opines, octopine and nopaline are found only in crown gall tumors. The genes required for their synthesis are present in the T-DNA from Agrobacterium tumefaciens

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Cointegration and binary vector system. The cointegration technique is based on in vivo recombination of two plasmids. One plasmid carries desirable DNA sequence, the other plasmid contains vir-genes and the border repeats of TDNA. The recombination of these plasmids leads to large Ti- plasmid which now can be used to transform plants. The binary vector system uses two separate plasmids: mini-Ti-plasmid to supply the disarmed T-DNA and the second having vir-genes. The mini-Ti -plasmid bears the gene construct that will be inserted into the plant genome, along with a eukaryotic selectable marker between T-DNA border sequences, so that both genes will be inserted as a unit. This plasmid when placed into an Agrobacterium strain containing a plasmid with virulence functions, the vir-gene products are able to drive the transfer of -DNA into plant cells, even though T-DNA is located on a separate DNA molecule. This is the most frequently used approach as mini-Ti-plasmids are very easy to manipulate using standard recombinant DNA techniques. This method is preferred over all other techniques as Agrobacterium is capable of transferring large fragments (50 kb) of DNA very efficiently without much rearrangements. Also, the gene transferred is stably inherited. Generation of transgenic plants and their identification The advent of recombinant DNA technique and transformation methods for plants has given agricultural scientists a powerful new way of incorporating defined genetic changes into plants and thus generating transgenic plants. The continued development of Agrobacterium based transfer systems for improving its efficiency and applicability to more crops is rapidly replacing other methods for generation of transgenic plants. Agrobacterium based generation of transgenic plants. The bacterial plasmid gives biotechnologists an ideal vehicle for transferring DNA. To put that vehicle to use, researchers often employ the leaf fragment technique. In this method, small discs are cut from a leaf. When the fragments begin to regenerate, they are cultured briefly in a medium containing genetically modified Agrobacter. During the exposure, the DNA from the Ti-plasmid integrates with the DNA of the host cell, and the genetic payload is delivered. The leaf discs are then treated with plant hormones to stimulate shoot and root development before the new plants are planted in soil. The major limitation to this process is that Agrobacter cannot infect monocotyledonous plants such as corn and wheat. Dicotyledonous plants – such as tomatoes, potatoes, apples and soybeans – are all good candidates for the process. There are few prerequisites for Agrobacterium mediated gene transfer which includes: –– In order to induce vir genes, plants must produce acetosyringone or Agrobacterium can be preinduced with synthetic acetosyringone.

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–– Following induction, the agrobacteria should have access to cells that are competent for transformation (Figure 69). Thus, wounded and dedifferentiated cells, fresh explants are used which have replicating DNA or are undergoing mitosis. –– Transformation competent cells should be able to regenerate in whole plants.

Figure 69. Agrobacterium mediated development of transgenic plants in tobacco The procedure for Agrobacterium mediated gene transfer is summarized in Figure 69. The explants used for inoculation or cocultivation with Agrobacterium carrying the vector include protoplasts, callus, tissue slices, sections of organs like leaf discs, etc. In practice, the procedure can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. So, a tissue segment like a leaf disc is excised and is incubated in Agrobacterium suspension for a few hours to 3-4 days followed by culturing on a media for bacterial growth to take place. Tissue explants are then transferred to media containing carbenicillin or cefotaxime which eliminate bacteria. After explants are inoculated with Agrobacterium carrying the vector with gene of interest, they are moved to media de-

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signed for selection of transformed plant cells. Selection is facilitated by selectable marker genes present in the vector which is usually resistance to herbicide or antibiotics like kanamycin. Following selection, the transformed calli is put in regeneration medium for development of shoots and roots. The second level of selection for transformed tissue is done by expression of a reporter gene or a scorable marker gene like GUS. Different selectable marker genes and reporter genes with their substrate and assays are summarized in tables 6 and 7. These transgenic plants (T0) are then tested for stable integration and expression of genes by PCR or by Southern hybridization. Table 6 Selectable marker genes used for gene transfer No 1 2 3 4

Selectable marker gene Hydromycin phosphotransferase (hpt) Neomycin phosphotransferase (npt II) Phophinothricin acetyltransferase (bar) Bromoxynil nitrilase

Substrate used for selection Hydromycin B G 418, kanamycin, neomycin L-phophinothricin (PPT), bialaphos Bromoxynil

In genetic transformation systems, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding nontransformed cells. However, simultaneous use of the gene encoding green fluorescent protein (GFP) and an antibiotic resistance gene facilitates the selection process, since it allows visible selection of transformed cells. Here, we report the development of a visual selection system for transformed cells using a GFP marker without selection against antibiotics after Agrobacterium-mediated transformation in rice. Both GFP protein levels and GFP fluorescence in calli isolated by visual selection were higher than in calli selected on hygromycin (Hyg), suggesting that transgenic calli hyper-accumulating GFP were efficiently obtained by selection using GFP fluorescence itself rather than Hyg resistance. Table 7 Scorable marker genes used for gene transfer No 1 2 3

Scorable marker gene β-glucoronidase (GUS) β-galactosidase (lac Z) Neomycin phospho-transferase (NPT II)

Substrate and assay X-gluc X-gal Kanamycin+P32 ATP

Identification Fluorescence Colony colour Radioactivity detection

Furthermore, gfp transcripts in calli isolated by visual selection were more abundant than under Hyg selection; in contrast, transcript levels of hpt in calli

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selected visually were comparable to those obtained under Hyg selection. These results suggest that there was no correlation between  hpt  and gfp expression levels, despite the fact that they are aligned in tandem on an integrated locus after selection by either GFP fluorescence or Hyg-resistance. This fact indicates that positional effects can influence the expression of each transgene differently, even when they are located in tandem at the same locus. In summary, based on our results, we discuss a model system for rice cell culture transformation for the production of recombinant proteins using visual selection. Engineered minimal chromosomes with sufficient mitotic and meiotic stability have an enormous potential as vectors for stacking multiple genes required for complex traits in plant biotechnology. Proof of principle for essential steps in chromosome engineering such as truncation of chromosomes by T-DNA-mediated telomere seeding and de novo formation of centromeres by cenH3 fusion protein tethering has been recently obtained. In order to generate robust protocols for application in plant biotechnology, these steps need to be combined and supplemented with additional methods such as site-specific recombination for the directed transfer of multiple genes of interest on the minichromosomes. At the same time, the development of these methods allows new insight into basic aspects of plant chromosome functions such as how centromeres assure proper distribution of chromosomes to daughter cells or how telomeres serve to cap the chromosome ends to prevent shortening of ends over DNA replication cycles and chromosome end fusion. Molecular Markers A genetic marker is any character that can be measured in an organism which provides information on the genotype of that organism. A genetic marker may be a recognizable phenotypic trait (e.g. dwarfism, albinism, altered leaf or flower morphology), a biochemical trait (e.g. proteins like isozymes) or a molecular trait (molecular or DNA based). Use of phenotypic markers is limited by their dependence on the expression of genes, which are influenced by environmental or developmental conditions. Molecular markers are defined as readily detectable DNA sequences whose inheritance can be easily monitored, which are independent of developmental stage or the environment and are numerous in number. Markers are used to study genetic diversity within species/strains or for marker assisted selection of desirable genes. The location of a desirable gene can be assigned by comparing the inheritance of a mutant gene with inheritance of the marker whose chromosomal location is known. Coinheritance of the gene of interest and markers suggests that they are physically close together on the chromosome. Therefore, the desirable features of a molecular marker are: –– Should be easy, fast and cheap to detect –– Should be reproducible

