Basics of Biotechnology: Plant Biotechnology: textbook 9786010421028

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REPUBLIC OF KAZAKHSTAN MINISTRY OF EDUCATION AND SCIENCE AL-FARABI KAZAKH NATIONAL UNIVERSITY

S. K. Turasheva

BASICS OF BIOTECHNOLOGY: PLANT BIOTECHNOLOGY Textbook

Almaty «Qazaq university» 2016

UDC 581.19 (075.8) T 95 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.1 from November 2, 2016 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.3 from June 2, 2016 y.) Ministry of Education and Science of the Republic of Kazakhstan (protocol No.1633-3531/17-6 from October 18, 2016) Peer reviewers: Dr., Professor K. Zhambakin Dr., Professor G. Djuskalieva Dr., Professor S.S. Kenzhebaeva

T 95

Turasheva S.K. Basics of Biotechnology: Plant Biotechnology: textbook / S.K. Turasheva. – Almaty: Qazaq university, 2016. – 188 p. ISBN 978-601-04-2102-8 In the textbook presented basic information on theoretical, practical and applied aspects of plant biotechnology. Each chapter includes relevant media protocols, methods, technologies and is profusely illustrated with schemas, diagrams and original photographs. The textbook includes 3 parts, 13 chapters and contains a complete bibliography and a glossary of terms commonly used in plant biotechnology literature. This textbook help students understand plant biotechnology, how the research is conducted, and how the technology may impact the future. The textbook proves to be an excellent text-material for undergraduate, postgraduate students, researchers in various fields of plant sciences, plant biotechnology and a useful reference book for those interested in the application of any aspect of plant technologies. Published in authorial release. В учебнике представлены основные теоретические и практические материалы, а также прикладные аспекты биотехнологии растений. В каждой главе рассматриваются протоколы питательных сред и методов, технологии, которые сопровождаются поясняющими схемами, диаграммами, а также оригинальными фотографиями. Учебник состоит из 3 частей, 13 глав, а также библиографических ссылок и глоссария терминов, общепринято используемых в биотехнологии растений. Книга будет полезна студентам для изучения основ биотехнологии растений, правильного проведения исследований и понимания того, каким образом технологии могут оказывать влияние на будущее развитие биотехнологии. Учебник предназначен для студентов, магистрантов, молодых научных сотрудников, занимающихся в области растениеводства, биотехнологии растений и для широкой аудитории читателей, интересующихся прикладными аспектами биотехнологии растений. Издается в авторской редакции.

UDC 581.19 (075.8) ISBN 978-601-04-2102-8

© Turasheva S.K., 2016 © Al-Farabi KazNU, 2016

INTRODUCTION Biotechnology is the application of scientific techniques to modify and improve plants, animals, and microorganisms to enhance their value. It is integrated use of biochemistry, molecular biology, microbiology to achieve technological application of the capabilities of biological agents. Therefore, biotechnology has emerged as a science with immense potential for human welfare ranging from food processing, human health to environment protection. 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 generally, culture in vitro are 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 potential to make 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 the 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. 5

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Basics of biotechnology: plant biotechnology

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 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 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 regenerate triploid plants difficult to raise  in vivo. The plant protoplasts (plant protoplasts are naked cells from which cell wall has been removed) can be fused to form somatic hybrids. Such fusion products are the result of the union 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 single set of chromosome, as that 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, method accelerates breeding by 3-5 years. This is particularly important for highly heterozygous, long-generation plant species. DH production is aimed for gene transfer into the homozygotic state in the first generation. Recessive mutations, important recombinations, and other genomic changes can be found in doubled haploid 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

Introduction

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to raise new improved varieties which may have of commercial value such as, new flower color, big canopy plants, large sized grains etc. 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 parent plant. Thus, it permits perpetuation of the parental characters of the cultivars among the plants resulting from micropropagation. Activelydividing 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. 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 rapid and can continue round the year, independent of the season and climate conditions; 3) reduced growth cycle and rapid multiplication as shoot multiplication has short cycle and each cycle results in exponential increase in 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 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.

Part I HISTORY OF BIOTECHNOLOGY

Chapter 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 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 embryo and then to whole plant. The idea that cell division may be initiated by a diffusible factor originated with plant physiologist G. Haberlandt. He demonstrated that vascular tissue contains a water-soluble substance or substances that will 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 about the requirements in media in experimental conditions which could possibly induce cell division, proliferation and embryo induction. G.Haberlandt is thus regarded as 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 (upto 1934), there was very little further progress in cell culture research. Within this period, an innovative approach to 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 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 was in 1930s, when progress in 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 can 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 10

Chapter 1.1. History of plant biotechnology

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salts supplemented with yeast extract. He later (1937) replaced YE by vitamin B namely pyridoxine, thiamine and proved their growth promoting effect. In 1926, Fritz Went discovered first plant growth regulator (PGR), indoleacetic acid (IAA). IAA is a naturally occurring member of a 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 by Nobecourt (1937), who could successful 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 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 a 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 heart shaped embryos by enriching culture media with coconut milk besides the usual salts, vitamins and other nutrients. This provided 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 whole plant. After 1950, there was an immense advancement in knowledge of 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 auxinic nature of milk. This prompted further research and so other classes of PGRs were recognized. A great many substances were 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, 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 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)

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Part I. History of biotechnology

who isolated ‘kinetin’- a derivative of adenine (or aminopurine) 6- furfuryl aminopurine. Kinetin and many such 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 an auxin. Letham (1973) isolated the molecule responsible for this activity and identified it as trans-6-(4-hydroxy-3-methylbut-2-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 culture media by differing essentially in mineral content. In this direction, Murashige T. and Skoog F.(1962) prepared a medium by increasing the concentration of salts twentyfive times higher than Knops. This media enhanced the growth of tobacco tissues by five times. Even today MS medium has immense commercial application in tissue culture. Having achieved success and expertise in growth of callus cultures from explants under in vitro conditions, focus now shifted to preparation of single cell cultures. Muir (1953-1954) demonstrated that when callus tissues were transferred to 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. Next step for realization of Haberlandt’s objectives was development of whole plant from the proliferated tissue of these cells. Vasil and Hilderbrandt were 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 finally demonstrating 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 also genetic engineering. For e.g. 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 virus 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 fertlization

Chapter 1.1. History of plant biotechnology

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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 has been practiced since the 1950’s. A considerable contribution toward the development of this method was carried out 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 result of abnormal development of the female gametophyte and subsequent embryo- or callus formation. Next breakthrough in application of 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 early 1970s, 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 first restriction enzyme from Haemophillus influenzae which was later purified and named Hind III. Same year witnessed 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 first recombinant DNA in vitro by combining DNA from SV40 virus with that of lambda virus. This led to construction of 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 crown gall disease in plants was recognized as early as 1907 by Smith and Townsend. However, it was in 1974, that Zaenen et al discovered that Ti- plasmid is the 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 development of techniques of genetic engineering in 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.

Chapter 1.2 THE DEVELOPMENT OF BIOTECHNOLOGY RESEARCHES 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 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 (Fig. 1).

М.A. 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 USR (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 was engaged in the search for study of the physical and chemical properties of informosomes in plant cells. Informosomes are the free cytoplasmic, polysome-associated and nuclear,

Chapter 1.2. The development of biotechnology researches in kazakhstan

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including RNA-binding proteins. He studied the physical and chemical properties of informosomes influent 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 USR. In 1983, M.A. Aytkhozhin organized the Institute of Molecular Biology and Biochemistry in Alma-Aty, then for four years he was Head of the Institute. Murat Aytkhozhin was the first scientist who introduced a course in Molecular Biology and several special courses for students at Kazakh State University in the Biology faculty. In 1987, he founded the Kazakh Agricultural Biotechnology Center and organized research into the plant cell and genetic engineering. In 1986-1987, he was President of the Academy of Sciences of the Kazakh USR. Under the leadership of M. Aytkhozhin a set of instruments for the automatization of experiments in Molecular Biology was designed (it was protected by 15 copyright certificates and 16 patents in leading countries). For the research series «Discovery informosome – a new class of intracellular particles» and «Molecular mechanisms of plant protein biosynthesis» he was honored with the Lenin Prize and awarded 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 Presidium of the Russian Plant Physiology Society and Vice-President of 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 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 me­ thodological guides/mannuals and 5 patents. Professor I. Rakhimbaev and breeders at the Institute of Plant Biology and Biotechnology together created 4 new wheat cultivars and 2 rice cultivars. Under his leadership, 30 PhD and 8 Doctor’s theses were defended in Kazakhstan and other foreign countries. In 1993-2007, he was Chief – Editor of the Scientific Journal «Biotechnology. Theory and Practice», «Proceedings of the National Academy of Sciences» and Chairman of the Dissertation Council on the Defence 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 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 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, alumnus of Professor I. Rakhimbaev work in leading research companies, the Research and Academic Institute, National Centres, Universities in Kazakhstan and abroad. Gulzhannet Zhansultanovna Valikhanova (1939) – Biologist, Professor of the De­ partment of Plant Biotechnology, Biochemistry and Physiology (Department of Bio­ technology) at Al-Farabi Kazakh National University. In 1961, she graduated from

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Part I. History of biotechnology

Kazakh State University in Alma-Ata and gained her 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 USR Academy of Sciences. In 1971-1981, G. Valikhanova was the senior lecturer at the Department of Plant Physiology and Biochemistry. In 1981, she was Associate Professor and in 1998 she was awarded the academic rank of Professor of Kazakh National University. In 1989-1990, Professor G. Valikhanova headed the Department of Plant Physiology and Biochemistry at the Biology faculty of 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 of 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 guides/mannuals on Biotechnology, a Test-set in the disciplines of Plant Physiology and Plant Biotechnology, education programs in the field of the obligatory basic biotechnological disciplines (Cell Biotechnology, Plant Biotechnology, Plant Physiology) and special courses/pacticums. Professor G. Valikhanova is 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. Erezhepov. Methodical gui­de­li­ nes 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. This led to further investigations by I.R. Rakhimbaev, B.B. Anapiyaev, S.V. Kushnarenko (from the Institute of Plant Biology and Biotechnology, IPBB), Dr. S.S. Bekkuzhina (from 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. Through the research of Professor N.K. Bishimbaeva et al (2006) was identified the way to regulate cytodifferentiation and morphogenesis in long-term cultured embryogenic tissues of barley and wheat, growing in normal conditions and under abiotic stress.

Chapter 1.2. The development of biotechnology researches in kazakhstan

17

Genotype-independent cell technology of plant regeneration during long-term sub­ cultivation 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 crops 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, 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. Methods of embryo culture have been improved by Professor A.E. Erezhepov, S.K. Mukhambetzhanov, L.N. Tuypina (from Al-Farabi Kazakh National Univesity and IPBB) for increasing the viability of wheat hybrid embryos with its wild relatives. Professor S. Svanbaev and Associate Professor E.D. Dzhangalina (from the Institute of Plant Physiology, Genetics and Bioengineering) received «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 leadership 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 head of 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 1999-2002 by head of Professor Z.R. Mukhitdinova, Professor M.A. Berdina, Associate Professor S.K. Turasheva and senior researcher N.K. Ismagulova. They have shown that the formation of gametes and embryo deve­ lopment in a culture of isolated ears of wheat can be realized in the zero-gravity of a space. It has been estimated that a significant damage and decrease in the rate of cell division, callus growth and cell secretory activity. During extended space flight and further cultivation under Earth conditions, somaclonal variants in a culture of somatic tissue of potato were obtained by head of Professor M.K. Karabaev, Dr. L. Ligay, R.M. Tur­ panova, Zh.T. Lesova from the Institute of Molecular Biology and Biochemisrty named M.A. Aytkhozhin. In 2002, the cultivar «Tokhtar» (the cultivar was called according name the first kazakh astronavt Tokhtar Aubakirov) was obtained from somaclones. In the application of tissue culture came the isolation and regeneration of wheat and maize protoplasts demonstrated by Professor M. Karabaev and Zh. Dzhardemaliev in 1995 at the Institute of Molecular Biology and Biochemisrty named M.A. Aytkhozhin.

18

Part I. History of biotechnology

A priority area in the development of biotechnology in Kazakhstan is genetic engineering. Scientists from the National Biotechnology Centre (NBC) and Institute of Molecular Biology and Biochemistry named M.A. Aytkhozhin as well as from the Institute of Plant Biology and Biotechnology and L.N.Gumilyov Eurasian National University Professor R.T. Omarov, Dr. Sh.A. Manabaeva, Professor A.A. Kakimzhanova, O.N. Kha­ pilina, Professor B.K. Iskakov, Professor N.K. Bishimbaeva and Dr. N.N. Galiakparov have created methods of stable genetic transformation and regeneration of transgenic crops, potato, cotton and grapes (2010-2013). Transgenic potato with genetically fixed Y-resistant virus has been obtained. A viral genome fragment was introduced into the potato genome in the antisense orientation. The genetic transformation of maize was 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 from NBC demostrated that tobacco is the best host for transient production of heterologous proteins compared to other plants from Solanaceae (2014). 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. Professor K.Zh. Zhambakin optimized the protocol for the production of transgenic canola (rapeseed plants) expressing the gene transcription factor HvNHX2 by use of Agrobacterium-mediated transformation (2015). 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 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 identifing specific plant traits. The use of molecular markers can reduce the labor and resources needed to analyze the sample. Academic K.R. Urazaliev 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 are applying molecular genetic analysis, QTL, MAS for improving and increasing the efficiency of breeding (2012-2014). The results, obtained by our scientists have been actively used for the identification of new genes associated with stress abiotic resistance, search of 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 quantitative trait loci (that are associated with yield, plant resistance and seed/grain quality) in the selection process and provide for the development of new varities 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 development of Biotechnology? 7. What do you know about biotechnology researches in Kazakhstan?

Part II TECHNIQUES AND METHODS OF CULTIVATION PLANT TISSUE IN VITRO

Chapter 2.1 ASEPTIC TECHNIQUE. TISSUE CULTURE TECHNIQUE 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, it can be transferred to ex vitro condition to allow continuous growth of the plantlets. 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: 1. Explant. 2. Aseptic environment. 3. Nutrient media. 1. 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 plant- root, stem, petiole, leaf or flower, choice of 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 initiate tissue culture. Therefore, the parent plant must be healthy and free from obvious signs of disease or decay. 2. Aseptic Technique – procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures. Aseptic technique is absolutely necessary for the successful establishment and maintenance of 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 plant cell division is slower compared to 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 20

Chapter 2.1. Aseptic technique. Tissue culture technique

21

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 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 the 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 minimize the possibility that microorganisms remain in or enter the cultures. Aseptic environment during culture 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 (Fig.2).

Figure 2. Laminar flow with HEPA filter («High Energy Particle Air»)

Laminar airflow hoods are used in commercial and research tissue culture settings. 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.

22

Part II. Techniques and methods of cultivation plant tissue in vitro

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. Sterilization and Use of Supplies and Equipment. Sterilization procedure involve: 1. Sterilizing tools, media, vessels etc. 2. Surfacesterilizing 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 volume in individual vessels and 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 (Fig.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 275350 °C and will destroy bacterial and fungal spores that may be found on 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 (large pressure cooker), less often by filtration and seldom by irradiation. The container with the medium should be properly closed and autoclaved at 121°C, 105 kPa, for 20 minutes. It 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 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),

23

Chapter 2.1. Aseptic technique. Tissue culture technique

30-40 min is 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 a medium. Too high temperatures or too long cycles can also result in changes in properties of the medium.

