Advanced Crop Improvement, Volume 1: Theory and Practice [1st ed. 2023] 3031281454, 9783031281457

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Aamir Raina · Mohammad Rafiq Wani · Rafiul Amin Laskar · Nasya Tomlekova · Samiullah Khan   Editors

Advanced Crop Improvement, Volume 1 Theory and Practice

Advanced Crop Improvement, Volume 1

Aamir Raina  •  Mohammad Rafiq Wani Rafiul Amin Laskar Nasya Tomlekova • Samiullah Khan Editors

Advanced Crop Improvement, Volume 1 Theory and Practice

Editors Aamir Raina Mutation Breeding Laboratory Department of Botany Aligarh Muslim University Aligarh, Uttar Pradesh, India Botany Section, Women’s College Aligarh Muslim University Aligarh, Uttar Pradesh, India Rafiul Amin Laskar Department of Botany Pandit Deendayal Upadhyaya Adarsha Mahavidyalaya (PDUAM), Eraligool Karimganj, Assam, India

Mohammad Rafiq Wani Department of Botany Abdul Ahad Azad Memorial Degree College Bemina, Cluster University Srinagar, Jammu and Kashmir, India Nasya Tomlekova Molecular Biology Laboratory Maritsa Vegetable Crops Research Institute Agricultural Academy Plovdiv, Bulgaria

Samiullah Khan Mutation Breeding Laboratory Department of Botany Aligarh Muslim University Aligarh, Uttar Pradesh, India

ISBN 978-3-031-28145-7    ISBN 978-3-031-28146-4 (eBook) https://doi.org/10.1007/978-3-031-28146-4 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Dedicated to Sir Syed Ahmad Khan, the Founder of Aligarh Muslim University, Aligarh, India. Sir Syed Ahmad Khan, born on 17 October 1817, in Delhi, has played a critical role in shaping the modern India. Sir Syed was a great social reformer, educationist, philosopher, and a pioneer in emphasizing the vital role of education in the empowerment of Muslim community. He worked selflessly in educating and igniting the minds of Muslims. He was the first to realize the need of imparting formal education to Muslims and acquiring proficiency in the English language and modern sciences. He established Scientific Society in 1863 to inculcate a scientific temperament into the Muslims and to make the Western knowledge accessible to Indians. Dr. Sir Mohammad Iqbal observes: “The real greatness of Sir Syed consists in the fact that he was the first Indian Muslim who felt the need of a fresh orientation of Islam and worked for it – his sensitive nature was the first to react to modern age.” On 24 May 1875, Sir Syed established the Madarsatul Uloom in Aligarh following the

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patterns of Oxford and Cambridge universities. His aim was to shape a college in line with the British education system but without compromising its Islamic values. During Sir Syed's own lifetime, The Englishman, a renowned British magazine of the nineteenth century, remarked in a commentary on 17 November 1885: “Sir Syed’s life strikingly illustrated one of the best phases of modern history.” He died on 27 March 1898, and lies buried next to the main mosque at AMU.

Preface

With the twin pressures of climate change and burgeoning population, achieving sustainable development goals in general and food security in particular is a daunting task. The human population is growing at a faster rate in developing nations, and food and nutrition available to feed the sky-high population is challenging task to scientists. As expanding arable land is not possible, the viable approach to enhance the food production is to create varieties with higher yielding potential and wide adaptability. So far, both conventional and new plant breeding approaches have contributed in terms of developing plant varieties with high yield and better resistance to biotic and abiotic stresses and in enhancing the food production. In both approaches, genetic variation is a prerequisite for improving yield and yield-attributing traits in crops. The major drawback of conventional plant breeding approaches is that more time is required for achieving the goals. However, new plant breeding technologies such as molecular breeding complement the conventional breeding approaches to obtain the desired food production. Recently developed tools and techniques such as molecular marker, genome wide association studies, Omics, TILLING, Eco-TILLING, and gene editing have made significant contributions in the crop improvement programs. These approaches have brought preferred set of traits in the varieties particularly high yielding potential, stress tolerance, nutrient quality and adaptability. This book provides insights into the concept, limitations, and role of conventional breeding approaches as well as latest developments in the modern plant breeding field. This book consists of two volumes: Volume 1 subtitled Theory and Practice and Volume 2 subtitled Case Studies of Economically Important Crops. This first volume comprises 18 chapters and the range of topics covered encompasses mutation breeding, molecular breeding, nanotechnology, transgenics, crop biofortification, forward and reverse genetics, RNA interference technology, doubled haploid production, TILLING and ECO-TILLING, genome-wide association study, genome editing, CRISPR/CAS, and Next-Generation Sequencing. The second volume consists of 19 chapters and covers detailed aspects of different crops such as capsicum, potato, carrot, buckwheat, cowpea, mung bean, lentil, chickpea, faba bean, maize, sunflower, and sorghum in addition to several techniques such as vii

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Preface

Raman spectroscopy, molecular markers, in  vitro embryo rescue techniques, genome-wide association study, and CRISPR/CAS. Each chapter contains different sections such as introduction providing background, present progress, and a detailed discussion and explanations. Each chapter ends with a conclusion and future directions and a comprehensive list of references to facilitate further reading. In addition, each chapter is supported by good-quality figures and tables. This book shall prove useful to researchers who intend to expand their plant breeding techniques. Besides, it will help practicing plant breeders working in government and private sectors. Moreover, the book shall be helpful for undergraduate and postgraduate students pursuing specialization of plant breeding, plant genetics, and plant biotechnology. Chapters were drafted by internationally recognized scientists, and each chapter was reviewed multiple times to ensure high-quality content and scientific integrity and accuracy. In this book, the experienced writers have put in a lot of effort in converting their vast experience and knowledge into useful guidelines for students, teachers, plant breeders, geneticists, policymakers, and other stakeholders. We are thankful to our contributors for nicely drafting the chapters and facilitating the publication of this two-volume book representing about 144 scientists from 19 countries. Despite our careful editing and reviewing, we might have missed some errors for which we seek reader’s indulgence and feedback. We the editors are proud in completing this book by working day and night amid tough times of Covid-19 pandemic. Lastly, we are thankful to Springer for providing us an opportunity to compile this book. Moreover, we are grateful to other staff members of Springer, particularly Kenneth Teng, Arun Siva Shanmugam, Alicia Richard, Vinesh Velayudham and Kate Lazaro for helping us in accomplishing the publication of two volumes of this book. Aligarh, Uttar Pradesh, India Srinagar, Jammu and Kashmir, India Karimganj, Assam, India Plovdiv, Bulgaria Aligarh, Uttar Pradesh, India

Aamir Raina Mohammad Rafiq Wani Rafiul Amin Laskar Nasya Tomlekova Samiullah Khan

Acknowledgments

We acknowledge the financial support from International Atomic Energy Agency, grant number BUL/5/016 and RER/5/024. ERA-NET CORE Organic “Diversifying organic crop production to increase resilience” (DIVERSILIENCE) (МОН  – КП06ДО-02/7). We acknowledge all original contributors whose works are cited in the chapters.

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About the Book

In the current scenario of an increased rate of urbanization and expanding cities, urban food security and nutrition is a great challenge. The human population is growing at a faster rate in developing nations, and that puts a lot of pressure on food systems. Besides sky high human population, food production is reduced due to climate change, dwindling arable lands, exhausting water resources, narrow genetic diversity, and shift in agriculture toward cultivation of few food crops. Food and Agriculture Organization predicted that, the human population will exceed 9.6 billion by 2050 and this means 70% more food would be required to feed the burgeoning population. As the expansion of land is not possible, the food production can be increased by developing crop varieties with more yielding potential. In this context, breeding approaches have played a pivotal role in developing crops with desired traits. Conventional breeding approaches are arduous and tedious; therefore, newer plant breeding techniques like omics and gene editing should be used to supplement the already existing breeding strategies in achieving the desired food production. New plant breeding strategies should be designed and implemented to accelerate the crop development and to bring preferred set of traits in crops of economic importance. In any breeding approach for crop improvement programs, genetic variability is an important prerequisite. The book entitled Advanced Crop Improvement is divided into two volumes with emphasis on role of breeding approaches in enhancing genetic variability in important crops. The first volume of the book covers topics such as mutation breeding, molecular breeding, nanotechnology, transgenics, crop biofortification, forward and reverse genetics, RNA interference technology, doubled haploid production, TILLING and ECOTILLING, genome-wide association study, genome editing, CRISPR/CAS, and Next-Generation Sequencing. The second volume covers detailed aspects of different crops such as capsicum, potato, carrot, buckwheat, cowpea, mung bean, lentil, chickpea, faba bean, maize, sunflower, and sorghum.

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About the Book

The basic concept of this book is to provide a broader view of collective role of breeding approaches (both conventional and modern) in advancing the crop improvement programs. The book provides a detailed background of breeding approaches and recent advancements as well as the role and land mark achievements in accelerating the crop development. This book may prove as a complete guide for plant breeders, geneticists, researchers engaged in plant breeding programs in addition to post- and undergraduate students specializing in the field of mutation breeding.

Contents

Plant Breeding from Classical Genetics to Molecular Approaches for Food and Nutrition Security ��������������������������������������������������������������������    1 Aamir Raina, Rafiul Amin Laskar, Mohammad Rafiq Wani, Nasya Tomlekova, and Samiullah Khan Nanotechnology in Agriculture ����������������������������������������������������������������������   33 Mohammad Faizan, S. Maqbool Ahmad, Lukman Ahamad, Chen Chen, and Fangyuan Yu  Contribution and Impact of Mutant Varieties on Food Security����������������   47 Joy Gilbert Manjaya  Mutation Breeding: Protocol and Role in Crop Improvement��������������������   75 Abdulwahid A. Saif  Transgenic Techniques for Plant Improvement: A Brief Overview������������   95 Lidia Stefanova, Slaveya Kostadinova, Atanas Atanassov, and Ivelin Pantchev  Mutagenesis and Transgenesis in Plant Breeding ����������������������������������������  111 Anurag Tripathi, Sudhir Kumar, Ashish Gautam, Biswajit Lenka, Jeet Ram Choudhary, and Pradipta Ranjan Pradhan Crop Biofortification: Plant Breeding and Biotechnological Interventions to Combat Malnutrition����������������������������������������������������������  143 Richa Sao, Parmeshwar K. Sahu, Ishu Kumar Khute, Samrath Baghel, Ravi Raj Singh Patel, Antra Thada, Deepika Parte, Yenkhom Linthoingambi Devi, Prabha R. Chaudhary, Suvendu Mondal, B. K. Das, and Deepak Sharma In Vitro Techniques in Plant Breeding������������������������������������������������������������  185 M. K. Sarma, Anwesha Ananya Sharma, Kajal Samantara, and Shabir H. Wani

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 Crop Improvement for Sustainable Food and Nutritional Security: Applications of Mutagenesis and In Vitro Techniques����������������������������������  217 Samuel Amiteye  Forward and Reverse Genetics in Crop Breeding����������������������������������������  257 Jeet Ram Choudhary, R. K. Bhavyasree, Seema Sheoran, Mukesh Choudhary, Subhash Chandra, Vineet Kaswan, and Shabir H. Wani Genetic Mutations and Molecular Detection Techniques in Plant Breeding����������������������������������������������������������������������������������������������  277 Samuel Amiteye RNA Interference (RNAi) Technology: An Effective Tool in Plant Breeding����������������������������������������������������������������������������������������������  309 Ankur Singh and Aryadeep Roychoudhury Doubled Haploid Production – Mechanism and Utilization in Plant Breeding����������������������������������������������������������������������������������������������  321 Ilknur Yel, Betül Ayça Dönmez, Binnur Yeşil, Merve Tekinsoy, Faisal Saeed, and Allah Bakhsh TILLING and Eco-TILLING: Concept, Progress, and Their Role in Crop Improvement������������������������������������������������������������  349 Sourav Ranjan Mohapatra, Prasanta Kumar Majhi, Kinjal Mondal, and Kajal Samantara Genome-Wide Association Study: A Powerful Approach to Map QTLs in Crop Plants��������������������������������������������������������������������������  379 Henri Desaint, Alexandre Hereil, and Mathilde Causse  Genome Editing: Mechanism and Utilization in Plant Breeding����������������  457 Muhammad Jawad Akbar Awan, Naveed Anjum, Komal Pervaiz, Muhammad Usman Ijaz, Muhammad Zuhaib Khan, Imran Amin, and Shahid Mansoor  CRISPR/CAS: The Beginning of a New Era in Crop Improvement����������  489 Yaswant Kumar Pankaj and Vinay Kumar Next-Generation Sequencing in Plant Breeding: Challenges and Possibilities������������������������������������������������������������������������������������������������  507 Ceyhun Kayihan, Hikmet Yilmaz, and Yelda Özden Çiftçi Index������������������������������������������������������������������������������������������������������������������  537

About the Editors

Aamir  Raina is currently working as Assistant Professor in Department of Botany, at Aligarh Muslim University, Aligarh, India. Dr. Raina obtained his Masters in Botany in 2013 with specialization in Genetics and Plant Breeding from the University of Kashmir, Srinagar, Jammu and Kashmir, India. Dr. Raina earned the Degree of Doctorate in 2018 for his research work on “Induced Mutagenesis in Cowpea” from the Aligarh Muslim University, Aligarh, India. His current research interests are the selection for novel mutations induced by mutagens in pulses, medicinal and aromatic plants and elucidation of physiological and molecular mechanisms in response to mutagens and looking for suitable germplasm donors for breeding purpose. Working on the mutagenesis of plants, Dr. Raina has found a significant role of mutagens in the regulation of plant growth and development and have suggested that mutagens could play an important role in improving the yield and yield attributing traits of crops. Recognizing the contribution of Dr. Raina in the field of mutation breeding, International Atomic Energy Agency (IAEA), Vienna, Austria appointed Dr. Raina as Technical Corporation Expert and Lecturer to conduct National level mutation breeding training in Sudan. The successful conduct of Dr. Raina in different IAEA projects as an expert supported his research in mutation breeding of crops and proved his ability to work in a multicultural environment. Considering all his achievements and outstanding contributions in the field of Plant Science, he has been conferred with various research

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fellowships including CSIR JRF, DBT-SRF, GATE, ICAR NET, JKSET and Nuffic OKP (recieved from Dutch Ministry of foreign Affairs, The Netherlands) and awards including National Environmental Science Academy (NESA) Young scientist and Bharat Vikas Award. He has been recognized as Young Scientist of the Year 2018, receiving the award from President of NESA, India. Dr. Raina has published more than 40 journal articles and 25 book chapters. Dr. Raina has edited special issues on research topic entitled “Legume Breeding in Transition: Innovation and Outlook” in Frontiers in Genetics and “Emerging Talents in Horticulture Breeding and Genetics 2022” in Frontiers in Horticulture. He is among the editorial board members of many scientific journals such as Frontiers in Plant Science, Frontiers in Genetics, Scientific Reports, PLOS One, and BMC Plant Biology. Dr. Raina has participated and presented several research papers in different national and international conferences. Dr. Raina has also attended many workshops, trainings, and short courses under scholarships from national and international funding agencies. Mohammad  Rafiq  Wani  is currently working as Assistant Professor (Selection Grade) in the Department of Botany at Abdul Ahad Azad Memorial Degree College Bemina, Cluster University Srinagar, Jammu and Kashmir, India. Dr. Wani did his Masters’ degree in Botany in 2003 with a specialization in Genetics and Plant Breeding from Aligarh Muslim University (AMU), Aligarh, Uttar Pradesh, India. After obtaining the Degree of Doctorate in 2008 for his research work on “Chemical Mutagenesis in Mungbean” from the same university, he joined the Department of Higher Education, Jammu and Kashmir Government in 2009. He teaches a range of bioscience-related subjects at undergraduate and postgraduate levels. Dr. Wani’s research interests are primarily focused on induced mutagenesis and molecular biology in food crops more particularly in pulse crops. As a part of his research endeavor, Dr. Wani has extensively researched and written on the issues of induced mutagenesis in food crops, with special reference to pulses. He has published more than 65 research papers in peer-reviewed journals and

About the Editors

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20 book chapters in various research-­oriented volumes. Dr. Wani, while constantly working for his academic and research interests, has so far edited six volumes of books on the subjects of Plant Stress Physiology, Induced Plant Mutagenesis, and Crop Improvement with reputed international publishers like Springer and Wageningen Academic Publishers, the Netherlands. Rafiul Amin Laskar  is Assistant Professor of Plant Sciences affiliated with the Department of Botany, Pandit Deendayal Upadhyaya Adarsha Mahavidyalaya (PDUAM), Eraligool, Karimganj, Assam, India. He received his M.Sc. in Botany with specialization in Genetics and Plant Breeding in 2012 and DBT-PDTC in Plant Tissue Culture and Micropropagation in 2013 from the Aligarh Muslim University, Aligarh, India. Dr. Laskar has obtained his Ph.D. in 2018 from the Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India, for his work on Induced Mutagenesis. After completing his Ph.D., he worked as a Guest Faculty at Nagaland University, Lumami for over 1 year and as an Assistant Professor at Bahona College, Jorhat for over 3 year. Dr. Laskar is actively engaged in research on mutation breeding in grain legumes for genetic improvement of qualitative and quantitative traits. He is a life member of the various educative societies and associations and had been active member of the interdisciplinary societies. To his credit are edited book, 10 book chapters published by reputed publishing companies like Springer and more than 50 other publications as research papers, editorials, popular science articles, review papers, and other reports. He has edited a special issue entitled “Legume Breeding in Transition: Innovation and Outlook” in Frontiers in Genetics and is serving as Editorial Board Member of many journals. He has presented a number of research papers in various national and international conferences and symposiums. He has attended many workshops and short courses under scholarship from national and international bodies. He has acted as the co-coordinator of the Institutional Biotech Hub, Bahona College funded by Department of Bio­ technology, India. Dr. Laskar has served as the Co-Principal Investigator for the project titled “DBTNER Institutional Biotech Hubs at Bahona College,

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About the Editors

Jorhat, Assam with a Focused Area of “Development of Infrastructure and Skilled Human Resources Through Science & Technology Intervention”. Currently he is maintaining an active student research program on induced mutagenesis of food crops, cytogenetics, and in  vitro tissue culture of economically important plants focusing on plant genetic resources of North East India. Nasya Tomlekova  is Professor and Head of Molecular Biology Laboratory in the Department of Breeding at the Maritsa Vegetable Crops Research Institute, Plovdiv, Bulgaria. She graduated from Sofia University “St. Kliment Ohridski” in Biology and did her Masters with specialization in Genetics and Biochemistry. She has been employed in the Institute in 1990; trained in the area of her research interests by 12 international fellowships; and she has a long postgraduate experience in research on the application of induced mutagenesis, molecular and in  vitro techniques, and other relevant biotechnologies related to crop improvement. Mrs. Tomlekova obtained the Degree of Doctorate at the Commission on Plant-Growing and Biotechnologies and her Habilitation was at the Commission on Breeding and Seed Production. She has published 5 books, including a methodological recommendation, and 11 book chapters, 121 scientific papers in peer reviewed journals and 43 international conference proceedings. Prof. Tomlekova teaches on several topics related to biotechnology and she has trained many students and fellows from different countries at Ph.D. and post- and undergraduate levels. She conducted dozens expert missions as a lecturer in different countries from Asia and Africa. Dr. Tomlekova acted as lead editor of 2 books by Wageningen Publisher, and she is a member of 3 journal editorial boards, a reviewer of more than 30 international bioscience-­related journals and projects, and a member of 5 professional scientific societies, like EUCARPIA, International Carotenoid Society, and International Pepper Network. The successful participation of Prof. Tomlekova in nine different IAEA and an EU projects as coordinator/scientific holder, and supported her research in mutation breeding of vegetable crops and potato and proved her ability for a work in a

About the Editors

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multicultural and multilanguage environment from and outside Europe. Also, she has managed projects and participated in many research teams of national and international projects. Her research focuses on the field of molecular biology with applications in agriculture, induced mutagenesis, genetics, biochemistry, and molecular marker selection. Samiullah  Khan  is currently working as Professor and Head of Genetics and Plant Breeding, and Plant Biotechnology in the Department of Botany at the Aligarh Muslim University, Aligarh, India. Dr. Khan obtained his Ph.D. in 1990 from Aligarh Muslim University, specializing in Genetics and Plant Breeding. He has 29 years of teaching experience in Embryology of Angiosperms, Genetics and Plant Breeding and supervised a number of Ph.D., M. Phil., and M.Sc. projects. He has edited several books and published more than 100 research papers in journals of national and international repute. He has attended many conferences in India and abroad including XVIth International Congress of Genetics at Toronto, Canada. He is a member of editorial board of many scientific journals. He is a fellow of the Indian Society of Genetics and Plant Breeding.

Plant Breeding from Classical Genetics to Molecular Approaches for Food and Nutrition Security Aamir Raina, Rafiul Amin Laskar, Mohammad Rafiq Wani, Nasya Tomlekova, and Samiullah Khan

Abstract  Concerns over global food security have a significant influence on the UN’s Sustainable Development Goals, which have been primarily centered on ending hunger by 2030. According to the 2019 Global Food Security Index, 88% of nations claim to have enough food supply. Still, the fact is that one out of every three countries has an insufficient food supply, implying that more than 10% of the population is malnourished. Plant breeding approaches have been used since ancient times to achieve food security by creating crop varieties with high yield and wide adaptability. Different conventional breeding approaches such as mutagenesis, which involves treating seeds or whole plants with mutagenic chemicals or high-­ energy radiation in the hopes of creating phenotypic enhancements; this, too, resulted in unforeseen and undiscovered genetic implications from which the plant breeder picked the advantageous features. These breeding efforts are also aided by biotechnological methods, such as marker-assisted selection. Lately, techniques have been developed that allow the transmission of particular and well-defined A. Raina (*) Mutation Breeding Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Botany Section, Women’s College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India e-mail: [email protected] R. A. Laskar Department of Botany, Pandit Deendayal Upadhyaya Adarsha Mahavidyalaya (PDUAM), Eraligool, Karimganj, Assam, India M. R. Wani Department of Botany, Abdul Ahad Azad Memorial Degree College Bemina, Cluster University, Srinagar, Jammu and Kashmir, India N. Tomlekova Molecular Biology Laboratory, Maritsa Vegetable Crops Research Institute, Agricultural Academy, Plovdiv, Bulgaria S. Khan Mutation Breeding Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, Uttar Pradesh, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_1

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genes or small chunks of genes encoding specific characters, along with a reliable assessment of the resultant phenotypic and genotypic features, which is referred to as “transgenesis” or “genetic engineering” because genes are transmitted out of a benefactor to a beneficiary. In this chapter we briefly discuss the current state and future prospects of food security, role of different breeding approaches such as plant introduction, hybridization, mutagenesis, doubled-haploid production, transgenics, tissue culture, marker assisted selection for diseases, insect pests, drought, heat, salt and submergence stresses and next generation sequencing in achieving the desired food production. Keywords  Food security · Nutrition security · Yield · Hybridization · Mutagenesis · Tissue culture · Marker-assisted selection

1 Introduction

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Food and Agriculture Organization defines food security as everybody enjoying the constant availability of food they require to live a healthy and active life. The notion denotes achieving food autonomy and ensuring that it will remain so in the future. Food security entails achieving a productive development compatible with the farmers’ economic situation and environmental preservation (World Food Summit, 1996). The stability of production and availability of food to all individuals in the society are the criteria that define the degree of food security in any region, nation, or zone (FAO, 1998). In recent decades, there has been an unprecedented increase in cereal production and, as a result, a reduction in global hunger. Since 2000, the intensity of the food shortage has dropped substantially (Fig. 1), bringing the total

Arable land (% of land area) Prevalence of undernourishment (% of population) Cereal yield (kg per hectare)

Fig. 1  Time trend in arable land, cereal yield and prevalence of undernourishment. (Source: World Bank (2022))

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number of underfed people to around 700 million, still a significant figure. Although excellent, this historical pattern may provide the misleading impression that food security is improving right now. On the other hand, recent evidence indicates the number of undernourished persons has grown both at objective and subjective levels since 2014. Today, Africa, Western Asia, and Oceania have a more significant number of malnourished individuals than they had a decade ago. Population expansion and climate change are the two most urgent issues to food supply presently and for the future. Rapid population growth (UNPD, 2017) fuels worldwide demand and puts pressure on land due to urbanization (Satterthwaite et al., 2010; UNPD, 2018). Economic growth is critical to food access, and it is still significant in decreasing hunger (Gödecke et al., 2018) and poverty (Dollar et al., 2013), even though it may not be enough to reduce hunger and malnutrition as quickly as it might (FAO, 2012). Climate change is commonly regarded as a severe danger to food security in the future. Although it is impossible to forecast the specific effects of climate change, the consensus is that world food production would suffer (Wassmann et al., 2009; Lobell & Gourdji, 2012; Zhao et  al., 2017; Van Oort & Zwart, 2018). Increased CO2, temperature (IPCC, 2014a, b; Nelson et al., 2009; Peng et al., 2004), pests and disease outbreaks (Newton et  al., 2011), and decreased harvest and post-harvest yield, as well as quality attributes (Sreenivasulu et al., 2015), can all have negative consequences. Severe climatic patterns such as floods and droughts are becoming more common due to climate change (Mirza, 2011; Hay et al., 2016). As a result, food security in the future confronts multiple issues of mismatch demand-supply ratio and the necessity for resilient and sustainable production (FAO, 2010; Smith, 2013). Moreover, such factors are predicted to magnify the overall cost of food insecurity and the resulting need for food system change due to their interaction and collective reinforcement (Reardon & Timmer, 2014). While increased food supply may appear frightening, it is not uncommon. The overall production of rice and wheat has more than tripled since 1960 (FAOSTAT, 2018). The food shortage reduction was achieved by increasing cereal productivity, as shown in Fig. 1. Future constraints in the changing environment will make maintaining the present productivity growth challenging. However, the current rate of yield improvement may not be enough to meet future grain crop demands (Ray et al., 2013). Most studies that estimate the annual production increase required to keep up with demand predict a rise of more than 1%, which is higher than the year past yield growth trend (World Bank, 2007; Cassman et al., 2003; Bruinsma, 2003; Tilman et  al., 2011; Seck et  al., 2012). With demand outstripping supply, the number of undernourished individuals could rise again, reversing worldwide hunger reduction gains. Furthermore, even if supply meets demand on average, abrupt changes in climate and environment may weaken incentives for food system investment and reduce food security. Growing demand and climate change will define the future state of food security to a large extent and may stall or reverse progress toward a world without hunger (Beddington, 2010; Wheeler & Von Braun, 2013). In this light, recent studies have claimed that current food production practices are insufficient and that the food system must be transformed. (i) A dietary shift (Davis et al., 2016), (ii) food waste reduction (Godfray et al., 2010), (iii) reducing yield gaps through improved agronomy (Mueller et al., 2012), (iv) an increase in

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arable land (Kampman et al., 2008), and (v) higher productivity (Cassman et al., 2003) are all possible ways to improve future food availability. Productivity gains are dependent on both the development and distribution of new technology. Dissemination and acceptance of innovative technologies are sometimes impeded, especially in developing countries, limiting their potential to increase food security. Owing to the genetics of different crops, plant breeding is a time-consuming procedure. A new rice variety takes at least 10 years to develop and release (Acquaah, 2007). Hybridization, line fixation, and field trials are the three stages of the breeding process (Lenaerts et al., 2019). Plant breeding is an extensive operational activity that necessitates many plants at the beginning, which are reduced to a limited number of improved breeding lines by the end of the process. Roughly 98% of the original beginning material in a breeding program is rejected and abandoned due to selection during the breeding process. Most nations have an impartial government-­ led mechanism for comparing “best” advanced breeding lines to current ones, usually taking 2 years to complete. It is critical to emphasize essential factors about the time it takes to develop a new variety when it comes to variety development. The “line fixation” stage is time-­ consuming due to the heterogeneous nature of preliminary breeding material until further advancement into the 6th to 8th generation. For advanced field trials, homozygous lines are necessary. Furthermore, because seed from a new breeding line comes from only one plant, it takes time to produce enough seed throughout the breeding process (i.e., for further field trials). A new field crop variety takes around 10 years to produce, albeit variances occur in crop species and varietal testing requirements across countries (Acquaah, 2007). The breeding cycle is another aspect of variety development. This is the amount of time it takes for breeders to start developing a novel plant variety, commonly known as the “from across to cross” time. Breeders have historically employed new methods and instruments to generate new kinds and have long debated the benefits and drawbacks of various breeding procedures, mainly breeding speed (Forster et al., 2014). Many technical advancements in various fields relevant to agricultural research have occurred in recent decades, including genomics, transgenics, phenomics, and geographic information systems (GIS) (Tester & Langridge, 2010; Xu et al., 2017). In the following sections, we researched literature to evaluate established technology for fast breeding that may be used in public breeding programs in the near future. The scope of our investigation includes methods that involve genetic improvements of crop species. According to fundamental selection theory in plant breeding, reducing the time required for developing novel breeding lines increases the rate of heritability estimates, irrespective of the method used. Notably, one of the easiest and most successful techniques to generate new kinds adapted to present conditions to lessen climate change implications is to use quicker breeding and shorter breeding cycles (Atlin et al., 2017). Over the last three decades, using DNA markers as a selection technique in plant breeding has led to a significant efficacy and the eventual release of new cultivars (Collard & Mackill, 2008; Xu & Crouch, 2008). Molecular marker-­ assisted screening is generally more effective than traditional approaches, resulting

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in increased precision and savings of money or time, or the ability to screen for diseases that are not detectable using standard phenotyping methods (William et al., 2007a, b). One of the key benefits of utilizing markers is that homozygosity may be traced quickly. In private sector breeding programs, molecular breeding has been widely embraced on a huge scale, with data indicating rising rates of genetic gain (Crosbie et al., 2008; Eathington et al., 2007). Many reports of marker-assisted variety generation have emerged due to the widespread use of marker-assisted selection (MAS) in major crop breeding projects (Gregorio et al., 2013; Gupta et al., 2010).

2 Plant Introduction Germplasm is a critical resource for developing new plant kinds with desirable features that increase agricultural productivity and, as a result, enhance human nutrition. It powers many aspects of contemporary agriculture, including research, development, and output (Chang, 1987). Plant genetic resources have historically been used to address the possible utility of plant species within their primary and secondary gene pools. Crop development has become the axle of the wheel of human life, with the ever-increasing need for more food and more excellent nutrition. Plant breeders require more diversified germplasm; acquiring superior varieties from other locations is necessary (Allard, 1960). As a result, a thorough understanding of the ideas underpinning the plant introduction procedure is essential. Primitive man chose valuable plants from the wild and moved them close to his dwellings/home, eventually cultivating them. With the growth of civilization and improved human communication, the migration of these valuable plants began to spread, and they ultimately made their way into other regions. According to Frankel (1957), plant introduction is the transfer of a genetic entity from one environment to another that it is unfamiliar with. This includes the complete range of contemporary plant breeding techniques (such as gene addition or replacement, mutation, structural chromosomal change, and so on), with the ultimate objective of introducing superior plant types into cultivation everywhere on the planet. All such initiatives, known as organized plant introduction services, began in some of the world’s most advanced countries, such as the United States, the Soviet Union, Australia, and others, to carry on plant introduction by conducting organized explorations and collection of crop plants, or by procuring them through correspondence for use directly as varieties or as donors of valuable characters (yield, quality, and stress tolerance). Bennett (1965) defines “primary” plant introduction as the introduction of wild species into cultivation and the effective transport of cultivars to other settings with their genotypes intact and “secondary” plant introduction as everything else. Both are, nevertheless, equally important for the proper utilization of plant germplasm. The variety has acclimated well to the new environment in the primary introduction. It has been released for commercial production with no changes to the genotype. In contrast, in the secondary introduction, a variety introduced may be subjected to selection and hybridization to isolate a superior variety. Also, based on use: (1)

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direct introduction – creating a new variety requires no time; (2) indirect introduction – it takes time for a new variety to establish itself. For ages, plant introduction efforts have been carried out worldwide. Travelers, pilgrims, conquerors, explorers, and naturalists were the primary agents for plant introduction in the past. Due to geographic connectivity, plant migration inside old-­ world countries was feasible much earlier. Still, plant trade between the old world and the new world was only possible following Colombus’ discovery of the Americas in 1492 and the subsequent European colonialism. The United States, which is currently a wheat and soybean exporter, did not have old-world wheat and soybean 400 years ago, and the same was true of rice. Maize, potato, sweet potato, tomato, peanut, and other new-world crops have become vital crops in the old world. The old-world and new-world economic plants have been traded between the Western and Eastern hemispheres and between the nations that make up the two hemispheres. Over the last five centuries, there has been a lot of back-and-forths between the commercially beneficial flora of different parts of the world. Many plants were brought into various locations by the Portuguese, British, French, and Dutch during the colonial process in the sixteenth century and thereafter. Pilgrims and explorers made similar swaps, as did conquerors who brought plants with them. Muslim rulers in India, for example, introduced a variety of plants from Afghanistan and Iraq, such as cherries and grapes; by the seventeenth century, the Portuguese had introduced new-world crops such as Zea mays, Arachis hypogaea, Capsicum frutescens, Solanum tuberosum, Ipomoea batatas, Psidium guajava, Annona reticulata, Ananas comosus, Anacardium occidentale, and Nicotiana tabacum. The British East India Company had introduced Camellia sinensis, Litchi chinensis, Eriobotrya japonica from China, and Brassica oleracea var. capitata, Brassica oleracea var. botrytis, and other winter vegetables (Singh, 1963; Randhawa, 1964). The monumental work of Russian botanist/explorer N.I.  Vavilov (1926) raised awareness of the prevalence of plant genetic variety in eight phytogeographical zones – locations where crop diversity was discovered to be exceptionally strong for some species, which he referred to as “centers of origin.” As a result, various scientists from the United States, the Soviet Union, Europe, and Australia organized plant exploration and gathering. They ultimately brought the seeds/planting materials they had collected into their respective areas and examined them for various qualities. The dissemination of such findings piqued the attention of plant breeders worldwide in obtaining such material for use as primary or secondary introductions, which aided crop plant migration (Dadlani et al., 1981). The introduction of sunflower to the USSR from Central Mexico/United States, Chinese soybean to North America, Ethiopian coffee to Central and South America, Bahian cacao to West Africa, Amazonian rubber to Malaysia, African oil palm to Indonesia and Malaysia, Asiatic yams into tropical America and Africa, wheat from the Near East to the United States are all classic examples of new crop introductions that benefited other countries more than the country of origin. In India, the procedure of plant introduction includes (a) germplasm acquisition – any individual or organization can bring germplasm into India. However, all introductions must go via the National Bureau of Plant Genetic Resources (NBPGR) in New Delhi. Plant introduction can take one of two paths. In the first route, an

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individual or an institution directly requests a person or organization in another country. In the second phase, a person or institute files a request to import germplasm to the NBPGR: (b) quarantine – it is the process of isolating items to avoid the spread of diseases, weeds, and other pests. All new materials have been extensively screened for weeds, diseases, and insect pests. To prevent contamination, the materials are fumigated or treated. The materials are cultivated in isolation if necessary to observe diseases, insect pests, and weeds: (c) cataloging – a new material’s accession number is assigned during cataloging. Names of species and varieties and their origins, adaptations, and varied characteristics are all documented. The gene bank publishes cataloging of germplasm holdings. Exotic Collection, Indigenous Collection, and Indigenous Wild are the three sorts of materials offered. Each has its prefix: EC – Exotic Collection, IC – Indigenous Collection, and IW – Indigenous Wild. (d) Germplasm evaluation  – the newly introduced material is assessed to determine its potential and performance. At various substations, these materials are analyzed. The material’s disease and pest resistance are evaluated in a controlled setting, and the most promising is released as a variety or subjected to selection or hybridization. (e) Multiplication and distribution  – promising introductions or selections from introductions may be multiplied and released as varieties after the requisite trials. The majority of the introductions, on the other hand, are classified for desired characteristics and kept for future use. These materials are commonly used in crossing programs and are available from the bureau upon request. (f) Acclimatization  – it refers to the process of a plant or animal adapting to a new environment. Introduced plants typically perform poorly because they are not suited to their new habitat. The performance of a variety in a new environment can sometimes improve as the number of generations increases. Acclimatization occurs when those genotypes (found in the original population) that are more acclimated to the new environment multiply more quickly. As a result, acclimation is a form of natural selection.