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–– Should be polymorphic –– Should have codominant inheritance to allow discrimination between homo and heterozygotes in diploids [32, 42, 43, 44]. There is a wide range of molecular markers available to detect polymorphism like RFLP, RAPD, AFLP, SSR, etc. Restriction Fragment Length Polymorphism (RFLP) was termed by Botstein in 1980. RFLPs rely on the combination of a probe and restriction enzyme (RE) to identify polymorphic DNA sequence using Southern blotting. RFLP is generated by the presence or absence of a recognition site for one restriction endonuclease in the same region of chromosome from different individuals of a species. In this method, DNA is digested with RE, electrophoresed, blotted on a membrane and probed with a labeled clone. Polymorphism in the hybridization pattern is a result of the same RE producing fragments of different lengths representing region of same chromosome of different individuals. Single base pair changes, inversions, translocations, deletions, etc., may result in loss or gain of a recognition site, which in turn leads to restriction fragments of different lengths between different genotypes. Since this approach requires large amount of highly pure DNA and radioactive or non-radioactive detection method to identify polymorphic DNA bands, it is time consuming, laborious and expensive. Randomly Amplified Polymorphic DNA (RAPD) is a PCR based molecular technique. This involves amplifying DNA segments randomly distributed throughout genome by PCR using single decamer primers at low stringency. Polymorphism occurs as a result of presence or absence of complementary sequence to the primer in the genome. The RAPDs may be of different types viz. Arbitarily Primed-PCR, DNA Amplification Fingerprinting (DAF), etc. These techniques differ in the length of primers used, the amplification conditions and the resolution of PCR products. RAPD needs small amount of DNA (15-30ng). Since, it is PCR based, it is a quick and efficient technique. But since, it is not a codominant marker and also not reproducible, it is used only as an initial approach to identify polymorphism. Amplified Fragment Length Polymorphism (AFLP) is a combination of RFLP and RAPD techniques. It involves PCR amplification of genomic restriction fragments generated by specific RE and oligonucleotide adaptors of few nucleotide bases. Many potentially polymorphic fragments are generated by this approach which are separated on highly resolving sequencing gels and visualized using autoradiography or a fluorescent dye. This is a highly sensitive, reproducible fingerprinting technique. Simple Sequence Repeats (SSR) also called microsatellites are groups of repetitive DNA sequences that are present in a significant proportion of plant and animal genomes. They consist of tandemly repeated mono-, di-, tri-, tetra- and penta nucleotide units. The number of repeats at that locus varies in different

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individuals, thus displaying high levels of genetic variation. These SSR length polymorphisms are detected by PCR. Therefore, it is necessary to sequence the conserved flanking DNA to design PCR primers that will amplify the repeated sequences. Microsatellites provide reliable, reproducible molecular markers. Under the lsupervision of Professor N.N.Galiakparov at the Institute of Plant Biology and Biotechnology genotyping with the use microsatellite markers was tested on some varieties of Malus domestica. It revealed differences between the genotype of Golden Delicious imported from France and the same variety, taken from the nursery in Almaty. Wild population of M.sieversii has an exceptional level of polymorphism resulting from different ecological and environmental growth conditions and interspecies hybridization. They present a much broader genetic pool of important horticultural traits than the domesticated apples currently used in breeding programs [45]. Role of Biotechnology in Crop Improvement The last decade has witnessed a remarkable change which has taken plant biotechnology from the study of basic science to large-scale commercial applications. This is true for almost every aspect of plant biotechnology such as development of molecular markers to speed up plant breeding practices and using knowledge of genes and their expression to generate and commercialize transgenic crops. In general, the role of biotechnology in crop improvement can be divided into two categories: 1) Methods directed towards the same goals as conventional plant breeding like improved yield, quality, resistance to pests and diseases, tolerance to abiotic stresses, etc., by molecular breeding or production of transgenic crops. 2) Novel applications such as use of plants as bioreactors to generate pharmaceuticals, vaccines or biodegradable plastics. Genetic engineering techniques are only used when all other techniques have been exhausted and when: 1) the trait to be introduced is not present in the germplasm of the crop; 2) the trait is very difficult to improve by conventional breeding methods; 3) it will take a very long time to introduce and/or improve such a trait in the crop by conventional breeding methods (Figure 70). Molecular breeding. Molecular maps using markers RFLP, RAPD, SSR, ESTs for major crop species like rice, maize, tomato, etc., have been utilized very effectively in crop improvement programmes like marker assisted selection (MAS). MAS is a method of breeding which allows us to trace desirable agronomical traits during selection based on genomic rather than morphological analyses. The basic approach of the MAS is simple: if one knows the position of a gene(s) controlling any trait of interest, it is possible to monitor the inheritance of such a

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trait in breeding populations based on the gene-linked genetic markers instead of the trait itself. Such type of selection is most effective in work with agronomic characters controlled by not completely penetrant genes or with quantity traits controlled by groups of genes where each gene has well expressed effect on the trait.

Figure 70. Integration of conventional and modern biotechnology methods in crop breeding

The geneticists usually establish genetic control of a trait of interest; they have to find, map and annotate genes and to fit appropriate genetic markers for key alleles. The breeders should apply these markers integrating ones in conventional breeding schemes in order to optimize the selection. The MAS methods are developing continually and now there are many new plant varieties created by means of such approach. However, it should be noted that the MAS methods are mostly effective to monitor and select traits having monogenic control. There are few practical examples of working markers for quantitative traits in spite of very intensive worldwide research in the field of so called QTLs. It seems that the MAS approaches would be extremely demanded in the selection of plants with altered biochemical properties, which is practically impossible to achieve by means of conventional breeding. First of all, that goes for polyploidal cultivars. Also the MAS methods have good practical potential in fruit tree breading where due to long ontogenesis of these plants the breeding process usually requires many years.

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Many markers have been identified which are closely linked to genes for agronomic traits of interest. These include markers for genes coding for: 1) Pest and disease resistance 2) Quality traits for e.g. malting quality in barley, alkaloid levels, etc. 3) Abiotic stresses, e.g. tolerance to salinity or drought 4) Developmental traits for e.g. flowering time, vegetative period 5) Quantitative traits, e.g. high fibre strength in cotton 6) Micronutrient uptake, e.g. high iron and zinc uptake in wheat Pest and disease resistance (against viruses fungi, bacteria, nematodes, insects) for e.g. RFLP has been used to map Tm-2a locus in tomato resistant to tobacco mosaic virus (TMV), gene Mi in tomato for resistance against root knot nematode, two genes in rice for blast resistance and in many other crops. RAPD assisted selection has been done for pto-gene conferring resistance to Pseudomonas in tomato. IARI has developed new improved Basmati by marker assisted transfer of Xa13 and Xa21 genes against bacterial leaf bight [46, 47]. The role of molecular MAS for crop improvement is in increased speed and accuracy of selection, gene pyramiding, and reduced cost of field based selection. Thus rather than growing breeding lines in the field and testing for important traits over the growing season, it is possible to extract DNA from 50ng of seedling leaflet and test for presence or absence of a range of traits in that DNA sample in one day. Plants lacking the required traits can then be removed early in the breeding programme. With the availability of more validated molecular marker, MAS therefore, becomes a highly cost effective and efficient process. The same principle used in developing molecular markers can be applied for a range of molecular diagnostics and DNA fingerprinting viz.identification of breeding lines and varieties, characterization of genetic resources and study of phylogenetic relationships [42]. Transgenic Plants. The development of transgenic plants is the result of integrated application of rDNA technology, gene transfer methods and tissue techniques. With conventional plant breeding, there is little or no guarantee of obtaining any particular gene combination from the millions of generated crosses. Undesirable genes can be transferred along with desirable genes or while one desirable gene is gained, another is lost because the genes of both parents are mixed together and re-assorted more or less randomly in the offspring. These problems limit the improvements that plant breeders can achieve, eating time and funds along the way (Figure 71). In contrast, genetic engineering allows the direct transfer of one or just a few genes, between either closely or distantly related organisms. Not all genetic engineering techniques involve inserting DNA from other organisms. Plants may also be modified by removing or switching off particular genes and genetic controls (promoters).