A

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 a 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 is 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. 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

24

Part II. Techniques and methods of cultivation plant tissue in vitro

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 more 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. Generally 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 between 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. Sterilization techniques used in Plant Tissue Culture Technique

Table 1

Materials sterilized

Steam sterilization/Autoclaving (121°C at 15 psi for Nutrient media, culture vesels, glasswares and plas20-40 min) ticwares Dry heat (160-180°C for 3h) Instruments (scalpel, forceps, needles etc.), glassware, pipettes, tips and other plasticwares Flame sterilization Instruments (scalpel, forceps, needles etc.), mouth of culture vessel Filter sterilization (membrane filter made of cel- Thermolabile substances like growth factors, amino lulose nitrate or cellulose acetate of 0.45- 0.22 µm acids, vitamins and enzymes. pore size) Alcohol sterilization Worker’s hands, laminar flow cabinet Surface sterSodium hypochlorite, hydrogen peroxide, mercuric ilization chloride etc Explants

Calcium hypochloride is used for surface sterilization 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 of 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. The use of antibiotics

Chapter 2.1. Aseptic technique. Tissue culture technique

25

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. 3. 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 e.g. some species are sensitive to high salts or have different requirements for PGRs (Plant Growth Regulators). Some tissues show better response on solid medium while others prefer a liquid medium. Therefore, development of culture medium formulations is 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) This is a high salt medium due to its content of potassium and nitrogen salts. B5 medium works well for protoplast culture. It has lesser amounts of nitrate and particularly ammonium salts than MS medium. Nitsch’s medium developed for anther culture contains salt concentration intermediate to MS and White. 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 micronutrients 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 form of salts in media. Nitrogen is usually supplied in form of ammonium (NH4+) and nitrate (NO3-) ions. Nitrate is superior to ammonium as the sole N source but 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 are becoming less acid. When ammonium uptake, it 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 the amount of nitrogen in media have significant effects on cell growth and differentiation. pH controlling in the media is not the only reason of 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 thetwo nitrogen forms promotes cation–anion balance within the plant. Media containing high levels of NH4+ also inhibits chlorophyll

26

Part II. Techniques and methods of cultivation plant tissue in vitro

synthesis. 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 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. 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 quantity 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 at wide range of pH. When iron 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 approached this problem by adding iron together with citric acid or tartaric acid. Compounds such 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 thus are 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

Chapter 2.1. Aseptic technique. Tissue culture technique

27

oxygen is produced from water. Many enzymes require zinc ions (Zn2+) for their activity, and zinc may be required for chlorophyll biosynthesis n 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 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 disaccharide 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 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 medium as it is involved in carbohydrate metabolism. Rest vitamins are promontory. Addition of amino acids to media is important for stimulating cell growth in pro­ toplast cultures and also in inducing and maintaining somatic embryogenesis. This redu­ ced 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 form of mixtures as individually they inhibit cell growth. Complex organics are 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 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 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 the 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 for exploration of plant physiological processes, it needs the addition of effective

28

Part II. Techniques and methods of cultivation plant tissue in vitro

plant growth regulators. These two aspects can be considered to plant tissue culture the wings to take off. With the starting of common or specific media and the selection of appropriate plant tissue culture, enable induction of cell division, callus growth, differentiation of shoots, roots and embryos. Plant growth regulators (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 is must to the culture medium Auxin was the first hormone to be discovered in plants and is one of an 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 concentration, 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), indolebutyric 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-hydroxy-3-methylbut-2enylamino) 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 the 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 decides morphogenesis. If this ratio is high, leads to embryogenesis, callus initiation and root initiation whereas if ck/auxin is high, it gives rise to axillary and shoot proliferation (Fig. 4). Gibberellins and abscissic acid are lesser 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 dwarf and rosette species and grasses. Gibberellin induces transcription of the gene for α-amylase biosynthesis in cereal grain aleurone cells. Gibberellic 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 and to promote the adventitious shoots and absorb and prevent the phenolic production. Whereas, the ethylene one of the gases plant hormone, it is moved by diffusion around the plant rather than translocation. It has stimulates the final stage of fruit development and flower fall. The main function of ethylene in plant tissue culture, it can stimulate

Chapter 2.1. Aseptic technique. Tissue culture technique

29

the respiration, seed germination, peroxidase enzymes and regulates 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

Solidifying agents are used for preparing semi-solid tissue culture media to enable explant to be placed in right contact with nutrient media (not submerged but on surface or slightly embedded) to provide aeration. Agar is high molecular weight polysaccharide obtained from sea algae Gelidium amansii and 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 gel­ ling agent is added to the liquid medium before autoclaving. Gelling 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 easier to detect contamination which might develop during culture growth. Gelritet 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

30

Part II. Techniques and methods of cultivation plant tissue in vitro

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 volume by water, pH is adjusted and then 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. Table 2

Stock solutions of growth regulators Compound CYTOKININS** 6-Benzyladenine N 6 -(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

Abbreviations

mg/50 ml (1mМ or 10 -3 Molar)*

BA, C 12 H 11 N 5

11.25

2-iP, C 10 H 13 N 5 Kinetin, C 10 H 9 N 5 O ZEA, C 10 H 13 N 5 O TDZ, C 9 H 8 N 4 OS

10.15 10.75 10.95 11.00

IAA, C 10 H 9 NO 2 IBA, C 12 H 13 NO 2 NAA, C 12 H 10 O 2 2.4-D, C 8 H 6 ClO 3 МСРA, C 9 H 9 ClO 3 2.4.5-T

8.76 10.16 9.31 11.05 10.03 12.78

3,6-Dichloro-2-methoxy-benzoy acid Dicamba, C 8 H 6 C 12 O 3 4-Chlorophenoxyacetic acid 4-CPA, C 8 H 6 ClO 3 4-Amino-3,5,6-Trichloropicolen acid PIC, C 6 H 3 C 13 N 2 O 2 (Picloram) OTHER PGRs**** Gibberellic acid GA 3 , C 19 H 22 O 6 Abscisic acid ABA

9.33 12.06 17.32 13.20

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 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 volume. Dissolve picloram in DMSO; **** Dissolve in 95% ethanol or 1N NaOH; stir, heat gently, gradually add water to volume.

Chapter 2.1. Aseptic technique. Tissue culture technique

31

Appropriate quantities are taken from stocks and mixed to constitute basal medium. Make up the final volume of the medium with distilled water (according this formula (1)): Dilutions: required concentration x medium volume = volume of stock required concentration of stock solution

(1)

Required quantity 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 the 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 an even 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 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 (Fig. 5). Seed culture – growing seed aseptically in vitro on artificial media. The plant seed cultivate in vitro for following aims: – increasing efficiency of germination of seeds that are difficult to germinate in vivo;

32

Part II. Techniques and methods of cultivation plant tissue in vitro

– precocious germination by application of plant growth regulators; – production of clean seedlings for explants or meristem culture. Embryo culture – growing 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 embyo (usually immature embryo) cultivate in vitro on nutrient media because: it has been further developed for the 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. Organ culture – any plant organ can serve as an explant to initiate cultures, for examples, 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 for obtain the secondary methabolites accumulated in root cells. Some plants can produce methabolites by hairy root cultures in liquidphase bioreactors. By reproductive organ culture (ovary or ovule culture and anther/ microspore culture) may be obtained haploid plants. It is possible in vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth. Also, may be overcome abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers. Generative organs of plants use for production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines and also, for uncovering mutations or recessive phenotypes.

Figure 5. Modes of culture in vitro

Protoplast culture – the living material of a plant cell, including the protoplasm and plasma membrane after the cell wall has been removed. Protoplast culture use as a recipient model for gene transformation in genetic engineering, for obtaine somatic hybrids in cell engineering etc.

Chapter 2.2 METHODS OF CULTIVATION 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 of which are differentiated although may be 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, nondividing. Therefore, the differentiated tissue undergoes modifications to become meristematic that means having the characteristics of a meristem, especially high mitotic activity. Meristem 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 meristematic state to form undifferentiated callus tissue is called dedifferentiation. Callus culture The culture of undifferentiated mass of cell on agar media produced from an explant of a seedling or other plant part is called callus culture (Fig. 6). For callus formation, auxin and cytokinins, both are required.Callus can be subcultured indefinitely by transferring a small piece of the same 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, the usefulness of callus in experimental system is limited. The main use of callus culture is for purposes of maintaining cell lines and for morphogenesis. Morphogenesis – the 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 callus culture, the following factors are important: the origin of explants used for the establishment of callus culture, the cellular/tissue; differentiation 33

34

Part II. Techniques and methods of cultivation plant tissue in vitro

status, external plant growth regulators, culture media and culture conditions. Cellular competence to plant 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 and these metabolites have biological activity [2].

G

H

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

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 liquid medium and vessel is incubated on shaker (Fig.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 (Fig. 8). Cell suspensions are also maintained by subculturing of cells in early stationary phase to a fresh medium Their growth is much faster than callus cultures and hence need to be subcultured more frequently (3-14 days). Cell suspension cultures when fully

35

Chapter 2.2. Methods of cultivation plant tissue in vitro

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 which 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

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

Batch culture The culture medium and the cells produced are 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 fresh medium at regular intervals. The biomass or cell number of a batch culture follows a typical sigmoidal curve, where to start with the culture passes through lag phase during which cell number is constant,

36

Part II. Techniques and methods of cultivation plant tissue in vitro

followed by brief exponential or log phase where there is a rapid increase in cell number because of culture cell division (Fig. 9). Finally, the growth decreases after 3-4 generations which is the doubling time (time taken for doubling of cell number) and culture enters stationary phase during which 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 Figure 8. Cell suspension culture and hence, not used for studies related to aspects of cell behaviour. But batch cultures are convenient to maintain, hence used for initiation of cell suspension and scaling up for continuous cultures. Continuous culture In this type culture steady state of cell density is maintained by regularly replacing a portion of the used up medium with fresh medium. Continuous culture are further classified into two types: 1) Closed; 2) Open. In closed type, the used medium is replaced with fresh medium, hence, the cells from used medium are mechanically retrieved and added back to the culture and thus, the cell biomass keeps increasing. In open type, both cells and used medium are replaced with fresh medium thus maintaining culture at 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 upto a certain turbidity (decided on the basis of optical density) when a predetermined volume of the culture is replaced by fresh culture. On the other hand, in chemostat, the fresh culture medium to be added has one nutrient kept at a concentration so that it is depleted rapidly and becomes growth limiting while other nutrients are still in concentration higher than required. Increase or decrease in the concentration of growth limiting factor is correspondingly expressed by increase or decrease in growth rate of cells or density. Density of suspension culture has been calculated by this formula (2): X = М×n×1000, 3.2

(2)

where Х – number of cells, М – average number of cells in the chamber, n – dilution. Thus, the desired rate of cell growth can be maintained by adjusting the level of concentration of growth limiting factor with respect to that of other constituents.

Chapter 2.2. Methods of cultivation plant tissue in vitro

37

Chemostats are useful for the deter­mi­ nation of effects of individual nutrients on cell growth and metabolism. Single cell culture Free cells isolated from plant or­ gans or cell suspensions when grown as single cells under in vitro conditions thus producing a clone of identical cells is called single cell culture. – Isolation of single cell from plant organs. Leaf tissue in particular is utilized as it has homogeneous po­ pu­ lation of cells using either of the Figure 9. Growth curve for plant cell suspension grown in closed system. The four different growth fol­ lowing two methods: mechanical phases are labeled: Lag phase, Exponential phase, and enzimatic. 1. Mechanical. Small Linear phase, Stationary phase 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 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 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 and thus called nurse tissue. This technique of culturing single cell 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 petridish. 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. Other point to be taken care of here is that since light has detrimental effect on cell proliferation, single cells should be cultured in dark. Synchronisation of suspension cultures. Cell suspension is mostly asynchronous, different cells of different size, shape, DNA, nuclear content and also in different stages of cell cycle (G1, S, G2, M). This is not desirable in cell metabolism studies. Hence, it is

38

Part II. Techniques and methods of cultivation plant tissue in vitro

essential to obtain synchrony in suspension cultures and can be achieved by following methods: starvation, inhibition, 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 anormal 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 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 (Fig. 10). Evan’s Blue staining is the only dye which is taken up by dead cells. Therefore, evan’s blue is used usually to complement FDA.

A 60 rpm

B 120 rpm

C 240 rpm

Note: A) cellular clump at 60 rpm, showing unstained dead cells in the centre of cell aggregate and live cells fluoresce 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

The entire plant tissue culture techniques can be largely divided into two categories based on to establish a particular objective in the plant species:

Chapter 2.2. Methods of cultivation plant tissue in vitro

39

I. Quantitative Improvement – Adventitious shoot proliferation (leaves, roots, bulbs, corm, seedling- explants 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. 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? 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?

Chapter 2.3 BIOLOGY OF CULTURED PLANT CELLS The ability of mature cell to dedifferentiate into callus tissue and the technique of maintained 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 an entire 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, follow by redifferentiation which is the ability to reorganize into new organ (dedifferentiation); c) Competency – is the endogenous potential of a given cells or tissue to develop in a particular way. Dedifferentition is possible because the nondividing 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 multicellular plant is termed as cellular totipotency. To express totipotency, after dedifferentiation (when mature cells reverting to meristematic state to produce callus), the cell has to undergo redifferentiation or regeneration which is the ability of dedifferentiated cell to form 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 indirecty 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 whole plant. Organogenesis is a process involving redifferentiation of meristematic cells pre­ sent in callus into shoot buds and roots. These organs may arise out of pre-existing meristems or out of differentiated cells (Fig.11). Also, organogenesis is the ability of 40

Chapter 2.3. Biology of cultured plant cells

41

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 relies according 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 induce 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. Explants Organogenesis Shoot formation Rooting

Root formation Shooting

Whole plants Figure 11. Pathways of morphogenesis and plant regeneration in vitro

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 the 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 (Fig. 12).

42

Part II. Techniques and methods of cultivation plant tissue in vitro

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 an intervening callus phase (Fig.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 pre-existing vascular tissue developed from the callus Figure 12. Shoot differentiation from callus tissue (from website http://nptel.ac)

Under the optimal conditions, meristems formed from the callus are random and scattered. On transferring to the medium supporting organized growth promotes first the 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 plant tissue culture will be successful by maintaining various factors involved, including media factors and environmental factors. The stimulation of shoot

Chapter 2.3. Biology of cultured plant cells

43

bud differentiation in plants depends on many factors which differ for different plant species. The following factors affecting 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 explants are maintained. The environmental factors involved 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 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 total amount of nitrogen in the medium and it needs both of 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 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 plant form is the degree of apical dominance. Although apical dominance may be determined primarily by auxin, physiological

44

Part II. Techniques and methods of cultivation plant tissue in vitro

studies indicate that cytokinins play a 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 whre 2.4-D is replaced by IAA or NAA. Gibberellin GA3, which in general has inhibitory effect on shoot buds whereas many species show enhanced shoot regeneration due to abscissic acid. The variable responses of different plant species to the growth regulators is because the requirement of exogenous GRs depends on their 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 and would 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. Re­ge­ nerability 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 vs. mature, position of the explant on the plant and the explant size. The larger the explant (containing parenchyma, cambium and vascular tissue), more is likelihood of shoot bud formation. Orientation of the explant on the medium and the inoculation density may also affect shoot bud differentiation. Generally, the explants inoculated horizontally on 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 percent cultures showing regeneration with increasing age of the seedlings. Also, genotype of explant affects shoot regeneration as explant taken from different plant varieties of 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 genotype plays equally, if not more critical role as the growth regulator. Besides, there are certain other factors which 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 effect as blue light has been shown to induce shoot formation and root by red light in tobacco. Alternating light and dark period (diffused light,

Chapter 2.3. Biology of cultured plant cells

45

15-16 hrs) proved 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 while 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, 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: the swelling at the apical end of the specific protonemal cell. The optimum temperature is 25±2 °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 semi-solid medium give different degree of organogenesis. In 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 dif­fe­ rentiation of shoot-buds or somatic embryogenesis (Fig.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

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. For non-zygotic embryos use various terms. For example adventious embryos are somatic embryos arising directly from other organs or embryos; parthenogenetic embryos (apomixis) are somatic embryos which formed by the unfertilized egg; androgenetic embryos are somatic embryos formed in the male

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Part II. Techniques and methods of cultivation plant tissue in vitro

gametophyte culture. Somatic embryos are bipolar structures with radical and plumule is contrast to monopolar shoot bud with only plumular end in organogenesis. The gradation or change in 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 development and mostly they are 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 furthest from the original point of attachment i.e. the tip of the leaf or shoot or root, while proximal means nearest to the point of attachment. While developing into somatic embryo, the mersitematic cells break any cytoplasmic or vascular connections with other cells around it and become isolated. Therefore, unlike 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 embryogenic callus, maturation and germination process and relies by scheme of indirect somatic embryogenesis: Explant → Callus Embryogenic → Maturation → Germination. Regeneration in plant tissue culture will be successful by maintaining various factors involved, including media factors and environmental factors. Plant growth hormons such as auxines (2.4-D, NAA, dicamba) required for induction form proembryogenic masses and embryogenic callus. However, at the next step, auxin must be removed for embryo development because continued use of auxin inhibits embryogenesis. The following stages are similar to those of zygotic embryogenesis: Globular stage, Heart and Torpedo stages, cotyledonary (Fig. 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 long axis. This division creates two cells – an apical and a basal cell – that have very different fates. After the first division, the apical cell undergoes a series of highly ordered divisions, generating an eight-cell (octant) globular embryo. Additional precise cell divisions increase the number of cells in the sphere. The basal cell also divides, but all of 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. 4. The maturation stage embryo. Toward the end of embryogenesis. Axial polarity is established very early in embryogenesis. Maturation require complete development

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Chapter 2.3. Biology of cultured plant cells

with apical meristem, radicle, and cotyledons. In this stage sometimes obtain repetitive embryons (polyembryoids). For complete maturation often require ABA and necessary production of storage protein. Plant hormone such as ABA 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%.



