The latest technologies in agriculture and plant sciences: Improved techniques, methods, and yields 1774694158, 9781774694152

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

THE LATEST TECHNOLOGIES IN AGRICULTURE AND PLANT SCIENCES: IMPROVED TECHNIQUES, METHODS, AND YIELDS

Hazem Shawky Fouda

www.delvepublishing.com

The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields Hazem Shawky Fouda Delve Publishing 224 Shoreacres Road Burlington, ON L7L 2H2 Canada www.delvepublishing.com Email: [email protected] e-book Edition 2023 ISBN: 978-1-77469-682-8 (e-book)

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ABOUT THE AUTHOR

Dr. Hazem Shawky Fouda has a PhD. In Agriculture Sciences, obtained his PhD. from the Faculty of Agriculture, Alexandria University, 2008, MSc. In Agriculture Sciences from the Faculty of Agriculture, Alexandria University in 2004, Post-Grade Diploma in Cotton, 2001, BSc. in Agriculture Sciences, from the Faculty of Agriculture, Alexandria University, 1997, worked in Cotton Arbitration & Testing General Organization (CATGO) from 1999 till 2018. Was working in the International Cotton Training Center (ICTC) – Cotton Arbitration & Testing General Organization (CATGO) from 2000 till 2015, as a Lecturer & Classer’s Trainer for Egyptian and foreigner classers, technicians, ginners, spinners & traders in all cotton aspects. Besides that he was an editor and active member in the Research & Translation Committee, participating in issuing weekly, monthly and annually issues about the international & local cotton market including price trends and direction, recent developments & researches concerning cotton production, protection, harvesting, ginning, fiber testing, spinning & weaving since its foundation in 2000 till 2014 and from 2015 till 2018 he worked as an inspector, since 2018 till present works as a consultant.

TABLE OF CONTENTS

List of Figures ........................................................................................................xi Preface........................................................................ .......................................xiii Introduction .............................................................................................. 1 Chapter 1

Introduction to Advances in Plant and Agricultural Research ................... 3 1.1 Introduction ......................................................................................... 4 1.2 Technology .......................................................................................... 6 1.3 Artificial Intelligence ............................................................................ 7 1.4 Genomics .......................................................................................... 11 1.5 IPR..................................................................................................... 14 1.6 Precision Agriculture.......................................................................... 14 1.7 Bioactive Compounds ........................................................................ 17 1.8 Markers ............................................................................................. 21 1.9 Pyrosequencing ................................................................................. 23 1.10 Plant Breeding ................................................................................. 26

Chapter 2

Advances in Plant Research..................................................................... 29 2.1 Introduction ....................................................................................... 30 2.2 Imaging Technology ........................................................................... 31 2.3 Microbial Interactions ........................................................................ 33 2.4 Plant Growth- Promoting Bacteria ..................................................... 35 2.5 Genetic Code .................................................................................... 37 2.6 Pests and Diseases ............................................................................. 39 2.7 Sensitivity .......................................................................................... 40 2.8 Stressors............................................................................................. 42 2.9 Transgenic Crops................................................................................ 44 2.10 Proteomics....................................................................................... 45

Chapter 3

Genomics in Plant Research .................................................................... 51 3.1 Introduction ....................................................................................... 52 3.2 Model Organisms .............................................................................. 54 3.3 Molecular Markers............................................................................. 54 3.4 DNA Sequencing Technology ............................................................ 56 3.5 Exome Sequencing ............................................................................ 58 3.6 Greenphyldb ..................................................................................... 61 3.7 Phylogenomics .................................................................................. 62 3.8 Plant GDB ......................................................................................... 66 3.9 Plaza ................................................................................................. 68 3.10 Weeds ............................................................................................. 72 3.11 Herbicides ....................................................................................... 74 3.12 Weed Management.......................................................................... 76 3.13 Mutation Breeding ........................................................................... 78 3.14 Allergies .......................................................................................... 80

Chapter 4

Recombinant DNA Technology and Plants.............................................. 83 4.1 Introduction ....................................................................................... 84 4.2 Pesticide Resistance ........................................................................... 85 4.3 Biosafety ........................................................................................... 87 4.4 Gene Transfer..................................................................................... 90 4.5 Crop Enhancement ............................................................................ 91 4.6 Resistance.......................................................................................... 93 4.7 Markers ............................................................................................. 95 4.8 Genetic Engineering .......................................................................... 95 4.9 Synthetic Chemicals .......................................................................... 96 4.10 Agrobacterium ................................................................................. 97 4.11 Biotechnology ............................................................................... 101

Chapter 5

Microarray Technology ......................................................................... 107 5.1 Introduction ..................................................................................... 108 5.2 Microarrays...................................................................................... 109 5.3 Arabidopsis ...................................................................................... 113 5.4 Cdna Microarray .............................................................................. 115 5.5 Cotton Fibers ................................................................................... 119 5.6 Molecular Analysis .......................................................................... 120 viii

5.7 Transcript Profiling ........................................................................... 121 5.8 GM Crops ........................................................................................ 124 Chapter 6

Drought Resistant Plants ....................................................................... 127 6.1 Introduction ..................................................................................... 128 6.2 Drought ........................................................................................... 128 6.3 Stomata ........................................................................................... 130 6.4 Drought Resistant Crops .................................................................. 133 6.5 GM Technology ............................................................................... 135 6.6 Soybean........................................................................................... 136 6.7 Drought Stress ................................................................................. 137 6.8 Rainfall ............................................................................................ 139

Chapter 7

Disease Resistant Plants ........................................................................ 145 7.1 Introduction ..................................................................................... 146 7.2 Weeds ............................................................................................. 146 7.3 Growth and Reproduction ............................................................... 151 7.4 QTL ................................................................................................. 153 7.5 RNAi ............................................................................................... 156 7.6 Breeding Programs........................................................................... 158

Chapter 8

Sustainable Agriculture ......................................................................... 167 8.1 Introduction ..................................................................................... 168 8.2 Manures .......................................................................................... 169 8.3 Biochar ............................................................................................ 171 8.4 Drip Irrigation .................................................................................. 176 8.5 Conservation ................................................................................... 179 8.6 Ecological Security .......................................................................... 182

Chapter 9

Climate Resilient Agriculture ................................................................ 187 9.1 Introduction ..................................................................................... 188 9.2 Green House Gases (GHG).............................................................. 189 9.3 Climate Change ............................................................................... 190 9.4 Sustainable Agriculture .................................................................... 191 9.5 Water Availability ............................................................................ 194 9.6 Agricultural Productivity .................................................................. 199

ix

Chapter 10 Plant Breeding ....................................................................................... 211 10.1 Introduction ................................................................................... 212 10.2 Potatoes ......................................................................................... 214 10.3 Tomato........................................................................................... 216 10.4 Modern Genetics ........................................................................... 219 10.5 X-Rays ........................................................................................... 222 10.6 Transgenics .................................................................................... 225 References............................................................................................. 227 Index ..................................................................................................... 241

x

LIST OF FIGURES

Figure 1.1 Agriculture Figure 1.2 Drones Figure 1.3 Genomic technologies Figure 1.4 Precision Agriculture Figure 1.5 Bioactive compounds Figure 1.6 Molecular markers Figure 1.7 Pyrosequencing Figure 1.8 Small interfering RNAs Figure 1.9 Molecular plant breeding Figure 2.1 Imaging technology Figure 2.2 Microbial interactions with plants Figure 2.3 Plant growth-promoting bacteria Figure 2.4 Genetic code Figure 3.1 Retrotransposons Figure 3.2 Drosophila melanogaster Figure 3.3 DNA sequencing technology Figure 3.4 Exome sequencing Figure 3.5 GreenPhylDB Figure 3.6 Phylogenomics Figure 3.7 CoGepedia Figure 3.8 PLAZA Figure 3.9 Herbicides Figure 3.10 Genome editing techniques Figure 3.11 Mutation breeding Figure 4.1 Flavr Savr Figure 4.2 Biosafety frameworks Figure 4.3 Restriction endo-nucleases

Figure 4.4 Agrobacterium-mediated plant transformation Figure 5.1 Microarrays Figure 5.2 GeneChip high-density oligonucleotide probe arrays Figure 5.3 cDNA microarray Figure 6.1 Drought-resistant crop Figure 6.2 Drought-resistant crop Figure 7.1 Abiotic stressors Figure 7.2 Drought and heat stress Figure 7.3 Salinity and pathogen stress Figure 7.4 Quantitative trait loci Figure 7.5 CRISPR–Cas9 system Figure 7.6 Genome mapping Figure 8.1 Biochar Figure 8.2 Organic fruit farming Figure 8.3 Drip irrigation Figure 8.4 Ecological security Figure 9.1 Green House Gases Figure 9.2 Climate change Figure 9.3 Sustainable agriculture Figure 9.4 Destroying ozone (O3) Figure 9.5 Hydrological cycle Figure 9.6 Receding glaciers Figure 9.7 Cold-water fisheries Figure 9.8 Climate Smart Agriculture Figure 9.9 Economic growth theory Figure 10.1 Malnutrition Figure 10.2 Potatoes (GM) Figure 10.3 Tomato (GM) Figure 10.4 Modern landrace combinations Figure 10.5 Modern genetics Figure 10.6 Mendel Figure 10.7 Mendel Experiments Figure 10.8 X-rays Figure 10.9 Extranuclear mutations xii

PREFACE

Agriculture is considered one of the essential elements that humans relied on for several centuries. Though the traditional approaches in agriculture are producing limited results and there is a need to include latest technologies that can enhance yields for the growing population. Drones Agricultural research is an important sector of science and technology that is to be improved on a regular basis. As the technology progresses, there is always scope for improvement and this is evident in agricultural and plant research. Artificial intelligence and drones are playing a crucial role in the management of crops. Genetic Engineering of plant species is yet another improvement in the farming sector. Though some environmental problems surfaced, they are effectively handled with the technology at hand. The intensification of agriculture always called for extreme but manageable measures and this need is satisfied by the progress in agricultural research all over the world. Genomics is another important area that helped many farmers worldwide in decision making about their cropping patterns and farming choices. The disease resistant varieties are being grown in several countries with less problems. Most of the crop yields are positive due to improvement in their genotypes. Food safety is always a concern among populations that are solely connected with the traditional routes of farming. Due to severe economic shifts, it has become obvious that the growers need to include these advancements in their farming business. Precision agriculture is one such an attempt that can lower the risk of ineffective farming with technology. Though some of the issues are always on the table, it is the advancements in plant research that determine and ensure food security across the continents. Bioactive compounds have become popular these days due to emerging diseases previously unknown to humans. This is due to the fact that these compounds work as a medicine with limited side effects. This book emphasizes the advancements of plant and agricultural research. Special focus has been made in illustrating genomics, rDNA technology and Microarray technology. The topics of disease and drought resistance in plants are explained in detail. The role of sustainable agriculture and the importance of climate resilient agriculture is illustrated. The plant breeding techniques with a special focus on contemporary research tools are explained.

This book can help students who are interested in pursuing courses in plant and agricultural sciences. It is expected that the readers may have minimal knowledge in biology and agriculture to understand some of the topics in this book better.

INTRODUCTION

Agriculture is an integrated field that allows us to experiment in any area. It plays a crucial role in human life and its existence. There are many molecular breeding techniques that can be used in the improvement of agriculture. There has been improvement in the transformation techniques to enhance the phenotype of the crops. Agrobacterium mediated transformation is one such important method that can be used in crop improvement. Plant molecular analysis has led to the development of plant genomics and this has raised the research standards in silico. This has enabled several researchers to collaborate real time and get the benefits of technology. Recombinant technology is another important area that gave use genetically modified plants with better yields. This technology is satisfying the needs of the ever-growing population worldwide. This technology allowed us to use both natural combinations and traditional breeding techniques for the enhancement of crop traits. Microarray technology is another essential technology that can be used to analyze and store huge amounts of biological data. This can be considered as an important breakthrough in plant and agricultural research. The data stored can be shared with several scientists all over the globe via the internet and some of the databases are free to use. The high yielding varieties can be observed in the drought and disease resistant plants and this comes with the advancements in genetic engineering and associated tools. Agriculture is meant to satisfy the food demands of the present generation and should also help the future generations in the same manner and this purpose can be satisfied by practicing sustainable methods of agriculture. Though

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

technology improves the quality of life, it is essential to follow sustainable agriculture. Climate change is decreasing agricultural yields and this is an important area that needs to be addressed. It left us with limited options and we cannot cope up with the detrimental effects of climate change with the technology we have. It is important to opt for climate resilient agriculture to keep the cycle going forward. We are in need of integrated technological solutions that can enhance crop yields with limited effects on climate. This can be done with the use of environmentally friendly tools and techniques frequently. Plant breeding exists in almost all the areas of crop science and research and this can be treated as an important approach towards improvement of the crop breeds and yields.

CHAPTER

1

INTRODUCTION TO ADVANCES IN PLANT AND AGRICULTURAL RESEARCH

CONTENTS 1.1 Introduction ......................................................................................... 4 1.2 Technology .......................................................................................... 6 1.3 Artificial Intelligence ............................................................................ 7 1.4 Genomics .......................................................................................... 11 1.5 IPR..................................................................................................... 14 1.6 Precision Agriculture.......................................................................... 14 1.7 Bioactive Compounds ........................................................................ 17 1.8 Markers ............................................................................................. 21 1.9 Pyrosequencing ................................................................................. 23 1.10 Plant Breeding ................................................................................. 26

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

1.1 INTRODUCTION In today’s world, technology has played a significant role in how we live, communicate, travel, and interact. The rapid advancement of technology is having a significant impact on all industries, particularly agriculture. Most developing economies rely heavily on agriculture. Agriculture is undertaking a major transition as a result of digitalization today. Robots, drones, and machine learning are among the new technologies that are meant to assist farmers increase productivity and yield (Roldán et al., 2017). Emerging technologies have already shown to be an important factor in agriculture’s long-term viability and profitability. For most countries, agriculture has been the most important industry for economic development. It includes everything from tiny enterprises to giant corporations to multinational corporations. Agriculture encompasses more than just farming and ranching, which are its primary sources of income. Agribusiness is also the industry that converts agricultural commodities into consumer goods (Macrae, Henning, & Hill, 1993). Food processing, packaging, shipping, retail, preparation, and consumption at home all play a role in getting goods to consumers. Agriculture has gone through a number of changes. Mechanization has recently changed farming by replacing horses with tractors. Today, technology is being accepted at an increasing rate, to the point where it has become an unavoidable requirement for every farmer, particularly in wealthy countries. There is insufficient land on the planet to support today’s global population utilizing yesterday’s technologies.

Figure 1.1: Agriculture. Source: https://www.croplife.com/management/get-ready-for-agriculture-3-0/

Introduction to advances in Plant and Agricultural Research

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Agriculture, (Figure 1.1) like every other element of our modern life, is being influenced by technology. In its most elementary form, technology can be defined as a set of skills that enable us to create items and machines to meet our requirements. Agriculture technology, commonly known as AgTech, has revolutionized the business in recent years. Farming technology is assisting farmers in increasing efficiency and production. Harvest automation, autonomous tractors, planting and weeding, and drones are just a few of the primary technologies used by farms. Technology is altering the field of livestock management, which operates poultry farms, dairy farms, cattle ranches, and other livestock-related agribusinesses, according to recent developments. Livestock provides us with essential renewable natural resources that we require on a daily basis. In the coming years, emerging technologies have the potential to completely revolutionize the agricultural environment. On a small and large scale, emerging technologies ranging from robots to machine language have totally altered modern agriculture. They'll take farming to new heights. Farms are finding it cost-effective to strategically deploy sensors around their land in order to reap a variety of benefits. Farmers can observe their crops from anywhere in the world thanks to sensors and image recognition technology. Agriculture benefits from sensors since they allow for real-time traceability (Ko, Kwak, & Song, 2014). They would provide a real-time picture of the current state of a farm, forest, or body of water. They assist in the management and monitoring of livestock and crop production. They also contribute to the farm's environmental sustainability by conserving water, controlling erosion, and lowering fertilizer levels in local rivers and lakes. Vertical farming has made its way to the city. Indoor vertical farming is the process of producing vegetables in a closed and regulated environment, layered one on top of the other (Benke & Tomkins, 2017). Artificial lights are utilized in place of natural sunlight in the growing process. Within a decade, vertical farming will not only be technically possible, but also commercially viable. It is a type of urban agriculture that produces food in layers that are vertically stacked. It isn't only restricted to metropolitan settings. It can be applied to any condition in order to make better use of available land. Vertical farming can boost agricultural yields, overcome land constraints, and lessen farming's environmental impact. This is often referred to as "ecological agriculture” (Kiley-Worthington, 1981). Organic farming is environmentally benign and does not cause harm to the environment. It is thought to be a greater alternative to chemical-based farming. Organic farming, often known as organic agriculture, is a type of farming that

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

avoids the use of artificial fertilizers and pesticides to a considerable extent (Leifeld, 2012). It's a system that keeps soils, ecosystems, and humans healthy. It bans genetically modified and animal cloned items, as well as industrial herbicides and fertilizers. It has a one-of-a-kind place in the world's agricultural systems. Organic farming's major purpose is to create businesses that are both sustainable and environmentally friendly. Crop rotations, crop diversification, crop residues, animal manures, legumes, green manures, off-farm organic wastes, bio-fertilizers, and mechanized cultivation are all encouraged in organic farming.

1.2 TECHNOLOGY Technology has continuously played an important part in how we live our lives. It has an impact on how we interact, travel, and even eat. Agricultural technological advancements are transforming the way we grow food and manage its production. In agriculture, the ultimate purpose of technology is to increase yields, shorten harvest periods, and reduce expenses and environmental effects. Emerging technologies have a significant impact not just on small-scale farming, but also on the large-scale food distribution system. As new technology is integrated into modern farming, output improves and supply chain management becomes easier. Automation is the actual emphasis of agricultural technological advancements, and it is already in use on farms all over the world. Modern automation has gone a long way since the days of mechanical timers, and it now requires relatively little human intervention. From seed to sale, systems are being developed to monitor, feed, and harvest crops. Automation incorporates a wide range of sensors, computers, feeding mechanisms, and, of course, robots. Complete automation is a nearly self-contained system that can manage all of the farm's day-to-day operations (Edan, Han, & Kondo, 2009; Jha, Doshi, Patel, & Shah, 2019). It virtually eliminates the need for human staffing, which can be beneficial or detrimental depending on your perspective. A huge network of sensors is one of automation's most valuable assets. The backbone of future automated farming is likely to be crop, air, and soil sensors. While today's sensors can determine fundamental parameters such as

Introduction to advances in Plant and Agricultural Research

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pH, sensors of the future will be able to do much more. Not only will soil and crop sensors be able to read nutrient levels and EC, but they will also be able to undertake more extensive analysis utilizing infrared, electromagnetic, and acoustic methods. More data allows crop growers to break from traditional feed plans and embrace a more as-needed approach, saving time and money. Equipment sensors will also be utilized to convey information from smart technology to a central control unit in order to warn of potential mechanical breakdowns. A sensor will be constantly interacting with a centrally managed artificial intelligence system for almost any metric that can be measured. Artificial intelligence, or AI, will improve the adaptability of automated systems to changing environments. Furthermore, AI agricultural systems will be capable of analyzing, diagnosing, and prescribing suitable crop treatment regimens at a level of efficiency unequalled by humans. When we talk about AI, we're not talking about The Terminator. For the time being, AI is merely a sophisticated computer system that can adapt to new inputs. Agriculture AI technologies help farmers better coordinate mechanical systems, establish feed plans, identify sickness, and boost yields and productivity. Drones are one of the more intriguing technologies that AI will coordinate in agriculture.

1.3 ARTIFICIAL INTELLIGENCE Drones (Figure 1.2) are becoming more common and are presently controlled by the Federal Aviation Administration (FAA) in the United States (Puri, Nayyar, & Raja, 2017). They appear to have far more practical applications than anyone could have expected. Farmers may use a surveillance drone to fly over acres of crops and gather images and video in real-time. In the winter, they can also be used to monitor crop temperatures. Drones are currently being utilized on farms not only for observation but also for application. Drones that spray crops are one of the newest types of unmanned aerial vehicles (UAVs) that can be found on today’s farms. Drones that spray pesticides or fertilizers on crops are unaffected by difficult terrain (Adão et al., 2017). Modern farming is also being influenced by autonomous robots.

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

Figure 1.2: Drones. Source: india/

https://archive.factordaily.com/drones-for-precision-agriculture-in-

Farms of the future may not require people to cultivate crops at all. Drone-like autonomous robots are now being employed to undertake duties such as seed planting, crop tending, and harvesting. Drones are beginning to appear on the market in a number of configurations. Drone tractors, microseed planters, and weed-eating robots are all progressively making their way into the agricultural mainstream. The concept is to construct a group of autonomous robots controlled by a central AI that eliminates human error and adjusts to changing conditions to maximize yields, reduce time, and boost efficiency. Farming devices that are automated work similarly to self-driving cars. GPS technology, which accurately controls their locations and functions, keeps them in sync. Precision agriculture is a larger trend in farming that includes the use of GPS technology. Satellite farming and site-specific crop management are two terms used to describe precision agriculture (SSCM)(Ahmad & Mahdi, 2018). Precision agriculture combines the most precise topography data with sensor data on the ground to produce a precise picture of crop requirements. It is divided into four stages: data collection, variable analysis, strategy development, and practice implementation. Finally, precision agriculture aims to maximize efficiency by analyzing data precisely and using cutting-edge technologies.

Introduction to advances in Plant and Agricultural Research

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Changes in the global economy are converting American agriculture into a mission to improve public health, social well-being, and the environment in addition to efficient food and fiber production. Recent technological advancements will make it easier for agriculture to realize its full potential in terms of providing a wide range of societal benefits. However, in order for that vision to become a reality, the agricultural research system must seize new possibilities and form new collaborations, as well as possess the leadership necessary to meet agriculture’s complex and diversified duties in the twenty-first century. Food and fiber production has been the primary public need addressed by the United States agriculture during the previous century, and agricultural research has centered on increasing the productivity of agronomical important crops and livestock. The productivity of that endeavor can be seen in such productivity gains as the tripling of corn yields over the last 50 years and a 2.5-fold increase in overall productivity, as well as the low average percentage of consumer income spent on food in the United States (Ning & Reed, 1995)outflow, and reinvestment. Cultural linkages, trading blocs, host market size, tax considerations, exchange differentials, and host market growth rates are found to be significant determinants of DFI in food manufacturing. Wage rate differentials were found to be important in the position and reinvested equations, but not in the outflow equation. Thus, cheap labor may not be as important in attracting DFI as in the past. © 1995 by John Wiley & Sons, Inc.”,”container-title”:”Agribusiness”,”D OI”:”10.1002/1520-6297(199501/02. Those increases have been fueled by scientific breakthroughs in plant and animal genetics, plant and animal nutrition, and livestock health, as well as the practical application of those breakthroughs in production systems. Simultaneously, significant advances in public opinion have widened the scope of agricultural research to encompass environmental, human health, and community aims. Agriculture’s link to the food and fiber system, the environment, and the fabric of American society will be altered by changing public attitudes and wants, which will provide new market opportunities. Agriculture’s capacities will be revolutionized as the speed of scientific discovery and technological advancement accelerates. The link between agriculture and public health has never been more obvious, necessary, or promising. The increased public concern in food safety indicates an understanding of this connection. Foodborne disease is becoming more common in the United States. Furthermore, due to the rising percentage of the US population over 65, a growing number of people infected with HIV, and a growing number of people receiving bone

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

marrow or organ transplants, as well as patients receiving chemotherapy or immunosuppressive drugs, a growing percentage of the populace is becoming vulnerable to opportunistic infections, including foodborne pathogens (Shiels et al., 2011)an increasing number of HIV-infected people are at risk of nonAIDS-defining cancers that typically occur at older ages. We estimated the annual number of cancers in the HIV-infected population, both with and without AIDS, in the United States.Incidence rates for individual cancer types were obtained from the HIV/AIDS Cancer Match Study by linking 15 HIV and cancer registries in the United States. Estimated counts of the US HIV-infected and AIDS populations were obtained from Centers for Disease Control and Prevention surveillance data. We obtained estimated counts of AIDS-defining (ie, Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. Increased mobility of animals, people, and products into the food system is introducing new and unknown dangers. Some animal production methods, such as those with larger animal densities and mechanized systems that disseminate food, water, and other inputs and outputs, may increase human exposure to infectious disease, according to epidemiologic studies. Because of problems with food security, and the fragility of our agricultural resources, public awareness of food safety is at an all-time high. This sensitivity is part of a bigger trend in which people are becoming more interested in where their food comes from. Several well-publicized foodborne pathogen outbreaks have raised concerns about disease origins and mitigation. The discovery of genetically modified corn in human food products that have been approved for human consumption has raised concerns about food traceability and accountability. These and other factors have contributed to the explosive growth in consumer demand for organic and low-input agricultural goods during the last decade. Another significant shift is taking place in American society’s perception of the relationship between agriculture and the environment. Through the twentieth century, a number of public policies were established in an attempt to mitigate the negative environmental effects of agricultural intensification and widespread pesticide and fertilizer use. The public, on the other hand, is now demanding agriculture to go further and provide environmental advantages. Lands are projected to play a larger role in supplying clean water, mitigating global climate change, protecting biological diversity, and

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maintaining rural amenities like open space and recreational activities. Indeed, in some locations, the national demand for environmental and recreational services from the land is likely to overtake food needs. There is also a higher level of public awareness and concern about global environmental change and concerns, such as natural-resource depletion, desertification, climate change, and biodiversity loss.

1.4 GENOMICS The use of genomic technologies (Figure 1.3) is being employed to investigate the genetic propensity to environmental variables that cause disease in humans and animals. The techniques will also be used to characterize the effects of food chemical components on disease conditions, leading to a better knowledge of the relationship between human health and nutrition. Collaborations between nutritionists and health-care specialists, as well as plant scientists, will increasingly lead to the development of foods that combat diseases and disease predispositions. In the 1990s, advances in nutrition research broadened our understanding of important nutrients and their involvement in disease genesis (Milner, 2000). This paved the way not just for the recent increase in “functional” foods with specialized nutritional properties, but also for future biotechnology-based creation of nutritionally fortified foods. Advances in animal nutrition and genetics have resulted in significant gains in efficiency and quality in the dairy, livestock, poultry, and industries, which are predicted to boost US animal agriculture’s future competitiveness. Animal feeds should be developed to match the genetics of the animals as cloning of farm animals progresses to commercial use, resulting in more effective growth and meat production, augmented compatibility of meat with human nutritional needs, and reduced waste and pollution from animal production facilities. Advances in disease identification and control, such as the introduction of vaccinations and other preventives in feed, will reduce bacterial, fungal, and viral contamination of animal products, boosting production efficiency and food safety even further.

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

Figure 1.3: Genomic technologies. Source: https://www.sciencedirect.com/science/article/abs/pii/ S0167779917300318

Global data on natural resources, as well as the tools for managing, manipulating, and using them, are fast evolving, allowing ideas that could not previously be tested to be explored. Farms, forests, and rangelands will benefit from tools that incorporate spatially referenced and satellitebased remotely sensed data into decision support systems. Large new datasets have provided the foundation for better epidemiologic techniques to understanding, preventing, and reducing disease outbreaks. Massive datasets are now routinely transferred and manipulated among academics. Synthetic data analysis that was before unachievable has become possible thanks to Internet access to various databases at the same time. Advances in the social sciences have led to a more comprehensive knowledge of the social and economic linkages between the agricultural and non-farm sectors. New analytic and modeling approaches, for example, have made it possible to compare the effects of various policy alternatives in addressing a wide range of social goals. Trade and immigration trends may now be studied demographically, economically, and environmentally thanks to the growth of information resources. Modeling approaches for assessing how changing economic conditions affect land-use decisions and ecologic conditions are

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expected to yield significant insights into the determinants of environmental quality and the effectiveness of various policy approaches. Food safety and food acceptance have gained a new human dimension because of the social and communication sciences. The mixing of biomedical and social sciences can be seen in risk assessment, risk communication, consumer education, and human behavior and attitudes. With the introduction of genetically modified crops and animals, the human components of contemporary agriculture and related studies have become even more important. Few contemporary economic shifts have matched the effects of globalization in the last quarter of the twentieth century. In addition to handling their highly productive resource base, American farmers must also respond to changing consumer demands for products and services, as well as manage technology, capital, and labor in internationally integrated marketplaces. Even if global population growth slows, demand for animal products will skyrocket as incomes rise in less-developed nations, creating new market opportunities and global problems for agricultural systems. Agriculture in the United States will need to maintain its technological leadership and long-term productivity increases to remain competitive in this global economy. This will necessitate the development of new and more complex information management technologies and systems. Advances in information technology and genomic sciences have opened up new research opportunities to help agriculture provide higher-quality products and services. However, as the global nature of possible threats posed by new technology becomes better known, a more thorough assessment of such risks is required. The global trend of countries exporting and importing an increasing share of products, services, production inputs, and intellectual property will have significant implications for national economies, society, and the environment. In order to offer a solid, scientific basis for policies and programs that address such consequences in the United States, more research is required. Such research must be comprehensive, looking at all of globalization’s implications as well as the environmental, social, and economic trade-offs that policymakers will face. One of the most important concerns that research should address is the relative advantages and costs of investing in various types of research, including societal and environmental research. The task of correcting policy distortions that bias incentives in global agriculture is a second concern.

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

1.5 IPR Improved knowledge of how global changes in IPR laws affect the public research agenda is a related field of research. Changes in technology, legal judgments, and international agreements have enhanced the return on investment and international spill overs from privately supported agricultural and food research. In agricultural research, partnerships, joint ventures, and other collaborations between public and private institutions are becoming more widespread. Such collaborations increase funding for some types of research and improve the chances of commercialization and application of new technologies, but they also raise questions about whether private-sector interests are playing an excessive role in determining research priorities. Although such concerns are not unique to agriculture, the rapid pace of development in agricultural research institutes and biotechnology poses a number of unanswered difficulties. In order to increase the efficacy and specificity of gene-transfer technologies, the public sector must also invest in research. The development of techniques for modifying plant and animal genomes, the construction of models and systems that integrate basic knowledge about plants and animals into gene selection, and the synthesizing of findings on gene mapping and the protein expression related with quantitative traits are all examples of important research. The current state of knowledge about physiologic systems and metabolic pathways does not allow for precise genetic modifications. Greater precision and predictability are required due to the expenses of genetic alterations, particularly in animals. To build quantitative and dynamic models of interactions in physiologic and metabolic systems, collaboration between experimentalists and modelers will be critical; this will allow scientists to make particular changes and better comprehend the ramifications for the entire organism. Finally, the application of genomics-based techniques to environmental challenges is unlikely to be a high commercial priority, thus it should be included in the public sector’s portfolio. Advances in agricultural genomics as a result of the abovementioned research will create new information resources and needs, hence expanding the use of bioinformatics in agriculture for acquiring, processing, storing, distributing, analyzing, and interpreting biologic data.

1.6 PRECISION AGRICULTURE Precision agriculture (Figure 1.4) is another cutting-edge technology that has the potential to boost productivity while also benefiting the environment.

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Tracking productivity and adapting inputs to fit the specific demands of sub acre areas in individual fields are part of this spatially explicit approach to crop management. Precision agriculture technology has advanced faster than its practical implementation in recent years. We need practical decisionmaking tools that allow farmers to alter the timing and quantity of seed, fertilizer, water, and pesticides to maximize productivity while minimizing waste and negative environmental effects. Integrating experimental findings into decision-support systems and underlying models for crop, animal, and environmental systems will require close collaboration among experimental scientists, statisticians, economists, engineers, and systems analysts.

Figure 1.4: Precision Agriculture. Source: https://www.arcweb.com/blog/iot-steps-smart-farming-precision-agriculture

Understanding the whole range of potential repercussions of new technologies and practices pertaining to social, economic, health, environmental, and ethical as well as their worldwide implications, is critical for quality research and technology transfer. New technologies frequently hold great promise for improving people’s lives. They do, however, present critical considerations concerning environmental and health dangers,

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reward and risk distribution, and public principles and ethics. Exploring such queries early in the R&D process will help to focus technological development efforts on those that are most likely to benefit the public. The development of genetically modified food has brought new questions about the proper amount of health and environmental scrutiny, product labeling, and public communication. Differences in attitudes and values among different parts of society, as well as among scientists with varying levels of competence, have been highlighted in public discourse. Similar challenges will arise as new technology and methods emerge. The usage of recombinant bovine somatotropin (Growth hormone) in dairy cattle, the development of antibiotic resistance as a result of antimicrobial use in the livestock and dairy industries are some examples. Because research on transgenic crops has far outpaced research on pleiotropic and other unintended consequences, there is widespread public and scientific support for establishing a government-sponsored program to investigate queries about food allergens and toxicants that are improbable to be pursued by the private sector.

Figure 1.5: Bioactive compounds. Source: https://www.researchgate.net/publication/349348618_Natural_Bioactive_Compounds_Useful_in_Clinical_Management_of_Metabolic_Syndrome/ figures

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The development of an animal model of food allergenicity in humans, for example, would be accelerated by a vigorous federally financed study. Once these questions are answered, it may be feasible to pinpoint the mechanisms by which certain proteins induce allergies or harmful consequences, as well as devise novel ways to mitigate the risk associated with these proteins. Developing biotechnological ways to inactivate allergic or harmful chemicals in meals could be one of the strategies. In an increasingly integrated global economy, scientific advancements offer new options to control plant and animal health. They include novel epidemiology, risk assessment, and risk management methods to better comprehend the threats posed by wildlife or growing international plant and animal trade. Using genetic approaches to improve disease resistance in plants and animals could result in significant savings in processing and manufacturing costs, as well as a reduction in the usage of antibiotics in animal production. Basic research on biotechnology applications will be required for such applied research.

1.7 BIOACTIVE COMPOUNDS Carotenoids, flavonoids, plant sterols, omega-3 fatty acids, allyl and diallyl sulphides, indoles, and phenolic acids are examples of bioactive components found naturally in many foods, particularly fruits and vegetables(Rice-Evans & Miller, 1996). These food components (Figure 1.5) require a scientific understanding of their chemistry, metabolism, and health impacts. In order to estimate and track dietary intakes, it is also necessary to quantify the amounts of these components in foods and incorporate the information into food-composition databases. The Agricultural Research Service should keep putting together databases on carotenoids, flavonoids, and other bioactive chemicals. Human genetics-based nutrition research will lay the groundwork for better understanding the metabolic fate of nutrients and the biochemical roles of dietary components such as macronutrients, vitamins, minerals, bioactive components, and pharmacologic agents. It will also explain how and why people’s requirements for and utilization of specific food components differ. Disease prevention and reducing exposure to physiologically hazardous chemicals in plant and animal products are major applications of this knowledge. The genetic underpinnings of such variance are unknown. Researchers have discovered a small number of particular genes that influence how the human body uses certain food components. Many features of how genes interact with one another and the environment to produce certain nutritional or disease consequences are also unknown.

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Improved knowledge of how genes influence individual dietary status and disease risk could help shape public-health policies in the future. A deeper understanding of how genes affect the body’s storage and use of food calories, for example, would substantially aid attempts to design effective food and nutrition policies aimed at correcting our nation’s obesity epidemic(“Obesity — United States, 1999–2010,” n.d.). Given the likely rapid introduction of transgenic foods into the marketplace in the coming years, and the possibility that some of the intended and unintended compositional changes may disproportionately affect genetically susceptible populations, there is a pressing need to speed up research into gene-bioactive compound interactions in food and dietary supplements. Indeed, there may be value in arranging this research in some way that prioritizes researching genetic interactions with chemicals that are consumed by the biggest number of people or have the highest potential for causing negative outcomes. The public’s perception of the relationship between agriculture and the environment is changing dramatically. Whereas previous public policies aimed to reduce the negative environmental effects of agricultural practices, such as pollution, today’s and tomorrow’s policies will go even further in realizing agriculture’s potential to provide broad environmental benefits, such as clean water, carbon sequestration, and biodiversity conservation. Agricultural research must thus fulfill two roles: establishing environmentally-friendly farming practices and advancing innovative land and natural resource management practices that will improve the environment. Integrating recent conceptual advancements from the ecologic and social sciences can help both pursuits. In the face of rising pressures on global land, water, and genetic resources, as well as global environmental change, agricultural research conducted in the United States can help bring worldwide environmental benefits and inform international environmental agreements. The use of genomics to generate new crop cultivars and livestock strains has the potential to improve environmental compatibility in a variety of ways. Improving plant nutrient usage, as well as the efficiency of nutrient digestion and use in cattle, could reduce fertilizer requirements and keep surplus nitrogen and phosphorus out of waterways. Increasing the efficiency with which main crops use water would lower agricultural water demands. Pesticide and fungicide application rates should be reduced for plants and animals that are more resistant to pests or diseases. Related research should focus on applying biotechnology to environmental concerns as well as assessing related environmental dangers, such as the possible spread of

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novel genes and phenotypes into native microbes, plants, and insects. The transfer of novel genes to microorganisms, in particular, poses a significant threat to agricultural and natural landscape ecology. The future of agricultural research will be difficult to predict. The rising economic, social, and environmental demands on agriculture provide a difficult context in which to plan research. Although these demands increase the potential for increased societal returns from agricultural research, they also place a strain on the system’s ability in a variety of ways. With limited resources, there will be trade-offs between research goals that must be handled. Traditional and new players in the agricultural research system will send contradictory messages. Occasionally, research is required to address trade-offs or perceived trade-offs among the numerous demands placed on the agricultural system. Researchers may be enlisted to help mitigate the unintended consequences of food and agriculture policies. Established agricultural research methodologies and partnerships must develop to meet new demands without losing their unique value. Only by long-term vision, leadership, and political will can those tensions in the research agenda be managed. For the improvement of plant varieties, plant breeders primarily rely on phenotypic selection. However, the introduction of molecular markers made it possible to more directly pick favorable features. Molecular markers, primarily DNA markers, are segments of an organism’s genome that are used to identify a larger portion of the genome. The genome is the totality of genes in an organism, and genomics is the study of the entire genome, which includes both structural and functional aspects of genomics. Restriction fragment length polymorphism was the first DNA-based genetic marker. A molecular marker can be found within a gene of interest or related to a gene that controls a trait of interest. Plant breeding can now utilize easily detectable DNA markers for marker-assisted selection (MAS) to generate superior varieties based on the genotype of plants rather than only the phenotype. Furthermore, DNA markers are employed in germplasm evaluation, genetic diagnostics, phylogenetic analysis, genome organization research, and transformant screening. Marker-assisted backcrossing, marker-assisted recurrent selection, advanced backcross-quantitative trait loci, and gene pyramiding are the most common MAS breeding procedures for introgression of genes from breeding lines or wild relatives to cultivated species. Disease and pest resistance are the primary breeding objectives for which MAS is currently used in crops, followed by yield improvement, quality attributes, and abiotic stress resistance.

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Plant genomics encompasses both scientific and applied aspects, and includes structural and functional genomes. The comprehension of genes to genomes has improved due to the rapid evolution of novel technologies, particularly the introduction of bioinformatics. Genomic analysis, proteomics, transcriptomics, genome sequencing, and metabolomics are examples of ‘omics’ technologies (Morgante & Salamini, 2003). Genomic technology, in particular, is revolutionizing breeding practice, resulting in ‘genomics-assisted breeding,’ which improves the efficiency of breeding for the enhancement of agronomical desirable traits. Plant breeding using genomics technologies is sometimes referred to as molecular breeding. Furthermore, genomics facilitates plant biotechnology by increasing the number of native target genes. Many agronomic features are controlled by genes whose functions are unknown, but which can be mapped and cloned using genetic mapping. In addition, various novel technologies have been developed, such as next-generation sequencing (NGS), Solexa Illumina, applied biosystems (ABI) solid and high-throughput marker genotyping technologies]. Marker-assisted selection (MAS) has been a technology used by agricultural businesses and research institutes to generate new superior varieties, allowing for a breeding program based on plant genotype rather than only phenotypic. Genomics is a technique that uses molecular characterization and whole genome cloning to understand the structure, function, and evolution of genes in order to answer all fundamental biological issues. It involves combining biology, engineering, bioinformatics, and statistics to solve the sequence of a complicated genome and then mining the sequence data in silico to extract biological insights. Fine-scale genetic mapping and efforts to discover the whole DNA sequence of organisms are both parts of this field. Although the sequence of DNA is important in genomics, it is only the beginning of a large-scale genome investigation. The placement/sequence of bases in a DNA strand is known as the genome/DNA sequence. The life cycle of an organism, its developmental program, disease resistance or susceptibility, and how similar various organisms are all revealed by genome sequence information. Genomes are chosen for sequencing based on a variety of factors, including genome size, cost, and relevance to human–animal disease transmission and agriculture, among others.

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Figure 1.6: Molecular markers. Source: https://link.springer.com/chapter/10.1007/978-94-017-9996-6_2

1.8 MARKERS The first step in molecular marker (Figure 1.6) research is the isolation of DNA. Fresh, lyophilized, preserved, or dead material can all be used to extract DNA; however, fresh material is best for extracting high-quality DNA. At the end of each DNA isolation procedure, there are three possible outcomes: (i) There is no DNA; (ii) DNA has been degraded; and (iii) DNA appears as whitish thin threads and that means good-quality DNA or brownish threads (DNA oxidized by pollutants such phenolic chemicals). The most commonly employed hybridization-based DNA marker is RFLP. RFLP markers were originally utilized in 1975 to detect DNA polymorphisms for genetic mapping of adenovirus serotypes with temperature-sensitive

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mutations. These markers were later employed for human genome mapping and, more recently, plant genomics. Restriction enzymes can be used to cut genomic DNA and identify RFLPs. When the DNA is digested, each of these enzymes recognizes a distinct recognition sequence in the genome, which is often palindromic and results in restriction fragments of a specific length. Changes in these sequences, such as point mutations, insertions, and deletions, result in DNA fragments of various sizes and molecular weights. Agarose gel electrophoresis is used to separate these fragments based on their size, and Southern blots with specific probes are used to analyze them. The co-dominant nature of RFLP markers and their great repeatability are their key advantages. However, RFLP analysis has various drawbacks, including the need for relatively high quality and quantity of DNA, the need for probe libraries, the inability to automate the process, the fact that it is arduous and time-consuming, and the need for radioactively labeled probes. RAPD markers are made by using PCR on random DNA segments with single, often 10-mer primers of any nucleotide sequence. The primers attach to complementary sample DNA sequences, and a stretch of DNA is amplified when the primers bind to the sample DNA in close proximity for effective PCR. Gel electrophoresis is used to visualize the DNA amplification results. No previous information of the genome sequence is mandatory because the primers are chosen at random. The genome is supposed to be sampled at random, and this approach is particularly beneficial when testing loci throughout a full genome. The technical simplicity of RAPD markers, as well as their independence from any preceding DNA sequence information, are two of its biggest advantages. While the polymorphisms are simply recognized as the presence or lack of a band of a specific molecular weight, there is no information on heterozygosis, i.e., dominant inheritance, and RAPDs have some repeatability issues. RFLP and RAPD methodologies are combined in the AFLP methodology. It is based on restriction fragment amplification using selective PCR. After digesting genomic DNA, oligonucleotide adapters or specified short oligonucleotide sequences are ligated to both ends of the restriction fragments. Second, the fragments are amplified selectively, with the adapter and restriction site sequences serving as primer binding sites in future PCR operations. Because the ends of the primers extend 1–4 bp into restriction fragments, only those fragments whose ends are fully complementary to the ends of the selective primers are amplified. Finally, gel electrophoresis is used to separate the amplified fragments, which are then seen by autoradiography, silver staining or fluorescence. In comparison to RFLPs, AFLP technology

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allows for the detection of higher amounts of polymorphisms. AFLPs are also more reproducible than RAPDs. However, AFLP is a difficult technique to master because it necessitates the use of polyacrylamide gels for detection, as well as higher equipment investments. Because diploid homozygous candidates create a more intense peak than heterozygous individuals for a single character, scoring AFLP polymorphisms as co-dominant marker locus is achievable in some situations. It will take specialized algorithms and software packages to detect such markers and score them as co-dominant. The technique of establishing the precise order of nucleotides within a DNA molecule is known as DNA sequencing. It refers to any technology that is used to determine the order of four bases in a strand of DNA: adenine, guanine, cytosine, and thymine. Rapid DNA sequencing tools have sped up biological and medical research tremendously. Modern DNA sequencing technology has aided in the sequencing of entire genome sequences because of the high speed of sequencing(Dabney & Meyer, 2012). The primer attaches to the end of the sequenced DNA. The primer-coated DNA is separated into four reaction mixes. All four dNTPs and one of the four dideoxy analogues or ddNTPs are present in each reaction combination. Because the hydroxyl in the dideoxy sugar has been replaced by a hydrogen moiety, the chain cannot be extended further. As a result, the dideoxy analogue serves as a unique chain-termination reagent. Depending on the amount of ddNTP in the mixture, variable-length fragments are generated. Using a tagged radioactive or fluorescent primer is also known.

1.9 PYROSEQUENCING Pyrosequencing (Figure 1.7) detects individual nucleotides added to nascent DNA by using luciferase to emit light, and the combined data is utilized to generate sequence readouts. Mostafa Ronaghi and Pal Nyren invented the approach in 1996 at the Royal Institute of Technology in Stockholm (Mikeska, Felsberg, Hewitt, & Dobrovic, 2011). It varies from Sanger sequencing in that it uses pyrophosphate on the nucleotide base rather than chain termination with dideoxynucleotides to detect pyrophosphate release. The sequencing in pyrosequencing is done by extending a primed template with polymerase. At each cycle, single nucleotides are added. The integration of the nucleotide supplied to the polymerase reaction with the nucleotide on the template creates a luciferase-based light reaction. After that, the reaction chamber is cleansed, and the cycle is repeated. Functional genomics is a branch of genomics that studies the roles and interactions of

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genes and proteins. Its goal is to learn how the genome works at different stages of development and in diverse environments. Functional genomics focuses on gene transcription, translation, and protein–protein interactions, i.e., it uses high-throughput approaches rather than a more traditional 'geneby-gene' approach to answer questions regarding the function of DNA at the level of genes, RNA transcripts, and protein products.

Figure 1.7: Pyrosequencing. Source: https://www.researchgate.net/publication/306030603_Current_molecular_methods_for_the_detection_of_hepatitis_B_virus_quasispecies/ figures?lo=1

The use of antisense RNA technology to limit gene expression is a potent technique. Synthetically manufactured complementary molecules attach to messenger RNA (mRNA) in this method, effectively preventing the last step of protein production. An antisense nucleic acid sequence base couples with its matching sense RNA strand and stops it from being translated into a protein, according to the theory. The original sequence of the DNA or RNA molecule is referred to as sense.’ The complementary sequence of DNA or RNA molecules is referred to as ‘antisense.’

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Figure 1.8: Small interfering RNAs. Source: https://www.researchgate.net/publication/333048593_Oncogenic_Signaling_in_Tumorigenesis_and_Applications_of_siRNA_Nanotherapeutics_in_ Breast_Cancer/figures?lo=1

Small interfering RNAs (siRNAs) (Figure 1.8) and microRNAs (miRNAs) are small RNAs that are produced by processing longer doublestranded RNA (dsRNA). RNase III, a member of the RNase III family of dsRNA-specific ribonucleases, cleaves dsRNA in an ATP-dependent manner (Kesharwani, Gajbhiye, & Jain, 2012). Dicer enzymes are essential for the production of these two RNAi effectors. Exportin-5 transports the precursor miRNA (pre-miRNA) to the cytoplasm, where it is cleaved into miRNA duplexes by Dicer, a dsRNA-specific ribonuclease. The mature singlestranded miRNA is integrated into an RNA-induced silencing complex when the duplexes’ strands are separated. A DNA microarray is also known as a DNA chip, genomic chip, or biochip is a solid-surface collection of tiny DNA patches. Each DNA patch on the solid surface comprises probe picomoles, which are picomoles of a specific DNA sequence. Southern blotting and hybridization, in which target DNA is bonded to a substrate and then probed with a known DNA sequence, gave rise to microarray technology. The essential premise of DNA microarray is a hybridization between two DNA strands, which takes advantage of the ability of single strands of DNA to establish hydrogen bonds between

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complementary base pairs. To count the relative abundance of nucleic acid sequences, hybridization of the probe to the target is commonly identified and quantified by detecting fluorophore, silver, or chemiluminescence-labeled targets. A DNA microarray can be used to perceive SNPs by measuring changes in expression levels. Thousands of genes are simultaneously tracked in gene expression profiling tests to examine the impact of various therapies. By comparing gene expression in infected cells or tissues to that in uninfected cells or tissues, microarray-based gene expression profiling can be utilized to recognize genes whose expression varies in response to pathogens of other organisms. By comparing genome content in different cells or closely related organisms, a DNA microarray may aid in comparing different genomes.