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Transgenic plants have both basic and applied role in crop improvement. 1) Genes have been successfully transferred to many crops for resistance to various biotic stresses such as the biopesticidal gene of bacterium Bacillus thuringiensis (Bt) has been incorporated into cotton for resistance against bollworm, maize against European corn borer, tomato, tobacco against Bt cotton is already in markets and the rest are in process.

Figure 71. Conventional breeding vs. genetic engineering

Bt proteins are naturally occurring insecticides produced by the soil bacterium, used to control crop pests such as larvae of butterflies and moths, beetles, and mosquitoes since the 1920s. The crystalline, inactive insecticidal Bt proteins, form bodies inside the bacterium and become active when they are eaten by the target insect larva and cleaved. The active peptides bind to specialized receptors in the midgut of the insect, creating holes in the gut membrane that cause contents to leak and kill the larvae. The precision of different Bt proteins for their targets resides is in the specificity of their tight binding to companion receptors in the insect gut. In recent years, a variety of safety studies was conducted specifically on native Bt proteins to show that they do not have characteristics of food allergens or toxins. Data on Cry1Ab in maize and cotton and Cry1Ac in tomato, maize and cotton have been carefully reviewed by regulatory agencies in numerous countries, including the U.S., Canada, Japan, UK, EU, Russia, and South Africa. Also, plant derived insecticidal genes like protease inhibitors, lectins, etc., have also been integrated into many pulse crops which are under field trials. Also, transgenic plants have been especially successful for control of viral diseases. The approach followed is to identify those viral genes or gene products, which when present for a wrong time or in improper amount, will interfere with the normal functions of the infection process and prevent disease development. A few examples of transgenic plants with viral resistance genes are in tobacco against TMV, rice against rice yellow mosaic virus, potato against potato virus Y and po-

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tato leaf roll virus, etc. [47, 48]. In addition, cytokinin production could be linked to damage caused by predators. For example, tobacco plants transformed with an ipt gene under the control of the promoter from a wound-inducible protease inhibitor II gene were more resistant to insect damage. The tobacco hornworm consumed up to 70% fewer tobacco leaves in plants that expressed the ipt gene driven by the protease inhibitor promoter. 2) Genes resistant to abiotic stresses like herbicide resistance. Herbicides are the method of choice to control weeds and hence maximize crop yields by reducing competition from weeds. This has necessitated development of safer, biodegradable herbicides and development of crop plants resistant to those herbicides. Also, because herbicide resistance genes are also effective selectable marker genes in culture, herbicide tolerant crop varieties were the first major transgenic trait to be produced and commercialized. Based on either expression of a herbicide insensitive gene, degradation of herbicide or overexpression of herbicide target gene product, engineered resistance is now available to a range of herbicides, for example, transgenic petunia plants resistant to glyphosate of Roundup herbicide was developed by transfer of a gene for EPSPS (5-enol-pyruvyl-shikimate-3 phosphate synthase) that overproduces this enzyme. Transgenic tomato and maize plants using herbicide detoxifying gene GST (glutathione-S transferase) have been successfully used. Also, extensive research is going on for producing transgenic crops against salt, drought, chilling stresses in rice, wheat, tomato, etc. With genetic engineering, more than one trait can be incorporated into a plant, and they are called stacked traits. These are currently corn, cotton, and soybean crops with both herbicide and insect tolerance traits. Transgenic crops with combined traits are also available commercially such as the herbicide tolerant and insect resistant maize and cotton. Stacking different genes for one trait makes the crop more durable to resist the pest/disease and tolerate more herbicides. In most cases, advances to generate stress tolerant plants by traditional breeding are slow because of involvement of many genes and physiological processes. For cold and drought tolerance, recent research has shown that a series of functionally different cold and drought response genes show common promoter regulatory sequences. These results are significant because they show that introduction of a single regulatory gene confers tolerance to different stresses. Cultivation of GE HT (herbicide-tolerant) crops has also had other positive effects on the environment, i.e. increases in low-or no-till practices and use in combination with integrated pest management schemes, which were made possible because early season pesticide sprays could be eliminated, allowing beneficial insects to establish. Most reports indicate pesticide use and cost decrease following adoption of Bt varieties. In Argentina, numbers of herbicide applications increased with HT soybean but use shifted to more environmentally friendly herbicides. Reduction in pesticide use can also be achieved by using the best

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methods and tools available, including integrated pest management, biocontrol, organic production methods, and GE organisms to reduce environmental impact while achieving adequate production levels. 3) Gene transfers to improve quality of food products – Bruise resistant tomatoes were developed which expressed antisense RNA against polygalactouronase which attacks pectin in the cell walls of ripening fruit and thus softens the skin. This transgenic tomato was commercialized under the name of Flavr Savr. Also, tomatoes with delayed ripening were developed by using gene for ACC deaminase to degrade ACC which is immediate precursor to ethylene. This increases shelf life of tomato. Starch content in potatoes could be increased by 20-40% by using a bacterial ADP glucose pyrophosphorylase gene. To date, commercial GM crops have delivered benefits in crop production, but there are also a number of products in the pipeline which will make more direct contributions to food quality, pharmaceutical production, and livestock feeds. Examples of these products include: “Golden rice” – rice with higher levels of iron and beta carotene – an important micronutrient which is converted to vitamin A in the body (Figure 72) [49]; long life banana that ripens faster on the tree and can therefore be harvested earlier; maize with improved feed value; delayed ripening papaya; tomatoes with high levels of flavonols, which are powerful antioxidants; edible vaccines from fruit and vegetables.

Figure 72. Components of a gene construct used in developing Golden Rice (from Ye, X, Al-babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science. 287: 303-305 [49])

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GE foods are tested in comparison with conventional counterparts in terms of the nutritional composition: levels of protein, carbohydrate, fat, vitamin, mineral, fiber, moisture, and phytochemicals, and analyzed if the composition is substantially equivalent. GE crops and conventional crops should have been grown in comparable conditions to eliminate the effect of the environment in the nutritional composition. GE foods with altered nutritional traits must be labeled to indicate nutritional differences; one example is VistiveTM, a low-linoleic oil from GE soybeans that can be used instead of trans fat-containing oils. Such crops should be tested for substantial equivalence to compounds unrelated to the introduced trait. In July 2007, the European Food Safety Authority released statements on the fate of genes and proteins in food and feed: “After ingestion, a rapid degradation into short DNA or peptide fragments is observed in the gastrointestinal tract of animals and humans” and “To date, a large number of experimental studies with livestock has shown that recombinant DNA fragments or proteins derived from GM plants have not been detected in tissues, fluids or edible products of farm animals” [50]. If a GE food is significantly different from its conventional counterpart, the food must be labeled to indicate the difference. Instances where the nutritional profile changes are included, for example if the GE food is created using genetic information from a previously recognized allergenic source, such as peanut, soy, or wheat, or if the new proteins have characteristics of known allergens. For example, oils made from GE soybeans and canola varieties with changes in fatty acid composition must be labeled; foods containing those oils must be labeled and companies producing that oil must use a new name. For example, Monsanto is using the name VistiveTM, to market its low-linoleic acid product from GE soybean oils. If a food contains a new potentially allergy-causing introduced protein, the label must state that the product contains the allergen and name its source [41, 50]. Data and information from peer-reviewed science on the safety of these products should be a part of the information considered when growing and consuming foods from these crops. 4) Male sterility and fertility restoration in transgenic plants which is required for hybrid seed production. For many crops, it is difficult to generate commercial hybrid seed by conventional means. Hybrid seed is attractive to seed companies as farmers must purchase new seed from them each year, since hybrid varieties do not breed true. It is now possible to engineer male sterility by expression of a ribonuclease gene (barnase) specifically during development of the tapetal layer that nourishes developing pollen grains. Developmental regulation of the ribonuclease by the TA29 tapetum specific promoter kills the tapetal cells leading to male sterility. Male sterile plants can be used as the female parent to produce hybrid seeds. Fertility can be restored by expression of the barstar gene, which