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

In generally, 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 (Fig. 16); – Maturation: In this phase somatic embryos develop into mature embryos by differentiating from globular to heart shaped, torpedo to cotyledonary 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 were covered most of surface of the callus (Fig. 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 (Fig. 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 appearance. The inner tissue of the callus consisted

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Part II. Techniques and methods of cultivation plant tissue in vitro

mostly of parenchyma cells and vascularized zones (Fig.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 (Fig.16 D). A close-up of this embryogenic tissue shows two spherical cells at the time of cytokinesis (Fig.16 E) and demonstrates how the daughter cells of the embryogenic tissue remain as a coherent tissue (Fig.16 F). The expression of embryogenic program could subsequently be observed when the proembryogenic clusters developed into the first visible globular embryos.



Note: 1A-Somatic embryos on the surface of the callus (arrows) (Bar = 1.25 mm); 1B- Pro-embryogenic cells with densely stained cytoplasm indicating high metabolic activity (Bar = 1 mm); 1C- Crosssection 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 aspect of the callus with embryogenic cells, and histological sections of 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])

Chapter 2.3. Biology of cultured plant cells

49

Somatic embryogenesisis influenced by following factors: – Growth regulators. The presence of auxin (generally 2.4-D) in the medium is essential for 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 (Fig.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; – Nitrogen source. NH4+ form of nitrogen is essential for induction of somatic embryogenesis while NO3- form is required during maturation phase; – Other factors. Like shoot bud differentiation, explant genotype has influence on somatic embryogenesis also. In cereals, use of maltose as carbohydrate source promotes both somatic embryo induction and maturation.

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) 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 elliptical shape showing the procambium (arrow). (Bar = 60 μm); e) 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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Part II. Techniques and methods of cultivation plant tissue in vitro

Histological analyses of callus material Cleome rosea revealed heavily stained cells located on the surface of the callus (Fig.17). Callus induced by 2.4-D presented a nodular appearance (Fig. 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 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 (Fig. 17 b). The globular embryos presented a protoderm formed by cells dividing in anticlinal plane (Fig. 17 c). 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 (Fig. 17 d). Mature embryos loosely attached to the callus surface were observed 15 days after transfer to medium with reduced 2.4-D concentration (Fig. 17 e). These embryos showed a bipolar structure with the presence of procambial strands connecting the root and the shoot apices and with no vascular connection to the callus tissue (Fig. 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 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 have plant cells growing in vitro? 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 are affecting organogenesis and shoot-bud differentiation? 6. What is somatic embryogenesis? 7. What differences between direct embryogenesis and indirect embryogenesis? 8. What factors influence on somatic embryogenesis?

Part III INDUSTRIAL AND AGRICULTURAL APPLICATIONS OF IN VITRO CULTURE

Chapter 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 important source of variety of chemicals used in pharmacy, medicine and industry. The past two decades plant cell biotechnology has evolved as a promising new area within the field of 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 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 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 watersoluble 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, quinine, vinblastine, atropine, scopolamine and digoxin, one has so far not been able to come to a commercially feasible process. In contrast with 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 52

Chapter 3.1. Production of secondary metabolites in plant cell culture

53

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. The 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. Presently research particularly focuses on the possibilities to apply 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 and 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 insect, pest 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, but because of their pharmaceutical importance to deal with 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. Typically primary metabolites are found in all species within broad phylogenetic groupings, and are produced using the same metabolic pathway (Fig.18). As secondary metabolites provide industrially important natural products like colour, insecticides, antimicrobials and 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 cosmetics. Artabotrys hexapetalus, a climbing herb, secretes oil which is 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 fulfil demands for this valuable secondary metabolite. Various in vitro methods for enhancement of

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Part III. Industrial and agricultural applications of in vitro culture

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 have been 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)

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.

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Chapter 3.1. Production of secondary metabolites in plant cell culture

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 suspensions cultures. Table 3 Classification of secondary metabolites

Terpenes



Type Monoterpenes Sesqui-terpenes Diterpe-nes

Example Farnessol

Type Lignan

Example Lignan

Nitrogen and/or sulphur containing compounds Type Example Alkaloids Nicotine

Limonene Taxol

Tannins Flavo-noids

Gallotan-nin Anthocya-nin

Atropine Glucosino-lates

Triterpe-nes

Digitogenin Carotene Spina-sterol

Couma-rins

Umbellife- rone

Tetra-terpenoids Sterols

Phenols

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 growth is very active. On the other hand, when 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 growthassociated 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 comparable properties to hairy roots, genetic stability and good capacities for secondary metabolite production. They also provide the possibility of gaining a link between growth and the 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 the Central Kazakhstan) carry out the complex development of the original domestic phytodrugs: search biologically active substances, cultivation of

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Part III. Industrial and agricultural applications of in vitro culture

medicinal raw material, processing and production of pilot lots of medicinal forms of new phytodrugs [8]. One of the basic scientific directions of activity of the Holding «Phytochemistry» is the study of natural raw material for the obtaining of new materials and drugs as well as carrying out of research works on development high technologies for production the medical drugs corresponding requirements of international standards GMP and their introduction into industry. The Head of Holding «Phytochemistry» is an Academic of National Science Academy of the Republic of Kazakhstan, Doctor, Professor S.A. Adekenov. The main scientific directions of Holding are: – search for new biologically active compounds from natural sources and deve­ lopment of methods for their extraction, determination of molecules structure and investigation of the properties of the obtained compounds; – development of methods for the synthesis of organic compounds and obtaining 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 researches of original phytopreparations; – development of methods for deriving the substances and standard samples of medicinal drugs, phytopreparation biotechnology and introduction of quality control in pharmaceutical production; Analysis of species diversity and perspectives using of Kazakhstan medicinal flora in official and folk medicine allowed 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 the Kazakhstan 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 and etc. In the collection in vitro of holding «Phytochemistry» supports 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 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 playback of endemic, rare and endangered plant species [8, 9]. Commercial demand of 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 also 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

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Chapter 3.1. Production of secondary metabolites in plant cell culture

produced in the in vitro grown plants through plant tissue culture. In addition, stereoand 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 rapidity of production are its added 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 the creation herbal remedies number.The most important and promising species introduced medicinal plants include: Artemisia glabella Kar.et Kir. extract which is used to create phytopreparation «Arglabin» having the property to stimulate the immune system (Fig. 19), Salsola collina Pall., extract which is used to manufacture phytopreparation «Salsocollin» possessing anti-inflammatory, anti-oxidant effect (Fig. 20), Serratula coronata L. based on the extract which is obtained phytodrug «Ecdyphyt» possessing anabolic and tonic properties (Fig. 21) and Ajania fruticulosa Ledeb, is used to obtain phytopreparation «Aefrol» has wound-healing properties.

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/

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Part III. Industrial and agricultural applications of in vitro culture

Arglabin, a new herbal anti-tumor medicine, is developed and used in Kazakhstan. The method of the obtaining of an antitumor preparation «Arglabin» was 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 has been studied chemically most extensively. Most studies have been devoted to flavonoids, sesquiterpene lactones, and essential oils components from different regions of the geographic range. These compounds are responsible for some sorts of biologically activity. 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. Identification of polyprenols can be achieved by the application of HPLC. Comparing the chromatographic profiles of samples of lipophilic extracts was identified 20 paraffin hydrocarbons with normal and branched structure and chain length С16-С36, aliphatic and triterpenic alcohols were also found. Some species contain polymethoxylated flavonoids [10].

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); phytodrag «Salsocollin» (D) /from website www.phyto.kz/

Artemisia annua L. is one such 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

Chapter 3.1. Production of secondary metabolites in plant cell culture

59

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 lead compounds, more than 200 rupestonic acid derivatives have been synthesized and the in vitro activities against influenza viruses A and B were assayed. Sesquiterpene lactones (SLs) are a varied group of secondary plant metabolites, occurring widely within the families Asteraceae and Apiaceae. SLs, including recog­ nized 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 contains a large amount of chamazulene. Oils may find application as a source of new medicines and dietary supplements and have 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 synthonsin the preparation of practically useful compounds. Components of essential oils have all elopathic activity. Chemical compounds of Artemisia halophila Krasch (white wormwood) and Artemisia arenaria Dc. (desert wormwood) essential oils were observed by researchers from 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-octadien-1-ol, 3,7-dimethylacetate, trans-geraniol, chrysanthenone, camphor, chrysanthenyl acetate and sabinyl acetate [12].

Figure 21. Chemical structure of polyhidroxisteroid – basic compound of phytodrag «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 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 (1.5-3%), 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

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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» the significant differences of callus formation frequency, total weight andcallus growth during cultivation cycle between the species were detected. Artemisia kasakorum showed the maximum intensity of callusgenesis with growth index RI ranged from 2.37 to 4.98 depending on the medium hormonal composition. The callus cultures had the similar S-shaped growth curve with the latent (first 10 days), logariphmic stage (from 11 to 20-25 days), slow growth and stationary growth (from 26 to 40 days) phases. The obtaining callus cultures were differed on callus biomass accumulation [13]. For isolation of several phenolic acids, phenolic acid glycosides and esthers, triterpenic acids, a cardenolide glycoside and a cardenolide aglycone use following scheme: plant material extracte 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 subject to the column chromatography on Sephadex LH-20, elute with methanol. The sub-fractions further separate by RPHPLC (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. Biological 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 doxorubicininduced toxicity in cardiomyocytes, antimicrobial, antivirus, cytotoxicity and antioxidant activities. In the research 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 CHCl3 layer was separated using a silica gel column, a sephadex LH-20 column, and preparative HPLC to afforded 7 known compounds in which four flavonoids together with Oleanolic acid, Ursolic acid, and Stigmasterol were isolated for the first time from the plant 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 countries 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 raw material. For detection of phenolic compounds use alcohol-water extracts and ethyl acetate butane fraction. 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.

Chapter 3.1. Production of secondary metabolites in plant cell culture

61

To determine aglycones belonging to the flavonoid glycosides use authentic samples in solvent systems: chloroform-acetic acid-water (13:6:1) and benzene-ethyl acetateacetic 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 plants 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. The 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 N2fixing 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 play a significant role in insect-plant interactions. Plant-based pesticides are not accumulated in the food chain as by 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 and to treat inflammatory and rheumatic diseases [15]. Leaves and fruit of sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae) are a sources of valuable biologically active substances. They are used in folk medicine for diseases of the skin, 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 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 recommend 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 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 of medicines, food additive and cosmetic [10]. 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 the antitumorals 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 charac­ te­ristics, such as sensitivity, site of action, and developmental stage. At times, toxi­ city effects can be both harmful and beneficial depending on the ecological or phar­

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ma­cological 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 of the alkaloid itself during controlled attacks of pathogens and herbivores or upon the 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. Conducted primary biological tests of natural alkaloids and their derivatives on the physiological activity showed that many of them exhibit antiviral, antibacterial, antiinflammatory, cytotoxic and phagocytosis stum action, allowing you to set them on the basis of new drugs that would increase the range of the domestic pharmaceutical products. Thus, the analysis of data on chemical study alkaloids plant flora of Kazakhstan, including the selection, establishment 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 lists the major alkaloid types and their amino acid precursors. Nearly all alkaloids are also toxic to humans when taken in sufficient quantity. 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 the chemical transmitters, others affect membrane transport, protein synthesis, or miscellaneous enzyme activities. 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

Table 4

Human uses

Nicotine

Stimulant, depressant, tranquilizer

Atropine

Coniine

Quinolizidine Isoquinoline

Lysine (or acetate) Lysine Tyrosine

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)

Lupinine Codeine Morphine

Restoration of heart rhythm Analgesic (pain relief), treatment of coughs Analgesic

Indole

Tryptophan

Psilocybin Reserpine Strychnine

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

Cocaine Piperidine

Chapter 3.1. Production of secondary metabolites in plant cell culture

63

The biosynthetic capacity of the hairy root cultures is equivalent or sometimes more to the corresponding plant roots. Therefore, hairy root cultures have been developed as an alternate source for the production of root biomass and to obtain 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 the successful transformation of a plant with Agrobacterium rhizogenes. They have received considerable attention of plant bio­ technologists, 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 to the profusion of lateral roots (Fig. 22). This growth can be assimilated to an exponential model, when the number of generations of lateral roots becomes large. Hairy roots have following properties: 1) high degree of lateral branching; 2) profusion of root hairs; 3) absence of geotropism; 4) they have 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 consequently 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. The 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 inducesd 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 effective biotechnological protocol for large-scale artemisinin production was established by cultivation of Artemisia annua hairy roots in nutrient mist bioreactor (NMB). Artemisinin is used for the treatment of cerebral malaria. It was extracted from hairy root culture which 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 gasphase NMB cultivation [19].

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Native roots of Scutellaria baicalensis Georgi

Hairy roots culture of S. baicalensis Georgi

Hairy roots culture of Rubia tinctorum

Freeze-dried hairy roots of Rubia tinctorum

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

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.). 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 volume per minute) and 70 rpm, respectively (Fig. 23). The production of tropane alkaloids, especially scopolamine, in bioreactors instead of from native plants, is an efficient way because the process is performed under clean and controlled circumstances, which consequently controls or decreases the diversity in component quality and the alkaloid yield [20].

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Chapter 3.1. Production of secondary metabolites in plant cell culture

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 roots culture obtained in bioreactors. Bioreactor with agitation 70 rpm and aeration 1.75 vvm (A) and bioreactor with agitation 110 rpm and 1.75 vvm (В) [20] Figure 23. Hairy roots culure

The hairy root system is stable and highly productive under hormone-free culture conditions. The fast growth, low doubling time, easy maintenance and 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 the poor or insignificant synthesis of secondary metabolites. Hairy roots are also a valuable source of photochemical that is useful as pharmaceuticals, cosmetics and food additives. These roots synthesize more than a single metabolite, prove economical for commercial production purposes. 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 the 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 densely packed root beds and reduces mass transfer (both oxygen and nutrients). Root thickness, root length, the number

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of root hairs and root branching frequency are some of the factors which should be taken into consideration for hairy root cultures in bioreactors (Fig.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. Also 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 added 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. Largescale 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 aspects 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 metabolites compounds in many studies. Growing a plant outside its natural environment under ideal conditions may therefore, result in being unable to produce the desired bioactive substances, hence the need for prior evaluation. Chemical and 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 (Fig.24). In the past years new approaches have been developed: the culturing of differentiated cells (e.g. shoots, roots and hairy roots),

Chapter 3.1. Production of secondary metabolites in plant cell culture

67

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 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 also the culture condition like temperature, light etc influence the production of metabolites. For e.g. 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 compound 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 hold the ability of enhancing secondary metabolite accumulation in plant cells and their quality production in cell suspension cultures. – Permeabilisation. Secondary metabolites produced in 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 medium to absorb secondary metabolites. – Immobilisation. Cell cultures encapsulated in agarose and calcium alginate gels or entrapped in membranes are called immobilised plant cell culture (Fig.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 number of species. Elicitors can also be added to these systems to stimulate secondary metabolism. – Addition of precursors. Precursors are the compounds, whether exogenous or endogenous, that can be converted by living system into useful compounds or secondary metabolites. It has been possible to enhance the biosynthesis 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, 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

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of rosmarinic acid and decreased the production time as well. Phenylalanine also acts as precursor of the N-benzoylphenylisoserine side chain of taxol. Supplementation of Taxus cuspidata cultures with phenylalanine resulted in increased yields of taxol. The 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. Plant screening for production of secondary metabolites

Systematic chemical screening of particular plant part (explant) to be used as sample, for higher production of metabolites

Callus induction from screened plant part for cell biomass production

Subculture cycle to increase cell biomass

Screening of cell lines for metabolite productivity

Cell suspension culture (biosynthetic pathway and elicitation)

Bioreactor studies. Cultivation high yield productive cell lines in bioreactors

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

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 the 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, that 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 of genes by antisense genes. In all cases the respective biosynthetic pathway has to be known on the level of products, enzymes and genes, as

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Chapter 3.1. Production of secondary metabolites in plant cell culture

well as the regulation on all these levels, including aspects as compartmentation and transport.