3 Hybridization In plants, animals, and fungi, natural hybridization is acknowledged as a crucial evolutionary process (Whitney et al., 2010). At least among plants, hybridization has been regarded as the norm rather than the exception. Several studies on the importance of natural hybridization in shaping the earth’s biodiversity have been published in recent decades (Wissemann, 2007). In this regard, inter-specific hybridization has been discovered to significantly impact the genetic diversity of a single population (Arnold, 2006). Hybridization may also be a factor in the extinction of unique or uncommon plant populations (Levin & Francisco-Ortega, 1996). On the other hand, it may result in the emergence of new species (Arnold, 2006). Hybridization refers to a process of developing new crop varieties by crossing two parents with genetically distinct characters. It can be employed in both self and cross-pollinated species. The primary goal of hybridization is to provide variety. Based on the kind and connection of the plants to be crossed, the hybridization

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process is divided into the following categories: (a) inter-varietal hybridization, (b) intra-varietal hybridization, (c) inter-specific or intra-generic hybridization, (d) inter-generic hybridization. Hybridization objectives include creating a single variety containing all of the positive characteristics and beneficial variants by introducing character recombination and utilizing hybrid vitality. The selection of parents from accessible material with desired characteristics is the initial stage in hybridization. The selfing of parents is the next stage in achieving homozygosity in desired traits. For years, sexual hybridization has been the standard strategy for improving the properties of cultivated plants. The main drawback in sexual hybridization is that it can only be done within a plant species or between closely related species. This limits the amount of progress that may be made in plants. Somatic cell fusion, which can generate a viable hybrid, can circumvent the species limitations for plant improvement in sexual hybridization. It is the process of creating hybrid plants by fusing somatic protoplasts from two separate plant species or types. Somatic hybridization is the process of fusing isolated protoplasts in vitro to form a hybrid cell, which then develops into a hybrid plant. It (protoplast fusion) is a very recent and adaptable technology for inducing or promoting genetic recombination in many prokaryotic and eukaryotic cells (Bhojwani et al., 1977).

4 Mutagenesis The initial stage in plant breeding is to find acceptable genotypes with the desired genes among existing cultivars or to develop one if none exists. Mutations are the primary cause of variation in nature, and plant breeding would be impossible without them. In this context, the primary goal of mutation-based breeding is to create and enhance well-adapted plant types by changing one or two main features to boost production. Physical and chemical mutagenesis are used to create mutations in seeds. The first generation is used to select agronomic features, with most mutant lines being removed. The economic characteristics are validated in the second and third generations by phenotypic stabilization, with further analyses in later generations. In advanced generations,  only attractive mutant lines are chosen as a new variety. It has been believed that the history of plant mutation may be traced back to 300 BC in China when tales of mutant crops were first reported (Kharkwal, 2012; Van Harten, 1998). Hugo de Vries discovered mutations as a technique for producing diversity in the late 1800s while testing on the “rediscovery” of Mendel’s rules of heredity (Kharkwal, 2012). He saw this variation as heritable modifications caused by means other than segregation and recombination. He defined this phenomenon as rapid alterations in hereditary organisms, which had a rather significant influence on the external features of the organism. He then coined the term “mutation” and gave an integrated idea describing the occurrence of a rapid, sudden alteration in existing attributes that lead to the emergence of new ones. After Stadler discovered the mutagenic effects of X-rays in Zea mays, Hordeum vulgare and Triticum aestivum, radiation- mutagenesis as a technique for producing

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unique genetic diversity in plants advanced as a field (Stadler, 1930), tobacco generated the first marketable mutant cultivar in 1934. Acquaah (2006) reported 77 cultivars developed using mutagenesis before 1995, which increased to 484  in 1995. Since then, the number has risen dramatically, with new mutant types being reported on a regular basis around the globe. In the 1950s, many crops and ornamental plant species were primarily treated with radiations to promote trait diversity, and mutagenesis became famous as a breeding approach (Oldach, 2011; Leitao, 2012). Mutation breeding has resulted in the development and official release of high-­ yielding mutants that have benefited mankind in achieving the desired food production (Raina et al., 2016; Khursheed et al., 2019). Millions of hectares of land have been devoted toward the cultivation of mutant varieties that resulted in the profit of billions of revenues. Mutation breeding allows the breeders to select mutant lines with desired combination of traits (Raina et al., 2018a, 2021; Raina & Danish, 2018; Wani et al., 2021a, b). Several workers have used various mutagens for developing varieties with useful traits in crops such as Cicer arietinum (Laskar et  al., 2015; Raina et al., 2017, 2019), Lens culinaris (Laskar et al. 2018a, b), Vigna unguiculata (Raina et al., 2018b), Vigna radiata (Goyal et al., 2020a, b, 2021; Wani et al., 2017), Vicia faba (Khursheed et  al. 2018a, b, c), Trigonella foenum-graecum (Hassan et  al., 2018), and Nigella sativa (Amin et  al., 2016, 2019; Tantray et  al., 2017). Mutagenesis have been successful in enhancing the  agronomic traits like yield, maturity, adaptability, and resistance to stresses (Khursheed et  al., 2015, 2016; Laskar et al., 2019; Goyal et al. 2019a, b; Raina & Khan, 2020; Raina et al. 2020a, b).

5 Doubled Haploid Production The doubled haploid (DH) populations are produced from haploid cells such as pollen grains followed by chromosomal doubling, substantially reduces the line fixation step since totally homozygous lines are formed instantly (Mishra & Rao, 2016). This is only achievable in species that are receptive to tissue culture and is done in tissue culture laboratories (e.g., cereal species including rice). This strategy has been employed in rice breeding for decades (Pauk et al., 2009) and, is a tried-and-­ true breeding technique that has resulted in the release of several rice varieties. However, compared to the japonica subspecies, it has proven more challenging to establish doubled haploid populations for the indica subspecies due to biological considerations (Grewal et al., 2011).

6 Transgenic Approach A transgenic approach can be used to develop plants with desired combination of traits. It uses an infinite pool of genes to transfer and express desired features from one species to another, even if they are evolutionary and taxonomically diverse. Adding new genes and/or altering the expression profiles of existing genes often

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result in the creation of transgenic plants (Malik & Maqbool, 2020). Although traditional plant breeding approaches supplemented with modern biotechnological selection tools delivered significant success in plant breeding over the past few decades, however, selective breeding has certain drawbacks such as poor heritability, lack of genetic diversity, and linkage drag. These drawbacks have been overcame in transgenics that has proven as a more viable technique for improvement of staple crops. The role of genetically modified crops in food security is a contentious issue. Genetically modified crops may influence food quality and nutritional composition, increasing food production and availability (Qaim & Kouser, 2013). When utilizing genetically modified crops as part of a more extensive food security plan, appropriate legal and regulatory frameworks are required to ensure that the needs of impoverished farmers and consumers are satisfied and that negative social consequences are avoided. Transgenic crops have been developed to mitigate the yield losses in major food crops. These transgenic approaches have been employed to create the crop varieties that can withstand different environmental stresses. For instance, Kumar et al. (2009) have reported increased salt tolerance in transgenic rice harboring a mutagenized gene P5CS (Δ1-pyrroline-5-carboxylate synthase). Xiao et al. (2007) and Duan and Cai (2012) developed transgenic rice with improved tolerance to abiotic stress. Up to now a large number of transcription genes conferring tolerance to drought, heat, and salinity have been identified and characterized (Kumar et al., 2013). Many genes isolated from genetically related or distinct crops have been stacked into widely accepted varieties using different modern breeding techniques. For instance, transgenic rice showing overexpression of HvCBF4, ZmCBF3, OsDREB1F, and OsDREB2A isolated from Hordeum vulgare, Zea mays, and Oryza sativa, respectively, revealed an improved survival under salt and drought stress (Wang et al., 2008; Mallikarjuna et al., 2011).

7 Tissue Culture The culturing of plant cells, tissues, or organs on the artificially prepared nutritional medium is known as tissue culture or in vitro propagation (Ahloowalia et al., 2004). A whole plant can be regrown using a single cell with all requirements available. It is a technique that has been available for more than 50 years and is a critical technology for developing disease-free, and uniform plants. Tissue culture can be utilized to enhance the amount of planting material available for large-scale planting and distribution. A significant number of plants may be generated in a short span of time. Tissue-cultured plants are known to develop faster with consistent production cycle than traditionally propagated plants (Bertrand et  al., 2005). Plant cells and tissues can now be used to regenerate virus-free, herbicide-resistant, salt-tolerant, disease-­resistant, high protein-content plants, and genetically altered plants with desired characteristics. Secondary metabolites, a rich source of many therapeutic and commercial goods, have also been widely produced (Ragavendran & Natarajan, 2017). Different explants such as embryo, anther, endosperm, protoplast, etc. are all

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used in plant tissue culture (Basavaraju, 2005). Tissue culture offers several benefits to plant breeders such as  rapid and large-scale production  of  disease free plants, regardless of season, space, and time constraints. Nutrient preparation, proliferation, multiplication, rooting, and hardening are the basic steps of micropropagation, according to Murashige (1974). Tissue culture has played a central role in the development of crops with desired traits and could play a role in achieving goals of plant breeding in the context of food production.

8 Marker-Assisted Selection The concept of marker-assisted selection (MAS) was first introduced by C. Smith and P. Simpson (1986) and by Soller and Beckmann (1983). The first studies were based on isozymes and proteins (Krishna & Mitra, 1988; Lu & Pickersgill, 1993). In less than a half-century, the concept led to the discovery of several DNA markers such as restriction fragment length polymorphism (RFLP; Botstein et  al., 1980), followed by Random Amplified Polymorphic DNA (RAPD; Williams et al., 1990; Halward et al., 1991, 1992; Hilu & Stalker, 1995; Subramanian et al., 2000), Inter-­ Simple Sequence-Repeats (ISSRs; Zietkiewicz et  al., 1994), Amplified Fragment Length Polymorphism (AFLP; Zabeau & Vos, 1993; Vos et al., 1995; He & Prakash, 1997; Gimenes et al., 2000, 2002; He & Prakash, 2001; Herselman, 2003; Tallury et  al., 2005), and more recently single nucleotide polymorphism (SNP; Hopkins et al., 1999). In the process of evolution of molecular markers, a rise and fall in different markers are evident and reflect a continuous improvement in the way we evaluate genetic variability. This also led to developing a repertoire of molecular markers and fine-tuning the indirect selection process of elite genotypes. Molecular markers can distinguish between genotypes relevant to traits of interest such as stress tolerance, disease resistance, high yield. This distinction is not necessarily based on the presence or absence of a particular trait. Molecular markers are not influenced by environmental factors and developmental stages. These markers have resolved significant shortcomings of conventional breeding methods. Molecular markers provide information about DNA level variations that cannot be detected using traditional breeding methods. In MAS, a molecular marker is employed to indirectly select a genetic determinant involved in the expression of agriculturally essential traits (Prabhu et  al., 2009). By virtue of its qualities, as mentioned above, MAS has substantially improved the effectiveness or efficiency of selection for the traits of interest by considering the markers that are genetically associated with the genes of interest. This validates it as a tool for crop improvement and has been employed in a wide range of crops. MAS has evolved as a promising technique for identifying gene markers and subsequent selection of plants with elite traits. Further MAS is neutral in phenotypic reactions; that is, they lack pleiotropic effect and are unaltered in their segregation and inheritance by the environmental flux.

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Moreover, MAS can be employed at all growth stages; therefore, it substantially reduces cost and the number of individuals evaluated by the breeder. It also offers the possibility of selecting plants irrespective of the season as in the phenotypic selection and is more convenient to the breeder. Many genes from different crops can be combined into one cultivar in MAS approaches. With the advent of molecular biology sciences, a repertoire of molecular markers and high resolution of genetic maps has made the application of MAS possible for agronomically essential traits controlled by significant QTLs (Choudhary et al., 2008). It is gaining popularity in crop improvement programs due to its ability to manage (i) traits with low penetrance or complex inheritance effectively in cost- and time-effective ways; (ii) traits that are influenced by environmental factors and developmental stages; (iii) improving pace and precision of backcross breeding; and (iv) pyramiding multiple monogenic traits (such as pest and disease resistance) or several QTL for a single target trait with complex inheritance (such as drought tolerance). It is also critical as it effectively facilitates the transfer of genetic determinants of traits of interest and speeds up the recovery of the recurrent parent genome (Wijerathna, 2015). It also provides opportunities to gather target traits in the same genotype more accurately in fewer selection cycles. The interest of breeders in MAS is engrossed primarily in the traits such as resistance to biotic and abiotic stresses.

8.1 MAS for Biotic Stresses The emergence of new diseases and pests are the major threats that substantially reduce the productivity of major food crops in all parts of the world. Even though conventional breeding approaches have contributed immensely to the development of cultivars that showed improved tolerance to biotic stresses. However, the emergence of new pests and pathogens require a quick pyramiding of multiple genes into high-yielding variety background to combat the single or concurrent stresses. MAS has played a vital role in crop improvement and has enabled crops to survive in unfavorable conditions and escape or withstand the pathogen and pest attacks (Hasan et al., 2015). However, the process of developing crops with improved biotic stresses requires a broader understanding of pathogenic mechanisms and the identification of host plant genes that confer resistance. At the International Maize and Wheat Improvement Center (CIMMYT), a large-scale level MAS was initiated and succeeded in developing wheat varieties with improved stress resistance (William et al., 2007a, b). Similarly, MAS Wheat Consortium, United States, worked extensively to implement MAS to incorporate 22 different disease and pest resistance genes in wheat (Dubcovsky, 2004). In the present era, incredible advancement in the execution of MAS approaches for cultivar development has been accomplished. The use of different resistant crop varieties could be the most reliable and cost-effective way to alleviate the detrimental effects of the pest attacks and disease outbreaks. Therefore, the development of varieties with improved tolerance against diseases and insect

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pests is one of the vital aims in the breeding programs (Divya et al., 2014). Different techniques have been employed to identify, characterize, and pyramid resistance genes into new cultivars. To date, hundreds of R genes (that confer resistance to biotic stresses) have been isolated from different plant species and introduced into widely cultivated crops (Sanseverino et al., 2010). For instance, ASC1 and RPW8 genes confer resistance to Alternaria stem canker and mildew diseases, respectively, in Arabidopsis thaliana (Xiao et al., 2001; Brandwagt et al., 2000). 8.1.1 MAS for Disease Resistance MAS has been successful in developing several crops with improved disease tolerance. For instance, soybean cultivars resistant to cyst nematode (Concibido et al., 1996), two rice varieties (Angke and Conde) resistant to bacterial blight (Bustamam et al., 2002), common bean variety (USPT-ANT-1) resistant to anthracnose (Miklas et al., 2003), pearl millet (HHB 67 and HHB 67-2) tolerant to downy mildew resistance (Navarro et al., 2006), peanut varieties (COAN and NemaTAM) resistant to root-knot nematode (Garcia et al., 1996; Simpson & Starr, 2001; Simpson et al., 2003), peanut variety (Tifguard), a nematode-resistant cultivar (Holbrook et  al., 2008). In rice, blast is one of the most severe and dreadful diseases that incurs yield loss from 50% to 80% (Babujee & Gnanamanickham, 2000). Das and Roa (2015) successfully transferred genes to confer tolerance to blast (Pi2, Pi9) in a rice variety CRMAS2621-7-1 known as Improved Lalat using conventional backcross breeding till BC3F1 generation and MAS starting from BC1F1 onward (Singh et al., 2001). MAS has been successfully used for pyramiding R genes such as xa5, xa13, and Xa21 for bacterial leaf blight disease in a susceptible indica rice cultivar (PR106). In India, rice variety such as Pusa Basmati is a bacterial leaf blight-resistant variety developed using MAS approaches. In Korea, another rice variety harboring three R genes (Xa4, xa5, and Xa21) showed resistance to the dominant races (Jeung et al., 2006). Recently, marker-assisted backcross (MAB) breeding has been used to pyramid R genes such as xa21, xa13, and xa5 in a deepwater variety Jalmagna (Pradhan et al., 2015). MAS has also been employed to increase resistance to sheath blight in rice (Tan et al., 2005; Pinson et al., 2005; Wang et al., 2005). Fusarium head blight (FHB), MAS has successfully pyramided genes associated with Fusarium head blight (a severe disease causing a significant yield loss) resistance, such as Fhb1 and Qfhs.ifa-5A in wheat (Miedaner et al., 2006). Yadav et al., 2015 were also successful in the introgression of genes such as Sr25, SrWeb, and Sr50, conferring resistance to rust in Indian wheat. Wheat lines with increased resistance to powdery mildew, leaf rust, and tan spot have also been developed using MAS (Miedaner & Flath, 2007; Zwart et al., 2010). In addition to rice and wheat, MAS has been implemented in maize to increase resistance to diseases such as downy mildew, northern corn leaf blight, turcicum leaf blight, Polysora rust banded leaf, and sheath blight disease (Singh et al., 2016). With the advent of MAS, QTLs associated with disease resistance have been identified and stacked into susceptible lines of maize (Lohithaswa et  al., 2015; Prassana et  al., 2010). Zhao et  al. (2006) succeeded in

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pyramiding genes conferring resistance to banded leaf and sheath blight disease. Kyetere et  al., 1995; Welz et  al., 1998; Wisser et  al., 2006 were able to identify major genes (Msv1, Mv1, Wsm1, Wsm2, Wsm3, and Mdm1) conferring resistance to maize streak virus. Recently, incomplete tolerance to maize stripe virus and mosaic virus was achieved using SSR markers (Jacques et al., 2014). 8.1.2 MAS for Insect Pest Resistance Insect pests persistently evolve and implement new ways to overcome host-plant resistance and cause significant food and monetary loss. For instance, Asian rice gall midge, Orseolia oryzae, is a dreadful pest of rice in India, causing substantial yield loss (in the range of 10–100%), of about 477,000 tons of grain or US$80 million in India and $550 million in Asian continent (Biradar et al., 2004). The best way to mitigate the detrimental effects of gall midge is to increase the tolerance in the existing rice cultivars or develop cultivars with improved tolerance to gall midge. MAS provides an effective, fast, and reliable way to enhance gall midge resistance (Singh et al., 2015). Das and Roa (2015) successfully transferred QTLs to improve resistance to gall Midge (Gm1, Gm4) in a rice variety CRMAS2621-7-1 as Improved Lalat using conventional backcross breeding till BC3F1 generation and MAS starting from BC1F1 onward. In rice, two SSR markers, such as RM3754 and RM3761, have been identified to play a role in conferring resistance to the green rice leafhopper and could be helpful in breeding programs for insect resistance (Fujita et al., 2006). In wheat, MAS has been implemented for developing insect resistance against Hessian fly, Russian wheat aphid, and green bug. Moreover, markers associated with green bug resistance genes Gbx 1, Gba, Gbb, Gbc, Gbd, and Gbz on 7DL have been identified and pyramided into wheat (Zhu et al., 2005). In maize, MAS has achieved a remarkable success in developing resistance against southwestern corn borer (Diatraea grandiosella) (Khairallah et  al., 1998); Mediterranean corn borer (Sesamia nonagrioides L.) (Samayoa et  al. (2015); and stem borer [Scirpophaga incertulus (Walker)] (Bergvinson & Hoisington, 2001). In pigeon pea, insect pests cause a significant loss in yield and resulted in the static average global productivity over the 30 years (Choudhary et al., 2013). However, several cultivated pigeon pea lines are resistant to insect pests (pod fly) that could be used as donors to increase insect pest tolerance in susceptible lines using MAS (Durairaj & Ganapathy, 1997; Lal & Rathore, 2001). The best way to mitigate the adverse effects of insects and pests is to increase the tolerance in the existing cultivars or develop cultivars with improved tolerance.

8.2 MAS for Abiotic Stress Abiotic stresses such as heat, drought, water logging (submergence), and salinity adversely affect the productivity of crops worldwide and result in a huge loss in terms of food and revenue. In contrast to biotic stress tolerance, which is usually

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governed by a single gene, abiotic stress tolerance is controlled by multiple genes with a complex mode of inheritance, low heritability, unpredicted epistasis, and environmental influences. The development of crop varieties with improved abiotic stress tolerance is a challenging task due to the role of multiple genes, dependence on the developmental stage, and the impact of environmental factors that impede the repeatability and precision of direct selection in the natural field. Each developmental stage requires different screening methods, and traits are also influenced by plant ontogeny. Among the crop improvement approaches, MAS has proven itself to be a coherent tool in developing varieties with improved resistance to the different abiotic stresses and is being used for selection for resistance to heat, drought, submergence, and salinity. Crops with enhanced stress tolerance are less dependent on pesticides, herbicides, insecticides, etc., deter the risk of yield loss from stresses, and facilitate more stable crop production across diverse and adverse environments and poor soil conditions. MAS has enabled plant breeders to identify quickly, followed by characterizing QTLs associated with stress tolerance during different developmental stages. The use of MAS has led to the development of crops with enhanced stress tolerance throughout the plant ontogeny and overcame the limitations generally encountered in phenotypic selection. 8.2.1 MAS for Drought Stress Drought is also considered major abiotic stress causing a significant yield loss (Devi et al., 2017). It has been estimated that 65% of the world’s population will face a dearth of water scarcity by 2025 (Gantait et al., 2019). Drought impacts vital processes that eventually influence normal plant growth, development, food production, and productivity. Consequently, plant breeders are required to develop new breeding approaches to create cultivars with improved water stress tolerance. A lot is dependent on high-yielding stable cultivars with improved drought tolerance in making food accessible to the burgeoning human population that is expected to rise to nine billion by 2050. Among the breeding approaches, MAS is the most suitable and cost-effective strategy to develop drought-resistant cultivars in a short period (Nezhadahmadi et al., 2013; Tuberosa, 2012). The development of cultivars with improved drought tolerance could alleviate the adverse effects of drought. To date, hundreds of drought-resistant cultivars in crops such as common bean, sunflower, peanut, chickpea, wheat, barley, wheatgrass, and maize have been developed (Ashraf, 2010). Several QTLs associated with drought tolerance have been mapped in various crops such as maize, cotton, wheat, sorghum, barley, and rice (Quarrie et al., 1994; Sari-Gorla et al., 1999; Sanchez et al., 2002; Nevo & Chen, 2010). Babu et al. (2003) identified two QTLs on chromosomes 4 and 9 for drought tolerance which has pleiotropic effects on yield. In rice, QTLs such as RZ19-RZ909, RG938-RG620, RG978-RG598, and R41-RM242 were found to be associated with drought resistance traits (Kamoshita et al., 2008; Kanagaraj et al., 2010). Jongdee et al. (2006) employed MAS to pyramid drought tolerance genes in a single variety, using selected lines from a doubled haploid population as drought donors. Courtois

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et al., 2003 also implemented MAS to pyramid four drought-associated QTLs from a japonica upland cultivar into “IR 64.” Further, Marker-assisted backcross (MAB) was also employed to enhance drought resistance of an upland rice variety, “Kalinga III” (Roberto & Silvio, 2006). While working on drought tolerance in maize, Messmer et al. (2011) reported 32 and 25 QTLs for chlorophyll and senescence, respectively. They concluded that high chlorophyll and low senescence are valuable traits for selecting drought-tolerant lines. Ulemale et al. (2013) reported that parameters such as chlorophyll stability index, chlorophyll content, relative water content in leaves, proline buildup, NR activity, and membrane injury index play a vital role in the selection of drought-tolerant chickpea lines. In wheat, a microsatellite marker, wmc89, was identified with a QTL impacting grain yield and drought susceptibility index using SSR markers. In chickpea, 10,996 high-quality drought-­ responsive expressed sequence tags (ESTs) were developed from root tissue cDNA libraries (Varshney et al., 2009). Yu et al. (2012) employed the Solexa Illumina array and identified 1092 drought-responsive genes, and drought tolerance-associated genes such as BnLAS, BrERF4, AnnBn1, BrECS, and BnLEA4-1 could be utilized for breeding of oilseed Brassica species (Zhang et al., 2014). Schneider et al. (1997) identified four markers for QTL associated with drought tolerance in one population and five in the second population of common bean. Further advancement in MAS is required to develop a competent approach for drought tolerance. 8.2.2 MAS for Salt Stress Salt stress is one of the detrimental abiotic stresses that affect almost all food crops, particularly rice, triggering more than 50% yield losses (Molla et al., 2015; Ahmad et al., 2019). It is a significant obstacle in achieving the desired production across nearly all rice-producing areas. It has been documented that salt stress affects over 150 million hectares of rice land worldwide (Dissanayake & Wijeratne, 2006). This may be partly attributed to the least level of salt tolerance available in the existing rice cultivars. However, with the advancement in molecular breeding, several workers have developed rice varieties that can withstand a high level of salt in the soil and the water. QTLs associated with salt tolerance have been mapped in A. thaliana (Quesada et  al., 2002), Hordeum vulgare (Ellis et  al., 1997, 2002), Oryza sativa (Prasad et  al., 2000; Lin et  al., 2004; Lee et  al., 2006), Glycine max (Lee et  al., 2004), Triticum aestivum (Ma et al., 2007). In addition to QTL mapping, several crops have been created with enhanced tolerance to high levels of salts and produce economically acceptable yields. For instance, transgenic wheat expressing a vacuolar Na+/H+ antiporter gene, AtNHX1, from A. thaliana (Xue et al., 2004; Ashraf & Akram, 2009). Das and Roa (2015) successfully transferred genes/QTLs to confer tolerance to salinity (Saltol) in a released rice variety Improved Lalat. The recent identification of major QTLs like Saltol (Bonilla et al., 2002; Nejad et al., 2010) has achieved remarkable breeding resistance for salinity stress. At Central Rice Research Institute, India, salt-tolerant rice lines were developed by transferring Saltol QTL from Pokkali into FL478. The lines having Saltol QTL showed improved tolerance

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to salt. Chankaew et al. (2014) reported a single major QTL associated with salt tolerance in Vigna marina subsp. oblonga that could be utilized in improving salt tolerance in mungbean. Sehrawat et al. (2014) reported novel SSR markers associated with genes that govern salt resistance in mungbean genotypes. Further, Hayat et al. (2011) studied salinity tolerance in B. juncea genotypes based on photosynthetic attributes and recommended Varuna cultivar in Indian soils with high salt concentration. Tomato QTLs associated with salt tolerance traits such as better seed germination and good vegetative and reproductive growth (fruit size) were identified (Foolad, 2004). Over the last three decades, numerous research programs worldwide have identified and reported hundreds of QTLs associated with different stress tolerances and could be used in future breeding programs. 8.2.3 MAS for Submergence Tolerance In addition to the salt stress, submergence is also major stress that causes significant yield losses and is one of the critical issues in flood-prone rice producing areas (Iftekharuddaula et al., 2015). Submergence is mainly governed by a major gene (Sub1). The discovery of major QTLS like Sub 1 (Xu et al., 2006) has played a vital role in conferring tolerance against submergence. Indica cultivar FR13A is a highly tolerant rice variety to submergence owing to a Sub1 on chromosome 9 (Xu & Mackill, 1996; Manivong et  al., 2014). At International Rice Research Institute (IRRI), Philippines, a submergence tolerant variety Swarna Sub 1 was developed by transferring Sub 1 QTL from FR 13A. Further, Das and Roa (2015) also reported the efficacy of Sub1 QTL as the pyramids having the Sub1 showed improved resistance to submergence. In the near future, as the molecular markers gain wide acceptability, applicability, rapid selection, and reduced cost, MAS will be used to improve the submergence of crops in flood-prone areas. 8.2.4 MAS for Heat Stress Current analysis carried out by scientific organizations, including NASA’s Goddard Institute for Space Studies, reported that CO2 concentrations have increased from ~280 to 414.38 ppm from the preindustrial era (CO2 Earth, 2020). As atmospheric CO2 is a crucial contributor to global warming and has been linked with a global temperature rise of 0.85  °C during the 1880–2000 period, it is assumed that the frequency and intensity of heatwaves will be more in the near future (IPCC, 2014a, b; Bourgault et  al., 2018). Anthropogenic activities increase CO2 concentration, which is expected to rise to 800 ppm, further increasing the temperature from 0.3 to 4.8 °C by the end of 2100 (IPCC, 2014a, b). This significant temperature rise would have devastating effects on food production worldwide. Crop production must be doubled by 2050 to maintain the food security of an expanding human population and meet the challenges of the Agriculture Organization’s planned “No Hunger Zone.” Abiotic stresses in general and heat stress in particular are the main obstacles

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in achieving desired goals of crop production. Heat stress affects crops more frequently and severely and is considered most detrimental to cool-season food legumes (Singh et  al., 2016, 2017). Lens culinaris Medik (Lentil) is an essential cool-season legume and highly sensitive to heat stress, grown as a rainfed crop in India (Rajendran et al., 2020). At temperatures above 32 °C, the growth and development of lentil is severely hampered and causes a substantial reduction in yield (Singh et al., 2016). Like lentils, wheat is also sensitive to heat stress and is adversely affected by heat-induced physiological and biochemical changes. Vijayalakshmi et  al. (2010), while working on post-anthesis heat tolerance, identified 16 QTLs associated with heat tolerance that could be useful in wheat breeding programs. Similarly, Lopes et al., 2013 while phenotyping wheat under hot irrigated conditions, reported 16 QTLs associated with yield and canopy temperature under heat stress. Lucas et al. (2011) identified four QTLs viz., Cht-1, Cht-2, Cht-3, Cht-4, and Cht-5 associated with heat tolerance in Vigna unguiculata using recombinant inbred lines generated from CB27 × IT82E-18. Dong et al. (2015) identified differentially and specifically expressed genes and markers in oilseed Brassica exposed to heat stress. The development of varieties that can withstand high temperature with a minimum yield reduction is a prime concern of plant breeders.

9 Next-Generation Sequencing (NGS) Plant breeders have employed different molecular markers to achieve crop improvement goals, such as identifying QTLs/genes and subsequent pyramiding desired genes into a single cultivar (Varshney & Tuberosa, 2007). However, molecular markers have drawbacks such as their reliance of DNA fragments on electrophoretic separation that restricts the detection of genetic polymorphism. Further, molecular marker-based genotyping of a large plant breeding population is time-consuming and costly. The drawbacks of marker-based genotyping have been overcome by discovering NGS. This low-cost sequencing technology produces massive data sequences quickly and cost-effectively and enables breeders with a quick and effective method of marker identification (Church, 2006). Such markers aid in the indirect selection for agro-economic traits using QTL mapping and genome-wide association studies (GWAS). In addition, whole-genome sequencing technologies have shifted the perspective of marker identification from fragment to sequence SNP. NGS is now recognized as a powerful tool to detect numerous DNA sequence polymorphism-based markers quickly and cost-effectively. NGS plays a critical role in crop improvement by providing information about the gene functions, evolutionary processes, and gene regulatory mechanisms (Bevan & Uauy, 2013). It compares whole-genome sequences of different individuals within a species and provides data about millions of SNPs and InDels (insertions/deletions). By virtue of having a high frequency, more stability, and high-throughput capability over other DNA markers, SNPs and InDels are preferred (Henry & Edwards, 2009). Large-scale SNP markers have been developed in various crops, including wheat (Akhunov et  al., 2009),

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grapes (Myles et al., 2010; Arai-Kichise et al., 2011; Gopala Krishnan et al., 2011; Yamamoto et al., 2010), maize (Yan et al., 2010), and soybean (Kim et al., 2010). The NGS technologies are also used to predict breeding values in the genomic selection that could evolve as a powerful tool in crop improvement programs (Poland et  al., 2012). It can assist as a worthy tool for the next-generation plant breeders to alleviate the growing demand for food and fodder in the near future.

10 Conclusions The widespread food insecurity and nutrition reflects the failure of current agriculture in achieving sustainability. Further this has worsened under the climate change. To achieve the food security or sustainable agriculture various breeding strategies have been implemented from time to time by the plant breeders. Conventional breeding approaches have contributed a lot in achieving the desired food production, however, these approaches suffer from drawbacks such as time consuming, laborious and tedious. These drawbacks have been overcome in modern breeding approaches, especially molecular markers and sequencing technologies. However still a lot is required to design breeding approaches to address the food security and climate change issues.

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Nanotechnology in Agriculture Mohammad Faizan, S. Maqbool Ahmad, Lukman Ahamad, Chen Chen, and Fangyuan Yu

Abstract  This chapter explains numerous opportunities offered by nanotechnology to boost the agriculture sector by addressing the issues of agricultural sustainability. The increase in the population required more and more food demand all over the world. To overcome the food safety problem, there is an imperative requirement to improve agricultural production through sustainable approaches. In this regard, nanoparticles’ (NPs) effects on plants through several changes occur at the morphological, physiological, and biochemical levels; therefore, it is necessary to understand the effective role of NPs in plants and their subsequent use in the development of a tool to ameliorate agriculture part. Nanotechnology is a speedily growing field, which has potential to take forward the agri-sector and food industry. Nanotechnology with novel apparatus provides protection for the crops from pests and stresses as well as increases the foodstuff construction in a sustainable way. However, nanotechnology is in concert a key function which deals with a huge variety of ecological issues by providing effective and innovative explanations. The involvement of nanotechnology in the agriculture sector will extend great help to the farmers and enable them with better agriculture management practices. Keywords  Nanotechnology · Biochemical · Food industry · Stresses

M. Faizan (*) · S. M. Ahmad Botany Section, School of Sciences, Maulana Azad National Urdu University, Hyderabad, India L. Ahamad Plant Pathology Lab, Department of Botany, Aligarh Muslim University, Aligarh, India C. Chen · F. Yu Collaborative Innovation Centre of Sustainable Forestry in Southern China, College of Forest Science, Nanjing Forestry University, Nanjing, China © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_2

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1 Introduction To tackle the rising defy of sustainable manufacture and foodstuff safety, key technical progressions and novelties have been completed in current years in the farming division (Xiao et al., 2013; Dwivedi et al., 2016; Kou et al., 2018). Such incessant farming modernisms are vital to convene the growing food order of ignition world population with the exercise of usual and artificial resources. Nanotechnology has the potential to offer effectual resolutions to various farming problems. To bridge the gap among large materials and atomic or molecular structures, NPs give immense scientific interest. Nanotechnology appears to be the optional that might transform this pasture of cultivation as the whole nanotechnology industry had grown to $1 trillion in 2015 (Harper, 2015). Nanotechnology can effort in several magnitudes to advantage agriculture as it has the impending to play a vital effect in the manufacture, security, and safety of food (Fig. 1). Agro-nanotechnology presently takes attention on goal farming connecting the employ of NPs in sort to improve crop efficiency (Batsmanova et al., 2013). Due to the sole characteristic of NPs, they have been used in all disciplines of agricultural making in speckled forms and actions like crop development (Tarafdar et al., 2014), plant defense components (Corradini, 2010), nano-fertilizer for balance crop nutrition (Abobatta, 2017), observing the superiority of agricultural product (Rameshaiah et  al., 2015), bioprocessing of NPs for farming use (Tarafdar et al., 2014), plant growth regulators (Choy et al., 2007), and agricultural trade features (Gonzalez-Melendi et al., 2008). Impacts of nanotechnology on agriculture like seed germination, shoot and root length, crop improvements, and food industry have been discussed briefly, while the role of nano-fertilizer, nano-­pesticide, nano-herbicides, and nano-sensors has also been discussed throughout the chapter.

Nanotechnology

Crop protection

Crop improvement

Crop production

Crop processing

Disease management

Nanobiotechnology

Nutrient management

Food processing

Pest management

Seed management

Weed management

Food preservation

Fig. 1  Diagrammatic representation of effect of nanotechnology on agriculture

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2 What Is Nanotechnology? Nanotechnology is a compound interdisciplinary branch of science which includes nano-science, nano-chemistry, nano-physics, nano-materials, nano-electronics, nano-meterology, etc. (Madkour, 2019). It is a relatively emerging technology that is executed at the nanoscale and has various applications in the natural world. The NPs with range of 1–100 nm have a special place in the nanotechnology, not only for their particular properties but also for different promising building blocks of more complex structures (Benelmekki, 2015). Because of their special physical and chemical properties, they can be utilized in various applications for the welfare of the society (Bhushan, 2017). In this regard, nanotechnology provides various advantages to the public sectors such as textile, materials, health care, information and communications technology (ICT), and energy. It also has a wide range of opportunities in pharmaceuticals, medicine, and electronics as well as in agriculture.

3 Possible Application of Nanotechnology in Agriculture The agriculture business is recognized as the spine of many emerging countries, which have about 60% of their people depending on it for their day-to-day life (Raliya et  al., 2017). Many efforts have been taken to develop new strategies to boost food production. It was known that the world population is sketchy to grow approximately by 48% by 2050. The agricultural land decreases with the expansion of urbanization, and increases in food demand, which give rise to monoculture practices at broader scale of many crop varieties that, cannot be sustained without the application of agrochemicals. The use of agrochemicals (fertilizers, pesticides, growth hormones, etc.) throughout the world has reached about $266 billion by 2021, with an estimated 4.5% rate of growth annually (Wiseguyreports, 2018). In spite of the necessity and benefits for agriculture, application of agrochemicals guides the generation of large amount of dangerous substances into the surroundings (Nuruzzaman et al., 2016). Many other confronts arise such as the application of pesticides to manage pests, ecosystem contaminations, and toxic residues from the food animal/human consumption (Carvalho, 2017). In agri-food sector, nanotechnology plays a crucial responsibility in plant construction, food dispensation and wrapping, water purification and surrounding remediation, food security, crop defense, and improvement. Nanotechnology has the power to positively impact agriculture by reducing the negative impact of agricultural practices on environmental health and human beings as well as to increase food security and productivity (Fraceto et al., 2016). It has a broad range of possible applications in agri-food sector, foremost to strong research at together industrial and academic levels (Parisi et al., 2015). The expansion of the new technology in agriculture sector with the use of nano-tools could be an outstanding scheme to build a rebellion in agricultural practices with an aim to reduce the effect of current

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farming on the surroundings as well as to increase the quantity and quality of crop yields (Sekhon, 2014). It also serves as a significant device in farming to boost plant development, yield, and superiority attributes of crop plants with an increase in nutrient-use efficiency (NUE) and a decrease in the fertilizers waste and the cultivation cost (Pirzadah et al., 2020).