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inactivates barnase. This technology can be used to produce hybrids of crops such as maize, sugarbeet or canola. It is not possible to produce hybrid canola conventionally but hybrid canola can exhibit hybrid vigor and increased yields. Molecular Farming. A major role of biotechnology is the use of transgenic plants as factories for manufacturing special chemicals and pharmaceuticals such as transgenic tobacco plants carrying mannitol dehydrogenase gene from E. coli which is used for increasing production of mannitol. Similarly, production of Polyhydroxy butyrate (PHB) in plants provides an attractive source of biodegradable plastics at low cost. Two genes which catalyse two steps in production of PHB, acetoacetyl CoA reductase (phb B) and polyhydroxy butyrate synthase (phb C) have been successfully transformed and expressed in Arabidopsis thaliana. Efforts are on creation of transgenic plants as sources of edible vaccines, antibodies, pharmaceuticals. Plant-derived pharmaceuticals and vaccines for common diseases such as hepatitis B, pneumonic and bubonic plague, as well as against allergy sufferers, asthma, seasonal allergies and atopic dermatitis have been developed since the early 1990s. Plant vaccines have the advantage of being readily consumed with limited or no processing without the need for cold storage. To study regulated gene expression. Transgenic plants have proven to be particularly useful tools in studies on plant molecular biology, gene regulation, identification of regulatory sequences involved in differential expression of gene activity. Many cold stress–induced genes are activated by transcriptional activators called C-repeat binding factors (CBF1, CBF2, CBF3; also called DREB1b, DREB1c, and DREB1a, respectively) CBF/DREB1-type transcription factors bind to CRT/ DRE elements (C-repeat/dehydration-responsive, ABA-independent sequence elements) in gene promoter sequences CBF/DREB1 is involved in the coordinate transcriptional response of numerous cold and osmotic stress–regulated genes, all of which contain the CRT/DRE elements in their promoters. CBF1/DREB1b is unique in that it is specifically induced by cold stress and not by osmotic or salinity stress, whereas the DRE-binding elements of the DREB2 type are induced only by osmotic and salinity stresses and not by cold. The expression of CBF1/ DREB1b is controlled by a separate transcription factor, called ICE (inducer of CBF expression). ICE transcription factors do not appear to be induced by cold, and it is presumed that ICE or an associated protein is posttranscriptionally activated, permitting activation of CBF1/DRE1b, but the precise signaling pathway (s) of cold perception, calcium signaling, and the activation of ICE are presently under investigation. Transgenic plants constitutively expressing CBF1 have more coldup-regulated gene transcripts than wild-type plants do, suggesting that numerous cold–up-regulated proteins that may be involved in cold acclimation are produced in the absence of cold in these CBF1 transgenic plants. In addition, CBF1 tansgenic plants are more cold tolerant than control plants.

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T-DNA and transposable elements are used as molecular tags to produce mutations by becoming inserted within genes thus making it nonfunctional. Using this approach regulatory sequences of many structural genes have been cloned like light inducible genes like ribulose biphosphate carboxylase (rbcS), chlorophyll a/b binding protein (Cab), heat shock protein genes, etc. The genes for seed storage proteins provide an excellent example of a cell and tissue specific expression. Although, biotechnology alone will not be able to generate sustainable food production, however, the “gene revolution’ will have immense role in crop improvement and agriculture. Control questions: 1. What are the benefits of genetic engineering in agriculture? 2. How does genetic engineering differ from traditional biotechnology? 3. What is plant transformation? 4. What is a vector and its essential elements? 5. Describe the type of the vector which is used in plant transformation. 6. What is the plant virus vector? 7. Describe the position of regulatory sequences in the plant gene. 8. What type of bacteria is Agrobacterium? 9. What are Ri and Ti plasmids? 10. What are the essential segments in Ti plasmis? 11. Describe the main steps of Agrobacterium mediated gene transformation. 12. What are the roles of vir genes in Agrobacterium mediated transformation? 13. What is the basic principle of particle bombardment? 14. Describe the parameters considered in in vitro electroporation techniques. 15. What is the role of electric field in electroporation techniques? 16. What are the possible risks associated with using transgenic crops in agriculture? 17. Can the viral genetic sequences inserted in the genetically engineered crops create a human risk?

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Chapter 10. Cryoconservation

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10

CRYOCONSERVATION The main task for the future is to work at abating negative tendencies and securing the most optimal conditions for save ex situ, in situ and in vitro conservation of plant genetic resources, promotion of fundamental and applied research in the sphere of biodiversity, increasing the capacity in collecting valuable genetic diversity, and enrichment of national germplasm holdings through targeted collecting missions all over the world. The continuing search for high yielding varieties of crop plants with resistance to biotic and abiotic stresses necessitates conservation of germplasm of different crops and their wild and weedy relatives. Germplasm Conservation. The genetic material, especially, its molecular and chemical constitution, inherited and transmitted from one generation to the other is referred to as germplasm. In other words, the total sum of all the genes present in a crop and its related species constitute its germplasm. It is generally represented by a collection of various strains and species. Germplasm is valuable because it contains diversity of genotypes needed to develop new and improved genetic stocks, varieties and hybrids. Therefore, germplasm is the basic indispensable ingredient of all breeding programmes and great emphasis is placed on collection, evaluation and conservation of germplasm. In 2013, in the framework of the National program “Botanical variety of Kazakhstan wild congeners of cultivated plants as a source of enrichment and preservation of gene pool of agrobiodiversity for realization of the Food programme” the Institute of Botany and Phytointroduction created seed bank of wild congeners of cultivated plants. The National Plant Germplasm collections include grains, vegetables, fruits and nuts, oil and fiber, root and tuber and forage crops. Over 40 active sites store, regenerate and distribute plant germplasm to qualified users throughout the world. The germplasm may be stored as seeds, growing plants, tissue cultures or cryogenically as pollen, shoot tips or seeds. At present the most priority way of preservation of genetic variety of plants is their preservation as a part of natural communities in situ, and the ex situ method of preservation biological diversity components out of their natural habitats is also used. In vitro methods in combination with traditional approaches of in situ and 409