1.10 PLANT BREEDING Molecular plant breeding (Figure 1.9) strives to increase crop variety in terms of yield, quality, and resistance by utilizing the most recent advances in genetics and genomics. With the use of genomics techniques, we are learning more about the relationship between genotype and phenotype. The identification of genetic markers linked with the trait of interest is one of the most important uses of genomics directly related to breeding. Molecular characterization is vital for unearthing species’ histories, such as their evolution from wild ancestors, and classifying them into appropriate groups based on genetic diversity, distinctiveness, and population structure. This is especially crucial when morphological markers have failed to provide correct results or the results have been deceptive. For example, morphological features have classed the rice lines Azucena and PR 304 as indices, despite the fact that they behave like japonicas in crossing tests. When molecular markers are used to analyze these samples, it is evident that they are japonicas (Yang, Su, Wu, Wang, & Hu, 2011). Quantitative variation is regulated by multiple QTL, each having a modest effect, in the bulk of traits of interest. In diverse habitats and seasons, minor QTL have an uneven QTL effect. Even if the effect of these minor QTL is consistent, MABC introgression into the desired genotype becomes incredibly challenging because more progenies are needed to select acceptable lines. MARS can be used to pyramid superior alleles at several loci/QTL in a single genotype in such instances. MARS is based on the discovery of trait-associated markers and the estimate of their impact. This method entails a series of marker-based selection cycles, which include (i) identifying F2 progenies with favorable alleles for most QTL;

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(ii) recombination of the selected progenies with the selfed ones; and (iii) repeating these two cycles (Swamy, Vikram, Dixit, Ahmed, & Kumar, 2011). When prior QTL information is known, MARS is used more frequently, and the response reduces as knowledge of the several minor QTLs associated with the characteristic of interest declines. Genomic advances have the potential to accelerate the development of crops with promising agronomic features. Agriculture genomics is the use of genetics in agriculture to increase crop and livestock production productivity and sustainability.

Figure 1.9: Molecular plant breeding. Source: https://www.researchgate.net/publication/346580221_Conventional_ Breeding_Molecular_Breeding_and_Speed_Breeding_Brave_Approaches_to_ Revamp_the_Production_of_Cereal_Crops/figures?lo=1

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There has been a great increase in genomic resources available, including expressed sequence tags (ESTs), BAC end sequence, genetic sequence polymorphisms, gene expression profiling, whole-genome (re) sequencing, and genome-wide association studies, thanks to the combination of traditional and high-throughput sequencing platforms. With the advent of genomic sequencing and the development of bioinformatics tools, we are moving away from single gene studies and toward whole-genome analysis, which provides a more comprehensive understanding of how all genes interact. Almost every species-specific genome can be read for a reasonable price, opening up a world of possibilities for targeted crop breeding. In addition, genomics is playing an increasingly important role in biodiversity conservation. Advanced genomics aids in the identification of the genome segments that are responsible for adaptability. It can also increase our grasp of microevolution by better comprehending natural selection, mutation, and recombination. Understanding the structure, organization, and dynamics of genomes in plant species can reveal how genes have been altered to respond to environmental restrictions through natural and artificial selection, as well as the potential for manipulating them for crop development. In agriculture, traditional breeding relies solely on phenotypic selection. Comparative genomics approaches were successful for discovering homologues/ orthologues or cloning species-specific genes utilizing sequence conservation from model plant systems before genomic sequence for model plants became available. RNA transcription, protein modification, and calcium signaling were among the functional categories assigned to genes encoding MRPs. In Arabidopsis and soybean, MRPs were discovered to be primarily responsible for drought and salinity stress. It can be challenging to predict gene function purely based on similarity to other genes. In addition to generating reference genomes, high-throughput sequencing technology has made it possible to resequence genomes from the same species but different accessions in order to detect genomic diversity. Largescale marker segregation data on mapping populations have been generated using genotyping technologies, resulting in detailed genetic maps. We can use the genome sequence to find genome-wide molecular markers such as functional markers, candidate genes, and breeding prediction markers. Duckweeds are promising plants for removing pollutants from wastewater and digesting them into renewable biofuel. Varied ecotypes of the same duckweed species have been found to have different biochemical and physiological features.

CHAPTER

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ADVANCES IN PLANT RESEARCH

CONTENTS 2.1 Introduction ....................................................................................... 30 2.2 Imaging Technology ........................................................................... 31 2.3 Microbial Interactions ........................................................................ 33 2.4 Plant Growth- Promoting Bacteria ..................................................... 35 2.5 Genetic Code .................................................................................... 37 2.6 Pests and Diseases ............................................................................. 39 2.7 Sensitivity .......................................................................................... 40 2.8 Stressors............................................................................................. 42 2.9 Transgenic Crops................................................................................ 44 2.10 Proteomics....................................................................................... 45

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2.1 INTRODUCTION In the modern molecular breeding of crops, plant genetic engineering has become one of the most essential molecular techniques. Significant progress has been attained in the development of novel and efficient transformation methods in plants over the previous decade(Ghosh et al., 2018). Despite the availability of a number of DNA delivery techniques, both Agrobacteriumand biolistic-mediated transformation remain the most popular. Particularly impressive progress has been made in Agrobacterium-mediated transformation of cereals and other resistant dicot species. Other transgenicenabling technologies, such as marker-free transgenics, gene targeting, and chromosomal engineering, have evolved in the meantime. Although the transformation of some plant species or elite germplasm remains a challenge, further progress in transformation technology is expected because the mechanisms governing regeneration and transformation processes are now better understood and are being applied in designing better transformation methods enabling technologies. Advances in plant biotechnology are already aiding developing countries and should continue to do so in the future. Cotton that is insect-resistant and contains a natural pesticide protein from Bacillus thuringiensis (Bt cotton) is giving increased yields, lower insecticide costs, and less health hazards to millions of farmers. Many additional valuable plant biotechnology products that can benefit farmers and consumers are in the research and development pipelines of developing-country institutions, and should be available to farmers in the near future. At all levels of organization, from single to large multicellular organisms, biological systems are dynamic in space and time. Understanding the mechanisms of life requires being able to visualize and measure how biological processes develop. Imaging tools are continually being created across the complete spectrum of biological organization, revolutionizing how we observe the inner workings of cells, tissues, organs, and whole organisms. Many improvements in imaging techniques were first developed in animal systems, with numerous benefits to the area of plant imaging. Plants, on the other hand, differ from animals in a number of ways: their development is normally slower, their cells do not migrate, and their cell walls, plastids, and vacuoles present unique imaging issues. As a result, plant-optimized methodologies have been developed to enable functional imaging of plant processes at various scales, allowing researchers to gain a better understanding of how plants work as multicellular animals.

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2.2 IMAGING TECHNOLOGY The evolution of imaging technology (Figure 2.1) requires the development and use of novel microscopy methods. Light sheet imaging of tissues and organs, as well as two-photon microscopy enabling long-term threedimensional and deep-tissue imaging, are examples of exceptional breakthroughs(Lu, Dao, Liu, He, & Shang, 2020). Other key topics that have been discussed elsewhere include super-resolution and single-particle imaging, as well as artificial intelligence–based denoizing.

Figure 2.1: Imaging technology. Source: https://medium.com/remote-sensing-in-agriculture/hyperspectral-imaging-in-agriculture-befa83cafaa7

Quantification of cell characteristics in time-resolved datasets that can be very large is at the cutting edge of functional plant imaging. Their method uses neural networks to evaluate images captured by a light sheet microscope,

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revealing that during the first two rounds of lateral root formation, cells integrate growth and division to accurately partition their volume upon division. In many species, including human brains, two-photon imaging is one of the most potent approaches for whole organ and deep-tissue cellular imaging. Laser cell ablation for two-photon imaging employing a multiphoton laser is also described as a tool for studying individual cell functions and resolving cell-to-cell communications in deep tissues. Vacuole dynamics are controlled by several genetic pathways, and this dynamic behavior is crucial for their directional migration from the apical to the basal region of the zygote, according to genetic and pharmacological dissection using quantitative two-photon microscopy. Users investigating plant development and physiological responses at the cellular level benefit greatly from the invention and implementation of innovative sensors, reporters, and probes. Until now, genetically encoded sensors and indications have served as effective molecular tools. Oxygen sensors are divided into two types: direct and indirect. Direct sensors bind oxygen, whereas indirect sensors require additional components. Internal oxygen concentrations are not in balance, resulting in strong O2 gradients in plant tissues. Oxygen sensors are thus extremely useful for studying O2 responses in a variety of physiological processes and throughout plant development. A subset of root meristem cells was discovered to have a rapid increase in cytosolic calcium levels among cell populations. When investigating plant development, seeing the cell cycle of individual cells in plant tissues has been a major challenge. While various histochemical techniques have traditionally allowed for the recording of cell-cycle status in fixed tissues, new genetically encoded reporters such as Cytrap and Plant Cell Cycle Indicator now allow for cell cycle progression to be tracked in living plant tissues such as root and shoot meristems(Yokoyama, Hirakawa, Hayashi, Sakamoto, & Matsunaga, 2016)we focused on the proliferating cell nuclear antigen (PCNA. Chemistry-enabled imaging is another promising area of research, with innovative and widely applicable fluorescent compounds being produced at the intersection of biology and chemistry. N-aryl pyrido cyanine (N-aryl-PC)

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derivatives, also known as the Kakshine series, are super, cell-permeable DNA staining dyes that are useful for two-photon, super-resolution, and time-gated imaging of living cells. Chemistry-enabled probes utilizing fluorescent chemicals, especially in plant cells, have challenges with precise labeling and cell permeability when compared to genetically encoded probes using fluorescent proteins. Fluorescent substances, on the other hand, may be able to overcome issues such as photostability and the availability of excitation wavelengths for fluorescent molecules. The development of chemistry-enabled probes will allow for novel plant imaging methodologies. Plant biotechnology is now at the forefront of fields such as alternative energy, which includes biogas production, bioremediation, which involves the use of natural products to treat human diseases, sustainable agriculture, which includes organic farming practices, and genetic engineering of crop plants to make them more productive and effective in dealing with biotic and abiotic stresses. Biotechnology’s major tool set includes genomics, proteomics, metabolomics, and systems biology, among other molecular biology techniques. Its goal is to create a cost-effective way to produce specially designed plants that can be cultivated in a safe environment and used in agricultural, medical, and industrial applications.

2.3 MICROBIAL INTERACTIONS Microbial interactions with plants (Figure 2.2) have both good and detrimental effects, and they play an important part in ecosystem processes. Plant diseases are caused by negative interactions between microorganisms (bacteria and fungus), which pose a global danger to agriculture. Positive interactions, on the other hand, have positive consequences in pharmacological, biotechnological, and agricultural applications. Research has recently concentrated on understanding the complicated molecular mechanisms of host-pathogen interactions in order to produce microbebased fertilizers (bioprotectants), phyto sanitizers, and rhizosphere microbe management for increased nutrient absorption and disease control. The current research is aimed at elucidating the mechanisms of host-pathogen interaction in order to promote sustainable agriculture.

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Figure 2.2: Microbial interactions with plants. Source: 1385(20)30272-7

https://www.cell.com/trends/plant-science/fulltext/S1360-

Pesticide use that is both consistent and judicious results in insect resistance, the elimination of beneficial species, and an increase in residual problems, posing a hazard to human health and its ecological partners in the living biome. The need of the future is to develop an environmentally friendly approach to combating insect pests that can regulate pest populations by exploring naturally occurring botanicals such as plant extracts, insecticidal plants, and plant essential oils that can be used as repellents, antifeedants, insecticides, molluscicides, and other pest control agents. The findings of this study overwhelmingly validated the action of a significant number of plants. As a result, these observations will be valuable in the collecting of plants for laboratory and field research studies, which could eventually lead to the commercialization of plant biopesticides.

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Figure 2.3: Plant growth-promoting bacteria. Source: https://microbewiki.kenyon.edu/images/0/06/Nrmico1129-f1.gif

2.4 PLANT GROWTH- PROMOTING BACTERIA Plant growth-promoting bacteria (PGPB) (Figure 2.3) and plant symbionts have been used to improve plant performance for centuries. However, inoculation with microorganisms was not linked to increased plant growth. Rhizobia inoculants have been economically manufactured for almost 120 years, since the discovery of rhizobia in 1886, primarily in developed countries (Furseth, Conley, & Ané, 2012). Inoculant technology, particularly with PGPB, has minimal or little impact on family farm productivity in the

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vast majority of developing nations because inoculants are not used, are of poor quality, or are handmade. Surprisingly, and most likely as a result of the potential for small companies to produce inoculants at a lower cost than expensive chemical fertilizers and pesticides, many practical studies of a variety of crops were conducted in developing countries, such as the Indian subcontinent, Vietnam, and on cereals and legumes in Latin America, primarily in Argentina and Mexico, as well as in Africa. For most PGPB species, the bacteria population rapidly drops once suspensions of bacteria are introduced into the soil without a suitable carrier. This characteristic, when combined with low bacterial biomass production, difficulty maintaining movement in the rhizosphere, and the physiological phase of the bacteria at application time, can hinder the rhizosphere from building up a large enough PGPB population. The natural variability of the soil is a major problem, as introduced bacteria frequently fail to find an open niche in the soil. These unprotected, inoculated bacteria must compete with the local microflora, which is frequently more suited, and survive predation by soil microfauna. As a result, one of the important functions of inoculant formulation is to offer more favorable niches well as physical protection for a long time, in order to inhibit the spread of introduced bacteria. Fieldscale inoculants must be designed to give a consistent source of bacteria that survives in the soil and becomes available to crops when needed. Many inoculants do not accomplish this, despite the fact that it is the primary goal of inoculant development. When it comes to inoculating plants with PGPB (including rhizobia), the first goal is to locate the optimal bacteria strain or microbial consortia for the desired effect on the target crop. The next stage is to develop a customized inoculant formulation for the target crop as well as a feasible application method that takes into account the producers’ restrictions. In practice, the inoculant’s potential success is determined by the formulation and application method used. Many beneficial strains have been identified in the scientific literature but have yet to be commercialized, possibly due to incorrect formulation. The majority of the research focused on specific genera, such as Rhizobia and Azospirillum, field performance of several PGPBs, availability of diverse PGPBs and their modes of action, fertilizer reduction through inoculants, and possible marketing. Despite the fact that some evaluations covered formulations and practical elements of inoculants briefly, none of these recent reviews focused on that area. Bacterial isolates are particular bacterial strains like PGPB or rhizobia that can boost plant development after inoculation. The abiotic substrate like

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solid, liquid, or gel employed in the formulation process is referred to as the “carrier.” Formulation refers to the process of combining the carrier with the bacterial strain in a laboratory or industrial setting. The term “inoculant” refers to the finished result of a formulation that includes a carrier, bacterial agent, or a group of microorganisms. The practice of measuring defined quality characteristics of the inoculant is referred to as “quality control.” “Quality assurance” is a broad assessment of whether quality control procedures and strategies are accomplishing their goals. The amount of viable and effective cells capable of nodulating plants and fixing nitrogen of the intended strain given by the inoculant at point-of-sale is defined as the quality of the inoculant in legumes. Similar characteristics apply to PGPB, with a greater focus on contaminant-free inoculants. Plant biology in the twenty-first century is and will remain substantially different from that of the twentieth century. The use of genomics approaches to uncover the genetic blueprints for plant species, as well as resolving genome differences in thousands of people at the population level, has been a driving force behind this. Since the first plant genome sequence, that of Arabidopsis thaliana, was published in 2000, genomics technology has advanced significantly(Platt et al., 2010). Researchers in plant genomics have adopted new algorithms, tools, and methodologies to generate genome, transcriptome, and epigenome datasets for model and crop species, allowing for deep conclusions into plant biology. The capacity to create de novo transcriptome assemblies offers another way to get around these stubborn species' complicated genomes and into their gene space. The field of genomics is being propelled forward by technological advancements in sequencing platforms; nevertheless, software and algorithm development have lagged behind lower sequencing costs, increased throughput, and higher quality. Sequencing platforms are expected to enhance the length and quality of their output, as well as the complementing algorithms and bioinformatics software required to manage huge, repetitive genomes. Our understanding of plant biology will continue to increase exponentially in the future.

2.5 GENETIC CODE The genetic code (Figure 2.4) is the foundation of all biological life. As a result, access to the core DNA sequence, i.e., the genome, and how genes are encoded within it has become an important resource in biology. Despite

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the fact that plant genome sequencing lags behind that of microbial and mammalian systems, genomics and the associated data are widely used in plant science sub-disciplines such as agronomy, biochemistry, forestry, genetics, horticulture, pathology, and systematics. In addition to de novo genome sequencing, sequencing technologies and associated bioinformatic and computational processes enable the determination of the transcriptome and epigenome or modified DNA and chromatin state, as well as genome such as the complement of exons and regulatory regions.

Figure 2.4: Genetic code. Source: https://www.genome.gov/sites/default/files/tg/en/illustration/genetic_ code.jpg

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Challenges in genomics, particularly for plants, are presented in order to promote awareness of the field's current limits among the larger plant community. To familiarise readers with these resources, new computational methodologies for addressing the bulk of sequencing data sets are explored. The introduction of automated sequencing methods that combined dideoxy chain termination with fluorescent molecules, often known as Sanger sequencing, in the early 1990s signaled the beginning of genomics (Loit et al., n.d.). The first large-scale gene discovery attempt by sequencing, i.e., expressed sequence tags (ESTs), was made possible by this technology. The finding of genes from discrete tissues of interest was made possible by single-end sequencing of cDNA clones. Plant biologists accepted this strategy, which included sequencing ESTs from the model species Arabidopsis thaliana, despite its initial controversy. The National Center for Biotechnology database of ESTs or dbEST now has sequences for 733 plant species, demonstrating the interest in and utility of single-pass sequences for transcribed genes in plants(Sayers et al., 2011). The bacterium Haemophilus influenzae had the first de novo sequenced genome of a free-living organism. This ground-breaking achievement was made possible by Sangerbased sequencing technology, which showed that whole-genome shotgun sequencing or WGS, in which the genome was randomly fragmented, cloned into a plasmid vector, paired-end sequenced, and the reads were assembled using computational algorithms, was possible. This was shortly followed by a second bacterial WGS effort using Mycobacterium genitalium, proving that this method could be replicated. Although WGS was successful for small microorganisms, it was not possible to apply this approach to larger eukaryotes due to assembly issues. Thus, in the 1990s, early eukaryotic genome WGS studies required the fragmentation of the genome into huge portions such as cosmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC), which were then shotgun sequenced per clone (Balloux et al., 2018).

2.6 PESTS AND DISEASES Plants are constantly exposed to biotic challenges such as pests and diseases, as well as abiotic stresses such as intense light, UV radiation, drought, salt, and extremely high or low temperatures. Surprisingly, earlier exposure to most stimuli makes plants more tolerant of later exposures, a process known as acclimatization. The memory of most stresses is linked to epigenetic modifications, according to research conducted over the previous two

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decades. Heat stress induces cell injury and death by damaging membrane proteins, denaturing and inactivating different enzymes, and accumulating reactive oxygen species. Thermosensors are installed in plants to detect particular changes and trigger protection mechanisms. Phytochrome and calcium signaling are important in detecting abrupt temperature changes and activating signaling cascades that lead to the synthesis of heat shock proteins (HSPs), which keep protein unfolding under control. Heat shock factors (HSFs) are transcription factors that detect thermosensor activity and cause HSP production. HSF epigenetic changes are thought to be a crucial component of thermal tolerance acquisition (TAT). Despite breakthroughs in understanding the mechanism of thermomemory development, it is unknown if plants have systemic activated thermal protection, such as that seen in pathogen infection in the form of systemic acquired resistance (SAR). Bioreactors are devices that can maintain a biologically active environment while conducting aerobic or anaerobic biochemical processes. Bioreactors are a good alternative to traditional plant tissue and cell culture (PTCC) methods because of their stability, operating ease, better nutrient uptake capacity, time and cost-effectiveness, and significant amounts of biomass output. Bioreactors are used in a variety of plant research applications and have evolved over time. Such advancements in technology have resulted in outstanding breakthroughs in the field of PTCC.

2.7 SENSITIVITY Plant sensitivity to mechanical stress has long been thought to be an artefact of plant hardening in harsh settings, a curiosity of useful plants adapted for insectivory, or an avoidance of grazing herbivores. Wind, rain, hail, and animal movements are examples of mechanical stress vectors seen in nature. Pruning, pinching, and clipping are all physical injuries to plants used in production agricultural and landscape operations. Air turbulence created by urban high-rise structures permanently entrains trees and shrubs to the growth behaviors of natural plants found on seacoasts and mountain slopes. Wind as a powerful element restricting plant development has taken a long time to gain acceptance. Because wind and precipitation are not constantly present and because numerous environmental stress variables mix with the wind in the outside environment, it is easy to ignore the mechanical impact of wind and precipitation on plant structure and development habits. Mechanical stress is frequently masked or negated by the effects of other environmental stress factors on plants. Airborne sea salt, desiccation, and

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evaporative chilling are all natural causes that can cause distress. It is only possible to separate the effects of mechanical stress per se from those of other environmental stresses such as heat, cold, drought, flooding, and mineral deficiencies if controlled mechanical stresses like shaking, handling, flexing are applied to plants growing in the wind-protected confines of a greenhouse or growth chamber. Plants have a general impact of delaying internode elongation and inhibiting leaf development, which shrinks plants in size and mass depending on the stress dose. Internode compression and lateral enlargement of stems are the hallmarks of thigmotropism. Seismic stress causes plant responses that are similar, but not always identical, to thermal stress. The plants grew substantially more slowly than undamaged controls when the branch ends of potted chrysanthemums were manually flexed for a few seconds each day. The flowers of the shorter, stressed plants were not smaller, but they used far less water than the taller controls, which had a larger surface area for transpiration. Laboratory shakers can be fitted with platforms to handle numerous plants to standardize seismic treatment applications for research, but growers still can’t afford to manually load and unload a restricted number of platform shakers. In the mid-1970s, Purdue University constructed a series of automated mechanical oscillatory shaking (AMOS) devices to serve as a prototype for shaking devices with commercial potential(Latimer, 1998). Early morning was the most efficient period for shaking, according to research on chrysanthemum height control using AMOS, and isomorphism obeys the law of reciprocity. Plants of the ‘Alaska’ pea (Pisum sativum L.) was shrunk by daily shaking on a gyratory platform shaker, which also reduced the number of pods generated and the quantity of seeds per pod Shaken pea plants’ seed output was only half that of undisturbed controls as a result of the combined effect. Mechanical stress has reduced yield in every crop species studied thus far, either due to a delay in flowering or a reduction in the size, number, or mass of harvestable components. It was found that striking the stem tips or shaking the entire shoot of potted potato plants reduced the size and mass of tubers formed during treatment, but not the number. Mechanical stress tests with potatoes indicate that shoots, which receive the major mechanical stress stimuli, can transfer those signals to below-ground plant components. The fact that localized rubbing of stem tips has the same growth-retarding effects on potato tubers as more general shaking treatments applied to the entire shoot calls into question any claim

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that stress effects on roots and tubers are caused solely by physical forces transmitted from shoots to below-ground plant parts. Plant molecular farming or PMF is a novel field of plant biotechnology in which plants are genetically modified to produce vast amounts of recombinant medicinal and industrial proteins. PMF is still struggling to obtain social recognition as an emerging subset of the biopharmaceutical business, compared to the well-established production methods that create these high-value proteins in microbial, yeast, or mammalian expression systems.

2.8 STRESSORS Plant growth and development are hampered by abiotic and biotic stressors, which have a negative impact on crop output. Plants have evolved stressspecific adaptations as well as simultaneous responses to a combination of abiotic and pathogen stressors. Stress-induced adaptive responses are dependent on the activation of molecular signaling pathways and intracellular networks via changing the expression, abundance, and/or post-translational modification (PTM) of proteins that are predominantly connected with defense mechanisms. Advanced quantitative proteomic techniques have improved total proteome and sub-proteome coverage from small amounts of starting material, as well as characterized PTMs and protein–protein interactions at the cellular level, providing thorough evidence on organ and tissue specific regulatory mechanisms responding to a variety of individual stresses or stress combinations during the plant life cycle. We can focus on tissue-specific signaling networks that are localised to various organelles and are involved in stress-related physiological plasticity and adaptive mechanisms like photosynthetic efficiency, symbiotic nitrogen fixation, plant growth, tolerance, and common responses to environmental stresses. We also highlight the present challenges and limitations of proteomics techniques and data interpretation for non-model organisms, as well as the advancement of proteomics with main crop species. To accomplish substantial successes in genomics-driven breeding of key crops for high productivity and stress tolerance, it is critical to understand all levels that regulate adaptation mechanisms and the resilience of crop plants in the context of climate change. Agricultural production systems have already been impacted by a new pattern of often occurring extreme weather events. The development of bioinformatics techniques and analytical instrumentation, in addition to the growing genomic information available for both model and non-model plants, has made proteomics an essential

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approach for revealing major signaling and biochemical pathways of plant life cycle, interaction with the environment, and responses to abiotic and biotic stresses. Quantitative profiling, analysis of dynamic post-translational modifications or PTMs, subcellular localization and compartmentalization, protein complexes, signaling pathways, and protein–protein interactions are all examples of high-throughput proteomic studies. Plant development and productivity are inevitably influenced by extreme environmental variables such as drought, heat, salinity, cold, or pathogen infection, which can delay or induce seed germination, restrict seedling growth, and diminish crop yields when grown in the field or in the lab. Proteomics research can help uncover nearly every element of cellular function in plant stress responses, as well as putative links between protein abundance and/or alteration and plant stress tolerance. Studies using model plants like Arabidopsis, and sorghum explained the involvement of proteomics to understand the molecular mechanisms of plant responses to stresses and signaling pathways linking changes in protein expression to cellular metabolic events. Major monocotyledonous cereals and dicotyledonous legumes like maize, wheat, etc. have been widely used to study quantitative changes in protein abundance related to climate change due to improvements in diverse proteomic technology platforms that united classical two-dimensional electrophoresis and gel-based techniques with mass spectrometry (MS) based quantitative approaches. Crop plants are subjected to a complex mix of abiotic and biotic stressors in the agricultural environment. Evidence shows that simultaneous incidence of multiple stresses affecting crop growth, yield, and physiological traits can alert plants to activate intricate metabolic pathways involved in specific programming of gene expression that uniquely respond to different combinations of stresses, in addition to studying the effects of various stresses applied individually under laboratory-controlled conditions. Transcriptome and proteome analysis of various crop plants subjected to various stress combinations revealed several different signaling pathways involved in multiple stress-responding mechanisms, implying a complex regulatory network orchestrated by hormone signals, transcription factors, reactive oxygen species (ROS), and osmolyte synthesis. Fundamentally, crop growth is dependent on the efficient synthesis of energy and nutritious substances, which is regulated by several organs, each of which is equipped with its own set of cytosolic proteins, hormones, and metabolites. Plant cells respond differently to abiotic stressors in

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

various tissues. Organ-specific proteomics, in combination with subcellular organelle proteomic studies of developmental mechanisms from leaf to root, can provide more detailed information about cellular mechanisms that regulate stress response and signal transduction in various organelles. By assessing spatial and functional sub-proteomes, tissue-targeted seed proteomic studies of distinct developmental phases under abiotic challenges have contributed to enhancing our depth of knowledge about the processes driving seed development, dormancy, and germination. Pests and diseases have a negative impact on agricultural output and quality, as well as resource efficiency. Crop protection measures that prevent such damage and loss can boost productivity and contribute significantly to food security. The identification of novel disease agents will be aided by rapid sequencing of nucleic acids from affected plants. Pest outbreaks can also be detected through biomarkers of infections or crop damage, such as volatile compounds. Biosensors linked to information networks will allow for real-time monitoring and surveillance of crops or stored produce, as well as early detection of emergent problems and invading species. Although the rapid expansion of the internet, mobile phones, and other communication networks will give new opportunities, challenges remain in the transmission of new technologies and information to resource poor farmers in developing countries. Identifying the genetic and molecular basis of innate plant immunity has been a significant step forward in plant biology, with the potential to uncover new targets for intervention using innovative chemical or genetic manipulation (GM).

2.9 TRANSGENIC CROPS There should also be options for selecting more responsive crop genotypes or developing transgenic crops that respond to specific chemical signals or molecular patterns that can be used to diagnose specific biotic concerns. The genome sequencing of important crop species and their wild relatives will vastly extend the gene pool and diversity of genetic resources available to plant breeders. It should be possible to uncover genomic areas and genes that give greater long-lasting, quantitative pathogen resistance. High-throughput phenotyping and efficient selection of resistance features utilizing withingene markers will speed up the breeding cycle. Pyramiding or combining resistance genes with diverse specificities and modes of action will be easier in using GM methods, lowering the possibility of virulence directional selection. Genome analysis of plant pathogens and invertebrate pests is

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already revealing new genes, gene families, and mechanisms involved in host colonization and pathogenicity. Comparative genomics of species with distinct host ranges, eating patterns, and pathogenic lifestyles can help researchers find new targets for pest control and produce new antimicrobial medications. Knowing the natural ecology of pests and pathogens, such as the factors that determine host placement and interactions with other organisms, will help us influence behavior or exploit natural enemies or other antagonists of pest species. Volatile signals will be more frequently exploited to modify pest behavior, whether they come from natural plant sources or are produced in transgenic crops. It may also be feasible to alter microbial communities that regulate pathogen populations and activity, allowing more effective biocontrol agents to be retained. New knowledge on pest species suppression methods will be gained by studying the natural variety and activity of soil and microbial populations in the zones surrounding roots and seeds. Because of the complexity and diversity of the soil system, fully effective interventions are improbable, but progress toward integrated control regimes combining more resistant crop genotypes with focused management of natural suppressive processes should be made. Improved understanding of the variables driving pest and pathogen adaptability and evolution will be required to use new technology and knowledge to build more durable resistant crops and sustainable disease and pest management systems. More focus must be placed on translational research and delivery, as well as devising tactics that are fit for lower-input production systems.

2.10 PROTEOMICS Proteomics is becoming increasingly relevant for the study of many different aspects of plant functioning, thanks to the avalanche of genetic data and advances in analytical technology. Protein studies are critical for revealing molecular mechanisms underpinning plant growth, development, and interactions with the environment since proteins are crucial components of major signaling and metabolic pathways. The proteome of plants is extremely complex and dynamic. Although much progress must be made toward the ultimate goal of characterizing all of the proteins in a proteome, current technologies have opened the door to a plethora of high-throughput proteomic research to include quantification, PTM, subcellular localization, and protein–protein interactions. The focus is on recent advancements in plant protein functional analysis, which pave the way for comprehensive

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integration of transcriptomics, metabolomics, and other large-scale “omics” into systems biology. Plants are constantly attacked by herbivores and diseases, and as a result, they have developed both constitutive and induced defenses over time. Both herbivore-induced factors like elicitors, effectors, and wounds and plant signaling via phytohormone and plant volatiles in response to arthropod factors trigger and drive the sophisticated signaling network for plant defense responses. Interactions between plant and herbivore-derived elicitors and effectors, followed by fast activation of sophisticated plant signaling cascades, result in coordination of defense activities against attacking pests. However, the molecular mechanisms in the hosts that control the balance between resistance activation and repression are unknown. Despite the large number of documented plant responses to herbivores, animal-derived defensive elicitors are rare. Volicitin, a hydroxy fatty acid-amino acid conjugate (FAC), was discovered in the oral secretions of the beet armyworm and was the first fully defined herbivore-derived elicitor. The biological roles of FACs on plants, as well as FAC variation patterns in Lepidoptera species, have been extensively researched. FACs injected into wounds during feeding, for example, are rapidly degraded by lipoxygenases in the octadecanoid pathway, resulting in the formation of additional active elicitors. Due to their amphiphilic nature, FAC-type elicitors can cause plasma trans-membrane potential (Vm) depolarization in plant cells. As a result, detergent-like potential ion fluxes induced by oral secretions cause Vm depolarization and, as a result, the opening of voltage-dependent Ca2+ channels to transmit the signal. Research has been conducted on herbivore secretions, with some success in identifying other elicitors and herbivore-associated molecules, such as caeliferins, glucosidase from the cabbage white butterfly, benzyl cyanides from Pieris brassicae, disulfonyl fatty acids from the American bird grasshopper. Inceptin and similar peptides are generated from the regulatory areas of chloroplastic ATP synthase gamma. These peptides cause the generation of phytohormones to occur quickly and sequentially, resulting in volatile emissions. In contrast to caterpillars, however, nothing is known about sucking arthropod oral elicitors like spider mites and aphids. The release of aphid elicitors like oligogalacturonides due to cell wall destruction by gel saliva enzymes has recently been postulated to cause Ca2+ influx. Furthermore, plant responses may be elicited by egg deposition. The egg or egg-associated components of several insects cause plant defensive

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responses, though the reaction chemistry has only been identified in bruchid beetles: long-chain, -monounsaturated C-22 diols and, mono- and diunsaturated C-24 diols, mono- or diesterified with 3-hydroxypropanoic acid. Similarly, powerful elicitors secreted by herbivorous arthropods upon tarsal contact with a plant may exist. Although certain diseases inhibit these mechanisms by interfering with defense-related communication pathways, evidence of such interference in herbivores is sparse. Herbivorous arthropods, on the other hand, cause a complex and interconnected array of molecular and physiological reactions in plants, whether by defoliation or feeding on specific tissues like phloem or xylem. These reactions may lower host resistance and even limit photosynthesis. Suppression of host defenses and phenotypic changes in host plants are common in a wide range of plant-pest particularly plantpathogen interactions, and entail the production of chemicals that affect host cell processes. With Lepidoptera salivary glands, massive proteome and transcriptome analyzes were conducted, and some of the major components of saliva were identified. Helicoverpa zea mandibular glands release salivary glucose oxidase, an enzyme that acts as an effector suppressing the host plant’s induced defenses by contributing to the initial oxidative burst of H2O2 observed in herbivore-damaged leaves. Scientists discovered a link between host range breadth and GOX activity, with more polyphagous species exhibiting higher GOX levels than species with a more limited host range. In Arabidopsis, it was recently discovered that egg-derived elicitors cause the inhibition of defenses against chewing herbivores. Salicylic acid (SA) is involved in this mechanism, as indicated by the lack of gene repression and increased sensitivity in sid2-1 mutants. Distinct plant responses may be triggered by herbivore species belonging to different feeding guilds, such as parenchymal cell content feeders and phloem feeders. SA-responsive gene transcripts accumulated locally and systemically in Arabidopsis plants infested by the phloem-feeding silver-leaf whitefly, whereas JA and ethylene dependent RNAs were suppressed or not altered. B. tabaci was also reported to interfere with Lima bean plants' indirect defense against spider mites (Tetranychus urticae) by inhibiting the JA signaling pathway triggered by the latter. Tetranychus evansi inhibits the induction of the SA and JA signaling pathways in tomato, which are important in induced plant defenses. Furthermore, significant variations in features that lead to resistance or susceptibility to JA-dependent defenses

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of a host plant, as well as attributes relevant for induction or repression of JA responses, have been demonstrated within a single herbivore species, the spider mite T. urticae. Aphids, like plant diseases, send effectors into their hosts to control host cell processes, allowing plants to be successfully infested. Plant disease resistance proteins that detect plant pathogens and those that confer aphid resistance have a nucleotide binding site domain and leucine rich repeat (LRR) regions in common (Martin, Bogdanove, & Sessa, 2003). A functional genomics approach based on common features of plant pathogen effectors was recently devised for the identification of potential aphid effector proteins from the aphid species Myzus persicae. M. persicae has 46 potential secreted proteins, according to data mining of salivary gland expressed sequence tags (ESTs)(Hogenhout & Bos, 2011). Mp10, for example, generated chlorosis and mildly induced cell death in Nicotiana benthamiana, as well as suppressing the oxidative burst induced by the bacterial PAMP flagellin. Furthermore, two potential effectors namely Mp10 and Mp42 have been identified as lowering aphid performance using a medium throughput experiment based on transient overexpression in N. benthamiana, whereas MpC002 improved aphid performance. Overall, aphid-secreted salivary proteins have characteristics that are similar to plant pathogen effectors, suggesting that they could act as aphid effectors by disrupting host cellular processes. A nitrile-specifier gut protein found in the larvae of various lepidopteran species, notably Pieris rapae and P. brassicae, detoxifies the breakdown products of glucosinolates, which are the principal insect deterrents in Arabidopsis. Furanocoumarins are degraded by the cytochrome P450 monooxygenase gene superfamily in Papilio butterflies, while pyrrolizidine alkaloids are degraded by the flavin-dependent monooxygenase system of the arctiid moth Tyria jacobaeae. Plant susceptibility is caused by nematode effectors. A nematode-secreted peptide and a plant-regulatory protein were discovered to interact directly. The induced plant defenses against herbivores appear to be the result of an integrative "cross-talk" between signaling molecules such as Ca2+ ions, reactive oxygen species (ROS), protein kinases, JA, cis-12-oxophytodienoic acid (OPDA), SA, ethylene, and yet unknown octadecanoid family members. Phytohormones, such as those mentioned above, play a key part in signal transduction in a variety of signaling pathways. SA, JA, and ethylene are three phytohormones that are important in monocot and dicot defense. The SA pathway, which is involved in both locally expressed basal resistance and systemic acquired resistance (SAR), has been demonstrated to be activated predominantly in response

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to biotrophic diseases or insects that cause little damage, such as phloemfeeding aphids and spider mites. The JA/ethylene route, on the other hand, is activated in the presence of necrotrophic infections, wounds, and tissuedamaging insect feeding. JA is a signaling substance that regulates induced plant responses to herbivores and pathogen infection by activating several sets of defense genes. While herbivore resistance is known to be mediated by JA, pathogen resistance is mostly mediated by SA in plants. Plants, for example, respond to piercing-sucking herbivores such as aphids, whiteflies, and spider mites by up-regulating both SA and JA responses simultaneously. In several species of plants affected with aphids, mRNAs encoding putative proteins that may be involved in the synthesis of JA and SA are up-regulated, leading to a variety of plant defense responses, including aphid-dependent blends of plant volatiles caused by the feeding of various aphid species. Furthermore, JA and SA are antagonistic and both need for the induced response in response to herbivore feeding or pathogen attack. JA and SA have antagonistic interactions, and JA–SA crosstalk is an outstanding illustration of the intricate regulatory networks that allow the plant to fine-tune individual responses to distinct pathogens. Although various studies have suggested that JA and SA have negative interactions in defensive signaling, this crosstalk is highly dependent on concentration and timing. In N. attenuata, it was discovered that ethylene plays a key role in the activation of JA-regulated plant defenses against herbivores. After herbivore attack, JA-ethylene crosstalk limits local cell development and growth, allowing more resources to be directed to induce herbivore defenses. By modifying the sensitivity to a second signal i.e., Ca2+ signal and its downstream responses, ethylene is required for the simultaneous induction of JA or other signals. After herbivore injury, ethylene appears to act as a switch, limiting the production of constitutive defensive chemicals like nicotine while increasing the production of JA and volatiles. Ethylene has also been shown to contribute to herbivoryinduced terpenoid biosynthesis in Medicago truncatula by influencing both early signaling events such as cytoplasmic Ca2+ influx and downstream JAdependent terpenoid biosynthesis. Some of these volatiles have the ability to activate defense genes, which is likely mediated by well-known signaling pathways such as Ca2+ influx, protein phosphorylation and dephosphorylation, and ROS activity. GLVs with an unsaturated carbonyl group have been postulated to trigger defense by acting as reactive electrophile species, while other GLVs that have been reported to be biologically active lack this pattern. When mechanically

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

wounded and stimulated with caterpillar regurgitation, corn seedlings previously exposed to GLVs or terpenoids from nearby plants produced much more JA and volatile sesquiterpenes than seedlings not subjected to GLV. Early signaling steps in the cellular response to stress are mediated by changes in Vm, and exposure to numerous GLVs altered membrane potentials in intact leaves. It's tempting to think that the intra-membrane connection of volatiles with membrane proteins, presumably analogous to insect odorant-binding proteins, causes changes in transmembrane potentials and, as a result, gene activity. Except for the gaseous hormone ethylene, nothing is known about such sensory proteins for plant volatiles. Plants typically respond differently to structurally identical substances, implying that plants can respond selectively to distinct chemical molecules or even compounds that differ solely in their stereochemistry. The low-molecularweight, lipophilic nature, along with their wide structural variation and high vapor pressures at ordinary temperatures of many VOCs account for their significance as chemical information carriers. Next-generation sequencing (NGS) is transforming genetics and revealing new information on genome organization, evolution, and function. The number of plant genomes being sequenced is increasing. However, obtaining whole genome sequences in large genome species remains problematic for the time being, owing to the fact that short reads produced by NGS platforms are insufficient to deal with repeat-rich DNA, which makes up a substantial percentage of these genomes. In polyploids, which dominate the plant kingdom, the problem of sequence redundancy is exacerbated. Flow-cytometry can be used to reduce the complete nuclear genome to its individual chromosomes, which can help overcome some of these challenges. This DNA has proven to be suitable for a variety of applications, including PCR-based physical mapping, in situ hybridization, DNA array formation, DNA marker creation, BAC library construction, and positional cloning. When chromosome sorting and NGS are combined, it opens up possibilities for studying genome organization at the single chromosomal level, comparative analysis between related species, and validation of whole genome assemblies. Apart from the primary goal of lowering the template's complexity, using a chromosome-based technique allows multiple teams to work in parallel, each analyzing a distinct chromosome.

CHAPTER

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GENOMICS IN PLANT RESEARCH

CONTENTS 3.1 Introduction ....................................................................................... 52 3.2 Model Organisms .............................................................................. 54 3.3 Molecular Markers............................................................................. 54 3.4 DNA Sequencing Technology ............................................................ 56 3.5 Exome Sequencing ............................................................................ 58 3.6 Greenphyldb ..................................................................................... 61 3.7 Phylogenomics .................................................................................. 62 3.8 Plant GDB ......................................................................................... 66 3.9 Plaza ................................................................................................. 68 3.10 Weeds ............................................................................................. 72 3.11 Herbicides ....................................................................................... 74 3.12 Weed Management.......................................................................... 76 3.13 Mutation Breeding ........................................................................... 78 3.14 Allergies .......................................................................................... 80

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3.1 INTRODUCTION Until recently, plant molecular analysis was frequently limited to single genes. Recent technological advancements have shifted this paradigm, allowing for the study of genomic organization, expression, and interaction. Genomic science is the study of how genes and genetic information are structured inside the genome, as well as the methods for collecting and interpreting this data and how this arrangement influences their biological performance (DellaPenna, 1999). Genomic techniques are pervading every part of plant biology, and because they rely on DNA-coded information, they enable multi species molecular investigations. Plant genomics is reversing the old paradigm, which focused on locating genes behind biological functions, and instead focused on identifying biological functions behind genes. It also bridges the gap between phenotype and genotype, allowing researchers to better understand how a gene in the genetic environment and the genetic networks with which it interacts can affect its activity.

Figure 3.1: Reterotransposons. Source: https://en.wikipedia.org/wiki/Retrotransposon#/media/ File:Retrotransposons.png

Plant genomes contain a variety of repetitive sequences as well as retrovirus-like retrotransposons (Figure 3.1) with lengthy terminal repeats and other retroelements like long interspersed nuclear elements and shortinterspersed nuclear elements. Retroelement insertions are responsible

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for the substantial size disparity between collinear genome segments in different plant species, as well as the 50 percent or more variability in overall genome size among species with big genomes, such as maize (Van Bel et al., 2012). In plants with smaller genomes, they contribute a smaller fraction of genome size. When other repetitive sequences are taken into consideration, the corn genome contains about 70% repetitive sequences and 5% protein encoding regions. 70-80 percent of flowering plants are thought to be the result of at least one polyploidization event (H. Tang et al., 2008). Many economically significant plant species, including corn, wheat, potato, and oat, are polyploids, meaning they have more than one, and in the case of wheat, three, homologous genomes inside a single species. A considerable portion of the rice genome is made up of duplicated sequences. The Arabidopsis genome is split into 24 duplicated segments, each measuring more than 100 kb(Platt et al., 2010). Through gene duplication and gene silencing, ancestral polyploidy leads to the creation of genetic variation. In plants, genome duplication and subsequent divergence is a major source of protein diversity.

Figure 3.2: Drosophila melanogaster. Source: https://www.theguardian.com/science/2017/oct/07/fruit-fly-fascination-nobel-prizes-genetics

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3.2 MODEL ORGANISMS Drosophila melanogaster (Figure 3.2), Caenorhabditis elegans, and Saccharomyces cerevisiae are model organisms that provide genetic and molecular insights into the biology of more complex species. The plant scientific community has embraced model organisms because the genomes of most plant species are either too vast or too complicated to be thoroughly studied. They have common characteristics such as being diploid and suitable for genetic study, being amenable to genetic transformation, having a relatively tiny genome and a short growth cycle, having widely available tools and resources, and being the subject of much research. Although tissue culture techniques encouraged the use of tobacco and petunia, rice and Arabidopsis are increasingly employed as model organisms for monocotyledonous and dicotyledonous plants, respectively. Arabidopsis is a tiny Cruciferae plant that produces seeds in only 6 weeks after being planted. It has a short genome of 120 Megabytes and only five chromosomes (Lenoir, Cournoyer, Warwick, Picard, & Deragon, 1997). For genomic analysis, whole genome sequence, Expressed Sequence Tags (ESTs) collections, point mutants, and huge populations mutagenized using insertion elements or transposons or Agrobacterium T-DNA, there are numerous techniques available. Agrobacterium tumefaciens and biolistics can be used to genetically modify Arabidopsis on a wide scale. Saturated genetic and physical maps are two other techniques accessible for this model plant. Rice, unlike Arabidopsis, is one of the most important cereals on the planet. Rice is produced in excess of 500 million tonnes per year and is the staple food for half of the population. Rice is divided into two subspecies: japonica is grown primarily in Japan, whereas indica is grown primarily in China and other Asia-Pacific countries. Rice also has a lot of genetic maps, physical maps, whole genome sequences, and EST collections from various tissues and stages of development. It has 12 chromosomes and a genome of 420 megabytes, and it can be transformed using biolistics and A. tumefaciens, just like Arabidopsis(Goff et al., 2002). Although some recent successes have been reported, efficient transposon-tagging systems for gene knockouts and gene detection have yet to be developed for saturation mutagenesis in rice.

3.3 MOLECULAR MARKERS The development of molecular markers has enabled the creation of complete genetic maps for the majority of economically significant plant species.