3.1 Nano-fertilizer The applications of chemical fertilizers are inevitable for the fertility of soil, plant growth, and productivity of crops (Keller et al., 2013). A precise management of soil nutrients (fertilizers) is needed to attain sustainable production in agri-food sector (Li et al., 2018; Davarpanah et al., 2016). To fulfill the increasing food demand, conventional agricultural practices lead to exhaustion of soil nutrients and reduction in the yield of crop plants (Huang et al., 2017). It was noted that about 40% of agricultural land in the world face a drastic loss in soil fertility due to conventional exercises being followed in agriculture (van Dijk & Meijerink, 2014; Dubey & Mailapalli, 2016). The excessive use of fertilizers affects the nutrient status of the soil and toxic waste from the fertilizers leached into water bodies which causes contamination in groundwater (Kale & Gawade, 2016). Nano-fertilizers prove to be a significant development in agri-food sector. They are defined as any product made up of NPs or used in nanotechnology to improve the nutrient efficiency and enhance plant nutrition over conventional use fertilizers (Mikkelsen, 2018; Pirzadah et al., 2020). The engineered nanoparticles (ENPs) at nanoscale have the potential to be fit in the context of sustainable agriculture and also overcome the uncertainty in the agri-food sector with very less accessible resources (Solanki et al., 2015; Godfray et al., 2010). Nano-fertilizers have immense potential to increase growth and nutrients of crop plants. Depending upon the requirements of the crop, they are released in a controlled manner to prevent differential loss. The use of nano-fertilizers not only increases the nutrient-use efficiency (NUE), but also decreases the nutrients leaching in soil water. Furthermore, nano-fertilizers increase the tolerance against abiotic stress and in combination with various microorganisms to provide more benefits to the plants (Zulfiqar et al., 2019). The porous nanomaterials (NMs) (clay, zeolite, or chitosan) efficiently reduce the nitrogen loss and increase plant uptake mechanism (Miao et al., 2015; Millán et al., 2008). The nano-fertilizers are available in different forms. Based on their action mechanism, they are classified as nanocomposite fertilizers, slow-release fertilizers, or magnetic fertilizers to deliver a range of micro- and macronutrients in plants with advantageous properties (Aziz et al., 2016). Nanofertilizers (NFs) have a wide range of applications for crop productivity. The carbon NPs along with fertilizer can increase the productivity in terms of grain yields of rice (10.29%), wheat (28.81%), soybean (16.74%), spring maize (10.93%), and vegetables (12.34–19.76%) (Subramanian et al., 2015). NFs improve the uptake of nutrients, or utilization of

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endocytosis/ion channels, or assist complexation with root exudates or ion transporters through formation new pores (Liu et al., 2009). Therefore, the NFs provide a balanced source of crop nutrition throughout the developmental stage of plant that ultimately improves agricultural production.

3.2 Nano-pesticide Plant pests, which includes nematodes, mites, insects, and pathogens, are the main limiting factors for the agricultural productivity that need to be controlled efficiently. Utilization of chemical-based pesticides has become one of the inevitable parts in agro-food sector. These chemicals with higher dosage on per-hectare basis have led to many environmental and health hazards (Rajna et  al., 2019). Many approaches have been taken into consideration for the management of agriculturally harmful pests in a sustainable manner in the last few years. Currently, there have been remarkable expansions in the field of nano-science and nanotechnology that have opened up new chances in agriculture sector. The development of new areas such as nanotechnology offers sustainable and environmentally safe alternatives for the management of crop diseases and has many advantages in comparison to conventional chemical-based pesticides. ENPs are receiving an increasing concern in the pesticide industry with the development of a range of products for crop protection. Nano-pesticide products symbolize a growing technological expansion relative to the use of pesticides and propose a wide variety of benefits through augmented durability, efficiency, and decrease in the quantity of energetic components that require to be used. Number of nano-pesticide formulations have been recommended such as nanoemulsions, nanocapsules, and products containing ENPs (Kookana et al., 2014). Different NPs are used in agriculture but most extensively studied NPs include titanium, zinc, silica, silver, copper, gold, aluminum, chitosan, and sulfur (Sabir et  al., 2014). NPs are not only inhibiting the enlargement of many plant pathogens but also have the capability to improve the development of plants (El-Argawy et al., 2017). NPs have shown strapping antimicrobial latent adjacent to plant pathogens and supposed to preserve nutrient grade of soil (Ponmurugan et al., 2016). Many reports are directed toward pathogen suppression using metallic oxide/ metalloid NPs as fungicides to enhance the health of plants. At recent times, plant disease has involved the use of metalloids/metallic oxides/non-metals either as fungicides to affect disease resistance (Datnoff et al., 2007). Due to smaller size of NPs and higher surface area in relation to conventional formulations, nano-pesticides have been shown to increase the stability and action of various fungicides (Campos et al., 2015), nematicides (Cao et al., 2015), insecticides (Chen et al., 2018; Wang et al., 2018), acaricide (de Oliveira et al., 2018), and bactericide.

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3.3 Nano-herbicides Weeds are a greater threat to our agriculture and cause significant economic loss in crop yields. Herbicides are the chemical substances which are utilized to discard the weeds in agriculture. Despite the benefits of herbicides, the use of herbicides can cause serious problems for human, animals, and the environment. The plant community with continuous application of different herbicides in different seasons makes the plants herbicide resistant and becomes uncontrollable through these chemicals. The simplest step to discard the agricultural weeds is to eliminate their seeds from the soil and stop them from germination. The action mechanism of herbicides includes absorption by the plant, translocation, and disruption or alteration of metabolic processes which ultimately leads to the plant death (Hashim et al., 2014). Nano-herbicides are a class of biological pesticides formulated by exploiting nanotechnology-based tools to make herbicide formulations. Due to being small in size, nano-herbicide has the ability to be mixed up with the soil, discard the weeds in a sustainable way without leaving the toxic residues, and halt the growth activity of weed species which become resistant to traditional herbicides (Prasad et  al., 2014). These nanoformulations could improve the herbicide efficacy and solubility and reduce the toxicity over conventional herbicides (Abigail & Chidambaram, 2017). The herbicide nanoformulations also offer exciting ways for averting a safe and effectual delivery and also overuse of herbicides (Gonzalez et al., 2014). The nanostructure herbicide could substantially decrease the rate of consumption of herbicides and improve the productivity of crops (Sekhon, 2014). Among the various nano-herbicide formulations, polymeric NPs are able to reduce herbicide mobility in soil but increased its herbicidal potential in relation to free atrazine (Pereira et al., 2014). Polymeric NPs encapsulated with atrazine were also found effective against tested Brassica species. Other studies also suggested that the use of polymer encapsulated with triazine herbicides including atrazine, ametryn, and simazine decreases the hazardous effect on environment produced by them (Grillo et al., 2010). The alginate or chitosan (Ag/Cs) NPs were selected for the encapsulation with paraquat herbicide and their possible use in agricultural applications (dos Santos Silva et al., 2011). The paraquat herbicide was also encapsulated with chitosan/tripolyphosphate NP and found effective with polymeric NP complex as well. The paraquat herbicide efficiency was not found reduced even after encapsulation with very low toxicity. Viability of cell culture and chromosomal aberration tests in onion (Allium cepa) verified to the enhanced safety of the polymeric herbicide complex against non-target organisms (Grillo et al., 2014). Till date, some works reported on polymeric NPs encapsulated with herbicides and give a safe basis for using herbicides by decreasing the harmful impact on human health and environment. In the recent past, the SiO2-NPs were discovered as inorganic herbicide transporters which are active substances with pH receptive. These SiO2-NPs preserve their optimal herbicide attention with attend lessening in occurrence of expenditure rate of herbicide.

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3.4 Nano-sensors Sensors are defined as sophisticated instruments, which have ability to respond to physico-chemical and biological aspects and transfer that response into a signal or output that can be used by human beings (NNCO, 2009). In relation to nanotechnology, it has a profound impact on the development of a new class of biosensors called nano-biosensors. The biosensors are compact analytical devices which are composed of biological makeup such as oligonucleotides, proteins, cells, or tissues. They have three major components (i.e., probe, transducer, and detector). The NP-based devices sense the specific type of signals such as force and electrochemical, or biological substances (Munawar et al., 2019). They can also be utilized for sensing a wide range of plant pathogenic microorganisms, moisture, fertilizers, and pH of soil with an aim to remove application of plant protection products, decrease the nutrients loss, and increase the yields of crop plants through better management of nutrients (Kaushal & Wani, 2017). Several NPs are designed as biosensors for disease diagnosis as well as delivery agents for probes, genetic material, and agrochemicals (Elmer & White, 2018). In addition to this, nano-sensors are very crucial for the analysis of biochemicals (Sertova, 2015; Viswanathan & Radecki, 2008; Fraceto et al., 2016) as well as to detect the presence of myco-toxins in various agri-­ foods (Sertova, 2015). The development of smart sensors in precision farming enhances crop production in agriculture by providing real-time information and helping the farmers to take better decisions.

4 Nanotechnology for Crop Improvements Crop productivity has become a concern during the last decades. The agricultural growth can be achieved only through enhancing crop production by the effective use of new techniques as land and water resources are very limited. The use of nanobiotechnology offers the tools and technical solutions to enhance crop productivity through the genetic improvement and delivery of drug molecules and genes to specific sites at cellular level (Ahmed et  al., 2013). The use of ENPs could be the upcoming solution to increase productivity of crops for the accelerating global population (Misra et al., 2016). Improved crop production was noticed by foliar application of NPs as fertilizer (Raliya & Tarafdar, 2013). Positive role of NPs in plants has been reported through enhanced percentage of germination of seeds (Lu et al., 2002), improved height shoot and root (Hafeez et  al., 2015; Liu et  al., 2005), enhanced fruit yield, increase in metabolites content (Kole et al., 2013), and a considerable increase in plant biomass in various crops (Table 1). Many studies also confirmed that metallic NMs enhance various physiological parameters such as increase in rate of photosynthesis and efficiency of nitrogen assimilation in crops including spinach (Gao et al., 2006), soybean (Agrawal & Rathore, 2014), and peanuts (Giraldo et al., 2014) (Table 1). Previously, it is reported that the application of

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Table 1  Effect of NPs on growth and physiology of plants S. no. NPs 1 Titanium oxide 2 3 4 5 6

Aluminum oxide

7 8 9 10

Copper oxide

11 12 13 14 15 16

17 18

19 20 21 22 23 24

Zinc oxide

Impacts Increased the plant growth, yield, and starch content Enhanced the chlorophyll content, growth, and yield Promoter effect on germination Increased plant growth Inhibited the leaf growth Significantly increased elongation Adverse effects on developmental processes Improved growth Enhanced uptake of plant micronutrient Decreased seed germination Inhibited plant growth Decreased yield and biomass Decreased the shoot and root length Adversely affected root length Inhibited root growth Seed germination increased

Plant species Triticum aestivum L. Linum usitatissimum L. Glycine max

Enhanced plant weight

Sesamum indicum L. Cucumis sativus

Reference Jaberzadeh et al. (2013)

Prasad et al. (2012)

Vigna radiata L. Chen et al. (2014) Zea mays L. Triticum Wulandari et al. (2014) aestivum L. Nicotiana Jaberzadeh et al. (2013) tabacum L. Glycine max L. Lactuca sativa L. Lactuca sativa L. Lemna minor L. Cucurbita pepo L. Triticum aestivum L. Gossypium barbadense Brassica napus Allium cepa L.

Significant increase in germination, decreased biomass Increased plant growth and Lycopersicum development esculentum Silver oxide Increased seed germination Solanum lycopersicum L. Promotion of root growth Crocus sativus Enhanced germination rate Trigonella foenum-graecum Enhanced plant growth Lolium multiflorum Glycine max L. Reducing germination of cytotoxic byproducts of glycolysis

Camp and Fudge (1945)

Laware and Raskar (2014) Ma and Yamaji (2006), Khodakovskaya and Lahiani (2014) Ding et al. (2009) Holleman and Wiberg (2001)

Chapman (1966)

(continued)

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Table 1 (continued) S. no. NPs 25 Silicon oxide 26

27 28

29

Impacts Enhanced seed germination Increased the fresh and dry weight, chlorophyll content Enhanced the seed germination and growth Increasing growth, antioxidant enzymes activities Increased root length and fresh weight

Plant species Solanum lycopersicum L. Ocimum basilicum L.

Reference Mahmoodzadeh et al. (2013)

Lens culinaris Medik. Vicia faba L.

Solanum lycopersicum L.

TiO2-NPs accelerates spinach growth by improving the activity of the enzyme Rubisco and absorbance of light (Hong et al., 2005; Yang et al., 2006). In addition, NPs-mediated delivery of molecules such as nucleotides or proteins has the potential to regulate the metabolism of plants, and their genetic modification. The beneficial function of NPs in crop plants has been evidenced through effectual expression of improved fraction in germination of seed, amplified length of root and shoot (Hafeez et  al., 2015), improved fruits yield, improvement in metabolite amount (Kole et al., 2013), and a considerable rise in the morphological attributes of several plants species. Same as the above, NPs also improved the other growth-related attributes in the presence of NPs, such as photosynthesis and nitrogen-use efficiency in various plants. In addition to this, nano-­biotechnology offers the tackle and skill stage to get better crop efficiency by hereditarily altering plants and conveying genes and drug molecules to testing positions at cellular phase.

5 Nanotechnology in Food Industry Nano food industry is an area of rising scientific stream that is generally applied in our daily life and it is very much challenging in the global market. It is largely impressive and has focused lots of food ventures concerned in expansion and promotion of nanomaterial-supported products and improving efficiency, food distinctiveness, flavor, and security (Pramanik et al., 2020). NPs are normally applied in each characteristic of food industry from food agriculture, processing, storage, and transportation (Srilatha, 2011). The Food and Drug Administration (FDA) controls on a product-by-product foundation and tips out numerous crops which are presently keeping pace construct of NPs (Weiss et al., 2006). FDA has conventionally coordinated several crops with particulate material in nano-scale but has not focused on applied technology for their preparation.

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6 Conclusion Nanotechnology remains in the first row as the substitution to alter the condition of the agricultural segment for the superior. The applications and benefits of nanotechnology are enormous. Nanotechnology empowers the plants to use pesticides, herbicides, and fertilizers much precisely and efficiently. The applications of nanotechnology in agriculture can perhaps express advantages to the farmers through food production. The significant nanotechnology enhances a latest revolution with minimized farming risk. However, there is still a large space in our understanding of the uptake, toxicity, and allowable limit of various NPs. Therefore, more research is required to fill this gap.

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Contribution and Impact of Mutant Varieties on Food Security Joy Gilbert Manjaya

Abstract  After the discovery of the mutagenic action of X-rays by Muller and Stadler, experimental mutagenesis in the past 90 years has created a vast amount of genetic variation of both quantitative and qualitative traits. The use of induced mutations over the past five decades has been a key factor in the development of superior plant varieties all around the world. The widespread use of the mutation breeding technique in 228 crop species has resulted in the development and release of more than 3332 mutant cultivars around the globe. The maximum number of mutant varieties are released in cereals, flowers/ornamentals, and grain legumes. Most of them are released in Asia, followed by Europe, North America, Africa, Latin America, Australia, and the Pacific. The varieties released in these crops include direct mutants or mutant derivatives through inter-mutant or cultivar-mutant hybridizations. Some of these mutants’ varieties have made a great economic impact in Australia (rice), Bangladesh (rice), China (rice, soybean, and wheat), Europe (Barley), Germany (ornamentals), India (rice, grain legumes, and ornamentals), Italy (durum wheat), Japan (rice, soybean, pear, and ornamentals), Mali (Sorghum), Malaysia (rice and banana), the Netherlands (ornamentals), Pakistan (rice, wheat, and cotton), Thailand (rice), Vietnam (rice and soybean), and the USA (rice, sunflower, and peppermint). Many mutants have made a transnational impact on the yield and quality of several seeds propagated crops since most of the mutant varieties are in cereals, pulses, and oilseeds crops and therefore had a direct impact on global food security and added billions of dollars to the national economy around the world. This chapter reviews the success story of mutation breeding in enhancing food production and the impact of mutation-derived varieties on food security around the globe.

J. G. Manjaya (*) Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India Homi Bhabha National Institute, Mumbai, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_3

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Keywords  Crop improvement · Food security · Induced mutations · Mutant varieties · Plant breeding

1 Introduction Food security is the physical, social, economic, and cultural access to food that satisfies everyone’s dietary needs, food preferences, and dietary requirements for an active and healthy life (UNFPA, 2012) The world’s agriculture has seen events such as the green revolution in the past, which helped many developing countries become self-sufficient (Conway, 1999). However, diminishing cultivable land, water resources, and climate change-associated environmental adversities are major challenges facing agricultural sustainability and food security (UNEP, 2002; Parry et al., 2005). The world’s population is expected to reach 9.9 billion by 2050 which demands 70% increased agricultural production (PRB, 2018). Therefore, to meet the global food demand and to fight the worsening crop production conditions due to climate change it is necessary that our agricultural research efforts need to be focused on increasing crop yields (Ronald, 2011, 2014). In the past, different methods of plant breeding have helped to increase the diversity of crop genetic stock by developing genetically better cultivars for cultivation. Step-by-step improvements in traditional plant breeding like distant hybridization, mutagenesis, tissue culture-based approaches, and molecular breeding has been applied to enhance agricultural production. However, to meet the increased demand for food, the existing germplasm resources may not be adequate (Tester & Langridge, 2010; Shiferaw et al., 2013). The susceptibility of crop varieties to various biotic and abiotic stresses, genetic similarities, and narrow genetic bases are the main reasons for low yields (Basey et al., 2015; Mba, 2013). In addition, some of the plant species of cultivated crops have rich genetic diversity; others have very limited genetic variation. The genetic improvement of crop plants is a continuous process and success depends on the availability of large genetic variability, which a plant breeder can combine to generate new varieties (Holme et al., 2019). Therefore, genetic variability is must for a crop improvement programmes and genetic research. In the past, naturally occurring mutations have played major roles in crop improvement. It was the introduction of the mutant dwarfing genes into breeding programmes of ‘Norin-10’ wheat and ‘Dee-Gee-Woo-Gen’ rice that contributed to the success of the Green Revolution. In nature, the occurrence of natural variability in the form of spontaneous mutations is not adequate for their use in intensive breeding programmes and the same can be enhanced to several folds by using ionizing radiations or chemical mutagens. After this discovery, Plant Breeders worldwide started practicing mutation breeding for developing better mutant varieties and also creating new genetic variability unavailable in the germplasm (Muller, 1928; Stadler, 1928; Roychowdhury & Tah, 2013: Jankowicz-Cieslak et  al., 2017). Stadler’s work on maize provided convincing evidence for the mutagenic effects of X-rays on plants and radiation-induced mutagenesis was used to develop mutants of barley. This was followed by the production of mutants in wheat, oats, lupin, flax, and mustard.

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Mutation breeding has been universally accepted as one of the plant breeding methods and a substantial amount of genetic variability has been induced by different mutagens (Brunner, 1995). The use of induced mutations over the past five decades has played a major role in the development of superior plant varieties in the entire world and the majorities are food crops. Crop improvement through mutation breeding has resulted in the development of improved varieties that are directly used for commercial cultivation or in recombination breeding by hybridizing mutant × mutant, mutant × cultivar, mutant derivative × mutant, or mutant derivative  ×  cultivar. Induced mutagenesis is the most efficient technique to greatly increase genetic variety in a short period of time and have been employed in various crops such as cowpea (Raina et al., 2022a, b; Sellapillai et al., 2022; Rasik et al., 2022) lentil (Laskar et al., 2018a, b; Wani et al., 2021) faba bean (Khursheed et al., 2015), fenugreek (Hasan et  al., 2018), mungbean (Wani et  al., 2017), urdbean (Goyal et al., 2019a, b), chickpea (Laskar et al., 2015; Raina et al., 2017), black cumin (Tantray et al., 2017; Amin et al., 2019), finger millet (Sellapillaibanumathi et al., 2022). Because natural mutations occur sporadically, artificial mutations are generated, and genetic gain is best achieved by using mutagens (Raina & Khan, 2020; Raina et al., 2018a, b). The incorporation of the mutation breeding technique in plant breeding programmes has helped to develop and officially release more than 3631  mutant varieties for commercial cultivation (Raina et  al., 2016; Sarsu et al., 2020; Ma et al., 2021; IAEA, 2021). The large numbers of mutant varieties are released in Asia, followed by Europe, North America, Africa, Latin America, Australia, and the Pacific. Maximum varieties were released in cereals, ornamentals, legumes and pulses. The mutant varieties developed are having improved agronomic traits, increased yield and quality and resistance to different types of stresses (Khursheed et  al., 2018a, b, c; Laskar et  al., 2019; Goyal et  al., 2021a, b; Raina et al., 2022d). Physical mutagens, mostly ionizing radiations (gamma rays, X-rays, neutrons, etc.) have been widely used for inducing mutations. Other mutagens like ion beam, electron beam, protons, cosmic rays, and space flight are now used as an alternate source of energy to induce mutation in addition to the conventional electromagnetic radiations, like X-ray and gamma-ray. The mutation frequency depends on the efficiency of the mutagen and therefore, the use of powerful mutagens having various mutation spectra is of utmost importance in inducing variability in plants (Ma et al., 2021; Luxiang et al., 2018). The importance of induced mutations in crop improvement and its role in sustainable agriculture is well known worldwide and some of these mutant varieties have helped to improve the socioeconomic status of the farmers and also contributed to the national economy by generating additional revenue (Khursheed et al., 2016; Sarsu et  al., 2020; Raina et  al., 2022c). The success stories of mutation-­ derived varieties developed and released in major crops all over the world have been published (Sarsu et al., 2020; Micke et al., 1990; Ahloowalia et al., 2004; Kharkwal & Shu, 2009; Oladosu et al., 2016; Raina et al., 2016). Among the mutant varieties, some classical success stories that have made a major economic impact include rice varieties in Australia, Bangladesh, China, and Thailand, groundnut, pulses, and ornamentals in India, soybean varieties in Vietnam, cotton in Pakistan, Japanese

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Table 1  Popular mutant varieties of the world Country China

Japan

Crop Rice Wheat Others Rice Soybean

India

Bangladesh Pakistan

Pear Rice Black gram Groundnut Rice

Wheat Cotton & others Vietnam Rice Soybean Thailand Rice Indonesia Sorghun Italy Durum Wheat Czechoslovakia Barley UK-Scotland

Mutant variety Zhefu 802 Luyuan 502 Other mutants Akihikari, Reimei, Kinuhikari, Haenuki, and Tsugaru-roman Mura-yutaka, Kosuzu, Akita-midori, and Ryuhou Gold Nijisseiki PNR-102 and PNR-381 TAU 1 TAG 24 Binasail, Iratom-24, Binadhan-6, and Binadhan 7 Jauhar 78, Soghat 90, and Kiran 95 NIAB-78 DT10 and VND95-20 DT84 & others RD6 and RD15 Pahat, Samurai-1, and Samurai-2 Creso Golden Promise and Diamant Golden Promise

Economic impact/ value or area US$ 500

US$ 937 million US$ 116 million US$ 30 million US$ 1748 million US$ 67 million/year US$ 1.18 million 795,000 ha US$ 6 billion

US$ 3.3 billion 2,429,361 ha 800,000 ha US$ 1.9 billion

US$ 417 million

pear in Japan, grapefruit in the USA, barley varieties in Europe, durum wheat in Italy, sunflower in the USA, sorghum in Mali and peppermint in the USA (Kharkwal & Shu, 2009; Bado et al., 2015) (Table 1). In this review, the worldwide achievements and role of induced mutations for sustainable agriculture and food security are presented in this paper.

2 Contribution of Mutagens for Enhancing Genetic Variability The mutations are broadly classified as spontaneous and induced mutations. Naturally occurring mutations are called spontaneous mutations and their occurrence is very low (about 10−6) and the same can be enhanced to several folds (~10−3) by using mutagens. Any agents used for artificially inducing mutations are called mutagens. It was only after the mutagenic effects of X-rays were successfully demonstrated in corn, barley, and wheat by Stadler, that the use of induced mutations to

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create plant genetic diversity began (Stadler, 1928). The artificially induced mutants and spontaneous mutants found in nature are the same and are an important source of genetic variability (Khursheed et al., 2017; Raina et al., 2021). Different types of mutagens have been used for inducing mutations in plants.

2.1 Plant Materials The selection of the plant material for inducing mutations depends upon the type of crop and facilities available. The plant material that has been commonly used for mutation experiments are pollen, seeds, cuttings, bulbs, corms, whole plants, tubers, stolons, organ tissues, or cells cultured in  vitro. The selection of plant material depends upon the objectives of the experiment. However, the seed is the most commonly used plant material because it can be irradiated at any time, easily handled, and transported (Bado et al., 2015).

2.2 Mutagens Many different types of agents have been used for inducing mutations in plants and are broadly classified as physical or chemical mutagens. 2.2.1 Physical Mutagens Physical mutagens consist of nuclear radiations and sources of radio-activity including several types of ionizing radiations, namely X- and gamma-rays, alpha and beta particles, protons and neutrons, and non-ionizing radiation (ultraviolet light), ion beam and laser beam irradiation, and cosmic rays (Mba et al., 2012; Mba & Shu, 2012). In the modern era, the use of physical mutagen for mutation breeding is broadly classified into classical, particle, and space mutation breeding (Ma et al., 2021). Physical mutagens, principally ionizing radiations (gamma rays, X-rays, neutrons, etc.) have been widely utilized for inducing mutations in the last eight decades, and 1703 officially released mutant crops are developed by gamma irradiation, accounting for more than 70% of the total varieties released (IAEA, 2021). The use of accelerated particles like protons or heavy ions for inducing mutations started in Japan in the 1990s (Tanaka et al., 2010). The accelerated particles are a powerful mutagen that has been considered for crop breeding since at relatively low radiation doses it shows better biological mutagenic effects (Tanaka et al., 2010). As there is a dense localized effect on DNA, the mutagens can induce novel mutations in the parent cultivar without affecting the other characters (Abe et al., 2015). In China and Japan, mutant varieties are successfully developed using the accelerated particles technique (Nakagawa, 2009; Wu et  al., 2005). Several

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mutant varieties of ornamental plants and crops have been developed in Japan using high-energy ion beam irradiation (Tanaka et al., 2010; Nakai et al., 1996; Kitamura et al., 2006). Space breeding also called spaceflight-induced mutation technology uses an aerospace environment to generate genetic variation. The space environment is defined as the area outside the atmosphere consisting of radiation, microgravity, alternating magnetic fields, and super vacuum. The plant materials are sent to aerospace using spacecraft or high-altitude balloons for irradiation and kept there for one or several weeks. Around 41 mutant varieties developed through space–induced mutation breeding in crop species like rice, wheat, cotton, sesame, pepper, tomato, and alfalfa (Wang et  al., 2004; Zhao et  al., 2001; Li et  al., 2000; Liu, 2012; Liu et al., 2021). 2.2.2 Chemical Mutagens The successful use of the chemical mustard gas as mutagens is documented way back in 1940 (Auerbach, 1946; Auerbach & Robson, 1946). The most commonly used chemical mutagens for practical and experimental plant mutagenesis are alkylating agents and sodium azide. The most commonly used alkylating agents in plant mutation breeding are sulphur mustards, nitrogen mustards, epoxides, ethyleneimines, ethyleneimine, alkyl methanesulphonates, alkylnitrosoureas, alkylnitrosoamines, alkylnitrosoamides, alkyl halides, alkyl sulphates, alkyl phosphates, chloroethyl sulfides, chloroethylamines, diazoalkanes, etc. (Luxiang et al., 2018). More than 106 mutant varieties are developed using EMS (ethyl methane sulphonate) followed by NEU (nitrosoethyl urea) (57), MNU (N-methyl N-nitrosourea) (53), colchicine (46), and EI (ethylenimine) (36). Using chemical mutagens maximum varieties are developed in cereals like rice, barley, wheat, maize, and grain legumes (Ingelbrecht et al., 2018).

3 Contribution and Impact of Mutant Varieties on Food Security Over the past 10,000 years, spontaneous mutations along with selection for desirable plant traits were responsible for crop domestication. The discovery of X-rays for inducing mutations by Muller and Stadler in drosophila and barley respectively were responsible for the success of modern mutation breeding and associated genetic studies. The Joint FAO-IAEA Division of Nuclear Techniques in Food and Agriculture supported the research, improvement, and application of nuclear techniques in food and agriculture for the past 55  years. The application of induced mutation methods in 228 crop species has resulted in the development and release of more than 3332 mutant cultivars around the globe [Mutant Variety Database (MVD) (http://mvd.iaea.org)]. The highest number of mutant varieties are released

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2318 Asia Europe North America Africa Latin America Australia and Pacific

10

53 82 209 959

Fig. 1  Number of mutant varieties released in different continents

in Asia (2318), followed by Europe (959), North America (209), Africa (82), Latin America (53), and Australia and the Pacific (10) (Fig. 1). The characteristics improved by induced mutations are improved plant ideotype (2981), quality and nutrition traits (1173), increased yield and yield components (1029), resistance to biotic stress (557), and tolerance to abiotic stress (248). Most of the mutant varieties developed and released are cereals, pulses, oilseeds, root and tuber crops, and ornamentals. The released plant mutant varieties are of cereal species (47.13%) with maximum rice mutants (25%), followed by flowers/ornamentals (21.9%) and legumes and pulses (13.8%). The review of the mutation-derived varieties and their contribution globally are well documented (Stadler, 1928; Sarsu et al., 2020; Mba et al., 2012; Liu et al., 2021).

3.1 Asia More than 64% of mutant varieties are developed and released in Asia and the top Asian countries that contributed a maximum number of mutant varieties are China, Japan, India, Bangladesh, Pakistan, Vietnam, Korean Republic, and Indonesia (IAEA, 2021). Rice is the vital source of food for more than 50% of the world’s population and 90% of it is produced and consumed in Asia. The increase in rice production in the Asia-Pacific region is due to the important role played by induced mutation techniques. In total, 823 rice mutant varieties are released in 30 Asian Countries and the maximum is in China (35.6%), Japan (26.8%), and India.

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3.1.1 China In China, crop improvement by induced mutations has been practiced since 1965 and up to 2018, more than 1030 mutant varieties from around 46 crop and ornamental species are developed and officially released for cultivation (Liu et al., 2021). Mutation techniques have played a crucial role in ensuring food security by developing new mutant germplasms and mutant varieties, which have generated a tremendous socioeconomic impact (Liu et  al., 2004). The mutant varieties covered more than 9 million hectares and produced more than 1.5 million tonnes of crop production annually, generating $500 million in revenue. In China, gamma-ray irradiation is the most popular mutagen commonly used for mutation induction and more than 80% of the released crop mutant varieties were derived directly from gamma-ray irradiation or in combination. Mutant varieties Yuanfengzao (rice), Lumia No. 1 (cotton), Tiefeng 18 and Heinong 26 (soybean), Shannongfu 63 and Chuanfu No. 1 (wheat), Luyuandan No. 4 (maize), Sandakan (legume forage), and JÍ7681 (mulberry) are some of the most famous mutants among the Chinese farmers. The direct rice mutant varieties ‘Yuanfengzao’, ‘Zhefu 802’, ‘Yangdao 6’, and super hybrid rice ‘Liangyoupeijiu’ played a very important role in increasing the production and productivity of rice in China. The early maturing mutant variety ‘Yuanfengzao’ was released in the year 1970 which was popular and was cultivated on more than 1.5 million ha. However, the early maturing mutant variety ‘Zhefu 802’ developed by the Institute of Nuclear Agricultural Sciences, Zhejiang Agricultural University using gamma rays has made a great impact on rice production in China. The mutant variety is having high yield potential, wide adaptability, resistance to rice blast, and is tolerant to cold it was widely cultivated in an area of 14 million ha in the Zhejiang, Jiangxi, Hunan, Hubei, Anhui and Fujian provinces of China (Shu et al., 1997). The success continues with the rapid expansion of the planting area of the new mutant variety ‘Yangdao 6’ released in the year 1997 having a high yield and superior grain quality. The mutant variety ‘Yangdao 6’ was used in indica rice genome sequencing and is the male parent of the super hybrid rice ‘Liangyoupeijiu’. Both the varieties have made a great impact on the area and production of rice cultivation in China and were grown on more than 3.5 million ha, and it is estimated that the planting area will be more than 2 million ha (Liu et al., 2004). Henan province of central China had developed 21 new mutant varieties of wheat. Some of the varieties like Taikong 5, Zhengmai 3596, Fumai 2008, Yufeng 11, Zhengpinmai 8, Yutong 843, and Fumai 2008 are known for high yield and good quality and have made a great contribution to the production of wheat in central China. The development of Luyuan 502, a new wheat mutant variety has been achieved by combining space mutagenesis with conventional breeding. It has wide adaptability, high productivity (12 t/ha), and stable yield characteristics. It is now the second-largest cultivated wheat variety in China and was cultivated on more than 5.13 million hectares by 2019 (Liu et al., 2021).

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3.1.2 Japan The mutation breeding experiments started in Japan in the year 1960, after the building of the gamma field at the Institute of Radiation Breeding (IRB) in Ibaraki (Yamaguchi, 2001). Different types of mutagens like physical, chemical mutagenesis, and in  vitro culture (somaclonal variation) were used for induced mutation studies in 79 species and 479 mutant varieties have been developed and registered. In Japan, since 1960, 332 mutant rice varieties were developed out of which 162 cultivars (48.8%) were derived from the semi-dwarf mutant variety Reimei. The mutant variety Reimei was released in the year 1968 and was responsible for increasing the area and production of rice in Japan. The other popular rice mutant varieties developed by using Gamma-rays are Kinuhikari, Haenuki, Tsugaru-roman, Yume-akari, Yume-tsukushi, Aichi-no-kaori, Asahi-no-yume, Mutsuhomare, Dontokoi, Yume-shizuku, Mine-asahi, Yume-hitachi, Yume-minori, Aki-geshiki, Aki-roman, Miyama-nishiki, and Tsukushi-roman. Among them, the mutant varieties Akihikari, Reimei, Kinuhikari, Haenuki, and Tsugaru-roman have been cultivated in more than 100,000 ha and annually added US$ 937 million in income to the Japanese economy (Nakagawa, 2018). The three mutant rice cultivars Reimei, Kinuhikari, and Mineasahi played a very important role in Japanese agriculture. The other important mutant varieties registered are chrysanthemum (64), rice (45), soybean (18), carnation (15), rose (15), wheat (4), and barley (4). Out of the 16 soybean mutant cultivars registered in Japan, cultivars Mura-yutaka, Kosuzu, Akita-midori, and Ryuhou are popular among the farmers. They were cultivated on an estimated area of about 136,700 ha and contributed US$ 116 annually to the farmer’s income (Nakagawa, 2021). 3.1.3 India In India, induced mutation experiments were initiated in 1930 and a few spontaneous mutants were released as new cultivars in the 1940s. India is the first country in the world to release radiation-induced mutant crop varieties in cotton (MA-9), wheat (NP-836), sugarcane (Co 6608), castor (Aruna), black gram (Co-4), mungbean (TAP-7), chickpea (Pusa-408), pearl millet (NHB-5), pigeon pea (Co-3), cowpea (V-16), lablab (Co-10), okra (MDU-5), turmeric (Co-1), bitter gourd (MDU-1), and ridge gourd (PKM-1). However, concerted efforts to use induced mutations for the genetic improvement of crop plants were initiated in the late 1950s. The Indian Agricultural Research Institute (IARI) in New Delhi, the National Botanical Research Institute (NBRI) in Lucknow, the Bhabha Atomic Research Center (BARC) in Mumbai, various State Agricultural Universities (SAUs) and the Indian Council of Agricultural Research (ICAR) institutes are among the country’s key research centres involved in crop improvement using induced mutations and have made substantial contributions by developing and disseminating of a large number of mutant varieties. More than 386 mutant varieties of 62 crop species are developed through induced mutagenesis (Fig. 2).