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ex situ conservation of plants allow us to solve the reintroduction and introduction problems. In situ conservation In situ (on-site) conservation refers to the maintenance and use of wild plant populations in the habitats where they naturally occur and have evolved without the help of human beings. Wild populations regenerate naturally and are also dispersed naturally by wild animals, winds and in water courses. There exists an intricate relationship, often interdependence, between the different species and other components of the environment (such as their pests and diseases) in which they occur. The evolution is purely driven by environmental pressures and any changes in one component affect the other. Provided that changes are not too drastic, this dynamic co-evolution leads to greater diversity and better adapted germplasm. Conservation of forests and other wild plant species is often carried out through protected areas such as national parks, gene sanctuary and nature reserves. However, this mode of conservation has certain limitations such as there is risk of loss of material due to environmental hazards. Ex situ conservation Ex situ (off-site) conservation of germplasm takes place outside the natural habitat or outside the production system, in facilities specifically created for this purpose. This is the chief mode of preservation of genetic resources for both cultivated and wild materials. The most convenient method of ex-situ germplasm conservation is in the form of seeds. Thus, the majority of field crops and vegetables which produce orthodox (dessication tolerant) seeds are conserved in gene banks by reducing their moisture content (3-7%) and storing under low humidity and low temperature. In case of crops with dessication sensitive or recalcitrant seeds (which lose their viability after being dried below a critical limit) and also in vegetatively propagated crops, in vitro methods are most useful for germplasm conservation. This tissue culture-based method has been mainly utilized for conservation of somaclonal and gametoclonal variations in cultures, plant material from endangered species, plants of medicinal value, storage of pollen, storage of meristem culture for production of disease-free plants and genetically engineered materials. In vitro Germplasm conservation Germplasm can be stored in vitro in a variety of forms including isolated protoplasts, cells from suspension or callus cultures, meristem tips, somatic embryos, shoot tips or propagules at various stages of development. Methods for in vitro germplasm conservation are classified into two groups based on culture growth: 1. Slow growth of cultures: where a limited growth of culture is allowed. This is a simple, effective and economic method, which can be used for all spe-

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cies where shoot tip/ nodal explants are available. In these techniques, growth is suspended by either cold storage or lowering oxygen concentration. Such methods require serial subculturing for periodic renewal of cultures. The storage of germplasm by repeated cultures has some disadvantages as during subculturing there is risk of contamination by pathogen, and genetic changes may also occur. 2. Cryopreservation: In this technique, any growth in the plant cell and tissue culture is brought to a halt, however, the cell/tissue still retains its viability by storing at ultra low temperature (-196°C) using liquid nitrogen. This method, also called freezepreservation, is most popular and effective for indefinite storage in the Djuar apparatus/equipment. Cryopreservation for germplasm purposes utilizes only shoot tips and buds but protoplasts, cells, tissues and somatic embryos are also cryopreserved for other tissue culture processes. Freezing injury is primarily associated with damage caused by ice crystals formed within cells and organs. Freezing-resistant species have mechanisms that limit the growth of ice crystals to extracellular spaces. Mechanisms that confer the resistance to freezing that is typical of woody plants include dehydration and supercooling. Cold stress reduces water activity and leads to osmotic stress within the cells. This osmotic stress effect leads to the activation of osmotic stress–related signaling pathways, and the accumulation of proteins involved in cold acclimation. Other cold specific, non-osmotic stress–related genes are also activated. Ice formation starts at –3 to –5°C in the intercellular spaces, where the crystals continue to grow, fed by the gradual withdrawal of water from the protoplast, which remains unfrozen. Resistance to freezing temperatures depends on the capacity of the extracellular spaces to accommodate the volume of growing ice crystals and on the ability of the protoplast to withstand dehydration. Several specialized plant proteins may help limit the growth of ice crystals by a noncolligative mechanism - that is, an effect that does not depend on the lowering of the freezing point of water by the presence of solutes. These antifreeze proteins are induced by cold temperatures, and they bind to the surfaces of ice crystals to prevent or slow further crystal growth. In rye leaves, antifreeze proteins are localized in the epidermal cells and cells surrounding the intercellular spaces, where they can inhibit the growth of extracellular ice. Plants and animals may use similar mechanisms to limit ice crystals: a cold-inducible gene identified in Arabidopsis has DNA homology to a gene that encodes the antifreeze protein in fishes such as winter flounder. Antifreeze proteins were first discovered in fishes that live in water under the polar ice caps. Antifreeze proteins confer to aqueous solutions the property of thermal hysteresis (transition from liquid to solid is promoted at a lower temperature than is transition from solid to liquid), and thus they are sometimes referred to as thermal

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hysteresis proteins (THPs). Another group of proteins found to be associated with osmotic stress is also up-regulated during cold stress. This group includes proteins involved in the synthesis of osmolytes, proteins for membrane stabilization, and the LEA proteins. Because the formation of extracellular ice crystals generates significant osmotic stresses inside cells, coping with freezing stress also requires the means to cope with osmotic stress. Sugars and some of the cold-induced proteins are suspected to have cryoprotective (cryo = “cold”) effects; they stabilize proteins and membranes during dehydration induced by low temperature. In winter wheat, the greater the sucrose concentration, the greater the freezing tolerance. Sucrose predominates among the soluble sugars associated with freezing tolerance that functions in a colligative fashion, but in some species raffinose, fructans, sorbitol, or mannitol serves the same function. A cryoprotective glycoprotein has been isolated from leaves of cold-acclimated cabbage (Brassica oleracea). In vitro, the protein protects thylakoids isolated from nonacclimated spinach (Spinacia oleracea) against damage from freezing and thawing. Plants develop freezing tolerance at nonacclimating temperatures when treated with exogenous ABA. Many of the genes or proteins expressed at low temperatures or under water deficit are also inducible by ABA under nonacclimating conditions. All these findings support the role of ABA in tolerance to freezing. The cell protoplast suppresses ice nucleation when undergoing deep supercooling. In addition, the cell wall acts as a barrier both to the growth of ice from the intercellular spaces into the wall, and to the loss of liquid water from the protoplast to the extracellular ice, which is driven by a steep vapor pressure gradient. Resistance to cellular dehydration is highly developed in woody species that are subject to average annual temperature minima below -40°C, particularly species found in northern Canada, Alaska, northern Europe and Asia. Factors affecting viability of cells frozen for cryopreservation –– Physiological state of material. Cells in the late lag or exponential phase are considered ideal for freeze preservation. After thawing, these cytoplasm rich cells are able to retain their viability and grow again from the actively dividing meristematic cell component. But in shoot tips, embryos, etc., tissue is large with highly vacuolated cells which get damaged by freezing and are unable to recover back. –– Prefreezing treatment. Conditioning treatment given to cells before freezing results in their hardening and increased survival rates. Such hardening treatments include growing culture in presence of cryoprotectant or growing at low temperature (4°C) (for cold dormant sp) or in presence of osmotic agents like sucrose. These treatments

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function by either changing the cell water content, metabolite content or membrane permeabilites. –– Cryoprotectants Cryoprotectants are chemicals imparting protection to withstand low temperature. For plants, most frequently used cryoprotectant is Dimethyl sulphoxide (DMSO). About 5-10% of DMSO is prepared and added gradually to prevent plasmolysis of the cells. Other commonly used cryoprotectants include glycerol, polyvinyl pyrollidone, polyethylene glycol (PEG) etc. –– Thawing rate and reculture For better survival of preserved samples, rapid thawing from - 196°C to about 22°C is recommended. By thawing rapidly, the damaging effects of ice crystal formation (crystallization of cell water while freezing) are minimized. These thawed samples during reculturing require special growth conditions, for enhanced recovery rates like dim light, high osmoticum, gibbrellic acid, and activated charcoal in the medium. Methods of Cryopreservation The sensitivity of cells to low temperature varies with the species. However, usually the sample to be preserved are treated with suitable cryoprotectant and then frozen by any one of the following methods: –– Rapid freezing. The vials with plant materials are directly dipped in liquid nitrogen. The temperature lowers very fast at the rate of 200°C/minute. It is a very hard treatment and hence survival rate is low. However, this method has been successful for germplasm conservation of a large number of species where plant material with small size and low water content has been chosen. –– Controlled freezing. The plant material is cooled stepwise from room temperature to intermediate temperature (-20°C) maintained at that temperature for thirty minutes followed with rapid freezing by dipping into liquid nitrogen. This is a reliable method and is applicable to wide range of plant materials including shoot apices, buds and suspension cultures (Figure 73) Advantages of Cryopreservation: –– Indefinite preservation as metabolism comes to halt –– Low maintenance as only liquid nitrogen needs to be replenished –– No contamination –– Applicable to all species amenable to tissue culture Limitations: –– Sophisticated equipment and facilities required –– Expertise needed –– Cells/tissues get damaged due to ice crystal formation or high solute concentration during desiccation.