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They detect genetic variation at the DNA level directly. There is a plethora of molecular marker systems available, however, describing them is beyond the scope of this work. A genetic map depicts the ordering of molecular markers along chromosomes as well as the genetic distances between adjacent molecular markers, which are usually expressed in centiMorgans (cM). Many experimental populations have been used to create genetic maps in plants, but the most common are F2, backcrosses, and recombinant inbred lines. Recombinant inbred lines provide a better genetic resolution and practical advantages, but they take longer to create. It only takes a few months to create a genetic map with a 10 cM resolution once a mapping population has been established. Genetic maps help us understand how plant genomes are organised, and once we have them, we can use them to develop practical applications in plant breeding, such as identifying Quantitative Trait Loci and Marker Assisted Selection. Plant features that are economically important, such as yield, plant height, and quality components, have a continuous dispersal rather than discrete classes and are classified as quantitative traits. Several loci, each with a tiny effect, govern these features, and different combinations of alleles at these loci might result in diverse phenotypes. The identification of genetic areas linked to the phenotypic expression of a particular characteristic is referred to as loci analysis. Individuals possessing chromosomal fragments associated with the expression of a certain phenotype can be assembled into designer genotypes once the location of such genomic areas is determined. As a result, the presence of the molecular marker is always linked to the existence of the desired allele. Genomic maps are also useful for isolating plant genes, because once the genetic position of a mutation is determined, positional cloning can be used to try to isolate it. In addition, genetic maps aid in determining the level of genome collinearity and duplication between species. Plants play a significant role in providing a large amount of food. Plants have also been utilized as model organisms to research transposable elements in heterochromatin and epigenetic regulation and have been chosen as model organisms to study transposable elements in heterochromatin and epigenetic control. Because of its importance, plant biology has been studied extensively since the beginning of human history. Plant biology research has advanced to new heights. High-throughput sequencing tools have allowed scientists to exploit the structure of genetic material at the molecular level, a process called genomics. Because of the rapid increase in sequenced genomes of many plant species, plant genomics has recently blossomed and

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has become the dominant focus in plant research. Plant genome research has a tremendous impact on the advancement of economically important plants as well as plant biology. Plant genetic information is open to the public and updated on a regular basis, creating a fruitful environment for plant research.

3.4 DNA SEQUENCING TECHNOLOGY The evolution of DNA sequencing technology (Figure 3.3) has been long and marked by numerous historical events. Nearly all DNA sequence generation in the last decade has been restricted to capillary-based, semiautomated applications of Sanger biochemistry and its variants. Over the years, the area of DNA sequencing has been resurrected and developed due to various scientific achievements. Due to a variety of factors, these technical developments finally encouraged the development of fresh experimental designs for this sector. Finally, in 2005, next-generation sequencing (NGS) methods were made public (Yohe & Thyagarajan, 2017). They're referred to as high throughput sequencing technologies because they parallelize the sequencing process, producing millions of sequences at once at a substantially cheaper per-base cost than traditional Sanger sequencing. DNA polymerase is used in the sequencing-by-synthesis platform to lengthen multiple DNA strands in parallel. This approach employs modified deoxynucleoside triphosphates or dNTPs with a terminator that stops further polymerization, allowing DNA polymerase to add only one base to each developing DNA copy strand. As a result, as extension progresses, the newly integrated nucleotide or oligonucleotide can be determined. The sequencingby-synthesis (SBS) approach underpins the pyrosequencing platform. It is dependent on the detection of pyrophosphate generated by DNA polymerase during nucleotide integration to support a series of enzymatic events that eventually produce light from the cleavage of oxyluciferin by luciferase. DNA ligase is used to create sequential ligation of dye-labeled oligonucleotides in the Sequencing-by-ligation platform.

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Figure 3.3: DNA sequencing technology. Source: https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/ genomics/sequencing/sanger-sequencing

The concealed sequence of the target DNA molecule is subsequently determined using the sensitivity of these amplified DNA fragments. The hydrogen ions generated during DNA polymerization are detected by an ion semiconductor-based non-optical sequencing device. The successive enzymatic breakdown of fluorescently tagged single DNA molecules, and the detection and identification of the liberated monomer molecules according to their sequential order in a microstructured channel is the basis for single-molecule sequencing. Amplification of DNA fragments is not required before sequencing with a single-molecule sequencer. Individual nucleotide sequences are identified by variations in the ion current when the DNA strand passes through a membrane-inserted protein nanopore, one base at a time.

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Figure 3.4: Exome sequencing. Source: https://www.researchgate.net/publication/235368577_Application_of_ Whole_Exome_Sequencing_to_Identify_Disease-Causing_Variants_in_Inherited_Human_Diseases/figures?lo=1

3.5 EXOME SEQUENCING Exome sequencing (Figure 3.4) can be used to explore biodiversity, study host–pathogen interactions, investigate the natural evolution of crops, test for genetic marker inheritance, provide large-scale genetic resources for crop improvement, identify genes, and establish the presence of functional gene sets that are involved in symbiotic or other co-existential systems. Furthermore, single-base resolution NGS approaches can give epigenomic information. For example, a study of the A. thaliana epigenome found that cytosine methylation was highly associated with the location and

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abundance of short RNA targets. Genotyping by sequencing or GBS, a highthroughput and low-cost method for optimizing genotype populations, is another application of plant genome sequencing. GBS provides a number of methods for improving genomic map building, particularly the detection of single nucleotide polymorphisms or SNPs. GBS showed that there is a favorable correlation between trait-related genes and 681,257 SNP markers from 2,815 maize inbred accessions (Romay et al., 2013). It is undeniable that NGSs have been successful in plant genome research. However, building computer tools for studying genomic sequences is difficult. Galaxy project is one of the software systems that allows researchers to employ analysis tools using web-based interfaces that contain massive amounts of freely available biological data. Artemis is another free programme accessible from the Sanger Institute. It has a genome browser as well as an annotation tool. The Broad Institute's Genome Sequencing and Analysis Program or GSAP provides various more genome sequence analysis tools. Furthermore, the significant decrease in the cost of genome sequencing necessitates the quick creation of large database storage and administration systems. In reality, to meet this demand, a growing number of plant genome databases have been created. Plant genomes from non-model and non-crop species can also reveal information on genome building and flowering plant evolution. Genomes from Utricularia gibba and Genlisea aurea, for example, can reveal a lot about genome size variation. Furthermore, the genome of Spirodela polyrhiza is equivalent in size to that of Arabidopsis, although it only requires 28% fewer genes to operate normally (W. Wang et al., 2014). In another case, the genomes of Selaginella moellendorffii and Amborella trichopoda offer key understandings regarding the trajectory of plant-specific gene families and the radiance of flowering plants, giving further insights into flowering plant evolution. Individual alleles can be recognized, classified, exploited, and tagged using genomic knowledge, as well as molecular markers can be promoted and manipulated to track desired alleles in breeding programs. Many genome sequencing projects in the field of horticultural crops have been carried out for these reasons, including the Tomato genome sequencing project, the Potato genome sequencing consortium, the Papaya genome sequencing project, the Grape genome sequencing project and, ideally, in the near future, many more will be released in the public domain for scientific purposes. These studies used sophisticated sequencing technologies in conjunction with traditional

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approaches to fully validate the development of high-quality sequences and cost-effective design. As a result, by providing important resources for comparative and functional genomic investigations, these whole-genome sequencing efforts may have a considerable impact on global food security and bio-energy progress. If present research continues, applications of genomic science resources to horticultural plant species could have a significant impact on worldwide human well-being. An increasing number of genes in plant species have been annotated using a comparative genomics technique. Several known stress-responsive transcription factors OR TFs in Arabidopsis and rice, for example, were utilized to properly predict stressresponsive TFs in soybean, maize, sorghum, barley, and wheat. Furthermore, not only within plant species, but also between plants and distantly related prokaryotes, comparative genomics can considerably infer the functionally linked genes. Knowing the function of the NiaP protein family in bacteria led to the discovery of its function in plants. Researchers can analyze gene annotation in freshly sequenced plant species using similar methodologies to discover functional genes among various plants using comparative analysis. To store and manage enormous genomic data, substantial computational resources are required in addition to tools and methodologies for analysis. Many online platforms for comparative genomic studies among different plant species have been developed, published, and made available. For example, the following plant genomic data platforms have recently been the most representative and widely used. Phytozome is one of the largest comparison databases for plant species. It offers information on the plant genome, gene families, and evolutionary history. Only 25 plant genomes were sequenced and annotated at the start. In its current state, this number has risen to more than 50 species(Goodstein et al., 2012)and the application of inexpensive next generation sequencing. To interact with this increasing body of data, we have developed Phytozome (http://www.phytozome.net. Phytozome also includes powerful tools for comparing sequences, gene structures, gene families, and genome architecture. With these tools and a complete web platform, Phytozome makes plant study accessible to scientists all around the world. PLAZA is the most user-friendly online platform for plant comparative genomics. It integrates functional and structural annotation of all currently available crop plant genomes. PLAZA offers a variety of interactive tools for studying genes, genome evolution, and gene function in addition to that massive data set. Pre-computed datasets, intraspecies dot plots, whole-genome multiple sequence alignments, homologous gene

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families, phylogenetic trees, and genomic collinearity between species are only a few of the tools available.

Figure 3.5: GreenPhylDB. Source: https://www.researchgate.net/publication/46413750_GreenPhylDB_ v20_Comparative_and_functional_genomics_in_plants/figures?lo=1

3.6 GREENPHYLDB GreenPhylDB (Figure 3.5) is a public-access web resource that is part of the South Green Bioinformatics Platform. GreenPhylDB is a database for plant comparative and functional genomics. At the current release version 4, this database comprises 37 complete genomes of Plants. Gene predictions of genomes give a catalogue of gene families from GreenPhylDB, which covers a large taxonomy of green plants. Its web interfaces are constantly being improved to make it easier to navigate through information about each gene or gene family, including gene composition, protein domains, publications, orthologous gene predictions, and external links. The most recent edition of this database now allows users to search the entire Gene Oncology database, which aids gene discovery. PlantsDB is one of the most widely utilized plant database sites for integrative and comparative plant genome research. Tomato, Medicago, Arabidopsis, Brachypodium, Sorghum, maize, rice, barley, and

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wheat have database instances in PlantsDB. Individual plant genomes are stored and made available through this platform. It’s also outfitted with cutting-edge bioinformatics tools for visualising synteny, transferring data from model systems to crops, and comparing and contrasting plant species. Repeat catalogues and classification systems for all plant species are other key analysis methodologies generated from PlantsDB.

Figure 3.6: Phylogenomics. Source: https://www.nature.com/articles/s41467-019-13443-4/figures/1

3.7 PHYLOGENOMICS Phylogenomics (Figure 3.6) is a type of molecular phylogenetic analysis that involves using sets of genomic databases to predict gene function and investigate evolutionary links across species. This definition of

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phylogenomics is based on early investigations from the late 1990s when a scientific hypothesis concerning protein function was published based on the evolutionary analysis of a gene and its homologs. Phylogenomics has also been described as a new era of phylogenetic analysis in which more entire genomes have been sequenced. Plant phylogenomics has an advantage over other species in that it can find hundreds of low copy number nuclear genes, making molecular systematics and evolutionary biology much easier to examine. Plant phylogenomics researchers may now use current NGS methodologies to learn more about plant genome diversity, such as the nature and frequency of genome duplication across a variety of plant lineages. There are two major objectives that phylogenomic research attempts to achieve. The first step is to use nuclear genomic data to uncover evolutionary patterns among plant species. The second goal is to come up with a new theory for the unknown function of plant genes linked to important divergence events in plant evolution. Genomic data have greater advantages in evolutionary research than morphological data, which can be readily misinterpreted, or fossil data, which is frequently fragmented. Phylogenomics also uses a set of orthologs derived from the genomic sequence in a phylogenetic context to identify gene and biological process hypotheses. The main difference between functional phylogenomics and traditional phylogenetic analysis methods and current functional genomic methods is that in phylogenomics research, genomic data is mined without taking into account the phylogenetic context when looking for orthologs or candidate genes of functional significance. However, creating the tree of life or phylogeny of all organisms using phylogenomics as the advanced method, which inferred evolutionary relationships, remains a contentious subject. Some studies revalidated the locations of certain plant species in biological taxonomy on a regular basis in order to obtain the most accurate tree. As a result, due to several restrictions, such as conflicting techniques and character sets and systematic errors from simply adding more sequences, determining how to construct a scientifically important topology remains a challenge. The most difficult aspect of phylogenomics is determining how to properly handle huge amounts of genomic data in order to avoid making systematic assumptions. However, statistical confidence or P-value, which is commonly used in such phylogenetic issues, was found to be incorrect. The magnitudes of differences and biological relevance should be given more attention in order to obtain reliable results. Another option is to improve existing phylogenetic

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methods so that phylogenomic links can be inferred with fewer technical biases and more computing power. There are two ways to conduct these searches. Searching the internet using key terms or looking through specific databases relevant to genetic studies are two options. Despite the fact that the information in these databases isn't always up to date, the second method is more suitable for obtaining the finest results. When it comes to plant genome information, there are four primary genomic project databases to choose from: GOLD or Genomes Online Database, NCBI genomes, CoGepedia, (Figure 3.7) and plaBi. Gold is a World Wide Web resource that provides full access to information about genome and metagenome sequencing efforts, as well as the metadata that goes with them. GOLD is presently hosted by the JGI DOE Institute, with version 5 being the most recent release. More than 20,000 studies, 60,000 biosamples, 60,000 sequencing projects, and 50,000 analytic projects are now stored in the database (Bernal, Ear, & Kyrpides, 2001). GOLD is unique in that it encompasses not just nuclear and organelle genome research, but also transcriptome, methylation, exome, and re-sequencing initiatives. Species from the phyla Chlorophyta and Streptophyta are involved in around 100 and 3,400 completed or continuing initiatives, respectively. GOLD is handcurated, with quality-controlled metadata, and fully supports and adheres to the Genomic Criteria Consortium (GSC) Minimum Information standards.

Figure 3.7: CoGepedia. Source: png

https://genomevolution.org/wiki/images/d/d9/CoGe_system_design.

NCBI Genomes is an NCBI (National Center for Biotechnology Information) database that organizes genome-related information such as sequences, maps, chromosomes, assemblies, and annotations. The NCBI

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Genome database gathers genome sequencing initiatives for a given species and links them to records in the BioProject, Assembly, Nucleotide, and Protein databases. More than 2,200 eukaryotic genomes have been deposited so far, with over 200 of them corresponding to plant species. Assembly, a new database from NCBI, was recently launched. The database keeps track of changes to assemblies as they are updated by submitting groups throughout time with a versioned Assembly accession number. The Assembly database contains metadata such as assembly names, simple assembly statistical reports, and assembly change history. Users can readily obtain sequence and annotations for current versions of genome assemblies from the NCBI genomes FTP site using links in the Assembly resource. CoGepedia is the wiki page for CoGe, a platform for comparative genomics research that offers a network of interconnected tools for managing, analyzing, and visualizing next-generation sequencing data. This data is organized in a phylogenetic tree made up of roughly 100 species that have been sequenced (Kim et al., 2016)draft sequences or pseudomolecules have been published for more than 100 plant genomes including green algae, in large part due to advances in sequencing technologies. Advanced DNA sequencing technologies have also conferred new opportunities for high-throughput low-cost crop genotyping, based on single-nucleotide polymorphisms (SNPs. plaBi is a plant genome database that includes an up-to-date tool for determining which plant species have been sequenced. This data can be viewed from either a chronological or phylogenetic standpoint. There are also links to the research publications where each plant genome has been published. Ensembl Plants is an integrative resource that provides genome-scale information on 39 sequenced plant species, including 12 dicots, 21 monocots, one moss, one pseudo fern, two green algae, one red alga, and the genome of Amborella trichopoda, a sister group to the other angiosperm species. Genome sequence, gene models, functional annotation, and polymorphism loci are among the data supplied. With the help of existing genome alignments, a comparative analysis may be done on the entire genome. Gene families are offered based on an all-versus-all BLASTP alignment. Under the Plant Compara section, you can find gene trees that depict the evolutionary history of each gene family. A genome browser with tracks displays genome sequence and assembly information, additional gene model and variation datasets, and precomputed sequence alignments including ESTs, RNA-Seq studies, repetition features, oligo-probe, and marker sets provides access to the data. Ensembl Plants is updated four to five times a year and was created in collaboration with the

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Gramene database and the transPLANT project, which aims to make it easier to exchange and integrate plant genome data from disparate sources while also developing common standards and protocols. Gramene is a curated online resource for comparative functional genomics in crops and model plant species, with 45 sequenced reference genomes currently available in build number 48. Since 2009, Gramene has collaborated with Ensembl Genomes’ Plants division to develop the genome browser described above, which uses the Ensembl infrastructure to provide an interface for exploring genome features, functional ontologies, variation data, and comparative phylogenomics. Genetic and physical maps with genes, and ESTs and QTLs location studies of proteins, plant pathways databases like BioCyc and Plant Reactome platforms, and descriptions of phenotypic features and mutations are just a few of the tools available in Gramene.

3.8 PLANT GDB Plant GDB is a website dedicated to the development of reliable genome annotation methodologies, tools, and standard training sets for plant genomes. Plant GDB has been providing access to sequence data from 29 plant species since 2012. Plant GDB also includes annotated transcript assemblies for over 250 plant species, with transcripts mapped to their relevant genomic context when possible, and is integrated with a number of sequence analysis tools and web services. Plant GDB is home to a plant genomics research site that provides easy access to a wealth of research and training resources. The Plant GDB’s funding was terminated in July 2015, and the website is no longer being updated. Plants DB is a database created by the Plant Genome Science Board’s plant genomics department. Plants DB, which presently supports 13 monocot and dicot species, intends to provide a data and information resource for individual plant species, particularly complicated Triticeae member genomes. Searches for sequence similarity can be performed against databases from 18 distinct species (Duvick et al., 2008)with transcripts mapped to their cognate genomic context where available, integrated with a variety of sequence analysis tools and web services. For 14 plant species with emerging or complete genome sequence, PlantGDB’s genome browsers (xGDB. PlantsDB also serves as a resource for integrative and comparative plant genome research. The database framework integrates genome data from model and crop plants and facilitates knowledge transfer using cutting-edge comparative genomics tools like CrowsNest, which was developed to visualize and investigate syntenic relationships between monocot genomes, and the Genome Zipper

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concept for an ordered gene annotation in cereals. Plants DB is a member of the transPLANT consortium. The Joint Genome Institute’s plant comparative genomics portal, Phytozome, is part of the Department of Energy’s Joint Genome Institute. Phytozome’s current release, v10.3, has access to sixty-one sequenced and annotated plant genomes, 47 of which have been grouped into gene families at 12 evolutionarily relevant nodes. It contains connections to the individual pages of the various genomes it hosts, as well as the specific species genomic sequences compiled in the DOE JGI. There are families of linked genes that reflect the current descendants of ancestral genes. They were created using an all-versus-all BLASTP alignment to calculate the evolutionary distance between every two proteins, reciprocal best hit or synteny analysis to identify orthologs, and outgroup scores to accrete paralogs. These families provide quick access to speces-specific orthology/paralogy linkages as well as information on alterations. Each gene has been annotated with the most recent PFAM, KOG, KEGG, PANTHER, and GO designations, as well as its evolutionary history at the sequence, gene structure, gene family, and genome level. Phytozome also makes the plant genomes it holds accessible using the JBrowse genome browsers, which are available for all genomes. PLAZA is a platform established at the University of Ghent for comparative genomics in plants.

Figure 3.8: PLAZA. Source: https://www.researchgate.net/publication/294430579_Integration_of_ genomic_data_to_study_genome_evolution_in_plants/figures?lo=1

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3.9 PLAZA PLAZA (Figure 3.8) includes data from Gene Ontology, MapMan, UniProtKB/Swiss-Prot, PlnTFDB, and PlantTFDB in its extensive structural and functional annotation of genes. Gene families and subfamilies have been identified from the over one million genes annotated in the genomes it contains. First, using an all-against-all BLAST to calculate protein sequence similarity, and then using graph-based clustering algorithms defined in TribeMCL and OrthoMCL. Phylogenetic trees to identify physiologically relevant duplication and speciation events, as well as extensive information about genome organization to reveal tiny and large genome duplication events, are also provided in this database. This collection also includes methods for transferring functional annotation from well-studied plant genomes to different plant species. Biodiversity International and the International Cooperation Center for Agricultural Research for Development collaborated to create GreenPhylDB, a comparative genomics database (CIRAD). The current version of GreenPhylDB 4.0 has 37 Plantae species, including one red alga, two green algae, one moss, one lycophyte, one conifer, the ancestral angiosperm Amborella, 10 monocots, and twenty eudicot species. Annotated sequences are also grouped into gene families in this database(Valentin et al., 2020). TribeMCL was used to cluster the data. This software uses a variety of pairwise similarity matrices derived from protein-protein BLAST searches with more rigorous standards. After that, a Markov cluster technique is used to arrange proteins in families at different levels of clustering using these matrices. The automatic clustering results are manually annotated with cross-reference databases and examined using a phylogenetic-based technique to infer homologous relationships. GreenPhylDB now has 8,347 clusters with more than 5 sequences at level 1, of which 2,939 are annotated and 4,788 have phylogenetic trees accessible. This database gives protein domains, orthologous gene predictions, and important external links for each gene cluster, as well as rapid access to the gene composition by species(Valentin et al., 2020). Plant breeding and genetics efforts have historically been driven by inadvertent plant selection and later cropping, as well as the need and desire for more food and feed items. Plant genome components were elucidated, and whole DNA sequences of plant genomes governing the entire plant life were decoded as a result of the work achieved toward this goal. Plant genomics aims to develop high-throughput genome-wide-scale technologies, tools, and methodologies to elucidate the fundamentals of

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genetic traits/characteristics, genetic diversities, and by-product production; to comprehend phenotypic development throughout plant ontogenesis with genetic by environmental interactions; to map important loci in the genome; and to speed crop improvement. Plant genomics research has increased steadily over the last 30 years, thanks to the development of low-cost, highthroughput DNA sequencing tools that have resulted in the sequencing of 100 plant genomes with far-reaching consequences for plant biology study and application. The Plant Kingdom is an important part of our planet's food chain. Plant domestication by humans happened early in human history, and subsequent agricultural activity and incidental and purposeful plant breeding resulted in the development of profitable crop species that provided food and nourishment for all living organisms, including humans. Plant species are extremely diverse, with over 300,000 species worldwide. To meet the human diet needs, humanity currently cultivates 2000 plant species on 15.5 million square kilometers of agriculturally appropriate land. Crop domestication, followed by breeding and cultivation, has resulted in the creation of 15 priority crop species that produce more than 90% of food (Rieseberg, 1997). Plants offer clothes and housing materials, and earth ecology, give medications and treatments for a variety of ailments, provide energy and biofuels, and have a variety of other important features and uses that help us comprehend life on our planet. Plant domestication, combined with the desire and demand for more food and feed products, has resulted in ongoing breeding and genetics initiatives. Primitive selection efforts led to the development of methods for shuffling traits and attributes between plant genotypes via controlled sexual crosses, which led to the discovery of the genetics of essential crop characteristics. These progressions have resulted in the creation of superior crop genotypes, which have aided in the rise of agricultural productivity. For the past 50 years, the cereal crop yield has increased 2.6 times due to the Green Revolution, efficient exploitation of plant genetic diversity and plant germplasm resources, novel cultivar development, and better and appropriate agrochemical technologies, whereas maize production has increased 5-fold. There are numerous examples of conventional breeding initiatives that have been effective. Despite this, food insecurity and human starvation are still widespread, and they will get substantially worse when the world human population grows to 9 billion by 2050, with 1 billion people at risk of going hungry (Evenson & Gollin, 2003). In an era of global climate change, everworsening environmental conditions on Earth, societal globalization, and

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technological breakthroughs, there is a desire and need to feed the growing human population, sustain agricultural production, and face newly emerging biosecurity challenges. These events motivated the plant breeding and genetics community to add precision techniques beyond conventional hybridization, selection, and cultivation or farming procedures to enhance and power traditional plant breeding and genetics methods. This is also dictated by the long history of traditional breeding and crop improvement, which has been hampered by limitations in phenotypic evaluations, masking the effect of the environment, the polygenic nature of traits with many unnoticed minor genetic components, negative genetic correlations between important agronomic traits, linkages, and distorted segregation problems in hybridization between diverse genotypes. To solve all of these issues, plant scientists have attempted to decode the molecular basis of genetic diversity by cloning and sequencing the genes expressing the desired feature, and then using these genes in plant breeding as tools for vertical or even horizontal gene transfers. Plant genome composition has been revealed, and the full DNA sequences of plant genomes controlling plant ontogenesis have been decoded. The term genomics was coined by Winkeler in 1920 and is derived from the term genome, which refers to a haploid collection of chromosomes (Greilhuber, Doležel, Lysák, &Bennett, 2005). Similarly, plant genomics is a plant science discipline that aims to decode, characterise, and analyze the genetic compositions, structures, organizations, and functions of a plant genome, as well as molecular genetic networks. Plant genomics aims to develop high-throughput technologies and methodologies to elucidate the fundamentals of genetic traits or characteristics, genetic diversities, and byproduct production; to understand phenotypic development and to accelerate genome-wide crop breeding and selection. Plant genomes research has developed steadily during the last 30 years. The number of scientific papers on plant genomics research has risen dramatically, reaching 17,210 in 2015, according to the PubMed database, with the first increase occurring in 2000-2001 and a large peak occurring after 2010. The model plant Arabidopsis had the first fully sequenced plant genome, which was released in 2000. By 2013, around 50 plant genomes had been fully decoded, and the plant sciences community had completed more than 100 plant genomes by 2015. Furthermore, the plant sciences community elaborated on a sequencing vision of 1001 Arabidopsis accessions and 1000 plant species that will have broad implications for areas as diverse as evolutionary sciences, plant breeding, and human genetics,

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while posing many unexpected challenges and grand tasks (Twyford, 2018). The reference genomes for plants, including specialized crops, have been sequenced, resulting in a new paradigm for modern crop development. Crop breeding has rapidly spread and grown ever more productive and efficient in the plant genomics era, aided and enhanced by molecular markers, genetic linkage maps, QTL mapping, association mapping, and marker-assisted selection approaches in the previous century. This is due to the availability of large-scale transcriptome and whole-genome reference sequences; high-throughput SNP markers and cost-effective technologies, which allow breeders to screen multiple genotypes in a short amount of time in identification and use of expression QTLs in breeding. The economical sequencing and resequencing potential for population individuals of genetic crossings and breeding lines has been the most powerful driving force for genomics-assisted crop breeding in the plant genomics era. This allows researchers to more precisely detect and link genetic differences to phenotypic expressions by accounting for rare allelic variations seen in crop line populations or germplasm resources. The availability of SNP markers and automated genotyping systems enabled genome-wide genotype-to-phenotype associations (GWAS) to be performed. Breeders utilizing GBS and HTS systems can also genotype their mapping population and give genomic selections for the specific crops of interest when wholegenome sequences are not available and SNP markers are only present in a restricted number. Although genomic selection was first used in animal breeding, it has lately been effectively applied to a number of plant species, including studies that used GBS in the context of genomic selection. Most notably, the use of accessible genomics tools, as well as a vast number of high-throughput DNA markers and next-generation genotyping platforms, has enabled breeding by design and virtual breeding approaches for effective crop development. The availability of genome sequences and a vast number of SNP marker collections has tremendously aided crop development efforts by allowing for the investigation of copy number variants (CNVs) in crop genomes and their linkages to key traits. Furthermore, despite the hurdles, post-genome sequencing developments have allowed for the integration and enrichment of genomic selection with critical proteome and metabolome indicators. This greatly aided and accelerated the development of complicated crop traits. As a result of the knowledge gained from plant genetics combined with proteomic and metabolomic breakthroughs, chemical genomics has

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permitted the birth of a novel approach to agriculture. This necessitates the translation of pharmaceutical industry knowledge and expertise on the development of personalized medicine to treat each person based on their reaction to medicinal medications into agriculture. Many plant compounds, such as herbicides, growth regulators and phytohormones, elicitors, low molecular metabolites like salicylic acid can be screened for a genetic response of individual crop genotypes and studied for their mechanism of action contributing to agricultural productivity. Once discovered, highly genotype-specific chemical compounds can be produced that have a greater impact than commonly used "fit-for-all" pesticides, growth stimulators, and fertilizers. A combination of chemical genomics, proteomics and metabolomics, genetic engineering, and genomic selection will pave the way for agriculture that ensures crop production sustainability.

3.10 WEEDS Weeds have constantly disrupted crop plants since their domestication, resulting in higher production losses than diseases and pests, necessitating the use of weed control techniques. Weed control is critical to ensure that enough food is available for a fast-growing human population. Weed management strategies that combine chemical weed control or herbicides with integrated weed management (IWM) approaches can be the most successful and dependable. The use of herbicides for weed control necessitates the development of herbicide-resistant (HR) crops as soon as possible. Recent advances in genome editing technologies, particularly CRISPR-Cas9, have opened up new opportunities for providing sustainable farming in the current agricultural business. To date, genome editing has resulted in the development of numerous non-genetically modified (GM) HR crops that can play a key role in combating weed problems while also enhancing crop output to meet rising global food demand. We discuss the chemical way of weed control, herbicide resistance development approaches, and the potential benefits and drawbacks of genome editing in herbicide resistance. By 2050, the global human population is predicted to increase to 10 billion people, putting significant pressure on present agriculture to produce 25–70 percent more food to meet the growing population's nutritional needs. Global food output must be expanded from 70% to 100% to meet

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human food demand. The world's total grain production is currently 2.1 billion metric tonnes, with a yield loss of 200 million metric tonnes, with weeds accounting for up to 10% of this loss(Hawkes & Lobstein, 2011). Weeds are the most common of all crop pests, invading crop areas year after year. Weeds provide a multifaceted problem in every cropping system, competing for water, space, nutrients, and sunlight, negatively affecting crop productivity. The most serious consequence is a reduction in the end product's quality and quantity. Weeds not only carry viruses and insects that affect crop plants, but they also harm native habitats, putting local animals and plants at risk. Weeds can quickly spread from their natural environment to different places around the world due to their rapid growth capability and adaptation to multiple environments, interfering with crop development and impairing ecosystem processes. They reduce input usage efficiency, induce the loss of very fertile soils, and raise cultivation expenses, in addition to direct and indirect losses. Weed competition and allelopathy are directly related to agricultural production reduction. Generally, a 1-kilogram increase in weed growth corresponds to a 1-kilogram decrease in crop growth(van Heemst, 1985). Weeds have thus been recognized as serious plant pests since antiquity. Weeds have always played a part in agricultural plant domestication, leading to the development of numerous weed management measures. Physical and manual weeding equipment were used to till the soil to manage weeds when weed problems first appeared in agriculture. Other strategies were afterwards adopted, such as biological and cultural approaches. Although these strategies aid in increasing agricultural output and reducing weed infestations, they have several drawbacks, including inconsistent weed control, lower labor availability, and higher labor costs. These approaches aren't always successful, aren't long-lasting, and can be costly. Weed management is critical in current agricultural systems to ensure adequate crop productivity, and the goal is to maximize yield while lowering costs. Herbicide application became an important aspect of weed management programs in agriculture as a result of ineffective weed control. Since its adoption, herbicide technology has provided an effective and relatively inexpensive means of weed management, reducing severe financial strain and contributing to increased average production.

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Figure 3.9: Herbicides. Source: https://www.intechopen.com/chapters/49524

3.11 HERBICIDES Herbicides (Figure 3.9) are currently used extensively as the major weed control method for agronomic crops. Herbicides can be taken up through leaf and root absorption, causing phytotoxic effects near the entrance point, or they can be translocated throughout the plant, depending on the application method. The active chemicals pass through multiple barriers after foliar application, including epicuticular waxes and leaf cuticles, before reaching the apoplast and entering the plant cells. Herbicides can also enter the plant through the stomata and reach the mesophyll cells. The root hairs and root tips are the most common sites for uptake in roots. Herbicide absorption in roots is a two-step process, with the first being quick uptake by bulk water flow and the second being herbicide diffusion along a concentration gradient, which is a non-metabolic process. The second step is linked to the metabolic process, which results in a slower entry and accumulation of material. Herbicides can be metabolized by a natural metabolic mechanism of plant detoxification, which comprises four steps, (a) conversion, which involves modifying the active components chemically through reduction, oxidation, oxygenation, and hydrolysis. The compounds become more hydrophilic and less phytotoxic after functional groups like COOH, OH, NH2 are introduced. This process is aided by the enzyme Cytochrome P450 monooxygenases (P450). (b) Conjugation, in which herbicide molecules or metabolites generated from conversion are conjugated with amino acids, sugars, or the tripeptide Glutathione, increasing their water solubility and

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lowering phytotoxicity. Conjugation with glutathione, homo-glutathione or glucose is one of the most important conjugation pathways seen in most plants. (c) Secondary conjugation takes place, resulting in non-phytotoxic molecules. Furthermore, conjugated metabolites are primarily carried into the vacuole by ABC transporters. (d) Compartmentalization, in which metabolites from the detoxification process are compartmentalized in the vacuole and may be associated to insoluble residues formed by cell wall components such as polysaccharides, lignin, pectin, and protein fractions (Vencill, 2002).

Figure 3.10: Genome editing techniques. Source: https://www.nature.com/articles/s41467-018-04252-2/figures/1

Genome editing techniques (Figure 3.10) have been successfully utilized to target genes in a variety of crop species to improve average crop yields in order to satisfy the rising needs of the current global food famine. They can give a cost-effective and environmentally sustainable agricultural programme to improve cultivars for better quality, higher yield, disease resistance, and HR. Due to its great efficacy, adaptability, simplicity, and consistency, this method has transformed the area of crop breeding in recent years. For HR development in plants, all modern genome editing technologies have been used, including transcription activator-like effector nucleases (TALENS), zinc finger nucleases (ZFNS), clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPRassociated (Cas) approaches. CRISPR-Cas9 systems are the most effective

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and frequently used gene editing approach for inducing trait improvement in crop plants, including HR. The most recent advancements in genome editing have resulted in new CRISPR-Cas9 tools, such as base editing, which is more accurate and efficient, and a promising tool that permits targeted point alterations via programmable nucleotide substitution. Techniques like CRISPR-Cas, particularly base editing, have the potential to create non-GM HR crops. In a few countries, non-GM plants generated with CRISPR-Cas systems have been exempted from GMO legislation. As a result, genome editing is now the most suitable alternative to transgenic and conventional processes for the creation of non-GM HR plants, which can give producers a cost-effective weed management solution.

3.12 WEED MANAGEMENT Chemical weed management is the most common approach used worldwide, therefore the evolution of HR weeds and certain environmental problems are the restrictions. The discovery of herbicide modes of action as a result of a revitalized interest in research and development initiatives in the agrochemical business, as well as academic and government organizations, is a beneficial development. Recent research has identified natural phytotoxins owing herbicidal action to novel MOA, after three decades without the identification of a new herbicide MOA. Lipid Biosynthesis, Plastoquinone Biosynthesis, and Imidazole Glycerol Phosphate Dehydratase (IGPD) are among the mechanisms involved in the new mode of action. Natural phytotoxins, such as sorgoleone, have many MOAs that can effectively reduce the development of resistance to the target site by inhibiting photosynthesis in developing seedlings and plants. It plays a role in the suppression of PSII in vitro, which results in a reduction in plant growth. It also impacts essential plant processes including water and solute uptake, inhibits HPPD, and inhibits mitochondrial activities. CRISPR-Cas9 systems and other genome editing methods have been known to target multiple genes. Base editing techniques are currently being utilized to modify TaALS and ACCase genes that provide resistance to various herbicides. This method could be useful for producing plants that are more resistant to a variety of herbicides. Bipyrazone is a newly developed candidate of HPPD inhibiting herbicides that has been reported to reduce broadleaf weeds in wheat growing areas in China. Bipyrazone was used as a post-emergence herbicide in the greenhouse and in the field, and it was found to be operative in controlling broadleaf weeds.

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Integrated weed management (IWM), a complete approach to controlling weeds that combines the application of complementary weed control measures such as biological control, herbicide application, grazing, and land fallowing, is now receiving research and financing. IWM has the potential to bring weed populations under control. It aids in minimizing herbicide resistance selection pressure, reducing the environmental consequences of specific weed control strategies, and boosting cropping system sustainability. Despite the fact that genome editing has resulted in the development of a number of HR crops, agricultural trials using these crops are rare. HR technology, on the other hand, can be a key component of IWM. HR crops can be used in IWM to create a long-term, environmentally beneficial, and economic weed control solution. HR crops have been the most effective way for farmers to control weeds during the past two decades. They were available at a period when weed management was becoming more expensive and time-consuming for contemporary agriculture, as farm sizes became larger and the number of farm laborers shrank. Thus, the capacity to manipulate biotechnology to develop HR crops was a significant scientific breakthrough that revolutionized weed management by providing a non-chemical weed control option. HR crops have been developed through a variety of mechanisms, including altering the plant to induce a mechanism that prevents the herbicide from reaching a molecular target site, introducing or enhancing herbicide deactivating or degrading enzymes into the plants, and changing the plant to induce a mechanism that prevents the herbicide from reaching a molecular target site. The principal method of natural crop resistance to targeted herbicides is metabolic inactivation or degradation. HR crops, including glyphosate-resistant cotton, maize, canola (Brassica napus), and soybeans (Glycine max), have had a significant impact on weed management. HR technology has quickly gained widespread usage due to its ability to contribute to considerable gains in yield and cost savings, as well as its efficacy and simplicity in weed management. For the creation of HR in plants, however, a variety of strategies or approaches have been used, including genome editing. HR crops, despite their efficiency, have some environmental consequences, such as influencing farming methods, agronomy, weed management, and biodiversity loss. Herbicides appear to be inescapable in the current weed environment. Weed management by a single method, such as herbicides, is, however, impossible. In combination with IWM, gene-edited HR crops harbouring novel and multiple MOA could be more successful at controlling weeds and reducing environmental consequences.

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3.13 MUTATION BREEDING Mutation breeding (Figure 3.11) is a process of inducing heritable mutations in an organism’s genetic material using chemical, physical (UV rays), or mobile genetic components. In mutation breeding, there are three types of mutagenesis: induced mutagenesis, where mutations are induced by radiations like X-rays, gamma rays, ion beam or chemical mutagen treatment; (ii) sitedirected mutagenesis, which is the method of creating specific mutations at target sites in a DNA molecule, mainly performed with PCR-based methods, traditional PCR, and inverse Induced mutagenesis has been used to produce novel genetic alterations since the 1930s. As a result, various crop plants with better monogenic features have been created. Chemical mutagenesis and subsequent herbicide selection create many herbicides tolerant (HT) crops, such as soybean tolerant to sulfonylurea herbicides, sunflower tolerant to imidazolinones and sulfonylurea, and wheat tolerant to sulfonylurea.

Figure 3.11: Mutation breeding. Source: https://www.mdpi.com/2223-7747/8/5/128#

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All commercial herbicide-tolerant crop varieties, on the other hand, have been generated via a single nucleotide change of genes encoding proteins or enzymes that are targeted by herbicides. Certain HT alleles are heterozygous for herbicide tolerance induction, while others tend to be homozygous. Except for the triazine-tolerant mutation, which has pleiotropic alleles, all commercial HT mutations have incompletely dominant alleles inherited maternally that are implicated in numerous agronomic aspects. By crossing with a trait donor, HT characteristics can be introduced into an elite variety. Induced mutagenesis has a number of flaws, including the fact that the approach is usually random and unstable, and advantageous mutants are few and mostly recessive. High population size and efficient mass screening procedures are required for picking uncommon variants. To reduce background mutations and eliminate chimaeras, a dense mutation burden necessitates extensive breeding. Multiple gene variants occurring at the same time are extremely rare. Furthermore, transcription factors are implicated in the regulation of many quantitative features, and changes in these genes can have an impact on the transcriptional function of their downstream targets, which could explain the effects of quantitative changes. Crop growers had long preferred mutations at specific places within the plant genome rather than random non-specific variants such as those caused by chemical or radiation mutagenesis. As a result, when genetic engineering technology became available, it became possible to precisely and quickly introduce particular mutations within the target gene to induce gene suppression or gene expression. Plant science has made significant progress in the development of novel biotechnology-based plant breeding strategies for modifying genetic and epigenetic variables. Site-specific nucleases and gene-targeting oligonucleotides can be used to create novel plant products (NPPs) through cisgenesis, intragenesis, and genome engineering. Zinc finger nucleases, and Cas9 nucleases linked with clustered, regularly interspaced, short palindromic repeats are among them. Improved plants and fruit trees free of transgenesis and cisgenes have resulted via reverse breeding procedures and backcrossing modified plants with natural kinds. NPPs devoid of viral sequences and antibiotic genes, and containing solely genetic material acquired from the species itself or from closely related species, are gaining in popularity. There is a need for harmonization in the rule and defining disparities between modified and non-modified plant genomes, as well as in the differences between regulation and regulatory authorities in various nations. Plants contain a minimal number of hormones compared to animal

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and insect organisms. The complexity of hormone crosstalks among them is to blame for this. Recent research using Arabidopsis and rice as model plants has revealed the interplay of a range of signaling molecules with phytohormone metabolism and signaling.

3.14 ALLERGIES Allergies in the Western countries in recent decades, and several protein families have been discovered as factors of allergenicity. So far, four allergen families (Mal d 1–4) have been found in apple fruit, one of the world’s most significant fruit crops, including pathogenesis-related proteins, thaumatinlike proteins, lipid transfer proteins, and profilins (Nomura, Morita, Ohya, Saito, & Matsumoto, 2012). Furthermore, it has been shown that patient sensitivity varies depending on apple variety, fruit tissue, culture, and preservation circumstances, making it more difficult to link genetic, molecular, and biochemical data to clinical test results. Allergens are structured in vast families with many distinct isoforms, the role of which to allergenicity is still largely unknown, according to mapping studies and the availability of the whole apple genome sequence. Antibody-based therapies and molecular farming are being investigated in a growing number of therapeutic modalities. There are currently a variety of designed formats for antibody molecules as well as many approaches for raising and adjusting binding specificities. Recombinant secretory IgA can be expressed in plant cells (sIgA). Humanized antibodies against the herpes simplex virus HSV-2 can be produced by transgenic soybeans. GM corn has been shown to produce human antibodies at yields of up to 1 kg per acre and to maintain antibody activity after 5 years of storage under normal conditions. A plant that is designed for large seed and high protein production is clearly favoured for seed production (de Vendômois, Roullier, Cellier, & Séralini, 2009)MON 810, MON 863. Transgenic tobacco chloroplasts can create human somatotropin or interferons at 100-fold higher protein levels than their nuclear transgenic counterparts, with somatotropin accounting for 7% of total plant protein output (Daniell, Lee, Panchal, & Wiebe, 2001)000 copies per cell. Because of their benefits in terms of cost, feasibility, and scalability of production, plants are attractive biotechnological instruments for the synthesis of medicinal proteins and vaccines. Virus-like particles (VLPs) constituted of single or multiple virus proteins with self-assembly capacity are one of the most appealing systems of antigen production in plants

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among biopharmaceuticals. This is because VLPs have a high immunogenic capability and are safe to employ as plant-made vaccines against a variety of human and animal diseases. Furthermore, VLPs do not require lengthy purification stages or the use of a cold chain, which are both limiting considerations in the manufacture of traditional vaccinations. These features enable VLPs to compete as excellent alternative candidate vaccines, either as simple products or as more complicated platforms for carrying heterologous immunogenic sequences on their surfaces. The Euphorbiaceae family is important because it includes commercially valuable crop species like Jatropha curcas, Manihot esculenta, and Ricinus communis, all of which have distinct applications. Jatropha is primarily a biofuel and pharmaceutical crop with the potential to reclaim marginal soils and reduce erosion and desertification risks, whereas cassava is primarily a staple food and provides food security, and Ricinus is a crop with growing economic importance, particularly in the chemical industry. Due to biotic and abiotic stressors, they are all facing significant challenges. The use of molecular markers to investigate genetic diversity throws light on nucleotide polymorphisms and provides information about adaptive processes and population history. The use of diverse molecular detection techniques to identify viral infections that affect Euphorbiaceae allows for the development of protective strategies in situations where multiple species are planted side by side. Furthermore, studies of miRNAs expressed by viral infections that affect Euphorbiaceae led to the discovery of RNA silencing suppressors. The mapping of their targets on the genomes of their host plants revealed that some of these targets are engaged in plant defense mechanisms.

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RECOMBINANT DNA TECHNOLOGY AND PLANTS

CONTENTS 4.1 Introduction ....................................................................................... 84 4.2 Pesticide Resistance ........................................................................... 85 4.3 Biosafety ........................................................................................... 87 4.4 Gene Transfer..................................................................................... 90 4.5 Crop Enhancement ............................................................................ 91 4.6 Resistance.......................................................................................... 93 4.7 Markers ............................................................................................. 95 4.8 Genetic Engineering .......................................................................... 95 4.9 Synthetic Chemicals .......................................................................... 96 4.10 Agrobacterium ................................................................................. 97 4.11 Biotechnology ............................................................................... 101

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4.1 INTRODUCTION Recombinant DNA technology provides the potential to create genetically modified plants with desirable features such as higher biotic and abiotic tolerance in plants, as well as improved flexibility for better survival. In comparison to natural recombination or traditional breeding methods, recombinant DNA technology allows for faster, cheaper, and more accurate insertion of specific features from many sources into the plant genome (Pappu, Niblett, & Lee, 1995). Furthermore, genetic alteration of both nuclear and eukaryotic and plastid or prokaryotic-like plant genomes has produced a transgenic plant with different properties. Genetic engineering has always been a contentious topic because the balance it seeks to strike between the benefits to people and the ethical implications is up for dispute. Concerns in the disciplines of agriculture, medicine, bioremediation, and biotechnology differ according to the discipline. The main source of concern, however, is the real or perceived environmental impact of recombinant DNA technology, particularly the release of genetically modified organisms into the environment. The transfer of genes across bacteria of the same Escherichia coli species pioneered the application of recombinant (r-) DNA technology to make genetically altered species in the early 1970s. Cohen and colleagues transferred an insulin production gene into an E. coli plasmid in 1978, resulting in the world's first genetically engineered organism (GMO)(Rousset et al., 2021). By 1982, national drug regulatory authorities, including the US Food and Drug Administration, had given their full approval to this protocol, allowing for the viable mass production of human insulin, a hormone that controls blood sugar levels and is produced naturally by beta cells in the pancreas. This permitted the widespread commercial availability of insulin at a cost that was reasonable to people with diabetes mellitus types 1 & 2, who either do not make or metabolize enough insulin. This demonstration of genetic modification's medical benefits sparked a trend for molecular cloning technologies to transfer genes expressing desirable features into another host organism, resulting in desirable qualities. This currently includes both prokaryotes like bacteria which are relatively easy to genetically change using r-DNA technology and eukaryotes like yeast, plants, insects, and mammals comparatively complex to manipulate via r-DNA technology. In agriculture, the production of genetically modified crops with the goal of increasing output while also increasing resistance to plant pests or herbicides appears to have achieved popular support and

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is already being used commercially in various countries. The genetically modified tomato was the first commercially cultivated, genetically edited crop product to get a license. This was created in 1994 to express the characteristic of delayed softening of tomato flesh as a practical way to reduce crop losses after harvest. Ironically, given its brand name, 'Flavr Savr,' (Figure 4.1) this flopped in the marketplace due to an apparent lack of taste rather than public apprehension over consuming a genetically altered food. Nonetheless, the introduction of a genetically modified fruit cleared the door for the usage of GMOs in food. In the United States, 88 percent of maize and 93 percent of soybeans are genetically modified, and most of this ends up in processed foods without being labeled(Kramer & Redenbaugh, 1994).