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171

76 60

China Japan

176 1030

180

India Russian Federation United States

209

South Korea Netherlands

216

Germany Bulgaria 386

479

Bangladesh

Fig. 2  Number of mutant varieties released in top 10 countries of the world

A total of 82 mutated varieties were released of cereals, followed by pulses (63) and oilseeds (55). The maximum number of mutant varieties were released in ornamentals (112) followed by other horticultural (21) and vegetable crops (15) (Jagadeesan & Ganapathi, 2021). The Mumbai-based (BARC) has been engaged in mutation breeding since the early 60s using physical mutagens like X-rays, beta particles, gamma rays, and electron beams. BARC has developed more than 49 varieties of different crops with improved traits in association with SAU’s and ICAR institutes (Badigannavar et al., 2020). The important cereal grown in India is rice, wheat, maize, sorghum, and pearl millet. In India, rice is the most important food crop and is ensuring food security for underprivileged people, reducing poverty and improving livelihoods through increased agricultural incomes. In India, induced mutations have been successfully used in the rice improvement which has led to the development of more than 42 high-yielding rice mutant varieties. The gamma radiation-­ induced rice mutant varieties of PNR series, IIT-48, IIT-60, K-84, Jagannath, Keshari, and Sattari released in India are among the most significant varieties of economic importance (Chakrabarti, 1995). The early maturing, aromatic mutant rice varieties, PNR-381 and PNR-102 were very popular among farmers and generated annual income worth 1748 million US dollars (Chakrabarti, 1995). Recently four high-yielding mutant rice varieties ‘TCDM-1’, ‘TKR’, ‘Kolam’, ‘Vikram-TCR (Trombay Chhattisgarh)’, and ‘CG Jawaphool Trombay’ have been developed by BARC and released for cultivation in Chhattisgarh and Konkan region of Maharashtra states of India. The varieties ‘TCDM-1’, ‘TKR’, and ‘Kolam’ are having dwarf plant stature and are tolerant to lodging and mature in 130–140 days. The varieties are having better milling and head rice recovery resulting in higher grain yield. ‘Vikram-TCR’ is dwarf, non-lodging, non-shattering, drought tolerant and high yielding, with long slender grain, and better-puffed rice-making quality.

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All these varieties are becoming very popular among the farmers of Chhattisgarh and the Konkan region of Maharashtra states of India. Basmati rice, unique aromatic rice from the Indian subcontinent, is one of only a few rice varieties with a distinct name that is traded internationally. Basmati rice is defined by its origin and by genotype known for its pleasant aroma, long slender kernels which elongate linearly by nearly twice their original length at the time of cooking. The traditional Basmati rice varieties are low-yielding with late maturity and lodging susceptibility and respond poorly to fertilizer application. To address these undesirable features, mutant variety ‘CRM 2007-1’ was developed from traditional Basmati rice variety Basmati 370 through gamma rays for the North Western Region of India by Central Rice Research Institute, Cuttack, India. It is semi-dwarf, early, and high yielding and gave a yield of 6.2 t/ha in the farmers’ fields of Punjab and Haryana. Based on its better performance in Orissa state, a non-traditional Basmati area, CRM 2007-1 was released under the name ‘Geetanjali’ for commercial cultivation. The variety was widely accepted by the farmers and consumers and gave higher economic returns to the farmers in the region (Patnaik et al., 2006; Rao et al., 2011). Wheat is the second important cereal crop grown in India and the success of the Green Revolution was the cultivation of the Mexican wheat strains. However, the Mexican wheat strains had red hard grains and were not accepted by Indian farmers and consumers. In order, to alter the grain colour, a mutation breeding programme was started and amber grain colour mutant varieties Sharbati Sonara and Pusa Lerma were developed from the variety Sonora 64 and variety Lerma Rojo 64-A, respectively. The mutant variety Sharbati was cultivated on large acreage because of its early maturity coupled with the desirable grain colour (Chopra, 2005). A high-­ yielding dicoccum wheat mutant variety ‘HW 1095’ having semi-dwarf stature, disease resistant, and nutritionally rich was developed by the Indian Agricultural Research Institute, Regional Station, Wellington using mutation technique. The variety was released for non-traditional areas of Tamil Nadu state of India for commercial cultivation and is becoming popular among the farmer’s community (Nirmalakumari et al., 2010). Pulses are an important source of protein for India’s large vegetarian population and they constitute a unique and essential component of the diet by supplementing the basic cereals with proteins, essential amino acids, vitamins, and minerals (Goyal et al., 2020a, b; Raina et al., 2020a, b). In India, induced mutagenesis, either alone or in combination with hybridization, has resulted in the release of 56 mutant crop varieties in pulses. Almost 50% of these mutant varieties have been developed and bred at BARC, Mumbai (Jagadeesan & Ganapathi, 2021). The mutation breeding programme at BARC helped in the release of eight varieties in mungbean (TAP-7, TARM-1, 2, 18, TMB-37, TJM-3, TM-96-2, and TM-2000-2) five each in urdbean (TAU-1, TAU-2, TPU-4, TU94-2, and TU-40) and pigeon pea (TT-6, TAT-10, TT-401, TJT-501, PKV-TARA) and two in cowpea (Khalleshwari (TRC-77-4, TC 901). The black gram variety TAU-1, developed in 1985 by BARC is still the most popular variety and is still cultivated on large acreage throughout Maharashtra state and occupies more than 50% of the area under black gram cultivation. The black gram YMV-resistant variety TU 94-2 and PM-resistant variety TU 40 is widely

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cultivated in the southern states. In Asia, BARC developed the first powdery mildew (PM)-resistant mutant mungbean variety TARM-2 through induced mutagenesis and subsequently, a series of PM-resistant mungbean varieties (TARM-1, 2, 18, TMB-37, TJM-3, TM-96-2 and TM-2000-2) were developed by using PM-resistant mutant. The mungbean mutant variety ‘TMB-37’ known for earliness and suitability for summer cultivation is widely cultivated by farmers throughout the country. The mungbean varieties TM96-2 and TM 2000-2 occupy large areas under rice fallows. The pigeon pea mutant variety TJT-501 is one of the leading varieties receiving more than 12% of national breeder seed indent and occupies almost 60% of the area under pigeon pea in the Madhya Pradesh state of India. The other pigeon pea varieties TT-401 and PVK-TARA are gaining popularity among farmers of southern and Maharashtra states respectively. Other pulses ‘MaruMoth-1’ of moth bean, ‘Co-4’ of black gram, Ajay, Atul, Girnar, Kiran, and Pusa-547 of chickpea are of economic importance (Jagadeesan & Ganapathi, 2021). Different oilseed crops are grown in India and the oilseed sector plays a significant role in the country’s agricultural economy. At BARC, 15 mutant Trombay groundnut (TG) varieties (TG 1, 3, 17, 22, 26, 37A, 38, 47, 51, TGS 1, TAG 24, TKG 19A, TPG 41, TLG 45, TBG 39) are developed for cultivation in different states of the country (Badigannavar et al., 2020, 2021). Most of the TG varieties are widely grown and are very popular among the farming community and have made an excellent impact on Indian agriculture. The varieties TAG 24, TG 37A, 38, 39, 51, TPG 41, and TLG 45 have become popular in different states of India and farmers have been able to achieve high yields of up to 5000–7000 kg/ha and profits up to 1200 US dollars/ha were earned by the growers. The variety TAG-24 is the most popular variety and commands a major share of the national breeder seed indent. TAG 24 was released in the year 1992 but is still used as a check variety in the national trials and is grown throughout the country. Almost 700 tonnes of breeder seed of TG varieties were produced and supplied by BARC to State Seed Corporations, National Seed Corporations, State Agriculture Departments, State Agricultural Universities, ICAR institutes, NGOs, seed companies, and farmers. The other oilseed mutant varieties are mustard, soybean, sunflower, and linseed developed by BARC is also popular among the farmer’s community. Recently, high yielding, low-linolenic acid mutant variety TL99 was developed by BARC and released for commercial cultivation. TL 99 has unique fatty acid composition having very low-linolenic acid (2–5%) and can be used as edible oil (Manjaya et al., 2020). 3.1.4 Bangladesh The Bangladesh Institute of Nuclear Agriculture (BINA), since its inception in 1961 has released more than 60 plant mutant varieties. The mutants of rice, wheat, lentils, chickpeas, peanuts, mustard, sesame, soybean, jute, and tomato account for about 8% of its total crop area, improving the socio-economic status of the farmers. In Bangladesh, about 40% of cultivable land is saline or prone to drought,

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submergence, or excesses of cold or heat which affects rice production. Rice being a staple food of Bangladesh, BINA has released improved varieties of rice through the induced mutation technique. Three rice mutant varieties Binasail, Iratom-24, and Binadhan-6 are grown on an estimated area of about 795,000 ha and have a high impact on food security in Bangladesh. The rice variety Binadhan-7 is released in 2007; it gives yields up to 3.5–4.5 tonnes per hectare and matures in around 115 days. The variety is extensively grown in the northern part of the country and provides income to the farmers and employment to 20% of people living in the northern region of Bangladesh (Azad et al., 2012; Jawerth, 2017). The new mutant varieties of rice have helped Bangladesh to increase its rice production three-fold in the last few decades. 3.1.5 Pakistan The induced mutation studies were initiated in Pakistan at the Nuclear Institute for Agriculture and Biology (NIAB) for the improvement of important food and fibre crops. A total of 59 mutant varieties in different crops, such as cotton (12), mung bean (11), rice (10), chickpea (9), wheat (6), lentil (3) oilseed brassica (3), mustard (1), sesame (2), groundnut (1), castor bean (1), and mandarin (1) were developed. The cotton mutant varieties ‘NIAB 78’ covered 80% of the cotton area in Punjab and Sindh provinces. The four improved mutant varieties of rice have improved the socio-economic conditions of farmers and the 43 mutant varieties developed by NIAB generated a profit of US dollars 6 billion in 2018 (Haq, 2009). 3.1.6 Vietnam In Vietnam, the induced mutation technique was successfully used for crop improvement, and 58 crop varieties predominantly rice (36) have been released to farmers. The other mutant varieties are soybean, groundnut, maize, chrysanthemum, Indian jujube, and field mint making significant contributions to national food security (Sarsu et al., 2020). In Vietnam, mutation breeding for crop improvement resulted in the release of 45 rice mutant varieties which are making significant contributions to the national food security. All the mutant varieties have well-supported food security and poverty alleviation in the country since the 1970s, generating profits of hundreds of millions of dollars for the farmers every year. The productivity of rice mutant varieties is around 6–8  t/ha and has helped Vietnam to become a rice exporter from 1980 to-1990s. The mutant varieties DT10 and VND95-20 originated from IR64, and are been cultivated in more than 1 million ha and 900,000 ha areas in the rice-growing belt of Vietnam. The mutant varieties TNDB-100, VND95-19, VND99-3, VN212, VN214, OM2717, and OM2718 are also grown in a large area. These rice mutant varieties have 10–20% higher yields in comparison to the parent varieties, with lodging resistance, tolerance to acid soils, salinity, tolerance to biotic stress, short duration, and better nutritional quality. Two early maturity mutants

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TNDB-100 and THDB with improved grain yield are extensively grown in the Mekong Delta region of Vietnam (Vinh et al., 2009; Do, 2009; Ham & Xuan, 2018; Khanh et al., 2021). Along with rice, soybean is one of the important traditional food crops and plays an important role in providing food for humans and livestock in Vietnam. Since the late 1980s, ten mutant varieties have been released and adopted by farmers. The most popular variety DT84 occupies more than 50% of the total cultivated area in Central and North Vietnam. Even after 20 years of release, no other soybean variety could overcome the popularity of DT84. The other popular soybean mutant varieties are DT90 and DT2008 and they occupy about 5000  ha/year and 10,000  ha/year areas respectively. The five leading mutant varieties are generating an income of US$ 3.3 billion and are improving the farmer’s income. The mutant varieties of soybean alone fetched US$ 3 billion thereby benefiting about 3.5 million farmers (Le & Pham, 2021). 3.1.7 Malaysia The mutation breeding programme first started in Malaysia in the year 1984 to develop Semi-Dwarf Mutants for Rice. The Malaysian Nuclear Agency having excellent research facilities in plant mutation breeding and biotechnology developed 53 mutant varieties which include rice (19), groundnuts (2), banana (1), orchids (6), chrysanthemums (7), hibiscus (3), roselles (3), and other ornamental and landscaping plants (12). Blast-tolerant rice mutant varieties MR219-4 and MR2199 having adaptation to different soil moisture conditions were released for commercial cultivation. High-quality rice MRQ74, commonly known as Mas Wangi, has a fragrance that was developed by cross-breeding and is grown on 1000 ha. The other success story is the release of the banana mutant variety ‘Novaria’ the most popular mutant variety from Malaysia which was planted in an area of about 566 ha. The farmers grew the mutant groundnut varieties KARISMA Sweet and KARISMA Serene resistant to Cercospora leaf spots on large areas. As evidenced by new varieties of orchids, hibiscus, chrysanthemums, and pot and landscaping plants such as petunias, cannas, etc., mutation breeding is especially successful in Malaysia (Ibrahim, 2018). 3.1.8 Thailand The experiments on induced mutation studies on rice were initiated in Thailand in the year 1965. Two aromatic indica glutinous rice varieties RD6 and non-glutinous early maturing variety RD15 derived from gamma-irradiated progeny of the popular rice variety ‘Khao Dawk Mali 105’ (‘KDML 105’), were released in 1977 and 1978 respectively. Both the varieties are grown extensively in the North and North-­ Eastern regions of Thailand and had played an important role in generating revenue worth billions of US dollars (Ahloowalia et  al., 2004; Kharkwal & Shu, 2009).

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The mutant variety RD6 was cultivated on 2,429,361 ha and occupies 26.4% of the cultivated area under rice in Thailand. 3.1.9 Myanmar In Myanmar, rice is the main crop cultivated on different agro-ecosystems. After the initiation of the mutation breeding programme in 1970s, four mutant rice varieties ShweThwe Tun (a mutant of IR 24), Shwe War Tun (a mutant of IR 24), Thukayin (a mutant of Manawthuka), and Yezin Lone Thwe (a mutant of Lone Thwe Hmwe) were officially released. The second-largest rice variety grown in Myanmar was Shwewartun and was cultivated on 17% of the total cultivated area under rice in Myanmar (Khin, 2006). 3.1.10 South Korea In Korea, the research on induced mutation studies was started at the Atomic Energy Research Institute, Rural Development Administration (RDA), and other universities in the early 1960s. In Korea, around 180 new mutant varieties have been developed and are officially registered as a result of extensive research. Fifteen sesame mutant varieties occupied 55% of the total cultivable land in Korea. New mutant varieties of flowers and ornamental plants have rapidly increased in area and are currently being commercialized by private companies and breeders on a larger scale (Kang et al., 2020). 3.1.11 Sri Lanka In Sri Lanka, mutation breeding was started by the Department of Agriculture (DOA) in the year 1960. The first drought-tolerant rice mutant variety MI 273 (a mutant of the H-4 variety) was released for general cultivation in 1971. Another rice mutant variety BW 372 moderately tolerant to blast, bacterial leaf blight, brown planthopper, gall midge, and iron toxicity was released which helped in increasing rice productivity by 3–4 t/ha in soils with iron toxicity. The groundnut mutant variety Tissa developed by gamma rays is the most popular variety and 80% of groundnut area is under this variety. The sesame mutant variety Malee (a gamma-ray mutant of MI-3) resistant to Phytophthora blight is also popular among the farmers because of its high yield (1.1–1.8 t/ha) (Parasuraman & Weerasinghe, 2021). 3.1.12 Indonesia In Indonesia, three sorghum mutant varieties Pahat, Samurai-1, and Samurai-2, were released for cultivation. The grain sorghum mutant varieties Pahat and Samurai-2 are been grown for food and sweet sorghum mutant Samurai-1 for sugar

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syrup production and bioethanol in Indonesia (Human & Indriatama, 2020). All the three mutant varieties Pahat, Samurai-1, and Samurai-2 are extensively cultivated on an estimated area of about 800,000 ha.

3.2 Europe The mutation breeding technique has become one of the important breeding methods since its initiation in the year 1920. The extensive use of the mutation breeding technique resulted in the release of 959 mutant varieties, particularly in wheat and barley (Sarsu et al., 2020). 3.2.1 Bulgaria In Bulgaria, experimental mutagenesis work started in 1950 using physical and chemical mutagenesis. The extensive use of the mutation breeding technique resulted in the development of 76 new cultivars in different crops like maize (26), durum wheat (9), tomato (6), barley (5), wheat (5), soybean (5), lentil (4), pepper (4), sunflower (3), bean (2), tobacco (2), chickpea and vetch (2), cotton (2), and pea (1). The maize mutant hybrid varieties Kneja 509 and Kneja 683A occupy 40–50% of the total cultivated maize growing area in Bulgaria. The high protein mutant hybrid Kneja 556, Kneja HP 633, Kneja HP 556, and Kneja МHP 556 are well known for silage making in the milk-corn belt areas of Bulgaria. In the 1980s, nine durum mutant varieties were developed through mutation breeding and significantly increased the productivity of durum wheat. All the wheat mutants are grown in the durum wheat-growing area and the variety Gergana has become the leading variety occupying up to 50% of the total cultivable area. In Bulgarian agriculture, all the mutant varieties have attained a special status because of their high productivity, resistance to biotic stresses, and better quality (Yanev, 2006). 3.2.2 Italy Italy is well known for basic and applied durum wheat research and production among the European Union (EU) countries. The application of the mutation breeding technique resulted in the development of 22 varieties including 6 direct selections [76]. The most important variety Creso was developed from this programme in 1974 and was cultivated on one-third of the durum wheat area in Italy and contributed to additional production of 0.9 t/ha. The variety has a significant impact on the Italian economy contributing to US$ 1.9 billion (Scarascia-Mugnozza et  al., 1991; Ahloowalia et al., 2004; Sarsu et al., 2020).

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3.2.3 Czechoslovakia The gamma-ray mutant-induced cultivar of barley ‘Diamant’ and ‘Golden Promise’ was officially released in Czechoslovakia in 1965. Both the varieties have made a major impact on the brewing industry in Europe and were used as parents in the hybridization and more than 150 cultivars were developed. Both the varieties have added billions of dollars to the value of the brewing and malting industry (Kharkwal & Shu, 2009).

3.3 North America In the USA, the gamma-ray-induced mutation experiments resulted in the development of the first semi-dwarf rice mutant variety Calrose 76 in California in the year 1977. In total, 209 mutant varieties have been developed in the USA. The mutant variety Calrose 76 was used in the cross-breeding programme and 25 semi-dwarf varieties were developed in California (13), Australia (10), and Egypt (2) (Rutger, 2008). A high-yielding wheat mutant Stadler having early maturity, resistant to loose smut, and leaf rust was released in Missouri. The wheat mutant was once cultivated on 2 million acres annually in the USA. The high-yielding barley mutant variety Luther was cultivated on 120,000 acres annually in the USA. Another high-­ yielding winter barley mutant variety Pennrad was released in Pennsylvania and was grown on more than 100,000  ha in the USA.  Three bean mutant varieties Sanilac, Gratiot, and Sea-way released in the USA were also popular and were grown on more than 87,000 ha and 160,000 ha respectively. Thermal neutron mutagenesis was used to develop grapefruit mutant cultivars Star Ruby and Rio Red. In Texas, both varieties are grown in 75% of the grapefruit planting area (Ahloowalia et al., 2004).

3.4 Latin America The mutation breeding programme in 18 Latin American countries got good support from IAEA and developed 53 mutant varieties in different crops for cultivation (Sarsu et al., 2020). In Argentina, rice mutant variety Puita INTA-CL covered 40% of the rice production area and was also successfully grown in other Latin American countries, such as Uruguay, Colombia, Chile, Costa Rica, Panama, Dominican Republic, Nicaragua, and Honduras. The mutant variety has contributed significantly to the economies of Latin American countries (Livore et al., 2018). The groundnut mutant variety has a high yield and oil occupies more than 80% of the groundnut area in Argentina.

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In Brazil, the induced mutation technique has been effectively used for the rice improvement programme and developed high-yielding herbicide-resistant rice mutant variety SCS118 Marques. Another herbicide-resistant rice mutant cultivar Clearfield rice was registered in 2002 (Croughan et al., 1996) and was cultivated on 1.1 million ha of arable land. The variety has increased the yield by 50%. Cuba has successfully applied induced mutation experiments to genetically improve crops and released 21 varieties of hibiscus (3), sugarcane (4), tomato (3), rice (9), and soybean (2) for cultivation. The high-yielding and salt-tolerant rice mutant variety LP7 and tomato mutant varieties Maybel and Domi have significantly increased yields under drought conditions (Gonzalez et al., 2008; González-­ Cepero et al., 2020). In Peru, the mutation breeding work for the crop improvement started in 1978, and barley mutant variety ‘UNA La Molina 95’ was released in 1995 for its earliness, naked grains, and higher protein content. In 2002, another barley mutant variety Centenario was released and is cultivated at high altitudes up to 5000 m above sea level. Both the varieties are contributing significantly to the food security of the country (Gomez-Pando et al., 2009; Gómez-Pando et al., 2020).

3.5 Africa In total, 82 mutant varieties are released in different countries in Africa (Sarsu et al., 2020). In Egypt, semi-dwarf mutant rice varieties viz., Giza 176 and Sakha 101, were developed during the 1990s, which increased yield level from 3.8  t  ha−1 to 8.9 t ha−1. The mutant variety Giza 176 is cultivated as the most popular and promising variety having a yield of 10 t ha−1. Five high yeilding and better quality sesame varieties and two safflower varieties were developed in Egypt (Sarsu et al., 2020). In Ghana mutagenesis has helped to develop Africa Cassava Mosaic Virus (ACMV) tolerant cassava mutant variety having high dry matter content (40%) and used in preparing famous traditional dishes (Kharkwal & Shu, 2009). Similarly, the four sorghum and seven high yielding and drought tolerant cowpea varieties have been developed in Namibia (Horn et  al., 2018). In Sudan, mutagenesis have been employed to increase the yielding potential bananas, tomatoes, and groundnuts under stressful environments. A high yielding banana variety viz., ‘Albeely’ and a drought-tolerant peanut mutant variety, viz., ‘Tafra-1’, have been developed that ease the financial burden of Sudanese farmers (Sarsu et  al., 2020; Abdalla et al., 2018).

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3.6  Australia and Pacific Ten varieties have been released in Australia and the Pacific, consisting of nine rice varieties (Amaroo, Bogan, Echua, Harra, Illabong, Jarrah, Langi, Millin, and Namaga) and a lupine mutant variety named Tanjil-AZ-33. The rice mutant variety Amaroo was widely cultivated, covering 60–70% of the rice cultivation area in Australia (Kharkwal & Shu, 2009).

4  Next-Generation Mutagens For the last 90 years, a variety of physical and chemical mutagens have been widely utilized to induce mutations in plants, resulting in the release of 3332 mutant varieties of commercially cultivated crops, including cereals, pulses, oil, root and tuber crops, and ornamentals (Fig. 3). Physical mutagens, such as gamma rays, X-rays, and neutrons, have been extensively employed to induce mutations, with over 70% of mutant varieties being developed through physical mutagenesis. The sustainability of agriculture and food security is threatened by challenges such as shrinking cultivable land, limited water resources, and environmental adversities associated with climate change. With the world’s population expected to reach 9.9 billion by 2050, there is a need to increase agricultural production by 70% (PRB, 2018). Therefore, agricultural research should prioritize increasing production to meet the growing demand for food globally. Agricultural research needs to be focused to increase crop production to meet the world food demand. Modern technology like gene editing or molecular breeding techniques based on a few genes will not help to improve complex quantitative traits. In such conditions, mutation breeding research needs to be strengthened by using new mutagens like accelerated particles, such as heavy ions or protons (Tanaka et al., 2010). They induce excellent biological mutagenic effectiveness at relatively low radiation doses compared with gamma rays and X-rays because accelerated particles with high linear energy transfer (LET) cause high-density ionization, causing a large amount of damage to DNA in a small area resulting in more variation with excellent traits. The radiation mutagenesis technology based on advanced particle accelerators originated in Japan in the 1990s and was used for the improvement of ornamental plants, and new mutant cultivars were developed. The research on variety improvement using accelerated particles started in the twenty-first century. The space environment contains radiation, microgravity, and alternating magnetic fields. In space breeding, the mutation frequency can reach up to 10%. Microgravity and radiation in space affect living organisms. Solar cosmic rays and galactic cosmic rays produce radiation that causes mutations. Many new germplasm resources have been created and a large number of new plant varieties have been released in China using space breeding technology (Ma et al., 2021).

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18% 16%

Ornamental crops Cereals

24%

6% 4%

Pulses Oilseeds Horticultural crop Vegetable crop

32%

Fig. 3  Number of mutant varieties released in India

5 Contribution of Joint FAO/IAEA Division in Popularizing Mutation Breeding Since 1964, the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture has supported financially numerious projects aiming at developing mutant varieties with preffered set of traits. The IAEA provides assistance for Technical Cooperation Projects (TCPs) aimed at integrating plant tissue culture techniques, advanced molecular methods, and induced mutations into national plant breeding and conservation programmes through Coordinated Research Projects (CRPs). This support helps to characterize plant genetic resources, expand plant genetic diversity, and identify and introduce commercially useful traits for agriculture. As a result of the programme’s efforts, there was progress and increased interest in the field of mutation breeding, leading to the creation of superior varieties in both developed and developing nations. The FAO/IAEA recognized the contributions of different member countries to plant mutation breeding and awarded outstanding awards in 2014 and 2021. The outstanding achievement award was given to many countries for their contribution to developing and popularizing the mutant varieties (IAEA Newsletter, 2021).

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6 Conclusion Mutation breeding is one of the tools in the hands of plant breeders to improve crop plants. The main objective of induced mutations is to create genetic variability and rectify the lacunae in the well-adapted variety. Plant breeders rely on genetic diversity to develop new and improved varieties with desirable traits by means of hybridization, recombination and mutations (Spontaneous or induced). The spontaneous mutations are not adequate for their utilization in plant breeding programmes because of less frequency and the frequency can be increased by using different mutagens. The widespread use of induced mutants in plant breeding programmes throughout the world has led to the official release of more than 3332 mutant varieties that have been registered and released for commercial cultivation in different parts of the world. The mutant varieties developed are having improved agronomic traits, increased yield, and improved quality and resistance to biotic and abiotic stresses, and some of the mutant varieties have immensely helped to increase the productivity and economy worldwide. Among the mutant varieties, some classical success stories that have made a major contribution and economic impact include rice varieties in Australia, Bangladesh, China, and Thailand, groundnut, pulses and ornamentals in India, soybean varieties in Vietnam, cotton in Pakistan, Japanese pear in Japan, grapefruit in the USA, barley varieties in Europe, durum wheat in Italy, sunflower in the USA, sorghum in Mali, and peppermint in the USA.  The positive impacts of mutant varieties can be seen on food security and farmers’ livelihoods in many countries of the world. Many more mutant varieties are successfully grown throughout the world but the information about the acreage and production is not documented or the varieties are not registered. In many countries, the supply of seeds is not carried out through the proper channel and the seed of mutant varieties disseminates from farmer to farmer without proper documentation. It is sure there may be many more success stories about the mutant varieties in different parts of the world. However, the world is still facing a food and energy crisis of unprecedented proportions and therefore induced mutations programme needs to be accelerated for crop improvement. A large number of breeding traits are complex quantitative traits, and gene editing or molecular breeding techniques based on a few genes are not suitable for the improvement of quantitative traits. In such conditions, mutation breeding research needs to be strengthened by using new mutagens like accelerated particles, such as heavy ions or protons. In space breeding, the mutation frequency can reach up to 10% and should be exploited to induce novel mutations. In the future, mutation breeding will continue to be the leading plant breeding technique for crop improvement and taking care of food security.

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Mutation Breeding: Protocol and Role in Crop Improvement Abdulwahid A. Saif

Abstract Mutation breeding is a well-established breeding strategy for crop improvement in enhancing the quality and quantity and resistance to biotic and abiotic stresses of important food crops. In mutation breeding, various physical and chemical mutagens have been used to accomplish the goals of crop improvement. Numerous research articles have been published on the role of mutation breeding in crop improvement. However, very little is known about the steps involved in the mutation breeding strategy. In this chapter, I reviewed the protocol and important aspects of mutation breeding, emphasizing selection techniques for disease and drought-resistant mutant lines. Keywords  Mutation breeding · Physical mutagens · Chemical mutagens · Selection · Screening

1 Introduction The discovery that physical and chemical mutagens could induce genetic alterations in organisms opened a new strategy for crop improvement. Plant breeders have exploited this capability of mutagens to develop desired varieties in different crops such as cowpea (Raina et al., 2018a; Rasik et al., 2022), lentil (Laskar et al., 2018a, b; Wani et al., 2021), faba bean, fenugreek (Hasan et al., 2018), mungbean (Wani et al., 2017), urdbean (Goyal et al., 2019a, b), chickpea (Laskar et al., 2015; Raina et al., 2017), black cumin (Tantray et al., 2017; Amin et al., 2019), and finger millet (Sellapillaibanumathi et al., 2022). Induced mutagenesis is the most efficient technique to increase genetic variation in a short period of time and has been employed in various crops. Because natural mutations occur sporadically, artificial mutations are generated, and genetic gain is best achieved using mutagens  (IAEA, 1961; Muller, 1927; Raina & Khan, 2020). Mutation breeding has proven a coherent tool A. A. Saif (*) Agricultural Research & Extension Authority (AREA), Sana’a, Yemen © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_4

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for the improvement of food crops (Khursheed et al., 2015; Goyal et al., 2020a, b; Raina et  al., 2022a, b, c, d; Rasik et  al., 2022; Sellapillai et  al., 2022; Sellapillaibanumathi et  al., 2022). Different physical and chemical mutagens in varying doses have been employed to achieve the aims of crop improvement program. Hence, mutation breeding could complement and supplement existing cultivars that have low-yielding potential and are susceptible to environmental stress. Mutagen treatments may create a novel combination of alleles, increase the genetic variability, and modify gene linkages (Laskar et al., 2019). By virtue of this mutation, breeding is widely used for improving a single trait without altering the entire genetic constitution, unlike genetically modified crops that lack social acceptance and face strong criticism. Mutation Breeding has been successful in the development and official release of thousands of mutant varieties that belong to hundreds of plant species (Raina et al., 2016; Khursheed et al., 2018a, b). Such mutant varieties are being cultivated on millions of hectares of land and generate billions of dollars (Raina et al., 2021; Khursheed et al., 2018c). Almost all plants belonging to different usage categories such as pulses, cereals, vegetables, fruits, oil crops, ornamental, and medicinal plants have been mutated. Till now, 3604 mutant varieties have been developed with one or more improved characters.

2 History of Mutation Breeding The history of mutation breeding dates back to the beginning of the twentieth century when the first artificial genetic alteration was claimed, but experimentally it was proven only in the late twenties by Muller and Stadler using X-rays as a mutagen (Muller, 1927). Although Muller, as an entomologist, assumed that induced mutations could be pivotal in the crop improvement programs, Stadler being a plant breeder became rather dubious about such predictions on observing deleterious effects of mutations in maize and barley (Stadler, 1928a, b). Among the pioneer researchers who employed mutagenesis for improving mildew resistance in barley were Freisleben and Lein in Halle, Germany. Later they also developed a practical mutation breeding protocol; however, such work remains unattended due to the World War II crisis (Freisleben & Lein, 1942a, b). In the meantime, Sweden plant geneticists such as Nilsson-Ehle, Gustafsson, Hagberg, Gelin, and Nybom continued to work with mutation breeding using X-rays and evaluated the optimum doses, mutation frequency, and spectrum (Gustafsson, 1947). Further comparative studies on X-rays effects and ethylenimine were also carried out systematically. Even though the work was fundamental without a detailed information on important aspects of mutation, yet few important mutants were developed in barley, wheat, oats, pea, soybean, flax, mustard, and grape (Sigurbjornsson & Micke, 1974; Linqing, 1986; Wang, 1996; Shu et  al., 1997; Bhatia et al., 1999; Maluszynski et al., 2009). By 1950, mutation breeding gained momentum and started in other countries including the USA, Italy, USSR, the Netherlands, and Japan. Initially, gamma rays

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were employed using 60Co as source of gamma rays or neutrons fitted in newly developed nuclear reactors. However, plant breeders were challenged with the dearth of literature on important aspects, namely, radiation dose and duration. For more than a decade, continuous efforts were put to establish a working protocol with details on optimum doses, treatment duration, and precautionary measures. Meanwhile, factors that could influence the outcome of mutation breeding were identified and included water, oxygen, and time; however, their deliberate control only brought about quantitative differences, without any substantial role in useful methodology (Scarascia-Mugnozza, 1965; Sigurbjornsson & Micke, 1969; Saeed Iqbal et al., 1991; Laskar et al., 2019). In the meantime, developing countries particularly in Asia started using mutation breeding as a main strategy for crop improvement. This led to the development of new rice varieties with desired attributes and wide acceptance. In the initial stages, few mutagens such as X-rays were known; however, as the work in mutation breeding intensified, new mutagens were discovered and used to induce mutations. The remarkable efforts put forth by Joint FAO/IAEA Division in 1969 and the publication of the first edition of the Manual on Mutation Breeding were pivotal in the establishment of mutation breeding for crop improvement. The rigorous training organized by FAO/IAEA equipped the breeders with the new techniques required to conduct successful mutation breeding. Even today FAO/IAEA is organizing such training to facilitate the mutation breeding across the world (IAEA, 1965). Therefore, mutation breeders consider 1969 as the birth year of mutation breeding as a new strategy to develop high-yielding mutants with other desired traits such as improved quality and resistance to environmental stresses (IAEA, 1969).

3 Physical Mutagens The mutation breeding used as a crop improvement technique gained momentum at a fast pace and also delivered outstanding products in the form of elite mutant cultivars that are cultivated on a million hectares of land and generate billions of dollars (Awan, 1991; Maluszynski et al., 1999). In this process, several mutagens, namely, ultra-violet, X and gamma rays, alpha and beta particles, and protons and neutrons have been recognized and made available to the plant breeders to employ them in different crop improvement programs. These mutagens are able to interact with the tissues while interacting deposit energy that varies in amount among the mutagens (Maluszynski et al., 1995; Raina et al., 2022a; Goyal et al., 2021a, b). Further, each mutagen is unique in inducing mutation, degree of biological damage, and mechanism of interaction with the tissues (Kawai & Amano, 1991). For instance, X-rays are electromagnetic waves with high energy, short wavelength, and high penetrance which are able to pass through many materials opaque to light. Similarly, gamma rays possess high energy (more energy per photon than X-rays), shorter wavelength and. Gamma radiation is usually obtained from radioisotopes such as Cobalt-60 and cesium-137. Ultraviolet radiations are also electromagnetic waves with energy and

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frequency beyond violet-colored visual light. Ultraviolet light has limited penetration and is used primarily to treat spores and pollen grains. Besides rays, subatomic particles like neutrons, one of the primary constituents of atomic nuclei, do not have any net electric charge; however, they have mass slightly greater than that of a proton. Neutrons are also capable of inducing mutations and have been exploited in the crop improvement programs. Whole plants are most easily irradiated in gamma field or gamma gardens. However, seedlings can easily be irradiated by X-rays or by gamma sources in a greenhouse or shielded rooms. Moreover, seeds are the most preferred plant material for irradiation due to easy maintenance and cost-effective treatment compared to other materials (Micke et al., 1987; Rutger, 1992; Khursheed et al., 2016). Seeds can be maintained for longer durations at room temperature and do not require any sophisticated system for storage. In the dry form, seeds are biologically inert and least affected by severe environmental conditions and easy to handle in transportation. However, high radiation doses are required to produce sufficient genetic effects than other plant materials irradiated. Besides seeds, pollen grain can also be mutagenized in breeding programs that aim at producing chimeras (mixture of genetically different tissues). The frequency of chimera formation is more in pollen grains compared to seeds.

4 Chemical Mutagens Initially, mutation breeding was tilted toward the physical mutagens however, at present chemical mutagens are equally used, and in some cases, preferred over the physical mutagens (Khursheed et  al., 2017; Raina et  al., 2022d). The number of chemical mutagens is rising and new chemicals are being reported capable of inducing mutations. Chemical mutagens have been classified on the basis of type of mutation and mode of action. However, class alkylating agents including mutagens such as ethyl methanesulphonate (EMS), diethyl sulphate (dES), ethyleneimine (EI), ethyl nitroso urethane (ENU), ethyl nitroso urea (ENU), and methyl nitroso urea are effective in inducing higher frequency of mutations. Another class including mutagens such as sodium azide is one of the most effective mutagens. Using chemical mutagens must be taken into consideration a dose/concentration of LD50 allowing the treated material to produce M2 progenis (Salnikova, 1993).

4.1 Treatment Procedures Several plant propagules, namely, seeds, cuttings, and floral buds are often treated either in the dormant state or in the active metabolizing stage (Awan, 1999). In the methodology, seeds, buds, and dormant cuttings may be dipped in a mutagenic solution. A shallow cut is often made in the plant stem and then dipped in the mutagenic solution to facilitate the absorption of mutagen from a test tube or from a piece of

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cotton saturated in the chemical agent. In another method, a mutagen is added in low concentrations to the growth medium containing plantlets. This method is used to study chronic mutagen exposures and sensitivity of plantlets at different growth stages. However, the methodology used to mutagenize pollen grains is slightly different. Pollen in a monolayer is exposed to the mutagen vapors in a closed humid chamber. The success of chemical mutagens is often determined by the optimum dose that intern depends on several factors such as selection of mutagen concentration, treatment duration, and to some extent temperature during treatment (Raina et  al., 2020a, b).