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А

В

С

D

Note: A – controlled freezing; B, C – Djuar apparatus/equipment; D – rapid freezing by dipping into liquid nitrogen Figure 73. Cryopreservation of plant material

Equally with traditional methods of plant preservation ex situ application of isolated tissue and organ cultures has become more actual. The storage in vitro of valuable plant forms is a highly efficient way for maintenance of plant collections and conservation of plant biodiversity. Under the supervision of Dr. S.V. Kushnarenko at the Institute of Plant Biology and Biotechnology protocols for preservation of South-East Kazakhtan’s rare species Lonicera iliensis Pojark from three natural populations were developed. These protocols include seed storage, in vitro culture and cryopreservation of L.iliensis germplasm. Dried seeds were cryopreserved in liquid nitrogen (LN) and no significant changes in germination were apparent following LN exposure for 1 h and 3 months. L.iliensis seed collections were stored at +4° C, -20° C and -196° C. In vitro plantlets were propagated on MS medium added 1 mg/l 6-benzylaminopurine. The protocol of shoot tips cryopreservation was optimized: duration of cold acclimation of plants (3 weeks at +4°C, 8 h light/16 h darkness or 10 μmol/m2/s), 2 days of shoot tips preculture on MS medium with 0.3 M sucrose (or 5% DMSO), PVS2 treatment period about 80 min. The optimum storage conditions in the genetics bank of aseptic cultures have been found for 3-7° C. The special role in plant conservation in vitro is played by osmotic and physical factors of cultivation, retardants, temperature and light intensity. At creation of gene bank in vitro primary importance is given to representation and preservation of genetic stability [51]. Potato is one of the most important crops worldwide. Genetic resources of potato (Solanum tuberosum L. ssp. tuberosum) and related cultivated species are conserved through storage of tubers, in vitro plants and in cryopreservation. Cryopreservation, storage in or above liquid nitrogen, is the best option to maintain vegetatively propagated plants in the long term. The present review gives comprehensive information about various cryopreservation techniques for potato published from 1977 until the present. It discusses factors that affect the process

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415

and success of cryopreservation, such as donor culture conditions, preculture, cooling, warming and post-culture treatments. Studies are presented that analyse the histological and ultrastructural changes after different cryopreservation steps and morphological pathways during regeneration of plants after rewarming. Maintenance of genetic stability in potato after cryopreservation has also been demonstrated by various phenotypic and molecular methods. The first thermal analyses on potato shoot tips are presented using differential scanning calorimetry to analyse the state of water during cooling and warming. Biochemical analyses of different compounds, such as soluble sugars and proteins, have been performed to understand and improve existing cryogenic methods. Potato is an example where successful virus elimination has been obtained via cryopreservation of shoot tips (cryotherapy). There are already cryopreserved collections of potato shoot tips in Germany, Peru, Czech Republic, South Korea and USA, but additional experiments on fundamental aspects of potato cryopreservation will help to improve understanding of the different cryopreservation methods, start new collections in the other countries and build up existing cryocollections of potato [52]. Cryopreservation is the most suitable long-term storage method for genetic resources of vegetatively maintained crops like potato. In the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) the DMSO droplet method is applied, and so far more than 1000 accessions are cryopreserved with an average regeneration rate of 58%. New experiments with four potato accessions using alternating temperatures (22/8°C day/night temperature, 8/16 h photoperiod prior to cryopreservation) showed improved regeneration. The influence of this preculture on the shoot tips was studied for two wild, frost resistant species Solanum acaule and S. demissum and for two cultivated, frost sensitive potatoes S. tuberosum ‘Désirée’ and ‘King Edward’. Comparison of liquid and solid media after cryopreservation showed improved regeneration on solid media with higher regeneration percentages, less callus formation and better plantlet structure. In comparative analyses biochemical factors like soluble sugars, starch, and amino acid concentrations were measured. Shoot tips after constant and after alternating temperature preculture were analyzed. Total concentrations of soluble sugars (glucose, fructose, and sucrose) were higher for all accessions after the alternating temperature preculture, which could be the reason for improved cryopreservation results. At the Kazakh Research Institute of Potato and Vegetable Growing the investigations on the collection, conservation and study of the gene pool of vegetable crops have been conducted since 1995. The most number of accessions collected have come to cucurbits – 3472 accessions on 13 crop plants of 12 botanical species. Solanaceous plants also have made a large group of the Institute’s gene pool – 3231 accessions of 7 crops, most samples of them have been in tomato

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(2266) and pepper (728). Onion crop plants in the gene pool have been represented by 797 samples of 36 botanical species; the most have been accounted for garlic (285) and onions (205) accessions. Plant genetic resources provide the biological basis of the Food security, contribute to the sustainable development of the economy and are also essential for the welfare of each country. Genetic variations are the strategic resources of breeding programs to create new varieties and hybrids of plants, including vegetable and melon crops [52]. Seed banks and in vitro plant collections are considered as a part of the nature protection system. An important prerequisite of successful application of in vitro approach is availability of reliable propagation methods. Shoot tips of plant species representing 15 families were used as an initial material for propagation and further conservation. Explants were placed on MS media supplied with 5 mg/l ABA or 20 g/l mannitol and cultivated at the standard conditions. Mannitol is more effective growth retardant than ABA for a great number of species. Application of 5 mg/l ABA led to maximum subcultivation intervals of 3, 4 and 4 month. The collections of Solanum species and interspecific hybrids deposited in vitro with more than 567 individual numbers including representatives of 42 species and the basic in vitro collection of potato varieties of Kazakhstan origin should be noted [52, 53]. Encapsulation technology has recently revolutionized the production and conservation program of elite and threatened germplasm throughout the globe. This technology has made the exchange program possible between different laboratories at an ease. Synthetic seed production not only scaled up extensive and commercial production of plants but also economized the requirements of space and time. Synseed technology has been utilized for commercial and industrial production of different agricultural, ornamental, medicinal and woody climbers. Germplasm conservation of pineapple (Ananas comosus (L.) Mer.) is crucial to preserve the genus genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, which is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes.