Figure 4.1: Flavr Savr. Source: https://alchetron.com/Flavr-Savr#flavr-savr-e610022c-006f4709-bad5-b8bd685bd7f-resize-750.jpg

4.2 PESTICIDE RESISTANCE In some areas, the introduction of pest-resistant brinjal also known as eggplant or aubergine was met with opposition, despite the popularity of pest-resistant cotton at the time. The same crystal protein gene (Cry1Ac) from the soil bacteria Bacillus thuringiensis (Bt) was inserted into the

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genome of the host plant expression of which synthesises so-called Bt toxins that confer resistance to predation by Lepidoptera insects in both attempts at implementation. However, of the two uses as food and clothing, human eating was the one that raised concern among the general public. In order to overcome the initial unpopularity of ingesting Bt-brinjals in developing nations such as India, Bangladesh, and the Philippines, the benefits of employing Bt toxin should be emphasised. This would minimize public scepticism based on the erroneous belief that consuming a plant product containing a ‘toxin’ is hazardous to humans, regardless of the toxin’s unrelated target and benign method of action. The emergence of novel genetic modification techniques (nGMs), sometimes known as new breeding techniques in other sources, has sparked debate about their regulation around the world. nGMs are covered to varied degrees by existing regulatory frameworks for genetically modified organisms (GMOs). The extent to which nGMs are covered is largely determined by the regulatory trigger. In general, two different trigger systems can be recognized, depending on whether the development method was used or the features of the final product. One important topic is whether regulatory frameworks based on process or product-oriented triggers are better for regulating nGM applications. The varying criteria and exceptions utilized to execute the triggers in the various regulatory frameworks are more decisive for the regulation of organisms or products, notably nGM applications. In certain countries, there are talks regarding whether legislative changes are required to achieve the desired level of nGM regulation. It was identified that there are five ways for countries wishing to regulate nGM applications for biosafety, ranging from using existing biosafety frameworks without changes to enacting new stand-alone laws. International harmonization will allegedly not be reached in the foreseeable future due to differing degrees of nGM legislation. Transparency about the regulatory status of specific nGM products is important in international trade. In most countries, biosafety frameworks established by particular legislation oversee genetically modified (GM) crop plants developed using recombinant DNA (rDNA) technology.

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Figure 4.2: Biosafety frameworks. Source: https://www.researchgate.net/publication/311821383_The_Biosecurity_Approach_A_review_and_evaluation_of_its_application_by_FAO_internationally_and_in_various_countries/figures

4.3 BIOSAFETY These biosafety frameworks (Figure 4.2) are typically based on the FAO and WHO. The OECD’s core principles for food and feed safety, as well as environmental risk assessments of crops produced by modern biotechnology are important (Sensi, Fao, Brandenberg, Gosh, & Sonnino, 2011). The Cartagena Protocol on Biosafety (CPB), formed under the Convention on Biological Diversity, is particularly significant for the creation and international harmonization of biosafety regimes. When creating national biosafety rules, the Parties to the CPB, currently 171 nations, are required to adopt the Protocol’s stipulations. Genetically engineered plants, particularly in agriculture, have boosted resistance to hazardous agents, increased product yield, and demonstrated increased adaptation for better survival. Furthermore, recombinant medications are now being utilized with confidence, and commercial approvals are being obtained quickly. Bioremediation and the treatment of serious diseases are additional common uses of recombinant DNA technology, gene therapy, and genetic changes. Because of the rapid growth and wide range of applications in the field of

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recombinant DNA technology. Three elements have a significant impact on human life: food scarcity, health problems, and environmental concerns. Aside from a clean and safe environment, food and health are essential human needs. Human food requirements are rapidly increasing as the world’s population grows at a faster rate. Humans demand food that is both safe and affordable. Several human-related health conditions cause a substantial number of deaths around the world. Despite significant efforts, present global food production falls well short of human needs, and healthcare facilities in third-world countries are far worse. Rapid industrialization has increased environmental contamination, and industrial wastes are permitted to mix directly with water, affecting aquatic marine life and, indirectly, humans. As a result, modern technology must be used to overcome these difficulties. Unlike traditional approaches to overcoming agriculture, health, and environmental issues through breeding, traditional medicines, and pollutant degradation through conventional techniques, genetic engineering makes use of modern tools and approaches, such as molecular cloning and transformation, which are faster and produce more reliable results. In contrast to conventional breeding, which transfers a large number of both specific and nonspecific genes to the recipient, genetic engineering simply delivers a small block of desired genes to the target by various methods such as biolistic and Agrobacterium-mediated transformation. Homologous recombination-dependent gene targeting or nuclease-mediated site-specific genome editing are both used to modify plant genomes. Site-specific genome integration mediated by recombinases and oligonucleotide-directed mutagenesis can also be utilized. Changing genetic material outside of an organism to get enhanced and desired features in living creatures or as their products is referred to as recombinant DNA technology. This approach entails inserting DNA fragments from a number of sources into a suitable vector with a desired gene sequence. Manipulation of an organism’s genome can be done by adding one or more new genes and regulatory elements, or by recombining genes and regulatory elements to reduce or prevent the expression of endogenous genes.

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Figure 4.3: Restriction endo-nucleases. Source: http://www.agrilearner.com/restriction-endonuclease/

Using restriction endo-nucleases (Figure 4.3) for specific target sequence DNA sites, enzymatic cleavage is used to generate distinct DNA fragments, which are then joined using DNA ligase activity to fix the desired gene in the vector. After that, the vector is delivered into a host organism, which is cultured to create multiple copies of the integrated DNA fragment, and then clones containing a relevant DNA fragment are selected and harvested. Paul Berg, Herbert Boyer, Annie Chang, and Stanley Cohen of Stanford University and the University of California, San Francisco created the first recombinant DNA (rDNA) molecules in 1973. Regulation and safe use of rDNA technology were debated in “The Asilomar Conference ‘’ in 1975 (Micklos, Freyer, & Crotty, 2003). Recombinant DNA technologies to encourage agricultural and medication development took longer than planned due to unexpected obstacles and barriers to get adequate results, contrary to scientists’ expectations at the time of Asilomar. Since the mid1980s, however, a growing range of goods such as hormones, vaccinations, therapeutic agents, and diagnostic tools have been produced to improve health. Recombinant DNA technology provides a rapid way to examine the genetic expression of mutations induced into eukaryotic genes via cloned insulin genes inserted into a simian virus fragment.

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For nearly two decades, molecular approaches have been utilized to introduce novel traits into agronomically important plants, such as disease, insect, and herbicide resistance. Antibiotic resistance genes were exploited as selection markers in the development of several transgenic plants. The widespread usage of transgenic plants in agriculture results in a substantial amount of recombinant DNA in the environment. It has been mentioned as a concern that an accidental transfer of recombinant genetic material into the soil microbiota may occur, resulting in increased antibiotic resistance in bacteria, including human pathogens. Simultaneously, the recombinant and thus distinct nucleotide sequences of genetically modified organisms’ DNA allowed for quantitative tracing of DNA from transgenic organisms in the environment via PCR amplification. High molecular weight DNA has been detected in areas where free DNA or plant material has been deposited, and it has been reported to remain in non-sterile soils for months. DNA produced by eukaryotic and prokaryotic cells is thought to form an extracellular gene pool that can be accessed by biologically competent bacterial cells that pick-up DNA and integrate it into their genomes or natural transformation. Transformation was discovered in non-sterile soils in microcosm tests. There has been no evidence of recombinant DNA transmission from transgenic plants to soil microorganisms.

4.4 GENE TRANSFER Gene transfer from transplastomic tobacco plants to Acinetobacter sp. strain BD413 was demonstrated when the plants were experimentally coinfected with Acinetobacter and Ralstonia solanacearum. The nptII gene, which is found as a selection marker gene in the genomes of various transgenic plants, was previously utilized to evaluate the conditions for horizontal plant DNA transfer into competent bacteria. When the recipient cells supply DNA homology for transgene integration by homologous recombination, recombinant plant DNA can change competent cells to antibiotic resistance. In the absence of homology, integration could not be detected. Transgenic potato plants harbouring nptII as a selection marker was evaluated and the presence of recombinant DNA in their environment to determine the level, frequency, and dynamics of DNA spread from plants during growth was studied. A biomonitoring test based on natural transformation of Acinetobacter sp. strain BD413 to detect recombinant DNA was employed. During the DNA uptake process, this species does not distinguish between its own DNA and foreign DNA. The technique has been used to detect nptII

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genes in leaf DNA extracts from a variety of transgenic plants, including potato, rape, tobacco, tomato, and sugar beet plants. It’s also been used to detect recombinant DNA from transgenic sugar beet plants in environmental samples recently. A biomonitoring genetic system was used to make it more specific for a certain recombinant construct, in this case, the DNA of a transgenic potato with a nptII-tg4 terminator fusion. All features are defined by their genetic makeup and how it interacts with the environment. An organism’s genetic makeup is its genome, which is made up of DNA in all plants and animals. Genes, or DNA sections that carry the instructions for generating proteins, are found in the genome. These proteins are responsible for the plant’s appearance. Genes that convey the instructions for generating proteins involved in producing the pigments that color petals, for example, determine the color of flowers. Transfer of DNA into a plant cell is the initial step in creating a GM plant. One way for transferring DNA is to cover the surface of small metal particles with the appropriate DNA segment and then bombard the particles into plant cells. Using a bacteria or virus is another option. Many viruses and bacteria transfer their DNA into a host cell as part of their usual life cycle. The most commonly utilized bacterium for GM plants is Agrobacterium tumefaciens. The desired gene is introduced into the bacterium, and the bacterial cells subsequently transfer the new DNA to the plant cells’ genome. The plant cells that successfully took up the DNA are then developed into a new plant. Because individual plant cells have the ability to create complete plants, this is possible. In rare cases, DNA transfer can occur without the need for human intervention. The sweet potato genome, for example, contains DNA sequences that were transferred thousands of years ago from Agrobacterium bacteria.

4.5 CROP ENHANCEMENT Genetic crop enhancement is achieved through both conventional plant breeding and genetic modification (GM). For thousands of years, genetic improvement has been a key component of increased agricultural output. Plants that can compete for light, water, and nutrients with neighboring plants, defend themselves from being eaten and digested by animals, and disseminate their seed across large distances are favored by natural selection. These features are in direct opposition to agricultural goals, which call for plants to devote as much of their resources as possible to producing nutritious, easy-to-harvest goods for human consumption. Because of the

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striking disparity between what natural selection has generated and what produces a productive crop, we’ve utilized traditional breeding methods to convert plants that compete well in the wild to plants that perform well in agriculture for thousands of years. As a result, we now have crop types that are significantly more productive and nutritious than their wild parents, but less useful in the wild. Crops can be genetically modified or conventionally modified to add new traits. This allows us the question whether a plant breeder should use a GM method rather than a traditional one. Only two conditions must be met for GM to be used to introduce a new trait into a crop. First, the trait must be able to be introduced with only a minimal number of genes, and second, it is required to determine which gene or genes are involved. We understood a lot less about which plant genes do what when GM technology was introduced, which limited the number of useful applications for GM in crops. We now know numerous genes that could help increase sustainable food production, thanks to advances in our understanding of which plant genes do what. In certain circumstances, traditional breeding that is, cross-breeding with the plant that possesses the genes that provide these qualities will be the ideal strategy to deploy these genes. In some circumstances, GM, in which scientists extract a gene and insert it directly into a plant, may be the simplest or only option to use them. There are two key reasons why GM may be the better option. To begin with, the desired gene may not exist in a species that may successfully cross with the crop. It’s possible that the gene came from a different kingdom, such as a bacterium, or from a different plant species. Many plant species in nature respond to shade by growing taller, allowing them to compete for light. The capacity to change their height is based on a specific protein that blocks stem elongation, and the plant can fine-tune its development by adjusting the amount of this protein in the stem. As part of the so-called Green Revolution in the 1960s, dwarf wheat cultivars were developed, drastically increasing yields. The dwarf wheat types take advantage of a mutation in the gene encoding the height adjusting protein, which increases the quantity of the protein in the stem, preventing stem growth. As a result, wheat types devote more resources to their seeds rather than their stems. As a result, they yield more and are less likely to be flattened by the wind, which is a primary cause of yield loss known as lodging. Concerns have been raised that simply adding new DNA into a plant genome by genetic modification could have unforeseeable repercussions. However, as our understanding of genomes has grown, it has become evident

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that comparable insertion events occur in all plants on a regular basis. Some bacteria and viruses, for example, introduce new genes into the genomes of the plants they infect. Plant genomes have a large number of so-called ‘jumping genes,’ which travel about the genome, re-inserting themselves in different locations. As a result of these processes, all new crop varieties, regardless of how they are created, may contain genes placed in previously unknown locations in the genome and novel genes that have never been found in the food chain or come from non-plant species. This means that both GM and non-GM crop varieties may have unintended repercussions on occasion.

4.6 RESISTANCE Resistance to the herbicides (glyphosate) in soybeans was the first GM trait to gain widespread adoption. Herbicide-tolerant crops can also be grown without using genetically modified seeds. Resistance to wide herbicides which would normally kill both weeds and crops allows for effective weed control since the herbicide may be given while the crop is growing without harming it. Without herbicide-tolerant crops, a variety of herbicides may be required to eradicate all weeds prior to sowing the crop. Herbicide-tolerant crops also have the advantage of being able to be planted in weedy fields because the weeds may be controlled with herbicide. This eliminates the need for ploughing, resulting in reduced soil erosion. The farmer must purchase a special herbicide to match the herbicide-tolerant crop, and this form of control goes against efforts to lessen agriculture’s reliance on chemical inputs. The Bacillus thuringiensis (Bt) bacteria creates a toxin family of proteins that are poisonous to certain insects but not to beneficial insects or other mammals. In organic farming, Bacillus thuringiensis is utilized as an insecticide spray. GM has introduced genes for various Bt toxins into several crops. The usage of Bt toxin genes in crops has prevented the administration of 450,000 tons of insecticide during the last 20 years, according to estimates. When weed management is especially effective, insect biodiversity is lost, according to a large farm scale evaluation of herbicide-tolerant GM crops undertaken in the UK between 1999 and 2006 (Tu et al., 1998). It didn’t matter if the crop was GM or not; what mattered was how many weeds remained in the crop. If a tiny portion of agricultural land is left aside for biodiversity, wildlife damage can be reduced. Another issue is the growing problem of weeds developing resistance to herbicides

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as a result of their misuse. Because recurrent growth of the same herbicidetolerant crop requires repeated application of the same pesticide, herbicidetolerant crops, whether GM or non-GM, can produce this problem. Rotating crops resistant to different herbicides or alternating herbicide use with other weed management techniques is one solution. Genetic use restriction technology (GURT) is based on seed germination prevention and was patented by the US government in the 1990s and licensed by private corporations, including Monsanto. In actuality, the technology was never proved to perform reliably. Because the plants would be unable to generate fertile seeds, the notion became known as ‘terminator seed’ technology(Lombardo, 2014). Because of concerns about the potential economic impact on farmers who would be unable to conserve seed for future planting, the United Nations Convention on Biological Diversity imposed an international embargo on the use of GURTs in 2000. When there are license limitations in effect, saving seeds for both GM and non-GM crops is not permitted. Furthermore, farmers and gardeners will be aware of F1 hybrid types, which are created by crossing two or more parents and from which seed cannot be stored since they do not breed true. A 3.5-kb mini-binary vector (pCB301) was created that can be used to clone DNA segments for transfer into the plant genome. However, conjugation cannot be used to introduce this into A. tumefaciens since crucial DNA sequences essential for conjugal transfer have been removed. As a result, electroporation is used to introduce this. Minivector pCB301 was used to create a number of derivatives(Xiang, Han, Lutziger, Wang, & Oliver, 1999)the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS. Despite the fact that A. tumefaciens -mediated gene transfer methods are effective in a variety of species, monocot plants such as rice, wheat, and maize are not easily changed. However, protocols for the transformation of maize and rice by A. tumefaciens -bearing Ti plasmid vectors have been developed by improving and carefully managing circumstances. Immature corn embryos, for example, were immersed in an A. tumefaciens cell suspension for a few minutes before being cultured at room temperature for many days in the absence of selective pressure. After that, the embryos were placed in a medium containing a selective antibiotic that allowed only altered plant cells to proliferate. For a few weeks, these cells were kept in the dark. Finally, the mass of transformed plant cells was transferred to a different growth medium containing plant hormones to encourage differentiation and incubated in the light, allowing entire transgenic plants to be regenerated.

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4.7 MARKERS A plant selectable marker gene, the target gene, right border, an E. coli origin of DNA replication, and a bacterial selectable marker gene are all present in the cointegrate vector. Within A. tumefaciens, the cointegrate vector recombines with a modified or disarmed Ti plasmid that lacks both the tumor-producing genes and the right border of the T-DNA. To generate a recombinant Ti plasmid, the whole cloning vector is inserted into the disarmed Ti plasmid. The disarmed helper Ti plasmid and the cointegrate cloning vector both have homologous DNA sequences for homologous recombination. The cloning vector becomes part of the disarmed Ti plasmid, which contains the vir genes, after recombination. The genetically altered T-DNA region can be transmitted to plant cells in this cointegrated arrangement. When employing binary vectors, one practical issue is that their vast size (>10 kb) makes manipulating them in vitro difficult and cumbersome. Furthermore, bigger plasmids tend to contain fewer distinct restriction sites for cloning reasons. For these reasons, developing and using smaller binary vectors is useful. Based on the DNA sequence of a regularly used binary vector, pBIN19, it was estimated that more than half of the DNA could be erased without affecting the vector’s functionality. E. coli and A. tumefaciens origins of DNA replication or a single broad host range origin of DNA replication are both present in the binary cloning vector. Before the vector is delivered into A. tumefaciens, all of the cloning stages are completed in E. coli. The recipient A. tumefaciens strain possesses a modified Ti plasmid that contains all of the vir genes but lacks the T-DNA section, preventing the T-DNA from being transferred. In this system, the vir gene products are synthesised by the faulty Ti plasmid, which also serves as a helper plasmid. This allows the T-DNA from the binary cloning vector to be introduced into the chromosomal DNA of the plant. Because T-DNA transfer begins at the right border, the selectable marker is normally put adjacent to the left border. Two plant selectable markers, one adjacent to the right border and the other adjacent to the left border, have been created into a few binary vectors.

4.8 GENETIC ENGINEERING Genetic engineering isn’t merely a continuation of traditional breeding. In truth, it is vastly different. Conventional breeding, in general, uses a selection method to create new plant varieties, with the goal of achieving expression of genetic material already existing within a species. Traditional breeding

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makes use of natural processes like sexual and asexual reproduction. The outcome of traditional breeding accentuates specific traits. These features, however, are not unique to the species. The features have existed for millennia within the species’ genetic potential. The primary method of genetic engineering is the insertion of genetic material, albeit gene insertion must be followed by selection. This type of insertion isn’t found in nature. A gene gun, a bacterial vector or a chemical or electrical treatment inserts genetic material into the host plant cell, which subsequently inserts itself into the chromosomes of the host plant with the help of genetic components in the construct. In order for the inserted gene to express itself, engineers must also include promoter genes from a virus. Even though the primary purpose is just to implant genetic material from the same species, this procedure, which uses a gene gun or a similar technique and a promoter, is vastly different from conventional breeding. Beyond that, the approach allows for the insertion of genetic material from hitherto unknown sources. It is now feasible to incorporate genetic material from species, families, and even kingdoms that were previously unavailable as sources of genetic material for a specific species, as well as custom-designed genes that did not exist in nature. As a result, we can construct synthetic life forms, which would be impossible to achieve through traditional breeding. It›s intriguing to compare this development to the breakthroughs that led to the development of synthetic organic compounds in the early 1900s.

4.9 SYNTHETIC CHEMICALS Synthetic chemicals could be considered a continuation of basic chemistry, and in some ways they are. Because we hadn’t co-evolved with them for millennia, many of the effects were negative. PCBs and vinyl chloride, both carcinogens, were discovered, as were various organochlorine pesticides, which were discovered to be carcinogens, endocrine disruptors, immune suppressors, and other issues. These breakthroughs are not identical in many respects, but the experience with synthetic organic molecules highlights the potential for unanticipated outcomes when fresh substances are introduced into the biosphere. This is in contrast to traditional breeding, which only allows genetic material to be transferred between distinct variants within a species, between closely related species, or between closely related taxa. Even extensive crosses and hybridization can’t transport genetic material much beyond these limitations. The great majority of hybrid crops are created by mating two genetically pure, i.e., homozygous for all alleles, lines

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of the same crop to produce a heterozygous line. To develop a mixed line, hybrid corn is merely the crossing of two pure corn lines. The trait that is put into the genome of a host plant can be controlled rather precisely via GE. However, it is still unable to precisely regulate where the characteristic is put into the genome or assure stable transgenic expression. Transformation is the process of inserting foreign genetic material into the host plant genome via GE and expressing that material. Currently, transformation is carried out using a number of primitive approaches that result in a random distribution of genes. Manipulation of bacteria belonging to the genus Agrobacterium is a common transformation approach. These bacteria are among the few known to be able to transfer genetic material from a kingdom/phyla to another. By attaching to plants, transferring bacterial DNA into the plant, and having that DNA incorporated into the host plant genome, these bacteria cause disease in plants.

Figure 4.4: Agrobacterium-mediated plant transformation. Source: https://bioone.org/ContentImages/Journals/arbo.j/2017/15/tab.0186/ graphic/f01_01.jpg

4.10 AGROBACTERIUM Agrobacterium-mediated plant transformation (Figure 4.4) entails removing disease inducing genes, keeping the bacterial transfer DNA (T-DNA), and

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adding the genetic traits or elements to be transferred into the Agrobacterium. This genetically modified Agrobacterium, often known as a bacterial truck, is then mixed with the desired plant cells and allowed to transform or infect them. Agrobacterium-mediated transformation is most commonly used on dicots and is difficult to do with grains. Almost all GE-derived agricultural plants have a potent promoter from the Cauliflower mosaic virus (CaMV 35S promoter), which causes disease in mustard plants in nature. Genes in plants typically have their own promoters, which ensure that the gene is turned on at the appropriate time in development and expressed at the appropriate level, resulting in the production of the desired gene product. Because viruses are genetic parasites that may infect a plant cell and hijack its cellular machinery to create several copies of themselves in a short amount of time, a promoter from a plant virus is employed. The CaMV 35S promoter is chosen because it is a potent promoter that leads to transgene hyper-expression, with transgenes expressed at levels 2 to 3 orders of magnitude higher than the organism’s own genes. As a result, GE requires the use of a foreign promoter, which is not required in traditional breeding, which includes hybridization and extensive crosses, and so distinguishes GE from conventional breeding. Indeed, using the naturally occurring promoter for most of the genes being transferred, the plant would never be able to recognize and express the inserted gene. As a result, a plant viral promoter must be used; hence, the widespread use of a plant viral promoter. Most plant promoters fail to get the gene expressed at a high enough level to complete the job, which is why the CaMV 35S promoter was used. Because of what we’re learning about how plants routinely turn many genes “off” through a phenomenon known as gene silence, CaMV’s ability to turn genes “on” is of special concern. Gene silencing appears to be an important defense against foreign DNA infiltration, especially from diseasecausing organisms, as well as a regulator of normal gene expression. In the last 5-10 years, scientists have discovered that genetic material can be transferred between organisms that are unable to reproduce. Horizontal gene flow or transfer of genes from parent to progeny is a type of lateral movement of genetic material that occurs in nature more frequently. Horizontal gene flow has been observed in microorganisms, and it is one of the primary mechanisms through which antibiotic resistance or pathogenicity is transmitted between bacteria. Viruses can also insert themselves into the genomes of their hosts. Horizontal gene flow in plants was formerly thought to be infrequent or non-existent. Scientists stated in 1998 that genes from a

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fungus had infiltrated 48 of the 335 species of terrestrial plants studied, and that this had happened in 32 distinct cases. They calculated that these genes had infected higher plants over 1000 times via horizontal transfer. Genomes are complex; research on other complicated systems has demonstrated that introducing additional pieces can cause the whole genome to destabilize. Indeed, normal plant development necessitates an exquisite coordination of genes, with the appropriate set of genes being turned on at the appropriate time during development. The plant’s regulation system should include a method to prevent or minimize unintended disruptions of such a complexly coordinated system. Post-integration strategies include gene silencing techniques. Gene silencing was first observed in transgenic plants, and it was assumed that it only happened with transgenes. Because it causes instability, it is a substantial hurdle to genetic engineering. Hyper-methylation of genetic material is one strategy linked with inhibiting transcription, although the predominant mechanism for posttranscriptional silencing is the production of aberrant RNA molecules, with occasional DNA methylation. Indeed, transgene silencing is becoming a more common occurrence. Several factors have been demonstrated to impact transgene inactivation, including the insertion of numerous copies of the transgene, transgene hyper-expression due to the use of the CaMV 35S promoter, and environmental conditions. Gene instability is far more common when there are several copies of the transgene and multiple insertion sites. Because these are hallmarks of direct gene transfer technologies, which are often utilized on cereals, we can predict more issues in these crops. The work done in Germany with petunias that were modified with a single gene from corn to produce a new salmon red bloom was perhaps the earliest, and most widely studied example of such unstable transgene silencing. The scientists worked with a line that carried a single copy of the inserted gene at a single insertion site after converting the petunias. Outside, 30,000 transgenic petunias with a single gene imparting the salmon red flower color trait were cultivated and differences were noticed. Initially, the scientists were looking for naturally occurring mobile elements that would jump into the color gene, disrupting it and causing a different color to emerge. These mobile elements were thought to occur at a frequency of 1 in 100 to 1 in 100,000(Meyer et al., 1992). The discovery that a considerable majority of the plants were either poorly colored, white, had variegated colors, or had distinct colored sectors of the flower was an unexpected outcome. Because petunias can produce up to 50 flowers during the growth

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season, any alterations in individual plants are immediately visible. During the season, the quantity of non-salmon red flowers also increased. Horizontal gene flow is nature's version of genetic engineering. However, only a small number of microbes appear to be capable of inserting DNA into plants, and plants have evolved defenses against this. Furthermore, each insertion is a one-time event, whereas with GE, instead of a single mutant individual arising, the environment is flooded with many altered plants, each having DNA from sources that bacteria would never carry normally. GE is, once again, a quantum leap beyond natural phenomena. The modified genome is destabilized by the simultaneous introduction of the CaMV promoter gene to override silencing. The nearly universal adoption of marker genes that code for antibiotic resistance is another noteworthy difference between conventional breeding and GE. Such marker genes are required to aid in the identification of the relatively uncommon occurrences of effective genetic transformation. The widespread usage of antibiotic resistance genes raises the possibility that such genes could be horizontally transferred to bacteria, making them resistant to the antibiotic in question. It is a way of transferring genes directly into a cell, tissue, organ, or organism utilizing bacteria. The genes found on the plasmids of the transformed bacterial strains are transported to the cells and expressed. Gene delivery can take place either intracellular or extracellular. It has the ability to express heterologous proteins like antigens, toxins, hormones, enzymes, etc. encoded by plasmids in a variety of cell types. Invasive strains with higher cell-to-cell dissemination are more efficient. Integrin receptors can help improve the effectiveness of bactofection-mediated gene transfer. Integrin receptors are transmembrane surface receptors found on the surface of mammalian cells. Lipofectamine-mediated bactofection is another approach that has been used to improve gene transfer efficiency in E. coli strains, notably in the transfer of large intact DNA for gene expression. This approach works with a variety of commonly used bacterial vectors, including L. monocytogenes and S. typhimurium. A receptor-mediated procedure allows virus particles with candidate gene sequences to enter the cell and then into the nuclear genome. The vector genome goes through a series of intricate steps that result in ds-DNA, which can either survive as an episome or integrate into the host genome, and then the candidate gene is expressed. The cell membrane is a film of amphipathic molecules that separates cells from their surroundings. Only the controlled exchange of materials between the different components of

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a cell and with its immediate surroundings is possible with these physical structures. DNA is a greater molecular weight anionic polymer that is hydrophilic and susceptible to nuclease breakdown in biological matrices. Unless they are helped, they cannot readily penetrate the physical barrier of the membrane and enter the cells. To assist DNA transport directly to the cell, a variety of charged chemical compounds can be utilized. These synthetic substances are delivered near recipient cells, disrupting the cell membranes, expanding the pore size, and allowing DNA to enter into the cell. They transfected human cells defective in the enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT) with entire uncloned genomic DNA. Selection on HAT media revealed a small number of HPRT-positive cells with pieces of DNA containing the functional gene. The real method of DNA uptake had remained a mystery until then. The development of a tiny DNA/calcium phosphate co-precipitate, which settles onto the cells and is then internalized, was later discovered to be the key to effective DNA transfer.

4.11 BIOTECHNOLOGY Biotechnology can be viewed as a continuation of past plant and animal breeding techniques that date back thousands of years. Compared to traditional breeding approaches, the technology produces faster, more exact findings and allows access to a larger genetic base. When combined with standard breeding methods, it becomes a powerful weapon. Gene technology’s precision is enabled by the ability to pinpoint the particular region of a chromosome that determines any desired attribute. Traditional breeding operations can be accelerated with this capability by locating seeds or progeny at an early stage using gene marker technology and breeding exclusively from them. Genes can also be extracted from one organism and introduced into another. Transgenesis, or the transfer of genes from one species to another, allows for the transmission of beneficial genes from any source to other species or organisms. While conventional breeding techniques have enhanced pest and disease resistance in Australian crops, the natural germplasm of these crops lacks resistance to some problems, according to the Cooperative Research Centre (CRC) for Tropical Plant Pathology. The promises of improved production and cheaper input costs are currently the key attractions of GM crops for farmers. Crops that are disease and pest resistant require less spraying, and animals that are disease and pest

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resistant require less care. As a result, significant chemical, labor, and energy input costs are reduced. Weed control is improved with herbicide-tolerant crops, which boosts productivity. It would be feasible to make greater use of the land if animals and crops were better matched to local conditions and climate, for example, by being more tolerant of drought, salt, and acid. Crop types that can make better use of soil nutrients or fix nitrogen could cut fertilizer expenses. Growers boost their marketing possibilities by providing higher-quality food to processors and consumers. The National Farmers’ Federation (NFF) stated in its response to the inquiry that herbicide-tolerant soybeans have reduced overall pesticide consumption by 33% in the United States. In Canada, herbicide-tolerant canola exhibited increased quality and yield increases of 10-20% above conventional types. Insecticide use has decreased by 40-50 percent in Australia because of Bt. cotton(Kilpatrick, 1996). This has resulted in higher survival of beneficial predators and parasites, as well as a lower risk of endosulfan contamination of cattle on neighboring ranches, which has previously resulted in their rejection by export markets. The fast uptake of GM crops in recent years demonstrates the benefits of GM crops to farmers. By the end of 1998, 17 nations had approved GM crops for planting and commercialization(Nap, Metz, Escaler, & Conner, 2003)the global area of commercially grown, genetically modified (GM. They consisted of 56 different crops, the most popular of which were squash, corn, canola, cotton, and tomato. GM crops have been accepted in the United States considerably more quickly than any other technology, and are also being grown in other nations, including Argentina and Canada. Gene technology provides growers with new options in the form of new products derived from existing species. Plants, for example, could one day be genetically modified to create industrial chemicals. Trees could be bred to produce wood with qualities similar to wood substitutes such as steel, aluminum, concrete, and plastic. Farmers will also benefit from the application of gene technology to reduce pest animal species and exotic weeds. In the coming decades, the world’s population is predicted to grow significantly and become increasingly urbanised, resulting in increased food demand. According to the AWB, global wheat consumption will have increased by 38% from present levels by 2020 (Ahmadi-Esfahani & Stanmore, 1995). Concerns have been raised regarding how the rising demand for food will be satisfied. Some see GM crops as a way to boost food security and assist satisfy long-term global food demands that

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traditional farming methods can’t achieve. Many enhancements to the plant and animal meals we eat are possible because of gene technology. Taste, texture, appearance, consistency, storage properties, and nutritional content are all likely to be improved. Nutritional quality will be the most important enhancement of these traits. Pesticide-tolerant GM plants allow for greater herbicide use than conventional cultivars. This is already occurring, and it may contribute to a loss of diversity among all types of life on land, as well as in the water and soil surrounding GM plants. Crop plants that are herbicide-tolerant are more prone to escape into the wild. Pollen drift from herbicide-tolerant crops to similar wild species, such as canola, could result in the emergence of super weeds, which has already occurred in a few cases. Bt. is present all of the time in GM crops, whereas it is only present on rare occasions when applied as a spray; it is anticipated that the pesticide’s presence may lead to a faster build-up of pest resistance and more harm to nontarget and beneficial insects. If crop plants that are better suited to marginal agricultural areas are produced, more native vegetation may be cleared and biodiversity may be lost. If terminator technology is employed, terminator genes may spread to other organisms, resulting in the extinction of species. GM crops, like other modern crops, are farmed in monocultures. There are numerous environmental hazards associated with the use of herbicidetolerant crops, none of which are unique to GM cultivars. Herbicidetolerant crops may also develop weeds in non-farming areas or alternative agricultural systems. Integrated Weed Management decreases the use of herbicides, lowering the probability of the above-mentioned consequences. To more effectively control and minimize potential negative repercussions, it must be combined with early detection of herbicide-tolerant weeds. Crosspollination with closely related species is another way herbicide-tolerant crops may have an impact on the ecosystem. If the herbicide resistance trait is passed down to wild populations, it may encourage the growth of weeds. The Genetic Manipulation Advisory Committee (GMAC) has addressed concerns regarding the spread of GM material from GM to non-GM crops through cross-pollination by establishing buffer zones surrounding GM crops to reduce this risk. While buffer zones surrounding GM crops are decided on a case-by-case basis, buffer zones around GM canola fields are typically 400 meters in length. However, pollen from GM canola fields can be transferred up to 15 kilometers by bees and 160 kilometers by wind. Concerns have been expressed concerning Bt. cotton cross-pollinating with conventional cotton or comparable wild species. A genetic obstacle, according to the CSIRO study, limits the flow of genes from agricultural

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cotton to related wild species. Cotton is also naturally self-pollinating, thus the chances of it spreading to other locations are slim. Pest insects may evolve resistance to the Bt. gene, resulting in unintended repercussions for the natural environment. The development of resistance is slowed by the interbreeding of resistant and vulnerable bugs. Bt. cotton’s impacts on nontarget insects, birds, and animals in the surrounding natural environment are unknown, and they could harm regional biodiversity. Several members of the committee expressed worries about the health effects of eating genetically modified foods. Allergies to soybeans have reportedly increased in the United Kingdom after the introduction of GM soybean types. Antibiotic resistant marker genes, which are used with other genes to trace their transfer from one organism to another, are suspected of being transmitted to bacteria that cause serious disease. Virus particles injected to confer virus resistance may also recombine with others in the environment or in the gastrointestinal system, resulting in the emergence of new diseases. Increased herbicide use is feasible with herbicide-tolerant crops; nevertheless, some herbicides, such as glyphosate, are known to have negative effects on humans. The oestrogen content of soybeans is likewise altered by glyphosate. The safety of GM food is determined by the substantially equivalent to its non-GM counterpart. If it is, no additional testing is required. Only foods that are significantly different are thoroughly evaluated. Another disadvantage of adopting gene technology in agriculture is the potential negative influence on farm revenue and rural communities. Biotechnology is considered as the most recent cause of agriculture’s industrialization, which has resulted in lower pricing for agricultural products and forced farmers off their land. It is feared that the usage of genetically modified organisms (GMOs) would intensify these trends. The monopoly of a few multinational corporations over important gene technology allows them to extract premium pricing for GMOs. Monopolistic control of GM crops will also contribute to the global trend of declining agricultural biodiversity by reducing genetic reserves from which future crop varieties can be generated. It was also widely reported that Monsanto had begun development on a ‘terminator gene,’ which would prohibit GM plants from generating viable seeds. The terminator gene will put an end to it, and farmers will be compelled to buy new seeds every season. Although Monsanto has stated that terminator technology will not be used in their seed, major concerns have been raised regarding the impact

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of such a system on farmers, particularly in underdeveloped countries. There are differing perspectives on how to overcome these dangers and drawbacks. At one end of the spectrum of opinions on this subject is the belief that few of the hazards will materialize, and that if they do, they will almost certainly be remedied. Others are less optimistic about the impact of GMOs in agriculture. At least some of the negative repercussions of releasing genetically modified organisms into the environment are likely to be irreversible. It may be difficult to recapture GMOs once they have been unleashed, given their ability to self-replicate. Several responses to the investigation offered a more pessimistic assessment of GMOs’ consequences. They stated that more time is required to determine the long-term health and environmental repercussions. It is permissible to invoke the precautionary principle under these circumstances. This principle emphasizes that when substantial or irreparable damage is threatened, a lack of complete scientific confidence should not be used as a justification for delaying cost-effective measures. People’s apprehension about using gene technology in agriculture reflects, in part, their reaction to the novel and unexpected, as well as their coming to terms with the ramifications for how they and their society live. Some people believe that such processes are in violation of natural laws.

CHAPTER

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MICROARRAY TECHNOLOGY

CONTENTS 5.1 Introduction ..................................................................................... 108 5.2 Microarrays...................................................................................... 109 5.3 Arabidopsis ...................................................................................... 113 5.4 CDNA Microarray............................................................................ 115 5.5 Cotton Fibers ................................................................................... 119 5.6 Molecular Analysis .......................................................................... 120 5.7 Transcript Profiling ........................................................................... 121 5.8 GM Crops ........................................................................................ 124

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5.1 INTRODUCTION Recent technological advancements and the ability to capture, store, and analyze large amounts of complicated biological data have resulted in a breakthrough in our knowledge of the systems that control organisms’ development and responses to their surroundings. We’re gathering the knowledge we need to rationally modify these organisms for specific goals(Heller, 2002). Microarrays are an early illustration of how highthroughput technologies can be effectively used with biological research. Living organisms are currently thought to be made up of a huge number of diverse biological components that interact in a controlled manner for the purposes of development and reproduction, as well as mediating appropriate responses to the environment. The reductionist approach has resulted in a thorough enumeration and description of these components, which have been grouped at several levels like organs, tissues, cells, subcellular structures, genes, and so on. The study of how these components interact has sparked a lot of attention recently. In terms of data collecting and analysis, the imposition of specific treatments, and the development and implementation of specific phenotypic assays, a thorough description of these interactions relies on efficiencies of scale. If we approach the problem of defining the performance of a biological system in the most abstract way possible, we discover that collecting a large number of formally independent measurements of the system across as many different treatment states as is reasonably possible will eventually provide a comprehensive description of the regulatory mechanisms governing the system’s behavior and responses. On a practical level, this necessitates highly parallel, precise tests as well as assay platforms that can handle high sample throughput while remaining cost-effective. The availability of appropriate data processing and storage resources to handle the resulting data stream is also critical to success. Microarrays (Figure 5.1) are one of the earliest instances of an experimental platform that meets these criteria, and they have had an important impact on our understanding of live organisms. This effect has previously been observed in model organisms, and it is now spreading to agriculturally relevant species, such as crops. The sort of data obtained with this technology is determined by three factors: sequences immobilized on the arrays, the nature of the hybridization targets used, and the hybridization circumstances. Probes have traditionally been built using known or expected gene sequences, and hence are both particular to and

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limited by this information. Microarrays have recently been built so that individual oligonucleotide elements increasingly tile the chromosomal sequences, thanks to the availability of higher probe densities. This method gives unbiased information about the complete genome’s transcriptional activity, allowing for the discovery of new transcription units as well as the verification of established gene models. Recent estimates of the disparity between the 1% to 2% of all base pairs that correspond to the exons of annotated human protein-coding genes and the approximately 93 percent of genomic sequences that can be transcribed highlight the importance of this latter approach to probe design. The study of targets formed from transcripts has been the most popular application of microarrays. They’ve recently been used to hybridize targets obtained from genomic DNA, providing information on changes in transcriptional activity sites, promoter binding, chromatin state, and overall polymorphism across genotypes. Microarrays are used to profile gene expression by separating messenger RNA (mRNA) and turning it into fluorescent targets, such as DNA or RNA, with or without rounds of amplification and it depends on the amounts of starting materials. The microarrays are then hybridized with the targets. Hybridization is often done pair-wise, with targets tagged with spectrally distinct fluorochromes whose contributions to fluorescence hybridization may be separated by scanning at different excitation and emission wavelengths. The two-color approach is excellent for removing biases produced by changes between slides, such as spot sizes that are inconsistent. Expression profiling and comparisons are made across different treatments of biological samples; these treatments can represent different genotypes, individuals, developmental stages, organs, tissues, and cell types, or they can represent different biotic or abiotic treatments imposed on the biological samples. All parts of microarray experiments require appropriate statistical design and analysis, as well as proficiency in developing and using this platform.

5.2 MICROARRAYS Some scientists have deemed the usage of microarrays with predetermined array elements obsolete, citing recent developments in methods for detecting transcriptional activity. However, this conclusion is premature. These statements overlook the importance of the huge number of array elements in defining the cellular transcriptional state, as they give basically orthogonal or distinct signals. Indeed, taken to its logical conclusion, many applications

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do not require knowledge of which transcripts are represented on individual microarrays.

Figure 5.1: Microarrays. Source: https://www.researchgate.net/publication/26888549_Basic_Concepts_ of_Microarrays_and_Potential_Applications_in_Clinical_Microbiology/figures

The information provided by the array elements is adequate to cluster and so characterize different cellular and therapeutic states of that organism as long as they are specific to the experimental organism, produce statistically significant signals, and do not cross hybridize. Microarrays’ tremendous dimensionality calls into question efforts to standardize microarray experiments through the adoption of “MIAME” methods. Minimal standards, by definition, lack the richness and breadth of information seen in microarray data sets. Recently, academics have compiled a concise list of broad network deductions. Combining data types that indicate diverse aspects of functional connections between genes yields the most insightful results. Interactions inferred by orthologies in distinct organisms, and interactions implied by occupancy of the same metabolic pathways are among them. The genes linked with distinct metabolic pathways have higher transcript levels than genes associated with diverse metabolic pathways. Co-expression analysis was used to link a significant number of Arabidopsis genes of unclear function to clusters of known function genes. Expanding the term

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reaction beyond its conventional biochemical definition to include other interactions such as protein-protein and protein-DNA interactions, as well as translocation events involving the movement of molecules between subcellular compartments, allows for a more comprehensive representation of biological processes in Arabidopsis. This wide definition resulted in the production of curated pathways that span roughly 8% of the Arabidopsis proteome, with the objective of representing all major biological activities. This method can be used on any plant species (Mergner et al., 2020)proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000. The identification of potential genes involved in cell-wall biosynthesis, glucosinolate biosynthesis, and pollen development are only a few instances of practical findings made possible by clustering investigations. Through knock-out analysis and ectopic expression, the regulatory roles of these genes, which encode R2R3-Myb transcription factors, were further defined. Glucosinolates, which are found in the Brassicaceae family, are antioxidative, anticarcinogenic, and antimicrobial compounds that play an important role in pest and pathogen control. They are also of agricultural and biomedical interest due to their antioxidative, anticarcinogenic, and antimicrobial properties. The genes discovered in the first and third cases have ramifications for biofuel production and plant breeding techniques, respectively. The retrieval of relevant data from databases remains a challenge, and populating databases with various data kinds is difficult to automate. Format compatibility is still elusive. Importantly, progress in silico appears to be hampered by the fact that there is less information about the biological roles of proteins and genes accessible for plants than for mammals. Some scientists offered in-depth studies of the good and negative aspects of using coexpression analysis on Web-based microarray data sets. However, one of the greatest obstacles to implementing such approaches is the scarcity of genetic information for the vast majority of crop species. The way biologists do research has altered as a result of genomics. Whole genome sequence information from numerous species has become available in recent years. Not just model organisms, but also commercially important species such as crops, human and plant infections, are among these organisms. The focus of the post-genome era, with the help of genomics, will be on using systematic ways to speed gene discovery by connecting

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phenotypes with gene sequence and expression data in a high-throughput manner. The two most often utilized platforms for gene expression analysis are GeneChip high-density oligonucleotide probe arrays (Figure 5.2) and cDNA microarrays. Because of its reproducibility and precision, as well as the ability to analyze medium-throughput samples, GeneChip microarrays are widely employed for large-scale genome profiling. Because of their flexibility and inexpensive cost, cDNA microarrays are commonly utilized for more targeted expression monitoring and other applications. The transcriptome of plant model systems may be profiled on a massive scale using GeneChip technology. Profiling data reveal a possible relationship between a specific attribute and genes whose expression varies in that particular biological activity based on pairwise comparisons of samples. Furthermore, data mining across multiple experiments in a database aids in the discovery of gene expression regulatory networks and the assignment of probable roles to genes that have yet to be identified (Dalma‐Weiszhausz, Warrington, Tanimoto, & Miyada, 2006).

Figure 5.2: GeneChip high-density oligonucleotide probe arrays. Source: https://www.researchgate.net/publication/26888549_Basic_Concepts_ of_Microarrays_and_Potential_Applications_in_Clinical_Microbiology/ figures?lo=1

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As a result, they provide a plethora of opportunities for plant development using both traditional and molecular methods. Model systems are commonly utilized in agricultural genomics for gene finding investigations. Arabidopsis and rice are the model plants for this purpose. Arabidopsis is a dicotyledonous plant with a small genome, a short life span, and a high sensitivity to genetic changes; as a result, it has been widely employed in molecular genetics research. The recently completed genome map provides the first glimpse into the organization and regulation of the plant genome. Rice, too, has a tiny genome with a lot of sequence similarity and synteny to other monocotyledonous plants, particularly cereal crops. The recently finished genome sequence project not only allows researchers to investigate gene activities in this vital food crop, but it also allows them to adapt the genes discovered in wheat, maize, and barley to other cereal species.

5.3 ARABIDOPSIS One-third of the Arabidopsis genome is represented by the Arabidopsis genome array, which has 160,000 perfect and mismatched probe sets for 8300 genes. Each probe is a 25 mer oligonucleotide with a surface area of 24m2. All of the probes can now be arranged in a 1.28 cm2 area(Yamada et al., 2003). This array has been used to classify circadian regulated plant genes; to analyze transcription patterns and uncover constitutive and organ specific promoters, and to dissect the photoreceptor phytochrome. A signaling pathways. Increased gene coverage per array not only allows for whole genome expression analysis, but it also reduces the volume of material necessary for microarray tests, as well as the cost and labor associated with them. Genome expression analysis currently necessitates the use of a series of arrays to cover a genome, especially for organisms with more complicated genomes. Simulation research was done to evaluate outcomes with and without mismatch probes in the probe set in order to maximize the capacity of the GeneChip array. Because similar results were obtained as a result of this work, changes to the rice genome array design were made. Mismatch probes were removed from the rice GeneChip array, and the feature size of each probe was lowered from 24 to 20 m2. Due to these changes, probe sets for around 24,000 rice genes may now be found in the same array area (Dalma‐Weiszhausz et al., 2006). The quality of the original RNA samples has a significant impact on the microarray findings. Controlling sample quality from many laboratories becomes one of the most difficult tasks for a centralized facility. Biological

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samples are subjected to stringent quality tests to solve this problem. Varying RNA extraction procedures are known to produce different sample quality, which can alter microarray results. A uniform sample preparation protocol was devised to ensure the quality of the data, and it was recommended to globally spread collaborators. Both spectrophotometer assay and electrophoresis utilizing either conventional gel electrophoresis or a BioAnalyzer were used to monitor the RNA samples received. Other procedures, such as setting up biochemical reactions and data archiving, were monitored with additional quality assay steps. In addition, through a web-based interface, full sample information was collected and maintained with the expression data. The lack of available genetic information, especially for non-model crops, is the main difficulty in applying microarray technology to agricultural research. Three techniques can be used to overcome these obstacles. Because many essential or significant genes in plants are preserved, model systems can be utilized to identify them. Putative orthologs of these genes can be found and isolated in economically relevant crops using sequence similarity searches. Several vegetable crops, such as sugar beets and Brassicas, have been studied using this method to uncover genes. 2. Microarray technology is based on hybridization. Cross-species hybridization can be used to use a microarray developed for a model system for closely related species. The practicality of such a heterologous system is determined by the balance of specific and non-specific hybridization. The sequence similarity and abundance of the hybridising targets determine the hybridization efficiency, which is utilized to represent the abundance level of the hybridized transcripts. Genomic DNA can be tagged and applied to an array to find relevant probes, which often hybridise to both model species and near relatives, to remove the noise caused by sequence variations. Using this method, a considerable number of the probes in the rice GeneChip array were shown to be usable to detect gene expression in maize and barley. After administering plant hormones like gibberellin or abscisic acid to barley aleurone cells, changes in gene expression of landmark genes including GAMyb and a-amylases were identified. The application of genomic arrays to non-model crops broadens their scope.