4.2 Concentration The optimum concentration of a mutagen is one of the most important factors that determine the outcome of mutation breeding (Raina et al., 2022e). Initially, high concentrations of mutagen were applied to dormant seeds at room temperature for long periods of time (12–24 h). This often resulted in increased seedling injury and reduced plant survival. Therefore, mutagens at lower concentrations for longer periods at lower temperature revealed high mutation frequency with insignificant seedling injury.

4.3 Duration To ensure proper hydration and diffusion of mutagen inside the plant tissues longer treatment duration usually 6–9 h is must (Raina et al., 2018b). However, in case of pre-soaked seeds, treatment duration could be reduced as mutagen uptake is fast in hydrated seeds compared to dry and dormant seeds. Pre-soaking initiates metabolic activities and renders cell membranes more permeable to the mutagens.

4.4 Temperature The temperature of the mutagenic solution impacts the outcome of mutation breeding and has a great influence on the mutagenic chemicals, while the rate of diffusion of the chemical is very little affected by it (Scarascia-Mugnozza et al., 1993). Even though the diffusion rate of mutagen remains unaffected by the temperature, however, at lower temperature hydrolysis rate decreases, mutagens remain stable and interaction with the tissues lasts for longer duration. Therefore, it is better to maintain 20–25 °C while treating pre-soaked seeds. These conditions expedite the mutagen absorption, enhance seed metabolism and interaction duration between the mutagen and plant material.

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4.5 Post-treatment Washing Few precautionary measures must be adopted to achieve a significant reduction in post-treatment damage. After mutagen treatment seeds must be washed under running water for 30 minutes to get rid of excess mutagen adhered to the seed surface. After post-treatment water wash seeds should be immediately sown in the field.

4.6 Radio-sensitivity Prior to the beginning of mutation breeding experiment, a radio-sensitivity test must be performed to evaluate the optimum dose of mutagen for the variety that needs improvement. In this, seeds are treated with varying doses at low, intermediate, and higher concentrations followed by observing their effects on different traits.

4.7 Plant Injury and Lethality Mutagens induce three types of effects which are of special interest in genetics and plant breeding: (a) Physiological damage: These damages are usually confined to M1 generation. Physiological damage could be attributed to mutagen-induced changes in chromosomes or extra-chromosomal elements. If one mutagen dose induces high lethality and low mutation frequency, while other mutagen doses induces low lethality and high mutation frequency. Therefore, it may be concluded that the second mutagen dose induced comparatively less extra-chromosomal damage than the first. Irrespective of the reasons behind differential behavior of physiological damage and heritable changes, mutagen dose with low physiological effects and strong genetic effects are always desirable for breeding purposes (Saif & Alshamiri, 2012). (b) Point and gene mutations: These mutations arise due to variations in the genetic material and may be transferred from M1 to the subsequent generations. However, such mutations are not visible in M1 generation and require the use of special tester stocks or mutated haploid gametes. (c) Chromosomal aberrations: Chromosome mutations also arise as result of alterations in the structure of chromosomes and can be observed in M1 generation. In mutation breeding, it is important to assess the balance between mutation frequency and biological damage or injury (reduction in plant survival). Plant injury can be measured quantitatively in various ways, namely, germination percent, measurement of seedling height, root length, plant survival, number of fruits per plant, and number of seeds (Fig. 1).

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Fig. 1  Effect of mutagens on morphological traits (a) Curly flag leaf (b) Albino (c) Abnormal spike (d) Curly flag leaf

4.8 Seedling Height Evaluation of seedling height provides a quick and easy method to assess the mutagenic effect on plant growth and development (Saif & Alshamiri, 2012). Seedling height can be assessed by three commonly used methods, namely, flat, petri dish, and “growing-rack” method (Fig. 2). Flat method is one of the oldest methods in which mutagenized seeds are sown in boxes or pots containing soil in rows, at a depth of 1 cm deep, and distance of 3 cm between the rows. This method can be used for any plant species. The mutagen-­ induced germination delay is measured when the first leaf has stopped its growth, usually after 10–14 days. A similar test can be conducted by sowing mutagenized seeds in a sand bed on greenhouse benches. For instance, 45 seeds (12% moisture) of any cultivar treated with 100, 150, 200, 250, 300, 350, and 400  Gy doses of gamma radiation sown in three replicates of 15 seeds each on a sand/peat mixture along with the control. After 7  days of sowing, measurement of seedling height reduction with respect to the control will be recorded. In petri dish method seeds are sown on blotting paper kept in the petri dishes. Seedling height is determined after 7 days or even earlier. This method is usually followed in laboratories in cereals and legumes. In the growing rack, the seeds are placed between two wet blotters which are supported vertically between slots in PVC racks kept in plastic trays. Adequate amount of water is added to dip the lower edge of the filter paper. It must be noted that irrespective of any of aforesaid methods, the plants must be grown in a growth chamber with regulated temperature, light intensity, and humidity. Further, a minimum of 40 seeds per treatment must be experimented with at least three replications per treatment (Al-Musalli et al., 2011).

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a

S e e d l i n g h e i g h t a s p e r c e n ta g e o f c o n tr o l

b

120

y = -0.2089x + 112.38 2

R = 0.9427

100 80 60 40 20 0 0

100

200

300 Doses (Gy)

400

500

600

Fig. 2  Effect of different doses of gamma rays on (a) wheat seedling height along with the control (0 Gy) grown in laboratory, (b) actual data record

4.9 Survival The plants that reach maturity and produce flowers are counted at the time of harvest of Ml generation. Mutagenic doses may produce less effect at the time of germination, and could lead to 100% lethality at the time of harvest. Plant mortality can occur at any stage onset of germination and ripening however some phases of

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growth are critical and sensitive to mutagenic effect, therefore it is necessary to mention the time and stage of growth while reporting the survival percentage. Besides this, plant survival in field differs from the survival rate under laboratory conditions. This may be attributed to diverse factors that impact the plant survival in field.

4.10 Cytological Effect Mutagenic doses are capable of producing diverse effects on the structure and function of chromosomes in mitosis and meiosis stages. Such effects result in the production of cells with meiotic aberrations and provide an idea about the mutagenic potency. Meiotic analysis is laborious as compared to recording seedling height. However, meiotic analysis provides necessary information about the impact of new mutagenic dose whose potency is not known. Different aberrations are recorded in different meiotic stages. For instance, bridges and chromosome fragments are frequently found at anaphase. Translocations are usually found at metaphase. The frequency of meiotic aberrations is directly proportional to the mutagenic doses.

4.11 Sterility Mutagen-induced sterility or reduction in reproductive fertility could be due to the following reasons: 1. Severe stunting 2. Flowers lack necessary reproductive structures 3. Aborted pollens 4. Aborted embryos 5. Seeds fail to germinate

5 Selection and Handling the Mutagenized Population The mutation breeding program usually aims at improving one or few specific traits of an otherwise outstanding variety, to address the food security issues by improving the yielding potential of existing varieties. The success of mutation breeding depends on the following factors.

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5.1 Selection Criteria The selection of plant material is an important step in the mutagenesis program, therefore breeder should variety that is well adapted to the wide range of climatic conditions. However, it requires improvement in yield and yield-contributing traits.

5.2 Mutagen Dose Mutagen dose is another factor that affects the outcome of mutagenesis program. It is always good to use different doses starting with lowest and ending with highest. The lowest dose could be selected on the basis of literature that reported favorable effects with such dose. In addition, a combination of physical and chemical mutagens would be better than employing a single mutagen. While recording the observations a breeder must figure out the lethal dose (LD50) a dose that induced 50% mortality and growth reduction (GR30 and GR50) that induces 30% and 50% growth reduction, respectively.

5.3 Population Size The mutation frequency is very low and to obtain desired mutants, mutagenized population must be big in size. In addition to the decline in the M1 survival and random nature of mutations a minimum of 3000 seeds must be treated with the mutagens to obtain the desired mutants. After mutagen treatment, seeds must be sown immediately in the field in a randomized complete block design. All the recommended agricultural practices such as irrigation, fertilizers, pesticides, and weedicides must be followed to facilitate good growth of mutagenized population. In the initial generations care must be in reducing the contamination through mechanical mixing, outcrossing (pollen grains may be transmitted by wind or insects from varieties of the same species), and birds often damage the M1 material because the range of maturity in treated materials may be greater.

5.4 Harvest All the seeds from each M1 plant should be harvested and stored for raising subsequent generations. However, methods of harvesting the M1 population vary and depend from species to species depending on the pattern of ontogenetic development and the methods of screening of mutants.

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6 Screening Technique The wide variability induced by radiation or chemical mutagens requires suitable screening methods to select the desired resistant types. The following points are important in carrying out a mutation breeding program for traits like disease resistance.

6.1 Screening Techniques for Disease Resistance (a) The objectives must be clearly defined before any choice of host cultivar and pathogen culture is made since the basis for selection of mutations is the result of intimate association of the two organisms (Borojevic & Worland, 1990). (b) It is necessary to use the same inoculum and inoculums potential in all screening tests to avoid changes in pathogenicity. (c) In soil-borne diseases, screening in infested fields is often very useful. High populations of the pathogen may be maintained by proper management of environmental conditions. The assessment of disease resistance may be based on symptoms, infection type, epidemiological criteria, and/or yield. Selection for a specific resistance may be based on symptoms alone, particularly in the initial selection, and also on severity and time of appearance of disease and yield components. To evaluate material, screening field tests at different locations may be necessary in view of the variable pathogenicity of many disease organisms and the effect of local ecological conditions.

6.2 Time and Intensity of Selection When emphasis on mutation breeding program is both for disease resistance and other categories of mutants, screening and selection may be done simultaneously with the following exceptions: • When the disease is eliminated, screening for disease resistance should be done on the entire generation after other kinds of mutants have been selected since only resistant plant will survive when the pathogen and host are brought together. • When the disease may strongly reduce seed production, inoculations of plants can be adjusted in a given generation so that the incidence of disease does not reach damaging proportion prior to adequate seed set. The selection pressure that can be used in early generations will depend largely on the amount of variation for disease resistance. Care should be taken to retain mutants that show reduced establishment and development of the pathogen or give good yields in spite of severe infection (Saif et al., 2008).

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6.3 General Methodology for Selection of Disease-Resistant Mutant Under Artificially Created Epiphytotic Conditions in M2 Generation About one-half of seed is grown on M2 plant progeny basis in the disease-screening nurseries. The seed is dibbled in two rows of 3 m length for each progeny and a spreader (highly susceptible) variety is planted after every eight rows and artificially inoculated with a mixture of physiological races of the causal organism (Saif et al., 2007). Artificial epidemics should be established because natural epidemics are unreliable. The first inoculation is done at the seedling stage (10–25 days old plant) and later on at the start of spike/panicle/pod formation and then subsequently sprayed with 3 or 4 sprays at weekly intervals (Fig. 3a, b). Data on disease incidence are recorded 3 weeks after inoculation using the following scale: Moderately resistant Moderately susceptible Susceptible Highly susceptible

: No infection. 5% foliage infection/or few small lesions on stem. : 5.1–15% foliage infection/or stem lesions are common and easily observed. : 15.1–25% foliage infection/or stem lesions are very common and damaging. : 25.1–75% foliage infection/or stem lesions bigger in size than 6 mm.

6.4 Evaluation of Mutants In M3 generation, selected progenies of M2-resistant plants are sown in two row plots, in two replications and disease resistance tests are performed in M2 generation in the disease-screening nursery. Other ergonomically important characters are estimated. In M4 generation, selected progenies are planted in replicated micro-plots in

Fig. 3 (a, b) Inoculation of M2 population of wheat with spore rust disease in wheat field

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different locations. All the ergonomically important characters, including grain yield, are measured. Sample of each line is tested for resistance to the specific disease under artificial epiphytotic conditions as well as under natural condition. In M5 generation, promising disease-resistant lines are tested in micro yield trials in different locations. Elite lines selected are propagated and evaluated in subsequent generation for varietal release.

6.5 Screening Techniques for Drought and Salt Resistance Drought, a period of no rainfall or irrigation that affects plant growth, is a major constraint for crop production. Within a crop, some genotypes are most drought resistant than others, out-yielding those exposed to the same degree of water stress. While there is large genotypic variation in response to drought, the physiological basis of this variation is not well understood. Such an understanding could exist in identification of traits that increase yield in water-limited environments. Such traits may include a deep rooting system, radiation shedding by leaf rolling, and dehydration tolerance. Hence, the screening techniques should be based on the study of these traits. A screening technique used commonly for drought resistance is the visual drought score in which genotypes are subjected to water stress at seedling stages and plant response is scored based on the amount of leaf death. A possible reason for genetic variation in leaf death is variation in epidermal conductance. Low epidermal conductance allows maintenance of leaf water potential when stomata are closed.

6.6 Screening Under Simulated Drought Conditions The following stress treatments should be imposed to simulate the type of drought. The cemented tanks may be protected from rain. At maturity, grain yield/plant may be recorded, and selection is made on yield (gm) basis. Terminal drought Post-anthesis drought Pre-anthesis drought Control

: No irrigation water during entire crop growth : Irrigation applied at tillering stage : Irrigation applied at anthesis : Normal irrigation stage

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6.7 Screening Under Natural Drought Condition A portion of M2 is planted in the field under natural drought environment. Selection is usually made by visual observation with respect to emergence, plant height, productive tillers per plant, and grain yield (Fig. 4). Variants exhibiting maximum mean values of the above-cited attributes are selected. Confirmation of the selected mutants is made in M3 generation by growing them in drought condition and observing the seedling height (root length and shoot length) after 14 days of planting and other traits till maturity. The selected mutants are then multiplied for testing in advanced generations (i.e., M3–M4) (Fig. 5).

Fig. 4  Screening under natural drought conditions at seedling stage

Fig. 5  Screening under natural drought conditions at maturity stage

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7 Use of Statistical Parameters for Measuring the Mutation Product Statistical parameters have been used for the study of variation and relationship among various characters and their relationship to environmental factors. X2 is applied to test the goodness of fit to different segregation ratios. Phenotypic and genotypic variance, coefficient of variation, heritability, and genetic advance are estimated for different characters. Analysis of variance and co-variance followed by Duncan’s Multiple Range (DMR) test is applied for multiple comparisons of paired means. General Combining Ability (GCA) and Specific Combining Ability (SCA) effects are computed to find out good general and specific combiners. Genotypic x Environment interaction is studied in depth through stability analysis. LD50 and optimum dose are worked out for different genotypes. Path coefficients are estimated to determine direct and indirect effect of causal variables on resultant variable. Correlation and regression analysis is performed to express dependent variable as a function of independent variables. Coefficient of determination (R2) is computed to determine the contribution of causal variable toward the variation in resultant variable.

8 Conclusion and Future Directions Mutation breeding technique has contributed a lot in the crop improvement programs and is gaining more importance in achieving the food security. Thousands of mutants with improved traits have been developed and officially released for multiple uses. Therefore, minute details about the steps involved in mutation breeding are important for mutation breeders to conduct the mutagenesis successfully. This chapter provides the much-needed information and would be useful to future plant breeders and geneticists.

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Bhatia, C.  R., Nichterlein, K., & Maluszynski, M. (1999). Oilseed cultivars developed from induced mutations and mutations altering fatty acid composition. Mutation Breeding Review, 12, 1–36. Borojevic, K., & Worland, A.  J. (1990). Induced multiple disease resistance in wheat. MBNL, 35, 2–3. Freisleben, R., & Lein, A. (1942a). Uber side auffindung einer mehltauresistenten Mutante nach rontgenbestrahlung ainer anfalligen Linie von Somergerste. Naturwissenschaften, 30, 608. Freisleben, R., & Lein, A. (1942b). Vorarbeiten zur zuchterischen auswertung rontgeinduzierter Mutationen I. Z. Pflanzenzucht, 25, 235–254. Goyal, S., Wani, M. R., Laskar, R. A., Raina, A., Amin, R., & Khan, S. (2019a). Induction of morphological mutations and mutant phenotyping in black gram [Vigna mungo (L.) Hepper] using gamma rays and EMS. Vegetos, 32(4), 464–472. Goyal, S., Wani, M. R., Laskar, R. A., Aamir, R., & Samiullah, K. (2019b). Assessment on cytotoxic and mutagenic potency of gamma rays and EMS in Vigna mungo L. Hepper. Biotecnología Vegetal, 19(3), 193–204. Goyal, S., Wani, M. R., Laskar, R. A., Raina, A., & Khan, S. (2020a). Mutagenic effectiveness and efficiency of individual and combination treatments of gamma rays and ethyl methanesulfonate in black gram [Vigna mungo (L.) Hepper]. Advances in Zoology and Botany, 8(3), 163–168. Goyal, S., Wani, M. R., Laskar, R. A., Raina, A., & Khan, S. (2020b). Performance evaluation of induced mutant lines of black gram (Vigna mungo (L.) Hepper). Acta fytotechnica et zootechnica, 23(2), 70–77. Goyal, S., Wani, M.  R., Laskar, R.  A., Raina, A., Amin, R., & Khan, S. (2021a). Quantitative assessments on induced high yielding mutant lines in urdbean [Vigna mungo (L.) Hepper]. Legume Science. https://doi.org/10.1002/leg3.125 Goyal, S., Wani, M.  R., Raina, A., Laskar, R.  A., & Khan, S. (2021b). Phenotypic diversity in mutagenized population of urdbean (Vigna mungo (L.) Hepper). Heliyon, 7(5), e06356. Gustafsson, A. (1947). Mutation in agricultural plants. Hereditas, 33, 1–100. Hasan, N., Laskar, R. A., Raina, A., & Khan, S. (2018). Maleic hydrazide induced variability in fenugreek (Trigonella foenum-graecum L.) cultivars CO1 and Rmt-1. Research & Reviews: Journal of Botanical Sciences, 7(1), 19–28. IAEA. (1961). Effects of ionizing radiations on seeds. Proceedings of a symposium (p.  655). International Atomic Energy Agency. IAEA. (1965). The use of induced mutations in plant breeding, (Report of the FAO/IAEA. Technical meeting, Rome, 1964). Pergamon Press. IAEA. (1969). Induced mutations in plants (p. 748). IAEA. Kawai, T., & Amano, E. (1991). Mutation breeding in Japan. In Plant mutation breeding for crop improvement (Vol. 1, pp. 47–66). IAEA. Khursheed, S., Laskar, R. A., Raina, A., Amin, R., & Khan, S. (2015). Comparative analysis of cytological abnormalities induced in Vicia faba L. genotypes using physical and chemical mutagenesis. Chromosome Science, 18(3–4), 47–51. Khursheed, S., Raina, A., & Khan, S. (2016). Improvement of yield and mineral content in two cultivars of Vicia faba L. through physical and chemical mutagenesis and their character association analysis. Archives of Current Research International, 4(1), 1–7. Khursheed, S., Raina, A., Parveen, K., & Khan, S. (2017). Induced phenotypic diversity in the mutagenized populations of faba bean using physical and chemical mutagenesis. Journal of the Saudi Society of Agricultural Sciences, 18, 113–119. https://doi.org/10.1016/j. jssas.2017.03.001 Khursheed, S., Raina, A., Laskar, R. A., & Khan, S. (2018a). Effect of gamma radiation and EMS on mutation rate: Their effectiveness and efficiency in faba bean (Vicia faba L.). Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 71(4), 397–404. https:// doi.org/10.1080/00087114.2018.1485430 Khursheed, S., Raina, A., Amin, R., Wani, M. R., & Khan, S. (2018b). Quantitative analysis of genetic parameters in the mutagenized population of faba bean (Vicia faba L.). Research on Crops, 19(2), 276–284.

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Khursheed, S., Raina, A., & Khan, S. (2018c). Physiological response of two cultivars of faba bean using physical and chemical mutagenesis. International Journal of Advance Research in Science and Engineering, 7(4), 897–905. Laskar, R. A., Khan, S., Khursheed, S., Raina, A., & Amin, R. (2015). Quantitative analysis of induced phenotypic diversity in chickpea using physical and chemical mutagenesis. Journal of Agronomy, 14(3), 102–111. Laskar, R. A., Laskar, A. A., Raina, A., Khan, S., & Younus, H. (2018a). Induced mutation analysis with biochemical and molecular characterization of high yielding lentil mutant lines. International Journal of Biological Macromolecules, 109, 167–179. Laskar, R.  A., Wani, M.  R., Raina, A., Amin, R., & Khan, S. (2018b). Morphological characterization of gamma rays induced multipodding mutant (mp) in lentil cultivar Pant L 406. International Journal of Radiation Biology, 94(11), 1049–1053. Laskar, R. A., Khan, S., Deb, C. R., Tomlekova, N., Wani, M. R., Raina, A., & Amin, R. (2019). Lentil (Lens culinaris Medik.) diversity, cytogenetics and breeding. In J.  M. Al-Khayri et  al. (Eds.), Advances in plant breeding: Legumes (pp.  319–369). Springer. https://doi. org/10.1007/978-­3-­030-­23400-­3_9 Linqing, W. (1986). Development and achievements in mutation breeding of plants in China. In Proc. International symposium of plant breeding by inducing mutation and in-vitro biotechniques, Beijing, China, 16–20 October 1985, pp. 7–14. Maluszynski, M., Ahloowalia, B.  S., & Sigurbjornsson, B. (1995). Application of in  vivo and in vitro mutation techniques for crop improvement. Euphytica, 85(1–3), 303–315. Maluszynski, M., Ahloowalia, B., Ashri, A., Nichterlein, K., & van Zanten, L. (1999). Induced mutations in rice breeding and germplasm enhancement. In Proceedings of the 19th Session of the International Rice Commission (pp. 194–204). FAO. Maluszynski, M., Szarejko, I., Bhatia, C.  R., Nichterlein, K., & Lagoda, P.  J. L. (2009). Methodology for generating variability. Part four: Mutation techniques. In Plant breeding and farmer participation (pp. 159–194). FAO. Micke, A., Donini, B., & Maluszynski, M. (1987). Induced mutations for crop improvement – A review. Tropical Agriculture (Trinidad), 64(4), 259–278. Muller, H. J. (1927). Artificial transmutation of the gene. Science, 66, 84–87. Raina, A., & Khan, S. (2020). Increasing rice grain yield under biotic stresses: Mutagenesis, transgenics and genomics approaches. In C.  Aryadeep (Ed.), Rice research for quality improvement: Genomics and genetic engineering (pp.  149–178). Springer. https://doi. org/10.1007/978-­981-­15-­5337-­0_8 Raina, A., Laskar, R. A., Khursheed, S., Amin, R., Tantray, Y. R., Parveen, K., & Khan, S. (2016). Role of mutation breeding in crop improvement-past, present and future. Asian Research Journal of Agriculture, 2(2), 1–13. Raina, A., Laskar, R. A., Khursheed, S., Khan, S., Parveen, K., & Amin, R. (2017). Induce physical and chemical mutagenesis for improvement of yield attributing traits and their correlation analysis in chickpea. International Letters of Natural Sciences, 61, 14–22. Raina, A., Khursheed, S., & Khan, S. (2018a). Optimisation of mutagen doses for gamma rays and sodium azide in cowpea genotypes. Trends in Biosciences, 11(13), 2387–2389. Raina, A., Laskar, R. A., Jahan, R., Khursheed, S., Amin, R., Wani, M. R., Nisa, T. N., & Khan, S. (2018b). Mutation breeding for crop improvement. In M. W. Ansari, S. Kumar, C. K. Babeeta, & R. K. Wattal (Eds.), Introduction to challenges and strategies to improve crop productivity in changing environment (pp. 303–317). Enriched Publications. Raina, A., Laskar, R. A., Tantray, Y. R., Khursheed, S., & Khan, S. (2020a). Characterization of induced high yielding cowpea mutant lines using physiological, biochemical and molecular markers. Scientific Reports, 10, 3687. Raina, A., Parmeshwar, K., & Khan, S. (2020b). Increasing rice grain yield under abiotic stresses: Mutagenesis, transgenics and genomics approaches. In C. Aryadeep (Ed.), Rice research for quality improvement: Genomics and genetic engineering (pp. 753–777). Springer. https://doi. org/10.1007/978-­981-­15-­4120-­9_31

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Raina, A., Sahu, P.  K., Laskar, R.  A., Rajora, N., Sao, R., Khan, S., & Ganai, R.  A. (2021). Mechanisms of genome maintenance in plants: Playing it safe with breaks and bumps. Frontiers in Genetics, 12, 675686. https://doi.org/10.3389/fgene.2021.675686 Raina, A., Laskar, R. A., Wani, M. R., Jan, B. L., Ali, S., & Khan, S. (2022a). Comparative mutagenic effectiveness and efficiency of gamma rays and sodium azide in inducing chlorophyll and morphological mutants of cowpea. Plants, 11, 1322. https://doi.org/10.3390/plants11101322 Raina, A., Laskar, R. A., Wani, M. R., Jan, B. L., Ali, S., & Khan, S. (2022b). Gamma rays and sodium azide induced genetic variability in high yielding and biofortified mutant lines in cowpea [Vigna unguiculata (L.) Walp.]. Frontiers in Plant Science, 13, 911049. https://doi. org/10.3389/fpls.2022.911049 Raina, A., Laskar, R. A., Wani, M. R., & Khan, S. (2022c). Plant breeding strategies for abiotic stress tolerance in cereals. In C. Aryadeep (Ed.), Omics approach to manage abiotic stress in cereals (pp. 151–177). Springer. https://doi.org/10.1007/978-­981-­19-­0140-­9_8 Raina, A., Laskar, R. A., Wani, M. R., & Khan, S. (2022d). Chemical mutagenesis: Role in breeding and biofortification of lentil (Lens culinaris Medik) mutant lines. Molecular Biology Reports. https://doi.org/10.1007/s11033-­022-­07678-­6 Raina, A., Laskar, R. A., Wani, M. R., & Khan, S. (2022e). Plant breeding strategies for abiotic stress tolerance in cereals. In Omics approach to manage abiotic stress in cereals (pp. 151–177). Springer. https://doi.org/10.1007/978-­981-­19-­0140-­9_8 Rasik, S., Raina, A., Laskar, R. A., Wani, M. R., Reshi, Z., & Khan, S. (2022). Lower doses of sodium azide and methyl methanesulphonate improved yield and pigment contents in vegetable cowpea [Vigna unguiculata (L.) Walp.]. South African Journal of Botany, 148, 727–736. https://doi.org/10.1016/j.sajb.2022.04.034 Rutger, J. N. (1992). Impact of mutation breeding in rice – A review. Mutation Breeding Review, 8, 1–24. Saeed Iqbal, R. M., Chaudhry, M. B., Aslam, M., & Bandesha, A. A. (1991). Economic and agricultural impact of mutation breeding in cotton in Pakistan. In Plant mutation breeding for crop improvement (Vol. 1, pp. 187–201). IAEA. Saif, A., & Alshamiri, A. (2012). Development of different mutant lines of local barley variety (T. Hordeum vulgare. cv. Bakkur) with improved quantities and qualitative traits under rainfed conditions. In Eleventh Arab conference on the peaceful uses of atomic energy, Sudan, under press. Saif, A., Hazza, A., Almaktari, A., Zaid, N., Alshamiri, A., & Abdulhabib, A. (2007). Improvement of some important crops in Yemen through induced mutations. In Seventh Arab conference on the peaceful of atomic energy, Sana’a, Yemen. pp. 256–287. Saif, A., Daoud, A., & Alshamiri, A. (2008). Evaluation of yield and agronomic characters of local wheat mutants under rainfed conditions. Yemeni Journal of Agricultural Research and Study, 18, 13–27. Salnikova, T. V. (1993). Chemical mutagenesis for crop breeding – Achievements in the former USSR. MBNL, 40, 11–12. Scarascia-Mugnozza, G. T. (1965). Induced mutations in breeding for lodging resistance. In The use of induced mutations in plant breeding (pp. 537–558). Pergamon Press. Scarascia-Mugnozza, G.  T., D’Amato, F., Avanzi, S., Bangnara, D., Belli, M.  L., Bozzini, A., Brunori, A., Cervigni, T., Devreux, M., Donini, B., Giorgi, B., Martini, G., Monti, L.  M., Moschini, E., Mosconi, C., Porreca, G., & Rossi, L. (1993). Mutation breeding for durum wheat (Triticum turgidum ssp. durum Desf.) improvement in Italy. Mutation Breeding Review, 10, 1–28. Sellapillai, L., Dhanarajan, A., Raina, A., & Ganesan, A. (2022). Gamma ray induced positive alterations in morphogenetic and yield attributing traits of finger millet (Eleusine coracana (L.) Gaertn.) in M2 generation. Plant Science Today, 9(4), 939–949. Sellapillaibanumathi, L., Dhanarajan, A., Raina, A., & Ganesan, A. (2022). Effects of gamma radiations on morphological and physiological traits of finger millet (Eleusine coracana (L.) Gaertn.). Plant Science Today, 9(1), 89–95.

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Shu, Q., Wu, D., & Xia, Y. (1997). The most widely cultivated rice variety “Zhefu 802” in China and its geneology. Mutation Breeding Newsletter, 43, 3–5. Sigurbjornsson, B., & Micke, A. (1969). Progress in mutation breeding. In Induced mutations in plants (pp. 673–698). International Atomic Energy Agency. Sigurbjornsson, B., & Micke, A. (1974). Physiology and accomplishments of mutation breeding. In Polyploidy and induced mutations in plant breeding (pp. 303–343). International Atomic Energy Agency. Stadler, L. J. (1928a). Mutations in barley induced by X-rays and radium. Science, 68, 186–187. Stadler, L. J. (1928b). Genetic effects of X-rays on maize. Proceedings of the National Academy of Sciences, 14, 69–75. Tantray, A.  Y., Raina, A., Khursheed, S., Amin, R.  U., & Khan, S.  A. (2017). Chemical mutagen affects pollination and locule formation in capsules of black cumin (Nigella sativa L.). International Journal of Agricultural Science, 8(1), 108–117. Wang, L. Q. (1996). Present status of crop mutation breeding in China. In Q. Liang (Ed.), Plant mutation breeding in Asia (pp. 11–20). China Agricultural Scientech Press. Wani, M.  R., Dar, A.  R., Tak, A., Amin, I., Shah, N.  H., Rehman, R., Baba, M.  Y., Raina, A., Laskar, R., Kozgar, M. I., & Khan, S. (2017). Chemo-induced pod and seed mutants in mungbean (Vigna radiata L. Wilczek). SAARC Journal of Agriculture, 15(2), 57–67. Wani, M. R., Laskar, R. A., Raina, A., Khan, S., & Khan, T. U. (2021). Application of chemical mutagenesis for improvement of productivity traits in lentil (Lens culinaris Medik). Annals of Biology, 37(1), 69–75.

Transgenic Techniques for Plant Improvement: A Brief Overview Lidia Stefanova, Slaveya Kostadinova, Atanas Atanassov, and Ivelin Pantchev

Abstract  Food supply, quality and security are among the keystones of modern civilization. The enormous product choice on shelves depends on successful agriculture. In turn, agriculture mainstays are improved plant species and long record of methods and approaches for their production is available. Since 1990, transgenic techniques gradually became one of the main tools for obtaining more productive species. Outside the commercial applications, transgenic plants have also played a quite significant role in elucidation of a number of cellular processes and corresponding mechanisms. It is too presumptuous to make a comprehensive review of all accomplishments in the field. Instead, we are presenting a brief overview of fundamentals, main applications and current achievements of the transgenic technologies in plant biotechnology. Keywords  Transformation · Agrobacterium · Particle bombardment · Vectors

1 Introduction Plant improvement is among the key priorities of modern agrobiology. Modern world is facing a decrease of arable land and fresh water shortage. One of the viable solutions to feed constantly increasing population is to develop more productive varieties. To achieve required improvements breeders need not only a set of

L. Stefanova · S. Kostadinova Department of Biochemistry, Faculty of Biology, Sofia University, Sofia, Bulgaria A. Atanassov Joint Genomic Center Ltd., Sofia, Bulgaria I. Pantchev (*) Department of Biochemistry, Faculty of Biology, Sofia University, Sofia, Bulgaria Joint Genomic Center Ltd., Sofia, Bulgaria e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_5

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molecular tools but also deeper knowledge on plant biochemistry, physiology, genetics, to name a few. Classic plant breeding was depending on phenotype-based selection only. During the twentieth century, classic genetic approaches as well as physiology-related chemical and physics methods were implemented. Next major breakthrough took place in the early 1970s after the development of first molecular biology methods. Development of transformation approaches in 1980s led to a boost in plant manipulation and paved the way to commercial plant biotechnology (Lurquin, 1987). Advancements in molecular biology reflect on plant biology and biotechnology during the next decades in many ways (Hiwasa-Tanase & Ezura, 2016): • Extensive genome characterization, originally linked to development of an array of molecular marker techniques and finally by implementing next-generation sequencing techniques • Cloning and characterization of numerous genes • Better understanding of plant metabolism • Integrated knowledge of regulatory networks • Improved plant in vitro culture techniques • Establishment of transgenic technologies • Current achievements in gene and genome editing Progress in plant physiology and ecology should also be considered. The boost in plant biology research relied on numerous approaches and transgenic techniques were among the most important ones. The use of transgenic technologies had the main impact on plant improvement through: • Revealing mechanisms, processes and their regulation in plants • Introduction of non-native genes • Manipulation of plant metabolism as well as designing novel metabolic pathways or processes • Precise manipulation of genes When critical mass of knowledge was accumulated, transgenic technologies had resulted in development of novel cultivars with improved properties  – molecular breeding was born. Transgenic technologies generally combine two approaches – gene delivery and plant regeneration (Fig. 1).

2 Plant Regeneration Plant regeneration methods had a long history of development. Since first reports for successful regeneration from tissue fragments or single cells, protocols for almost all plant species were available. Also, whole plants can be regenerated from a variety of tissue or cell types including haploid cells and protoplasts. For more detailed review on plant in  vitro culture technologies, readers are advised to see Thorpe (2007).

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Fig. 1  A diagram model of a procedure to create transgenic plant

3 Gene Delivery Approaches The main obstacle for DNA delivery in plant cells is the presence of a rigid cell wall. To overcome it, several methods for plant transformation were developed.

3.1 Transfection Transfection of plant cells is possible only upon removal of the cell wall by enzyme treatment. The resulting wall-less cells called protoplasts can uptake DNA from the environment (Cocking, 1977). For higher efficiency DNA must be condensed with PEG, Ca2+ and/or biogenic amines (i.e. spermidine) to form microparticles (Krens et al., 1982). Most likely DNA enters plant cell through endocytosis pathway. Main disadvantages are related to protoplast production, protoplast maintenance and subsequent plant regeneration efficiency (Paszkowski et al., 1984).

3.2 Lipofection DNA can be delivered into protoplasts by lipofection (Gad et al., 1990). For that purpose, DNA is mixed with phospholipids to form liposomes. Delivery into the cell occurs upon fusion between liposome with cell membrane. The main advantage is that DNA is better protected from degradation in the culture medium.

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3.3 Electroporation Upon its introduction, electroporation became an important tool for transformation of bacterial, yeast and animal cells. It is a robust, efficient and reproducible method of DNA delivery even of large molecules (500–700 kbp). Plant transformation by electroporation is achievable only in protoplasts (Fromm et al., 1985). DNA enters plant cell through transient lesions in cell membrane. Transfection, lipofection and electroporation depends on working procedures for protoplast production. Since the establishment of such procedures are time- and labour-intensive, these approaches for plant transformation did not gain popularity and are used by a relatively small number of researchers. For that reason, the search for DNA delivery methods that do not require cell wall removal continued.