A

B

D

E

C

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] )

For examples, in Tabernae montana species mevalonate is used for the m-terpene biosynthesis after elicitation, channelling away this precursor from the alkaloid pathway [7]. Blocking such pathways by means of antisense genes might be of interest to increase the availablity of these precursors for the alkaloid biosynthesis. Catabolism is another important factor in the accumulation of the alkaloids. Identification of the enzymes involved and the cloning of the encoding genes is thus of interest to eventually use antisense gene technology to block catabolism and thus increase the production. Current progresses have been made in the field of molecular biology through the alteration in metabolic skeleton of 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 being transformed. Antisense technology has been emerged as an additional alternative for enhancement of secondary metabolites. In  Tylophora indica enhancement in kaempferol, an antioxidant compound was observed by using precursors like salicylic acid, ornithine, cinnamic acid, tyrosine and phenylalanine [18]. Metabolic engineering offers new perspectives for improving the production of secondary metabolites by the over expression 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

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the foreign genes that encode enzyme activities not normally present in a plant. This may cause the modification of plant metabolic pathways. At present, analytical tools based on genomics, proteomics, metabolomics, bioinformatics, and other twenty-first-century technologies are accelerating the identification and characterization of natural products. On the other hand, many new bioactive compounds are failing due to a lack of efficacy in the clinic, which demands 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. Controll questions: 1. How secondary metabolites in the plant cells are produced? 2. What is the importance of plant cell culture in production of secondary metabolites? 3. What are the steps involved in the production of secondary metabolites from plant cell? 4. How production of secondary metabolites is 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?

Chapter 3.2 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. Micropropagation is a tissue culture method developed for the 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 key element to select the propagation method. Using micropropagation techniques in plant biotechnology applications are costlier than conventional propagation methods. Propagation by using in vitro techniques instead of conventional methods offer some advantages like utilizing small pieces of plants (explants) to maintain the whole plant and increase their number. The main point is to evolve new strategies to lower the time and cost consumed per plant. In tissue culture applications selection of initiating 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, long storage periods make micropropagation preferable to propagate plants in short time. There are also some disadvantages of micropropagation. Adaptation of cultured plants to the environmental conditions need transitional period to allow the plants to produce organic matter by photosynthesis. Summarized above the advantages of micropropagation over conventional pro­ pagation 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 less medium; 71

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Part III. Industrial and agricultural applications of in vitro culture

– 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 short cycle and each cycle results in exponential increase in 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: – involves 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 biochemicals which have otherwise not been detected in whole plants. 2. Production of synthetic seeds. Synthetic seed is a bead of gel containing somatic embryo or shoot bud with growth regulator, nutrients, fungicides, pesticides etc needed for development of complete plantlet. These are better propagules as do not need hardening and can be sown directly in the field (Fig. 26). 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

73

Chapter 3.2. Micropropagation in vitro

divides faster than disease-causing virus, clean materials are propagated and hundreds of uniform plantlets are produced in a short time.

A

В

С

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

Methods of in vitro propagation The main methods of in vitro propagation can be classified in 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 sur­ rounded 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 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 ensurable method to have the highest genetic stability during in vitro propagation of plants. Apical meristem

Axillary bud

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

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Part III. Industrial and agricultural applications of in vitro culture

Adventitious shoots or adventitious somatic embryos are established directly or indirectly. Somatic cells cultures are directly started with the excised explants from the mother plant tissues for organogenesis or embryogenesis. If shoots or embryos regenerate on previously formed callus or in cell culture, they are called as indirect organogenesis or embryogenesis. When propagation occurs via an indirect callus phase, the genetic identity of the progenies decreases. This is an important problem in commercial propagation 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. Origin of the callus also causes somaclonal variation.

Figure 28. Micropropagation from axillary and apical buds/meristems

Micropropagation involves following major stages: Stage 1. Selection and maintenance of stock plants for culture initiation (about 3 months) – stage of selection and preparation 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 (324 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 (1-6 weeks) – stage of rooting of microshoots. Stage V. Transfer of plantlets to sterilized soil for hardening under greenhouse environment – stage of acclimatization (Fig. 29). These stages are necessary for a 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

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conditions. Stage 4 is responsible of 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.

introduction in vitro

rooting of microshoots

in vitro culture establishment

shoot multiplication

stage of acclimatization Figure 29. The stages of plant micropropagation

Genotype and explant type. The genotype of mother plant plays an important role in in vitro propagation. The different genotypes had different responses to the same culture conditions. For this reason, it is necessary to establish a suitable procedure for each varieties of plants that can be adapted to commercial production. The success of tissue culture is related to the correct choice of explants. Shoot or shoot tips and node cultures are the most commonly used culture types in micropropagation of plants (Fig. 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, using young leaf explants are important for the success of 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].

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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 highly affected by the features of nutrients included. 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 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.

Note: A-I stages of clonal micropropagation technique Figure 30. Clonal micropropagation of Guava (Psidium guajava L.)

Plant growth regulator levels. In culture conditions, using synthetic chemicals with similar physiological activities as plant hormones have capabilities to induce plant growth as desired. Auxin and cytokinins are the most important hormones regulating growth and morphogenesis in plant tissue culture. Their combinative usage promote growth of callus, cell suspensions, root and shoot development and have capability to regulate the morphogenesis. Different combinations and concentrations of plant

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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 Karatau moutaine of the Southern Kazakhstan [25]. Endemic species are defined as «which grows 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 sm 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 an age and a cultivar. The compound of a medium influence on an efficiency in vitro cultivation. A rapid multiplication rate could be obtained from leaf explants by combining the phytohormones in MS medium (Fig.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 mean numbers as well as 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 have 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. It is reliable technique is currently available for large scale micropropagation of rubber plant on a commercial scale. Under the leadership of Professor K.K. Boguspaev and Associate Professor S.K. Turasheva at Al-Farabi Kazakh National University the extensive experiments to enhance 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, role of sucrose and abscisic acid on embryo induction and carbohydrate types have also been evaluated. In these research the explants isolated (leaf, root, apical meristems of shoots) from 1-3 year old seedlings 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 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 using young leaf explants are 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

B

C

D

F

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 tau-saghyz; plantregenerant 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 plants breeding, the current propagation method of grafting on to 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 are 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 later problem has been over come 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 overcome different problems and for the introduction of specific agronomically important traits without disrupting their otherwise desirable genetic constitution. A reproducible plant regeneration system for each genotype of rubber plants through tissue culture is essential for crop improvement programmes. 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 the cell proliferation in the presence of

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cytokinins. Similar findings were reported in medicinal plants like Tylophora indica, Holostemma annulare, Holostemma adakodien, Spilanthes acmella (Fig. 32). Nodal segments bearing two opposite axillary buds Washed in tween-20 and 1% savlon for 20 min Rinsed in distilled water Sterilization

Quick rinse in 90% ethanol before surface sterilizing with 0.1% mercuruc chloride for 6 min. Rinsed thrice with sterile distilled water Inoculate the explant vertically in the 1/2 MS medium with sucrose and 5 µM BA Incubate the cultures at 250 C, 50-60% humidity and diffused light. Screening the cultures for 5 week Terminal 3-4 cm long portions of shoots after 5 weeks plated in MS medium for rooting (1/2 ������������� М������������ S+50 g/l sucrose +1 µM NAA). The remaining portions of the shoots can be cut into single node segments and utilized for further multiplication Rooted shoots were transferred out of culture, acclimatized by covering the pots with polythene bags and irrigated with major salt solution of MS medium. After 25 days, the acclimatized plants were transferred to a shaded area under natural conditions at a temperature range of 20-25°C with photoperiod of 12/12 h (light/ dark).

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

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.

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Over exploitation for its antidiabetic, anti-inflammatory, antiviral, antioxidant properties concentrated in roots and stem has caused it to be endangered, thereby 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 regula­ tors. 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 rootinduction could be successfully applied for development of high quality planting stocks [13]. Sometimes the complex of biological active substances influent on root formation as well as plants growth regulators. For example, the research of kazakh scientists show 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. Under the leadership of Dr. V.K. Mursalieva at the Institute of Plant Biology and Biotechnology the growth-regulatory activity of Bergenia crassifolia (L.) extract which consist from hydrolysable and condensed tannins (20.87%), flavonoids, phenolic acids, amino acids and carbohydrate compounds was defined. In the samples of Sanguisorba officinalis the highest content of hyrolysable tannins (21.08%), phenol acid, amine compounds, flavonoids and steroids were defined [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 transfere to MS for further growth and finally washed before transfer to small pots containing soil + vermicompost (2 : 1) for hardening and acclimation. The pots cover 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 for rooting of shoot also use the hydroponic set. The technique of growing plants with their roots immersed in nutrient solution without soil is called solution culture or hydroponics. Successful hydroponic culture (Fig. 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 also critical may be achieved by vigorous bubbling of air through the medium. 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

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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.

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

Acclimatization. Acclimatization process carry out while the plants still under in vitro condition. A few days before the process was to be carried out, the cover of test tube 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 rose from 70 to 90%. The plantlets expose to the normal environment in stages as they will wilt due to rapid changes of relative humidity and light intensity. In vitro plantlets that reached 3-5 cm height take out from culture tubes and the excess media rinse to avoid contamination. They put into plastic pots and planted out in soil at a ratio of 1:1:1 for garden soil, sand and loam. Plantlets 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 (Fig. 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 purpose of Saintpaulia ionantha, various substrates were used, such as autoclaved mixed soil (compost, sand, and black soil in the ratio 1:1:2) and non-autoclaved 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.

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Successful micropropagation of plants which can survive under the natural environmental conditions depends on 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 transplanted in an adequate substrate such as peat Figure 34. Different substrate for plantlets 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. 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 so production is optimized and profits always exceed losses. Plant virus diseases are critical problems in agriculture and 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 sample with virus type analyze on Cucurbita pepo L., Nicotiana benthamiana Domim. and Datura stramonium Thunb. The virus inoculum (Papaya ringspot W, PRSV-W), Zucchini yellow mosaic 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 to study expression of symptoms. Normally, multiple infections cause severe symptoms such as leaf distortion, mosaic, bubbles, narrowing

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leaf, leaf rolling, necrosis, spur and underdevelopment, ruffled edges, blistering, leaf narrowing, shoestring, leaf rolling, necrosis, stunting (Fig.35-37) [28].

A. Mosaic with chlorotic rings. B. Bubbles. C. Narrowing leaf Figure 35. Symptoms of viral infections of leaves watermelon

Signs – indications of presence of disease causing organism – e.g., fruiting body or mycelium of fungi. Symptoms – change in host – exudations, resinosis, necrosis (death of tissue or tree), hypotrophy (dwarfing), hypertrophy (overgrowths -galls, witches brooms). Organisms causing biological disease: 1. Fungi and fungus-like organisms. 2. Vi­­ru­ ses, 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 more of a problem on soft tissued plants. Perhaps a million species of fungi, but only 100,00 are known. 10,000 are plant pathogens. Viruses – organisma 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 also, can become part of plant genome. The symptoms of viruses infection are 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 wood, shoot blight, bleeding cankers, galls. In addition to the crown gall bacterium, Agrobacterium tumefaciens, 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 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 a styletto pierce plant cell walls. Cause injury by feeding, toxins. They are the vectors of other diseases, contributing factors

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in declines. Evolution towards plants 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 (A); 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 pa-thogens 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 planted to food crops are destroyed [29].

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

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of the causal organism, accurate estimates of the severity of disease and 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 an important role to play that 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. Establishment of pathogen free plants The plants infected with bacteria and fungi can be treated by bactericidal and fungicidal compounds, there is no commercially available treatment to cure virusinfected plants. A large numbers of viruses are 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 (Fig. 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 undifferentiated 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, composed mostly of small, thin-walled cells, with a dense cytoplasm, and lacking large central vacuoles. apical meristem apical meristem intercalar meristem

leaf primordial

lateral meristem

procambium

leaf rudiments

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

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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 the 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 a 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 rather 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, has 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 meristem culture is the best possible means of virus elimination and produces a large numbers of plants in a short span of time. The term «meristem culture» specially means that a meristem with no leaf primordial or at most 1-2 leaf primordial which are excised and cultured (Fig. 39). The pathway of regeneration undergoes several steps. Starting with an isolated explant, with de-differentiation followed by re-differentiation and organization into meristematic centers.

Note: 1 – 3: Preparation of apical meristem from axial buds; 4 – 6: Cultivation and multiple shoot formation; 7: Rooting in vitro; 8: Regeneration of intact plants Figure 39. Scheme of obtaine virus free plants in vitro

Factors affecting eradication of virus through meristem tip culture Culture medium, explant size and incubation conditions affecting plant regeneration from meristem-tip cultures have 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

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success is achieved from MS medium which promoted healthy, green shoot development compare to 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 on 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 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 which promotes only callusing. NAA and IAA are widely used auxins and NAA being preferred due to better stability. The role of GA3 is also emphasized by few authors which is suggested to promote better growth and differentiation and suppresses callusing from meristem explants. Both liquid and semi-solid media have been tried for meristem–tip culture but, agar medium is generally preferred. Explant size. The survival of the meristem tips, under the controlled condition, is determined by the size of the explant. The larger the explant, the greater are the chances of plant regeneration. However, the survival of the explants can not be treated independent of the efficiency with which virus elimination is achieved that is inversely related 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 meristems domes develop bipolar axes very quickly during reorganization. Once the rootshoot axis is established further development follows the same pattern as that of seedlings. Physiological conditions of the explant. Meristem-tips should be collected from actively growing buds. In few cases, the tips taken from terminal buds proved 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 (Fig.40). The time excision of buds is also critical, specially for the 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, the meristemtip cultures can be raised during spring only for increased success rate. 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 are 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.

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A

В

D

С

E

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

Thermotherapy Often, apical meristems are not always free of virus and it can’t 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 interrupts the growth of the meristematic tissue. In such cases also it has been possible to obtain virus-free 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, shoottip 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 later case, continuous exposure to 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

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both plant and animal, 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 proportion of the cultures yield virus free plants. Therefore, it is required to test all plants, regenerated through meristem-tip or callus cultures, for specific viruses before being used as 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 diseased tissues do especially for biological agents. The lactophenol blue staining technique could be used and prepared slides examined at X 400 magnification. 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, separate from other plants. It may take 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 enzyme-linked immunosorbant assay (ELISA) is 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 viruses identification 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 (Fig. 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) place on a nitrocellulose membrane. The membrane dry at room temperature for 30 min and incubate with blocking solution of 0.5% PBS plus 3% non-fat Molico milk under gentle agitation for 3 h. Subsequently, the membranes is placed in PBS solution containing specific antibodies for each isolated virus at a dilution of 1 μg/ml. The second antibody of goat anti-rabbit IgG alkaline phosphatase conjugate use 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 use according to the manufacturer’s protocol.

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Figure 41. Dot-ELISA assay

Through micropropagation, it is now possible to provide clean and uniform planting materials in plantations – oil palm, plantain, pine, banana, abaca, date, rubber tree; field crops – eggplant, jojoba, pineapple, tomato; root crops – cassava, yam, sweet potato; and many ornamental plants such as orchids and anthuriums. 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 is the term 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 trueto-type plants? Why is it important to have virus-free seed stocks? 4. What is the source of microbial contamination for in vitro plantlets? 5. Why the rate of virus elimination is higher in meristem cultures than the nodal segment culture? 6. How does heat act as a therapeutic treatment?

Chapter 3.3 CELL ENGINEERING The application of protoplast technology or cell engineering for the 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 ‘presence of cell wall in plants’ paved 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 cell wall has been removed are termed protoplasts. Figure 42 shows the microscopic view of protoplasts isolated from tabaco 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 important status in plant biotechnology.