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Figure 5.3: cDNA microarray. Source: https://www.mun.ca/biology/scarr/cDNA_microarray_Assay_of_ Gene_Expression.html

5.4 CDNA MICROARRAY To create a cDNA microarray (Figure 5.3) for a given crop, it is important to follow certain steps. Because they do not need previous knowledge of genomic sequences, cDNA microarrays are beneficial for non-model systems. Whole genome expression analysis may be difficult with this method due to constraints in source clones and fabrication; it does give a fast way to examine gene expression at the transcription level in parallel. Furthermore, when comparing expression data among various kinds in species with a high polymorphism frequency in their genome, such as maize, the bigger probes used in the DNA microarray are preferable to the oligonucleotide probes used in the GeneChip microarray. In any facility, a system with highthroughput potential for cDNA microarrays was established. Another example of how the cDNA microarray can be used is to quickly confirm differentially expressed genes discovered using various transcription profiling techniques. cDNA fingerprinting has a wide gene coverage, excellent sensitivity, and is not dependent on genome sequence knowledge. It has been widely utilized for non-model systems due to these advantages.

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However, confirming that the gene segment reporting differential expression has been found is still a time-consuming process. To speed up the validation process, the discovered differentially expressed gene fragments were cloned and spotted onto a glass slide using a cDNA microarray in a huge parallel test. The results revealed a strong link between the two technologies. The cDNA microarray confirmed 41 of 48 randomly selected differentially expressed gene segments, whereas three were slightly confirmed. DNA microarrays were first described in 1995 for analyzing large-scale gene expression patterns at the same time(“Use of a CDNA Microarray to Analyze Gene Expression Patterns in Human Cancer,” 1996). They’ve risen to popularity in several fields of biological research since then, and are currently playing an increasingly vital role in diagnostics, genetics, pharmacology, cancer, and other biomedical research, among others. The use of DNA microarrays in plant disease detection and identification, particularly viruses, viroids, and phytoplasmas is evident. They are particularly dangerous because of their difficulty in detecting and identifying them. Many quarantine and certification procedures exist, particularly in industrialized nations, to avoid the entry of these infections into a country during international germplasm movement and to regulate and decrease their spread within a country. Biological indexing, immunological techniques, molecular hybridization, polymerase chain reaction (PCR), and/or reverse transcription (RT)-PCR are now used to detect these infections. DNA microarrays were first unveiled in 1995, and biologists have been fascinated by this technology ever since. It is a great tool for genetic study since it can display the expression of thousands of genes at the same time. It is now used in genomics for sequence analysis, gene expression studies, gene typing, and large-scale polymorphism screening; biomedical research for understanding cancer, infectious diseases, and genetic diseases; clinical diagnostics; and drug discovery and development. It was anticipated that there will be simultaneous detection and identification of many plant viruses, viroids, and other plant diseases using DNA microarrays due to the explosion of information resulting from plant virus and viroids sequencing. These approaches show how to use probes that are detected by a signal, whether radioactive or fluorescent. However, in recent years, a number of technologies have been developed that allow massive parallel investigation of hybridization events by applying vast numbers of DNA oligonucleotides to surfaces in ordered two-dimensional arrays. Along with techniques such as quantitative PCR and microarrays were presented as a tool for assessing the levels of gene expression of numerous genes in a high throughput

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analysis. The underpinning idea of DNA microarray is base-pairing of complementary sequences via hybridization. A target DNA or RNA can hybridise to a complementary DNA probe on the array due to DNA binding. Thousands of cDNAs or oligonucleotides make up each probe, each one specific for a gene, DNA sequence, or RNA sequence of interest. An array is a collection of samples arranged in a logical sequence. It provides a platform for comparing known and unknown nucleic acid samples using base-pairing criteria and automating the identification process. A few nano-itres of DNA probes are deposited on a solid support to create each array. The printing is done by a robot, which enables for repetitive spotting of identical spots. The size of the sample spots determines whether an array is classified as a macroarray or a microarray. Macroarrays have sample spot sizes of 300 metres or more. Microarray sample spots are typically less than 200 m in diameter, and these arrays frequently have thousands of dots (Brazma et al., 2001)one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME. Microarrays having a density of less than 100 spots per cm2 are sometimes referred to as low-density. Low-density arrays are available from a variety of commercial sources or can be made in-house by research organizations. For low-density arrays, a glass surface is frequently used. Plain glass, which is usually prepared before use, binds poorly to nucleic acids. Poly-L-lysine is frequently employed for this purpose because it binds molecules through ionic interactions. The glass substrate can alternatively be coated with saline, which binds to the probe DNA covalently and prevents it from being removed throughout the hybridization and washing procedures. Glass slides for detecting radioactively tagged nucleic acids can be coated with a nylon membrane to which DNA probes are covalently cross-linked using UV light. The DNA put to the array’s surface might be plasmids with 500-5,000 bases, complementary DNA with several hundred bases, PCR products with 100500 base pairs, or synthetic oligonucleotides with an amino or Thiol group on their 5’ end (Quackenbush, 2001)2001. Microarrays with a density of 1,000-10,000 spots per cm2 or greater are referred to as high-density. The phrase DNA chip is commonly used to describe such high-density arrays. Chemically modified glass or silicon are commonly utilized as the immobilization substrate for high-density DNA arrays. The technological breakthrough represented by DNA microarray technology is its massive parallel nature, or the ability to apply thousands

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of nucleotides in an ordered array to a surface, allowing simultaneous investigation of thousands of distinct sequences. Short or long oligonucleotides, cDNAs, chromosomes, entire organisms, or others with known identities can all be used as probes. Standard technologies like restriction enzymes, cloning, and PCR can be used to make the probes. They can also be bought individually or mounted to chips, glass slides, or nylon supports. Purchased and generated cDNA or oligonucleotide probe libraries are available. Thousands of these cDNAs or oligonucleotides make up each probe, each one specific for a gene, DNA sequence, or RNA sequence of interest. The baker’s yeast Saccharomyces cerevisiae was used to create arrays containing oligonucleotides representing known genes. More than 65,000 DNA synthesis characteristics were included in oligonucleotide arrays. Because oligonucleotides are intrinsically only of known sequences, cDNAs can have the advantage of being unknown, an experiment utilizing cDNAs could potentially help identify the function of hitherto uncharacterized and un-sequenced genes, including viral genome genes. Microarrays have become a significant tool for studying gene expression in people, animals, plants, and microbes on a global scale. cDNA and oligonucleotide arrays, when used in the context of a well-designed experiment, can allow high throughput, concurrent analysis of transcript abundance for hundreds, if not thousands, of genes. Despite widespread acceptance, however, the use of microarrays as a tool to better understand processes of interest to plant physiologists is still under investigation. Microarrays are being used to evaluate gene expression in plants exposed to experimental manipulations of air temperature, and aluminum concentration in the root zone based on the results. Characterizing transcript profiles for various post-treatment sampling periods and categorizing genes with common response patterns using hierarchical clustering approaches are important parts of analysis. Furthermore, microarrays are revealing developmental changes in gene expression linked to fiber and root extension in cotton and maize, respectively. Microarrays are a powerful tool for plant physiologists interested in the characterization and identification of individual genes and gene families with potential applications in agriculture, horticulture, and forestry, despite the fact that much work remains to be done.

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5.5 COTTON FIBERS Cotton fibers are single-celled trichomes that distinguish them from the developing cotton ovule’s epidermal layer. They proceed through a rapid cell expansion phase, then a secondary cell wall deposition phase, and eventually maturity. Cotton fibers are thus unique in that they are one of the longest and fastest-expanding plant cells, making them a useful experimental system for studying fundamental processes in plant biology. Cotton fiber elongation begins on the day of anthesis and ends around 21 days later, overlying with the onset of secondary cell wall generation, according to research. During this time of development, many genes are known to be transcriptionally regulated. However, progress in identifying genes responsible for cell elongation has been modest. The considerable time it takes to regenerate transgenic cotton lines and the absence of adequate transient assay techniques have hampered such research. Using mutants with altered fiber elongation kinetics is one technique to get around these problems and study more about the molecular mechanisms that control cell elongation in growing cotton fibers. The difference in mature fiber length in the Pilose mutant was related mostly to changed growth rate, not to early termination or late commencement of the elongation period, according to fiber length measurements taken throughout development. The researchers employed cDNA microarrays to explore the underlying molecular pathways that were responsible for the decreased growth found in Pilose fiber using this as their model system. Cotton cDNA microarrays contained 4875 cDNAs from isolated fibers 7–10 days after anthesis, constituting a unigene collection of expressed sequence tags (ESTs)(Wu et al., 2007). On each slide, each probe was duplicated twice. Cotton and non-plant sequences were also spotted in each replication to assess non-specific hybridization aided by the cDNA probes’ poly-T tails. One of the most stimulating discoveries was that four separate targets on the cotton cDNA array representing expansins exhibited a fivefold decrease in expression for Pilose mutant fibers. Expansins are cell wall proteins that affect the mechanical characteristics of cell walls through pHdependent mechanisms, resulting in cell wall loosening and turgor-driven cell expansion. According to the findings, expansins are one of the most significantly repressed gene families in elongating Pilose fibers as a result of lower cell development rates. Two expansin genes called GhExp1 and GhExp2 produced transcripts that are unique to growing cotton ovules, suggesting that

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GhExp1 may be involved in cell wall extension during cotton fiber formation. However, the question is whether the suppression of expansins is the major and exclusive determinant of shorter fiber length in the Pilose mutant using data from their microarrays. Profilin, an auxiliary protein of actin filaments, was decreased in Pilose. Profilins have a variety of biological functions and influence the actin cytoskeleton in different ways. This finding backs up the theory that the Pilose phenotype is caused by a combination of processes, including both expansins and cytoskeleton dynamics. These concepts will need to be explored more in future studies. To better understand the mechanisms that underpin the changed phenotype, plans are now underway to construct comprehensive time-series expression profiles for both control and Pilose mutants. The researchers at the University of Missouri presented an overview of a project in which microarrays were used to characterize gene expression for root elongation in maize for understanding the molecular mechanisms of fiber elongation in cotton.

5.6 MOLECULAR ANALYSIS The molecular analysis of gene expression under stress exposure of plants is one of the most common applications of microarrays in plant physiology. Under uniform light and moisture conditions, cold stress (5°C) and a heat shock (32°C) treatment were used. At 1, 2, 4, 8, and 12 hours after the start of each treatment, leaf samples were obtained from treatment and control plants, and mRNA was extracted, labeled, and hybridized to the array. Four distinct groups of reactions emerged from the hierarchical clustering of genes that showed at least an eight-fold shift in response to cold stress. The upregulated genes were separated into two groups: one in which transcript levels increased at first and subsequently declined over time, and the other in which transcript levels increased continuously during the treatment period. Cold stress, circadian rhythms, and drought tolerance, lipid metabolism, and different membrane transporters and transcription factors were among the genes identified. Downregulated genes were also split into two groups: one in which transcript levels progressively declined and subsequently increased, and the other in which transcript levels decreased continuously throughout time. Photosynthesis, cell and cell wall development, cellular signaling, and several genes with unclear functions were all downregulated. Hierarchical clustering of genes with a 4-fold change in expression revealed four separate classes of reaction in the instance of heat shock. Upregulated genes were

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separated into two categories, the first of which had the highest transcript levels during the first hour of treatment and thereafter declined. The majority of these genes were heat shock proteins. After 2 hours of treatment, transcript abundance was also high, and many of the genes in this second group were connected to defense. Genes involved with growth inhibition or auxin-repressed protein and cell signaling were among the 30 transcripts that were downregulated. Northern blot tests confirmed many of these expression patterns, the function of particular genes up- and downregulated by light freezing and heat-shock treatments will be investigated further in future investigations. These findings point to the prospect of discovering new genes involved in heat and cold temperature responses, as well as the temporary nature of these responses. The highly up- and downregulated proteins with unknown functions are of special relevance in this regard. Furthermore, these studies could provide insight into the coordinated, multigenic responses to temperature stress, as well as the identification of master regulators that could be used as future genetic engineering targets.

5.7 TRANSCRIPT PROFILING Transcript profiling (Figure 5.4) can help researchers find candidate genes underpinning quantitative trait loci (QTLs) for yield and other agronomic variables in agricultural plants grown under a variety of environments. There’s a lot of interest in utilizing DNA microarrays to track gene expression profiles in crops that have limited soil water availability. Stressinducible genes can be easily found in plants exposed to fast dehydration. Researchers identified 44 drought-inducible genes in Arabidopsis following a 2 h dehydration using microarrays containing 1300 full-length cDNAs. Some researchers recently used cDNA microarrays to identify large-scale changes in transcript abundance for barley exposed to rapid drought shock treatments (van Dongen et al., 2009). Research that imposes slower, more realistic rates of dehydration, as well as studies that compare the ensuing gene expression profiles to those from plants exposed to drought-shock therapies, are needed. Plants were grown in sand until they reached the four-leaf stage, at which point water was withheld by removing them from the sand and allowing them to dehydrate for six hours. In a second, more traditional ‘water-stress’ experiment, potted plants were grown in a greenhouse until they reached the four-leaf stage, then water was incrementally withheld for 11 days until the moisture content of the soil mix dropped to around 40% of maximal waterholding capacity.

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Figure 5.4: Transcript profiling. Source: https://www.semanticscholar.org/paper/Microarray-technology%3Abeyond-transcript-profiling-Hoheisel/5198b8b18713f831e321b3b9ac9bbb21c 9a03e86/figure/1

mRNA was extracted, labeled, and hybridized to microarrays comprising 1463 DNA elements generated from barley cDNA libraries from tissues of treated and control plants. Signal intensity differences between control and treatment plants greater than 2.2 times in two replicated tests were considered significant. Microarray analysis of mRNA isolated from water-shock and control tissues generally verified the alterations in transcript expression levels. Under rapid drought stress, over 15% of all transcripts were either upregulated or downregulated. Jasmonate-responsive, late-embryogenesis-

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abundant (LEA), and ABA-responsive proteins were among the transcripts that showed considerable overexpression. Aluminum toxicity inhibits root elongation as its principal consequence. Different species or crop cultivars may react to aluminum in different ways, and these reactions may have a genetic or molecular basis. Plants (some) detoxify aluminum in the rhizosphere by producing organic acids that chelate the metal before it is absorbed by the roots, while others detoxify aluminum internally by building complexes with organic acids. Unfortunately, the precise process by which plants tolerate metals in general, and aluminum in particular, remains completely unknown for the vast majority of plants. cDNA microarrays were used to assess differences in gene expression for tolerant and susceptible cultivars exposed to aluminum in understanding the molecular mechanisms associated with aluminum tolerance in wheat (Triticum aestivum L.). In hydroponic culture, seedlings of the cultivar Chisholm that are Al sensitive and its isogenic line Chisholm-T (Al-tolerant) were developed. Chisholm-aluminum T’s tolerance is developed from the Al-tolerant cultivar. Three-day-old seedlings were given an aluminum concentration of 10 mg/l, which was high but not fatal. The seedlings were cleaned and moved to deionized water after twenty-four hours. The roots of aluminum-stressed and control plants were stained with haematoxylin for 15 minutes. The deposition of a high level of aluminum in roots is indicated by high-intensity staining. The roots of Chisholm-T and Atlass 66 were mildly stained 24 hours after exposure to aluminum, whereas the roots of the Aluminumsensitive cultivar Chisholm were quite black. Suppression subtractive hybridization was performed to identify transcripts that changed between cultivars following exposure to aluminum, which was used to produce the microarrays used. This method was employed to create subtracted cDNA libraries by combining normalization and subtraction into a single step. The array revealed sequence tags from 1628 differentially expressed cDNA clones. At 6 hr, 1 day, 3 days, and 7 days following aluminum stress, the arrays were hybridized with cDNA from Chisholm-T and Chisholm roots. In a single reaction, the microarray approach is utilized to detect numerous diseases infecting plants simultaneously. As a probe, virus-specific oligos immobilized on a membrane or glass slide are used in this procedure. Total RNA from infected plants is transcribed to cDNA and amplified by PCR using pathogen-specific primers, which are then labeled with appropriate molecules for detection. After that, the amplified and labeled products are

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placed on the array and allowed to hybridize. Following washing, the array will be produced according to the label applied, and the outcome will be visualized using a CCD and appropriate software.

5.8 GM CROPS The debate about genetically modified (GM) plants and their potential influence on human health contrasts with the implicit acceptance of other modified plants that aren’t classified as GM products like varieties raised through conventional breeding such as mutagenesis. We can compare the level of transcriptome alteration that occurs during transgenesis versus mutant breeding for rice improvement. The observed modification was more extensive in mutagenized plants than in transgenic plants in all of the situations studied. The safety evaluation of modified plant types can be done on a case-by-case basis, rather than being limited to foods obtained through genetic engineering. Thousands of years ago, plant breeding began with the unintentional selection of seeds from plants with superior quality and productivity. People began to use deliberate interbreeding or crossing of closely or distantly related species to develop novel crops with desirable traits when sexual plant reproduction was discovered in the 17th century. Plant breeders and geneticists began to employ mutagenesis to rapidly develop and increase diversity in crop species and ultimately change plant features after discovering that x-rays induced mutations in the fruit fly Drosophila melanogaster and barley at the turn of the twentieth century. Classic mutagenesis’ high efficiency has been well documented, and its global influence on agricultural development has also been assessed. Advancements in molecular biology techniques have paved the way for the development of genetic engineering since the 1970s, allowing agricultural cultivars to reach the next level of genetic gain. This technology allows researchers to identify, isolate, and transfer a gene of interest from any type of organism to plant cells. Tissue culture is used to regenerate transformed plants from these cells. In contrast to the widespread acceptance of food items derived from traditional plant breeding, the potential benefits of this new technology have been largely ignored due to the intense debate over food safety. Despite the lack of universal methods for assessing the potentially harmful effects of genetic modification, the FAO and the European Food Safety Authority recommend that macro, micro, and anti-nutrients, toxins, allergens, and secondary metabolites are evaluated using targeted approaches. Some molecular profiling methods have also been developed to

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boost the odds of finding unwanted consequences. Microarrays are one of the profiling approaches described. This method enables the simultaneous monitoring of thousands of genes. Pearson’s correlation between samples revealed that duplicate samples always clustered together and modified genotypes always grouped with their respective unmodified controls after hierarchical clustering of microarray data of transgenic, mutagenized, and control plants. Genetically stable samples like the transgenic single-chain variable fragment or ScFv and mutant Estrela A are more closely associated with their corresponding controls than unstable samples, regardless of the type of breeding method used. Transgenic Nipponbare is also more closely connected to its control in unstable lines than the line acquired with 100-Gy-irradiation. In the unstable mutagenized rice line, 11,267 genes displayed differential expression, whereas only 2,318 genes were discovered in the non-stable transgenic line, as seen in volcano plots (Nishizawa et al., 1999). Because down-regulation of a nitrilase-associated protein was discovered in the mutant line, some differentially expressed genes found in the stable Estrela A line can be linked to a lower indole-3-acetic acid (IAA) content. Nitrilases are enzymes involved in the production of IAA, a plant hormone that belongs to the auxin family of plant growth regulators. Because the phosphatidylinositol signaling pathway is also involved in plant responses to hormones like auxins, the enzyme phosphatidylinositol 3-kinase, which belongs to the signal transduction functional group, could be linked to this predicted lower IAA level. It was discovered that, a group of genes involved in protein modifications whose altered transcription could be linked to the lower IAA concentration. This group included one F-box domain-containing protein and the ubiquitin carboxyl-terminal hydrolase, both of which are involved in ubiquitination. F-box proteins are adaptor components of the SKP1-CUL1-F-box protein complex, a modular E3 ubiquitin ligase that engages in phosphorylation-mediated ubiquitination. Protein ubiquitination is a precise technique for regulating gene function by sending tagged proteins to the proteasome for degradation, and it has been proposed as a key control system in desiccation resistance. Because auxin regulates transcription by stimulating the degradation of a family of transcriptional repressors known as Aux/IAA proteins, which is dependent on an ubiquitin protein ligase known as SCF, the downregulation of these 2 proteins could be explained by a drop in auxin levels. The F-box protein TIR1 attaches to the Aux/IAA proteins in the presence of auxin, resulting in their ubiquitination and destruction.

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One copy of the CBF1 gene is present in the unstable transgenic line Nipponbare GM. C-repeat binding factors (CBFs) regulate the expression of several stress-inducible genes by interacting with the cis-acting dehydration-responsive element-DRE. Despite the fact that the BCBF1 gene is controlled by a stress-inducible promoter. As a result, the differential expression of stress-related genes seen in research might be due to either the stress induced by the Agrobacterium-mediated genetic change or, at least in part, the inserted CBF1 transcription factor. To further elucidate this point, we can look for DRE core motifs in the promoters of the top 50 differentially expressed genes. We can observe that nearly all of the top 50 genes had several DRE core motifs in their promoter regions. As a result, it appears that the differential expression of these genes is primarily due to the transgene incorporated. This finding emphasizes the significance of carefully examining transformants with inserted transcription factor-coding genes. Microarray technology is increasingly being used in the agro-food branch, as it is in other disciplines in the biological sciences, to address basic and applied research concerns. The goal of this chapter is to provide an overview of microarray technology’s application in agricultural research by focusing on one of the most pressing challenges in modern agriculture biotechnology: the generation of genetically modified crops. Microarrays can be used to investigate any form of interaction in which the arrayed material can be properly deposited and the binding reagent can be hybridized and directly or indirectly labeled, or its binding can be identified in another way. Microarrays were first used to mimic Southern and northern blot investigations, but they have a lot of potential for studying other kinds of interactions, such as those between proteins and proteins and other substrates. Protein-based microarray applications were originally developed shortly after DNA-based microarrays were released, but their progress has been much slower since then. Developing protein microarrays as part of our functional genomics study has long been a goal of mine. However, like many others, we initially focused on nucleic acid-based microarrays because we thought they were more well-established and easier to perform.

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DROUGHT RESISTANT PLANTS

CONTENTS 6.1 Introduction ..................................................................................... 128 6.2 Drought ........................................................................................... 128 6.3 Stomata ........................................................................................... 130 6.4 Drought Resistant Crops .................................................................. 133 6.5 GM Technology ............................................................................... 135 6.6 Soybean........................................................................................... 136 6.7 Drought Stress ................................................................................. 137 6.8 Rainfall ............................................................................................ 139

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

6.1 INTRODUCTION Drought is the leading source of agricultural loss around the world, posing a serious danger to food security. Plant biotechnology is now one of the most promising fields in terms of producing crops that can provide high yields in water-scarce environments. The key response pathways to drought stress have been discovered through studies on Arabidopsis thaliana plants, and numerous drought resistance genes have already been put into crops(S. Wang et al., 2004). So far, most plants with increased drought resistance have had lower agricultural yields, indicating that new techniques to uncoupling drought resistance from plant development are still needed. Brassinosteroid (BR) hormone receptors use tissue-specific pathways to mediate various developmental responses during root growth. Boosting BR receptors in vascular plant tissues offers drought resistance without compromising growth in Arabidopsis, providing a unique opportunity to examine the mechanisms that confer drought resistance in plants with cellular specificity.

6.2 DROUGHT Drought is described in agronomy as the absence of water that impacts plant development and survival, resulting in lower crop yields. The broadest definition of drought stress in plant science is the same as the definition of water deficit, which occurs when transpiration exceeds water intake. This will be due to a deficiency of water, but it could also be due to higher salinity or osmotic pressure. The water loss from the cell, or dehydration, is the first process during drought stress, according to molecular biology. Dehydration normally causes osmotic and hormone-related signals, with abscisic acid (ABA) playing a key role in the latter. These signals trigger a response that can be divided into three categories. They are drought escape (DE), dehydration avoidance (DA), and dehydration. DE is a plant’s attempt to speed up flowering time before drought threatens its existence. This response is prevalent in annual plants, including the model species Arabidopsis thaliana (Arabidopsis), and cereal plant breeders use it to their advantage. Even amid water scarcity, the plant can maintain a high relative water in dehydration avoidance. This is accomplished by physiological and morphological responses such as reduced transpiration via ABA-mediated stomatal closure, deposition of cuticular waxes, and a slower life cycle for the plant. Dehydration avoidance usually results in plant survival by delaying plant growth and, as a result, senescence and mortality. This approach

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arose in response to moderate, transient drought stress, in which the plant falls into developmental hibernation until the next rain. While dehydration avoidance is helpful in boosting plant survival, it often comes with growth and yield losses, which are, of course, substantial drawbacks for crop breeders. Dehydration tolerance, on the other hand, is the ability of a plant to sustain its activities in a dehydrated state by increasing the production of sugars, osmoprotectants, antioxidants, and reactive oxygen species (ROS) scavengers through modulation of plant metabolism. The modulation of the GA-signaling molecule DELLA, a pathway that integrates multiple hormones and stress-related pathways usually activates these responses. DE and early flowering cultivars with quicker life cycles are attractive from an agronomic standpoint because an expected changeover to the reproductive stage could allow grain filling prior to the beginning of the seasonal terminal drought. A shortened crop season also minimizes the demand for agricultural inputs such as fertilizers and pesticides and may make double cropping possible i.e., the farming of two different crops in the same field within the same year. Crops that flower too early, on the other hand, will have their yield reduced. Despite the fact that DE is a growing research subject in crop science, no biotechnologically enhanced crops that use DE as a drought resistant characteristic exist. Still, it’s been suggested that DE may be used to produce fast-growing, early-flowering grain varieties, which would be particularly advantageous in temperate climates like the Mediterranean, where terminal dryness is likely to damage plants near the end of the crop season. Furthermore, OsFTL10, one of the 13 Blooming locust-like (FTL) genes listed in the rice genome, has recently been found to be triggered by both drought stress and GA, and when overexpressed in transgenic rice plants, promotes early flowering and enhances drought tolerance. However, because these transgenic rice lines were not evaluated in a field trial, it is unknown whether altering FTL genes could result in grain kinds that perform well in both dry and wet circumstances and provide high yields(Sharma et al., 2019). Nonetheless, manipulating the DE route could be a novel and effective method, especially given the extremely changeable nature of water availability. Due to the fact that DE includes specific tissues like leaf, phloem and cell types like phloem companion cells, FD-expressing SAM cells, it may be able to build drought-resistant plants by manipulating these plant components and tailoring DE to the various climatic conditions.

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6.3 STOMATA Stomata, which are apertures on the surface of a plant’s aerial section, are surrounded by two specialized guard cells that change their turgor pressure to open and close the pore. Stomata are important for CO2 uptake in photosynthetic organs and are tightly controlled by a molecular process that permits plants to take in CO2 while limiting water loss. Manipulation of stomatal quantity, size, and control was one of the first tactics used by scientists to create drought-resistant plants. In water-stressed situations, the major hormone signal that causes stomatal closure is ABA. The CLE25 peptide is translocated to the leaves, where it binds to barely any meristem (BAM) receptors, causing ABA buildup and stomatal closure in the leaves. Increasing stomatal responses in response to drought by manipulating ABA sensitivity could help plants survive. Condensed photosynthetic activity as a result of low CO2 uptake, on the other hand, is usually deleterious to carbon assimilation and has a negative impact on crop yield. Furthermore, evaporation of water through stomatal pores keeps plants from overheating. Reduced stomata capacity may not be a viable way to improve drought resistance while maintaining yield and biomass production in a natural environment because drought is likely to be followed by warm temperatures. It was discovered that constitutive expression of AtNF-YB1 in Arabidopsis enhanced the survival rate of transgenic seedlings in an early attempt to generate drought-resistant plants. Nuclear factors Y (NF-Y) are heterotrimeric transcription factors that regulate a variety of developmental pathways, including stomatal responses via modulation of the ABA signaling pathway, and have conserved functions in Arabidopsis and cereals during flowering. Under the direction of the rice actin 1 promoter, one maize homolog of AtNF-YB1, ZmNF-YB2, was expressed constitutively. In a greenhouse experiment, maize transgenic plants had a higher survival rate, showing the functional conservation between Arabidopsis and maize NF-YBs. Due to a combination of greater stomatal conductance, cooler leaf temperatures, higher chlorophyll content, and a delayed onset of senescence, the transgenic plants were also drought resistant in field trials. Nonetheless, despite encouraging findings in field testing, with the highest performing line having a 50% increase in yield relative to controls under extreme drought circumstances, these transgenic lines were never commercialized, possibly because yield in watered conditions was severely affected. Manipulation of stomatal kinetics, or more precisely, enhancing the speed of stomatal responses, could prevent the trade-off between stomatal conductance and drought tolerance. The expression of a synthetic blue

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light–induced K+ channel 1 (BLINK1) under the control of the strong guard cell–specific promoter pMYB60 recently improved plant stomatal dynamics (Cosentino et al., 2015). This significantly sped up stomatal responses, resulting in plants that responded to changing light conditions more quickly. When compared to control plants, Arabidopsis WUE i.e., biomass per transpired water was improved without reducing carbon fixation rates, resulting in a 2.2-fold increase in total biomass in transgenic plants maintained in water-deficit circumstances. It is yet to be investigated if this strategy would be effective in open-field crops, or whether the extra biomass would result in a higher yield. Overall, altering stomata physiological behavior is a great breakthrough that has yet to be applied to crops (Kenney, McKay, Richards, & Juenger, 2014). In the Alxa Desert of China, the distribution characteristics of Na+, K+, and free proline were studied in succulent xerophytes Haloxylon ammodendron and Zygophyllum xanthoxylum; xerophytes Artemisia sphaerocephala and Caragana korshinskii; and mesophytes Agriophyllum squarrosum and Corispermum mongolicum. The findings revealed that mesophytes and xerophytes were salt-free species, with Na+ values ranging from 1.5 to 3.8 percent lower than succulent xerophytes. K+ concentrations were 1.3–2.7 times higher in Agriophyllum squarrosum and Corispermum mongolicum than in Artemisia sphaerocephala and Caragana korshinskii. Agriophyllum squarrosum and Corispermum mongolicum had K+ concentrations in their stems that were 1.8 and 2.2 times higher than in their roots, respectively. Water transport over a soil–plant gradient may be aided by mesophytes accumulating substantial amounts of K+ in their stems. Large amounts of K+ and free proline were collected by the xerophytes. Their proline concentrations were 6.0–16.0 times greater in the total plant than in mesophytes, and 1.8–25.0 times higher in succulent xerophytes. Proline concentrations increased by 3.1 and 10.5 times in Artemisia sphaerocephala from roots to stems and stems to leaves, respectively. Caragana korshinskii had a similar pattern. As a result, xerophytes’ accumulation of K+ and free proline may play a role in drought adaptation(S. Wang et al., 2004). Haloxylon ammodendron and Zygophyllum xanthoxylum, two succulent xerophytes, were found as salt diluting species that absorbed a lot of Na+ from their roots and carried it to the leaves and photosynthesizing branches. Even at low soil salinities, succulent xerophytes gathered more Na+ than K+ for osmotic adjustment, resulting in the root systems having the lowest selective absorption and selective transport capacities. This implies that accumulating Na+ rather than excluding it could be one of the most successful methods for succulent xerophytes to adapt to arid settings.

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The main reason for C. polygonoides productivity differences appears to be changes in soil water content and nutrients. The competitive effect of D. sindicum grass, which appears to have a larger competitive effect on resource usage, was blamed for lower plant growth in FGP and SDP habitats. The variation in D. sindicum grass density and root biomass, which was negatively linked with growth increments and soil water, was explained by differences in growth and biomass production. The findings show that surface vegetation has an impact on C. polygonoide productivity through modifying soil resource availability. As a result, proper water management will be useful to increase the productivity and population of this species under the afforestation programme.

Figure 6.1: Drought-resistant crop. Source: https://www.frontiersin.org/files/Articles/483633/fpls-10-01676-HTML-r1/image_m/fpls-10-01676-g001.jpg

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6.4 DROUGHT RESISTANT CROPS Fully drought-resistant crop plants (Figure 6.1) would be beneficial, but selection breeding has not produced them. Genetic modification of species by the introduction of genes is claimed, predominantly, to have given drought resistance. Experiments in soil with cessation of watering demonstrate drought resistance in GM plants as later stress development than in wildtype (WT) plants. This is caused by slower total water loss from the GM plants which have smaller total leaf area (LA) and/or decreased stomatal conductance (gs), associated with thicker laminae or denser mesophyll and smaller cells. Non-linear soil water characteristics result in extreme stress symptoms in WT before GM plants. Then, WT and GM plants are overwatered: faster and better recovery of GM plants is taken to show their greater drought resistance. Mechanisms targeted in the genetic modification are then, incorrectly, considered responsible for the drought resistance. However, this is not valid as the initial conditions in WT and GM plants are not comparable. GM plants exhibit a form of ‘drought resistance’ for which the term ‘delayed stress onset’ is introduced. Claims that specific alterations to metabolism give drought resistance are not critically demonstrated, and experimental tests are suggested. Small LA and gs may not decrease productivity in well-watered plants under laboratory conditions but may in the field. Optimization of GM traits to the environment has not been analyzed critically and is required in field trials, for example of recently released oilseed rape and maize which show drought resistance, probably due to delayed stress. Current evidence is that GM plants may not be better able to cope with drought than selectionbred cultivars. Molecular biology techniques have offered the possibility of directly altering the genomes of higher plants for more than 30 years to change their metabolism and improve growth and yield under adverse environmental conditions to better serve human needs. One of the main areas has been to overcome abiotic environmental factors that restrict agricultural productivity, such as long generation times. Drought, or a shortage of water, has a significant detrimental influence on plant and crop productivity. The goal has been to genetically engineer drought-resistant plants. The terms genetic engineering or modification have been used to describe the processes of modifying plants. Hybridization, cell fusion, and tobacco transformation with a drought-inducible histone gene were all used to try to find and transmit genes responsible for DR between species. Drought-induced gene

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expression in plants has influenced the course of drought research ever since. As a result, candidate genes that are likely to confer DR in crop species have been found. Many types of genes and their control, such as gene promoters and transcription factors, are thought to have a wide range of consequences and benefits. Proponents of genetic engineering believe that the mechanisms impacting agricultural output caused by drought are well understood, and that the constraints can be alleviated by correct metabolic improvements via genome modification (GM). GM makes a strong case for being based on precise knowledge of mechanisms and the ability to manipulate key metabolic processes to produce a precise outcome and to improve both absolute and relative crop production per area of land surface, as well as to improve water use efficiency (WUE) when water is scarce and thus mitigate or even eliminate drought effects. Despite scientific and practical evidence to the contrary, there is a general, pervasive ethos in the GM literature that natural selection has not adequately provided DR plants and that GM is the only way to achieve the desired changes because selective breeding is incapable of doing so in any reasonable time scale. Claims in the GM literature that GM plants have resulted in the development of DR plants must be investigated. The social and economic importance of increasing productivity under water scarcity is huge, and producing true drought-resistant plants would be a major accomplishment. The world’s present human population is 7 billion, and it is predicted to grow to 10 or possibly 12 billion by 2050, necessitating the production of food, fiber, and energy(Bloom, 2011). Agriculture is practiced in many regions across the world where water availability is typically insufficient compared to evapotranspiration from crops, resulting in crops receiving insufficient water to reach their genetic potential production. Drought is a big issue in the long run, but it also reduces agricultural output in the near run, even when other conditions are favorable, and can have severe economic and social effects. Water supply varies greatly at different times during the growth and development of specific crops, and the consequences can be highly unique. Plant processes, from genome to growth and production and that means total biomass as well as economic yield and quality of crops are highly reliant on water and are extremely vulnerable to drought; losses are difficult to estimate but are undoubtedly in the millions of tonnes with significant economic value.

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During the introduction of agriculture, advantageous plant traits, as well as the discovery and application of applicable technology, increased yields of basic crops. Despite what may have been a tendency to select for productive genotypes in dry environments, crops nevertheless rely considerably on water. More recent selection breeding based on scientific principles has not resulted in drought-resistant crops, but has resulted in smaller improvements; for example, using carbon isotope differentiation in wheat breeding improved yields by 5% with a 50% yield reduction and by 10% with a 75% yield reduction(McLaughlin & Adams Kszos, 2005)a native perennial warm-season grass, as a dedicated energy crop is reviewed. The programmatic objectives were to identify the best varieties and management practices to optimize productivity, while developing an understanding of the basis for long-term improvement of switchgrass through breeding and sustainable production in conventional agroecosystems. This research has reduced the projected production cost of switchgrass by about 25% ($8– 9Mg−1. Evolved systems allow crops to continue to produce despite severe water shortages, and they lay the groundwork for further improvement through selective breeding and molecular genetics data. In contrast, certain GM publications give the idea that GM plants are ‘DR’ and thus unaffected by water scarcity.

6.5 GM TECHNOLOGY GM technology has been justified based on its potential to produce DR crops faster and more efficiently than selective breeding, hence alleviating food shortages. The potential for GM to boost food production in droughtprone locations, such as developing economies in Africa, has received special attention, despite the fact that the chances of even moderate success are slim. There have been a lot of claims that GM would produce DR crops. They stand in contrast to the belief that GM technology will not improve drought tolerance. Despite this, the prospect for speedy fulfillment of the objectives has resulted in significant adjustments in research funding and education in favor of GM technology. Drought has not resulted in greater yields despite massive investments in GM by public entities and, in particular, major corporations. There is still a body of opinion emphasizing the need to understand the effects of water supply and deficits on plant mechanisms from genome to yield including GM technology, of altering plant and crop responses and thus improving plant processes like photosynthesis and crop production. As evidenced by analysis of quantitative trait loci in DR

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breeding, the latter is clearly a function not merely of the genome, but of the entire complex plant system. Understanding requires a focus on the details of mechanisms, but it is also necessary to consider the entire system. However, very specific genome interventions produce drought resistance in the lab but have yet to produce clear evidence of significant improvements in crops under drought in the field, and those concerned with crops have high doubts about the ability of GM approaches to provide drought resistance. Drought, salinity, cold, and other abiotic stressors have a significant impact on plant output. Plant yields of essential major crops can be reduced by up to 50% as a result of these stressors(Mahajan & Tuteja, 2005). Abiotic stress-related genes or other transcription factors (TFs) have multiple functions, such as increasing proline content, decreasing transpiration rate by closing stomata, increasing the production of some important stressrelated protective enzymes, and so on, and thus increasing abiotic stress tolerance. Many transcription factors (TFs) and other stress-related genes have been discovered, described, and applied to a variety of essential cultivated plants to protect them against drought and other abiotic stresses. Transgenic plants outperform non-transgenic plants in terms of morphobiochemical and physiological performance. Wheat, rice, tomato, soybean, cotton, and a variety of other genetically modified plants have been designed to withstand drought stress. Researchers are increasingly turning to the effectively engineered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system to modify plant genomes for natural resistance to a variety of abiotic stressors. It is the leader in genomic editing by precise methods, with little or no effect on plant growth and development.

6.6 SOYBEAN Soybean is the economically important oilseed crop on the planet. Soybeans that have been processed are also the most common source of vegetable oil and protein feed. Soybeans contain secondary metabolites such as isoflavones, saponins, phytic acid, oligosaccharides, and phytoestrogens in addition to macronutrients and minerals. In 2007, global soybean production was estimated to be around 219.8 million metric tonnes (mmt). The United States produced the most soybeans (70.4 million tonnes), followed by Brazil (61 million tonnes), Argentina (47 million tonnes), and China (14.3 million tonnes)(Manavalan, Guttikonda, Phan Tran, & Nguyen, 2009). While soybeans have long been used in Japan to make traditional foods like tofu,

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miso, shoyu, and vegetable oil, consumption of soybean-based products is increasing worldwide due to reported health benefits such as cholesterol reduction, cancer prevention, diabetes and obesity prevention, and protection against bowel and kidney disease. Soybean is also seen as a promising crop for biodiesel production. It may also fix atmospheric nitrogen, using only a small amount of nitrogen fertilizer, which is often the single largest energy input in agriculture. Plants are exposed to abiotic and biotic stimuli that have an impact on their growth and development. Water scarcity, in particular, is expected to remain a major abiotic issue affecting worldwide crop production. Onethird of the world's population lives in water-stressed areas, and with rising CO2 levels in the atmosphere and expected climatic changes, droughts may become more common and severe in the future. It was believed that the resilience of legume crops to current climatic extremes, such as drought, excess water, heat, cool weather during grain filling, and early frost, can forecast their response to future climate change. Drought affects soybean yields by roughly 40%. During the growing season, soybeans consume roughly 450–700 mm of water, depending on hybrid traits. It was found that the most crucial phase for water stress in soybeans is during the blooming stage and the period following flowering.

6.7 DROUGHT STRESS Drought stress causes plants to utilize a variety of methods to cope. Drought escape allows the plant to finish its life cycle while there is enough water available prior to the commencement of the drought. In most cases, the life cycle is shorter, and plants produce a few seeds rather than a complete crop failure. The Early Soybean Planting System, which is now commonly employed in the southern United States, is an example of drought resistance. Short season cultivars are planted in March–April in places where later maturing cultivars have previously been produced in this way. Early maturing cultivars flower in late April to early May and set pods in late May, finishing the reproductive stage before the potential drought period of July–August. Drought avoidance is the second mechanism, and it entails techniques that help the plant maintain a high-water status during times of stress, either by efficient water absorption from the roots or by minimizing evapotranspiration from aerial portions. Drought tolerance is the third mechanism that permits the plant to sustain turgor and metabolism even when the water potential is low, for example by protoplasmic

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tolerance or the manufacture of osmoprotectants, osmolytes, or suitable solutes. One of the ways for increasing soybean production is to maintain optimal transpiration, which leads to higher WUE. Phenology, photoperiod sensitivity, developmental plasticity, leaf area index, heat tolerance, osmotic adjustment, early vigor, rooting depth, and rooting density are the features that have been studied. The huge diversity of PI lines and cultivars available around the world provides valuable resources for identifying germplasm that can be employed in root trait breeding. The time-consuming techniques required in root separation, as well as the lack of quick screening procedures, are the key roadblocks to genetically improving root features. Because the most potential genetic mechanism for enhancing soybean drought tolerance is a deep taproot system with a moderate quantity of lateral roots to collect soil water, study in this area is critical. Overall, root characteristics have a lot of potential for breeding to improve drought resistance. Selection based on root phenotypic measurement, on the other hand, would be extremely challenging. Molecular tagging, on the other hand, will make it easier to breed for root-related features. Another alternative methodology is the candidate gene approach, which entails selecting a candidate gene for root traits from public data, obtaining primer sequences to amplify the gene, uncovering polymorphisms, developing a simple procedure for large-scale genotyping, identifying a population for association studies, conducting an association study of the candidate gene with trait phenotype, and finally verifying the uncovered associations. Under low moisture conditions, this method proved successful in identifying candidate genes associated with root number in rice. Understanding the physiological mechanisms and genetic regulation of root drought adaptation would aid in the identification of relevant genes and metabolic pathways for gene-based marker selection or genetic engineering to generate soybeans with improved root-related attributes. Drought-induced nitrogen fixing: Thenitrogen (N2) fixation by legumes is highly sensitive to soil dryness. Soybean not only loses CO2 accumulation and leaf area development during drought, but its symbiotic N2 fixation is also at risk. It was found that in dry soils, the supply of N2 for protein formation, which is the plant’s important seed product, is reduced, resulting in lower crop yields. Reduced oxygen availability, reduced carbon flux to nodules, decline in nodule sucrose synthase activity, and an increase in ureides and free amino acids have all been linked to the inhibition of N2 fixation during drought.

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During the first four days of dryness, nitrogenase activity dropped by 70%, whereas photosynthesis only dropped by 5%. This means that water stress affects nitrogenase activity in a way that is independent of the photosynthetic rate. Water deficiency was also discovered to have a direct impact on nodule activity by increasing resistance to oxygen transport to the bacteroid. Increased oxygen diffusion resistance, decreased nitrogenaselinked respiration and enzyme activity, accumulation of respiratory substrates and oxidized lipids, and up-regulation of antioxidant genes all show that the respiratory activity of bacteria is impaired in drought, and that oxidative damage occurs in nodules before any effect on sucrose synthesis or leghaemoglobin. Soil drying also causes ureides to accumulate in soybean leaves, which are hypothesised to be nodulation inhibitors. The sensitivity of N2 fixation in response to soil drying was discovered to have a lot of genetic diversity. When comparing soybean germplasm with and without the ability to maintain tissue turgidity, and thus leaf and nodule function, under drought conditions at the reproductive stage, the germplasm with the ability to maintain tissue turgidity had the lowest grain yield reduction. The first step in identifying soybean lines whose N2 fixation is more tolerant of soil dryness is to test for petiole ureide levels. The variety Jackson, which is more resistant to N2 fixation in drying soils, has been employed as a parent for creating drought-tolerant lines. It’s uncertain if N2 fixation is only regulated at the whole-plant level via a systemic nitrogen feedback mechanism or if it might also happen at the local nodule level during drought. There is evidence that a N2 feedback system involving shoot N2 status can give signaling for biological N2 fixation. A combination of ureide and aspartic acid levels in nodules, as well as the transfer of various amino acids from the leaves, may be implicated in a feedback inhibitory process in soybean. It was found that N2 fixation activity under drought stress is primarily controlled at the local level rather than by a systemic N2 signal in a partially droughted split-root system. More research should be done to better understand the molecular genetics of the processes that restrict and regulate N2 fixation under drought conditions.

6.8 RAINFALL Rainfall is becoming less and less irregular as a result of human-caused climate change, especially in areas where food security is extremely low. The poor in rural and dry places will be the hardest hit, and will require low-cost, easily accessible ways to cope with unpredictable weather. This adaptation

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will have to account for not only a lack of water and droughts, but also an increased likelihood of extreme occurrences such as floods. Ecological approaches to making farms more drought-resistant and adaptable to harsh events rely on biodiversity and healthy soil. Resilience is the tendency to cope with and recover from change. Farm output and income are more resilient and stable when soils are better able to hold soil moisture and decrease erosion, as well as when biodiversity is increased in the system. Building healthy soil is critical to assisting farmers in coping with drought. There are a variety of proven soil-building strategies accessible to farmers right now. Cover crop residues that protect soils from wind and water erosion, as well as legume intercrops, manure, and composts that build soil rich in organic matter and improve soil structure, are all ways to improve water infiltration, hold water once it gets there, and make nutrients more accessible to plants. It is critical to boost productivity in rain-fed places where poor farmers apply existing water and soil conservation knowledge in order to feed people and ensure ecological resilience. Under a drier and more irregular climate, ecological farms that work with biodiversity and are knowledge-intensive rather than chemical input-intensive may be the most resilient options.