3.4 Agrobacterium-Mediated Plant Transformation One such method was based on the machinery of plant pathogenic bacteria Agrobacterium. Agrobacterium is a parasitic organism with unusual mechanism to exploit host plant resources. It possesses a modified Type IV secretory system that delivers a DNA fragment (T-DNA) into the plant host – a native genetic engineering mechanism. There are several excellent reviews on Agrobacterium biology (Gelvin, 2003; Nester, 2015) and we will focus on which features were important to create one of the most popular techniques for plant transformation. First feature was that bacteria does not need removal of cell wall to deliver T-DNA. Instead, it binds to plant cell surface and build a channel (a modified pillus) through the wall and cell membrane. The process depends on bacterial genes only and plant role is minimal and can be mimicked by some cell wall components (i.e. acetosyringone). Agrobacterium can deliver T-DNA not only to different plant species, but also to fungi and animal cells. Second feature regards T-DNA organization. T-DNA is a sequence delimited by two border elements, that is natively carried by a large plasmid. It was demonstrated that T-DNA can be relocated to another plasmid and produced in trans. Also, the native internal sequence does not play role in T-DNA production and delivery and can be replaced by any other sequence of interest. These features allowed for creation of so-called disarmed strains and binary vectors. The disarmed strains contain Ti plasmid without its native T-DNA locus but retain all necessary genes of secretory apparatus. The binary vectors contain T-DNA border sequences with convenient cloning sites and control elements (promoter and terminator). The size of T-DNA is limited only by the ability of bacteria to maintain its stability. Agrobacterium-mediated plant transformation was first reported in the late 1970s (Marton et  al., 1979). Soon after convenient sets of binary vectors and disarmed strains were developed (Hoekema et al., 1983; de Framond et al., 1983). The method

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was further simplified (Horsch et al., 1985) and was adopted by a large number of research groups. Agrobacterium-mediated transformation has several advantages: • • • • •

Does not require specialized equipment and extensive training Can be applied on cells, tissues or whole plants Efficient and reproducible upon established protocols Can be adapted to different species Can be used to deliver large DNA sequences (several genes at once)

Despite that, successful transformation was demonstrated for large number of species, some of them including economically important ones like wheat, proved to be recalcitrant. Nevertheless, protocols were developed even supposing lower efficiency. Another drawback is that Agrobacterium cells can persist in regenerated plants and their seeds. Such persistence makes confirmation of transgenic events by PCR impossible; instead, restriction analysis followed by hybridization must be performed. Attempts to develop PCR-based methods for event detection are made but they are not yet popular. Persistence In progeny might be a problem for biosafety reasons especially for varieties to be released into the environment. This problem can be solved by in vitro micropropagation from bacteria-free tissues of T1 or T2 plants. Recently, a binary vector modification for bacterial cell elimination after transformation was reported. The vector carries sacB gene which confer toxicity to the bacterial cell on sucrose-containing media. A limitation is that T-DNA is delivered as single-stranded linear molecule and might be truncated within the plant cell. Delivered T-DNA is either transiently expressed and then degraded or stably integrated into the genome. It is not possible to deliver DNA that is supposed to be independently replicated and maintained in the cell. An interesting subject is generation of “hairy root” cultures. It is a result of transformation with Agrobacterium rhyzogenes, carrying wt Ri plasmid. T-DNA region of Ri plasmid carries on a set of genes encoding regulatory proteins as well as enzymes for hormone synthesis. As a result, transformed cells differentiate as roots, forming specific morphology – “hairy roots”. One consequence of the transformation is that these cells have deregulated secondary metabolism and can serve as “bioreactors” for production of specific compounds.

3.5 Particle Bombardment Since its introduction in mid-1980s, particle bombardment method became one of the main tools for creating transgenic plants (Klein et al., 1987). The method solves the cell wall problem in a simple and straightforward way. First, DNA is condensed by Ca2+ and biogenic amines (spermidine) and then ethanol-precipitated on the surface of high-density microparticles (usually gold, rarely tungsten). Then, particles are accelerated toward the target cells or tissue using high-pressure gas (helium,

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80–110 bar) in a vacuum (0.8 bar). Particles obtain sufficient energy to penetrate cell wall and DNA is delivered into the cells. The technique has many advantages: • • • • • • •

Robust, efficient and reproducible Applicable to almost any plant species, even ones recalcitrant to Agrobacterium Can deliver large DNA fragments Not time- or labour-intense Usually does not need complicated procedures for preparing DNA vectors Can deliver circular and linear DNA (critical for some experiments) Allows for transformation of chloroplasts and mitochondria On the other hand, it has some drawbacks:

• Needs specialized equipment • Can inflict unwanted genome rearrangements due to mechanical damage • Inconvenient to transform cell suspension cultures Despite these drawbacks, particle bombardment is still a method of choice for many groups. It is still the only viable method to transform chloroplasts (Svab et al., 1990). A detailed comparison between particle bombardment and Agrobacterium-­ mediated delivery is well presented by Jackson et al. (2013).

3.6 SiC Whiskers An alternative to particle bombardment was the use of SiC whiskers (Kaeppler et al., 1992). It is significantly simpler, does not require specialized equipment and can be used in situ. Nowadays, it is largely abandoned due to health concerns.

3.7 Sonication Another alternative method was sonication, where the introduction of DNA is achieved upon treatment with ultrasonic waves (Joersbo & Brunstedt, 1990). The method works on tissue fragments and cells but its efficiency and reproducibility are questionable. All methods for transgene delivery can be employed to introduce several genes in the same genome – the so-called gene stacking (Halpin, 2005). This approach allows creation of complex artificial gene networks (i.e. novel metabolic or signalling pathways) (Zhu & Liu, 2021).

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4 Vector Designs The choice of the transformation method and experimental strategy dictate the design of the vector used. Vectors can be broadly divided into two categories – integrative and for transient expression. Most frequently vectors are based on plasmids but linear DNA fragments can also be used for transformation. Plasmid vectors can generally be used for both transient expression and genome integration. Linear DNA fragments are used mainly in experiments aiming for stable integration into the genome (Balazs et al., 1985). Transient expression can be readily achieved using any kind of DNA possessing all necessary control elements (Wang et al., 1988). On the other hand, integration of the heterologous DNA is often one of the goals (Halfter et al., 1992). When a plasmid is used this might be achieved by microhomologous recombination. When long regions are included in the vector, integration could be directed to a specific locus via homologous recombination mechanisms. In this case, linear DNA can be used resulting in sufficient transformation efficiency albeit lower as compared to plasmid. Since the dawn of transgenic technologies, achieving targeted gene integration was among the top priorities. First approaches were soon developed for model organisms like A. thaliana (Halfter et al., 1992). Since then several novel systems were developed based on megarestrictases, Zn fingers and TALEN but proved to be out of reach for most laboratories. The real breakthrough in this field was achieved with the development of CRISPR/Cas technology (Dong & Ronald, 2021).

4.1 General Vector Design Regardless of whether vector is for transient expression or stable integration, it contains some obligatory elements (Fig. 2) described in the very first vector designs (Balazs et al., 1985). An overview of typical vector designs is shown in Fig. 3. It should be noted that these designs are schematic and do not represent all known vector designs described in the literature. First group is the modules allowing for gene expression (i.e. promoters and terminators). Most widely used promoters are derived from CaMV 35S, nopaline synthase, ubiquitin and actin. These promoters allow for constitutive expression in most plant cell and tissue types. For regulated expression other promoters are available (light or temperature controlled). Another example is artificially designed promoters. Also, in some cases, bacterial regulatory elements can be employed like tetracycline regulons. It was also demonstrated that operon-like gene organization might be achieved in plants (Vonbodman et al., 1995). Very useful and efficient vectors can be designed using plant viruses as a base (Zaidi & Mansoor, 2017). Transcription terminators are the second important regulatory element. Most widely nopaline synthase and ribosomal operon terminators are used due to

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Fig. 2  Schematic description of main vector elements

Fig. 3  Overview of typical vector designs. (a) Plasmid vector used for transient expression. (b) Plasmid vector for targeted integration via homologous recombination. (c) A binary vector design. Abbreviations: AbR – antibiotic resistance (bacteria); ori – origin of plasmid replication (bacteria); P  – Promoter (eukaryotic); selectable marker  – a gene allowing selection of transformed cells; gene-of-interest – the heterologous gene to be delivered into plant cell; HR-1/HR-2 – homology regions targeting integration; LB/RB – T-DNA left and right border sequences

predictive effect and short sequence. A polyadenylation signal should also not be omitted. In most standard vectors it is already presented but might be missing in some designs. For efficient nuclear expression, artificial splicing site is frequently considered. Its presence significantly increases expression levels. Introduction of such a site is a complicated procedure requiring extensive optimization of the gene design. For most common genes (i.e. reporters, resistance, Cas9) such modifications are already available. In cases when chloroplasts are the object of transformation, the gene regulation elements resemble prokaryotic ones. In fact, genes optimized for expression in E. coli under bacterial or even T7 promoters can be expressed in chloroplasts.

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4.2 Reporters, Markers, Marker-Less Systems Reporter genes are used for visual detection and quantification of transformation efficiency. The key requirement to use a gene as a reporter is that its activity is unique and no inborn analogues exist in the transformed cells. Historically, the first successful reporter gene was one encoding beta-galactosidase. Its main disadvantage is that analysis is destructive and transformed cells cannot be propagated further. For that reason, fluorescent proteins became a viable alternative and gradually gain wider use. Most standard vectors contain markers for further selection of the transformed cells. Several groups of selectable marker genes were developed. First and most widely used are genes allowing resistance to antibiotics (i.e. kanamycin, chloramphenicol) or herbicides (i.e. 2,4-D, 2,4,5-T, glyphosate). Another group of marker genes allows for resistance to toxic components like D-amino acids, or allows for tolerance to stress conditions (osmotic stress). Selective marker genes can also allow for cell division in absence of hormonal stimuli. Ability to utilize unusual carbon source can also be used for selection of transformed cells. Advances in transgenic technologies opened the way for commercial applications which in turn raised questions about biological and environmental safety among all. One concern was related to possible horizontal gene transfer between transgenic plants and wild relatives where resistance marker genes (for expression in plant cells or from vector backbone) were considered as a main risk. During the last decades some approaches for removing marker genes after transformation event were developed. Most of them relied on site-specific recombination systems like Cre/lox. The other alternative was transformation with cassette without selectable marker at all followed by time-consuming and laborious identification of a few transgenic regenerates among the numerous non-transgenic ones. All aspects briefly outlined above were discussed in much more detail in several excellent reviews (Imani & Kogel, 2020; Sanagala et al., 2017).

5 Applications for Plant Improvement Initially, transformation experiments were related to delivery of genes conferring antibiotic resistance or visual identification of transgenic event in a proof-of-­concept manner (Paszkowski et  al., 1984). Soon after the potential was recognized and transformation became just one of the methods in more complex experiments in plant biology (Lurquin, 1987). An uncomprehensive overview of areas affected by transgenic technologies is depicted in Fig. 4. One of the first examples of applying transgenic techniques for analysis of molecular processes was on photo regulation of small RuBisCO subunit in pea (Nagy et al., 1985). Later, transgenic technologies became a useful tool for functional analysis of regulatory elements (Dean et al., 1988: Yamamoto et al., 1995).

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Fig. 4  An overview of transgenic technology applications

First successful experiments in the field sparked the interest to express heterologous genes for recombinant protein production in transgenic plants (Pautot et al., 1989; Hiatt et al., 1989). The ability to introduce and express novel genes opened the way to modify plant metabolism (Yun et al., 1992). A particularly interesting result was the development of Golden Rice variety based on metabolic engineering of carotene biosynthetic pathway in rice (Burkhardt et al., 1997). Initial attempts relied on introducing single genes but soon after it became possible to design more complex or entirely new pathways (Ma et al., 1995; Dasgupta et al., 1998). Several approaches were developed. They include co-transformation of several plasmids (Hadi et  al., 1996), operon-like design (Vonbodman et al., 1995) and design of self-processing preprotein (Marcos & Beachy, 1994). For a detailed review, see Dafny-Yelin and Tzfira (2007). Another topic of interest was pathogen resistance. Early experiments demonstrated that expression of viral proteins confer resistance to infection (Powell et al., 1989). Since then transgenic technologies were widely used to develop disease-­ resistant varieties for commercial applications (Lindbo & Dougherty, 1992; Raina & Khan, 2020; Raina et al., 2020). At the same time, it became clear that transgene technologies can also be used to confer resistance to bacterial and fungal phytopathogens (Neuhaus et al., 1991). Further experiments allowed for the validation of “gene-for-gene” hypothesis (Van den Ackerveken et al., 1992). Transgenic technologies became a sound base for applying RNA interference in plants. It did not take too long after first experimental proofs (Ecker & Davis, 1986; Zaccomer et al., 1993) to develop convenient tools (Wojtkowiak et al., 2002) and to outline the main directions of application (Lawrence & Pikaard, 2003). Currently,

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RNAi technology along with CRISPR/Cas editing is among the most promising tools for crop improvement (Rajput et al., 2021). A quite unexpected area of RNAi technology was its use as pesticide via external application (Huvenne & Smagghe, 2010) – an approach with significant potential for commercial purposes (Cagliari et  al., 2019). Readers are advised to see the recent review of Dalakouras et al. (2020). The role of transgenic technologies for gene editing is unquestioned. All components of the existing systems – Zn fingers, TALEN and CRISPR/Cas, are delivered by transformation (Laforest & Nadakuduti, 2022). A vast number of vectors were designed during the last decade (Alok et al., 2021). An interesting and promising development in the field is to design gene editing approaches that do not lead to transgenic organisms, the so-called DNA-free editing (He et al., 2021). It is hard to make a comprehensive overview on the role of transgenic technologies in plant science. A brief search returns over 20,000 publications. In this minireview we can only scratch the surface of a such data massive and are well aware that our work is far from completion by any way. For that reason, we are recommending several reviews: Laforest and Nadakuduti (2022), Reed and Bargmann (2021), Yan et al. (2021), Peng et al. (2022), Kumar and Ling (2021), Ozyigit and Yucebilgili Kurtoglu (2020), Tyurin et al. (2020), Metje-Sprink et al. (2018), Guo et al. (2019), Peng et al. (2022).

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Mutagenesis and Transgenesis in Plant Breeding Anurag Tripathi, Sudhir Kumar, Ashish Gautam, Biswajit Lenka, Jeet Ram Choudhary, and Pradipta Ranjan Pradhan

Abstract  Mutagenesis gives an avenue of possibilities for developing improved cultivars in crops with limited or no variability in a specific trait. Mutagenesis has been used in plant breeding to induce mutations within the crop’s DNA. The expansion of genomic techniques in applied breeding, particularly mutational breeding, has stemmed from recent advances in genomics technology. Because of technology like high throughput sequencing, plant genome sequencing is now quite inexpensive. TILLING (Targeting Induced Local Lesions in Genomes), zinc finger nuclease-­ mediated mutagenesis, and meganuclease-mediated mutagenesis have all allowed us to produce targeted mutations in agricultural plants in order to decode gene function and develop superior cultivars. The application of genetic engineering to modify the genome of crop plants for human economic gain is known as transgenesis. Although transgenic plants have been reported in a number of crops, commercialization has been limited to a handful of them, including cotton (Gossypium hirsutum L.), maize (Zea mays L.), soybean (Glycine max (L.) Merr.), and canola (Brassica napus L. and B. rapa L.). In the first generation of transgenic cultivars, plant genetic engineering was employed to create herbicide tolerance, host plant resistance to insects and viruses, and extended shelf life. This chapter examines several aspects of mutagenesis and transgenesis in plant breeding, as well as their relevance to crop improvement. Keywords  Mutagenesis · Transgenesis · Plant breeding A. Tripathi (*) · P. R. Pradhan School of Agriculture Science, G D Goenka University, Sohna, Gurugram, Haryana, India S. Kumar Division of Plant Biotechnology, ICAR-Indian Institute of Pulses Research, Kanpur, India A. Gautam Department of Genetics and Plant Breeding, GBPUA&T, Pantnagar, Uttarakhand, India B. Lenka Department of Genetics and Plant Breeding, Junagadh Agriculture University, Junagadh, Gujarat, India J. R. Choudhary Division of Genetics, ICAR-IARI, New Delhi, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_6

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1 Introduction Induced mutagenesis has been a rich resource in genomic research in the last decade, and genomic and molecular technologies have become a vital aspect of plant mutagenesis research (Raina et  al., 2021). Plant-induced mutagenesis is frequently employed in fundamental research and practical breeding programs, although plant scientists and breeders rarely discuss it separately. The basic principles of experimental plant mutagenesis and applications in plant research and breeding are all covered in this book. Mutagenesis means the alteration in genetic makeup of an organism in a stable manner (Khursheed et  al., 2019; Raina et  al., 2020a). The term “mutation breeding” was conceptualized by Freisleben and Lein (1944) to describe the intentional induction and growth of transgenic mutant lines for agricultural improvement. Historical Development  In plant breeding techniques, mutations are the primary source for bringing variability and diversity in the genetic makeup of any organism (Kharkwal, 2012). Mutation breeding is the technique of producing smart crop varieties by bringing genetic variability using certain chemical or physical mutagens. The concept of mutation breeding came at the start of the twentieth century with the discovery of the techniques of using different chemical and physical agents to bring genetic variability (Shu et al., 2012). During the last five decades, the discoveries of induced mutations led to the development of smart crop varieties all around the world. In this era, mutation breeding is one of the most powerful tools in the hands of plant breeders among other modern breeding techniques such as transgenic and recombinant breeding techniques. However, Novak and Brunner (1992) described that in seed-propagated plants, breeding through induced mutations can be a better way to achieve desirable traits in plants. However, in seedless crops (e.g., seedless grape and banana), mutation breeding is the only way to achieve new varieties with novel characters (Ahloowalia & Maluszynski, 2001; Broertjes & Van Harten, 1988). Moreover, for crops that propagate through roots or tubers and in ornamental plants, cross-breeding has many constraints of clonal identity and time consumption (Predieri & Virgillo, 2007), and mutational breeding is used to produce variation in colors and other traits (Kondo et al., 2009). A brief description of contributors toward mutation breeding is described in Table 1.

1.1 Mutations Arise in Two Ways Spontaneous Mutation: It occurs when untreated organisms cause exogenous mutations. Spontaneous mutation, perhaps the ultimate source of natural genetic variation observed in a population, explains the “background rate” of mutations. Spontaneous mutations can result from replication errors, spontaneous injury, or translocation of transposable factors during cell growth. Spontaneous mutations are extremely rare and recessive.

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Table 1  Historical timeline of mutation breeding Period I: Early spontaneous mutants: their origins 300BC Early mutant crop originated in China. Seventeenth A spontaneous mutant imperial rice in China. century 1859 Origin of Species book published by Darwin. Period II: Conceptualization of mutation and mutation breeding 1895–1900 Different types of mutagenic radiations like X-rays, α-rays, β-rays, and γ-rays are discovered. 1901 Mutation term first time coined by Hugo de Vries. 1901–1911 By using radiation to induce chemical mutation suggested by Hugo de Vries. 1904–1905 By using radiation to artificially induce mutation suggested by Hugo de Vries. 1910 Drosophilamelanogaster was first time used in mutation experiment by Thomas Hunt Morgan. 1920 Law of Homologous Variation in crops proposed by N. Vavilov’s. Period III: Identification of induced mutations and the launch of the first commercial mutant varieties 1927 Datura mutagenesis is described by Stuart Geiger and A.F. Blakesley. G. J. Müller’s convincing evidence of X-ray mutagenesis suggests that X-rays could be used to create genetically superior plants, animals, and humans. 1928 Lewis John Stadler successfully induced mutation after examining barley and maize. 1937 Colchicine effect for chromosome duplication in plants. 1941 Chemical mutagenesis: C. Auerbach, I.A. Rapoport, F. Oehlkers and others. 1942 In barley, X-ray-induced resistance was reported for the first time. 1951 The controlling elements (later identified as transposable genetic elements or transposons) was first time reported by Barbara McClintock. 1956 E.R. Sears used radiation-induced translocation to transfer resistance from Aegilops to wheat. 1958 In higher plants, use of chemical mutagens.. Period IV: Large-scale use of mutation breeding 1964 In 1964, the Joint FAO/IAEA Division on Nuclear Technology in Food and Agriculture was established in Vienna, Austria. A multinational research initiative on mutant breeding was initiated. 1969 The First Classification List of Mutant Varieties was published at the Pullman Symposium on Mutations Induced in Plant Breeding. 1990 A joint FAO/IAEA workshop in Vienna, Austria, looked at the results of 25 years of mutant-assisted breeding. Period V: Plant mutation is being integrated with biotechnology and genomics 1983 The transposable regulatory elements Ac and Ds were extracted and they were used to do transposon mutagenesis. 1997 Reactivation of retrotransposons through rice tissue culture, resulting in a large collection of Tos 17 mutations. 2000 The genome of the first plant (Arabidopsis) was sequenced. TILLING is a method for targeting the screening of induced mutations that was developed. 2002–2005 Indica and Japonica rice species genomes were sequenced. 2005 TILLING and T-DNA insertion mutant populations were established in crop plants for functional genomics study. 2008 In the genomics era, the International Symposium on Induced Mutations in Plants, held in Vienna, Austria, reviewed the applications of incite mutation in mutation research and breeding.

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Induced Mutation  It is caused by the use of mutagens on a plant or any plant part, such as seed, stem cuttings, pollen, and ovules. As the frequency of spontaneous mutation is very rare and insufficient to meet the requirements of crop improvement, it necessitates the induction of mutations artificially through mutation breeding (induced mutation). Types of Mutation: There are two types of mutation: (i) Micromutations—Mutations which lead to changes in polygenic traits and can be measured at the population level are examples of micromutations. Plant breeders pay close attention to micromutations because they cause genetic variability in the quantitative traits of crops. (ii) Macromutations—This is associated with large changes in properties, can be detected without the use of tools, and can be measured at the individual plant level. Mutagenic Agents  A mutagen is a substance that causes abnormal mutations. There are two types of mutagens: physical mutagens and chemical mutagens. Certain formulations differ in ease of use, safety considerations, efficacy in inducing specific genetic changes, suitable organization, and cost, among others. Physical Mutagens  Since the development of mutation breeding, physical mutagens, especially ionizing radiation are being most widely used. Mba et al. (2012) stated that more than 70% of mutant varieties have been developed only through these mutagenic agents. All types of ionizing radiation may be used, including gamma rays, cosmic rays, and X-rays (Mba, 2013). Radioactive particles of gamma rays are mostly obtained from radioactive isotopes of cobalt-60 and caesium-137. Subatomic particles such as alpha particles, beta particles, protons, and neutrons were also broadly used to induce mutations. Despite all their benefits, they can also cause hereditary aberrations. Physical mutagens require complicated tools and facilities to induce mutations. Some of the physical mutagens being used in mutation breeding with their characteristic features are given in Table 2. Chemical Mutagens  The most widely used chemical mutagens are a group of alkylating compounds. On the mutant varieties database, among the registered Table 2  Physical mutagens and their characteristics Mutagens Alpha particles Beta particles X-rays Gamma rays Ultraviolet rays Neutrons Ion beams

Penetrance Very shallow Shallow Penetrating Very penetrating Penetrating Up to several centimeters Very penetrating

Source Radioisotopes P32 and C14 X-ray machine Co60 and Cs137 UV source lamp U235 Particle accelerator

Hazardous effects Very strong Mild Strong Strong Strong Very strong Strong

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varieties more than 80% have been developed using alkylating agents. These agents are mostly found in classes of compounds including alkyl halides, alkyl phosphates, alkyl sulfates, alkylnitrosoamides, diazoalkanes, mustards of nitrogen and sulfur, ethyleneimines, ethyleneimides, epoxides, alkyl methanesulfonates, and many others. The most effective alkylating agents are 1-methyl-1-nitrosourea, 1-ethyl-1-­ nitrosourea and ethyl methanesulfonate (EMS). Wani et al. (2014) described that among the alkylating agents, some are capable of reacting with DNA to alkylate the phosphate group, purines, and pyrimidines of nucleic acids. In addition, base analogs such as 5-bromouracil are widely used to induce mutations by binding during DNA replication. In many plants, chemical mutagens have generated many phenotypic modifications by affecting the activities of the proteins (Laskar et al., 2015, 2019; Wani et al., 2021). In crop plants, resistance against the herbicide glyphosate has been developed through mutation induction which altered the protein binding sites (Bradshaw et al., 1997; Wakelin & Preston, 2006). Similarly, in a mutant variety of leguminous plant Medicago truncatula, resistance against the herbicide sulphonyl urea has been generated through mutation breeding (Oldach et al., 2008). Some antibiotics such as mitomycin C, azaserine, and streptonigrin are also used as chemical mutagens. Thus, chemical mutagens are a class of compounds that have brought great advances in mutation breeding programs of crops. Chemical mutagens have an advantage over physical mutagens not only due to their milder effects, but they are also relatively easier in their application. Using chemical mutagens, the mutation induction rate is relatively higher, and are thus more effective than physical mutagens (Acquaah, 2006). Although most of the chemical mutagens are synthetic in origin, a few are biological in origin (e.g., the strong mutagen streptozotocin has been isolated from the bacterium species Streptomyces achromogenes). They are usually applied to the source material for mutation induction only by soaking it in the solution of the respective mutagen. However, they are usually hazardous to health and extra care is needed for their application. Before the application of the chemical mutagenic agent, its choice, method of application, hazardous effects, and methods of disposal are the important factors to which the breeder must give due consideration (Khursheed et al., 2018a, b, c). Several chemical mutagens are known, but only a few have been used in plant breeding programs for mutation induction. Some of the widely used mutagens with their characteristic features are given in Table 3. Chemical mutagens cause less damage to chromosomes as compared to physical mutagens (Goyal et al., 2021a, b). These mutagens are also the source of high rate Table 3  Chemical mutagens and their characteristics Name of mutagen Alkylating agents Base analogs Antibiotics

Mode of action React with bases by adding methyl or ethyl groups which induces mutation during DNA replication Replaces nitrogenous bases during DNA replication Chromosomal aberrations.

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of mutation along with allelic mutations at target sites, and they mostly cause point mutations which are usually uniformly spread over the whole genome. Another advantage of using chemical mutagens is the availability of standardized protocols for mutation induction in almost all-important agronomic crops which can either be applied to seeds, in vitro grown calli, or any other tissue. Chemical mutagenesis is the most common, but due to its high mutation rate, selecting the desired mutation is time-consuming because it necessitates multiple backcrosses. In woody tissues, mutation induction is relatively difficult due to the low penetration of these mutagens in the plant tissue. Moreover, these tissues have low regeneration capacity. Mutation induction in recalcitrant seeds as well as in dormant tissues is also difficult. In dormant seeds, some pretreatments are often required to break the dormancy. Chemical mutagens are not capable of inducing large chromosomal mutations which are heritable, and thus have limitations in their use. Additionally, they have safety concerns, not only in their usage but also in their storage and disposal due to their carcinogenic or toxic properties.

1.2 Mutation Breeding for Crop Improvement Mutation selection is the process to incite mutations to improve crops. In mutation selection, desired mutations are induced in crops by physical or chemical mutations (Raina et  al., 2017; Laskar et  al., 2018a, b; Khursheed et  al., 2015, 2016; Sellapillaibanumathi et  al., 2022). The induced mutations are released into new varieties or used as parents in subsequent hybridization programs (Goyal et  al., 2019a, b). Mutation breeding programs should be large enough to screen large populations with well-planned and appropriate conditions (Wani et  al., 2017; Raina et al., 2016, 2020b, 2022a, b, c, d; Goyal et al., 2020a, b; Sellapillai et al., 2022). Plant breeders have since long developed crops to meet mankind’s need for food, fiber, and fuel through conventional breeding approaches. Gene pool concept for exploiting genetic variability in breeding program was given by Harlan and de Wet (1971). (GP-1) or primary gene pool encompass those species that are easily cross-­ compatible with each other, while the secondary gene pool (GP-2) covers those crop species that cross with a different combinable species. Crossings in the tertiary gene pool (GP3) produce viable but sterile hybrids with the crop species of interest. The quaternary gene pool (GP4) prevents introgression between cultures or other organisms of interest through sexual recombination. In this case, it is necessary to use transformation to insert the DNA sequences of any species known as transgenes into the crop of interest. Transformation, also known as genetic modification, is the process of introducing, integrating, and expressing a transgene into a host plant to produce a transgenic plant or genetically modified organism. Nowadays, with the help of rDNA technology, gene transfer technology, and tissue culture technology, transgenics are efficiently developed for a variety of crops(Gosal & Gosal, 2000; Chahal & Gosal, 2002; Altman, 2003; Grewal et al., 2006; Kerr, 2011; Nayak et al., 2011; Bakshi & Dewan, 2013; Kamthan et al., 2016; Arora & Narula, 2017; Cardi et al., 2017; Tanuja & Kumar, 2017).

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Transgenic breeding involves the transfer of agronomically important genes from a donor to a generation free of unwanted genes, eliminating the need to re-­ cross the recipient’s parents. The main goal of transformation is to induce genetic changes in plants to increase herbicide resistance and pest resistance, bacteria, fungi, and viruses. This is because these agents cause significant losses in crop production (James, 2007). Other goals of transformation include creating crops known as “designer crops” with higher nutritional value, extending the shelf life of crops, and the biosynthesis of pharmaceuticals and secondary metabolites, antibodies, vaccines, and industrial enzymes. Regulatory agencies around the world are responsible for introducing genetically modified crops that produce pharmaceuticals and other important molecules (Ramessar et al., 2008). But there are still some ethical issues in public domain related to the adoption and production of transgenic crops (Ming et al., 2008). A revolutionary and strategic improvement in crop research has been possible through genetic engineering. Genetic engineering is a well-established and widely used biotechnology tool that uses transformation techniques to clone genes and transfer specially designed genetic constructs into plants. Recombinant DNA (r-DNA) is a molecule obtained by joining together two or more DNA segments of interest known as DNA inserts. Paul Berg combined the SV40 monkey virus and ʎ virus to produce recombinant DNA called ʎ bacteriophage (Jackson et al., 1972). In 1985, a polymerase chain reaction was invented for in vitro DNA amplification. In vitro culture and regeneration protocol were developed for several crops. In 1996, Monsanto developed and commercialized the first insect-resistant transgenic Bollgard I variety of cotton using Cry1Ac gene (Purcell & Perlak, 2004), followed by glyphosate-tolerant Roundup Ready cultivar of soybean which became the most extensively grown transgenic crop globally (Konduru et al., 2008; Green, 2009). Herbicide-resistant Bt maize was first time grown in the United States, Canada, and Europe in 1997 which was commercially used in 11 different countries by 2009 (James, 2016). The launch of Bt cotton for profit-making in BT MECH 12, BT MECH 162, and BT MECH 184  in 2002 was recognized as a milestone in Indian Cotton Improvement History (GEAC) by the Government of India’s Genetic Engineering Approval Committee (James, 2016). Later in 2007–08, In India, a sum of 131 varying Bt hybrids were grown (James, 2007) and today most of the world’s corn and cotton are of Bt varieties. Ten years of efforts by Peter Baird and Ingo Potricus became reality in 2002, when β-carotene rich rice cultivar known as Golden Rice, was produced by introducing two phytoene synthase (psy) genes from the daffodil plant and a bacterial phytoene desaturase (crtI) gene from Erwiniaamylora (Ye et al., 2000; Burkhardt et al., 1997; Beyer and Hoa et al., 2003). This is known as the first generation of golden rice or Golden rice1 (GR1) and became the first biofortified GM food crop in the world (Welch, 2002). Second generation of golden rice 2 (GR2) was produced to elevate the amount of β-carotene (provitamin A) in GR1 by using genes (psy1) from Zea mays (Zmpsy1), the carotene desaturase I (crtI) gene from Pantoeaananatis. Phosphomannose isomerase (pmi) gene was used as a marker isolated from Escherichia coli (Paine et al., 2005). Golden Rice2 contains 31 μg/g β-carotene compared to 1.6 μg/g in Golden Rice1 (Al-Babili & Beyer,

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2005; Bollinedi et al., 2014). The worldwide area under transgenic crops has been recorded as 191.7  million hectares in 2018, indicating a 113-fold increase from 1.7 million hectares in 1996 (ISAAA, 2018). Thus, transgenesis has been considered as a rapidly adopted technique in crop improvement for the last three decades.

2 Transformation Techniques or Gene Transfer Methods Transformation is the uptake of foreign DNA or transgene by plant cells, followed by stable integration into the cell’s nuclear genome (Sharma et al., 2005). In the age of scientific research, many transformation methods for the production of these plants have emerged (Rasul et al., 2014). All such methodologies for genetic transformation can be classified into two heads: (i) Agrobacterium-mediated (Sun et al., 2006) and (ii) Direct gene transfer (Yao et al., 2006). The transformation techniques are associated with gene construct, a vector to carry the DNA being transferred, and reliable shoot regeneration protocols. Agrobacterium-mediated gene transfer and particle gun method using particles coated with plasmid DNA are the most commonly used transformation methods for the development of transgenic plants (Chung et al., 2000; Sharma et al., 2005; Davey et al., 2008; Widholm et al., 2010).

2.1  Agrobacterium-Mediated Gene Transfer The first transgenic plants were created by gene transfer of A. tumefaciens (Barton & Chilton, 1983; Fraley & Horsch, 1983). This method delivers DNA into dicot and monocot plants (Kunik et al., 2001). Agrobacterium tumefaciens and Agrobacterium rhizogenes are commonly used for gene transfer to produce transgenic plants. These soil bacteria cause diseases of crown gall disease (A. tumefaciens) and hairy root (A. rhizogenes) in dicotyledonous plants due to infection (Levee et al., 1997, 1999). Auxin and cytokinin synthesis genes are found on plasmid Ti which are responsible for tumorigenesis in plant cells. Ti and Ri plasmids have little symmetry but are functionally nearly identical. PlasmidTi is a large junctional plasmid about 150–250 kb in size and contains two elements: a TDNA segment and a vir region (Goodner et al., 2001; Wood et al., 2001). T-DNA segment is 23 kb in size, flanked by direct repeat border sequences of 24 bp, and contains genes for tumor induction and opine biosynthesis. Because the T-DNA segment is transferred to plant cells, Agrobacterium becomes a natural genetic engineer (Merlo et al., 1980; Zambryski et al., 1982; van Haaren et al., 1988). The T-DNA segment consists of three genes, two of these iaaM and iaaH genes encode an enzyme that simultaneously converts tryptophan to indole-3-acetic acid (IAA) and the third is the ipt gene, which encodes an enzyme for cytokinin production. Two border sequences, namely, right border (RB) and left border (LB) are responsible for the transfer of the T-DNA segment and become integrated into the

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nuclear genome of the plant cell. The virulence (vir) genes are the Ti plasmid’s second most important component. The virulence region’s VirA and VirG genes mainly help to control the activation of vir operon through transcription (Stachel & Zambryski, 1986).

2.2 Agroinfection Agricultural inoculation or Agroinfection (Grimsley et  al., 1986; Grimsley & Bisaro, 1987) is the introduction of infectious plant components using Agrobacterium. This method may be used to distribute viral DNA in two separate ways and is relevant to molecules that can replicate independently of plant chromosomal DNA. In the first method, viral DNA is juxtaposed with bacterial T-DNA, and the virus distributes systemically to the host plant following inoculation. It is not necessary to prepare nucleic acids or insect vectors for this approach. In another method, viral nucleic acid sequence can be integrated with Agrobacteria which helps to transfer genes to the nuclear genome of each cell of the transgenic plant. When the cauliflower mosaic virus (CaMV) genome was originally introduced, it was extremely infectious to turnips when deposited on T-DNA but not to other plasmid clones, indicating that the infection was not caused by Agrobacterium lysis (Grimsley et al., 1986). Basic features of virology, TDNA recombination, and transport have all been studied using agro-infection as a DNA delivery.

2.3 Direct DNA Transfer Direct gene transfer entails the introduction of external DNA (naked DNA) into the plant nucleus. The outer membrane of a plant cell is first disrupted, letting foreign DNA to move through. The majority of direct gene transfer procedures are straightforward and successful. Gene expression in these transgenic plants, on the other hand, can be altered either transiently or permanently. The two most common types of direct gene transfer methods are physical and chemical gene transfer.

2.4 Physical Method of Gene Transfer 2.4.1 Particle Gun Method The biolistic (biological ballistic) microprojectile method of DNA delivery was  developed as a solution to get over Agrobacterium’s host range limits (Weissinger et al., 1987). Microprojectile method or particle bombardment method has been most commonly used for plant transformation (Sharma et al., 2005; Davey

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et  al., 2008). High-velocity particles are used in the micro-projectile method to pierce cell walls and deliver DNA into cells, bypassing Agrobacterium’s host target area limitation. Hence, this method of genetic transformation is most adaptable, regardless of the type of cell and genotype, and used in transformation of much variety of plant cells. It has developed most of the resistant plants in legumes and also in cereals through transformation (Altpeter et al., 2005). 2.4.2 Electroporation All living cells’ lipid bilayers of cell membranes are reversibly permeabilized by short, high-intensity electric pulses. The electric pulses cause the plasmalemma to be compressed and thinned, causing transitory pore development in the plasma membrane (Neumann & Rosenhec, 1972). A wide spectrum of macromolecules, including proteins and nucleic acids, can diffuse through the pores that form transiently. The electroporation apparatus is quite straightforward. Protoplasts, cells, and tissues contained in a liquid culture media enclosed in a discharge chamber are subjected to high electric fields. A capacitor discharge generates the electric field, which can reach voltages of up to 2500 V in some commercial devices. In plant cells and tissues, electroporation has been utilized to transfer DNA. Corn, tobacco, soybean, sugarcane, rice, and oilseed rape have all been shown to have electroporation-­ mediated gene transfer into plant protoplasts (Fromm et al., 1986; Shimamoto et al., 1989; Dhir et al., 1991; Chowdhury & Vasil, 1992; Bergman & Glimelius, 1993). 2.4.3 Microinjection Injecting DNA straight into the nucleus is unique and the most direct method of gene transfer. The insertion of small glass needles into the nucleus of certain cells to transfer DNA is controlled by electromechanical systems (Spangenberg et  al., 1986). Gels or poly-L-lysine-coated surfaces immobilize recipient cells or protoplasts in a holding capillary. The nucleus is visible, and then DNA is mechanically inserted. The recipient cells are cultivated and then grown on culture conditions with a selection agent to ensure that the inserted genes are integrated. By introducing Ti plasmid into alfalfa protoplasts, the microinjection method was used to make transgenic alfalfa (Reich et  al., 1986). The microinjection technique is time-­ consuming and labor-intensive, yet is effective. 2.4.4 Microinjection Microinjection is the use of a hypodermic syringe to inject plasmid DNA or uncloned native DNA into the lumen of a growing inflorescence. The DNA is believed to be picked up by microspores at a specific point of their growth. The first study on transformation in rye using the microinjection approach was published in

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1987 and it revealed a relatively low frequency of transformed plants (0.07%). However, sustained transformation was not achieved in subsequent tests with barley and wheat. 2.4.5 Fabre-Mediated DNA Delivery Silicon carbide fibers with a diameter of 0.6 m and a length of 10–80 m are used to transfer DNA into the cytoplasm and nucleus of cells. Silicon carbide is a naturally hard material that fractures easily to produce sharp edges capable of penetrating plant cell walls. DNA can be transferred into plant cells through the pores produced by the whiskers. By vigorous vertexing, whiskers of silicon carbide are combined with recipient cells and plasmid DNA before being plated on culture media. Following that, the cultivated cells are examined for DNA insertion and integration with nuclear DNA. DNA transfer by silicon carbide whiskers has been shown to produce stable altered plant cells (Kaeppler et al., 1994; Kaeppler & Somers, 1994).