Figure 42. Microscopic view of isolated protoplasts from tobaco

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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 use of 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 backbonenof 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; – Enzyme mixture used should essentially 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: 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 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 for its high reproducible potential for regeneration. Generally, the youngest, fully expanded leaves from young plants or seedlings are used as source of protoplast. Preconditioning plants in darkness or cold (4-10 °C) for 24-72h before protoplast isolation improves protoplast

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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 (30-50 rpm). Incubation time and temperature varies with species and time. The osmotic concentration of enzyme mixture and of subsequent media is elevated by adding sorbitol or mannitol to stabilize protoplasts or they will burst. Addition of 50-100 mM/l CaCl2 improves stability of plasma membrane. At acidic pH in the range of 5.0-6.0 is optimal. The buffer often contains phosphate at 3 mM to minimize shifts in pH during digestion. The most commonly used compounds 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 is, therefore, should be removed gradually from the medium otherwise 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 most widely used solution into which osmoticum or enzymes are added, some times culture medium used to grow cells or plants can also be utilized for protoplast isolation at one tenth concentration. Low concentration of culture medium is much more advantageous when compared with CPW. Salt mix of protoplast washing media solution (Cocking, Peberdy and White – CPW) Components KH2PO4 KNO3 CaCl2 x2 H2O MgSO4x7 H2O KJ CuSO4x5 H2O pH=5.8

Table 5

Concentration, mg/l 27.2 101 1480 246 0.16 0.025

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 low speed for 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 protoplast, their viability is estimated by staining with Fluorescein diacetate (FDA). Viable protoplasts

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exhibit green fluorescence under UV. It should be examined within 5-15 min after the FDA treatment after FDA dissociates. Some protoplasts have leaky membrane through which metabolites are leached out. Like cell cultures, the initial plating density of protoplasts has 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 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 conditional media or feeder layer are used. Conditional media is one where plant cells were already grown and so has metabolites leached into it. After filtering, this media is used for growing isolated protoplasts. Feeder layer is prepared by plating solid media with protoplasts followed by irradiation which inactivates the nucleus but protoplasts are viable. Protoplasts at lower densty can now be plated on this feeder layer. Protoplast Culture. The following techniques have been adopted in order to maintain number of protoplast population between minimum and maximum effective densities after plating up: Liquid method. This method is preferred in 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 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 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 low density, a feeder layer technique is adopted. A feeder layer of X-ray irradiated non-dividing but metabolically active protoplasts after washing are 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

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fast growing protoplast provide other species with diffusible chemicals and growth factors which helps in cell wall formation and cell division. The co-culture methods is gene­ rally used where calli arising from two types of protoplasts can be morphologically dis­ tin­guished. For example, proto­ plasts isolated from albino plants and green plants are easily dis­ tinguishable based on color where albino protoplast will develop non green colonies. Freshly isolated protoplasts are spherical because they are un­ bound by cell wall. Viable pro­ toplasts regenerate a new cell wall within 48 to 96 h af­ ter isolation which can be determined by stai­ ning with calcafluor white. Proto­plasts with new cell wall fluoresce bluish white under UV. Proto­ plasts that fail to regenerate a wall generally will not divide and die eventually. Also, all the Figure 43. Protoplast culture techniques healthy protoplasts may not di­ vide and therefore, plating efficiency is calculated to estimate cell vigor. Plating efficiency is number of dividing protoplasts/total number of protoplasts plated. The protoplasts capable of dividing undergo first division within 2-7 days after isolation (Fig.43). The delicate nature of protoplasts demand 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. 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 members of same species or closely related

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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 (Fig. 44). = chloroplast = mitochondria

Fusion

= nucleus heterokaryon

cybrid



hybrid

hybrid

cybrid

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

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 require 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 (Fig. 45). Most commonly reported fusion inducing chemical agents are sodium nitrate (used by Carlson), high pH/Ca2+ concentration and Polyethylene glycol (PEG) treatment. Sodium

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nitrate treatment results in low frequency of heterokaryon formation, high pH and high Ca2+ concentration suits 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 presence of high pH/ Ca2+ is reported to be most effective in enhancing heterokaryon formation and their survivability.

Note: A- two separate protoplasts; B- agglutination of two protoplasts; C, D – membrane fusion at 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 fusogen are toxic to some cell systems; 2) it produces random, multiple cell aggregates; 3) must be removed before culture. A more selective, simpler, quick and non toxic approach is electrofusion which utilizes electric shock or 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 is applied, which causes protoplasts to become aligned into chains of cells between electrodes. This creates complete cellto-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 pulses 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 (Fig. 46). 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.

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Part III. Industrial and agricultural applications of in vitro culture Parent 1

Parent 2

Culture of the ovule extracted from immature fruit

Glashouse plant

Embryogenic nucellar callus

In vitro plants

Sterilized leaf tissue

Embryogenic cell suspension Leaf segments Enzymatic digestion Enzymatic digestion

Embryogenic protoplast

Mesophyll protoplast Fused protoplast by PEG or electrofusion

Colony

Somatic embryos

Soil Allotetraploid somatic hybrids Figure 46. Schematic view of symmetric protoplast fusion producing somatic hybrids (from website http://nptel.ac)

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Chapter 3.3. Cell engineering Parent 1

Parent 2

Culture of the ovule extracted from immature fruit

Culture of the ovule extracted from immature fruit

Embryogenic nucellar callus

Embryogenic nucellar callus

Embryogenic cell suspension

Embryogenic cell suspension

Enzymatic digestion

Enzymatic digestion

Embryogenic protoplast (DONOR) γ-rays

Embryogenic protoplast (RECIPIENT)

Fused protoplast by PEG or electrofusion

Colony

Somatic embryos

Soil Alloplasmatic somatic hybrids (cybrids) Figure 47. Schematic view of asymmetric protoplast fusion using donor-recipient method resulting into creation of alloplasmic somatic hybrid or cybrids (from website http://nptel.ac)

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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 (Fig. 47) 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 non-viable nucleus or suppressed nucleus. 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, source tissue, culture medium and environmental factors influence 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 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 differing 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 analogs are 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

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cells having both RITC and FITC fluoresce yellow. Chromosome counting of the hybrid is an easier and reliable method to ensure hybridity as it also provides the information of ploidy level. Cytologically the chromosome count of the hybrid should be sum of number of chromosomes from both the parents. Besides 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 number. Multiple molecular forms of same enzyme which catalyses similar or identical reactions are known as isozymes. Electrophoresis is performed to study 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 incom­pa­ tibility 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 cyto­ plasm transfer to one year from 6-7 years required in 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 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 the protoplast can be used as a starting material for genetic manipulation. 2. What is the role of cell wall degrading enzymes?

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Part III. Industrial and agricultural applications of in vitro culture 3. Name the best source used for protoplast isolation. 4. Which chemical is considered to be a 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?

Chapter 3.4 CELL SELECTION Development of modern experimental biology is dependent on new model systems which allow estimation of the potential of plant cell in biotic and abiotic stress conditions (selective conditions). Conventional model system allow 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, it means the select at the level of the single cell. Selection strategies are: – Positive. – Negative. – Visual. – Analytical Screening. Positive selection allow to select the cells which have resistance to stress factors (biotic and abiotic). In positive selection into nutrient medium add a toxic compound (e.g. hydroxy proline, kanamycin and other stress agents), after 2-4 weeks select cells resistant to toxic substances. In this case only those 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 days work (possible to plate up to 1,000,000 cells on a Petri-dish). In negative selection into medium add an agent that kills dividing cells (for example, chlorate / BUdR, or treatment by mutagenic agents) then plate out leave for a suitable time, wash out agent then put on growth medium. In this case, all cells growing on selective agent will die leaving only non-growing cells to now grow. This selection strategy is useful for selecting auxotrophs. Visual selection useful only for coloured or fluorescent compounds e.g. shikonin/ Berberine/ some alkaloids. At this type of selection 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 days work. Salinity is one of the main abiotic stress that has been addressed by in vitro selection. High temperature, drought have detrimental effects on plant growth and development, 103

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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 result in some degree of stress. Water deficit, 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 deficit leads to the expression of sets of genes involved in acclimation and adaptation to the stress. These genes mediate the cellular and wholeplant responses. The 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 chillingsensitive 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 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, andna signaling pathway, the SOS pathway, regulating the expression of these genes involved in ion homeostasis hasnbeen 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

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level, plants alter metabolism in various ways to accommodate 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 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 e.g. the dry weight of shikonin produced from cell culture is 20% more than from plants. – In case of plant material facing threat of extinction or are limited in supply like L-erythrorhizon, in vitro production of secondary metabolites is saving option. – Controlled environmental conditions in cell culture ensure continuous supply of metabolites. In conventional system, 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 (Fig. 48). 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.

Figure 48. 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 more valuable product by single step reaction in vitro. – Production of novel compounds: Mutants 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

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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», 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 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 follow Mendelian inheritance pattern and transmitted to 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 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

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Amplified Polymorphic DNA (RAPDs), Restriction Fragment Length Polymorphism (RFLPs) and Inter-Simple Sequence Repeats (ISSR). 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 (Fig. 49). 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). Mechanisms of genetic changes (or spontaneous mutations) • Replication errors • Polimerase slippage • Oxidative DNA damage • DNA deamination and depurination • Chromosome breakage • Asymetrical crossingover • Activation of transposones and retrotransposons

Point mutations nucleotide substitutions insertions and deletions of single nucleotides Chromosome rearrangements deletions duplications inversions translocations Changes in tandem repeats number

Figure 49. Sources of somaclonal variation

Biochemical changes: 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 (Fig. 50).

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A

B

Figure 50. 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 represent 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 environmental effects which are 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 would 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 charateristics [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 breeder to have greater control on selection process as here they have the option to select from large amount of genetically uniform material. Therefore, this is the only approach for 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 variety of valuable traits like disease resistance, stress (salt, low temperature) resistance, improved yield and 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 do an single cell culture is advantageous over complete plant tissue culture? 2. Of what importance are stress-resistant plants for the crop improvements? 3. Explain factors, which influence the induction of somaclonal variants? 4. What are main causes of somaclonal variation? 5. What differences are between somaclonal variation in vitro and genetic variability in vivo?

Chapter 3.5 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 condition support the development embryo which use nutrient compounds from culture media that same with the composition of endosperm. Embryos isolated at the early stage of embryogenesis and 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 at 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 medium increased the frequency of ESM formation on initiation stage, but inhibited the embryos development and even led to their degradation in proliferation stage. Some factors contributed by the germinating embryo is required for the stimulation of mature and dried endosperms of few plant species. In general, it has been found that mature endosperm requires the initial association of embryo to form callus but immature 109

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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 had been low (7-22%). Moreover, the 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

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from proliferating callus (Fig. 51 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 and 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 51. 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 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 organogenesis was also reported in Jatropa panduraefolia, Putranjiva roxburghii, Codiaeum varie­ gatum, Malus pumila, Oryza sativa, Annona squamosa, Actinidia chinensis, Mallotus philippensis, Actinidia deliciosa, Morus alba, Azadirachta indica and Actinidia de­ liciosa. 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

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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 kinetn. 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 number of cultures showing shoot-buds and number of buds per callus cultures occurred in the presence of BA. The percent response was highest on BA and NAA containing medium. However, the number of shoots per explants was maximum when TDZ alone was used. Physical factors includes effect of temperature, light and pH on endosperm proliferation. Straus and La Rue (1954) observed that corn endosperms develop better in dark than light conditions. But in Ricinus reverse is the case where a 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 till 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 seem 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 differentition 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 had combined yield traits of the sativa parent with local adaptation traits from O.glaberrima.

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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 (Fig. 52). 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

D.Embryo culture in 1/4 MS medium

F. Germination

G.Rooting

Figure 52. Embryo Rescue (from http://www.warda.cgiar.org website of West Africa Rice Development Association /WARDA/)

Wild rices are 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 led by Dr. Kshirod Jena has been attempting to cross the two rices since mid 1990s and has only been successful fairly 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 is adventure of embryo culture?

Chapter 3.6 HAPLOID TECHNOLOGY Plants usually reproduce through sexual means – they have flowers and seeds to create the next generation (Fig. 53). 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 obtain 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 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 (Fig. 54) [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 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 only in a short time after the initial hybridization. 114

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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 53. Life Cycle of Higher Plants

The in vitro production of haploid plants can be achieved by many techniques like: – 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.

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– 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. Year 1

Parent A x Parent B

Year 2

F1

Anther culture Haploid plants (greenhouse)

Doubled haploid plants

Year 3

Selection Year 4-6

Superior progenies

Selection Superior progeny released as variety

A

B

Figure 54. Pure line (inbred line) development with conventional breeding (A) and anther culture derived haploid plants for hybrid sorting (B)

Production of a haploid plant where egg cell is inactivated and only male genome is present is called androgenesis. Similarly, production of haploid by development of 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 (Fig. 55).

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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 double (diploid) during culture. In some species however, 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 express 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. Torpedo-shaped embryo

Induced haploid production

Androgenesis in vitro

Haploid plant

Seeds

Gynogenesis in vitro

Interspecific crossing in vitro

Pollen of another species

Irradiated pollen technique

Irradiated pollen

Microspore culture

Anther culture

Degenerate Meiosis

Mitosis

Mitosis

Gametophyle development Mitosis

Pollen tube

Fertilization

Sperm cells

Androgenesis

Semigamy

Spontaneous haploid production

Meiosis Gametophyle development

Egg cell

Polyembryony

Seeds

Chromosome Elimination

Haploid plant

Gynogenesis n female gametic nucleus n male gametic nucleus 2n nucleus

Figure 55. 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. Testing for resistances to pests and abiotic stresses are conducted also at this time. Lines with desired traits and are rated intermediate to resistant/tolerant 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 and are 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 parents. 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 unablem to produce functional pollen and would rely on other pollen source to produce seeds. This greatly facilitates large scale hybrid seed production, bypassing 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 as 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 play 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. First time, haploid plants were 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 research team (K.Zh.Zhambakin, B.B.Anapiyaev, K.K.Boguspaev, S.K.Turasheva) led by Professor I.R.Rakhimbaev has been obtaine haploid plants of wheat, barley, rise, potato which used in breeding program for create 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 flower bud of right stage (varies with species) is excised.

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– Antiseptic treatment. Disinfestation, excision and culture of anther. – Surface sterilization of buds or inflorescences. – Surface sterilization of anthers. Flower buds are surface sterilized in 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 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 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 (Fig.56). 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 on to 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. 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 (Fig. 57). Cochicine is a microtubule-depolymerizing drug. The chemical modification of natural colchicine (analogs of colchicine, colchicine derivatives) use for induced diploidization of haploids, they are more efficient as chromosome-doubling agents and are less toxic. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile

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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.

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 56. The different steps from androgenic structure, embryoid induction to maturity

Colchicine

N-Cysteinocolchicine

N-deacetil-N-(β,γ-thio-epo­ N-deacetil-N-(β, γ-epoxi-propil) xipropil) aminocolchicine (N2) aminocolchicine (N19)

N-aminoethylthiouronium colchicine chloralhydrate (L5)

N-deacetil-N-(carboxy-amino) dioxiethylamino-colchicine (N5)

Figure 57. Colchicine and colchicine derivatives

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With the cultivars «Ciccio» and «Claudio» an average of 36.5 and 148.5 fertile plants were 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 plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named «Lanuza», has been 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]. 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 cultures aproach. 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 do 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 firstly 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

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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%. The influence of genotype, of the developmental stage of micro-, megaspores on the formation of haploid plants, nutrient medium composition and cultivation conditions have been showen 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 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 cultivtion. 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 difference 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 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 genotype. In Triticum aestivum, 15 cultivars were tested: 7 interspecific hybrids and 8 lines of intergenus hybrid (Triticum aestivum x Aegilops squarrossa). It was observed a high degree of callus formation in two varieties: Grecum-476 (29.3 %) and Kazakhstanskaya-126 (19.6 %) 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 anyone cultivar or species usually prove to be unsuitable for another and require considerable modification. Most of the families 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 compounded by a high frequency of albinos. In anther culture of wheat

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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 affects 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 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. The important factor for successful cultivation of female/male gametophytes is the stage of development of embryo sac/microspores.