Figure 6.2: Drought-resistant crop. Source: https://www.researchgate.net/profile/Ashkan-Jalilian/post/In_climate_change_will_the_temperature_increase_be_harmful_to_agriculture_Or_reduced_rainfall/attachment/5c61fd6acfe4a781a57efb82/AS% 3A725255265996800%401549925738200/image/climate-change.jpg

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Agriculture will have to adjust to changing rainfall patterns as the climate changes (Figure 6.2). Water is the most important component of food production. Human-caused climate change is already leading to less and more irregular rainfall, particularly in areas with low food security. Rain-fed farms, which cover 80% of the world’s agriculture, produce more than 60% of the world’s food. Rain-fed farms provide 95 percent of food in Sub-Saharan Africa, where climate unpredictability already affects agricultural production. Climate change will have an impact on river flow and groundwater in South Asia, where millions of smallholders rely on irrigated agriculture for survival. Mexico had its driest year in seven decades in 2009, with major social and economic consequences. It came after a ten-year drought that had already ravaged the country’s north and western states. Furthermore, climate models show that as a result of global warming, Mexico will continue to dry. Climate change will intensify conflicts over water allocation and the already precarious status of water availability as temperatures rise and rainfall becomes less and less unpredictable. The poor in rural and arid areas will be the hardest hit by these changes, and they will require low-cost, easily available ways to cope with the unpredictable weather. Since the Neolithic Revolution, which was triggered by the advent of agriculture 10,000 years ago, humans have been using natural biodiversity to adapt agriculture to changing conditions (Putterman, 2008). Diversity farming is the single most significant tool for adapting agriculture to a changing climate. The intrinsic diversity and complexity of the world’s farming systems prevent a single solution from being pursued. Agriculture under a changing environment necessitates a variety of tactics. We can look at the successes and evaluate the potential of traditional breeding approaches, such as markerassisted selection (MAS), to generate drought-resistant cultivars without the environmental and food safety issues associated with genetically modified (GE) crops. Plants have evolved natural ways to cope with drought over millions of years, with water scarcity being the principal constraint to plant growth. From improved root growth to control of water loss in leaves, these mechanisms are complex and diverse. Frequently, developing cultivated crops under ideal well-watered conditions results in the loss of characteristics that allow them to cope with less water. The diversity of plant characteristics available to deal with minimal water in industrially grown crops has been diminished in the rush to build greater industrial monocultures fuelled by agrochemicals and enormous irrigation. Crop varieties and wild relatives, on the other hand, retain this diversity.

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Drought and salt are two main environmental challenges that affect peanut production over the world. Drought stress is thought to be responsible for a $500 million loss in peanut production per year. Because major peanutproducing countries like China, India, Nigeria, and the United States are all experiencing significant water shortages for agricultural irrigation, it is possible that global peanut production could suffer in the future. Climate change predictions suggest that extreme weather, notably drought, would become more frequent in the tropical and subtropical regions of the world, making peanut production extremely difficult in the future. Peanut is a popular food in Africa, Asia, and North and South America because it is one of the most nourishing foods for oil and protein. Reduced peanut output will raise the price of peanuts and peanut-derived items like peanut butter, putting people in many nations in a difficult situation. The great challenge now is to maintain, if not increase, peanut production to meet the demands of our growing population at a time when the conditions for peanut cultivation are deteriorating. Due to the rarity of genes for drought and salt tolerance in existing peanut germplasms, the traditional method of breeding for drought and salt-tolerant peanuts has been slow. Gene for drought or salt tolerance is discovered in a wild peanut relative, introgressing the gene into cultivated peanut cultivars will be challenging due to the reproductive barrier, not to mention the lengthy time required for many generations of back-crossing. With the development of molecular biology, a biotechnology approach offers a powerful alternative for more efficiently producing drought and salttolerant peanuts. Indeed, several genes that confer drought and salt tolerance have been introduced into diverse crops and tested in laboratories and in the field over the last 20 years, with a few of those genes showing significant potential for commercial release. The DREB/ CBF family of transcription factor genes, for example, has been widely exploited to improve stress tolerance in a variety of crops. In the future, this group of genes could be valuable in developing heat and drought tolerant peanuts. Other sorts of genes, in addition to transcription factor genes, may be effective in peanut enhancement. ABA or cytokinin biosynthesis pathways, antioxidation metabolisms, and stress signal transduction pathways are among the genes encoded by these genes. In the future, some of these functional genes may be valuable in enhancing peanut stress tolerance. We must be able to modify peanuts in order to boost their stress tolerance using a biotechnology technique. Previous attempts to alter peanuts utilizing Agrobacterium-mediated gene transfer or biolistic bombardment

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were mostly successful. However, the efficiency of these efforts in terms of transformation was relatively poor, ranging from 0.3 percent to less than 10%. Researchers developed a peanut transformation process that raised peanut transformation efficiency to 55% or higher, making peanut transformation reasonably simple and repeatable. More crucially, this novel approach appears to be adaptable to a wide range of peanut ecotypes, despite the fact that it still uses Agrobacterium as a vehicle. Overexpressing the Arabidopsis vacuolar pyrophosphatase gene AVP1 in transgenic peanut plants increased drought and salt tolerance at the same time. The proton pump vacuolar pyrophosphatase generates a proton chemical gradient across vacuolar membranes. For their actions, several secondary transporters, such as the Na+/H+ antiporter i.e., AtNHX1, rely on the proton chemical gradient formed by proton pumps like AVP1. Proton pump activity may be increased by boosting Na+/H+ antiporter activity, resulting in increased salt tolerance. Overexpression of AVP1 in transgenic plants increased drought and salt tolerance. The strong root development produced by enhanced auxin polar transport in transgenic plants was responsible for the higher drought tolerance in the AVP1- overexpressing plants. AVP1 and its homologs are overexpressed in various plants, and in all cases, enhanced drought and salt tolerance have been found. In the greenhouse, AVP1-overexpressing peanut plants were drought and salt tolerant, producing more biomass and maintaining greater photosynthetic rates and transpiration rates under reduced watering and saline conditions. When plants are exposed to environmental challenges such as high salt or drought, defense systems are activated. Many investigations have demonstrated that these defense systems rely on protective processes triggered by changes in stress gene expression levels. Solanum tuberosum, an agricultural species, is an autotetraploid with a complex, quantitative inheritance pattern. Thus, traditional methods of breeding new potato cultivars that are tolerant of salinity and drought stress are laborious, complex, and time-consuming, taking between 10 and 15 years on average. Potato cultivars can be improved faster and more reliably using genetic engineering techniques. Numerous potato stress genes, including those that code for functional and regulatory proteins have been isolated and characterized using homologue gene screening, differential screening, microarray analysis, and proteome analysis as a first step toward developing drought and saline tolerant potato plants using molecular breeding methods. Abiotic stress genes encoding functional proteins including proline synthesis protein, osmotin-like protein, GPD, trehalose synthesis protein,

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and regulatory proteins like StEREBP, CBF, and StRD22 have been used in various attempts around the world to generate drought- and saline-tolerant potato plants. Water is a well-known environmental component and a crucial limiting factor for plant growth, development, and production. Due to constantly changing environmental circumstances, plants frequently have a fluctuating water supply throughout their life cycle. The variability of drought tolerance among plants of the same species has not been fully explained. Plant responses to water stress are influenced by plant species, age, growth and development phases, drought severity and duration, and physical characteristics. There are recognized differences in drought tolerance among genotypes of plant species, such as maize, wheat, and triticale. Plants use a variety of strategies like morphological, physiological, and biochemical to reduce or eliminate the negative consequences of stress. It was observed that adding polyethylene glycol (PEG) to a hydroponic solution causes osmotic stress, which causes changes in the water status of the tissues and a decrease in plant growth and biomass production. Droughtresistant genotypes, on average, collect more biomass in their leaves than susceptible genotypes. Drought-stressed physiological processes include leaf water content and gas exchange. Reduced CO2 availability owing to stomatal closure is usually thought to be the cause of a decline in photosynthesis during mild drought. When dryness lasts for a long time, however, non-stomatal mechanisms induce a decline in photosynthesis. Changes in photosynthetic activity are linked to mesophyll cell membrane degradation, a decrease in chlorophyll concentration, and disruptions in assimilate synthesis and transport. Photosynthesis limitations caused by stomatal and non-stomatal mechanisms are dependent on plant species, stage of development, and leaf age, as well as the duration and degree of drought stress.

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DISEASE RESISTANT PLANTS

CONTENTS 7.1 Introduction ..................................................................................... 146 7.2 Weeds ............................................................................................. 146 7.3 Growth and Reproduction ............................................................... 151 7.4 QTL ................................................................................................. 153 7.5 RNAi ............................................................................................... 156 7.6 Breeding Programs........................................................................... 158

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7.1 INTRODUCTION Plants face a diverse range of climate-induced biotic and abiotic stresses in the current era of drastic climate changes such as global warming, irregular rainfall, and depletion of arable land and water resources. Stress is a negative situation for plant growth and development that can be produced by environmental, biological, or both factors (Collinge, Lund, & ThordalChristensen, 2008). Concurrent occurrence of two or more different types of stresses such as drought and salinity, drought and heat are more harmful to global agricultural productivity under natural settings. Abiotic challenges that occur at the same time affect plant metabolism and reduce yield more than abiotic stresses that occur at various phases of growth. Drought and heat stress in the summer, or drought and salinity stress in the winter, are instances of coupled abiotic stresses. Pests, diseases, insects, and weeds are all regulated by abiotic stressors (Figure 7.1).

Figure 7.1: Abiotic stressors. Source: https://www.mdpi.com/2223-7747/10/2/186#

7.2 WEEDS Weeds outcompete crops under abiotic stress due to their increased water consumption efficiency. Abiotic stress has a significant impact on plant growth, resulting in significant output losses. In most plant species, the resulting growth reductions can be as much as 50%. The yield of maize can be lowered by up to 40%, and the yield of wheat can be reduced by up to 21% with a 40% water reduction. Cowpea yield is also reduced, with

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reductions ranging from 34% to 68% depending on developmental stage and drought stress (Ashraf & Foolad, 2007). The yield drops in cowpea, an important crop in Africa and a source of food for millions of farmers, varies greatly depending on the developmental stage and the intensity of drought stress. In 2002, it was estimated that soil salinity alone cost more than US$11 billion per year and destroyed about 10% of the world’s arable land, having a significant impact on global food production and being regarded as the primary stress affecting worldwide crop yield. Depending on the number of interacting components, stress is classified as single, multiple, concurrent, or repeating. Single stress is made up of only one stress component, whereas multiple individual stressors are made up of two or more stresses that happen at the same time without overlapping, and concurrent stresses are made up of two or more stresses that happen at the same time but with some overlap. Plants are subjected to single or numerous stresses, which are followed by recovery periods that can be short or lengthy in duration. At various stages of plant development, several hot days or repeated bouts of drought and heat stress may occur. The interplay of multiple stress factors can either increase the plant’s tolerance capacity or predispose it to a variety of pressures. Drought, for example, promotes the establishment of Macrophomina phaseolina in the roots of Sorghum bicolor, resulting in a significant decrease in productivity. Similarly, in North China, the occurrence of concurrent drought and cold stress reduces the production of Vitis vinifera. Plants growing in hot, dry environments, such as arid and semi-arid locations, are frequently confronted with the development of salinity and heat stress. Cold and light stressors are the most common in the Mediterranean region, and they affect plant growth and development. Concurrent cold and ozone stresses, as well as concurrent salinity and ozone stresses, lower the frost durability of Triticum aestivum and the production of Cicer arietinum, respectively. Similarly, the combination of salt and ozone stress contributes to lower yields in chickpea and rice varieties. Plants are exposed to a variety of concurrent biotic pressures, similar to abiotic stresses, and are harmed more severely by the combination of fungal and bacterial infections than by infections with these pathogens alone. Researchers studied the occurrence of multiple biotic stressors at the same time and their effects on plant growth and yield. Plants have evolved a perceptual network that allows them to perceive both biotic and abiotic stressors at the same time, allowing them to reduce the harmful effects of stress. Depending on the timing and severity of biotic stresses such as powdery mildew, rust, and wilt, the impacts of abiotic pressures

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such as drought or salinity may lead to either susceptibility or resistance of plants to biotic stresses such as powdery mildew, rust, and wilt. The type of the interactions between the stressors, as well as the length of stress exposure, can have a variety of consequences on plant growth and overall output. The magnitude of the impact on crop yield is also determined by the nature of the interconnections between stressors. For example, abiotic– abiotic pressures such as concurrent drought and heat stress can result in higher soil water evaporation, resulting in a lower crop yield. It was reported that weeds outcompete crops due to their efficient water use ability during concurrent drought and heat stress, and that the synergistic effects of drought and heat stress on physiological aspects of plant growth result in a substantial reduction in crop yield. In tropical and subtropical conditions, these concurrent stressors produce a significant decline in leaf water potential and transpiration rate, resulting in increased leaf and canopy temperature. Concurrent stress-induced increases in transpiration rate have been shown to alter important physiological processes in plants, according to several researchers.

Figure 7.2: Drought and heat stress. Source: https://www.frontiersin.org/files/Articles/388913/fpls-09-01705-HTML/image_m/fpls-09-01705-g004.jpg

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Drought and heat stress (Figure 7.2) have a significant impact on nutrient relationships, slowing growth by limiting nutrient mobility through diffusion and reducing root mass, number, and growth. Drought and heat stress damage photopigments and thylakoid membranes, resulting in decreased chlorophyll biosynthesis and increased chlorophyll breakdown, or a combination of the two processes. Light reactions in the thylakoid lumen and light-dependent chemical reactions in the stroma are both affected by the damage caused by these concurrent stressors. Photosystem II is particularly susceptible to concurrent stressors, and its activity is considerably altered or even decreased to zero under severe heat stress. Heat stress promotes pathogen growth and leads to the emergence of a wide range of bacterial and fungal diseases, such as tomato wilt caused by Ralstonia solanacearum, seedling blight and bacterial fruit blotch of cucurbits caused by Acidovorax avenae, and panicle blight in rice caused by Burkholderia glumae. Heat stress inhibits plant growth and development while promoting pathogen growth and reproduction. Heat stress stimulates the growth of many vectors, boosting the incidence of vector-borne diseases in addition to its promoter effects on pathogen growth.

Figure 7.3: Salinity and pathogen stress. Source: ures/2

https://link.springer.com/article/10.1007/s40502-019-00483-7/fig-

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Salinity and pathogen stress (Figure 7.3) are another example of concurrent biotic–abiotic stressors. Pathogen virulence, plant physiology, and microbial activity in the soil are all influenced by salinity. Salinity produces increased sporulation in fungus and causes severe Fusarium wilt in tomatoes. The root system architecture refers to the root system’s spatial architecture or RSA. In cereals, the genetic regulation of the RSA and its link to higher output under stress has been thoroughly demonstrated. Roots facilitate water and nutrient intake, develop symbiotic relationships with fungi and bacteria, provide anchoring, and serve as storage organs, all of which are important in crop production. They also serve as the primary interface for interactions between plants and numerous stress elements, and they are critical in reducing the damaging effects of stress on plant growth and development. The organization and structure of the roots, such as their length and density, dictate the types of interactions that occur between roots and stress stimuli. Increased root length density (RLD) and a large root diameter are associated with drought resistance in rice varieties. Droughtresistant rice varieties have a higher RLD which facilitates access to moisture in the deeper layers of the soil. Maize with a higher RLD and fewer lateral roots had a higher photosynthetic rate, a better plant water status, and more stomatal conductance under drought stress than maize with a lower RLD and more lateral roots. The presence of fewer but longer lateral roots resulted in greater use of water available in deeper levels of the soil due to improved rooting, allowing the plant to function better under drought stress. The RSA is also important for preventing pathogen infection in plants. T. aestivum lines with longer roots were less susceptible to fungal infection with Pythium debaryanum and Pythium ultimum. The fungal pathogen Rhizoctonia solani reduced root length, root branching, and root tips, resulting in reduced water absorption from deeper soil layers. It may be argued that increasing the RLD can significantly minimize pathogen infection. The RSA is important in crop plant responses to drought stress and pathogen attack; however, in field circumstances, drought and pathogen stress frequently occur simultaneously, resulting in higher plant damage owing to RSA disruption. Plants that experienced progressive drought with 2 and 4 days of Ralstonia solanacearum infection were classified as experiencing short-duration (SD) and long-duration (LD) stress stresses, respectively, in a study of chickpea plants exposed to concurrent drought and infection with the pathogen Ralstonia solanacearum (Sinha, Gupta, & Senthil-Kumar, 2017).

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7.3 GROWTH AND REPRODUCTION The pathogen’s growth and reproduction were reduced by SD combined stress, while there was no difference in LD combined stress. Drought and the fungus Fusarium solani both inhibited the growth of Phaseolus vulgaris. Root rot induced by the pathogen is the reason for the lowered growth, which limited water intake from deeper layers of the soil. Plant size, leaf area, hydraulic conductance, photosynthetic and transpiration rates have all been documented to decline when drought and pathogen stress coexist. Plants’ molecular responses to coupled drought and pathogen stress have recently been studied in a few major molecular studies. This research has shown several viable possibilities for improving plant tolerance to mixed stimuli as well as provided light on plant defense systems against combined stresses. Methionine gamma lyase and azelaic acid induced 1 are some of the major candidate genes identified thus far. Genes implicated in the cross talk between the drought-associated and pathogen infection-associated signaling pathways contribute to tolerance to combined drought and pathogen stress. Some studies have suggested that proline and polyamine metabolism play a role in combined drought and pathogen stress tolerance in A. thaliana and V. vinifera. The identified candidate genes can be regulated in the right way to improve tolerance to these combined stresses. Genome editing technologies such as the CRISPR/Cas9 system can be used to make the changes. By guiding catalytically inactive dead Cas9 (dCas9) or dCas9 coupled with transcriptional repressors/activators to the promoter of a gene, CRISPR/ Cas9 can also be utilized to control the transcription of genes of interest. More study in this field, utilizing various functional genomic techniques, could reveal plant responses to coupled drought and pathogen stress. Plants in the field are exposed to a variety of abiotic and biotic challenges, and to counteract these impacts, they have evolved sophisticated signaling networks. Plant growth may be affected negatively or favourably by interactions between the two types of stress conditions. A concurrent drought, for example, might regulate the interaction of different diseases and plants in different ways, resulting in pathogen development being suppressed or increased. As a result, studying the interplay between the two types of stresses is critical in order to better understand the overall impact of stress combinations on plants. Several important diseases, such as dry root rot, powdery mildew, and charcoal rot, are significantly influenced by concurrent drought conditions, and a mechanistic understanding of the interactions between pathogen and drought stress can aid in the identification and development of superior cultivars. Improved crop performance under

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combined drought and disease stress necessitates a deeper understanding of the issues. Transcriptomic research has already begun in an attempt to comprehend the interconnections. Drought and pathogen stress on plants have also been combined in welldesigned studies, revealing some elements of drought–pathogen interactions. To find superior germplasm lines, plant genotypes can be tested for features like root system architecture, leaf water potential, leaf turgidity, leaf pubescence, and leaf cuticular waxes. It is critical to develop studies that can highlight distinct features of interactions between the two different forms of stress in order to vividly assess the effects of different stress combinations on plants. A well-designed stress imposition methodology that is similar to pressures that occur in the field, together with relevant physiological assays and recently developed genomic technologies, can aid in the discovery of plant responses to stress combinations. Breeders and field pathologists can use the knowledge gained from studies on plant responses to combined drought and disease conditions to better examine the performance of tolerant genotypes. In areas where the two pressures coexist, further development of crop simulation models containing a mix of drought and pathogen stress could aid disease predictions. As a result, collaborative efforts by crop modeling experts, agronomists, field pathologists, breeders, physiologists, and molecular biologists can lead to the development of combined stress tolerant crops that perform well in the field. Plant diseases are responsible for significant yield losses in most crop species, putting global food security and sustainability at risk. Plants defend themselves against pathogen invasion in one of two ways: qualitative or vertical or total resistance mediated by disease resistance (R) genes, or quantitative or horizontal or partial resistance regulated by multiple genes or quantitative disease resistance (QDR) genes. To manage disease incidence and yield losses, crop resistance to pathogens can be improved by conventional breeding, marker-assisted breeding (MAB), and transgenic development. As a result, we need to figure out which genes are involved in both qualitative and quantitative disease resistance. Major resistance genes encoding cytoplasmic proteins with nucleotide-binding and leucine-rich

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repeat domains or NLR proteins are frequently used to provide qualitative or complete resistance. These NLR proteins detect pathogen derived chemicals termed effectors, which are delivered into the host cell by a pathogen and thereby facilitate infection, either directly or indirectly. After effector recognition, an NLR protein mediated defense response is triggered, which includes a hypersensitive response (HR); rapid, localized programmed cell death at the point of pathogen penetration; and other responses such as ion flux, an oxidative burst, lipid peroxidation, and cell wall fortification.

7.4 QTL In other words, many quantitative trait loci (QTLs) (Figure 7.4) govern QDR, which interact with each other as well as the environment. QTL-mediated resistance has smaller individual effects than R gene-mediated resistance, but it is broad-spectrum or non-race specific, and thus is seen as a promising alternative to less durable race specific resistance for crop improvement. Pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) are the two major innate immune responses seen in plants. Activation of mitogen-activated protein kinases (MAPKs), induction of reactive oxygen species (ROS), callose deposition, and stimulation of pathogenesis-related (PR) genes are all part of the PTI response. An early response to pathogen infection is a ROS burst, which strengthens cell walls by cross-linking glycoproteins and activating defensesignaling components. To block the host PTI response and produce a favorable host cell environment, the pathogen releases effector chemicals into plant cells. Intracellular sensors encoded by resistance (R) genes with a nucleotide-binding site and leucine-rich repeats (NBS-LRRs) in plants detect pathogen effectors directly or indirectly, leading to ETI. ETI imparts significant resistance to specific pathogens, particularly for a specific race, and elicits a hypersensitive response; but, due to the rapid evolution of disease effectors, this is not long-lasting.

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Figure 7.4: Quantitative trait loci. Source: https://www.nature.com/articles/35072085

Non-host resistance, or the phenomenon in which most plants are resistant to most microbial diseases and thus contribute to quantitative resistance, is influenced by PTI. To find the genetic loci controlling multiple disease resistance loci, often known as QDR loci, researchers have utilized linkage analysis, a nested association mapping (NAM) technique, and genome-wide association studies (GWAS). This technique identifies loci that contain hundreds of genes and several candidate genes, making it challenging to pinpoint the causal gene. Multiple connected genes have been found to underpin a single QDR locus in some circumstances such as clusters of functionally related defensive genes involved in secretory processes and cell wall reinforcement. QDR is difficult to genetically dissect, as are other quantitative traits, and the link between phenotypes and molecular mechanisms is not well understood. Due to minor genetic effects, variability in disease severity across different geographical areas, and lack of uniformity in the evaluation of disease symptoms, map-based cloning of resistance conferring QTLs has proven to be extremely difficult. Disease resistance durability is difficult to assess in a short period of time, and evaluating durability is complicated if the QTLs have distinct genetics.

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When a resistance QTL is changed in a highly susceptible background, for example, it may have a greater effect. Despite the fact that some of these genes have been used, their usage in elite cultivars has been limited due to their close association with genes that govern undesired agricultural properties. Wheat Lr34 lines, for example, produce less grain than Lr34-free lines, the recessive barley mlo mutant induces early senescence-like leaf chlorosis. Cloning QDR loci has been difficult due to the minimal influence of numerous QDR loci and the difficulties of phenotyping disease features consistently. Tremendous progress has been made in narrowing down mapped QDR locations to the individual gene level. In recent years, advances in DNA, RNA, and protein sequencing technology, as well as bioinformatic analysis of sequencing data, thus allowing us to quickly locate QTLs and identify candidate genes in many crops for a variety of traits, including disease resistance. Gene mapping through bulked segregant RNA-seq (BSRseq), MutMap, target-enriched X-QTL, genotyping by sequencing (GBS), indel-seq, and exome aide from these, new genome-editing techniques, such as zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and the CRISPR–Cas9 system, have been shown to be promising in simplifying the process of gene deletion, editing, and insertion in plants, thereby assisting in the validation of identified candidate genes for traits.

Figure 7.5: CRISPR–Cas9 system.

Source: https://www.researchgate.net/publication/325336648_CRISPRCas9_System_A_Breakthrough_in_Genome_Editing/figures?lo=1

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The CRISPR–Cas9 system (Figure 7.5) is now widely regarded as the preferred way for improving numerous crops for a variety of qualities, as well as for finding genes of interest. The concept of sustainable agricultural production was developed to solve the issues posed by the world’s rapidly increasing human population by boosting crop plant yield and productivity while avoiding negative environmental effects. The greatest impediment in realizing the full potential of improved genotypes is environmental stresses, both biotic and abiotic. Plant diseases, among the different biotic stressors, pose a significant danger to long-term crop production sustainability. To acquire persistent resistance against disease-causing infections, various approaches have been consistently combined, including the use of pesticides, better agronomic practices, conventional molecular plant breeding, and genetic modification procedures. Enhancing the resistance of crop plants, on the other hand, has been proved to be the most successful, long-term, and cost-efficient technique for dealing with infections.

7.5 RNAI Plants have evolved numerous complicated strategies to strengthen their own defensive mechanisms against these diseases during the course of evolution. When a pathogen attacks, surface-localized pattern recognition receptors (PRRs) recognize pathogen associated molecular patterns (PAMPs), activating events that lead to the pathogen’s eradication. As a result, PAMPtriggered immunity (PTI) is regarded as the first and most important line of protection against pathogens. The identification of genes implicated in the pathways responsible for plant–pathogen interactions is complicated by the huge number of genes involved in PTI. Once the candidate genes have been identified, they must be introduced into elite germplasms using either traditional or molecular breeding methods. Modern omics technology has allowed identifying susceptibility or resistance genes in any species possible, resulting in a vast number of prospective crop protection targets. However, the lack of a quick, precise, and efficient gene targeting mechanism in plants has hampered attempts to confirm these potential genes. Various ways have been used to transfer genes from wild relatives to domesticated species over the years. Traditional breeding, on the other hand, takes about 8–10 years to cascade numerous disease resistance genes into a cultivar. Due to high pathogenic diversity and quick mutation rates, this extended lifetime might promote the rapid breakdown of resistant cultivars. Through the silencing of transcription factor genes, RNA interference (RNAi) based techniques have

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been discovered to be effective in regulating the expression of numerous disease related genes. However, RNAi transgenics have a number of significant problems. Transgene expression levels change amongst transgenic lines, so large populations of plants must be investigated to appropriately identify the set of plants in which the transgene is highly expressed over generations. Other problems include the insertion of transgenes into non-target places in the genome and the introduction of undesired characteristics. Furthermore, because plants developed using RNAi-based approaches are classified as transgenics, they must go through stringent regulatory processes before being commercialised. As a result, more novel biotechnological strategies that provide crop plants with enhanced plant immunity and permanent broadspectrum resistance against pathogens with minimal loss are needed(Dutta, Banakar, & Rao, 2015)new avenues for engineering resistance have opened up, with RNA interference being one of them. Induction of RNAi by delivering double-stranded RNA (dsRNA. Recent advances in sequence-specific nucleases (SSNs) for introducing double-strand breaks at target loci have resulted in very precise genomeediting tools. High-fidelity homologous recombination (HR) or error-prone nonhomologous end-joining (NHEJ) methods are utilized to repair genespecific DNA double-strand breaks (DSBs) induced by SSNs. Furthermore, unlike RNAi, SSN-based genome editing allows for full knockdown without the use of foreign DNA. Zinc-finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins are all examples of SSNs (Cas). CRISPR/Cas9 has been proven to be the most effective SSN among these technologies. CRISPR was first identified in Escherichia coli in 1987 by Ishino et al., who discovered a collection of 29 nucleotide repeats split by non-repetitive short sequences. CRISPR/Cas systems are part of bacteria and archaea’s adaptive immune systems, which protect them from invading nucleic acids like viruses by cleaving foreign DNA in a sequencedependent way. The invading pathogen’s DNA or RNA is targeted by CRISPR/ Cas. Wheat is the world’s second most important cereal crop, behind rice or maize, and is the most important source of calories and protein in human food, especially in underdeveloped countries. Over 700 million tonnes of wheat are harvested annually from approximately 215 million hectares around the world, which is more than any other crop. Wheat has evolved to a variety of

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climates and is cultivated at a variety of elevations and latitudes in irrigated, severe drought, and moist circumstances. Wheat demand is expected to rise by 60% by 2050 as the world’s population grows and people’s lifestyles change. As a result, worldwide average wheat yields per hectare will need to rise to around 5 tonnes per hectare from the present 3 tonnes per hectare. Efforts to continuously improve yield and quality are fraught with difficulties. Unexpected abiotic and biotic challenges, such as abrupt environmental changes or disease translocation, have posed a threat to wheat production. Furthermore, urbanization has reduced the amount of appropriate farmland for wheat farming. Pathogen resurgences have emerged from the monoculture of current wheat cultivars with little genetic diversity, posing a threat to wheat supplies. Fifty diseases and pests out of approximately 200 have been identified as economically important due to their potential to harm crops and reduce farmer income. Diseases are expected to cause 18 percent grain output losses, with actual losses of 13 percent with present disease control(Young et al., 2013). Pathogenic fungi, among other biotic stressors, pose a substantial threat to wheat production. On the basis of their lifestyle, pathogenic fungi can be divided into two categories: biotrophic and necrotrophic fungi. Race-specific resistance also known as seedling resistance and race non-specific resistance are two types of genetic resistance to rusts. More than 200 rust resistance genes in wheat or wild relatives have been identified and formally classified; the majority of them confers race-specific resistance and identified at least 60 of these genes as SR resistance genes. Sr31 was once one of the most extensively used race-specific SR resistance genes; however, after testing against Ug99 races in Kenya, its presence at the International Maize and Wheat Improvement Center (CIMMYT) has been dramatically reduced. With the introduction of Ug99, virulence against Sr31 evolved, resulting in susceptibility to SR in the majority of wheat produced around the world.

7.6 BREEDING PROGRAMS Breeding programs around the world will benefit from new technologies such as marker-assisted selection (MAS), genomic selection, transgenics, and gene editing. MAS has been widely employed in the selection of disease resistance in wheat in high-income nations, for example, rust resistance in Australia. Because of the high cost, the lack of diagnostic and reliable markers, and the high phenotypic selection accuracy, breeding programs around the world rely on phenotypic selection. Furthermore, using MAS

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in conjunction with phenotypic assays is usually recommended to avoid false positives and poor, agronomically weak plants. In wheat, MAS aids in the selection of race-specific resistance genes. The transfer of two or more disease resistance genes in wheat using MAS and traditional backcrossing methods is known as gene pyramiding. This method takes time and is a slow method of transferring resistance genes; it is reliant on the availability of reliable, breeder-friendly markers. Another new wheat technology is the transfer of gene cassettes or gene stacks also known as gene stacking). As gene cassettes or gene stacks, desirable combinations of efficient resistance genes can be assembled and turned into wheat. This could lead to speedier disease resistance advancements in present high-yielding cultivars. However, whether the resulting gene-stacked wheat is a cisgenic or transgenic product is still up for debate. Gene-stacked wheat should be regarded as cisgenic rather than transgenic because the genes used to build gene cassettes or gene stacks originate or are derived from wheat or its relatives. Humans consume or use a variety of cereals, pseudo-cereals, pulses, oil-yielding plants, fiber-yielding plants, spices, and medicinal plants around the world. In the fields, all of these diverse groups of plants are cultivated on a larger scale. These plants are constantly subjected to a variety of environmental challenges in the field, all of which have an impact on their growth, development, survival, and subsequent production. These environmental stresses, which can be divided into two categories: biotic factors and abiotic variables, have a significant detrimental impact on crop productivity around the world. Living organisms that have predatory or symbiotic connections with the host plant are included in the biotic factor. Fungi, bacteria, nematodes, weeds, insects, parasites, rodents, birds, and viruses are all members of this class. Phytopathogens cause a variety of diseases in non-resistant plants by disrupting the plant’s metabolism at the cellular, molecular, hormonal, and physiological levels, among other biotic stressors. The most important biological limitation affecting the food usage component is the vast number of plant diseases. This is evident from the fact that plant diseases account for global agricultural losses between 20 percent and 45 percent which is supplemented by another 5–10 percent during postharvest storage with both direct and indirect consequences. As a result, in order to increase food production, it is critical to develop and improve already existing high-yield, disease-resistant crop types in the fields. Furthermore, breeders have improved a variety of crops around the world for higher yields and stress tolerance, including common bean, sorghum, wheat, barley, sugarcane, and rice

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Breeders have used traditional breeding tactics to add desirable features from related species to high-yielding variants for ages. Traditional breeding methods, on the other hand, have a number of drawbacks, including (i) Time intensive, (ii) being focused on phenotypic evaluation and selection, and (iii) being difficult to transmit traits with polygenic inheritance. Agricultural scientists have been using marker-assisted breeding (MAB) to overcome the limits of traditional breeding strategies. Advances in genomic methodologies and sequencing, as well as the availability of genome sequences, internet databases, and a variety of bioinformatics tools, have all aided this MAB approach. The MAB approach has become indispensable over time because it focuses on improving overall performance, yield stability, traits such as stress tolerance, and farmer acceptance. Crop losses due to disease infestation cost farmers a lot of money all over the world. To manage diseases in crop plants, scientists and farmers have long used a variety of traditional and chemical approaches. Chemicals, primarily fungicides and insecticides, have been used extensively to manage the disease infestation to some extent, but they come at a cost. Chemical use not only raises production costs, but it also has a harmful influence on the environment and the health of both farmers and consumers. Frequently, pathogens acquire resistance to the fungicides used on them, rendering them ineffective in controlling the damage that affects agricultural output and quality. As a result, developing disease-resistant crops is one longterm method to lessen the impact of crop yield and quality loss due to plant diseases. Disease resistance breeding has been an important source of disease control. The basic definition of disease resistance breeding is the introduction of disease resistance genes into disease-infected plants. The resistance genes can come from a natural or artificial source. Disease resistance is divided into two types: qualitative and quantitative. Quantitative resistance is based on oligogenic or polygenic inheritance and is governed by additive or partial dominant genes and is generally race-nonspecific, whereas qualitative resistance is based on a single dominant or recessive gene, is race-specific, and usually confers a high level of resistance. Plant breeders place a higher value on quantitative disease resistance since it is more persistent and has a broader specificity. Gene-for-gene interaction theory is one of the most well-known hypotheses about disease resistance and susceptibility. The hypothesis of gene-for-gene interaction was developed using flax as the host plant and the fungal rust disease Melampsora lini as the pathogen. Disease resistance requires a dominant or semi-dominant resistance R gene in the host

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plant and a corresponding avirulence (Avr) gene in the pathogen. R genes in plants are important for detecting pathogen compounds that are particular to the Avr gene, leading to downstream signal cascades that create defensins, which drive defense. The hypersensitivity response is typically thought of as a defense mechanism that initiates a pathogen-host incompatibility reaction. Disease susceptibility is typically seen in biotrophic pathogens such as fungus, bacteria, viruses, and nematodes when the R gene or the Avr gene is mutated or lost completely. In general, the R gene generates proteins that identify pathogen effectors or modify plant proteins that are the effectors’ targets. The nucleotide binding, leucine-rich repeat (NB-LRR) amino acids sequence motifs, which are involved in pathogen recognition and related functions, are the most well-known of the six known classes of R-genes. Understanding the structural and functional roles of these R genes can help plants become more disease resistant. Several R genes have been discovered, isolated, and cloned. Hm1, a maize R gene that causes resistance to the leaf spot fungus Cochliobolus carbonum, was the first R gene discovered. Hm1 is a reductase enzyme that detoxifies the HC-toxin from C. carbonum. R-genes confer an effective defense response in response to biotrophic pathogen invasion, usually by involvement in a hypersensitive response, in which tissue directly adjacent to the infection’s location undergoes rapid programmed cell death. Pto gene, which encodes a serine/ threonine kinase and protects tomato against Pseudomonas syringae, was another early cloned R gene. RPS2 in Arabidopsis, an NBS/LRR protein family, is another cloned gene for Pseudomonas syringae. Traits that can be quantified and have continuous variations are known as quantitative or metric traits. Quantitative trait loci are loci that influence the genetics of certain qualities (QTL). The constant variety is due to the polygenic inheritance of genes with mostly minor additive effects, which are modified by the environment. Because Mendelian methods of genetic analysis are ineffective for dissecting these quantitative features, other quantitative methods are employed to investigate and comprehend them. The physical localization or mapping of polygenes began with the discovered relationship between seed coat color and seed size in common beans. The ability to generate the genomic map of a given species, together with the development of the linkage idea, leads to the creation of QTL mapping. Paterson et al. used the restriction fragment length polymorphism in tomato to map the first QTL in 1988. The use of a mapping population, usually a bi-parental population derived from the cross of two genetically diverse parents, a dense marker linkage map for a particular species and genotypic

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data (SNPs, SSRs), standard phenotypic measurement, and suitable software programs, such as R/QTL, QTL Cartographer, and so on, are all common methods. Various infections in the environment pose a constant threat to plants. Some wild plants have an inherent resistance to attack that helps them survive in the wild. Plants that were domesticated and subsequently developed by humans to yield good qualities eventually lost their resistance and became prone to disease attacks. Despite the presence of particular resistance genes, newly developed pathogen strains can overcome genetic resistance. This ongoing co-evolution of agricultural plants and their pathogen necessitates long-term plant breeding efforts to develop new crop varieties or boost resistance genes in well-adapted kinds. Another source of concern is the projected rise in climate variability, which could increase pathogen occurrence in a given area. For environmental and economic reasons, host plant resistance is often the most preferable control technique. Until now, conventional plant breeding methods have aided in the solution of this problem. However, demand for newer, more resistant crop varieties must be met quickly. Molecular breeding, also known as marker-assisted breeding (MAB), has the ability to alleviate this problem and solve it more effectively and quickly than traditional breeding. Furthermore, without disease manifestation in the field in MAB, the selection of resistant plants is simple. Marker-assisted gene pyramiding is a popular strategy for gene stacking within an adapted variety that avoids the requirement for several pathogen races to be tested in different conditions. They are separated into tropical pulse crops like pigeon pea, mung bean, urd bean, cowpea, common bean, and so on and temperate pulse crops like pigeon pea, mung bean, urd bean, cowpea, common bean, and so on based on their climatic conditions for growth like chickpea, lentil, pea, etc. Plant pathogens such as viruses, bacteria, fungi, and pathogenic weed species cause damage to these pulses. Yellow vein mosaic virus, for example, is a prevalent issue in tropical legumes such as mung bean, urd bean, and cowpea. Ascochyta blight causes significant damage to chickpea and lentil crops. Chickpea and pigeon pea are both susceptible to fusarium wilt. In subtropical pigeon pea farming, sterility mosaic caused by a virus is an endemic concern. The creation of resistant cultivars in the above pulse crops is an immediate requirement to save pulse production from these plant infections. Recent genome sequencing efforts in major pulse crops have resulted in massive amounts of marker data and a molecular breeding or genomics platform. The use of these has aided in the production of less

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enhanced variants and has a bright future in the development of diseaseresistant pulse crops. Previously, great effort was put into improving grain production, agronomic features, and disease resistance in oat breeding. The complex oat breeding program’s major goal is to generate new high-yielding winter and spring types with good grain quality and resistance to the oat disease complex. Because oats are self-pollinated, the basic breeding technique of selection, introduction, and hybridization followed by selection is used all over the world. The oat cultivars grown in the United States during the 1930s were all introductions or selections from previously introduced cultivars from other regions of the world. After that, oat breeders used hybridization extensively to generate high-yielding grain quality with disease resistance to a wide range of diseases.

Figure 7.6: Genome mapping. Source: https://www.genome.gov/sites/default/files/tg/en/illustration/genetic_ map.jpg

Gene and genome mapping, (Figure 7.6) as well as understanding chromosomal behavior and evolution, were all considerably aided by large crosses between different ploidy species. Oat diseases are still the main cause of yield and grain quality losses. To date, the main goal of oat breeding has been to restore disease resistance diversity in cultivated oats by introducing resistance genes that remained unselected from wild progenitors due to genetic constraints during domestication. As a result, phytopathological

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investigations among the wild oat complex should be prioritised in order to uncover novel sources of resistance for widening the genetics of farmed oats(Lee, Tang, Wang, & Paterson, 2013)owing to their propensity for chromosomal duplication or even triplication in a few cases. Duplicated genome structures often require both intra- and inter-genome alignments to unravel their evolutionary history, also providing the means to deduce both obvious and otherwise-cryptic orthology, paralogy and other relationships among genes. The burgeoning sets of angiosperm genome sequences provide the foundation for a host of investigations into the functional and evolutionary consequences of gene and GD. To provide genome alignments from a single resource based on uniform standards that have been validated by empirical studies, we built the Plant Genome Duplication Database (PGDD; freely available at http://chibba.agtec.uga.edu/duplication/. Traditional breeding relied on controlled hybridization to generate new genetic combinations, followed by phenotypic selection in segregating populations. Traditional breeding techniques are often focused on phenotypic selection for desirable trait combinations with desired gene combinations. Traditional breeding strategies have boosted the yielding capacity of major food crops while also reducing the effectiveness of phenotypic selection and hindering the identification of superior genotypes due to genotypeenvironment interactions. Furthermore, due to the complexity of genes involved in disease resistance, pathotyping for disease resistance becomes a tough challenge for plant breeders. Traditional hybridization is a complex and time-consuming process for introducing a resistance gene into a susceptible variety from a resistant one. Genetic diversity among agricultural species has declined throughout time as a result of developmental activities and industrialization, while diseases and insect pests have evolved continuously as a result of climatic change. As a result, host resistance has deteriorated, necessitating the development of breeding variants that combine high yield and resistance. The advent of genomics methods and resources has opened up new horizons in plant breeding since they allow for a more accurate investigation of the genotype and its interaction with the phenotype, which is especially crucial when dissecting complex features. Plant breeders use molecular markers to better understand complex polygenic traits, dissect genes responsible for desired traits, characterize plants, create a genetic linkage map that aids in gene tagging and gene mapping, and develop new cultivars using various marker-assisted selection (MAS) schemes, such as markerassisted backcross breeding (MABB), marker-assisted gene pyramiding,

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and marker-assisted recurrent selection. The development of a wide range of DNA marker technology and genetic mapping in important crops has aided in the identification of a source of variation and acted as a reliable tool for disease resistant individual identification and selection. Soybean is the source of protein and oil. It’s a versatile crop that’s been utilized for human food, protein feed ingredients, and industrial purposes. Various biotic and abiotic stressors pose a threat to soybean production. After brown spot, charcoal rot is the second most cost-effective soybean disease. Macrophomina phaseolina causes soybean charcoal rot, which is an economically significant disease all over the world. In addition to soybeans, this virus infects a variety of crops, including sorghum and maize. The severity of the disease grows as the temperature of the soil and air rises. Synergistic yield losses occur owing to both environmental stress and charcoal rot disease when soil moisture is low. Due to the confounding effects of drought, estimating the yield loss caused by the occurrence of charcoal rot disease is challenging. Charcoal rot disease causes 6–33 percent output loss in vulnerable cultivars in irrigated conditions, indicating the disease’s relevance even under irrigated conditions. Fermentation technology is regarded as mankind’s most crucial invention, second only to the control of fire. This viewpoint is relevant to beer production and barley malt, a technology that has been used for centuries around the world. The first evidence of barley beer consumption dates from around 3350–3000 BC. Many cereal sources were domesticated by mankind for livelihood as human settlement spread over the globe. One of the first multipurpose cultivated grains was barley. In contrast to rice and wheat, cultivated barley is an annual, self-pollinating temperate grass that requires little fertilization. It blooms in both the winter and spring seasons all over the world, with different spike morphologies. It is one of the four primary crops produced worldwide, according to the Food and Agriculture Organization Corporate Statistical Database, with global production of 145.96 million metric tons (Kaur et al., 2012). Barley is currently the world’s fourth most significant cereal crop, with large use in feed, beer production, spirit production, and the food value chain. In both tropical and temperate settings, a surge was observed in both usage and production of both types of barleys. Stress resistance, yield stability, and quality traits are currently the focus of barley breeders. Due to the integration of conventional breeding with DH production, genomic tools, and molecular marker technologies over the decades, there has been a

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favorable progress in the augmentation of lasting resistance against a variety of relevant diseases. The use of non-traditional technologies has decreased the time between the first cross and the introduction of disease-resistant cultivars. The availability of genomic sequences of rice, Brachypodium, sorghum, and wheat, high-density maps, map-based cloning, genome-wide transcript profiling, genome editing techniques, and various bioinformatics tools to exploit the synteny between barley and these species has boosted this even further. Using molecular breeding procedures, more phytopathogen resistance genes, alleles, and QTLs will be identified, isolated, mined, transferred, and transformed into elite-susceptible cultivars in the near future to improve disease resistance. Taking all of this information into account, all of these advancements have improved disease resistance breeding efforts for barley.

CHAPTER

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SUSTAINABLE AGRICULTURE

CONTENTS 8.1 Introduction ..................................................................................... 168 8.2 Manures .......................................................................................... 169 8.3 Biochar ............................................................................................ 171 8.4 Drip Irrigation .................................................................................. 176 8.5 Conservation ................................................................................... 179 8.6 Ecological Security .......................................................................... 182

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8.1 INTRODUCTION Water is vital for human health, as well as our food and farming systems’ longterm ecological and socioeconomic resilience. Because the agricultural sector is responsible for a major portion of water consumption and contamination, it must take the lead in saving and safeguarding water resources. Chemical pesticides and fertilizers are used in food production, resulting in ongoing deterioration of water quality and increased societal expenditures. Efforts to decrease agricultural contamination of ground and surface waterways remain a persistent problem. Although there are many different water treatment procedures available, not all of them are cost-effective or feasible for small farmers, resulting in the usage of low-quality water in agricultural fields (Reganold, Papendick, & Parr, 1990). Despite considerable progress, bad water management practices continue to have a negative influence on water quality. Organic farming is said to have the potential to deliver benefits in terms of environmental protection, non-renewable resource conservation, food quality enhancement, surplus product reduction, and agricultural reorientation toward regions of market demand. Governments have noticed these potential benefits and responded by providing financial incentives or indirect assistance for research, extension, and marketing campaigns to encourage farmers to embrace organic agricultural practices. Farmers’ judgments about whether or not to switch from conventional to organic farming, on the other hand, have not been thoroughly researched. The use and release of water in both animal and plant farming is a major source of water pollution. When water is swapped in a fish pond, for example, effluent is discharged into the adjacent surface waterways. The wastewater contains a variety of contaminants, as evidenced by the indicators chosen. Chemicals, fertilizers, and feed supplied to the ponds are the source of these contaminants. As a result, water pollution is decreased in organic farming since the eutrophication of chemical inputs used in conventional farming methods, such as nitrogen and phosphorus, is considerably reduced. Organic farms also have far better soil structure, which results in less nitrate pollution and is healthier for agricultural plants because it is chemical-free. As previously stated, the two agricultural nutrients of significant significance to water quality and human health are nitrate and phosphorus. Leaching occurs when nitrate, the most frequent type of nitrogen in soils, is released. Nitrate is negatively charged, unlike potassium, calcium, and magnesium, which are positively charged. Because most soil particles, including organic matter, have negative charges, positively charged nutrients are able to bond to them. However, negatively charged nitrate is attracted to negatively

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charged soil particles. As a result, it can easily pass through the soil profile and into the groundwater. Because phosphorus is limited in freshwater systems, it is the nutrient of greatest concern for runoff and erosion losses. Even a small amount of phosphorus added to lakes, rivers, or streams can induce nutritional imbalances that promote algae growth, limiting fish access to nutrients and oxygen. When nutrients are transferred beyond the reach of plant roots, leaching has an impact on crop growth. When nutrients are carried into groundwater, it is a source of problems for water quality. The National Organic Practice Standards specifically state that raw manure must be applied in a manner that does not contribute to the contamination of crops, soil, or water by plant nutrients, pathogenic organisms, heavy metals, or residues of prohibited substances to ensure that organic production practices are implemented in a manner that protects the environment. This criterion gives certifying authorities the authority to prohibit problematic actions including spreading manure on the ground or too close to water sources. The transfer of soil particles by wind or water is known as soil erosion. Erosion takes more topsoil, reactive clays, and organic matter than other soil components because these forces may easily move less dense particles. As a result, it destroys the soil by eliminating the most fertile elements. Soil erosion can also harm nearby farmland and contaminate nearby bodies of water. Transported sediments carry nutrients, pathogens, and other pollutants associated with them. These sediments influence fish habitats by clouding the water, changing the temperature, and becoming embedded in feeding and nesting regions along stream banks. Algal blooms, fish habitat degradation, and eutrophication are all caused by nutrients delivered by sediments. If lakes supplied by contaminated streams are used as a source of drinking water, pathogens clinging to sediments can decrease the quality of water for animal and human use and increase purification expenses.