2.5 Chemical Method of Gene Transfer 2.5.1 Polyethylene Glycol (PEG)-Mediated Plant protoplasts treated to polyethylene glycol absorb more DNA from their surroundings and integrate it into the chromosomal DNA of the plant (Mathur & Koncz, 1997). Protoplasts are then cultivated in circumstances that allow them to establish cell walls, begin dividing to form a callus, produce branches and roots, and regenerate complete plants. The specific process for chemical gene transfer is unknown, although it is thought that PEG increases osmotic pressure and promotes protoplast contraction, allowing the divalent cation/DNA complex to be endocytosed more easily (Lazzeri et al., 1991). 2.5.2 Lipofection Lipofection is one of the processes in which liposomes is used to insert DNA into cells. Liposomes are nothing but phospholipid vesicles that has a diameter from 0.2 to 1.6 mm, depending on extrusion operations and measurement methods (Olson et al., 1979; Szoka et al., 1980; Jousma et al., 1987). A fusiogenic substance, such as polyethylene glycol (PEG) or polyvinylalcohol, can be used to fuse the liposomes with the carrier molecules by a strong calcium ion treatment to induce protoplast fusion (Keller & Melchers, 1973). This liposome-mediated DNA transfer technique was equal to PEG-mediated operations, but less efficient than electroporation, according to the marker gene assay (Caboche, 1990). Only a few plant species have been studied for liposome-mediated DNA transfer to create transgenic plants, with some success reports.

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The true success and implementation of several transformation processes are uncertain. Some major challenges encountered in transferring genes via Agrobacterium-mediated delivery from rice to other cereals is an excellent example of how particular crops necessitate the specific modifications of transgenic plant methods (Shrawat & Lorz, 2006). However, advances in plant bioinformatics and gene sequencing, as observed in rice (Matsumoto et al., 2005), will make transformation techniques more generally applicable. The CRISPR/Cas9 system (Wang et al., 2016; Arora & Narula, 2017) and RNA interference (RNAi), in which RNA molecules neutralize targeted mRNA molecules to inhibit gene expression or translation (Kim & Rossi, 2008; Gupta et al., 2013; Younis et al., 2014), have recently broadened the scope of genome engineering.

3 Applications of Transgenesis in Crop Improvement To develop improved plant varieties having good agronomic traits with high level of resistance against different biotic and abiotic stresses is the primary aim of plant breeders. Through conventional plant breeding methods, there is little scope or no assurance of gaining particular target gene combination by millions of manual crosses generated. Many unwanted genes got transported side by side (Linkage Drag) with target genes; or, another situation is that one target gene is obtained while the other is lost due to the mixing of both parental genes and also due to more or less re-assortment of genes randomly in progeny. Hence, these problems create difficulties to plant breeders to achieve their goals for crop improvement. In the current era, genetic engineering emerged as a major technology for crop improvement because it allows direct transfer of one or few desirable gene(s) between closely as well as distantly associated organisms to achieve target traits.

3.1 Abiotic Stress Tolerance The world population is increasing exponentially which lead to continuous increase in the demand for food globally. But, abiotic stresses such as saline soils, water deficits, heavy metal contamination, and low- or high-temperature climatic challenges limit plant cultivation and productivity (Bartels, 2001). It is recorded that approximately more than 50% of yield reduction in crops is caused by abiotic stresses (Bray et al., 2000). In order to cope up abiotic stresses, plants produce some compounds called osmoprotectives, also known as osmolytes, such as glycine, betaine, sugars, alcohols, and some amino acids which enhance the osmotic potential and permit more influx of water; hence, plant cell stabilizes macro-molecular structures (Holmberg & Bulow, 1998). So, many approaches were made to enhance the osmolytes production in transgenic crops. In case of transgenic rice and tobacco, the level of disaccharide trehalose sugar was raised which imparts resistance against low-­ temperature stress, salinity, and drought (Garg et al., 2002). The aldose-aldehyde

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reduction gene responsible for ectopic expression in alfalfa crop transfer through Agrobacterium prevents the oxidative stress by eradicating aldehydes and make plant tolerant to heavy metal, salinity, and dehydration stress (Oberschall et  al., 2000). Similarly, in Festuca arundinacea Schreb (Tall fescue), AtHDG11 gene was introduced by Agrobacterium which provides tolerance against drought stress (Cao et al., 2009).

3.2 Biotic Stress Tolerance One way to secure food production is to reduce the loss caused by insect pest attacks, disease-causing pathogens, and weeds. Pathogens cause estimated production losses of 10–16% of global production (Chakraborty & Newton, 2011), whereas insect pests cause estimated production losses of 14–25% of global crop production (DeVilliers & Hoisington, 2011). 3.2.1 Resistance to Insects Bt corn has been modified using the cry 1 Ab, cry 1 Ac or cry 9 Ac in order to protect it from Ostrinianubilalis and Sesamianonagriodes, or with cry1F from Spodoptera frugiperda, and cry 3Ab, cry 34Ab and cry 35Ab from the infestation of rootworms of the genus Diabrotica (James, 2012). Commercially, cultivated Bt cotton contains either cry1Ac or a combination of cry1Ac and cry1Ab (James, 2013). Bt potatoes carrying the gene cry3Aa, which are resistant to the pest Leptinotarsa decemlineata, have been successfully farmed commercially in Europe and North America (Coombs et al., 2002). In 2008, a genetically modified (GM) Bt eggplant intended for management of Leucinodesorbonalis was released in India. Bt crucifer vegetables targeted against Plutellaxylostella are under development (James, 2012). In future, commercialization of Bt rice expressing the Bt-toxin is planned (James, 2012). Various GM rice cultivars have completed field and environmental tests, and four paddy cultivars underwent a field experiment in 2001. For the first time in Iran, Bt alfalfa was produced utilizing the gene cry3a against Hyperapostica (Tohidfar et al., 2013). The Bt trait was introduced into the soybean crop utilizing two cry genes from cry 1Ab, cry 1Ac and cry 1F (James, 2013). The brown plant hopper (Nilaparvatalugens), a significant pest of high-yielding paddy types, has exhibited resistance to transgenic paddy expressing the Galanthus nivalis agglutinin (GNA). Allium leaf agglutinin (ASAL) present in several plants possesses an insecticidal property. Transgenic rice plants introduced with ASAL gene has shown resistance to hopper pests (Saha et al., 2006). The alpha-amylase inhibitor gene of Phaseolus vulgaris origin has been introduced into gram through Agrobacterium-mediated indirect gene transfer (Ignacimuthu & Prakash, 2006). Seed extracts from Coffea arabica plants have been genetically modified with the alpha-amylase inhibitor gene and show reduced amylolytic enzyme activity up to 80% (Barbosa et al., 2010).

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3.2.2 Diseases Resistance Resistance to Fungal Diseases In recent years, scientists have investigated the possibility of transforming plants using enzymes derived primarily from plants, such as 1, 3-glucanase and chitinase. Transgenic crops with improved fungal disease resistance were developed by inserting chitinase genes from both plant and microbial origins into the plants. Cotton (Tohidfar et al., 2012), peanut (Rohini et al., 2000), and grapevine are examples of such plants (Yamamoto et al., 2000). Many plant cell walls contain polygalactouronase inhibiting protein (PCIP), a type of glycoprotein that inhibits the fungal action of fungal endo polygalactouronases (Oelfose et al., 2006). Resistance to fusarium disease was demonstrated in wheat kernels from transgenic plant which is developed through modification of L3 gene (Di et al., 2010). Numerous R genes (resistance genes) for Phytophthora infestans, the late blight pathogen, have been discovered from a variety of sources (Ballvora et  al., 2002; Pel et  al., 2009). Co-expression of LpiO (as an effector) and Rpi-blb1 (as a resistance gene) in Nicotiana benthamiana discovered Rpi-sto1 and Rpi-pta1 as late blight-resistant genes (Vleeshouwers et al., 2008). According to another study, when three broad-­ spectrum Potato R genes (Rpi), Rpi-blb3 (Solanum bulbocastanum), Rpi-vnt1 (Solanum venturii), and Rpi-sto1 (Solanum stoloniferum), were stacked into the susceptible cultivar “Desiree,” over 4% of transformants showed horizontal resistance to Phytophthora. Thionins, osmonins, and lectins are all PR-proteins (pathogenesis-related proteins/peptides) that have been connected to the establishment of plant disease resistance (Gentile et al., 2007; Jing et al., 2009; Bashir et al., 2015; Punja et al., 2016). Activating phytoalexins is another approach for giving resistance to disease-causing pathogens in plants. Rice resistance to Pyriculariaoryzae was increased by inserting a stilbene synthase gene (STS) from Vst1, a key enzyme in the production of phytoalexin in grapes (Coutos-Thevenot et  al., 2001). Powdery mildew resistance was also improved in barley (Liang et al., 2000). In response to UV and blast infestation, the mitogen-activated protein kinase (MAPK) cascade, specifically OsMKK6, regulates the genes involved in phytoalexin synthesis in rice (Wankhede et al., 2013). According to the study, mitogen-activated protein kinase is the most important component in the MAPK cascade (MAPKK). They looked at the expression pattern of rice MAPKKs in response to UV radiation and were able to create transgenic rice lines with the phytoalexin-producing OsMKK6 gene. Resistance to Viral Diseases So far, some 700 plant viruses have been discovered that attack crops and cause global food loss. Virus infections were traditionally handled by removing sick plants from the standing crop, eliminating vectors chemically (no effective viricides exist), and using virus-free plant material. The generation of viral resistance via coat

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protein is one of the most significant achievements in genetic engineering. Two significant crops that have been conferred viral resistance using this technique are potato event HLMT15-15, which is resistant to Potato virus Y (PVY), and potato event RBMT21-350, which is resistant to Potato leaf roll virus (PLRV) (James, 2013). To confer resistance to the Papaya ringspot virus (PRSV), the viral coat protein sequence was employed as a transgene (Gonsalves, 2004). Otang Ntui et al. (2014) used CMV-derived dsRNA to create transgenic tobacco. The virus’s faulty mobility protein (MP) induces the replication-associated protein generated by AC1 (African cassava mosaic virus replication-associated) or the Cl gene of gemini virus through transgenic plants (Hallwass et al., 2014; Peiro et al., 2014). Through the expression of antibodies, a unique technology known as antibody engineering has been created to make pathogen-resistant plants (Cardoso et al., 2014). Resistance to Bacterial Diseases Bacterial leaf blight (BLB) is now a severe disease caused by the pathogen Xanthomonas oryzaepv. Oryzae is in cultivated rice (Oryza sativa). Pathogenesis-­ related genes (PR) are regulated by ethylene-responsive transcription factors (ERF) (Grennan, 2008). Many phytopathogenic bacteria need the Hairpins (hrp) genes to induce a hypersensitive response (HR) in non-host or resistant host plants. Transgenic bacterial disease-resistant plants have been developed exploiting this strategy. The chromosome in Erwinia amylovora causing fire blight disease in apple and pear contains a gene hrpN encoding HairpinNEa (HrpNEa). Several studies have revealed that increased levels of HrpNEa in transgenic plants boost bacterial resistance (Malnoy et al., 2005). Plant transformation with a pathogen-derived gene encoding a toxin-detoxifying enzyme could also be used to create plant resistance to bacterial infections. Pseudomonas syringe pv. tabaci generates tabtoxin, which is transformed to tabtoxinine—lactam in plants, inhibiting glutamine synthase and causing lethal ammonia buildup. The pathogen uses the tabtoxin resistance gene (ttr) to acetylate the tabtoxin in order to defend itself against the toxin. Transgenic tobacco with ttr gene showed reduced symptoms of disease (Batchvarova et al., 1998). 3.2.3 Herbicide Tolerance There are 222 occurrences in the 14 marketed herbicide-tolerant crops introduced by James (2013). Glyphosate-tolerant maize, soybean, canola, and cotton are the most frequent lines among these crops (Table 4). Gat, bxn, surb, dmo, epsps, 2mepsps, and other herbicide tolerance genes are widely found in imported crops. Glyphosate is the active component in Roundup. It’s a commonly used non-­selective post-emergence herbicide. Glyphosate, like enolpuruvate, binds to and inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is involved in the shikimic acid pathway and produces chorismate-derived metabolites like

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Table 4  Transgenes transferred for different biotic, abiotic stresses, and quality improvement in major crops Crop Rice

Gene/protein OsMYB3R-2 (DNA-­ binding domain) cry2AX1 p35S-Xa5-nost OsDREB1A, OsDREB1B, AtDREB1A, AtDREB1B and AtDREB1C AMV5’UTR-CYP2B6 CYP1A1, CYP2C19-nost AHAS mutant gene

Wheat

Avidin ZmAGPase CspB and CspA Ace-AMP1

Maize

Acetohydroxy acid synthase (ahas) mutant bxn

Cotton

betA (glycine betaine) cryIAb (Bt toxin) Hpa1Xoo Anti-sense coat protein AtRAV1/2 Isopentenyl transferase (IPT) gene NPR1

Targeted trait Enhanced cold tolerance To control Helicoverpaarmigera Resistance to blight disease

Reference Ma et al. (2009)

Tolerance to drought, high salt, and cold Enhanced tolerance toward many herbicides Dual herbicide tolerance (bensulfuron methyl and glufosinate) Resistance to Sitophilus granaries Higher grain yield

Ito et al. (2006)

Drought-resistant (Drought Gard) Enhanced antifungal activity Tolerance to herbicides (e.g., sulfonylurea and imidazolinone) Tolerance to bromoxynil Drought tolerance Cotton bollworm Resistance against Verticillium dahlia Resistance against cotton leaf curl disease Resistance to drought stress Enhancing salt stress tolerance with delayed leaf senescence Resistance against Verticilliumdahliae, Fusarium oxysporum f. sp. vasinfectum, Rhizoctoniasolani, and Alternariaalternata

Gonzalez et al. (2019)

Udayasuriyan et al. (2010) Jiang et al. (2006)

Kawahigashi et al. (2006) Fartyal et al. (2018)

Abouseadaa et al. (2015) Smidansky et al. (2007)

Roy-Barman et al. (2006) Le et al. (2010)

Tan et al. (2006) Quan et al. (2004) Tohidfar et al. (2008) Miao et al. (2010) Amudha et al. (2011) Mittal et al. (2014) Liu et al. (2012)

Parkhi et al. (2010)

(continued)

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Mutagenesis and Transgenesis in Plant Breeding Table 4 (continued) Crop Soybean

Gene/protein ATPG8 and ATPG10

Targeted trait Reference Increased seed yield Cho et al. (2019) and delayed senescence

GmFad2-1b

For increased oleic acid Zhang et al. (2014) content (up to 51.71%) Tolerance to glyphosate Mathesius et al. (2009)

GAT4601, GM-HRA SbDV-CP-specific siRNA Overexpressed oxalate decarboxylase (OXDC) Arabidopsis AtCSP3 (Cold shock protein) Brassica codA (Glycine betaine) juncea L. Alfalfa cry3a (Bt toxin) Viral coat protein Tobacco

coat protein (CP)

coat protein (CP)

Soybean dwarf virus (SbDV) resistance Sclerotinia stem rot resistance Freezing tolerance

Tougou et al. (2006)

Tolerance to salt stress conditions Insect resistance

Parsad et al. (2000)

Cunha et al. (2010) Kim et al. (2009)

Tohidfar et al. (2013)

Alfalfa mosaic virus Gomase and Kale (2015) (AMV) Resistance to Cowpea Mundembe et al. (2009) aphid-borne mosaic virus Resistance against Jacquemond et al. (2001) Cucumber mosaic virus

aromatic amino acids. Glyphosate could damage plants by deactivating this enzyme, making protein synthesis difficult (due to the absence of aromatic amino acids). Glyphosate tolerance in genetically engineered plants was obtained by introducing a mutant EPSPS synthase gene that can distinguish between its native substrate enolpyruvate and glyphosate (Stalker et  al., 1985). Glyphosate resistance was increased by up to 200  mM when the EPSPS gene from Pseudomonas stutzeri A1501 was used (Aimin et al., 2008). Another plant currently being investigated for glyphosate resistance is Amaranthus palmeri (Gaines et al., 2010). The phosphinothricin acetyl transferase (PAT) gene encodes a protein that acetylates the free NH2 groups of phosphinothricin (PPT), a herbicide component, rendering it inactive. As a result, a transgenic line that produces PAT, such as one that expresses the bar gene in sweet potatoes, can be created. Two other genes that can inactivate glyphosate and glufosinate are the glyphosate oxidoreductase (GOX) gene from Ochrobactrumanthropi strain LBAA and the pat gene, homologous to bar, from Streptomyces viridochromogenes, which produces N-acetyltransferases (Green & Owen, 2011). Tobacco received the GmGSTU gene from soybean. The GmGSTU4 is a catalytic isoenzyme for the diphenylether herbicide fluorodifen/alachlor (Benekos et al., 2010). Maize has just

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received an imidazolinone resistance (IR) XA17 gene. Herbicides imazaquin and nicosulfuron were discovered to be resistant in transgenic lines. In these lines, weed management has been employed to drastically minimize crop losses (Menkir et al., 2010). Introduce the glyphosate N-acetyltransferase (Gat) from Bacillus licheniformis into crop plants as another way to deactivate glyphosate (Siehl et al., 2007). Glyphosate resistance was achieved by genetically modifying soybean and corn plants using GAT gene (Castle et al., 2004). In addition to the genes stated above, Table 4 contains the other genes that have been turned into plant species.

3.3 Modified Product Quality In plant Transgenic for Better Nutrition, heterologous systems of two genes “phaseolin” and Ama-1 have been used. The AmA-1 gene was introduced into the potato and resulted in an increase in yield and protein content. The antisense RNA approach was used to mute the polyglygactonase gene, which is responsible for fruit rot, and FlavrSavar tomato was created. “Golden rice” is produced by the introduction of provitamin A gene and carotene genes. Transgenic plants that produce vitamins have also been developed (Herbers, 2003), and multigene engineering is becoming more popular (Daniell & Dhingra, 2002). Transgenic plants that produce specialty chemicals and biopharmaceuticals have also been developed for molecular farming and pharmaceutics (Fischer & Emans, 2000). The main goal of these crops is to add value to foods like tomatoes with high lycopene, flavonols as antioxidants, cavity-­ fighting apples, rice enriched with carotene and vitamin A (golden rice), iron-­ pumping rice, vitamin E-rich canola (golden brassica), proteinaceous potatoes, edible vaccines, and decaffeinated coffee, which are some of the most promising genetically modified foods for the future (Doshi et al., 2013). Hepatitis B viral vaccine was introduced into transgenic tobacco and potatoes. Several important agronomic genes have, therefore, been identified from various organisms, cloned, and suitable plant transformation constructs were produced. Agrobacterium and “particle gun” technologies for genetic transformation of a wide range of field, fruit, vegetable, forest crop, and ornamental plant species have been developed and are currently being utilized for genetic transformation of a wide range of species.

3.4 Pollination Control System Crossing two distantly related kinds of the same crop plant produces hybrid crops. Plant pollination must be managed for success. Before pollen is released, the male flower components are normally removed by hand. In addition, sterilized plants have been genetically modified using a gene from the Bacillus amyloliquefaciens bacteria (barnase gene). For male sterility, this gene is dominant. Male sterility is caused by the introduction of the bacterial barnase gene, whereas the formation of

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the restorer line is caused by the introduction of the bacterial barstar gene into another plant. The hybrid that results is totally fertile. In maize and oilseed rape, this method has been economically utilized (Wu et  al., 2016). As a result, Brassica napus hybrids are being tested in the field in India. Similarly, in rice and wheat, hybrids are produced (Ray et al., 2007).

4 Achievements To suppress lepidopirus pests, the insect-resistant transgenic cultivars Bolgard I (Cry1Ac genes) and Bolgard II (Cry1Ac and Cry2Ab genes) were developed in cotton (Shelton et al., 2002; Head & Dennehy, 2010). Mahyco Seed Company launched three Bt. cultivars commercially in 2002–03: MECH 12, MECH 162, and MECH 184. In India, a total of 131 different Bt hybrids were cultivated in 2007–08 (James, 2007), and Bt cultivars now account for the majority of the world’s corn and cotton. The FlavrSavr tomato variety was created to prevent fruit softening. Dhara Mustard Hybrid-11, often known as DMH-11, is a genetically engineered mustard variety produced by Delhi University’s Centre for Genetic Manipulation of Crop Plants utilizing the barnase/barstar male sterility system; however, it is yet to be licensed for commercial production. If GEAC approves it, will be the country’s second GM crop, following Bt Cotton, and the first transgenic food crop. By introducing a single gene, the EPSPS (5-enol-pyruvylshikimat-3-phosphate synthase) pathway, a glyphosate-resistant, roundup-ready transgenic soybean variety has been generated. Liberty link transgenic cultivars for Glufosinate-ammonium tolerance have been developed in maize and soybean. Cotton BXN has been genetically modified to be resistant to the herbicide bromoxynil. Golden Rice (GR1) was first manufactured in 2002. In 2005, Syngenta released “Golden Rice 2,” an enhanced type of golden rice that contains 31 g/g to-carotene, compared to 1.6 g/g in Golden Rice 1 (Al-Babili & Beyer, 2005; Bollinedi et al., 2014). Moondust is a Carnation variation with a different blossom hue. The HarvXtra alfalfa cultivar was created to have a different lignin production. The Golden Delicious and Fuji transgenic apple varieties were developed to prevent fruit from browning. Laurical canola has been developed in Brassica napus to produce more lauric acid. In 23 years of commercial cultivation (1996–2018), the global area of biotech/ GM crops reached 2.5 billion hectares. In 2019, 29 countries grew biotech/GM crops including 24 developing and 5 developed countries. In 2019, 190.4 million hectares of biotech crops were planted, helping to provide food security, mitigate climate change, and improve the livelihoods of up to 17 million biotech farmers and their families around the world. The United States, Brazil, Argentina, Canada, and India were the top five countries having the most biotech crops. In 2019, 1.95 billion individuals were affected by high biotech adoption rates in the top 5 biotech countries. The entire worldwide biotech crop area was grown by 54% in developing countries. A total of 44 countries (18 EU countries + 26 non-EU countries) have formally imported biotech/GM crops. Soybeans (95.9  million hectares), maize

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(58.9  million hectares), cotton (24.9  million hectares), and canola (24.9  million hectares) were the four biotech/GM crops with the greatest hectares in 2019. Alfalfa, sugar beets, papaya, squash, eggplant, potato, apples, pineapple, and sugarcane are among the other transgenic crops cultivated in 2019. Herbicide tolerance was still the most common characteristic in soybeans, canola, maize, alfalfa, and cotton. The area under biotech/GM crops with stacked features increased by 3%, or 2.8 million hectares, from 77.7 million hectares in 2017 to 80.5 million hectares in 2018. Regulatory agencies granted 4349 licenses for 387 biotech events involving 27 biotech crops between 1992 and 2018. Biotech crops received such licenses for food use (2063), feed use (1461), and environmental discharge or cultivation (2063). Thousands of farmers in 26 countries have made autonomous decisions to plant transgenic crops in the previous 23 years. Pesticide consumption has been cut by 670 million kg thanks to biotech crops. Biotech/GM crops assisted up to 17 million small farmers and their families, totaling more than 65 million people, in overcoming poverty (ISAAA, 2018).

5 Future Scope Although transgenic breeding has made tremendous and quick progress in basic plant research, the efficiency of transformation techniques is still very low, except for the Agrobacterium-mediated and biological methods. Transgenic plants are still not permitted for commercial cultivation by regulatory authorities and transgenic products are not accepted by consumers. Recently, Indian researchers concluded that the genes required to produce golden rice have involuntary effects on plant growth and yield. They transferred the GR2-R1 gene in the background of the high-­ yielding and agronomically superior Indian rice variety Swarna, which showed yellowing and stunted plant growth and the yield was one-third of non-GM Swarna, and thus was unsuitable for cultivation (Wilson, 2017). Therefore, researchers must integrate functional genomics to characterize the transgene (or gene construct) to assess their expression in the host plant’s genome background, and to determine whether the transgene changes the expression of a host genome in any negative way, and to analyze the risk associated with human health when integrated into the genome. Genome editing tools like TALENs (Transcription Activator-like Effector Nucleases), ZFNs (Zinc Finger Nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) provide an alternative tool for transgenic breeding that has recently been developed in the twenty-first century. It is possible to produce biotic and abiotic stress-resistant plants by modifying or editing the plant’s own genome with the use of potential genome editing technology rather than transferring foreign genes into plants. Researchers have modified the tobacco and corn plants using ZFNs for herbicide tolerance and rice plant with TALENs to knock out the bacterial blight susceptible gene. However, the use of these editing techniques in crop improvement ultimately depends on the wealth of the functional

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genomics data that is becoming available. In addition, plants designed through genome editing technology are free of transgene and have no ethical issues in public acceptance as they are considered to have no negative impact on human health.

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sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnology Journal, 14, 1046–1054. Yamamoto, T., et al. (2000). Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Reports, 19, 639–646. Yao, Q., Cong, L., Chang, J. L., Li, K. X., Yang, G. X., & He, G. Y. (2006). Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. Journal of Experimental Botany, 57(14), 737–749. Ye, X., Al-Babili, S., Kloti, A., et  al. (2000). Engineering the provitaminA (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Annual Review of Plant Biology, 66(5451), 303–305. Younis, A., Siddique, M. I., Kim, C. K., & Lim, K. B. (2014). RNA interference (RNAi) induced gene silencing: A promising approach of hi-tech plant breeding. International Journal of Biological Sciences, 10(10), 1150–1158. Yuan, D., Dashevskaya, S., Twyman, R. M., Capell, T., & Christou, P. (2012). Crop plants transformation methods. In R. A. Meyers (Ed.), Encyclopedia of sustainability science and technology. Springer. https://doi.org/10.1007/978-­1-­4419-­0851-­3_107 Zambryski, P., Depicker, A., Kruger, K., & Goodman, H.  M. (1982). Tumor induction by Agrobacterium tumefaciens analysis of the boundaries of transferred DNA. Journal of Molecular and Applied Genetics, 1, 361–370. Zhang, L., Yang, X., Zhang, Y., et  al. (2014). Changes in oleic acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing. International Journal of Genomics, 2014, 921950. https://doi.org/10.1155/2014/921950

Crop Biofortification: Plant Breeding and Biotechnological Interventions to Combat Malnutrition Richa Sao, Parmeshwar K. Sahu, Ishu Kumar Khute, Samrath Baghel, Ravi Raj Singh Patel, Antra Thada, Deepika Parte, Yenkhom Linthoingambi Devi, Prabha R. Chaudhary, Suvendu Mondal, B. K. Das, and Deepak Sharma

Abstract  Human race in all eras faced a common challenge of hunger and malnutrition. Despite much efforts at national and international level, hidden hunger is a major predominant in many countries of the world due to improper and unbalanced diets, lack in desirable minerals, vitamins, amino acids, iodine, proteins, etc. A report was published by FAO (2020), 690 million people were undernourished in the world in the year 2020 while the pandemic of COVID-19 has added around 83–120 million people to the undernourished category which is expected to reach 840 million by the year 2030. One intervention that has been proposed to combat the hidden hunger is the improvement in nutritional qualities of staple foods crops. This can be achieved by biofortification of crops which enhances the bioavailability of essential nutrients than slandered crop variety through conventional plant ­breeding, biotechnology, and agronomic approaches. It is an imperishable and viable method to alleviate the nutritional shortcomings in major staple crops generally consumed by a large population who have limited access to diversified food products and proper healthcare facilities. Interestingly, crop biofortification is a cost-­ effective and one-time investment which provides long-term and sustainable way to battle against malnutrition because once the biofortified crop varieties are developed; there are no need of buying the nutrient supplements and also no need to add Richa Sao and Parmeshwar K. Sahu contributed equally with all other contributors. R. Sao · P. K. Sahu (*) · I. K. Khute · S. Baghel · R. R. S. Patel · A. Thada · D. Parte Y. L. Devi · P. R. Chaudhary · D. Sharma Department of Genetics and Plant Breeding, Indira Gandhi Krishi Vishwavidyalaya, Raipur, India S. Mondal · B. K. Das Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. Raina et al. (eds.), Advanced Crop Improvement, Volume 1, https://doi.org/10.1007/978-3-031-28146-4_7

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them to the food supply during processing. Furthermore, mutation breeding, heterosis breeding, molecular marker-based breeding, and transgenic breeding have made development of biofortified varieties easier. In these ways, concerted efforts have been made by the national and international organizations to reduce malnutrition by providing sufficient, safe, and nutrient-rich foods to the marginal and poor peoples. Till now, numbers of biofortified crop varieties have been developed for direct consumption and are making significant impact on human health in positive direction. According to CGIAR, more than 30 countries have released or made biofortified crops available to 15 million farming households, and another 16 countries are evaluating these crops. Therefore, this chapter deals with the breeding and biotechnological aspects for developing biofortified crop varieties to reduce the impact of malnutrition throughout the world. Keywords  Biofortification · Malnutrition · Conventional breeding · Molecular markers · Biotechnology

1 Introduction Population explosion after independence led to food crises in India but no considerable boost in food grain production occurred to nourish the hunger population. Later, by the introduction of green revolution (1965–1970), semi-dwarf and high-­ yielding varieties (HYVs) were released which required high nutrient inputs and responded pretty well to fertilizers. This ensured regular and sufficient supply of food grains escalating the food grain production by 80%, from 50.8 to 257 mt by the year 2030 (Jena et al., 2019). Before the commencement of green revolution, scarcity of money was the major concern but later it shifted to undernourishment and nutritional deficiencies now which is also known as hidden hunger. At present, it has become a major issue, as in the developing countries of the world, more than 2 billion people endured from hidden hunger due to lack of nutritious foods in daily diet. This problem needs to be addressed quickly in the sustainable ways (Thai et al., 2020). The deficiencies of micronutrients, namely, iron (Fe), zinc (Zn), magnesium (Mg), selenium (Se), and iodine (I), vitamins, amino acids, and proteins are the major nutritional components which result in moderate to severe health complications and played crucial role in the augmentation of malnutrition (Harding et al., 2018). Among them, anaemia caused by Fe deficiency has been most commonly reported micronutrient disorder in human populations (Bailey et  al., 2015). Furthermore, due to Zn malnutrition, the immunity of the children (below 5 years) has been severely affected and they became highly susceptible for infectious diseases such as diarrhoea and pneumonia which causes death over 116,000 children every year (Rao et al., 2020). Although, iodized salt has been in use for decades, deficiency of iodine in human population is still prevailing in a number of countries. Furthermore, vitamins are the major components for various metabolic activities in human body and deficiencies of those vitamins may produce different kinds of difficulties in human (Simpson et al., 2011; Maggini et al., 2017).

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Usually, the minerals, vitamins, proteins, amino acids, etc., have been distributed among the poor and marginal households through nutrient supplementation programmes conducted by World Health Organization for eradication of hidden hungers. However, this programme requires lots of external funding and whose availability is not guaranteed from year to year. Furthermore, poor people may not be able to purchase nutrient supplements, inability to access the markets and health-­ care systems, and most importantly poor attentiveness towards sustainable health benefits of nutrient supplements (Perez-Massot et al., 2013; Gilani & Nasim, 2007). Therefore, among the available approaches, ‘Crop Biofortification’ is an effective process to increase the nutritional components, namely, micronutrients, vitamins, proteins, and amino acids, in food crops. The word ‘biofortification’ refers to the enhancement in the bio-availability of nutritional contents of commonly consumed crops through various plant breeding and biotechnological approaches. This is a sustainable and feasible strategy to alleviate the nutritional deficiencies in major staple crops generally consumed by a large population who do not have the means and monetary conditions to consume diversified foods. Interestingly, crop biofortification is a cost-effective and one-time investment which provides long-term and sustainable way to battle against malnutrition because once the biofortified crop varieties are developed; the requirement of purchasing nutrient supplements becomes nil and furthermore the need of adding nutrients during processing is also eliminated (Hefferon, 2016; Hirschi, 2009). Therefore, development of nutritionally enriched or biofortified crop varieties is the major goal of scientific communities and organizations including World Health Organization and the Consultative Group on International Agricultural Research (CGIAR) (Garg et al., 2018). Plant breeding approaches involving selection methodologies among diverse germplasm lines and testing its elevated nutritional contents is a traditional and efficient way to develop high yielding varieties rich in nutritional contents (Yadava et  al., 2020). It is well accepted that conventional breeding methods have made significant enhancement in the nutritional quality and yield attributing traits of staple food crops by exploiting the available genetic variability in the crop genetic resources and wild relatives (Jha & Warkentin, 2020). Development of quality protein maize (QPM) is one of the major breakthroughs in conventional plant breeding towards crop biofortification which have taken decades of efforts to make available varieties among the farmers (Singh et al., 2016). Further, breeding approaches like mutation breeding makes use of variability present in the population, which is created by induced mutagenesis, is also prominent and useful in development of crop varieties with enhanced grain nutritional quality (Raina et al., 2016, 2017; Khursheed et al., 2018a, b, c; Laskar et al., 2018a, b). Moreover, heterosis breeding involving crossing of nutritionally enriched donor with the high yielding varieties and further selections could also be a good approach for producing high yielding and biofortified cultivars. ICRISAT has developed several biofortified pearl millet and maize hybrids by using heterosis breeding (Ganguli et al., 2019; CGIAR, 2018). Proceeding towards the molecular world, marker-assisted breeding is making its way rapidly as it drastically reduces the number of generations by three to four generations for developing a variety. Recent advancements in the molecular field of biology and

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marker techniques have allowed creation of high-resolution linkage map enabling the chances to identify major QTLs/genes involved in various biosynthetic pathways influencing nutrient composing of any crop and their associated markers (Jha & Warkentin, 2020). This has empowered breeders to reduce the breeding cycle by practicing early generation selection. Marker-assisted selection (MAS), marker-­ assisted gene pyramiding (MAGP), marker-assisted backcrossing (MABC), marker-­ assisted recurrent selection (MARS), and genomic selection (GS) are the major marker-assisted breeding approaches for developing biofortified crop varieties (Jha & Warkentin, 2020; Ganguli et al., 2019; Garg et al., 2018). In addition, transgenic approaches are the latest entries as tools for combating malnutrition along with cutting-edge approaches to transfer genes directly into elite cultivars or varieties. The source of these genes can be any type of organism including microorganisms, flora, or fauna, and are designed to improve the minerals mobilization efficiency in the soil, silencing of antinutrient pathways, increasing the level of nutritional enhancer, biochemical pathway engineering, etc. Several biofortified crop varieties have been developed in many crops especially in rice through transgenic approaches (Yadava et  al., 2020). Many indica and japonica rice cultivars have been biofortified worldwide, being popularly known as ‘high iron rice’, ‘low phytate rice’, ‘high zinc rice’, and ‘high carotenoids rice’ (golden rice) varieties (Majumder et al., 2019). Unfortunately, few countries do not accept the crop varieties developed through genetic engineering which is the major problem in dissemination of engineered biofortified crop varieties. However, market availability of such varieties could reduce ‘hidden hunger’, and a large population of the world could be cured from the nutritional deficiencies (Ganguli et  al., 2019). With the aforementioned views, this chapter will comprehensively elaborate and discuss all the breeding and biotechnological aspects of crop biofortification with their outcomes towards tumbling the malnutrition.