A

B



C

Figure 58. Uninucleate (A) and binucleate microspores, х640 (B) of wheat; pathways of division pollen grains (C)

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

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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 (Fig. 58). 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 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. 4. Pathway IV – Finally in 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 (Fig.59). 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, that are at an identical stage with cultivated ovules. In different species of Angiosperms plants, culture of different developmental stages of embryo sac may be optimal; however, induction of haploid plants apparently possible for the same genotype from ovules that have embryo sacs in different stages of development. For example, induction 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 as on ovaries containing early stages embryo sacs megaspore mother cells and tetrads upto 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 eight 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 recomendation can be given. Moreover, the conditions needed for sporophytic macrospore induction, for callus, embryo and plantlet formation normally differ. The importance of certain ingredients in nutrient medium are inorganic salts, organic additives, carbohydrates, growth regulators. For most donor hybrid plants influence of genotype on induction of morphogenesis and regeneration in anther culture more (43.75%) than impact of culture medium factor

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(20.24%). There is a positive correlation between the yielding capacity of the lines and the anther response. 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 59. Pathways of microspores development in vitro

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 developing of nonfertilized ovaries. The presence the ammonium ion is usually sufficient for the induction of em­ bryogenesis in callus or suspension cultures containing NO3-, but on media where NH4+ is lacking, casein hydrolysate, or an amino acid such as alanine, or glutamine, is often promotory [39]. For embryogenesis in wheat anther cultures, 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

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casein hydrolysate. Casein hydrolysate could be replaced by glutamine, glutamic acid or alanine. Suspensions of haploid cells (microspores) 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 compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher. However, the regenerant plantlets from anther culture were mainly albinos while 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 stimulate callus formation from somatic tissue and inhibit embryo formation from haploid elements of the embryo sac or anther. 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

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(21 days at 4 °C). In control, IAA predominated among Auxs (11–39 nmol g−1 DW), with IBA constituting only 1 % of 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 PGRs 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 +220...+260 C, but sometimes lower or higher temperatures for incubation were optimal (+20...+380 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 is possessed by 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, that was morphologically similar to the zygotic embryo. Regeneration of haploid plants by direct embryogenesis was observed in Triticum aestivum, Hordeum vulgare, Morus indica [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, photosynthetizing plants, 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 separate 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 key especially as induced mutations which are predominantly

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recessive and can not be detected until the second generation of selfing of putative mutants (M2 generation) at the earliest. 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 are 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, with which 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 has been achieved thanks to the results of genetic improvement, while the residual advance has been 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 result 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 may be used in breeding programme. For e.g. doubled haploids have been used for rapid development of inbred lines in hybrid maize programme. 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 (Fig.50). 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 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

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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. 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 e.g. 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 is 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 pos­ sibility 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 do an isolated microspore culture is advantageous over complete anther culture? 2. Of what importance are haploid plants for the crop improvements? 3. With the help of a flow chart briefly describe the early patterns of cleavage in cultured pollen grains and the different modes of subsequent development of the proembryogenic mass so obtained. 4. Explain briefly, the three crucial factors, which influence the induction of androgenesis? 5. Explain the factors, which influence the induction of ginogenesis?

Chapter 3.7 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 single DNA sequence from one organism by breakage of DNA molecule at two desirable places and then inserts it at a desired position in another DNA molecule from completely different organism to form recombinant DNA and the technique involved is called recombinant DNA technique. This has also been termed as genetic engineering because of the potential for creating novel genetic combinations. Using this technique, 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 recombinant DNA technique can be outlined as follows: 1. Isolation of desired DNA fragment or gene of interest 2. Insertion of the isolated gene in a s suitable vector. 3. Introduction of this recombinant molecule into host cell by transformation 4. Selection of transformed host cells 5. Multiplication and expression of introduced gene in the host (Fig. 60). Although there are many diverse and complex techniques involved in genetic en­ gineering, its basic principles are reaso­nab­ ly simple. It is however, very important to know the biochemical and physiological mechanisms of action, regulation of gene ex­ Figure 60. Recombinant DNA technique pression and safety of gene and gene product (Genomes. Garland Science, 2007) 130

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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 use for DNA isolation from fresh, frozen or dried leaves of barley and wheat seedlings. With this method the usual yield is approx. 100 mg 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. Poly­ acrylamide is preferred for smaller DNA fragments. Separation is achi­ eved by moving the negatively char­ ged nucleic acid molecules through agarose matrix with an electric field. Rate of migration of DNA molecules is inversely proportional to their mo­ lecular weight. Smaller molecules Figure 61. Agarose gel electrophoresis of DNA move faster than larger ones (Fig. 61). (ethidium bromide stained gel showing migration Conformation of the DNA mo­ of DNA molecules) lecule 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 (Fig. 62). 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).

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A

B

Restriction nucleases produce DNA fragments that can be easily joined together

C Figure 62. Mechanism of restriction with EcoRI, EcoRV endonuclease (A, B) and ligases (from http://www.ncbi.nlm.nih.gov /bookshelf/ br.fcgi.book)

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 selfreplicating genetic element: typically into a plasmid. The new recombinant DNA molecule can then 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 (Fig.63).

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Figure 63. Typical PCR reaction (from http://bitesizebio.com/2008/01/23/the-essential-PCRtroubleshooting-checklist)

Basic PCR components: – DNA template. – Primers flankingthe 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 lot of molecular biology experiments such as to introduce restriction enzyme sites, or to mutate (change) 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 allow for quantitative measurement of DNA or RNA molecules

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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 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, instead of DNA, RNA is used for blotting and 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. A western blotting is a method 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 an 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

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chemicals, and large amounts of radiolabeled DNA, whereas the chainterminator method uses fewer toxic chemicals and lower amounts of radioactivity. The key principle of the Sanger method was the use of dideoxynucleotides 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 elongation. 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 sum total of DNA inserts in this collection represent the entire genome of the concerned organism. The genomic library is prepared by shotgun approach where the total genomic DNA of 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 upto 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 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. 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. The

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isolation and identification of desired clone from 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 donot 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 (Fig. 64). chromosomal DNA gene B

gene A exon

intron

nontranscribed DNA

RESTRICTION NUCLEASE DIGESTION

gene A

gene B

TRANSCRIPTION RNA transcripts RNA SPLICING

DNA fragments

mRNAs DNA CLONING REVERSE TRANSCRIPTION AND DNA CLONING

genomic DNA clones in genomic DNA library

cDNA clones in cDNA library

Figure 64. DNA cloning (Genomes. Garland Science, 2007)

c. Chemical synthesis of 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 deduced base sequence can be synthesized chemically using automated DNA synthesizers. d. Gene amplification through PCR. The polymerase chain reaction technique amplifies 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 that has the ability to replicate in

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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 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 large population of cells that have not taken up foreign DNA. 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 (upto 12 kb). The circular plasmid DNA which is to be used as vector is first cleaved by restriction endonuclease (RE) to give linear DNA molecule. The foreign DNA to be inserted is also cut by same endonuclease foolowed by ligation (joining) of the linearised vector and insert 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 foreign DNA at a 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) A selectable marker (ampr-antibiotic resistance gene) tetr-antibiotic resistance gene

Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers)

Figure 65. Cloning Vectors

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2. Bacteriophage vector are viruses of bacteria thateither infect the cell 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 (2325kb) 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 vector are lambda (λ) and M13 phages. 3. Cosmid vectors are plasmids which contain a fragment of λ DNA including the cos site. Since 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) are selected as the distance between two cos sites must be between 38 and 52 kb for packaging. Therefore, cosmids can accommodate upto 45 kb long DNA inserts which is much more than a phage vector. Because of their capacity for 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 phage are known as phagemids. Under normal circumstances, the plasmid ori is used for replication but following a phage infection 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 vector 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 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 host cell for its multiplication and expression. The host cells are made competent or permeable by either Calcium chloride treatment for transformation or electrick 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 vector. A reporter gene produces a phenotype which permits either easy selection or quick identification of cells in which it is present. For e.g. genes conferring drug resistance or nutritional deficiencies are selectable markers which allow only cells which possess it to survive under selective

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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 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 a 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 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 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. has 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 where usually naked DNA is directly transferred. Therefore, also referred to as DNA mediated gene transfer. – Particle bombardment or Gene gun technique. The various physical methods for gene transfer are electroporation, 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.

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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 fine tipped (0.5-1.0 µm diameter) capillary glass needle or micropipettes. Through microinjection technique, the desired gene introduce into large cells, such as oocytes, eggs, and the cells of early embryo (Fig. 66).

Figure 66. 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 have incorporated the new DNA. In one common approach, they combine the gene of interest with a 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 cell and try to grow them in a medium that contains a specific antibiotic. Only the genetically transformed cells will survive. The antibiotic-resistante 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 (Fig. 67). 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

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wound 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 typically is 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 67. 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 (Fig.68).

A

B

Figure 68. 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 (Fig.69). 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 (Fig. 70). 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 an 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 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.

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

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Figure 70. 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

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 T-DNA. 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 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 T-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 rapididly replacing other methods for generation of transgenic plants.

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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. – Following induction, the agrobacteria should have access to cells that are competent for transformation (Fig. 71). 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 71. Agrobacterium mediated development of transgenic plants in tobacco

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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 leaf disc is excised and is incubated in Agrobacterium suspension for 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 designed for selection of transformed plant cells. Selection is facilitated by selectable marker genes present in 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 reporter gene or 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 S.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 non-transformed 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 S.No

Scorable marker gene

Substrate and assay

Identification

1

β-glucoronidase (GUS)

X-gluc

Fluorescence

2

β-galactosidase (lac Z)

X-gal

3

Neomycin phospho-transferase (NPT II)

Kanamycin+P ATP

Colony colour 32

Radioactivity detection

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Furthermore, gfp transcripts in calli isolated by visual selection were more abundant than under Hyg selection; in contrast, transcript levels of hpt in calli 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 expression of genes which are influenced by environmental or developmental conditions. Whereas molecular markers defined as readily detectable DNA sequences whose inheritance can be easily monitored, which are independent of developmental stage or 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 marker whose chromosomal location is known. Coinheritance of 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. – Should be polymorphic. – Should have codominant inheritance to allow discrimination between homo and heterozygotes in diploids [32, 42, 43, 44]. There are a wide range of molecular markers available to detect polymorphism like RFLP, RAPD, AFLP, SSR etc.

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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 quick and efficient technique. But since, it is not 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 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 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 repeat sequences. Microsatellites provide reliable, reproducible molecular markers. Under the leadership 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 have 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].

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Role of Biotechnology in Crop Improvement The last decade has witnessed remarkable change which has taken plant biotechnology from 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 commercialise transgenic crops. In general, the role of biotechnology in crop improvement can be divided into two categories: 1) those directed towards 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 trait in the crop by conventional breeding methods (Fig. 72). Molecular breeding. Molecular maps using markers RFLP, RAPD, SSR, ESTs for major crop species like rice, maize, tomato etc has been utilized very effectively in crop improvement programmes like marker assisted selection (MAS). MAS is a method of breeding which allows 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 trait in breeding populations based on the gene-linked genetic markers instead of the trait itself. Such type of selection 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 72. Integration of conventional and modern biotechnology methods in crop breeding

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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 intergrating 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 a little of practical examples of working markers for quantitative traits in spite of very intensive worldwide researches 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 practically impossible achive by means of conventional breeding. First of all that goes for polyploidal cultivars. Also the MAS methods have good practical potential in fruit trees breading where due to long ontogenesis of these plants the breeding process usually requires many years. 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, 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 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 crosses generated. Undesirable genes can be transferred along with desirable genes or while one desirable gene is gained, another

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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 (Fig. 73). 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). 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 rest are in process.

Figure 73. 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 in the specificity of their tight binding to companion receptors in the insect gut. In recent years, a variety of safety studies were 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 has been especially successful for control of viral diseases. The approach followed is to identify those viral genes or gene products which when present a wrong time or in improper amount, will intefere with the normal functions of the infection

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process and prevent disease development. 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 potato 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 e.g. 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 (glutathioneS 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 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 confer 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 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

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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 (Fig.74) [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 74. 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])

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

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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 have 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 has 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 lowlinoleic acid product from GE soybean oils. If a food contains a new potentially allergycausing 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 sterlity 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 donot breed true. It is now possible to engineer male sterlity bt 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 seed. Fertility can be restored by expression of the barstar gene, which 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 speciality chemicals and pharmaceuticals such as transgenic tobacco plants carrying mannitol dehydrogenase gene from E. coli is used for increasing production of mannitol. Similarly production of Polyhydroxy butyrate (PHB) in plants provide 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 to create transgenic plants as source 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.

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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 cold–upregulated gene transcripts than wild-type plants have, suggesting that numerous cold– up-regulated proteins that may be involved in cold acclimation are being produced in the absence of cold in these CBF1 transgenic plants. In addition, CBF1 tansgenic plants are more cold tolerant than control plants. T-DNA and transposable elements are used as molecular tags to produce mutations by becoming inserted within genes thus making it non functional. 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 are vector and its essential elements? 5. Describe the type of vector which is used in plant transformation. 6. What is plant virus vector? 7. Describe the position of regulatory sequences in plant gene. 8. Agrobacterium are which type of bacteria? 9. What are Ri and Ti plasmids? 10. What are the essential segments in Ti plasmis? 11. Describes 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. Describes 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?

Chapter 3.8 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 the conservation of germplasm of different crops and their wild and weedy relatives. Germplasm Conservation. The genetic material especially its molecular and chemical constitution that is inherited and transmitted from one generation to other is referred to as germplasm. In other words, the sum total of all the genes present in a crop and its related species constitutes its germplasm. It is generally represented by a collection of various strains and species. Germplasm is valuable because it contains diversity of genotypes that is 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. The use of the National programm «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» in 2013 at the Institute of Botany and Phytointroduction was created seed bank of wild congeners 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 genetic variety of plants is their preservation as a part of natural communities in situ, and 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 ex situ conservation of plants allow 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 156

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evolved without the help of human beings. The 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. The conservation of the 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 material. The most convenient method of ex-situ germplasm conservation is in the form of seeds. Thus, 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 the 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 sp., 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 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 cultures: where limited growth of culture is allowed. This is a simple, effective and economic method and can be used in all species where shoot tip/ nodal explant are available. In thses 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 like during subculturing there is risk of contamination by pathogen, genetic changes may also occur. 2. Cryopreservation: Any growth in plant cell and tissue culture is brought to a halt still retaining its viability in this technique 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 Djuar apparate/equipment. Cryopreservation for germplasm purposes utilizes shoot tips and buds only but protoplasts, cells, tissues and somatic embryos are also cryopreserved for other tissue culture processes. Freezing injury is associated primarily with damage caused by ice crystals formed within cells and organs. Freezing-resistant species have mechanisms that limit the

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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 hysteresis proteins (THPs). Another group of proteins found to be associated with osmotic stress are 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 function 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 a 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

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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 minimum 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 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 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 (Fig. 75)

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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 dessication.

A

В

С

D

Note: A – controlled freezing; B, C – Djuar apparate/equipment; D – rapid freezing by dipping into liquid nitrogen Figure 75. 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 leadership of Dr. S.V. Kushnarenko at the Institute of Plant Biology and Biotechnology developed protocols for preservation of SouthEast Kazakhtan’s rare species Lonicera iliensis Pojark from three natural populations. Thise protocol include seed storage, in vitro culture and cryopreservation of L.iliensis germplasm. Dried seed 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 +40 C, -200 C and -1960 C. In vitro plantlets were propagated on MS medium added 1 mg/l 6-benzylaminopurine. The protocol of shoot tips cryopreservation was optimized: duration the cold acclimation of plants (3 weeks at +40 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 af aseptic cultures have been found for 3-70 C. The special role in plant conservation in vitro play osmotic and physical factors of cultivation, retardants,temperature and light intensity. At creation of gene bank in vitro primary importance is given to representative and preservation of genetic stability [51].