8.2 MANURES In manure, pathogens are frequently discovered. Escherichia coli, Cryptosporidium, and Giardia are among the pathogens that pose the greatest threat to human health. These organisms cause gastrointestinal issues in humans who eat or drink contaminated food or water. To kill E. coli and ensure the safety of drinking water, municipal purification systems chlorinate it. Cryptosporidium and Giardia, on the other hand, produce resistant resting stages like oocysts and cysts, respectively that are not killed

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by basic water treatment techniques like chlorination. To remove these parasites from water, sand filters are required. Fresh manure applied to growing crops or applied shortly before planting can contaminate them with diseases. If animal production operations or septic systems upstream are not adequately managed and enable fresh waste to flow into the water, water from rivers or streams used for crop irrigation can contaminate plants with diseases. Organic agriculture methods rely mostly on measures such as the adoption of pest-resistant cultivars, cultural management approaches, and actions that improve pest-predator balances for pest and pathogen control. Pesticides are generally limited to biologically produced chemicals with low mammalian toxicity and are only employed as a last option. Some botanical pesticides, on the other hand, are poisonous to non-target organisms. Pyrethrum kills both helpful and disease-causing insects, and rotenone is harmful to fish. Because of its physically disrupting features, diatomaceous earth is used to manage insect pests, but it may also be a significant irritant to human lung tissue if not handled properly. Even low-toxicity plant fertilizers and chemicals can become pollutants if used in large quantities, adjacent to water sources, or during periods of high rainfall or flooding. Using strategies that store and recycle nutrients within the farming system, organic farmers can safeguard water from contamination. When applied as part of an integrated, systems-based approach, such practices are most successful and long-lasting. Maintaining nutrient balances within fields while decreasing off-farm water flows, retaining water inside fields, and catching any water that flows away from fields would conserve nutrients on the farm while safeguarding the environment. The utilization of a varied range of plants as rotation crops, cover crops, and intercrops improves soil quality, enables nutrient capture, and aids in the recycling of nutrients that would otherwise be lost in the soil. These crops also provide soil cover, which promotes water infiltration while reducing the risk of nutrient runoff and erosion. The accumulation of active organic matter and various communities of soil organisms will improve nutrient storage in the soil while reducing the likelihood for these nutrients to be transported to ground or surface waters. Fresh manure should be stored on concrete slabs or soils with a low leaching potential, with collection or treatment areas for contaminated runoff water. Because of reduced soil erosion, increased stored soil water and organic matter, carbon sequestration, and other ecosystem services, conservationtillage practices are replacing conventional-tillage practices throughout

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semi-arid regions in North America and beyond. In a large portion of the US northern Great Plains, for example, no-tillage (NT) management is currently employed to manage over 50% of the area used for dry land crop production. These findings have piqued the interest of farmers and academics in North America and Europe in replacing conventional tillage with NT farming methods in organic systems, so that the benefits of this conversion might be realized. Although there are currently few studies on the use of biochar in organic farming systems, there is much to be learnt from historical charcoal use in agriculture as well as current conventional agricultural research. Farmers have utilized biochar, the method of burying charcoal in soil to promote fertility, from citrus fields in Japan to basket willow stands in north Great Britain to the legendary Terra Preta soils of the Amazon Basin. Biochar has experienced a resurgence in modern agriculture, with this carbon-rich material now being widely employed to improve soil and encourage more sustainable agriculture.

8.3 BIOCHAR Biochar is a carbon-rich, stable solid material made by thermochemical decomposition of organic material in an oxygen-limited environment under controlled conditions. It differs from charcoal made during wildfires or for fuel because biochar is made specifically for use as a soil amendment, whereas charcoal is commonly used as an energy carrier. Forest or agriculture residues, municipal solid waste, or biosolids can all be used to make biochar. Biochar has been proposed as a technique of mitigating climate change by sequestering carbon when applied to soils because of its C-rich nature and unusual resistance to decomposition. Furthermore, the morphological qualities of biochar may modify soil hydrological parameters, affecting soil nutrient conversions. The Terra Preta soils in the Amazon River Basin were reportedly developed by primitive tribes thousands of years ago and are still some of the most fertile and biodiverse soils in the Amazon today. Terra Preta’s origin is unknown, but the large amount of char found in these soils makes it unlikely that it was created by biomass burning, but it’s unclear whether the biochar application was done on purpose or as a means of sanitary waste management in populated areas of the Amazon basin (AupNgoen & Noipitak, 2020).

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Figure 8.1: Biochar. Source: https://onlinelibrary.wiley.com/doi/10.1111/gcbb.12885

An old process known as formiguer, has a structure that is similar to that of a charcoal kiln, was widely employed in the Mediterranean region to make soil-fertilizing material with dried woody plants. Farmers in Japan have been pioneering the use of biochar in agriculture in conjunction with composting processes since the early twentieth century. Rice husks and other farming residues would be used to make charcoal in traditional earthen

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kilns, which would be used primarily as soil improvers or odor absorbents. Fire is a major source of disturbance in western US forest ecosystems. Over the last few decades, active fire suppression and a shift in forest management have resulted in an increase in the incidence of extensively stocked secondgrowth forests, which may alter wildfire behavior. In the western United States, forest restoration and fuel reduction techniques such as selected harvest paired with managed fire are being used to establish a more resilient forest structure. Normally, forest leftovers from timber harvests are piled and burned, resulting in emissions of gaseous air pollutants and volatiles, nutrient loss, and no net environmental gain. As a result, developing a valueadded strategy for wood harvest residue management could help catalyse restoration efforts in both private and public forest areas. Thinning treatments have become regular practice for foresters and landowners on the islands, as the region is mostly covered with intensively stocked woods. The dominating timber in these forests, on the other hand, has a poor value and significant transportation costs, resulting in the timber being primarily piled and burned. Simultaneously, small-scale organic farming on sandy loam soils created in glacial deposits and outwash across the islands supports a significant portion of San Juan County’s economy. It would be ideal to create a strategy that produces less pollution from forests diminishing while also adding to the soil fertility of local organic farms for food production. Biochar production from local timber harvest wastes in this region could provide a long-term solution for reducing wildfire hazard fuel loading while also enhancing soil health on organic farms nearby. Pests and diseases cost fruit growers a lot of money since they reduce fruit yield by 30–100 percent. They also degrade the cosmetic look, market value, quality, and nutritional content of fruits by sucking, gnawing, or drilling into various reproductive regions, generating stains, cracks, and holes, as well as rotting. For a long time, chemical pesticides have been used to manage insect pests and fungal diseases. Pesticides used on fruit fields leave hazardous residues in fruits that are consumed by humans, posing a health risk to consumers. Their sustained use has a negative impact on the environment, resulting in the development of resistance in many pests, the resurgence and outbreak of new pests, and health risks to production workers and farm laborers as a result of incorrect or lack of knowledge of pesticide handling and use, as well as pesticide poisoning. Many synthetic pesticides, such as organochlorines, organophosphates, carbamates, and organo phthalides, have been banned or restricted from use due to their negative environmental impact and high toxicity to non-target organisms such as beneficial insects,

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amphibians, fish, and birds, as well as humans. However, for the control of insect pests and diseases, efforts are now being made to replace chemical pesticides with eco-friendly compounds that are safe for humans and the environment.

Figure 8.2: Organic fruit farming. Source: https://www.fao.org/organicag/oa-home/en/

Organic fruit farming (Figure 8.2) is a method of producing fruit that is good for the earth, ecosystems, and people. Rather than using harmful inputs, it relies on biological processes, biodiversity, and cycles that are tailored to local conditions. Because it is based on low use of chemical inputs for crop health and protection against biotic threats, it is an ecologically sustainable fruit production method that safeguards biodiversity, physico-chemical, and biological health of our soils. Food production has always followed an arithmetic progression, whereas the human population has always followed a geometrical progression. Farmers have been obliged to apply pesticides as a result of the enormous pressure to provide food at affordable costs. These chemicals could be growth regulators, defoliants, desiccants, The use of pesticides to increase food production resulted in significant environmental contamination as well as a significant reduction in food output. Pesticides and their breakdown products are exceedingly harmful to

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humans and other living organisms alike. Aldrin’s photodegradation products are significantly more dangerous than the parent molecule, and they are transported through numerous natural resources, contaminating them in the process. The necessity for a significant amount of solvent, extended analysis times, the need for professional personnel, and expensive expenditures for executing the tests are all important drawbacks of chromatographic procedures. As a result of these constraints, novel spectroscopy-based pesticide detection and estimation techniques have been developed. These strategies have the significant benefit of causing less harm to the environment and other living things. Organic animal husbandry isn’t a one-size-fits-all approach. It is in the process of being improved. On production guidelines, there are distinctions between traditional, conventional, and organic animal husbandry. Organic animal husbandry is a significantly more sophisticated and knowledge-based method of animal production designed to protect not only human health but also animal welfare and the environment as a whole. Organic livestock farming has set out to build environmentally friendly production, maintain healthy animals, achieve high animal welfare standards, and provide high-quality goods. Organic animal husbandry is a system that promotes the use of certified organic and biodegradable inputs from the environment in terms of animal nutrition, health, housing, and breeding, while avoiding drugs, feed additives, and genetically engineered breeding inputs. When it comes to plant farming, this may not have major effects, but when it comes to animal farming, it can have serious consequences. The organic rule makes no mention of any understanding of how to administer and manage an animal farm. It doesn’t even say that the animals should live as naturally as possible. There are a variety of diseases that commonly affect our animals. Viruses, bacterial infections, parasitic infections, and fungal infections are all examples of infectious disorders. Mastitis, infertility, milk fever, lameness, metabolic abnormality, and ketosis are the most prevalent disorders in dairy cows. Organic livestock has a lower incidence of milk fever, lameness, infertility, and metabolic problems. Dairy cows in organic agriculture farming systems have adequate movement, which helps to decrease lameness and metabolic diseases. Furthermore, in the case of broilers, organic livestock production reduces hock burn, footpad dermatitis, skin burn, blisters, and obesity. In the case of inorganic farming, blisters and obesity are big issues with broilers. The occurrence of blisters and obesity is reduced as a result of proper activity, and the quality of meat is improved, resulting in a higher dressing percentage. Organic management decreases stress, lowers

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disease incidence, and improves animal comfort. Local purebred strategies are followed in organic agriculture; synthetic breeds and GMOs are not used. Local breeds have higher disease resistance than synthetic breeds. Diseases are less common in indigenous breeds, and they do not suffer from immunosuppression. Local breeds are more capable of coping with stressful situations than synthetic types. As a result, animal health is better in organic animal husbandry than in any other production style.

Figure 8.3: Drip irrigation. Source: https://afko.com.tr/drip-irrigation/

8.4 DRIP IRRIGATION Drip irrigation, (Figure 8.3) a method of providing water directly to the plant root zone, is distinguished by water application at slow rates but increasingly frequently throughout the crop growth season as crop water requirements. It is best suited to vegetable crops, fruit trees, and vine crops, but because of its water conservation potential and improved crop productivity, it is now being used on a wide range of row crops. As a result, drip irrigation has extended over an area of roughly 1.9 million hectares, possibly the largest drip irrigation area in the world. Another significant benefit of drip irrigation is the ability to use saline water. Because of the low matric stress in the

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root zone caused by frequent applications, the plants are able to withstand the high osmotic stress associated with saline water irrigation. Despite the copious evidence accumulated over the previous three decades or so, there has been little success in expanding the use of saline or alkali water with drip irrigation. Furthermore, the high capital costs and knowledge necessary to address various complicated difficulties in drip irrigation pushed technologists to design local drip irrigation alternatives. Several of them are not only similar to drip irrigation, but they are also very inexpensive and need less sophistication for resource-constrained farmers to implement. Food insecurity is linked to a decrease in household food supplies, less frequent fruit and vegetable consumption, higher unemployment, increased involvement in food assistance programs, and an increase in eating disorders in the US population. Food insecurity is a spectrum with varied degrees of severity depending on the level of hunger experienced within the home. Food insecurity without hunger is the mildest kind of food insecurity, and it describes families who are concerned about running out of food and will alter their purchasing or consumption habits to affect the quality of the food supply. In households with moderate levels of food insecurity, parents or adults may be hungry while their children eat nutritiously enough meals. At its most extreme, hunger affects all members of a home for long periods of time. According to a 2015 survey, about six million people in California, or 15% of the population, were food insecure. Food insecurity affects minorities disproportionately, particularly those whose members are recent or undocumented immigrants. Food insecurity is strongly linked to a lack of enough and consistent income. Many Mexican-American households, for example, go through periodic cycles of food poverty due to agricultural employment schedules. Although these advancements have had numerous positive consequences and eliminated many risks in agriculture, they have come at a great cost. Three key goals of sustainable agriculture are environmental health, economic profitability, and social equality. Concerns of animal welfare in farm enterprises that incorporate animals must also be addressed as part of stewardship considerations. Sustainability requires an understanding of agroecosystems and food systems. Agroecosystems are defined in the broadest sense, encompassing everything from small fields to farms to entire ecozones. Food systems, which comprise agroecosystems as well as distribution and food consumption components, extend from the farmer to the local community to the global population in a similar way. An emphasis on a systems viewpoint provides for a holistic picture of our

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agricultural production and distribution enterprises, as well as their impact on human communities and the natural environment. A systems approach, on the other hand, equips us with the skills we need to examine the impact of human society and institutions on farming and environmental sustainability (Martinez, Grandner, Nazmi, Canedo, & Ritchie, 2019). Studies of several natural and human systems have shown that systems that endure over time are frequently very robust, adaptive, and diverse. Because most agroecosystems face variables such as climate, insect populations, political situations, and others that are extremely unpredictable and rarely constant over time, resilience is essential. Adaptability is an important component of resilience because, while it may not always be possible or desirable for an agroecosystem to return to its original shape and function after a disturbance, it may be able to modify and take on a new form in the face of changing conditions. Since the more variety that exists within a food system, whether in terms of varieties of crops or social knowledge, the more tools and pathways a system will have to adapt to change, diversity typically aids in adaptation. A multi-pronged strategy to research, teaching, and action is also part of an agroecosystem and food system approach. Farmers, laborers, retailers, consumers, policymakers, and others with a stake in our agricultural and food systems all have important responsibilities to play in achieving greater agricultural sustainability. Finally, there is no clear, well-defined ultimate aim for sustainable agriculture. The scientific understanding of what constitutes environmental, social, and economic sustainability is constantly growing, and it is influenced by current concerns, viewpoints, and beliefs. Agriculture’s ability to adapt to climate change, for example, was not considered a serious concern 20 years ago but is now getting more attention. Furthermore, the specifics of what makes a sustainable system might vary depending on the circumstances, as well as from one cultural and ideological standpoint to the next, making the term sustainable, a contentious concept. As a result, rather than thinking of agricultural systems as either sustainable or unsustainable, think of them as ranging along a continuum from unsustainable to very sustainable. When food and fiber production deplete the natural resource base, future generations’ ability to produce and thrive is harmed. Natural resource deterioration through non-sustainable farming and forestry methods is thought to have influenced the fall of ancient civilizations in Mesopotamia, Pre-Columbian southwest United States, and Central America. Sustainable

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agriculture aims to use natural resources in a way that allows them to regenerate their productive potential while minimizing negative impacts on ecosystems beyond the field’s edge. Farmers are contemplating how to leverage existing natural processes or how to build their agricultural systems to incorporate critical functions of natural ecosystems as one method to achieve these aims. It is often possible to sustain an economically viable production system with fewer potentially hazardous interventions by constructing biologicallyintegrated agroecosystems that rely more on the internal cycling of nutrients and energy. Farmers who want to achieve a greater level of environmental sustainability, for example, can think about how they can reduce their usage of hazardous pesticides by relying on natural mechanisms to control insect populations. This can be accomplished by planting hedgerows along field margins or ground coverings between rows, which provide habitat for insects and birds that prey on pests, or by planting more diversified crop blends that confuse or deflect pests. Maintaining a high level of genetic variety by preserving as many crop varieties and animal breeds as feasible can provide additional genetic resources for breeding disease and pest resistant plants.

8.5 CONSERVATION Conservation of resources is important for agricultural output, but it also includes looking after soil, which is a complex and highly organized entity made up of mineral particles, organic matter, air, water, and living beings. Farmers that are committed to long-term sustainability place a high priority on soil care because they understand that healthy soil encourages healthy crops and livestock. Maintaining soil functionality frequently necessitates a focus on preserving or even growing soil organic matter. Soil organic matter serves as a vital source and sink for nutrients, as a microbial activity substrate, and as a buffer against changes in acidity, water content, pollutants, and other factors. Furthermore, the accumulation of soil organic matter can aid in the reduction of atmospheric CO2 and, as a result, climate change. Another key role of soil organic matter is to improve soil structure, which leads to better water penetration, decreased runoff, improved drainage, and higher stability, all of which reduce wind and water erosion. The internal cycle of important plant nutrients such as nitrogen and phosphorus has been isolated from agroecosystem functioning due to a significant reliance on artificial fertilizers. Although phosphate minerals for fertilizer are regularly extracted, worldwide stockpiles are only expected to last another 50 to 100 years. As a result, unless new reserves are discovered

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and new methods for recovering phosphates from waste are developed, phosphate prices are expected to grow. Recycling nitrogen and phosphorus at the farm and regional level, increasing fertilizer application efficiency, and depending on organic nutrient sources like animal and green manure are all significant aspects of sustainable agriculture. Diversified agriculture, in which livestock and agricultural production are more spatially integrated, facilitates nutrient recycling. As a result, broad mixed crop-livestock systems, especially in developing countries, could make a significant contribution to agricultural sustainability and global food security in the future (Cordell, Drangert, & White, 2009).Water for agriculture is in short supply in many regions of the world, and its quality is degrading. Overdraft of surface waterways causes riparian zones to be disrupted, while overdraft of groundwater supplies puts future irrigation capacity at risk. Water quality problems include salinization, nutrient overloads, and pesticide contamination. Water may be used more efficiently in sustainable agroecosystems by selecting and breeding drought and salt tolerant crop species and better animal breeds, using reduced volume irrigation systems, and managing soils and crops to prevent water loss. Agriculture in the modern era is primarily reliant on non-renewable energy sources, particularly petroleum. Although the usage of these non-renewable resources cannot be perpetuated indefinitely, immediately ceasing to do so would be economically disastrous. Without the knowledge, technical competence, and trained labor required to efficiently manage agro-ecosystems, they will not be sustainable in the long run. Agriculture’s ever-changing and location-specific character necessitates a diversified and adaptable knowledge base that incorporates both formal, experimental research and farmers’ own on-the-ground local knowledge. Agricultural production and long-term sustainability can be increased by social institutions that support farmer and scientist education, foster innovation, and promote farmer-researcher cooperation. Farmers lack the economic influence to negotiate better pricing for their inputs and crops as food manufacturers and marketers and farm input suppliers consolidate. As a result, farmers’ profit margins are squeezed, leaving them with little options for improving environmental and labor conditions. Farmers can strengthen their relative economic power by banding together in production, processing, or marketing cooperatives. Farmers

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can be protected in the long run by policies that regulate consolidation. Many rural communities have become poorer as a result of these economic constraints on farmers, as farms and other local agricultural firms have gone out of business. Economic development policies and tax arrangements that encourage more diverse agricultural production on family farms can help rural economies thrive. Consumers can also play a role within the constraints of the market framework; through their purchases, they send strong signals to producers, merchants, and others in the system about what they value, such as environmental quality and social equality. Some of the pressures that have harmed on-farm sustainability have also harmed social equity for low-income consumers, who are frequently left with little access to healthy food as traditional supermarkets relocate to more lucrative neighborhoods in order to boost thin profit margins. Community and household gardens, farmers’ markets, the utilization of fresh local farm products in school lunch programs, and local food cooperatives are examples of food production and marketing systems to strengthen community food security. Furthermore, a food systems approach considers the effects of farming techniques on the safety and nutritional quality of final food products that reach consumers, such as reducing or eliminating harmful residues. By 2050, the world population will be over 10 billion people, and as economies expand, the average wealth of people in emerging countries will rise, posing new problems for agriculture and the food supply chain. One of the great success stories of applied research to date has been the growth in food production to meet global demand. This success is at the expense of nature; biodiversity is rapidly dwindling, rivers, lakes, and aquifers are polluted, soils are polluted or eroded, forests are being converted to grow crops for food and energy, and agricultural greenhouse gas emissions are nearly equal to those from transportation. Things cannot continue as they are; future food security will require a focus on the long-term supply of food, as well as technological, supply-chain, and social science or economic innovation. Agriculture may and does benefit from ecosystem services provided by nature, and these benefits must be factored into biodiversity management and protection.

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Figure 8.4: Ecological security. Source: https://ars.els-cdn.com/content/image/1-s2.0-S1470160X1930086Xga1_lrg.jpg

8.6 ECOLOGICAL SECURITY Ecological security (Figure 8.4) is critical for both preserving ecosystem function and providing ecological services to human well-being. The impact of land use change and cover on ecological security has received a lot of attention, although cropland reclamation’s function is yet unknown. The loss of ecological land that occurs as a result of agricultural programs has an impact on ecological security. The land where ecological security is expected to improve is concentrated in locations with a large number of connected croplands. The fact that a greater number of places would worsen rather than improve implies that agricultural modification has a detrimental influence on ecological security that should not be overlooked. When comparing various scenarios, croplands returning to ecological lands have the greatest influence on ecological security, especially in the higher reaches, where steep croplands are concentrated. According to the International Institute for Applied Systems Analysis, ecological security refers to the state of a natural ecosystem that can maintain its order and function while also providing sustained ecosystem services. Due to growing urbanization and

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frequent human involvement, ecological security is under unprecedented strain today. Natural catastrophes, urbanization, and land use change/cover or LUCC have all had an impact on ecological security in the past. The idea of ecosystem health was first developed in the 1980s, and it was then used to assess ecological security. To assess ecosystem health, a framework of vigor–organization–resilience (VOR) was established, based on the stress ecology definition of health as system organization, resilience, and vigor, as well as the lack of indicators of ecosystem distress. Indicator assessments, pressure–state–response (PSR) and its expanded framework driving– pressure–state–influence–response (DPSIR), and footprint assessments have all been established to assess ecosystem health. Because the CVOR framework depicts ecological integrity and natural ecosystem quality at a grid level, it is superior to the other techniques and is thus used in this study to assess ecological security. Despite the fact that LUCC’s impact on ecological security has been well proven, agricultural reclamation is frequently overlooked. When croplands are lost due to urbanization in China, farmland reclamation with the same amount and quality is demanded in order to ensure food security. It has also been suggested that the indirect impact on ecosystem services caused by agricultural reclamation may be greater than the direct impact caused by urban growth. Farmland reclamation under a strong cropland conservation policy can result in the indirect loss of natural habitats, which is significantly more than the direct loss caused by urban expansion. Furthermore, the loss of carbon storage caused by agricultural reclamation between 2000 and 2010 was estimated to be 1.12 times greater than that caused by urban expansion during the same period. As agricultural reclamation continues, this suggests that the indirect impact of cropland reclamation on ecological security should not be overlooked (L. Tang, Ke, Zhou, Zheng, & Wang, 2020)forest, grassland, wetland. The indirect effect of cropland reclamation on ecological security, however, is unknown. The amount of arable land available is unlikely to change, and as the world’s population expands, producing more crops from limited resources will become a requirement. By 2050, the world’s population will have grown to 9 billion people, up from 7.7 billion today, and food production would need to expand by at least 70% to feed the growing population. Without a shift away from traditional agricultural techniques, future generations’ ability to produce and exploit our arable land would dwindle. Previously, the emphasis was on making the most of available land and producing as many crops as possible. There was little emphasis on sustainable agricultural

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production or land preservation, and there was a comparable lack of emphasis on profitability and land and resource conservation from a financial aspect. These issues are intertwined, and the answers will help to modernize agriculture. Farmers’ decisions to adopt more sustainable practices, such as organic farming, hedgerow restoration, or cover crop planting, have their own risks. Farmers’ decisions to adopt more sustainable practices are primarily business decisions, occur less frequently, often have long-term personal and economic consequences, may entail large investments and long-term commitments like participating in voluntary land conservation programs, and entail the provision of public goods. Farmers’ decisions to adopt more sustainable techniques can likewise be expected to be more controlled and well-thought-out as compared to the above-mentioned examples of consumer or citizen decision-making. This does not, however, imply that these choices are free of heuristics and biases, or that the result will be sensible. Although the assumption of rationality provides a broad, often statistically valid account of producer choices, it precludes a more nuanced understanding of facts, which is especially inappropriate when studying farmer-environment interactions. Because of the fundamental distinctions between the intended goals of a behavioral approach and farmers, not all behaviorally informed interventions designed for consumers and citizens will be relevant or effective in the context of farmers’ decisions to embrace more sustainable farming techniques. Individual variances in thinking, feeling, and acting patterns are known as personality traits. Personality traits are far removed from specific decision-making tasks because they consist of regular patterns of behavior. Extraversion, openness to new experiences, agreeableness, neuroticism, and conscientiousness are the personality traits that are commonly regarded. Farmers’ aversion to change has been mentioned as a possible cause for their failure to adopt more sustainable practices. To address the social issues impacting farmers’ adoption of sustainable practices, several policy recommendations can be made. In terms of descriptive norms, the approach to take is largely determined by the amount of adoption of sustainable practices in a given area. If the level is high, one useful policy option is to inform farmers that the majority of their neighbors have embraced sustainable farming practices. Consumers have been successfully motivated to conserve energy using a similar strategy. This approach is especially essential in the context of the CAP for voluntary schemes. When participants were told that 80%

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of other farmers planned to continue using sustainable farming practices even if their contract was not renewed, their chances of doing so more than doubled. Farmers would produce perennial crops for bioenergy against a reduced compensation premium if their neighbors did as well. However, a study conducted in the United States found that disclosing the popularity of a volunteer program had no effect on new or renewal sign-ups. On the other hand, if adoption is poor, articulating this descriptive norm could backfire. If the majority of farmers in a given area continue to utilize conventional procedures, presenting them with information on this descriptive norm is likely to dissuade, rather than encourage, them to convert to more sustainable practices. Farmers, for example, who are advised that they use less water than the majority of their peers, are more likely to increase their water usage. Economic incentives may be more appropriate in places where the adoption of sustainable practices is very low, in order to reverse this self-feeding low descriptive norm. Using collective compensation for enrolling in agrienvironmental programs is one known approach of enhancing farmers’ perceptions that adoption is the descriptive norm. Farmers’ expectations of others’ participation are raised by this monetary bonus, which is provided in addition to the agri-environmental scheme premium if a specified adoption level is met. As a result, farmers are more likely to sign up for a lower subsidy amount, resulting in improved budget efficiency. Farmers, on the other hand, do not value communal participation in agri-environmental projects, according to studies.

CHAPTER

9

CLIMATE RESILIENT AGRICULTURE

CONTENTS 9.1 Introduction ..................................................................................... 188 9.2 Green House Gases (GHG).............................................................. 189 9.3 Climate Change ............................................................................... 190 9.4 Sustainable Agriculture .................................................................... 191 9.5 Water Availability ............................................................................ 194 9.6 Agricultural Productivity .................................................................. 199

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9.1 INTRODUCTION Temperature, precipitation, soil quality, insect regimes, and seasonal growth patterns are all projected to change as a result of rapid global climate change. It will be difficult to foresee the exact kind and degree of these changes for any given place. While the agricultural industry is being affected by climate change, research shows that existing agricultural activities are a significant source of greenhouse emissions that exacerbate climate disruption. The amount of GHGs emitted by an agricultural operation is determined by the system and management of the operation. Agricultural systems must be resilient and adaptable to change in order to cope with climate change, which is anticipated to be both rapid and unpredictable. In the face of major exogenous shifts such as climate change and price volatility, resilient agriculture systems are more likely to preserve economic, ecological, and social benefits. In the face of unpredictability, food production systems that are diversified and relatively flexible, with livestock and crop production integration and coordination, should be built (Kashyap, Rai, Srivastava, & Kumar, 2017). Through energy conservation, lower levels of carbon-based inputs, lower usage of synthetic fertilizers, and other aspects that limit GHG emissions and trap carbon in the soil, sustainable and organic agriculture systems can help reduce agricultural GHG emissions. Agricultural land, particularly through soil carbon sequestration, can act as a sink for greenhouse gas emissions, potentially assisting in the mitigation of climate change. However, agricultural land can only act as a long-term GHG sink if agricultural systems are implemented that improve general soil quality and enable reasonably consistent GHG reduction or sequestration. Fertilizer use and efficiency, nitrogen sequestration, and overall GHG emissions of linked animal production systems are all characteristics of agricultural crop and forage production systems (Li et al., 2004).

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Figure 9.1: Green House Gases. Source: https://www.epa.gov/sites/default/files/2021-04/gases-by-source-z021caption.png

9.2 GREEN HOUSE GASES (GHG) GHGs (Figure 9.1) are released into the atmosphere due to burning fossil fuels, clearing forests, manufacturing cement, and a variety of other industrial and agricultural operations, increasing the quantity of radiation trapped near the earth’s surface and accelerating warming. This process, known as the enhanced greenhouse effect, is triggered by a forced release of GHGs from their terrestrial storage into the atmosphere. Global temperatures are rising, which is altering core climatic processes. Some of the modifications may be beneficial in some regions, but the majority is projected to cause more harm than good. Following the agricultural and industrial revolutions, the majority of these human impacts to climate change happened in the previous 200– 300 years. Many countries around the world are experiencing food crises as a result of conflict and natural disasters, while food security is being harmed by unprecedented price increases for basic foods, fueled by historically

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low food stocks, high oil prices, and rising demand for agrofuels, as well as droughts and floods linked to climate change. Food riots have already erupted in some nations as a result of rising international cereal prices.

Figure 9.2: Climate change. Source: https://www.iberdrola.com/environment/impacts-of-climate-change

9.3 CLIMATE CHANGE Climate change (Figure 9.2) will exacerbate food insecurity, especially among the resource poor in developing nations who are unable to achieve their nutritional needs through market access. Communities must prepare for the likelihood of a food crisis by making effective use of resources in order to protect livelihoods, as well as lives and property. It is critical to find and institutionalize measures that help the most vulnerable people cope with the effects of climate change. This necessitates collaborative thinking and responses to concerns arising from the intersection of food security, climate change, and sustainable development. Agriculture and food systems must adapt to climate change and natural resource demands in order to improve and ensure food security, and they must also contribute to climate change mitigation. Because these issues are intertwined, they must be addressed at the same time. Climate change offers complex concerns such as various abiotic pressures on crops and animals, water scarcity, land degradation,

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and biodiversity loss, necessitating concentrated and long-term study to find solutions. It is vital to put in place the required infrastructure to conduct basic and strategic research. Simultaneously, there is potential to improve agriculture’s resilience by applying existing knowledge and technology to farmers’ fields as a whole. As a result, improved technologies must be developed through short- and long-term research, as well as existing technologies must be shown on farmers’ fields to improve resilience.

Figure 9.3: Sustainable agriculture. Source: https://www.pmfias.com/sustainable-agriculture-organic-farming-biofertilizers/

9.4 SUSTAINABLE AGRICULTURE Sustainable agriculture (Figure 9.3) aims to transform agriculture into a climate-resilient, ecologically sustainable production system while also maximizing its full potential, ensuring food security and equitable access to food resources, improving livelihood opportunities, and contributing to economic stability. Climate resilient agriculture (CRA) aids in the attainment of long-term development objectives. By jointly tackling food security and

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climate concerns, it unifies the three elements of sustainable development like economic, social, and environmental. Land usage and changes in land use can have a significant impact on overall climate change. In most cases, vegetation and soils operate as carbon sinks, storing carbon dioxide ingested during photosynthesis. When the land is disturbed, the carbon dioxide, methane, and nitrous oxide trapped in the soil are released, re-entering the atmosphere. Clearing land can lead to soil degradation, erosion, and nutrient leaching, all of which can diminish the land's ability to operate as a carbon sink. As a result of the reduced ability to store carbon, more carbon dioxide may pump into the atmosphere, increasing the total amount of greenhouse gases. Direct anthropogenic alterations and indirect changes are the two categories of land-use change. Deforestation, reforestation, and afforestation, as well as agriculture, are examples of anthropogenic alterations. Climate or carbon dioxide concentration changes that force vegetation changes are examples of indirect alterations. On a global basis, carbon dioxide emissions from land-use changes account for about 18 percent of total annual emissions, with one-third of that coming from emerging countries and more than 60% coming from less developed countries. These factors determine the biosphere's carrying capacity for producing enough food for humans and domesticated animals. The balance of these effects will determine the overall impact of climate change on agriculture. Assessing the effects of global climate change on agriculture could aid in correctly anticipating and adapting farming to maximize agricultural output. Carbon dioxide enrichment would probably be beneficial to agriculture if there were no accompanying climatic changes. More severe warming, flooding, and drought, on the other hand, may diminish yields. Higher temperatures, on the other hand, may hasten the microbial breakdown of organic materials, reducing soil fertility over time. Higher temperatures speed plant maturity in annual species, decreasing the growth stages of crop plants, according to a study on the biophysical impact of climate change linked to global warming. In addition, research of the effects on pests and diseases reveals that rising temperatures may expand the geographic range of several insect pests that are currently restricted by temperature. Increased ultraviolet-B radiation has a negative impact on the productivity of some agricultural crops. Heat stress may put livestock at risk, as well as a reduction in the quality of their food source. Changes in water temperature will affect fisheries by shifting species ranges, making environments more conducive to invading species, and altering life cycle timing.

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The world climate is being influenced by rising CO2 levels in the atmosphere and accompanying temperature rises, which will affect plant growth, development, and function. Higher photosynthetic rate, enhanced light-use efficiency, reduced transpiration and stomatal conductance, and improved water-use efficiency are the principal impacts of increased CO2 concentration. Since the year 2000, these levels have been continuously increasing by 1.9 ppm each year, owing mostly to the burning of fossil fuels. Photosynthesis requires carbon dioxide. Even small increases in carbon dioxide cause higher plant growth, according to scientists. Increased CO2 levels are anticipated to result in higher harvestable crop yields. This, however, is highly dependent on the availability of sufficient water and nutrients for plant growth. Crops with reduced nutritional and protein levels, according to some scientists, will be one of the drawbacks of greater output. If extra fertilizers are not introduced into food production, this could have a significant and broad influence on long-term human health. Climate change has a favorable impact on agriculture and forestry because plants respond favorably to rising CO2 levels in the atmosphere. Higher CO2 levels stimulate plant development by increasing the rate of photosynthesis and improving the efficiency of water consumption in plants known as CO2 fertilization. Experiments showed that when CO2 concentrations increased by nearly 50%, they resulted in 15 percent growth increases in crops and 10–50 percent growth increases in tropical savanna grasses. Wheat yields increased by 10–50 percent, cotton biomass grew by 35 percent, whole boll yields climbed by 40 percent, and lint yields increased by 60 percent in tests where CO2 levels were increased up to 700 ppm. Increased evaporative demand, fluctuations in rainfall, and variations in river runoff and groundwater recharge, the two sources of irrigation water, will all have an impact on agriculture (Bond & Midgley, 2012). Because water is so important to plant growth, changing precipitation patterns have a big impact on agriculture. Because rain-fed agriculture accounts for more than 80% of overall agricultural production, estimates of future precipitation variations frequently influence the degree and direction of climate impacts on crop production. Due to strong dependencies on changes in atmospheric circulation, the impact of global warming on regional precipitation is difficult to predict. Even if there is an agreement in the sign of change in some places, these uncertainties imply distinct indications of precipitation change averaged over all croplands. One scenario that forecasts a rise in precipitation overall shows significant increases in the southern United States and India, but significant losses in the tropics and subtropics.

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The opposite scenario depicts decreases in low latitudes, but no significant rises in India. Seasonal precipitation changes could be more important to agriculture than annual mean variations.

9.5 WATER AVAILABILITY Water availability is influenced by more than just precipitation. Increasing evaporative demand as a result of rising temperatures and longer growing seasons might raise crop irrigation requirements globally by 5 to 20%, or possibly more, by the 2070s or 2080s, but with significant regional variations Southeast Asian irrigation requirements could rise by 15%. Irrigation demand is expected to rise throughout the Middle East and North Africa, according to regional assessments. Clearly, these estimates are also reliant on unknown precipitation changes. Precipitation is the most important element determining crop productivity because it is the principal source of soil moisture. While global climate models indicate an overall rise in mean global precipitation, their findings also point to the possibility of altered hydrological regimes in most regions. Total seasonal precipitation, its within-season pattern, and the between-season variability can all be affected by climate change. A change in the pattern of precipitation events may be more important than a change in the yearly total for agricultural productivity. Warmer temperatures, drier air, or windier circumstances may cause a spike in the daily rate of evapotranspiration and a shift in the seasonal pattern of evapotranspiration, putting crops’ water regime at risk. Drought conditions can also be exacerbated by less precipitation falling as snow and early snow melt. These effects may affect subsequent river discharge and irrigation water supplies during the growing season in arid locations, such as the Sacramento River Basin in California. High relative humidity, frost, and hail can all damage maize and other grains, as well as the quality of fruits and vegetables. Crop yields and yield quality are variable due to interannual precipitation variations. Droughts in the United States’ Great Plains lowered wheat and corn production by up to 50% in the 1930s. Droughts worsen wind and water erosion by reducing plant cover, lowering crop output in the future. If dry spells occur during important developmental stages such as reproduction, crop yields are most likely to decrease. Flowering, pollination, and grain filling are all very vulnerable to water stress in most grain crops. Because of climate change, the soil system responds to short-term events like rainfall as well as long-term changes like physical and chemical degradation (Otkin et al., 2018). The

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organic matter supply, temperature regimes, hydrology, and salinity are all potential implications of climate change on soil health. Because of lower net primary production, soil carbon levels are predicted to fall. Higher carbon mineralization following episodic rainfall and reduced yearly and growing season rainfall is anticipated to exceed any gains from increased plant water-use efficiency due to elevated CO2. Where the more inert components of the carbon pool predominate, the quality of soil organic matter may likewise vary. N mineralization increases when soil temperature rises, but availability may decrease due to increased gaseous losses from processes like volatilization and denitrification. There is yet to be a full examination of the influence of possible climate changes on soils. Higher temperatures may hasten the decomposition of organic materials by microbes, reducing soil fertility in the long run. However, higher rates of photosynthesis could offset these impacts by increasing root biomass. Higher temperatures could speed up nutrient cycling in the soil, and faster root growth could lead to higher nitrogen fixation. However, these benefits may be insignificant in comparison to the negative consequences of changing rainfall patterns. Greater rainfall, for example, in existing damp places could contribute to increased mineral leaching, particularly nitrates. The effects of GHG accumulation in the atmosphere and water are linked to a variety of physical phenomena, including slow temperature changes, acidification of water bodies, changes in ocean currents, and rising sea levels. These physical changes have an impact on aquatic ecological functions as well as the frequency, intensity, and location of extreme weather events. There will be a variety of direct and indirect effects on fisheries and aquaculture. In most tropical and subtropical oceans, seas, and lakes, ecosystem productivity is anticipated to decline. Productivity is expected to rise in high-latitude environments. Climate change has an impact on agricultural crops as well as their pests like weeds, insects, and infections. Climate has a big influence on the distribution and proliferation of weeds, fungus, and insects. Organisms become pests when they compete with, prey on, or induce disease in crop plants to the point where productivity is reduced. Climate influences not just the types of crops farmed and the severity of pest problems, but also the insecticides commonly employed to control or prevent outbreaks. Pesticide effectiveness, persistence, and transport are all influenced by rainfall intensity and timing in relation to pesticide application. Pests will become more prevalent as temperatures rise due to a number of interconnected mechanisms, including range extensions and phenological

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shifts, as well as faster population growth and migration. The equilibrium between pests, their natural enemies, and their hosts is anticipated to shift as a result of climate change. The increase in temperature will help pests develop and survive the winter. As a result of lower foliar nitrogen levels, rising atmospheric carbon dioxide concentrations may result in a drop in food quality for plant-feeding insects. Plant disease epidemiology will change as a result. In times of rapidly changing climate and inclement weather, forecasting disease outbreaks will be more difficult. Increased prevalence of harsh weather and environmental instability may impair the effectiveness of insecticides on targeted pests or result in more harm to non-target organisms. The photodegradation products of one class of atmospheric trace gases, chlorofluorocarbons (CFCs), have a significant extra influence on the atmosphere, destroying ozone (O3) in the stratosphere(Plummer & Busenberg, 2000)synthetic, halogenated alkanes, developed in the early 1930s as safe alternatives to ammonia and sulphur dioxide in refrigeration. Production of CFC-12 (dichlorodifluoromethane, CF2C12 (Figure 9.4). The stratospheric ozone layer acts to filter out much of the ultraviolet component of the solar spectrum before it reaches the earth’s surface, as ozone is a significant absorber of solar ultraviolet light. As a result of the decrease in stratospheric O3, more solar UV can reach the earth’s surface. Changes in precipitation and rising temperatures between 1981 and 2001 resulted in yearly cumulative losses of wheat, maize, and barley of around 40 million tonnes per year. While the scientists believe these losses are minor in comparison to technical yield advances over the same time period, the findings show that climate change is already having a negative influence on agricultural yields on a worldwide scale. The data show that food yields in developing countries are falling while yields in developed countries are rising. The production of the four crops would be insufficient to feed the world in all scenarios. However, this is only possible if food is transferred from wealthy northern countries to impoverished southern countries. Individual crops, on the other hand, respond differently to climate change, making generalizations difficult. It is necessary to do a crop-by-crop and region-by-region analysis. Moisture, on the other hand, is critical in all crop production, and a lack of it will undoubtedly be a key limiting issue in North America.

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Figure 9.4: Destroying ozone (O3). Source: https://biophysics.sbg.ac.at/atmo/o3-scans/chapman.jpg

Heat stress might also be a problem for crops like corn and potatoes. Due to expected increased rainfall in eastern and central Africa, the picture is different. More yields of 10–30% are conceivable with increased rainfall and improved farming methods. Even these expected improvements, however, will not be enough to feed Africa’s rapidly rising population. Long-term significant changes in the projected patterns of average weather in a specific region due to global warming are known as regional consequences of global warming. The greenhouse effect, which is produced by rising amounts of greenhouse gases, particularly carbon dioxide, is causing global average temperatures to rise. When the global temperature changes, climatic changes are not likely to be uniform around the globe. Land areas, in particular, change quicker than oceans, northern high latitudes change faster than the tropics, and biome region edges change faster than

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their cores. The nature of global warming’s regional effects varies. Some are the result of a more widespread global shift, such as rising temperatures, which have local consequences, such as ice melting. A change could also be linked to a shift in a certain ocean current or weather system. In such circumstances, the regional impact may be exaggerated, and the global trend may not necessarily be followed.

Figure 9.5: Hydrological cycle. Source: https://www.noaa.gov/education/resource-collections/freshwater/water-cycle

Melting, modifying the hydrological cycle of evaporation and precipitation (Figure 9.5), and changing currents in the oceans and air flows in the atmosphere are three primary ways that global warming will affect regional climate. The fast-rising gap between limited water availability and escalating demand for water from diverse economic sectors is a big concern. Egypt’s rate of water use has already hit its peak, and climate change will worsen this vulnerability. Reduced crop yields in several locations will put many millions of Asians at risk of starvation. Decreased freshwater availability will affect more than 100 million people in Central, South, East, and Southeast Asia, notably in large river basins like Changjiang. With a temperature increase of 2–3 °C combined with decreasing precipitation in the semiarid and arid zones, grassland production is anticipated to drop by as much as 40%–90%.

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9.6 AGRICULTURAL PRODUCTIVITY Agriculture productivity in northern areas may increase. Boreal forest in North Asia may expand northward, while forest fires are anticipated to increase in frequency and size, limiting forest extension. In Asian countries, there are a variety of contemporary water vulnerabilities. Some countries that are not currently at high risk of water stress are likely to encounter water stress in the future, with varying capacity for response. Increased river and coastal flooding are projected to be most severe in coastal areas, particularly heavily inhabited mega delta regions in South, East, and Southeast Asia. The interplay of climate change consequences with rapid economic and population expansion, as well as migration from rural to urban regions, is projected to have an impact on development across southern and eastern Asia. The development path, physical exposures, resource distribution, antecedent stresses, and social and government institutions all influence a society’s vulnerability. Adaptive capacities are unevenly distributed among countries and within cultures, despite the fact that all societies have the innate capacity to deal with certain variations in the environment. Historically, the poor and underprivileged have been the most vulnerable to the effects of climate change. Recent Asian studies suggest that marginalized, primary-resource-dependent livelihood groups are more vulnerable to climate change impacts if their natural resource base is severely strained and deteriorated by overuse, or if their governance structures are incapable of responding effectively. Water security issues in southern and eastern Australia, New Zealand’s Northland, and other eastern regions are expected to worsen by 2030. Land degradation issues including erosion and salinization are anticipated to worsen. Drought and fire are expected to reduce agricultural productivity across much of southern and eastern Australia, as well as sections of eastern New Zealand, by 2030. Due to a longer growing season, reduced frost, and more rainfall in north-eastern Australia and the main portions of New Zealand, substantial yield increases are possible. Heat stress, decreased pasture productivity, lower fodder quality, and the spread of animal diseases such as cattle ticks are all expected to affect livestock productivity in Australia. CO2 fertilization, more rainfall, and a longer growing season will benefit forests, but increased water stress, pests, fires, and erosion will have negative consequences. Despite their hydrological and geological differences, both Australia and New Zealand are already suffering water supply consequences as a result of recent climate change, both due to natural variability and human

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activities. The El Nino Southern Oscillation cycle is the most powerful regional driver of natural climate variability. Almost all of Australia’s eastern states and the southwest region have been in drought since 2002. This drought is on par with the so-called Federation droughts of 1895 and 1902, and it has sparked heated debate over climate change’s impact on water resources and long-term water management (Graham, Michaelsen, & Barnett, 1987)a new approach in geophysical modeling. Our results are of interest both as they show the dominant modes of evolution in the SLP and wind fields through the El Niño-Southern Oscillation cycle and with respect to the problem of predicting equatorial SSTs. This paper deals with the first issue above and describes some statistical composites that typify the development of features in the predictor fields over periods of years. The results of the EEOF analyses clearly show slowly propagating anomalies in both the near-global SLP and trade wind fields. The CCA analysis, which highlights the co-evolution of the two fields, suggests a strong coupling between the two and depicts the anomalous features as migrating centers of divergence and convergence that first appear over the eastern Indian Ocean. These features propagate slowly eastward, amplify as they expand into the western Pacific, decline as they cross the central ocean, then reamplify over the eastern Pacific. As reamplification takes place, new opposing anomalies appear in the Indo-Pacific. Descriptions of the predictor data sets and details of the statistical techniques used may also be found in this paper. The SST data, model validation techniques, and forecast model results are presented in a companion paper (Graham et al., this issue. Due to water constraints, increasing biosecurity threats, environmental deterioration, and social instability, farming of marginal land in drier locations are projected to become unsustainable. When irrigation water availability is restricted, cropping and other agricultural businesses that rely on irrigation are likely to be threatened. Reduced growth length for maize in New Zealand reduces crop water requirements, allowing development to be more closely synchronized with seasonal climatic conditions. Crop productivity will grow slightly, but this may be considerably overshadowed by technological advancement. Crops from southern Europe, such as maize, sunflower, and soybeans, will expand northward. Droughts and dry spells will impact Mediterranean crop productivity, resulting in lower yields, scrublands and deciduous forests, increased water demand for irrigation, fire danger, and diminished biodiversity.