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Under the supervision of Dr. I.Y. Kovalchuk et al. at the Institute of Plant Biology and Biotechnology germplasm preservation of fruit, berry crops and grape was implemented in two main directions: cold storage, limiting the growth of in vitro culture, at +3...+40 C, requires periodic subculturing and is effective for short and medium term storage. Cryopreservation, in conditions of ultra-low temperatures in liquid nitrogen or its vapor at a temperature of -165...-1960 C, allows indefinite preservation of viability and high regeneration potential and genetic stability. To date, medium-term cold storage in vitro and long-term tissue cryopreservation in liquid nitrogen are used in addition to field collections, reliably preserving the gene pool of fruit and berry crops in Kazakhstan. Medium-term cold storage of plants in vitro is conducted in a chamber under a 10-hour photoperiod (7 μmol m-2 s-1 ) at +40 C. Shoot cultures are stored in air-permeable plastic bag on MS medium containing: 0.5 mg/l 6-BA, 0.1 mg/l IBA, 3% sucrose or 2% sucrose+2% mannitol for fruit and berry crops; for grape, 1/2 MS containing 0.1 mg/l BA, 0.2 mg/l IBA and 3% sucrose. Cryopreserved germplasm is held using the methods of vitrification, encapsulation-dehydrotation, freezing of dormant and winter buds and direct immersion in liquid nitrogen. It is effective to freeze dormant, winter buds and meristems in apple, pear, apricot, cherry, black currant, raspberry. Seed and embryonic axes cryopreserved by direct immersion in liquid nitrogen can be used for wild species and selections. The cryopreserved collection at the Institute of Plant Biology and Biotechnology includes 126 apple, 21 pear, 42 tart cherry, 58 sweet cherry, 31 strawberry, 20 black currant, 31 raspberry and 4 wild forms of apricot (Prunus armeniaca L.) varieties and hybrids stored in liquid nitrogen [54]. Climbing plants are groups of plants that often show unique horticultural uses because of their beauty-imparting features. As the stems are weak, these plants have evolved various climbing devices in order to support growth and development. This climbing habit is predominantly seen in angiosperms and some members of ferns, and Gnetum is the only representative genus of gymnosperm. Several families such as Convolvulaceae, Cucurbitaceae, and Dioscoreaceae are exclusively climbers, while over 50 species of families like Rubiaceae, Fabaceae, Calastraceae, and Apocynaceae are also of climber types. Besides their aesthetic use, the plants are of high medicinal value as almost all contain pharmaceutically active bio-compounds like michellamines A and B (anti-HIV properties) present in Ancistrocladus korupensis, various saponins (Asparagus racemosus), diosgenin (Dioscorea deltoidea), colchicine (Gloriosa superba), cordifolioside A (Tinospora cordifolia), momordin (Momordica balsamina), protoberberine, syringin, shatavarin I–IV, asparagine, aglycones, etc. Many of these plants are widely used in folk and traditional medicines [55]. The prevalence of diseases and high cost of modern Medicare coupled with increasing load of human population across the globe have resulted in overexploitation of plants/climbers with

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extinction possibilities. A large number of plants including the above has already been endangered. Therefore, a balanced approach is needed in order to preserve germplasm of climbers for future uses. The application of biotechnological interventions and ex situ conservation approaches like in vitro cryopreservation and setting/strengthening germplasm or seed banks have opened a new vista for preservation of climbers. Control questions: 1. Define germplasm and briefly discuss its significance. 2. Briefly describe the various approaches for in vitro germplasm conservation, their advantages and limitations. 3. What do you understand by cryoprotectant? Name any two most frequently used cryoprotectants. 4. Write short notes on Vitrification, Cryoprotectants, Stepwise freezing

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Appendix

APPENDIX The composition of culture media Components

Murashige Skoog culture media Concentration, mg/1 L medium KNO3 1900 NH4NO3 1650 Ca (NO3)2 (NH4)2SO4 MgSO4 · 7 H2O 370 CaCI2·H2O CaCI2· 2 H2O 440 СаCI КН2РО4 170 NaH2PO4· H2O MnSO4·H2O MnSO4 · 4 H2O 22,3 ZnSO4 · 4 H2O 8,6 ZnSO4 · 7 H2O Н3ВО4 6,2 CuSO4· 5 H2O 0,025 Na2MoO4 · 2 H2O 0,25 CoCI2 · 6 H2O 0,025 FeSO4· 7 H2O* 27,85 Na EDTA·2 H2O 37,25 Myoinositol 100 Ascorbic acid Thiamine-HCI 0,5 Pyrodoxine-HCI 0,5 Nicotyne acid 0,5 Sucrose 30 000 Agar, Gelrite pH 5.6-5.8

Table 1

B5 medium (Gam-borg's B5 medium)

White culture medium

Nitsch’s medium

3000 134 500 150 150 10 2 3 0,075 0,25 20 000 5.5

81 142 74 65 12 2000 5.6-5.8

950 720 185 166 68 25 10 10 0,025 0,25 27,85 37,25 200 3 3 1 60 000 7000

*Iron, 500 ml Stock (200x): Dissolve 3.725 gm of Na2EDTA (Ethylenediaminetetra acetic acid, disodium salt) in 250 ml dH2O. Dissolve 2.785 gm of FeSO4x7H2O in 250 ml dH2O Boil Na2EDTA solution and add to it, FeSO4 solution gently by stirring

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Basics of biotechnology Table 2

The composition of culture media for plant cell culture Shenk-ChilderWPM Gelrigel culture brandt's (Lloyd, Mc medium culture medium Cown) Concentration, mg/1 L medium 2500 400 386 492 300 400 123 180,7 200 72,5 170 136 300 10,0 22,3 1,0 8,6 -

Components

KNO3 NH4NO3 Ca (NO3)2 NH4Н2РO4 MgSO4 · 7 H2O MgSO4 CaCI2· 2 H2O СаCI2 КН2РО4 NaH2PO4· H2O MnSO4·H2O ZnSO4 · 7 H2O Н3ВО4 CuSO4· 5 H2O KJ КCI Na2MoO4 · 2H2O CoCI2 · 6 H2O FeCI3

5,0 0,2 1,0 0,1 0,1 -

6,2 0,35 0,25 -

0,75 0,25

FeSO4· 7 H2O Na2 EDTA·2 H2O Myoinositol Thiamine-HCI Pyrodoxine-HCI Nicotyne acid Sucrose pH

15,0 20,0 1000 5,0 0,5 5,0 30 000 5.9

27,8 37,3 20000 5.6-5.8

-

Knops culture medium 25 1000 51 25 12 1 drop from 5% solution -

The composition of culture media for plants anther culture Components 1 КNO3 NН4NOз (NH4 )2 SO4

N6 2 2830 463

Culture medium, mg/ 1 L В5 L85D12 3 4 2500 1400 300 134 -

Table 3

Роtato 2 5 1000 100

421

Appendix 1 Са(NO3 )2·7 H2O МgSO4· 7 H2O СаС12 КС1 КН2 РO4 NaН2 РO4 · Н2O FeNa ЕDТА МnSO4 · 4 Н2O KJ Н3ВO3 ZnSO4 СuSO4 · 5 Н2O Nа2МоO4· 2 Н2O СоС12· 6 Н2O Myoinositol Thiamine-HCI Nicotyne acid Pyrodoxine-HCI Potato extract Glycine Sucrose 2,4-D NAA Kinetin рН

2 185 166 400 10 М 4.4 0.8 1.6 1.5 0.100 100 1.0 0.5 0.5 2.0 90000 1.0 5.6-5.8

3 250 150 150 10 М 10.0 0.75 3.0 2.0 0.025 0.25 0.100 100 10.0 1.0 1.0 20000 0.4 0.1 5.5

4 150 150 400 -

5 100 125 35 200 -

11.2 0.83 6.2 0.025 0.025 1.0 0.5 0.5 2.0 20000 0,75 1.5 5.6-5.8

1,0 + 90000 1,0 05 5.8 Table 4

Plant Growth Regulator stock

The heat-labile plant growth regulators are filtered through a bacteria-proof membrane (0.22 μm) filter and added to the autoclaved medium after it has cooled enough (less than 600 C). The stocks of plant growth regulators are prepared as mentioned below. Plant Growth Regulator Benzylaminopurine (BAP) Naphtalene acetic acid (NAA)

Nature Autoclavable

Mol.Wt. 225.2

Soluble in 1 N NaOH

Heat labile

186.2

Ethanol

The desired amount of plant growth regulators is dissolved as above and the volume is raised with double disstilled water. The solutions are passed through disposable syringe filter (0.22 μm). The stocks are stored at - 200 C.