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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 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 the morphological pathways during regeneration of plants after rewarming. The 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 other countries and also 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 (2266) and pepper (728). Onion crop plants

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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 also are 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 Kazakhstanian origin should be noted [52, 53]. Encapsulation technology has recently revolutionized the production and conservation programme of elite and threatened germplasm throughout the globe. This technology has made the exchange programme possible between different laboratories at an ease. Synthetic seed production not only scaled up extensive and commercial production of plants but also has 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, that 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. Under the leadership 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

Chapter 3.8. Cryoconservation

163

and long-term tissue cryopreservation in liquid nitrogen are used in addition to field collections reliable preserve 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 10hour 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 both 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 include 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 extinction possibilities. A large number of plants including the above have 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 cryoprotectant. 4. Write short notes on Vitrification, Cryoprotectants, Stepwise freezing

GLOSSARY Adventitious – Developing from unusual points of origin, such as shoot or root tissues, from callus or embryos, from sources other than zygotes. Agar – a polysaccharide powder derived from algae used to gel a medium. Agar is generally used at a concentration of 6-12 g/liter. Aseptic – Free of microorganisms. Aseptic Technique – Procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures. Autoclave – a machine capable of sterilizing wet or dry items with steam under pressure. Pressure cookers are a type of autoclaves. Auxin – a group of plant growth regulators that promotes callus growth, cell division, cell enlargement, adventitious buds, and lateral rooting. Endogenous auxins are auxins that occur naturally. Indole3-acetic (IAA) is a naturally occurring auxin. Exogenous auxins are auxins that are man-made or synthetic. Examples of exogenous auxins included 2.4-Dichlorophenoxyacetic acid (2.4-D), Indole-3Butyric acid (IBA), α-Naphthaleneacetic acid (NAA), and 4-Chlorophenoxyacetic acid (CPA). Callus – an unorganized, proliferate mass of differentiated plant cells, a wound response. Chemically Defined Medium – a nutritive solution for culturing cells in which each component is specifiable and ideally of known chemical structure. Clone – plants produced asexually from a single source plant. Clonal Propagation – a sexual reproduction of plants that are considered to be genetically uniform and originated from a single individual or explant. Contamination – being infested with unwanted microorganisms such as bacteria or fungi. Culture – a plant growing in vitro. Cytokinin – a group of plant growth regulators that regulate growth and morphogenesis and stimulate cell division. Endogenous cytokinins, cytokinins that occur naturally, include zeatin and 6-γ,γdimethylallylaminopurine (2iP). Exogenous cytokinins, cytokinins that are man-made or synthetic, include 6-furfurylaminopurine (kinetin) and 6-benzylaminopurine (BA or BAP). Differentiated – cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. Modifications of new cells to form tissues or organs with a specific function. Dedifferentiation – the phenomenon of mature cells reverting to meristematic state to produce callus Explant – tissue taken from its original site and transferred to an artificial medium for growth or maintenance. Gibberellins – plant growth regulator that influences cell enlargement. Endogenous growth forms of gibberellin include Gibberellic Acid (GA3). Horizontal laminar flow unit – an enclosed work area that has sterile air moving across it. The air moves with uniform velocity along parallel flow lines. Room air is pulled into the unit and forced through a HEPA (High Energy Particulate Air) filter, which removes particles 0.3 μm and larger. Hormones – Growth regulators, generally synthetic in occurrence, that strongly affects growth (i.e. cytokinins, auxins, and gibberellins). Internode – The space between two nodes on a stem In vitro – to be grown in glass (Latin). Propagation of plants in a controlled, artificial environment using plastic or glass culture vessels, aseptic techniques, and a defined growing medium. In vivo – to be grown naturally (Latin) Media – plural of medium Medium – a nutritive solution, solid or liquid, for culturing cells. Micropropagation – in vitro clonal propagation of plants from shoot tips or nodal explants, usually with an accelerated proliferation of shoots during subcultures.

164

Glossary

165

Morphogenesis – the 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. Node – a part of the plant stem from which a leaf, shoot or flower originates. Organogenesis – a process involving redifferentiation of meristematic cells present in callus into shoot buds. Passage – the transfer or transplantation of cells or tissues with or without dilution or division, form one culture vessel to another. Passage Number – the number of times the cells or tissues in culture have been subcultured or passaged. Pathogen – a disease-causing organism. Pathogenic – capable of causing a disease. Petiole – a leaf stalk; the portion of the plant that attaches the leaf blade to the node of the stem. Plant Tissue Culture – the growth or maintenance of plant cells, tissues, organs or whole plants in vitro. Regeneration – in plant cultures, a morphogenetic response to a stimulus that results in the products of organs, embryos, or whole plants. Redifferentiation – the ability of the callus cells to differentiate into a plant organ or a whole plant. Shoot Apical Meristem – 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. Somaclonal Variation – phenotypic variation, either genetic or epigenetic in origin, displayed among somaclones. Somaclones – plants derived from any form of cell culture involving the use of somatic plant cells. Stage I – a step in in vitro propagation characterized by the establishment of an aseptic tissue culture of a plant. Stage II – a step in in vitro propagation characterized by the rapid numerical increase of organs or other structures. Stage III – a step in in vitro propagation characterized by preparation of propagules for successful transfer to soil, a process involving rooting of shoot cuttings, hardening of plants, and initiating the change from the heterotrophic to the autotropic state. Stage IV – a step in in vitro plant propagation characterized by the establishment in soil of a tissue culture derived plant, either after undergoing a Stage III pretransplant treatment, or in certain species, after the direct transfer of plants from Stage II into soil. Sterile – (A) Without life. (B) Inability of an organism to produce functional gametes. (C) A culture that is free of viable microorganisms. Sterile Techniques – the practice of working with cultures in an environment free from microorganisms. Subculture – see «Passage». With plant cultures, this is the process by which the tissue or explant is first subdivide, then transferred into fresh culture medium. Tissue Culture – the maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function. Totipotency – a cell characteristic in which the potential for forming all the cell types in the adult organism are retained. Undifferentiated – with plant cells, existing in a state of cell development characterized by isodiametric cell shape, very little or no vacuole, a large nucleus, and exemplified by cells comprising an apical meristem or embryo.

THE TASKS FOR STUDENTS’ INDEPENDENT WORKS 1. 2. 3. 4. 5.

Plant Tissue Culture: applications and limitations Use of Biotechnology in Agriculture – Benefits and Risks Plant Progapagation by Tissue Culture Plant cell biotechnology for the production of secondary metabolites New ways in the discovery of bioactive substances and their chemical and biotechnological synthesis. 6. Medicinal plants ex situ, in situ and in vitro. Plant Toxins as Sources of Drugs 7. Role of modern technologies in development of high effective phytopreparations 8. Plant Propagation: basic Principles and Methodology 9. Somatic embryogenesis and plant regeneration from callus cultures of Monocotyledonous plants 10. Cell engineering for quality improvement in Crops 11. Somatic embryogenesis and plant regeneration from immature embryos of Dicotyledonous plants 12. In vitro strategies for the micropropagation and conservation of Horticultural plants 13. Callus induction, proliferation, and plantlets regeneration under abiotic stress conditions 14. Hormonal requirements for effective induction of microspore embryogenesis in cereals anther cultures 15. Steroid hormones of plants: role outside plant kingdom and prospects for medicine 16. Application of genetic engineering in Crop production 17. The safety of genetically-engineered crops and foods: safety and regulations 18. Molecular breeding and Marker-Assisted Selection 19. Micropropagation of endangered plants 20. Biotechnological strategies for the conservation of Higher Plants

166

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Appendix

APPENDIX

Table 1

The composition of culture media Components

KNO3 NH4NO3 Ca (NO3)2 (NH4)2SO4 MgSO4 · 7 H2O CaCI2·H2O CaCI2· 2 H2O СaCI КН2РО4 NaH2PO4· H2O MnSO4·H2O MnSO4 · 4 H2O ZnSO4 · 4 H2O ZnSO4 · 7 H2O Н3ВО4 CuSO4· 5 H2O Na2MoO4 · 2 H2O CoCI2 · 6 H2O FeSO4· 7 H2O* Na EDTA·2 H2O Myoinositol Ascorbic acid Thiamine-HCI Pyrodoxine-HCI Nicotyne acid Sucrose Agar, Gelrite pH

Murashige – B5 medium White Skoog culture (Gamborg’s B5 culture medium media medium) Concentration, mg/1 L medium 1900 3000 81 1650 142 134 370 500 74 440 150 65 170 12 150 10 22,3 8,6 2 6,2 3 0,025 0,075 0,25 0,25 0,025 27,85 37,25 100 0,5 0,5 0,5 30 000 20 000 2000 5.6-5.8 5.5 5.6-5.8

Nitsch’s medium

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: plant biotechnology Table 2

The composition of culture media for plant cell culture Components

KNO3 NH4NO3 Ca (NO3)2 NH4Н2РO4 MgSO4 · 7 H2O MgSO4 CaCI2· 2 H2O СaCI2 КН2РО4 NaH2PO4· H2O MnSO4·H2O ZnSO4 · 7 H2O

Shenk-ChilderWPM Gelrigel culture brandt’s (Lloyd, medium culture medium Mc 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 -

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 -

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Table 3

The composition of culture media for plants anther culture Components КNO3 NН4NOз (NH4 )2 SO4 Сa(NO3 )2·7 H2O

Culture medium, mg/ 1 L N6

В5

L85D12

Роtato 2

2830

2500

1400

1000

-

-

300

-

463

134

-

100

-

-

-

100

МgSO4· 7 H2O

185

250

150

125

СaС12

166

150

150

-

-

-

-

35

400

-

400

200

-

150

-

-

10 М

10 М

МnSO4 · 4 Н2O

4,4

10,0

11,2

-

KJ

0,8

0,75

0,83

-

Н3ВO3

1,6

3,0

6,2

-

ZnSO4

1,5

2,0

-

-

-

0,025

0,025

-

0,25

0,025

-

0,100

0,100

-

-

КС1 КН2 РO4 NaН2 РO4 · Н2O FeNa ЕDТA

СuSO4 · 5 Н2O Na2МоO4· 2 Н2O СоС12· 6 Н2O Myoinositol

100

100

-

-

Thiamine-HCI

1,0

10,0

1,0

1,0

Nicotyne acid

0,5

1,0

0,5

-

Pyrodoxine-HCI

0,5

1,0

0,5

-

Potato extract

-

-

-

+

Glycine

2,0

-

2,0

-

Sucrose

90000

20000

20000

90000

2.4-D

1,0

0,4

-

1,0

NAA

-

-

0,75

-

-

0.1

1,5

0,5

5,6-5,8

5,5

5,6-5,8

5,8

Kinetin рН

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Basics of biotechnology: plant biotechnology 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 60 °C). The stocks of plant growth regulators are prepared as mentioned below. Plant Growth Regulator Benzylaminopurine (BAP) Naphtalene acetic acid (NAA)

Nature Autoclavable Heat labile

Mol.Wt. 225.2 186.2

Soluble in 1 N NaOH 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 – 20 °C.

QUIZ 1. Who among the following coined the term Biotechnology? A. James Clarke B. Karl Ereky C. Paul Terasaky D. Clarke and Sommer E. Caesar Milstein 2. The scientists who made a big contribution to development of plant biotechnology A. Michurin S., Krebs M. B. Murasige S., Skug F. C. Keller C., Milstein G. D. Rox E., Lenindzher E. Butenko R. G., Rakhimbayev I.R. 3. Note the name of the nutrient mediums applied for cultivation plant cells: A. Dyulbek Nutrient medium B. Igl nutrient medium C. Nutrient medium of BM D. White nutrient medium E. Erlich medium 4. Development of plant biotechnology is connected with such fundamental and applied sciences as: A. Geography B. Zoology C. Physiology of plants D. Geology E. Valeology 5. Cellular and subcellular objects of plant biotechnology: A. Nanoparticles B. Culture of plant cells C. Microorganisms D. Plant DNA E. Culture of animal cells 6. Scientists who introduced the concept of «morphogenesis and regeneration of plants» in Plant Biotechnology are: A. K. Erecki B. F. Skug C. T. Shvan D. S. Murasige E. D. Jenner 7. What are the macronutrients used in plant cell culture medium (MS)? A. Cu, S B. N, K

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Basics of biotechnology: plant biotechnology C. Ca, B D. Zn, N E. P, S 8. What are the micronutrients used in plant cell culture medium? A. N, Cl B. Zn, Co C. Na, B D. Ni, Mg E. J, Cu 9. What are the macronutrients used in Murashige-Skug culture medium? A. N, Ni B. Ca, Mg C. Co, Zn D. B, P E. K, S 10. What are the micronutrients used in Murashige-Skug culture medium? A. S, Ca B. Mo, Cu C. Cr, N D. Ni, P E. B, J 11. What are the macronutrients used in plant cell culture medium? A. N, P, K, S, Ni B. N, P, K, S, Ca C. N, P, K, Ca, Cl D. N, P, Ca, B, Cl E. N, P, K, Ca, Mo 12. What are the micronutrients used in plant cell culture medium? A. N, Ca B. Cu, J C. P, K D. Na, S E. Cl, Ca 13. To maintain the pH of the culture A. Ammonium Salts Are Used B. Organic Acid Such As Citric, Fumaric, Malic And Succinic Acid Is Used C. Nitrogen Salts Are Used D. Sucrose Is Used E. Synthetic buffers such as Tris, MES or HEPS are used 14. Cellular totipotency is the property of A. Animals B. Plants C. Bacteria D. Fungi E. Human 15. The ability of the component cells of callus to form a whole plant is known as A. Dedifferentiation B. Redifferentiation C. Both Dedifferentation And Redifferentation D. Competention E. None Of These

173

Quiz 16. Which of the following growth regulator promote cell division? A. Auxins B. Cytokinins C. Gibberellins D. Brassinosteroids E. Abscises acid 17. Which of the following growth regulator promote cell division? A. Indole acetic acid (IAA) B. Naphthalenacetic acid (NAA) C. 2.4-Dichlorophenoxyacetic acid (2.4-D) D. Benzilaminopurin E. Gibberellins 18. Which of the following growth regulator cause plant cells to grow? A. Kinetin B. 6-benzylaminopurine C. Brassinosteroids D. Naphthalenacetic acid (NAA) E. Zeatin 19. Which of the following is not a cytokinin? A. 6-benzylaminopurine B. 2.4-Dichlorophenoxyacetic acid C. Zeatin D. Kinetin E. Naphthalenacetic acid (NAA) 20. Which of the following is not a cytokinin? A. Zeatin B. Indole butyric acid C. Kinetin D. 6 benzylaminopurine E. Indole acetic acid (IAA) 21. Which of the following is not an auxin? A. Naphthalenacetic acid B. Indole acetic acid C. 2.4-Dichlorophenoxyacetic acid D. 6-benzylaminopurine E. Indole butyric acid 22. Which of the following is not an auxin? A. 2.4-Dichlorophenoxyacetic acid B. Brassinosteroids C. Indole butyric acid D. Naphthalenacetic acid E. Gibberellins 23. Auxanometer is used for measuring A. Respiratory Activity B. Growth Activity C. Photosynthetic Activity D. Osmotic Pressure E. Atmosphere pressure 24. Which is the most common carbon source used in the plant cell culture media? A. Glucose B. Sucrose

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C. Fructose D. Maltose E. Galactose 25. Neutralized activated charcoal is occasionally added to young regenerating cultures to A. remove toxic phenolics produced by the stressed plant cell B. remove toxic phenolics produced by the stressed plant cell and help to remove plants growth regulators introduced at an earlier stage C. help to remove plants growth regulators introduced at an earlier stage D. maintain the pH of the medium E. maintain the osmotic pressure 26. Choice bactericide substance: A. EDTA B. ethanol C. Chloramphenicol D. Pyridoxine E. Chloramine 27. Choice the auxins A. 6-BAP, 3-IAA B. NAA, 2.4-D C. IBA, 6-BAP D. FEP, 2.4-D E. IAA, FEP 28. Note the vitamins involved in the standard medium MS A. Vitamin A B. Vitamin B1 C. Ca2 + -pantotenat D. Vitamin C E. Vitamin E 29. Callus tissue formed from the process: A. Rhizogenesis B. Dedifferentiation C. Differentiation D. Organogenesis E. Shoot formation 30. Which of the following growth regulator is added for short initiation during plant regeneration from callus? A. Auxins B. Cytokinins C. Gibberellins D. Brassinosteroids E. Abscises acid 31. Which of the following growth regulator is used to stimulate embryo or shoot development? A. Auxins B. Gibberellins C. Cytokinins D. Brassinosteroids E. Abscises acid 32. Which of the following growth regulator cause plant cells to grow? A. Cytokinins B. Auxins C. Gibberellins