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The risk of livestock diseases such as bluetongue and African horse sickness will rise. Northern Europe’s Forest productivity will rise dramatically. In boreal forests, there will be soil carbon losses and seasonal variations in frost damage. The most popular and planned techniques to adapt to increasing water stress are measures such as damming rivers to create instream reservoirs. However, environmental constraints and hefty investment costs are limiting the construction of new reservoirs throughout Europe. Other options, such as wastewater reuse and desalination, are becoming increasingly popular, although their popularity is hampered by health issues with wastewater reuse and high energy costs with desalination, respectively. Household, industrial, and agricultural water conservation, as well as the reduction of leaky municipal and irrigation water systems and water pricing, are all potential. Introduce crops that are more suitable to a changing climate to reduce irrigation water use. Regional and watershedlevel climate change adaptation strategies are being incorporated into plans for integrated water management, while national strategies are being constructed to fit into current governance institutions, as an example of a unique European strategy to adjusting to water stress. Some countries and regions like the Netherlands, the United Kingdom, and Germany are developing water sector adaptation procedures and risk management techniques that reflect the unpredictability of expected hydrological changes. Food security will be harmed in dry areas due to salinization and erosion of agricultural land, which will reduce crop yields and livestock output. By the 2050s, agricultural lands are extremely likely to be desertified and salinized to the tune of 50% in some locations. Crop yields may be lowered in certain locations, while they may increase in others. Increased temperatures and groundwater loss have resulted in habitat loss and species extinction in many locations, including tropical forests, affecting indigenous tribes in particular. Population expansion is expected to continue, resulting in increased food demand. Because most Latin American countries’ economies are based on agricultural output, regional diversity in crop yields is a critical concern. As a result of its geographical configuration, Latin America offers a wide range of climates. There are also extensive dry and semiarid areas in the region. From snow mountains to moderate and warm climates, the climatic spectrum is vast (Kang & Banga, 2013)caused by anthropogenic activities, is a universal phenomenon across the globe. There is general consensus that combating climate change will require a set of internationally coordinated policy interventions for reducing greenhouse gas (GHG.

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Figure 9.6: Receding glaciers. Source: https://www.dailymail.co.uk/sciencetech/article-4369402/Timelapseimages-reveal-Earth-s-receding-glaciers.html

In recent decades, glaciers have typically receded, (Figure 9.6) and some very minor glaciers have already vanished. The Amazon, Parana, and Orinoco rivers collectively convey more than 30% of the world’s renewable fresh water into the Atlantic Ocean. These water resources, on the other hand, are inequitably distributed, and broad zones have a very low water supply. When there is a lack of precipitation or greater temperatures, it puts a strain on water availability and quality. Droughts in many parts of Latin America are statistically linked to ENSO episodes, resulting in severe limits on water resources. Rainfed agriculture is expected to boost yields by 5–20 percent in the early decades of the century, with significant regional variation. Warming in the western mountains will alter water resources, resulting in decreasing snowpack, greater winter flooding, and reduced summer flows, aggravating competition for over-allocated water resources. Crops that rely on heavily consumed water resources, such as wine grapes, or those that are near the warm end of their acceptable range may encounter significant issues. Forest growth is expected to increase by 10–20 percent in the twentyfirst century as a result of longer growing seasons and increased CO2 levels, though there will be significant spatiotemporal variance. Depending on the emission scenario, increases in insect, disease, and wildfire disturbances, as well as associated losses, are predicted to influence forests.

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Figure 9.7: Cold-water fisheries. Source: ter-1380965

https://www.thesprucepets.com/what-fish-species-are-coldwa-

Cold-water fisheries (Figure 9.7) are expected to suffer, while warmwater fisheries are expected to benefit, with mixed effects for cool-water fisheries. Higher temperatures will cause species distribution to shift northward. Most regions of North America should expect changes in the time, volume, quality, and spatial distribution of freshwater accessible for human settlements, agricultural, and industrial uses as the rate of warming accelerates in the next decades. While some of the above water resource changes are true for all of North America, twentieth-century trends show that climate change impacts on runoff, stream flow, and groundwater recharge vary greatly by location. In both Canada and the United States, differences in wealth and geography lead to an uneven distribution of likely consequences, vulnerabilities, and adaptability. As animals migrate northward in response to warmer temperatures and a longer growing season, opportunities for agricultural and pastoral operations expand, but there are risks of invasive species, biodiversity loss, and the development of animaltransmitted diseases. Scrubland and woods might potentially replace 10– 50 percent of the tundra. Increased warmth, decreased sea-ice cover, and shifts in hydrological regimes would impact ecosystems, causing harm to numerous creatures, including migratory birds, mammals, and higher predators. Changes in ecosystems, decreased transportation and market

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access, and lower-quality drinking water will jeopardize the food security of some subsistence systems. They found, with moderate confidence, that the benefits of a milder climate were contingent on local factors. Increased agricultural and forestry potential were deemed to be one of these benefits. The broad seeding of barley, which had been impossible 20 years earlier, was now conceivable due to rising temperatures. Some of the warming was caused by a local transient effect caused by Caribbean Ocean currents, which had also harmed fish stocks. Sea-level rise, soil salinization, seawater intrusion into freshwater areas, and a loss in freshwater availability will all have an impact on agricultural land and, as a result, food security. Extreme occurrences will have an overall impact on agricultural productivity. Increasing sea surface temperatures, rising sea levels, and damage from tropical cyclones will have an impact on fisheries. Fishing incomes will be impacted by coral reef degradation and bleaching. Forests that have been damaged by harsh events will take a long time to recover. On some high-latitude islands, forest cover may increase. Because of the reduction in island size or complete inundation, the viability and thus sovereignty of some governments would be jeopardized.

Figure 9.8: Climate Smart Agriculture. Source: https://www.researchgate.net/publication/326847389_Assessment_of_ Climate-Smart_Agriculture_CSA_Options_in_Nepal/figures?lo=1

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Climate Smart Agriculture (CSA) (Figure 9.8) is a method for guiding agricultural management in the face of climate change. The concept was first introduced in 2009, and it has since evolved based on the inputs and interactions of numerous parties involved in its development and implementation. The CSA intends to develop globally applicable principles for managing agriculture for food security in the face of climate change, which might serve as a foundation for policy support and recommendations from multilateral agencies such as the United Nations’ FAO. The key aspects of the CSA approach were established in response to discussions and conflicts surrounding climate change and agriculture policy for long-term sustainability. It’s vital to understand the trajectory of global climate change policies in recent years in order to place CSA and its debates in context. He categorizes evolution into five distinct stages (Lipper et al., 2014). During this time, the main focus of global climate change policy was the need for global action to stabilize greenhouse gas (GHG) emissions, which would be supported and guided by the IPCC, a globally cooperative framework for scientific research, and with the understanding that developed and developing countries would bear different responsibilities for mitigating climate change. This necessitates taking preventive action even before full knowledge regarding human-induced climate change is established, as well as emphasizing acts that would be beneficial even if climate change did not exist. The Brundtland Commission Report on Sustainable Development was also significant in recognizing the links between climate change and sustainable development, as well as the benefits of studying them together. The CSA concept arose at a period when there was a lot of debate about the concept of sustainable agricultural development and how to approach it. Agriculture’s critical role in food security was not well articulated in the global climate change policy arena, and the discussion of adaptation and mitigation in two separate negotiation streams hindered the ability to establish synergies between them. The agricultural sector, according to these early formulations of the climate smart agriculture concept, is critical to climate change response not just because of its high vulnerability to climate change consequences, but also because it is a major contributor to the problem. It also stated that ensuring food security requires a long-term restructuring of the agricultural sector, and that climate change measures must be framed in this context. Recognition of potential trade-offs between the three aims, as well as the possibility to create synergy between them through policies, institutions, and finance, has been a significant component

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of the CSA idea since its conception. A requirement for locally specialized solutions was also a significant factor. FAO gave a broad framework for examining trade-offs and synergies, as well as various instances of sustainable land management methods and modern inputs. However, there was no particular direction on how to design a CSA practice or prioritize objectives in order to develop sitespecific solutions. The intricacy of linking together the three primary aims also prevented a clear conceptual conceptualization of the link between sustainable agriculture and CSA. The lack of a clear methodology, combined with the concept’s quick adoption, resulted in a great deal of variation. Climate change has many effects on agriculture, both in terms of space and time. The outcomes are unpredictable and varied. Agriculture innovation is certainly a critical answer for successful and equitable adaptation and mitigation, and we need to reconsider how we foster innovation to handle the variability and uncertainty of climate change consequences. In both developing and developed countries, innovation will be critical in advancing toward climate smart agricultural (CSA) systems. For both adaptation and mitigation, we will require more resilient agricultural systems as well as higher resource efficiency. Technological innovation will be critical, but it will not be sufficient. In dealing with the varied and unknown implications of climate change, managerial and institutional innovations are going to be even more critical. To build CSA practices, innovation can be combined with various forms of climate change adaptation. In particular, innovation can improve technology adoption, avoid or assist production/population movement, improve trade and aid, and improve insurance efficiency and inventory feasibility. Climate change is projected to raise global temperatures by 1–3 °C, which is equal to a shift of 300–500 km of weather patterns away from the equator and towards the poles, depending on the range of mitigation activities done in the next decades. Similarly, temperature variability will rise in higher-altitude places. While climate change may have a negative overall effect on agricultural production, the distributional effects are significantly more significant. As a result, crop production in some warm agricultural areas in Texas, Oklahoma, Mexico, and Western Africa will become unviable. At the same time, agricultural production will be possible in parts of Russia, Canada, and even the Arctic.

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In many parts of the world, the sea level rise may result in the loss of high-value agricultural land as well as critical infrastructure for exporting and importing food. Coastal zones i.e., at less than 10 m altitudes are home to an estimated 10% of the world’s population, with considerable variations in population share by country, accounting for 14% of worldwide GDP Most notably, about half of the populations of Vietnam, Bangladesh, and Egypt live in these zones, while China and India, despite having a much lower overall population, have over 200 million people residing in these zones. The population affected by SLR will vary greatly depending on the actual rise in sea level, ranging from 56 million people with a 1-m rise to 245 million people or 5.57 percent with a 5-m rise (Dasgupta, Laplante, Meisner, Wheeler, & Yan, 2009)and unexpectedly rapid breakup of the Greenland and West Antarctic ice sheets might produce a 3–5 m SLR. In this paper, we assess the consequences of continued SLR for 84 coastal developing countries. Geographic Information System (GIS. Furthermore, rising sea levels will endanger significant swaths of prime agricultural land, particularly in tropical areas. Given the diversity of locations, developing location-specific solutions is critical. Transformational innovation, rather than incremental measures, may be necessary in areas most vulnerable to SLR in order to stimulate adaptation and preserve vulnerable populations. Fragile coastal districts may be saved in some situations by investments in protective infrastructure such as dikes and dams, but in many others, vulnerable areas will have to be abandoned, generating displacement issues. Different methods of agricultural production may be possible in some locations, but this will necessitate innovation. Increased temperatures, in addition to changing precipitation patterns, will increase melting, reducing the feasibility of using water stored in snow accumulated during the rainy season for irrigation during the dry season. Additionally, floods may become more likely. Given the importance of irrigated agriculture during dry seasons in many regions of the world, this change could have a large impact on food supply unless certain actions are done to mitigate it. The conditions at each site determine these solutions. Investment in new forms of water inventories and storage, such as flood control and storage dams and water diversion to subterranean reservoirs, could be one solution. Variations in crop timing and selection may be required as a result of these changes in water availability. Changes in water availability may also have an impact on the availability of hydroelectric

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power for irrigation, affecting agricultural supplies. As a result of climate change, agricultural water resources will need to be rearranged and managed differently. As previously stated, the form of innovative climate change responses must adapt to two aspects of these consequences. The first is the concept of heterogeneity. Climate change affects different locations differently: for certain desert or low-lying coastal regions, climate change may be disastrous, while for others, it may be seen as “climate betterment.” These disparities in consequences, as well as disparities in benefits and losses from mitigation initiatives, may explain the varied responses and desire to participate in and contribute to coordinated efforts to avert or reduce climate change. Uncertainty is the second factor that influences taking action to solve climate change concerns. The exact dates, magnitudes, and locations of certain climate change impacts remain unknown. On the same hand, there is a large body of evidence suggesting that farmers and other agricultural operators operate in a risk-averse manner. Risk aversion limits the magnitude of activities made by risk averse firms as the dangers they confront an increase in a static framework. Heterogeneity and uncertainty will thus make identifying the full range of responses to climate change from observable data more difficult, especially now, when some of the impacts of climate change like migration of warm weather toward the pole and a significant rise in sea level, triggering of tipping points leading to irreversible changes are more likely to occur in the long run. Others, such as those that enhance the likelihood of extreme occurrences such as floods and droughts, may have already occurred or will do so in the near future. As understanding about the concerns of climate change grows, so will investment in new initiatives to address them. New technology and institutional choices, as well as changes in behavioral responses to climate change and related solutions over time, must be considered in the inventive approach. We can learn about some activities from the responses thus far, as well as the future potential to adapt to climate change and the elements that influence responses.

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Figure 9.9: Economic growth theory. Source: https://www.doorsteptutor.com/Exams/IEcoS/Paper-2/Questions/ Topic-Economic-Growth-and-Development-4/Subtopic-Theories-of-EconomicDevelopment-3/Part-1.html

Innovations can be classified in a variety of ways. According to economic growth theory, (Figure 9.9) technologies are classified based on their impact on inputs and outputs. For example, capital-saving, laborsaving, quality-improvement, and risk-reduction innovations can all be distinguished. Another method to categories innovations is by their form, such as technological, managerial, or institutional innovations. New machinery embodies technological innovations, which are further split into mechanical like tractors, biological, and chemical categories. Better procedures like Integrated Pest Management, enhanced pruning techniques, and crop rotation are examples of managerial advances that are not reflected in physical capital. New organizational structures such as cooperatives and trading arrangements are examples of institutional innovations. Because of the variability and randomness of climate change impacts, numerous types of innovation will be particularly helpful. Throughout history, practitioners have been a primary source of innovation. Practitioners identified and improved the cultivation of crops, and early farming procedures. In the present period, however, science and research are becoming key sources of new breakthroughs. Furthermore, in

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the case of climate change, it is critical to speed up the innovation process so that new solutions are available when and where climate change occurs. Scientific research has aided in the invention of new types of engines, electric appliances, new medications, fertilizers, and crop kinds. There are several stages to the innovation process. The process of technical innovation starts with research efforts that lead to the discovery of ideas, which are at the heart of new developments. Following that, concepts are polished, tested, and scaled up through more experimentation as part of the development process. The development process for many biological and chemical discoveries also entails government permission for usage prior to commercialization. Production and marketing operations are used to commercialize the product after it has been approved for feasibility. Consumers begin to use and evaluate the product, and their feedback leads to product improvement and additional developments. This linear characterization ignores feedback and interactions, but it does provide a useful foundation for thinking about some of the major obstacles that new technologies face. The innovation process in managerial and institutional innovation may also begin with research efforts that identify alternate solutions to a problem, such as economic research or decision theory.

CHAPTER

10

PLANT BREEDING

CONTENTS 10.1 Introduction ................................................................................... 212 10.2 Potatoes ......................................................................................... 214 10.3 Tomato........................................................................................... 216 10.4 Modern Genetics ........................................................................... 219 10.5 X-Rays ........................................................................................... 222 10.6 Transgenics .................................................................................... 225

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10.1 INTRODUCTION Plant breeding is an attempt by people to manipulate nature in order to benefit plants’ heredity. Plant modifications are both permanent and heritable. The desire of humans to improve specific qualities of plants in order for them to perform new tasks or enhance existing ones is driving this attempt to change the status quo. As a result, in modern society, the terms plant breeding and plant improvement are frequently interchanged. Plant traits, structure, and composition are thus manipulated in breeding to make them more beneficial to humans. It’s significant to note that not every plant attribute or characteristic is easily manipulated by breeders (Hayes, Immer, & Smith, 1955). Plant breeders are increasingly able to achieve amazing plant manipulations as technology improves, though not without controversy, as is the case with the invention and use of biotechnology to plant genetic manipulation. Transgenesis, the technology for transferring genes over natural biological boundaries, is one of the most contentious of these modern technologies. Plant breeding is required to increase the value of food crops by increasing the production and nutritional quality of their products for human health. Key plant foods are low in certain important elements to the point where disorders related to nutritional deficiencies are widespread when these foods make up the majority of a regular diet. Cereals have low levels of lysine and threonine, whereas legumes have low levels of cysteine and methionine. To advance the nutritional quality of food crops, breeding is required. Rice, a staple of the world’s diet, is deficient in pro-vitamin A. The Golden Rice project, which is currently being carried out at the International Rice Research Institute (IRRI) in the Philippines and parts of the world, aims to develop, for the first time, a rice cultivar capable of producing provitamin A or Golden rice, which has a 20-fold increase in pro-vitamin A, was developed by Syngenta’s Jealott’s Hill International Research Centre in Berkshire, UK.

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Figure 10.1: Malnutrition. Source: https://www.fao.org/news/story/en/item/1199760/icode/

Around 800 million people worldwide, including 200 million children, suffer from chronic malnutrition, which causes a slew of health problems. Malnutrition (Figure 10.1) is particularly common in underdeveloped nations. By lowering harmful components and increasing texture and other features, breeding can also make some plant products more digestible and safer to eat. The value of plant material for animal feed is reduced by its high lignin content. Alkaloids in yam, cyanogenic glycosides in cassava, trypsin inhibitors in pulses, and steroidal alkaloids in potatoes are examples of toxic compounds found in key food crops. Forage breeders are interested in enhancing feed quality i.e., high digestibility, high nutritional profile for cattle, among other reasons. The current phenomenon of global climatic change is partly to blame for changing the crop production environment. This necessitates the development of new crop cultivars for new production settings. While affluent economies may be able to mitigate the consequences of unseasonably warm weather by supplementing the production environment, poorer countries are easily destroyed by even brief bouts of bad weather. Drought resistant cultivars, for example, are useful to agricultural productivity in places with marginal or irregular

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rainfall regimes. Breeders must also create novel plant varieties that can withstand biotic and abiotic challenges in the producing environment. Crop distribution can be broadened by adapting crops to new production settings. The development of photoperiod-insensitive crop cultivars might allow previously photoperiod-sensitive species to be produced in greater quantities. Processed foods account for an important portion of the global food supply (Hussain, 2015). Fresh produce for the table has different quality criteria than fresh produce for the food processing business. One of the reasons why the “FlavrSavrTM” tomato, the first genetically modified (GM) crop approved for, failed was that the product was marketed as a table or fresh tomato when, in fact, the gene of interest was placed in a genetic background for developing a processing tomato variety. Plant breeders can address a variety of commercial needs in their endeavors.

Figure 10.2: Potatoes (GM). Source: https://www.bbc.com/news/science-environment-26189722

10.2 POTATOES Potatoes (Figure 10.2) are a versatile crop that may be utilized for both food and industrial purposes. Breeders are working on several kinds for baking, cooking, frozen fries, chipping, and starch. The size, specific gravity, and sugar content of these cultivars vary, among other things. Because sugar caramelizes under high heat, causing unwanted browning of fries and chips, high sugar content is undesirable for frying or chipping. This is a traditional strategy. Traditional or classical breeding is another term for conventional breeding. The major method for creating variety in flowering species is to

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cross two plants. The most desired recombinant is subsequently identified using a variety of breeding strategies to discriminate among the diversity. Before being released to producers, the genotype is raised and checked for performance. Plant features that are controlled by a large number of genes are more challenging to breed. Despite its age, the traditional technique to plant breeding remains the industry’s workhorse. It is accessible to the average breeder and reasonably simple to implement when compared to the unconventional method. The unorthodox approach to breeding comprises the application of cutting-edge technologies to generate novel variety that is sometimes impossible to obtain using traditional approaches. This method, on the other hand, is more involved and necessitates specialized technical abilities and understanding. It is also costly to carry out. Breeders gained a new set of strong tools for genetic study and manipulation with the introduction of recombinant DNA (rDNA) technology. Gene transfer can now occur across natural biological barriers, bypassing the sexual process, for example, Bt. products, which are made up of bacterial genes that are introduced into crops to give resistance to the European corn borer. Molecular markers can be used to help with the selection process, making it more efficient and effective.

Figure 10.3: Tomato (GM). Source: https://www.deccanherald.com/content/343645/gm-tomato-gets-purple-hue.html

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10.3 TOMATO The tomato can be used to demonstrate how different breeding objectives for a single crop may be developed. Tomatoes are a widely used fruit with a variety of applications, each requiring different attributes. Tomatoes are used whole in salads, so tiny sizes are desirable; tomatoes are sliced in hamburgers, thus round huge fruits are preferred. Tomato pulp for canning must meet certain specifications. Gardeners want a tomato cultivar that ripens over time so harvesting can be spaced because tomatoes are a popular garden plant. However, for industrial applications, like canning, the fruits on the commercial cultivar must ripen at the same time so that the field may be collected mechanically. Furthermore, while the look of the fruit is not a top priority for a tomato juice processor, the appearance of fruits is crucial in marketing the fruit for table usage. A breeding system has been developed into every successful breeding program. The breeding system regulates how breeding lines progress through the selection process and how much planting material is available for cultivar release. The selection procedure will take place over several years and under various environmental circumstances. Many thousands of distinct genotypes will be screened in the early stages of breeding programs. As a result, early screening is typically unsophisticated, including merely visual selection in many cases. The disease-resistant genotypes will be kept for further study after each round of selection, whereas the least adapted lines will be removed. This procedure will be repeated over a number of years, with the number of individual genotypes or populations being reduced at each stage and the value of each input being estimated with higher precision. The breeding strategy will be heavily influenced by the crop species and cultivar being developed. As a result, the overall idea for generating a clonal cultivar such as potato differs from that of a pure-line grain cultivar such as barley. Breeding selections are genetically fixed through vegetative propagation in the former, while there will be a low rate of planting material multiplication in the latter. Although the segregating character of the earlygeneration breeding lines may complicate the selection process, there will be a faster increase in planting material in the future. The most efficient breeding strategies will make use of a crop species’ beneficial characteristics while reducing problems that may develop during the selecting process. Barley, chickpea, flax, lentil, millet, peas, soybean, tobacco, tomato, and wheat are among the crops grown as pure-line varieties. Most inbred crop species were farmed as ‘landraces’ in agriculture one and a half centuries ago.

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Landraces were locally developed populations that consisted of a mixture of several different genotypes that were genetically and phenotypically varied. Farmers first developed pure-line or inbred cultivars from these landraces by selecting specific plants from mixed populations and keeping them isolated, encouraging selfed progenies, and eventually developing homozygous, or near-homozygous, lines. Because landraces had almost totally vanished in countries with advanced agricultural systems by the end of the 19th century, it is safe to believe that these homozygous lines were actually more productive than the original landraces (Newton et al., 2011). These early pure-line breeders took advantage of naturally occurring genetic variety within the landraces they were propagating, as well as inherent inclination to self-pollinate. However, because this strategy has limited potential for generating new variation, modern plant breeders must constantly generate genetic variation, which is why three-phase breeding schemes were developed to generate genetic variation, identify desirable recombinant lines within progenies, and stabilize and increase the desired genotype. It’s worth noting, however, that a number of plant breeders have recently returned to old wheat and barley landraces to explore their genetic diversity, as well as to testing line combinations in modern landrace combinations (Figure 10.4).

Figure 10.4: Modern landrace combinations. Source: https://www.flickr.com/photos/cimmyt/5755175171

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Unfortunately, most landraces that existed even 100 years ago are no longer available, resulting in the loss of potentially valuable germplasm and adapted pairings. To shorten seed-to-seed time, single seed descent entails periodically growing a large number of individuals from a segregating population, usually under high-density, low-fertility circumstances. Every plant is replanted with a single seed from its natural self when it reaches maturity. To generate homozygous plants, this procedure is performed several times. In a greenhouse, where a number of growth cycles may be possible each year, single seed descent is most suited for rapid generation expansion. Growing plants under stress conditions of high density, high light, restricted root growth, and low nutrient levels can speed up single seed descent in canola, wheat, and barley, resulting in stunted plants with only one or two seeds per plant, but in a shorter growing period than growth under normal conditions. When employing single seed descent, it is critical to ensure that no unintended selection for unfavorable characters takes place. Vernalization needs may be artificially overcome in a cold chamber in a single seed descent system in winter wheat where plants will require a vernalization time prior to commencing a reproductive phase.

Figure 10.5: Modern genetics. Source: https://en.wikipedia.org/wiki/Genetics#/media/File:DNA_sequence,_ sequences.gif

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10.4 MODERN GENETICS Modern genetics (Serra, 1966)(Figure 10.5) has one of the most well-defined beginnings of any science. As previously stated, early plant breeders were aware of some relationships between parent and offspring, and researchers had conducted experiments to investigate these relationships at various times throughout history. However, true experimental genetics began in the middle of the nineteenth century with Gregor Johann Mendel’s work, and was only truly acknowledged until the turn of the twentieth century. Because the blooms of pea plants are designed to encourage self-pollination, the bulk of Mendel’s lines were homozygous or near-homozygous genotypes. Mendel’s selection of peas as an experimental plant species provided a significant advantage over many other plant species he may have used. The characters he chose were also serendipitous in their differences. As a result, many modern scientists say that Mendel’s discoveries are based on a lot of luck because of the decisions he made about what to examine. Many have concluded that he must have already foreseen the results he expected to attain when this is combined with segregation ratios that are better than would be expected by chance. There are a few general remarks to make about Mendel’s experiments before we look at an example of one of his studies. Others had made intentional hybridizations or crosses within various animals before Mendel.

Figure 10.6: Mendel. Source: https://www.britannica.com/biography/Gregor-Mendel

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To begin with, Mendel (Figure 10.6) possessed a bright analytical mind that enabled him to interpret his findings in ways that outlined heredity principles. Second, Mendel was an accomplished experimenter. He knew how to design trials in such a way that the chances of getting useful results were increased. He knows how to make data more understandable. He chose individuals with radically differing traits as parents in his crosses. Finally, in the crosses he analyzed, he used true breeding lines as parents (Figure 10.7). Plant breeders try to make a wide range of predictions that will allow them to create genetic variation and select attractive genotypes in the most efficient way possible. Plant breeders need to be able to make accurate forecasts since they can foresee conditions that may not be realized or noticed in field evaluations for several years. The questions answered and the trust in the predictions generated will determine whether those forecasts lead to substantial genetic progress and the production of improved breeding lines. With that due to the environment, progress can be made but only slowly unless very large numbers are handled. In other words, the breeder will be selecting phenotypes that are superior much of the time, but because this is mostly due to the environment, it will not give a reliable indication of a superior genotype. The phenotype is a good reflection of the genotype, meaning that the majority of variation is genetic, then progress will be rapid because when a breeder selects a good phenotype and uses it as a parent, it will pass on the superior attributes via its genes to its offspring.

Figure 10.7: Mendel Experiments. Source: https://www.britannica.com/biography/Gregor-Mendel

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Controlled cross-pollinations between two or more chosen parents are by far the most prevalent process for creating genetic varieties in plant breeding. Induced mutation, interspecific species hybridization, protoplast fusion, and plant genetic transformation are just a few of the techniques that have been employed to increase the genetic variability accessible to breeders and thus their chances of success. All living plants and animals, including all crop species, show variety as a result of natural mutations at the DNA level, with subsequent recombination and selection taking place over millions of years. However, this has been followed by structural alterations in the genetic material, such as chromosome rearrangement within and between them. Within plant species, mutations occur in the development of extra genetic variety. Mutations occur at a frequency of 1 in 1,000,000 each generation per locus in nature (Haldane, 2004). Because the majority of these mutations are recessive and detrimental, these new alleles only survive at a very low frequency in nature. As a result, the frequency of the novel allele within the population increases over generations. During the last 90 years or so, mutations have been openly employed as an additional source of genetic variation. It was discovered in the mid-1920s that X-rays could be used to cause high mutation rates, which was first demonstrated in the fruit fly and then in barley. Plant breeders were quick to recognize the promise of induced mutation, and mutation breeding became routine practice in practically all crop species and many ornamental flower breeding programs. In general, two strategies have been employed to increase the frequency of mutations in plant species: radiation and chemical induction, with radiation-induced mutants having the highest frequency of mutation-derived cultivars. Radiation-induced mutations are caused by a range of factors, ranging from physical damage to the disruption of chemical interactions. In crop breeding plans, two forms of radiation have been used to cause mutation. The most commonly employed radiation source in plant breeding is gamma rays, which have been used to create 64 percent of radiation-induced mutant derived cultivars. The disintegration of radioisotopes produces gamma rays, which are high-energy electromagnetic radiation. Cobalt-60 and caesium-137 are the two main sources of gamma radiation for induced mutation. Plant breeding programs have used single-dose gamma-ray radiation treatments or have treated entire plants with long-term gamma radiation exposure.

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10.5 X-RAYS X-rays (Figure 10.8) were the first radiation mutagen, yet 22% of the cultivars released worldwide are as a result of mutation-induced breeding programs. When high-speed electrons collide with a metallic target, X-rays are created. High-energy ionizing radiation with wavelengths spanning from ultraviolet to gamma radiation is known as X-rays (Oladosu et al., 2016). Mutations are caused by exposing seeds, complete plants, plant organs, or plant parts to a certain frequency of X-ray radiation for a set amount of time. Because X-rays require the expertise of professional radiologists, they are not always readily available to plant breeders, who must frequently rely on medical facilities for mutagenic plant therapy. Neutrons, beta radiation, and ultraviolet radiation used primarily for inducing mutations in pollen grains are other types of radiation that have been used to induce mutations.

Figure 10.8: X-rays. Source: https://en.wikipedia.org/wiki/X-ray_generator#/media/File:X-ray_table.JPG

Sulphur mustards, nitrogen mustards, epoxides, ethylene-imines, sulphates and sulphites, diazoalkanes, and nitroso compounds are all alkylating agents that bond to cellular DNA and interfere with chromosome

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division. Ethyl-methane-sulphonate (EMS) and ethylene-imine (EI) have been the most widely employed chemical mutagens (EI). It should be mentioned that all mutagenic compounds are extremely poisonous and carcinogenic, hence they must only be handled by qualified individuals in appropriate facilities at all times. The oxygen level in plant material, water content, and temperature can all influence the frequency of mutation. Translocation is a chromosomal aberration involving the interchange of different non-homologous chromosomes. Inversions are the changes in the arrangement of the loci but not in their number, deficiencies, deletions, duplications, and fusions are all structural changes in the chromosomes. Gene mutations, also known as point mutations, are changes in a single gene that are caused by a single base-pair change in the DNA. Some point mutations have no effect on the amino acid represented by the triplet of nucleotides where the mutation occurred due to the repetitive nature of the genetic code.

Figure 10.9: Extranuclear mutations. Source: https://slidetodoc.com/chapter-16-mitochondrial-dna-and-extranuclear-inheritance-jones/

Extranuclear mutations (Figure 10.9) involve one of the cytoplasmic organelles. Because the DNA involved comprises plastids and mitochondria,

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the mutation is normally passed down through only one sex, usually the egg cells, from one generation to the next. Cytoplasmic male sterility, which is frequent in many crop species, is an example of this type of mutation. Because they modify the genetic makeup of plants and promote variety, mutagenic agents and radiation are effective. They will, of course, have a similar effect on DNA if they are exposed to them. As a result, the importance of following proper safety precautions when handling any mutagen cannot be overstated. As previously stated, the facilities for administering mutagenic treatments are not always readily available to the average plant breeder; in most cases, specialized operators or personnel are responsible for the actual radiation exposure. To utilize chemical mutagenic agents safely, a variety of safety features are required, as spelled out by particular safety protocols in many countries. Employees who work with these compounds should be informed of the dangers and safety precautions that have been recommended. Minimum safety will almost certainly necessitate the use of appropriate gloves, protective clothes, and safety glasses, as well as mandatory Good Laboratory Management Practice’ Procedures and equipment must also be in place to cope with proper chemical disposal and to contain and clean up any unintentional leaks of mutagenic chemicals. Plant breeders have traditionally used inbreeding, or the practice of selfing or mating between close relatives, to attain homozygosity, which is a time-consuming process. Many plant breeders have aspired to be able to generate plants from gametic, haploid cells because this technique can produce ‘immediate’ inbred lines once the haploids’ chromosomes are doubled. The time it takes to create completely homozygous lines can be cut in half, allowing for the faster development of novel cultivars. The higher expenses and complexity of developing double haploid lines are frequently justified by the faster breeding cycle they provide. The creation of haploid gametes by meiosis is a genetic event crucial for obtaining homozygous lines. The number of chromosomes is halved during this form of cell division, and each chromosome is only represented once in each cell. If such gametic, haploid cells can be induced to develop into plantlets, a haploid plant can emerge, which can then be treated to encourage the chromosomes to double, resulting in a completely homozygous genotype. Running a plant breeding program is no different than organizing a series of scientific tests, thus all components of the operation should be planned and handled with the same care and attention to detail as individual studies. Good experimental design leads to knowledge of accuracy, which is used to evaluate and select candidates. Whether a plant breeding effort

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is based primarily on traditional procedures or includes molecular-based technologies, the quality of information obtained is the most important aspect determining its success. The right application of statistically valid designs can improve the heritability of the trait under selection, i.e., the ratio of genetic signal to experimental noise, and hence the rates of genetic gain, response to selection, and the likelihood of success. Unfortunately, this is frequently neglected. Multiple environment testing refers to a set of trials used by a breeding program to acquire insight into the performance of advanced breeding lines in a target Population of Environments. Within the same trial, it is typical to compare breeding lines to existing cultivars. In some circumstances, only one cultivar is employed, but in most cases, multiple cultivars are used in the evaluation trials. The range and number of cultivars already being grown in the target region for new cultivars, the style of trial, and the number of evaluations to be made all influence the choice and number of control entries. An evaluation trial can include the highest yielding cultivar available to compare yielding performance, the best quality cultivar to provide a quality baseline, a disease-resistant cultivar to assess response under disease pressure, and so on. Several USDA breeding groups, as well as similar public breeding efforts elsewhere, offer the cultivars they develop to the farming community royalty-free, and thus they do not hold any rights to the new varieties. In other words, the cultivar can be multiplied and sold by others. However, obtaining some kind of exclusive protection of ownership of newly released cultivars is now fairly frequent. All commercial firms require proprietary ownership of the cultivars they generate in order to control the supply of seed, determine who can grow the crop, and benefit from seed sales or royalties from seed sales to fund further research and breeding. It is possible to have automatic proprietary ownership when the cultivar generated is a synthetic or a hybrid by simply keeping the parents that were used to develop the synthetic population or hybrid seed and not giving access to the parents. Finally, if a cultivar is not a hybrid, the most frequent method of preserving cultivars propagated by seeds is to file a Plant Variety Protection application in the United States or a Plant Variety Rights (PVR) application in other countries.

10.6 TRANSGENICS Transgenic crop development is a time-consuming and expensive process, and biotechnology-based seed firms are no different from traditional

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breeding enterprises in that they must recoup their expenses. There are currently an increasing number of academic and publicly supported efforts focused on generating commercial transgenic crops, both in developed and developing nations, which will face similar hurdles in terms of acceptance and deregulation as GM cultivars. It’s yet unclear whether the benefits of transgenic lines will be adequate to cover the expenses and whether investments will be repaid. The regulatory studies required to comply with the risk assessment procedure account for a significant portion of such costs. This circumstance has caused problems since it may delay access to the benefits of transgenic crops created by academic institutions to meet the demands of small farmers or crops with a little acreage. Many of the first transgenic plants in species like potatoes were created by genetic engineering of cultivars that were not protected by plant variety laws. To compete successfully in commercial contexts, genetic engineering companies must develop cultivars transformed for specific traits that cannot be readily produced by more traditional means, and collaborate with traditional breeding programs or companies to keep the development of the myriad of other characteristics increasing in performance. Plant transformation technologies supplement traditional plant breeding by increasing the range of genes and germplasm available for inclusion into crops and reducing the time necessary for cultivar production. Plant genetic engineering also provides an intriguing potential for the agrochemical, food processing, chemical, and pharmaceutical industries to develop novel products and production methods within crop species. However, it is exceedingly doubtful that these procedures will ever completely replace the traditional methods utilized in the past. Recombinant DNA technology, on the other hand, will expand the array of options available to plant breeders in the production of future cultivars. Projects that combine gene discovery, genetic engineering, the routine use of molecular markers, and effective breeding programs are successful biotechnology efforts. A well-run breeding program is uniquely positioned to play a critical role in assembling a cultivar desired by farmers, which requires tolerance to biotic and abiotic stress, and adaptation to the farming environment. Transgenic crops are no exception. No single technique can realistically provide a solution to all agricultural concerns. Nonetheless, they are yet another weapon in inventory for dealing with important concerns such as feeding a fast-growing population, reducing environmental impact, limited availability of new farming regions, and the growing demand for accessible food, fiber, and fuel.

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INDEX

A ABA-responsive proteins 122 Abiotic stress 146 abscisic acid (ABA) 128 Acidovorax avenae 149 Acinetobacter 90 Agribusiness 4 agricultural contamination 168 agricultural reorientation 168 Agriculture 4, 5, 7, 9, 13, 15, 27 Agriculture productivity 199 Agriculture technology 5 Agriophyllum squarrosum 131 Agrobacterium 30 Agrobacterium-mediated plant transformation 97 Agrobacterium tumefaciens 54, 91 agronomy 38 Alkaloids 213 Allergies 80 allyl 17 alternative energy 33 Amborella trichopoda 59, 65 Animal feeds 11 antifeedants 34 applied biosystems (ABI) 20

Arabidopsis 53, 54, 59, 60, 61, 70, 80, 110, 113, 121 Arabidopsis genome 53 Artemisia sphaerocephala 131 Artificial intelligence 7 artificial intelligence–based denoizing 31 atmospheric trace gases 196 aubergine 85 automated mechanical oscillatory shaking (AMOS) 41 B Bacillus thuringiensis (Bt) 85, 93 bacteria 150, 157, 159, 161, 162 bacterial infections 175 bactofection-mediated gene transfer 100 barely any meristem (BAM) 130 Barley 216 beta radiation 222 Biochar 171, 172, 173 biochemistry 38 biolistics 54 Biological indexing 116 biological systems 30

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biomedical research 116 Bioreactors 40 biosafety frameworks 86, 87 Biotechnology 101, 104 Brachypodium 61 Brassicaceae 111 Brassinosteroid (BR) 128 Burkholderia glumae 149 C Caenorhabditis elegans 54 callose deposition 153 cancer 116 Caragana korshinskii 131 Carbon dioxide enrichment 192 carbon sequestration 170 Carotenoids 17 Cartagena Protocol on Biosafety (CPB) 87 cell-wall biosynthesis 111 centiMorgans (cM) 55 Chemical pesticides 168 Chemical weed management 76 chemistry 17 Chemistry-enabled imaging 32 chickpea 216 chlorofluorocarbons (CFCs) 196 chromosomal engineering 30 Cicer arietinum 147 circadian rhythms 120 citationID 11 Climate change 141, 142 Climate resilient agriculture (CRA) 191 Climate Smart Agriculture (CSA) 205 clustered regularly interspaced short palindromic repeats (CRISPR) 75 Cochliobolus carbonum 161

Co-expression analysis 110 Cold-water fisheries 203 Conservation 179 Cooperative Research Centre (CRC) 101 copy number variants (CNVs) 71 Corispermum mongolicum 131 Cotton 30 Cotton fibers 119 C-repeat binding factors (CBFs) 126 Crop plants 43 Crop protection 44 crop yields 128, 138 Cryptosporidium 169 crystal protein gene 85 D dehydration 128 dehydration avoidance (DA) 128 diallyl sulphides 17 diazoalkanes 222 DNA fragments 57 DNA polymerization 57 double-strand breaks (DSBs) 157 Drip irrigation 176 driving–pressure–state–influence– response (DPSIR) 183 drones 4, 5, 8 Drosophila melanogaster 53, 54 Drought 127, 128, 132, 133, 134, 135, 136, 137, 138, 140, 142, 144 drought escape (DE) 128 drought-resistant crop plants 133 Drought tolerance 137 E Ecological security 182

Index

effector-triggered immunity (ETI) 153 eggplant 85 endogenous genes 88 environmental protection 168 epoxides 222 ethylene-imine (EI) 223 Ethyl-methane-sulphonate (EMS) 223 Euphorbiaceae 81 Exome sequencing 58 Expressed Sequence Tags (ESTs) 54 Extranuclear mutations 223 F Farming technology 5 fatty acid-amino acid conjugate (FAC) 46 fertilizers 168, 170, 179 flavonoids 17 flax 216 food allergenicity 17 Food processing 4 food quality enhancement 168 Food riots 190 Food safety 13 food security 180, 181, 183 Forage breeders 213 forestry 38 fungal infections 175 fungi 150, 158, 162 Fusarium solani 151 G gene targeting 30 genetically modified (GM) 72 genetically modified (GM) crop plants 86

243

genetically modified organisms (GMOs) 86, 104 genetic code 37 Genetic crop enhancement 91 Genetic engineering 84, 95 Genetic Manipulation Advisory Committee (GMAC) 103 genetic networks 52, 70 genetics 38, 50 Genetic use restriction technology (GURT) 94 Gene transfer 90, 215 Genlisea aurea 59 genome-wide association studies (GWAS) 154 Genomic Criteria Consortium (GSC) 64 genomic organization 52 genomics 33, 37, 38, 39, 42, 45, 48 Genomic science 52 genotype 52, 59, 71, 72 genotyping by sequencing (GBS) 155 Giardia 169 global warming 146 glucosinolate biosynthesis 111 Glucosinolates 111 greenhouse gas (GHG) 205 greenhouse gas (GHG) emissions 205 GreenPhylDB 61, 68 H Haloxylon ammodendron 131 health impacts 17 heat shock proteins (HSPs) 40 Heat stress 149 herbicide-resistant (HR) 72

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

Herbicides 74, 77 herbicides tolerant (HT) crops 78 hormone signals 43 horticulture 38 hypersensitive response (HR) 153 hypoxanthine guanine phosphoribosyl transferase (HPRT) 101 I Imidazole Glycerol Phosphate Dehydratase (IGPD) 76 immunological techniques 116 indole-3-acetic acid (IAA) 125 indoles 17 insecticides 34 insect regimes 188 Integrated Pest Management 209 integrated weed management (IWM) 72 Integrin receptors 100 International Rice Research Institute (IRRI) 212 J Jasmonate-responsive 122 Jatropha curcas 81 L late-embryogenesis-abundant (LEA) 122 lentil 216 leucine rich repeat (LRR) 48 lipid metabolism 120 M machine learning 4 Macrophomina phaseolina 147, 165 maize 53, 59, 60, 61, 69, 77

Malnutrition 213 Manihot esculenta 81 marker-assisted breeding (MAB) 152, 160, 162 marker-assisted selection (MAS) 19, 158, 164 Marker-assisted selection (MAS) 20 marker-free transgenics 30 Massive datasets 12 mass spectrometry (MS) 43 Medicago 61 messenger RNA (mRNA) 24, 109 metabolomics 33, 46 Microarrays 108, 109, 110, 112, 117, 118, 125, 126 Microbial interactions 33, 34 microRNAs (miRNAs) 25 microstructured channel 57 millet 216 mitogen-activated protein kinases (MAPKs) 153 Modern automation 6 molecular hybridization 116 Molecular plant breeding 26, 27 molluscicides 34 Mutation breeding 78 Mutations 221, 222 N Natural catastrophes 183 NCBI (National Center for Biotechnology Information) 64 nested association mapping (NAM) 154 Neutrons 222 Next-generation sequencing (NGS) 50 nitrogen mustards 222 nitroso compounds 222

Index

nonhomologous end-joining (NHEJ) 157 non-renewable resource conservation 168 no-tillage (NT) management 171 novel genetic modification techniques 86 novel plant products (NPPs) 79 nucleotide-binding site and leucinerich repeats (NBS-LRRs) 153 O omega-3 fatty acids 17 organic agriculture 5 Organic animal husbandry 175 Organic farming 5 Organic fruit farming 174 organic matter 168, 169, 170, 179 osmolyte synthesis 43 oxophytodienoic acid (OPDA) 48 Oxygen sensors 32 P PAMP-triggered immunity (PTI) 156 parasitic infections 175 Pathogen associated molecular pattern (PAMP) 153 pathogenesis-related (PR) genes 153 pathogen stress 149, 150, 151, 152 pathology 38 pattern recognition receptors (PRRs) 156 peas 216, 219 Personality traits 184 Pesticide 34 pharmacology 116 Phaseolus vulgaris 151

245

phenolic acids 17 Phenology 138 phenotype 52, 55, 71 Phylogenomics 62, 63 Phytochrome 40 Phytozome 60, 67 Pilose mutant 119, 120 Plant biology 37 plant biopesticides 34 Plant biotechnology 33 Plant breeding 212, 221 Plant genomics 20, 52, 68, 70 Plant growth-promoting bacteria (PGPB) 35 Plant Kingdom 69 Plant modifications 212 plant molecular analysis 52 Plants 30, 39, 41, 42, 46, 49, 50 Plant sensitivity 40 plant sterols 17 plant symbionts 35 plant tissue and cell culture (PTCC) 40 Plant transformation 226 pollen development 111 polyethylene glycol (PEG) 144 polymerase chain reaction (PCR) 116 post-translational modification (PTM) 42 Potatoes 214 precipitation 188, 193, 194, 196, 198, 202, 207 Precision agriculture 8, 14 pressure–state–response (PSR) 183 Proteomics 43, 45 Pyrosequencing 23, 24 Pythium debaryanum 150 Pythium ultimum 150

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The Latest Technologies in Agriculture and Plant Sciences: Improved Techniques, Methods, and Yields

Q quantitative disease resistance (QDR) 152 quantitative trait loci (QTLs) 121 R Rainfall 139 Rainfed agriculture 202 Ralstonia solanacearum 90, 149, 150 reactive oxygen species (ROS) 43, 48, 129, 153 Recombinant DNA technology 84, 89 Resilience 140 resistance (R) genes 152, 153 reverse transcription (RT) 116 Rhizoctonia solani 150 rice 53, 54, 60, 61, 80 Ricinus communis 81 RNA interference (RNAi) 156 Robots 4 root length density (RLD) 150 S Saccharomyces cerevisiae 54 Salicylic acid (SA) 47 Selaginella moellendorffii 59 sequence-specific nucleases (SSNs) 157 sequencing-by-synthesis (SBS) 56 site-specific crop management 8 Small interfering RNAs (siRNAs) 25 Soil erosion 169 soil microfauna 36 soil quality 188 soil water 170 Sorghum 61

Soybean 136, 137, 138 Spirodela polyrhiza 59 Stomata 130 sulphates 222 sulphites 222 Sulphur mustards 222 surplus product reduction 168 Sustainable agriculture 191 Synthetic chemicals 96 systematics 38 systemic acquired resistance (SAR) 40, 48 systems biology 33, 46 T Temperature 188 thermal tolerance acquisition (TAT) 40 Thermosensors 40 tobacco 216 Tomato 59, 61 Tomatoes 216 transcription activator-like effector nucleases (TALENS) 75 transcription factors (TFs) 136 Transcript profiling 121, 122 Transgenic crop development 225 U ultraviolet-B radiation 192 ultraviolet radiation 222 unmanned aerial vehicles (UAVs) 7 Utricularia gibba 59 V Vertical farming 5 vigor–organization–resilience (VOR) 183 Viruses 175

Index

X

Vitis vinifera 147 W Water 131, 134, 137, 139, 141, 144 water resources 146 water scarcity 128, 134, 135, 141 Weeds 72, 73, 146 wheat 53, 60, 62, 76, 78 wild-type (WT) plants 133

247

X-rays 221, 222 Z zinc finger nucleases (ZFNS) 75 Zygophyllum xanthoxylum 131