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CONTENT INTRODUCTION..........................................................................................................................3 PART I. MICROBIAL BIOTECHNOLOGY Chapter 1. HISTORY OF MICROBIAL BIOTECHNOLOGY..................................................6 Chapter 2. OBJECTS OF BIOTECHNOLOGY AND THEIR APPLICATION.......................9 2.1 Basic nature of cells....................................................................................................................9 2.2 Classification of living organisms: three domains of living organisms....................................11 2.3 Microorganisms in industrial microbiology and biotechnology...............................................13 Chapter 3. MICROBIOTECHNOLOGY....................................................................................16 3.1 Microorganisms – objects of biotechnology, their demands.....................................................16 3.2 Stages and kinetics of microbial growth...................................................................................17 3.3 Products of microbial fermentation and metabolism................................................................18 3.4 Asepsis, antiseptics and disinfection. Sterilization methods in biotechnology.........................20 3.5 Organization of biotechnological production...........................................................................23 3.6 Methods of microorganism cultivation.....................................................................................26 3.6.1 The aerated stirred batch fermenter................................................................................27 3.6.2 Systems of cultivation with full replacement...................................................................29 3.7 Obtaining of microbial synthesis products...............................................................................30 3.8 Purifying of microbial synthesis products................................................................................32 3.9 Modern methods of substances separation...............................................................................34 3.10 Methods of microbial culture storage.....................................................................................39 Chapter 4. SELECTION OF MICROORGANISMS – PRODUCERS OF IMPORTANT SUBSTANCES.....................................................................42 4.1 The choice of initial microorganism for selection....................................................................44 4.2 Preparation of the initial strain for selection.............................................................................45 4.3 Production of mutants...............................................................................................................47 4.4 Methods of selection mutants with high productive level......................................................49 4.5 Selection of random (unpredictable) mutations........................................................................49 4.6 Receiving practically important strains through genetic engineering.......................................50 Chapter 5. APPLICATION OF BIOTECHNOLOGICAL OBJECTS IN DIFFERENT INDUSTRIES...............................................................................52 5.1 Food biotechnology..................................................................................................................52

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5.1.1 Microorganisms used in the fermentation and production of yeasts...........................................................................................................52 5.1.2 Yeast production..............................................................................................................55 5.1.3 Beer fermentation. Conditions for beer making..............................................................56 5.1.4 Manufacture of dairy products. Microorganisms used in the production of dairy products. Sour-milk drinks and technological processes of their production.............................................................................57 5.1.5 Ayran (kefir) and its technology of production................................................................57 5.1.6 Technology of Kumys and Shubat production.................................................................59 5.2 Agricultural biotechnology.......................................................................................................62 5.2.1 Biopesticides....................................................................................................................63 5.2.2 Biochemical Pesticides....................................................................................................63 5.2.3 Microbial Pesticides........................................................................................................63 5.2.4 The nitrogen cycle and nitrogen fixation.........................................................................65 5.2.5 Mechanism of biological nitrogen fixation......................................................................66 5.2.6 Products obtained from nitrogen fixitation......................................................................69 5.3 Environmental biotechnology...................................................................................................69 5.3.1 Biodegradation................................................................................................................69 5.3.2 Anaerobic biodegradation of pollutants..........................................................................70 5.3.3 Role of microorganisms in biodegradation of pollutants................................................71 5.3.4 Biodegradable pollutants................................................................................................71 5.3.5 Types of bioremediation...................................................................................................74 5.3.6 Bioremediation technology..............................................................................................76 5.3.7 Natural attenuation..........................................................................................................77 5.3.8 Biostimulation..................................................................................................................78 5.3.9 Bioaugmentation..............................................................................................................78 5.3.10 Microbial metabolism....................................................................................................79 PART II. ANIMAL BIOTECHNOLOGY Chapter 1. THE SUBJECT, OBJECTS, TOOLS AND METHODOLOGY OF ANIMAL BIOTECHNOLOGY..........................................................84 Chapter 2. BIOSAFETY AND BIOETHICS ISSUES IN ANIMAL BIOTECHNOLOGY..............................................................................................95 Chapter 3. BASIC TECHNICS IN ANIMAL CELL CULTURE............................................102 Chapter 4. STEM CELLS TECHNOLOGY.............................................................................120 Chapter 5. REPRODUCTIVE TECHNOLOGIES IN ANIMAL BIOTECHNOLOGY............................................................................................141 Chapter 6. INDUCTION OF SUPEROVULATION................................................................147 Chapter 7. COLLECTION OF SPERM, OOCYTES AND EARLY EMBRYOS..........................................................................................................150 Chapter 8. ARTIFICIAL INSEMINATION.............................................................................159

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Chapter 9. IN VITRO FERTILIZATION.................................................................................165 Chapter 10. EMBRYO TRANSFEr............................................................................................177 Chapter 11. CRYOPRESERVATION OF ANIMAL CELLS AND TISSUES......................180 Chapter 12. CHIMERAS. XENOTRANSPLANTATION.......................................................188 Chapter 13. CLONING TECHNOLOGY TO PRODUCE ANIMAL CLONES...................195 Chapter 14. TRANSGENIC TECHNOLOGIES IN ANIMAL BIOTECHNOLOGY............................................................................................208 PART III. PLANT BIOTECNOLOGY Chapter 1. THE SUBJECT, OBJECTS, HISTORY AND METHODOLOGY OF PLANT BIOTECHNOLOGY..................................................236 1.1 History of Plant Biotechnology..............................................................................................239 1.2 The development of biotechnology researches in Kazakhstan...............................................243 Capter 2. TECHNIQUES AND METHODS OF CULTIVATION PLANT TISSUE IN VITRO...................................................................250 2.1 Aseptic technique. Tissue culture technique..........................................................................250 2.2 Methods of cultivation plant tissue in vitro............................................................................267 2.3 Biology of cultured plant cells................................................................................................275 Chapter 3. INDUSTRIAL AND AGRICULTURAL APPLICATIONS OF IN VITRO CULTURE..........................................................................................................288 3.1 Production of secondary metabolites in plant cell culture......................................................288 Chapter 4. MICROPROPAGATION IN VITRO......................................................................310 Chapter 5. CELL ENGINEERING............................................................................................333 Chapter 6. CELL SELECTION..................................................................................................345 Chapter 7. EMBRYO AND ENDOSPERM CULTURE...........................................................353 Chapter 8. HAPLOID TECHNOLOGY....................................................................................359 Chapter 9. GENETIC ENGINEERING....................................................................................378 Chapter 10. CRYOCONSERVATION.......................................................................................409 APPENDIX..................................................................................................................................419 REFERENCES............................................................................................................................422

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Еducational issue

Zayadan Bolatkhan Kazykhanuly Dzhansugurova Leila Bolatovna Turasheva Svetlana Kazbekovna

BASICS OF BIOTECHNOLOGY Textbook

Stereotypical publication Editor V. Popova

Typesetting U. Moldasheva Cover design Ya. Gorbunov Cover design used photos from sites www.fool.com

IB №13049

Signed for publishing 23.09.2019. Format 70x100 1/12. Offset paper. Digital printing. Volume 26,75 printer’s sheet. 100 copies. Order №5819. Publishing house «Qazaq University» Al-Farabi Kazakh National University KazNU, 71 Al-Farabi, 050040, Almaty Printed in the printing office of the «Qazaq University» publishing house.