Quiz

175

D. Brassinosteroids E. Abscises acid 33. In plant cell culture media, auxins and cytokinins are used in the range of A. 100-125μM B. l-50μM C. more than 125μM D. 50-100μM E. More than 200 μM 34. Concentration of sucrose generally used in plant cell culture media is A. 10-15 g/l B. 20-30 g/l C. 40-50 g/l D. 60-70 g/l E. 80-100 g/l 35. Very high sugar concentration (40-100 g/l) have been used A. Only in specialized secondary metabolite production B. In specialized secondary metabolite production and to adjust the osmotic potential of the media in short term treatment for regeneration C. Only to adjust the osmotic potential of the media in short term treatment for regeneration D. In male and female gametophyte culture E. All of the above 36. Which is/are the naturally occurring plant auxins? A. Indole acetic acid (IAA) B. Naphthalenacetic acid (NAA) C. 2.4-Dichlorophenoxyacetic acid D. All of the above E. Indole butyric acid 37. Which is/are the disadvantage/(s) of using IAA in plant cell culture media? A. It is unstable in solution B. It isn’t the naturally occurring plant auxins C. Gets easily oxidized D. Conjugated to inactive form by plant cells E. All of the above 38. Which of the following is not a cytokinin? A. 6-benzylaminopurine B. 2.4-Dichlorophenoxyacetic acid C. Zeatin D. Kinetin E. Furfurilaminopurine 39. Which of the following is not an auxin? A. Indole acetic acid (IAA) B. zeatin C. Naphthalenacetic acid (NAA) D. Indole butyric acid E. 2.4-Dichlorophenoxyacetic acid 40. Which of the following is not an auxin? A. Indole acetic acid B. 6-benzylaminopurine C. 2.4-Dichlorophenoxyacetic acid D. Indole butyric acid E. Naphthalenacetic acid

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41. Which of the following is not an auxin? A. Naphthalenacetic acid B. Kinetin C. 2.4-Dichlorophenoxyacetic acid D. Indole butyric acid E. Indole acetic acid 42. What is ‘nurse’ or conditioned medium? A. It is the media full of growth factors used for the growth of cells B. It is the liquid medium removed from the suspension of fast growing cells C. It is the media full of growth factors used for the growth of cells and the medium added to nurse the callus culture D. It is the medium added to nurse the callus culture E. none of these 43. Which of the following is not true about nurse or conditioned medium? A. It is liquid removed from the suspension of fast growing cells B. it is removed aseptically from the culture and is autoclaved before use C. It contains uncharacterized growth factor released by growing cells D. It is used in the culture of regenerating protoplast E. All of the above 44. Organogenesis is A. formation of callus tissue B. formation of root and shoots on callus tissue C. Formation of embryogenic cell complex D. formation of morphogenic callus E. Formation of embryo 45. What is meant by «Organ culture»? A. Introduction of a new organ in an animal body with a view to create genetic mutation in the progenies of that animal B. Maintenance alive of a whole organ, after removal from the organism by partial immersion in a nutrient fluid C. Introduction of a new organ in plant with a view to create genetic mutation in the progenies of that plant D. Cultivation of organs in a laboratory through the synthesis of tissues E. The aspects of culture in community which are mainly dedicated by the need of a specified organ of the human body 46. In a callus culture A. increasing level of auxin to a callus induces shoot formation and increasing level of cytokinin promote root formation B. increasing level of cytokinin to a callus induces shoot formation and increasing level of auxin promote root formation C. auxins and cytokinins are not required D. only auxin is required for root and shoot formation E. only cytokinin is required for root and shoot formation 47. Which of the following is considered as the disadvantage of conventional plant tissue culture for clonal propagation? A. Multiplication of sexually derived sterile hybrids B. Storage of propagates C. Less multiplication of disease free plants D. Less multiplication of disease free plants and storage, transportation of propagates E. Transportation of propagates 48. What is/are the benefit(s) of micropropagation or clonal propagation? A. Rapid multiplication of superior clones

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B. Multiplication of disease free plants C. Multiplication of sexually derived sterile hybrids D. All of the above E. none of these 49. A(n) ___________ is an excised piece of leaf or stem tissue used in micropropagation A. microshoot B. extant C. medium D. scion E. root 50. The phenomenon of the reversion of mature cells to the meristematic state leading to the formation of callus is known as A. redifferentiation B. dedifferentiation C. regeneration D. morphogenesis E. none of these 51. Subculturing is similar to propagation by cuttings because A. it uses scions to produce new microshoots B. it separates multiple microshoots and places them in a medium C. they both use in vitro growing conditions D. all of the above E. none of these 52. Clonal micropropagation of plants is realized by following ways: A. Culturing anthers B. Activation and cultivation of axillary meristems C. Culturing ovaries D. Culturing microspores E. By Biolistic method 53. Clonal micropropagation of plants is preferably carried out by activation of axillary meristems as: A. Axillary meristem cells free from microscopic fungi B. Cells of axillary meristem is genetic stability C. Axillary meristem cells are genetically unstable D. Cells axillary meristem free from bacteria E. From axillary meristems receive genetic copy of the donor plants 54. Protoplasts are the cells devoid of A. cell membrane B. cell wall C. both cell wall and cell membrane D. nucleos E. none of these 55. Which breeding method uses a chemical to strip the cell wall of plant cells of two sexually incompatible species? A. Mass selection B. Protoplast fusion C. Transformation D. Transpiration E. micropropagation 56. Cell fusion method includes the preparation of ______ A) plant cells stripped of their cell wall

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B. single plant cell stripped of their cell wall C. plant cells with cell wall D. plant cells with cell wall from different species E. none of these 57. The processes of somatic cell fusion is (select the incorrect answer): A. Asexual hybridization B. Traditional Hybridization C. Asymmetric hybridization D. Somatic hybridization E. Parasexual hybridization 58. Protoplasts can be produced from suspension cultures, callus tissues or intact tissues by enzymatic treatment with: A. proteolytic enzymes B. cellulotyic enzymes C. amilolytic enzymes D. endonucleorestiction enzymes E. ligation enzymes 59. In angiosperm, the endosperm is A. haploid B. triploid C. diploid D. tetraploid E. hexaploid 60. What are the various disadvantages of cross protection? A. Possibility of mutations in inducing mild virus strain B. Possibility of synergism between inducing bacteria and other bacterias C. None of these D. Possibility of synergism between inducing virus and other unrelated virus E. All of the above 61. To obtain plant protoplasts usually use the following enzymes: A. Cellulase, Catalase B. Cellulase, Pectinase C. Catalase, Peroxidase D. Proteinase, Lipase E. Restrictase, Ligase 62. Cytoplast is: A. Plant cells without a cell wall B. Protoplasts without nucleus C. The hybrid cell producing monoclonal antibody D. Heterokaryons E. Plant cells with a cell wall 63. Methods of analysis and selection of somatic hybrids (select the incorrect answer): A. Physiological complementation B. Expression of the foreign gene product C. Genetic complementation D. Biochemical complementation E. Hybridological analysis 64. Heterokaryon – is: A. A cell containing genetically different quality mitochondria B. A cell containing different nucleus C. A cell containing genetically different quality chloroplasts

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D. A cell containing mitochondria from one species and the chloroplast of another type E. A plant cell without nucleos and a cell wall 65. Which cell-based plant technology involves the combining of two cells without cell walls from different species? A. Mutant selection B. Somatic hybridization C. Haploid technology D. Clonal propagation E. Gene engineering 66. Resistant to ion stress cell lines under in vitro conditions can be obtained by follows: A. By electron microscopy methods B. Direct cell selection of resistant cells in media containing toxic concentrations of salts C. Using serological analysis D. By hybridological analysis E. Using the polymerase chain reaction 67. Embryoid is: A. The embryo formed from gametes B. The embryo formed from the somatic cells C. The embryo formed from a zygote D. Axillary meristem of leaves E. The embryo formed from fertilized ovary 68. Direct embryogenesis in vitro is: A. Formation embryoid from callus cells B. Formation embryoid from explant cells C. Formation callus from embryo D. formation embryoid from meristem cells E. Formation embryo in vitro from cultivated cells of explant 69. Somatic hybridization allows: (select the incorrect answer) A. Interbreed phylogenetically distant species B. Obtaining homozygous hybrids C. Obtaining the asymmetric hybrids D. Receive heterozygous hybrids from the extra-nuclear genes E. Eliminate progamy incompatibility 70. Effect of electroporation in Cell engineering is based on the following methodological approach: A. Deep freezing cells B. Fusion non-membrane cell structures C. Transformation genes D. Reaction precipitation E. Agglutination of cells 71. Protoplasts are cultured: A. In a solid agar medium B. in a drop of culture medium C. in liquid nitrogen D. On the filter papers E. On the sterile soil 72. For detect viable protoplast use following dye: A. Methylene orange B. Fluoresceindiacetate (FDA) C. phenolphtalein D. indigo E. Bromide ethidy

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Basics of biotechnology: plant biotechnology 73. The phenomena of somatic cell fusion is: A. Traditional Hybridization B. Asexual hybridization C. Sexual hybridization D. Parasexual hybridization E. Symmetric hybridization 74. The following steps are applied in cell engineering of plants: A. Injection of a foreign gene into the plant DNA B. Isolation and cultivate protoplasts C. Isolation and cultivate the anther D. Cultivation axillary meristem E. The fusion protoplasts derived from different plant species 75. The following manipulations are applied in cell engineering of plants: A. Hybridization of gametes B. cultivation of somatic hybrid cells In vіtro C. Receive recombinant DNA D. Treatment by DMSO E. Reconstructing the cells and create a new type of cell 76. Cell selection is: A. Method of producing hybrid plants B. Selection of cell lines in media containing the selective agent C. Protoplast fusion of the two plant species by electroporation D. Method of production of genetically modified organisms E. Method of obtaining somatic hybrids 77. Which of the following metabolites are implicated in stress tolerance? A. Citrate B. Proline C. Glucose D. Terpenoids E. Auxin 78. Find organism heterozygous for genes A and D: A. AA BB CC dd B. Aa BB Cc Dd C. AA BB CC DD D. AA BB cc dd E. AABbCcDD 79. Note homozygous organism: A. Aa BB CC B. AA BB cc C. Aa BB CC D. Aa BB Cc E. aaBbCc 80. The method that apply for obtaining haploid plants with a male genotype A. Ginogenesis in vitro B. Anther culture in vitro C. «Bulbosum»-method D. Ovary culture in vitro E. Partenogenesis 81. The method that apply for obtaining haploid plants with a female genotype A. Androgenesis in vitro B. Ovary culture in vitro

Quiz

181 C. «Bulbosum»-method D. Anther culture in vitro E. Somatic hybridisation 82. The method that apply for obtaining haploid plants in vitro A. Protoplast culture B. Anther culture in vitro C. interspecific hybridization D. Gene engineering E. Cell selection 83. For obtaining doubled haploid plants (for dihaploidization) use: A. prolin B. colchicine C. PEG D. DMSO E. pectinaza 84. Which of the following is true about Agrobacterium tumefaciens? A. It causes crown gall disease of plants B. It infect monocots C. It infect monocots and dicots D. It infects dicotyledonous angiosperms E. none of the above 85. Which of the following is commonly used as vector? A. Fungi B. Artificial chromosome C. tRNA D. Yeast E. Plasmid 86. Vectors are A. Molecules that help in replication B. Molecules that are able to covalently bond to and carry foreign DNA into cells C. Molecules that protect host cells from invasion by foreign DNA D. Enzymes that linked DNA parts E. Molecules that degrade nucleic acids 87. Which of the following is commonly used as vector? A. Polinucleotide B. mtDNA C. Fungi D. Yeast E. Nucleus 88. Which of the following enzyme is not used to covalently bond foreign DNA to a vector plasmid? A. DNA ligase B. DNA polymerase C. Ligase D. DNA helicase E. all of the above 89. Transgenic plants: A. Received by cell engineering B. contain foreign genes in their cells C. are plants that differ in geographical locations D. are genetic copies of donor plants E. Plants that received by cell selection

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90. Which of the following genes can be used for making resistances against viral infection? A. Yeast gene B. Genes for capsid protein C. Genes of resistant to bacterial diseases D. Gene for nucleocapsid protein E. Satellite RNA 91. Insect resistance in the transgenic plant has been achieved by A. Treatment by antibiotic B. Transferring genes for Bt toxins C. Transferring genes of resistance to antibiotics D. Tsferring genes of resistance to fungicids E. Transferring genes for protease inhibitors 92. Which of the following is a characteristic of a transgenic crop? A. No resistant to herbicide B. herbicide resistance C. Haven’t resistance to abiotic factors D. Sensibility to fungi diseases E. Sensibility to bacterial diseases 93. Genetic engineering is: A. Method of production of new organisms by recombination of paternal and maternal genes of one species B. Method as a result of its application is genetically modified organism / product C. Method of production of new organisms by Monohybrid breeding D. Method of obtaining new organisms through polyhybrid breeding E. Method of obtaining somatic hybrids 94. Opine synthesis is the property A. of normal plant cells B. conferred to plant cells when it transformed by Agrobacterium tumefaciens C. of necrosed plant cells D. determined by the bacteria Escherihia coli E. determined by the bacteria Agrobacterium tumefaciens 95. Agrobacterium tumefaciens is A. a fungi that is used to produce antibiotics in large amounts B. a bacterium that can be used to introduce DNA into plants C. a disease in humans that causes loss of weight D. an yeast that can be used to produce organic acid E. a bacterium that infects human 96. The controversy regarding the use of Bt corn is that it A. Can contaminate air B. is a potential allergen to humans C. can contaminate groundwater D. Can contaminate soil E. Can contaminate environment 97. The plasmid is: A. Circular protozoan DNA – plasmodium B. One of the genetic elements of bacteria C. Cytoplasmic gene of eukaryotic cells D. Viral DNA E. Circular bacterial DNA 98. «Sticky and blunt ends» in DNA fragments appear during the action of the enzyme: A. Ligase

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Quiz B. restriction enzyme C. Transferase D. Isomerase E. Catalase 99. For gene probes to be useful they must A. be small size B. be large enough to contain gene-specific sequences C. not be labeled D. be thermolability E. none of the above 100. Enzymes that recognize and cleave specific 4 to 8 base pair sequences of DNA are A. DNA ligase B. restriction endonucleases C. helicases D. DNA gyrase E. Tag-polimerase

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CONTENT INTRODUCTION........................................................................................................................... 5 Part I. HISTORY OF BIOTECHNOLOGY..................................................................................... 9 Chapter 1.1. History of plant biotechnology.................................................................................. 10 Chapter 1.2. The development of biotechnology researches in Kazakhstan.................................. 14 Part II. TECHNIQUES AND METHODS OF CULTIVATION PLANT TISSUE IN VITRO...................................................................................................................................... 19 Chapter 2.1. Aseptic technique. Tissue culture technique.............................................................. 20 Chapter 2.2. Methods of cultivation plant tissue in vitro............................................................... 33 Chapter 2.3. Biology of cultured plant cells.................................................................................. 40 Part III. INDUSTRIAL AND AGRICULTURAL APPLICATIONS OF IN VITRO CULTURE.............................................................................................................. 51 Chapter 3.1. Production of secondary metabolites in plant cell culture........................................ 52 Chapter 3.2. Micropropagation in vitro ......................................................................................... 71 Chapter 3.3. Cell engineering........................................................................................................ 91 Chapter 3.4. Cell selection........................................................................................................... 103 Chapter 3.5. Embryo and endosperm culture .............................................................................. 109 Chapter 3.6. Haploid technology.................................................................................................. 114 Chapter 3.7. Genetic engineering................................................................................................. 130 Chapter 3.8. Cryoconservation .................................................................................................... 156 GLOSSARY................................................................................................................................. 164 THE TASKS FOR STUDENTS’ INDEPENDENT WORKS..................................................... 166 APPENDIX.................................................................................................................................. 167 QUIZ............................................................................................................................................ 171 BIBLIOGRAPHY........................................................................................................................ 184

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Еducational issue

Turasheva Svetlana Kazbekovna

BASICS OF BIOTECHNOLOGY: PLANT BIOTECHNOLOGY Textbook

Typesetting and cover design: G. Kaliyeva Cover design used photos from sites www.Thinkstockphotos.com.com

IB No.10241

Signed for publishing 08.12.2016. Format 70x100 1/12. Offset paper. Digital printing. Volume 15.83 printer’s sheet. 500 copies. Order No.5717. 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.