2 Role of Plant Breeding in Developing Biofortified Crops Varieties Since the advent of plant breeding the major goal of breeders has been to improve economic yield. With the ever-rising population, problem of malnutrition increases in the world which gave an indication for enhancing the nutritional quality of foods along with the productivity. The simplest and most efficient answer to the problem of malnutrition is biofortification of crops. The major food crops which are a part of regular diet of a large percentage of population are unfortunately devoid of one or the other essential nutrients. It is next to impossible to provide dietary supplements or a balanced diet to every individual. According to WHO, 462 million people are underweight, 159 million are stunted and 50 million are wasted (Garg et al., 2018). Plant breeding played a foremost role in developing nutritionally enriched biofortified crop varieties in several ways as given in subsequent heads (Fig. 1).

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Fig. 1  Stages of plant breeding in developing nutritionally enriched biofortified crop varieties

2.1 Collection and Conservation Plant Genetic Resources as Base Materials for Biofortification PGRs are the base material for all the breeding activities as they have abundant variations for all the traits including grain nutritional and quality components. These variations can be utilized for enhancing the availability of minerals and vitamins in crops. PGRs may be utilized in pre-breeding programme, direct selection, or in hybridization programme based upon their specialties and requirements of the breeders.

2.2 Pre-breeding It is the process of identifying the genes or traits of interest from PGRs which may not be useful directly in breeding programmes with the help of their characterization and evaluation, and transferring those traits/genes in the set of accessible and useful materials for developing new cultivars. Pre-breeding is an essential step in developing biofortified crop varieties as PGRs and wild relative of many crops have

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several valuable genes/traits which may not be available in the cultivated species. By exploiting those genes, breeder can develop the biofortified genotypes. Therefore, pre-breeding is the first step of ‘linking genetic variability available in PGRs to their utilization in crop improvement’.

2.3 Hybridization/Recombination It is the basic and important aspect of plant breeding. The genotypes identified through pre-breeding may be utilized in recombination breeding for the purpose of creation of biofortified crop varieties. Through typical plant breeding methods, lines with sufficient nutrient content are used as parents and are crossed with an elite cultivar with desirable agronomic traits for multiple generations for generating plants with required levels of nutrients and agronomic traits.

2.4 Selection and Evaluation These are the most essential part of plant breeding programmes. Success of plant breeding programme mostly relied upon the accuracy and capability of selection of plant breeders. Selection and evaluation of genotypes having high nutritional values would be fruitful for developing nutritional rice crop varieties.

2.5 Adaptive Breeding Simultaneously, the pipeline materials may further used in mutagenesis, crossing and selection programmes to meet out the requirements of the farmers. Traditional breeding methodologies mostly depend upon the already available genetic variation in the gene pool. In certain scenarios, this drawback can be resolved by crossing to distant relatives and trying to transfer the trait slowly into the commercial cultivars. Alternatively, novel traits can be incorporated directly into commercial varieties by the method of mutation breeding (Raina et  al., 2020a, b; Goyal et  al., 2019a, b; Laskar et al., 2015, 2019).

2.6 Varietal Release, Notification, and Distribution Once the accession is stabilized and evaluated as per requirements then they may be submitted for release and notification and further distribution among the farmers.

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In between these processes, application of molecular marker technology, advanced genomic approaches, transgenic approaches make feasible and easier to the existing methods for developing biofortified crop varieties. Molecular markers and genomic approaches have proven role in identification, mapping and introgression of desired genes/QTLs in the genotypes with higher precision and less time requirement. Furthermore, transgenic approaches are useful in inserting the desired genes in the desired genotypes with help of genetic engineering approaches (Garg et al., 2018). Thereafter, the biofortified crop varieties developed through marker technology, genomic approaches and transgenics goes under various testing and evaluation stages of plant breeding. Ultimately, biofortified varieties developed through any approach would have to take help of plant breeding to reach up to farmers’ field. Therefore, plant breeding has been playing significant role in development of biofortified crop varieties. Since plant breeding is the most expedient method to improve the plants therefore, several international institutes have started several projects like The Health grain Project (2005–2010) by European Union and HarvestPlus programme by the CGIAR, International Center for Tropical Agriculture (CIAT), and the International Food Policy Research Institute; The Biocassava Plus programme by Center for Tropical Agriculture (CIAT) for development of health promoting and safe cereal foods and ingredients of high eating quality; breeding biofortified staple food crops and improvement of the nutritional status of cassava crop, respectively (Ganguli et al., 2019; CGIAR, 2018; Garg et al., 2018). With the help of plant breeding, several biofortified varieties have been developed in various crops, namely, rice (for Zn, Fe, Vit-A, glycemic index, phytic acid, etc.), wheat (for Fe, Zn, low gluten content, etc.), maize (for Fe, Zn, proteins, amino acids, etc.), sorghum (lutein, zea xanthin, beta carotene, etc.), millets (Fe, Zn, proteins, amino acids), lentils (Fe, Zn), cowpea (proteins), beans (proteins), potato (micronutrients, antioxidants), sweet potato (Vit-A), cauliflower (Zn), and cassava (vitamin A) (Garg et al., 2018; Singh et al., 2017; Zhang et al., 2010; Gregorio et al., 2000). Hence, it can be said that plant breeding has contributed greatly towards eradication of malnutrition by developing biofortified crop varieties.

3 Status of Biofortified Crop Varieties Developed in India and World A balanced diet is essential for every human to grow and develop properly as it provides energy, protein, essential fats, vitamins, antioxidants and minerals to maintain the metabolic requirements of the body and help in fighting against various diseases along with maintaining the biological metabolism of the body (Yadava et al., 2020). Unfortunately, few of the nutrients may not be synthesized in human body at enough quantity; hence, they need to supplemented from outside as food supplement or directly through diets. Indian Council of Agricultural Research

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(ICAR) has made significant efforts by joining hands with many Indian or overseas programmes for improvement in nutritional quality of high yielding cultivars of rice, wheat, maize, pearl millet, finger millet, small millet, lentil, groundnut, linseed, mustard, soybean, cauliflower, potato, sweet potato, greater yam, pomegranate, etc., with the help of plant breeding methods and biotechnological tools. In addition to these, several biofortified advance breeding lines are in pipelines and will be released soon for combating the malnutrition. It is believed that these biofortified varieties have made immense impact to attain the nutritional security of the country (Yadava et  al., 2020). Details of the most of biofortified crop varieties developed in India are given in Table 1. Global scenario of crop biofortification is majorly surrounded by World Health Organization and the Consultative Group on International Agricultural Research (CGIAR). Harvest Plus, the CGIAR Research Program on Agriculture for Nutrition and Health is the global leader for developing biofortified crop varieties to tackle hidden hunger and malnutrition. In addition, it is the leading body which is making an effort to explore the acceptability of biofortified crop varieties, educate about their competence and effectiveness, and increase their availability to people residing in villages, cities or towns, where there is a scarcity of diversified or fortified foods and nutritional supplements (Ganguli et  al., 2019; Bouis & Saltzman, 2017; HarvestPlus, 2014). More than 290 new varieties of 12 biofortified crops, namely, rice, wheat maize, pearl millet, sorghum, lentil, sweet potato, cassava, beans, cowpea, banana, and potato are being cultivated as varieties or are in pipeline for release in 60 countries throughout the world. Some of those biofortified crop varieties developed by various countries are presented in Table 2. In 2017, 3.2 million families of farmers cultivated crops using biofortified planting material, which brought the total estimated number of farming households worldwide to 10 million who benefited from biofortification programmes (Ganguli et  al., 2019; CGIAR, 2018). Besides this, several international organizations have taken the initiative of improving nutritional qualities of staple foods of world. In this context, the health grain project was initiated in Europe in which 43 organizations from 15 European countries participated. A number of peer-reviewed articles have been published related to crop biofortification and their impact on human health which demonstrated that foods which enhanced nutritional qualities had a positive effect on different human ailments and even exhibited improvements in conditions like anaemia and prevalence and duration of diarrhoea, ameliorated micronutrient status, eyesight, and cognitive and physical performance (Ganguli et al., 2019; Bouis & Saltzman, 2017; HarvestPlus, 2014).

S.N. 1.

Crop Rice

Special features High zinc (22–24 ppm) content in polished grain High zinc (25–27 ppm) content in polished grain High zinc (27.4 ppm) content in polished grain High zinc (20.9 ppm) and protein (9.29%) in polished grain Intermediate Glycemic Index (GI) (52–55%) level High protein (10.3%) content in polished grain High zinc (24.9 ppm) content in polished grain High protein (10.1%) and high zinc (20.0 ppm) content in polished grain High zinc (20.91 ppm) content in polished grain High zinc (25.2 ppm) content in polished grain High zinc (22.6 ppm) content in polished grain High zinc (22.8 ppm) content in polished grain High iron (21 ppm) content in brown rice Contains 40% more than zinc of common rice variety Possesses high zinc content

Variety Chhattisgarh zinc rice-1 Chhattisgarh zinc rice-2 Zinco Rice MS Protezin Madhuraj 55 CR Dhan 310 CR Dhan 315 CR Dhan 311 DRR Dhan 48 DRR Dhan 49 DRR Dhan 45 24760 Surabhi IR 68144 Jalmagna

BHU 1, BHU 3, BHU 5, BHU 6, BHU 17, and BHU 18

Table 1  List of biofortified crop varieties developed in India

(continued)

Developed by IGKV, Raipur, CG IGKV, Raipur, CG IGKV, Raipur, CG IGKV, Raipur, CG IGKV, Raipur, CG NRRI, Cuttack, Odisha NRRI, Cuttack, Odisha NRRI, Cuttack, Odisha IIRR, Hyderabad, Telangana IIRR, Hyderabad, Telangana IIRR, Hyderabad, Telangana Nuziveedu Seeds Pvt. Ltd IRRI, Philippines Crop Research Station, Ghagharaghat, UP BHU Varanasi, UP

S.N. 2.

Crop Wheat

Table 1  (continued) Special features High iron (40.0 ppm) and zinc (22.6 ppm) content High iron (40.0 ppm) and zinc (42.0 ppm) content High protein (12%), iron (42.1 ppm), and zinc (42.8 ppm) High protein (13%), iron (43.0 ppm), and zinc (35.0 ppm) High protein (14.7%), iron (46.1 ppm), and zinc (40.3 ppm) content High protein (14.7%), iron (39.5 ppm), and zinc (37.8 ppm) content Rich in zinc content (47 ppm) content Rich in zinc content (42.5 ppm) content Rich in protein (12.1%) and iron (43.1 ppm) contents Rich in iron (48.7 ppm) and zinc content (43.6 ppm) Rich in protein (13%) Rich in protein (12.8%) and iron content (40.4 ppm) High protein (12.4%), iron (41.6 ppm), and zinc (41.1 ppm) contents Rich in protein (12.4%) content Rich in zinc content (42 ppm) content Rich in zinc content (41.4 ppm) content Rich in iron content (43 ppm) content Rich in protein (12.5%) and iron (40.7 ppm) contents Rich in protein (12.1%) content Rich in protein (12.7%) and iron (40.1 ppm) contents Rich in protein (12.1%) content Rich in protein (13.8%) content Rich in Zinc (40 ppm) and protein (11%) contents

Variety HPWB 01 WB 02 Pusa Tejas Pusa Ujala MACS 4028

MACS 4058

HD 3171 HD 3249 HD 3298 HI 8777 HI 8802 HI 8805 HI 1633 PBW 752 PBW 757 PBW 771 Karan Vandana DBW 173 DBW 303 DDW 47 DDW 48 UAS 375 CG Hansa Wheat

Developed by PAU, Ludhiana, Punjab IIWBR, Karnal, Haryana IARI, Indore, MP IARI, Indore, MP Agharkar Research Institute, Pune, Maharashtra Agharkar Research Institute, Pune, Maharashtra IARI, New Delhi IARI, New Delhi IARI, New Delhi IARI, Indore IARI, Indore IARI, Indore IARI, Indore PAU, Ludhiana PAU, Ludhiana PAU, Ludhiana ICAR-IIWBR, Kernal ICAR-IIWBR, Kernal ICAR-IIWBR, Kernal ICAR-IIWBR, Kernal ICAR-IIWBR, Kernal UAS Dharwad IGKV, Raipur, CG

S.N. 3.

Crop Maize

IQMH 201 IQMH 202 IQMH 203 Shakti-1 (composite) Shaktiman-1, Shaktiman-2, Shaktiman-3, Shaktiman-4, Shaktiman-5 HQPM-1, HQPM-5, HQPM-7, HQPM-4, Pratap QPM Hybrid-1

Pusa HQPM 7 Improved

Pusa HM 4 improved Pusa HM 8 improved Pusa HM 9 improved Pusa VH 27 Improved Pusa HQPM 5 Improved

Pusa Vivek QPM 9 improved

Variety Vivek QPM9

(continued)

Special features Developed by High tryptophan (0.83%) and lysine (4.19%) content in endosperm protein ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora High Pro-Vitamin A and high tryptophan (0.74%) and lysine (2.67%) IARI, New Delhi content in endosperm protein High tryptophan (0.91%) and lysine (3.62%) content in endosperm protein IARI, New Delhi High tryptophan (1.06%) and lysine (4.18%) content in endosperm protein IARI, New Delhi High tryptophan (0.68%) and lysine (2.97%) content in endosperm protein IARI, New Delhi Rich in provitamin A (5.49 ppm) IARI, New Delhi Rich in provitamin A (6.77 ppm), lysine (4.25% in protein), and IARI, New Delhi tryptophan (0.94% in protein) Rich in provitamin A (7.10 ppm), lysine (4.19% in protein), and IARI, New Delhi tryptophan (0.93% in protein) High tryptophan (0.66%) and lysine (3.04%) content in endosperm protein ICAR-IIMR, Ludhiana High tryptophan (0.77%) and lysine (3.48%) content in endosperm protein ICAR-IIMR, Ludhiana High tryptophan (0.73%) and lysine (3.03%) content in endosperm protein ICAR-IIMR, Ludhiana High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico High protein quality (high lysine and tryptophan) CIMMYT, Mexico

Finger millet

Little millet Lentil

Groundnut

Linseed

5.

6. 7.

8.

9.

Table 1  (continued) S.N. Crop 4. Pearl millet

Rich in iron (91.0 ppm) and zinc (43 ppm) contents Rich in iron (73 ppm) and zinc (63 ppm) contents Rich in iron (87 ppm) and zinc (41 ppm) contents Rich in iron (83 ppm) and zinc (46 ppm) contents Rich in iron (84 ppm) and zinc (46 ppm) contents Rich in iron (83 ppm) contents Rich in iron (66.0 ppm) content Rich in iron (72.0 ppm) content Rich in iron (131.8 ppm) content Rich in iron (131.8 ppm) Rich in calcium (454 mg/100 g), iron (39.0 ppm), and zinc (25.0 ppm) contents Rich in iron and zinc contents Rich in iron (59.0 ppm) and zinc (35.0 ppm) content High iron (65.0 ppm) content High iron (73 ppm) and zinc (51 ppm) contents Rich in oleic acid (78.5% in oil) content Rich in oleic acid (78.4% in oil) content High in linoleic acid (58.9%) content

AHB 1269Fe

ABC 04

Phule Mahashakti RHB 233 RHB 234 HHB 311 Hybrid 7 Hybrid 12 VR929 (Vegawahi) CFMV1 (Indravati) CFMV 2

Chhattisgarh Sonkutki CLMV 1 Pusa Ageti Masoor IPL 220 Girnar 4 Girnar 5 TL 99

Special features Rich in iron (73.0 ppm) and zinc (41.0 ppm) content Rich in iron (73.0 ppm) content

Variety HHB 299 AHB 1200Fe

Developed by CCSHAU, Hisar, Haryana VNMKV, Parbhani, Maharastra VNMKV, Parbhani, Maharastra ARS, ANGRAU, Ananthapuramu MPKV, Dhule, Parbhani SKNAU, Jobner SKNAU, Jobner CCSHAU, Hisar, Haryana ICRISAT, Hyderabad ICRISAT, Hyderabad ARS, ANGRAU, Guntur ARS, ANGRAU, Guntur ARS, ANGRAU, Vizianagaram IGKV Raipur, CG ICAR-IIMR, Hyderabad IARI, New Delhi IIPR, Kanpur, UP ICAR-DGR, Junagarh ICAR-DGR, Junagarh BARC, Mumbai

Greater yam

Pomegranate Solapur Lal

15.

16.

Source: Yadava et al. (2020), Ganguli et al. (2019), Sarawgi et al. (2019), and Garg et al. (2018)

Da 340

Potato

Tubers have high amount of beta carotene (5.70 mg/100 g) Tubers have high amount of beta carotene (4.23 mg/100 g) Rich in anthocyanin (0.68 ppm) Rich in anthocyanin (1.0 ppm) Rich in anthocyanin (50.0 mg/100 g), crude protein (15.4%), and zinc (49.8 ppm) Rich in anthocyanin (141.4 mg/100 g), iron (136.2 ppm), and calcium (1890 ppm) This contains high iron (5.6–6.1 mg/100 g), zinc (0.64–0.69 mg/100 g), and vitamin C (19.4–19.8 mg/100 g) in fresh arils

Chhattisgarh Shakarkand Narangi Indira Madhur Kufri Manik Kufri Nilkanth Sree Neelima

14.

High anthocyanin (90.0 mg/100 g) content

Cauliflower Sweet potato

12. 13.

Bhu Krishna

Soybean

11.

Special features Low erusic acid ( 1%, but all SNPs were considered). They identified 21 significant signals across the seven diseases using a single locus test. This test was either a standard one degree of freedom (trend test) or two degrees of freedom (general genotype test) of case-control association (i.e. logistic regression) complemented with Bayesian factor calculation to strengthen frequentist results with a better estimation of effect size and SNP ranking. By combining datasets, seven additional associations were detected when considering geographical structuration and/or sex differentiation and the response to several diseases. Finally, 58 signals were highlighted with P-values between 10−5 and 5 × 10−7 and a multilocus approach was conducted through imputation to identify haplotypes in strong LD regions. These results showed the efficiency and relevance of performing GWAS on sufficiently large dataset when focusing on loci with modest effect. In conclusion, the authors initiated a discussion on a problem which will permeate further human GWAS. Indeed, they interrogate the composition and size of the sample, SNP filtering criteria, confounding effect of population structure, P-value threshold choices and/or calculation as well as ensuring study replicability. 2.1.4 Twenty Years of GWAS on Humans Gave a Better Understanding of Complex Trait Genetics In almost 20 years, 325,538 associations have been reported in 5527 publications as of the 21st of December 2021 (https://www.ebi.ac.uk/gwas/home, Buniello et al., 2019). Despite a start focusing on human disease, GWAS results have now been published for hundreds of complex traits across a wide range of domains such as morphological traits (e.g. height), genomic measures (e.g. gene expression, DNA methylation) or social and behavioural traits (Visscher et  al., 2017). Describing these results in detail is beyond the scope of this chapter, but several observations and issues are worth mentioning. To start with, GWAS findings are highly replicable, which is quite uncommon in complex trait genetics (Marigorta et al., 2018). Then, in these 20  years, human GWAS have benefited from methodology improvements, such as (i) a better modelling of population structure and relatedness between individuals, (ii) the use of GWAS summary statistics, (iii) methods of estimating and partitioning genetic (co)variance and (iv) methods to infer causality (Visscher et al., 2017). These advances allowed to alleviate most of the perceived

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failures pointed in the early years of the approach (Manolio et al., 2009; Visscher et al., 2012; Claussnitzer et al., 2020). Furthermore, for most traits, the mutational target in the genome appears large, as even traits traditionally described as Mendelian seem to be very polygenic (Visscher & Goddard, 2019); the number of detected loci increases with the size of the studied panels. For instance, in 2008, only 54 genome-wide significant SNPs had been identified for height using data collected on ~63,000 individuals, explaining 5% of heritability (Visscher, 2008). In 2014, the number of associated SNPs had increased to 697 SNPs detected, thanks to data on 253,288 individuals, explaining 20% of heritability (Wood et  al., 2014). The latest publication on human height reports over 3000 statistically significant loci explaining ~24.6% of heritability detected with a panel of around 700,000 individuals (Yengo et al., 2018). Finally, some questions remain, such as delineating the causal genetic variants and biological mechanisms underlying the observed statistical associations with disease risk (Gallagher & Chen-Plotkin, 2018), identifying more accurately the “causal variant” in a locus (Schaub et al., 2012; Edwards et al., 2013), explaining the fact that more than 90% of disease-associated variants are located in non-­ protein-­ coding regions of the genome and far from the nearest known genes (Maurano et al., 2012) and extending GWAS finding to population not only composed of individuals from European descent, in a more interesting and pertinent way to deal with population stratification (Martin et al., 2019). After its initiation on human to elucidate primordial issues such as hereditary diseases, GWAS has quickly been transposed to plant genetics. Since there are specificities and disparities between plants and humans, there was a dialogue between plant and human GWA studies, both domains benefiting from the advances achieved in these domains. In the next part, we will thus describe how GWAS became widely used in plant genetics.

2.2 A Method Adapted to Plant Genetics Most traits studied in plants (either cultivated crops or wild species) are controlled by multiple QTLs (i.e. complex traits) (Yu & Buckler, 2006). The study of these traits has capitalised on the methodology developed in the frame of human genetics. Therefore, plant quantitative genetics followed the same path, using the same two major approaches: linkage analysis and association mapping (Risch & Merikangas, 1996; Mackay, 2001). Since it is possible in plants to produce large progenies from controlled crosses on several generations and conduct replicated trials with immortal individuals, linkage analysis has had broader applications and provided better results in plants compared to human or animal genetics (Zhu et al., 2008). Although the focus of this chapter is GWAS in plants, we will briefly describe linkage mapping in plants since it can be considered as the direct precursor of association studies (Cortes et al., 2021).

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2.2.1 Linkage Mapping in Plants Linkage mapping can be classified into family-based mapping, a term originating from human genetics, which takes a slightly different sense regarding plants since, in this case, mapping is performed in progenies of biparental or multi-parental crosses. Before the generalisation of next-generation sequencing (NGS) technologies, linkage mapping presented an advantage over GWAS: the lower number of genetic markers needed to ensure causative locus detection. It is a consequence of the difference in terms of number of recombination events (Cortes et  al., 2021). Therefore, the first genome-wide QTL analysis was conducted in tomato in 1988 (Paterson et al., 1988), 14 years before the first GWAS in human (Ozaki et al., 2002). The main biparental populations derived from inbred lines are presented in Fig. 1.

Fig. 1  Different biparental populations used in QTL mapping. F2 population is a segregating population obtained by selfing an F1 plant resulting from a cross between two phenotypically contrasted parents. Fine mapping with a F2 population is limited because F2 plants are largely heterozygous and can therefore only be phenotyped once (with the exception of species with a vegetative reproduction system (Collard & Mackill, 2008)). RILs (recombinant inbred lines) correspond to the progeny of repeated selfing of F2 plants over several generations. As opposed to F2 populations, RILs are almost completely homozygous, thereby allowing the replication of RILs within an experiment and/or across several environmental conditions. Mapping resolution of RILs is however often coarse, with the identification of QTL regions of a few megabases covering hundreds of thousands of genes. NILs (near isogenic lines) result from repeated backcrosses of the F1 progeny with one of the two parental lines, leading to the introgression of a unique genomic fragment of one of the two parental lines into the genome of the other parental line. NILs are generally used to validate the physical position of QTL with small allelic effect (Keurentjes et al., 2011). (Adapted from Bergelson and Roux (2010))

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The power of QTL detection is affected by QTL effects and the type and size of the mapping population. Biparental mapping has proven its efficiency, as for the dissection of the key component of the flowering time pathway in Arabidopsis thaliana (Kowalski et al., 1994; Clarke et al., 1995; Alonso-Blanco et al., 1998), or its usefulness in crop breeding (Morrell et al., 2012). The main limitation of a biparental populations is that only a few recombination events occur during development of the population, allowing the localization of QTL with large intervals (Borevitz & Nordborg, 2003; Xu et al., 2017). To overcome these two limitations, the approaches presented in Fig.  2 have been designed with new types of mapping populations involving more parents. Another type of multi-parental mapping population not presented in Fig. 2 is the nested association mapping (NAM). It was first suggested in maize for dissecting

Fig. 2  New populations developed for QTL mapping. To increase the allelic diversity within a mapping population, multiparent populations have been set up by intercrossing several accessions before producing RILs. In this example, the MAGIC (multiparent advanced generation intercross) population involves crosses of four parental lines producing two F1 hybrids thereafter crossed together. The derived progeny then follows several generations of selfing to reach inbred MAGIC lines. MAGIC populations have been developed in a variety of plants: A. thaliana (Kover et al., 2009; Huang et al., 2011), tomato (Pascual et al., 2015), rice (Bandillo et al., 2013), wheat (Huang et  al., 2012a), chickpea (Samineni et  al., 2021), maize (Dell’Acqua et  al., 2015), cotton (Fang et al., 2014), sorghum (Ongom & Ejeta, 2018) and strawberry (Wada et al., 2017). The relatively low resolution of biparental population can be dramatically improved with several generations of random intercrossing between the F2 plants before several generations of crossing when establishing the RIL (recombinant inbred lines) populations, creating an AI (advanced intercrossed)-RIL (Darvasi & Soller, 1995; Balasubramanian et al., 2009). Another mapping population design is the BILs. It is created by backcrossing the F1 hybrid into the recurrent parent followed by several selfing generations. A single (or few) part of the genome of the wild recurrent parent is introgressed (Ofner et al., 2016). MAGIC multiparent advanced generation intercross, BILs backcross inbred lines, AI-RILs advanced intercross RILs. (Adapted from Bergelson and Roux (2010) and Rothan et al. (2019))

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the genetic architecture of flowering time (Yu & Buckler, 2006; Yu et al., 2008). The NAM population was created by crossing 25 maize lines to a single inbred line, chosen as a reference line, resulting in 5000 RILs from 25 families, with 200 RILs per family. Thus, the NAM population affords very high resolution and power for detecting QTLs. Finally, bulk segregant analysis (BSA) coupled with multi-sample sequencing appears as an alternative to conventional linkage mapping and can be used in the genetic mapping of simple qualitative traits or mutant mapping (Huang & Han, 2014; Xu et al., 2017). Several methods have been reported for this application, including SHOREmap (Schneeberger et al., 2009), next-generation mapping (Austin et al., 2011), MutMap (Abe et al., 2012) and MutMap-Gao (Takagi et al., 2013). With the development of high-throughput genotyping platforms, chip-based BSA has been successfully used to detect QTLs for traits of agronomic importance, such as rust resistance in wheat (Forrest et al., 2014), kernel row number in maize (Yang et  al., 2015a), salt tolerance and blast disease in rice (Takagi et  al., 2013, 2015). Once the trait of interest has been detected and its inheritance pattern is determined, the line harbouring the trait is crossed to another genotype (QTL-seq) or to a wild-type (non-mutagenised) parental line (mapping-by-sequencing, MBS) to map the QTL on an F2 progeny. Despite these efforts, the diversity of alleles, the allelic frequencies and recombination events in any of these populations are different from those found in natural populations (Weigel, 2012). Depending on the study objectives, it might not be a problem, but it narrows the diversity available in the natural populations, potentially limiting the understanding of the genetic and biological processes (Korte & Farlow, 2013). 2.2.2 Association Mapping in Plants Association mapping has emerged as an alternative to traditional linkage analysis, allowing to decipher complex trait variation at the sequence level, thanks to recombination events occurring at the population level through its history and evolution (Risch & Merikangas, 1996; Nordborg & Tavaré, 2002). It offered three advantages compared to previous strategies: (i) increased mapping resolution with smaller blocks of LD, (ii) increased power to detect genes with smaller effect, (iii) reduced research time since it eliminates the need to perform crosses to prepare populations and (iv) screened directly larger diversity and greater allele number (Lander & Schork, 1994; Risch & Merikangas, 1996; Yu & Buckler, 2006). However, these advantages require high-density genotyping, meaning that the first association studies, carried out before the development of NGS, could only focus on small subsets of the genomes (Cortes et al., 2021). Therefore, the first association study carried out in plant (Thornsberry et  al., 2001) focused on a single candidate gene involved in maize plant height and flowering time, which was already identified, thanks to previous mutagenesis and QTL

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mapping (Koester et al., 1993; Schon et al., 1994; Peng et al., 1999). Thornsberry and colleagues examined 123 polymorphisms in and near dwarf8 (i.e. the mutant version of the candidate gene) along with 141 genome-wide markers (SSRs) on 92 maize inbred lines. The 141 genome-wide markers were used to estimate the population structure (Pritchard et al., 2000) impact on association while the 123 polymorphisms allowed identifying haplotypes associated with flowering time. However, there was no significant association between Dwarf8 polymorphism and plant height and the dwarf8 mutants with severe height phenotypes resulting from alterations in the DELLA domain at the N terminus of the predicted protein. The DELLA domain was conserved in all the inbreds used in the study. Finally, the distribution of nonsynonymous polymorphisms suggested that the Dwarf8 region had been a target of selection. 2.2.3 GWAS in Plants First GWAS on Arabidopsis thaliana The first GWAS published in plants focused on A. thaliana flowering time and response to P. syringae (Aranzana et al., 2005). It was even before its application on livestock (Abasht & Lamont, 2007) or crop species (in maize, Beló et al. (2008)). They used a panel of 96 accessions (Nordborg et al., 2005). The panel was screened with a small number of markers to avoid inbred siblings or extensively heterozygous individuals. They used a direct PCR-based sequencing of genomic DNA to genotype the accessions, obtaining 876 alignments, representing 0.48 Mbp of the genome, on which they detected more than 17,000 SNPs organised in short haplotypes within which LD was nearly complete. The authors therefore used the following three haplotype-based association methods: • A single-fragment haplotype test where phenotypic associations were tested using either a Kruskal-Wallis test (for flowering date, since it is a continuous trait) or χ2 tests for resistance (binary trait). • A stepwise cladistic association test in which the authors generated a similarity matrix using the extent of pairwise haplotype sharing between all pairs of accessions, clustered the accessions using a standard hierarchical clustering algorithm and heuristically searched clade association with phenotype (Kruskal-Wallis or χ2 tests), this test being performed anew after removing previously detected associated clades. • The Voronoi test (Molitor et al., 2003), a Bayesian spatial clustering algorithm. This algorithm compares the length of similarity of each haplotype in a haplotype cluster to a prototypic haplotype and then associates a haplotype cluster to an observed phenotype on the basis of haplotype risk parameters measured through Markov Chain Monte Carlo (MCMC) method.

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The authors demonstrated that the known genes FR1, Rpm1, Rps2 and Rps5 could be detected using GWAS, even though the sample used was small and heavily structured (Aranzana et al., 2005). From this point onwards, GWAS in plants expanded to cultivated crops with a leading role of maize and rice, benefiting from the ever-­ changing methodology and decreasing cost of sequencing. Fast Development in Cultivated Crops (Mostly Cereals) It is just as difficult for crops as for human genetics to point out which was the first GWA study to be carried out. Two methodologies coexisted at the beginning of GWAS, candidate gene association and whole genome scan, the latter actually corresponding to GWAS. Candidate gene approach consists in testing whether there is a correlation between DNA polymorphism in a specific candidate gene and the trait of interest (Rafalski, 2010). However, both methodologies rely on the use of a diversity panel composed of unrelated individuals. As of 2008, only four studies exploited more than 1000 markers, hinting at a representative genome scan, given the size of the genomes studied: • The work of Aranzana et  al. on A. thaliana described in the previous section (~17,000 SNPs) and a sister study (Zhao et  al., 2007) working with the same panel but focusing only on flowering time. • A study on sugarcane response to four diseases (Wei et al., 2006). Working with 154 individuals characterised by 1599 polymorphic AFLP (amplified fragment length polymorphism) and 181 polymorphic SSR markers, the authors recognised that it did not provide an extensive genome coverage given the large genome size. However, thanks to the long LD measured in the panel, they were still able to identify ~20 markers explaining part of phenotypic variation, as well as numerous markers with lower effect size. • An analysis of maize oleic acid content genetic control, thanks to 553 maize inbreds characterised for 8590 SNPs (Beló et  al., 2008). A single locus with major effect was mapped. The development of GWA studies in a species was not necessarily a consequence of genome sequencing. For instance, A. thaliana genome sequence was first released in 2000 (The Arabidopsis Genome Initiative, 2000), 5 years before the first GWAS, and for maize, the B73 genome was released in 2009 (Schnable et al., 2009) just after. Sugarcane is a counter example due to the higher complexity of its genome; the sequenced genome was released in 2018 (Garsmeur et al., 2018), the GWAS being performed on unsaturated genetic maps. The sorghum (Sorghum bicolor (L.)) genome sequence (Paterson et al., 2009) was used as a model for sugarcane due to its genome-wide collinearity. GWAS was performed on sorghum for the first time in 2013 by Morris et al. (2013a, b) to decipher the genetic determinism of major agronomic traits and flavonoid pigmentation.

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Regarding other staple crops, rice was an ideal candidate for GWAS application since it is self-fertilising and beneficiated from a high-quality reference genome sequence (International Rice Genome Sequencing Project, 2005). Therefore, the first GWAS performed on rice studied 373 O. sativa indica lines, genotyped with 3,625,200 SNPs and phenotyped for 14 traits divided in 5 categories: morphological characteristics, yield components, grain quality, coloration and physiological features (Huang et al., 2010). Using a compressed mixed linear model (MLM), they detected 37 association signals. Among the staple crops, common bread wheat (Triticum aestivum) displays a complex genome (hexaploid) with an important proportion of repeated sequences. Therefore, the sequencing of its genome started in 2012 (Brenchley et al., 2012) covering only one-third of the genome. A sequence representing 96% of the genome was only released in 2017 (Zimin et al., 2017). It is not surprising that the study recognised as the first GWAS performed on bread wheat used only 813 DArT markers (diversity array technology) and 831 other markers (SSRs, AFLPs, RFLPs) to cover the wheat genome (Crossa et al., 2007). While they used a standard mixed linear model (MLM) to control for population structure (Yu et al., 2006) and detect association with several traits (grain yield, resistance to stem rust, leaf rust, yellow rust and powdery mildew), we can question the coverage and resolution of this study faced with the size of the wheat genome. In 2014, Cormier et al. used, for instance, 23,603 SNPs while studying 28 nitrogen use efficiency (NUE)-related traits in 241 European elite varieties. Regarding vegetables, methodological development was a bit delayed. For instance, tomato (Solanum lycopersicum L.) is the second most consumed vegetable after potato in the world (FAOSTAT, 2019), the seventh most important crop and a model species (Bergougnoux, 2014). However, the tomato genome was released in 2012 (The Tomato Genome Consortium, 2012). Sauvage et  al. (2014) released a GWAS for primary metabolism traits using 5995 SNPs obtained with a genotyping array. Despite a genome released in 2011 (The Potato Genome Sequencing Consortium, 2011), the cultivated potato, Solanum tuberosum group tuberosum, presents many genetic complexities (highly heterozygous, autotetraploid) which limited genetic studies to biparental populations, using diploid individuals or candidate gene approach (Sharma et al., 2018). The two first GWAS on tetraploid potato were published in 2014, studying the genetic determinism of late blight resistance (Lindqvist-­ Kreuze et al., 2014) and tuber quality traits (D’hoop et al., 2014). These are only examples, and in the 10–15 years since the first GWA studies, the methodology has much improved (for phenotyping, genotyping and statistical models) and the number of studies has increased. For instance, Gupta et  al. (2019) underlined the advances of plant GWAS and noticed the evolution of numbers of markers available for major crops: 26.5 million SNPs for maize, 3.6 million SNPs for rice, 23.8  million SNPs for tomato and finally a 90k SNP chip for wheat. Performing a quick bibliometric survey (Fig.  3) on the number of publications focusing on plant GWAS since 2005, we observe a first increase in 2011 with 106 papers in 1 year, while between 2005 and 2010, only 214 articles were published in

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Fig. 3  Evolution of the number and type of publications focusing on GWAS between 2005 and 2021. A bibliometric survey performed on Web of Science (12/01/2022) using the keywords (genome-wide association) and plant on all fields from 2005 to 2021 and considering only articles, data papers, proceeding articles and early access. 4885 hits were obtained in total. The bars correspond to the number of publications in each year while the curve displays the cumulative number of publications

total. A second acceleration occurred in 2019 with 776 studies and 789 studies in 2020. We performed the same approach by organism and found that about 62.6% of GWAS were carried out on maize, rice or wheat while 26.9% studied A. thaliana. This quick analysis as well as the history of GWAS in plants showed the disparity of knowledge among crops but also the opportunities that still exist for discovery on certain topics. Some of these disparities are structural, and due to difficult phenotyping or particular genetics, in other cases, it is a question of funds and interests. We will now look deeper into GWAS methodology, identifying the potential for GWAS amelioration.

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3 Methods Used for GWAS GWAS consists in evaluating the association between the genotype at each marker and a trait of interest in a panel consisting of a large number of individuals (Korte & Farlow, 2013). This method can be divided into five steps: (i) collecting a sample population including elite cultivars, landraces, wild relatives and/or exotic accessions; (ii) phenotyping the population for the trait of interest, estimating the broad-­ sense heritability (H2) and determining the genome-wide genotypes of the sample population components; (iii) quantifying the LD decay extent in the selected population; (iv) identifying the proportion of the phenotypic variance explained by the population structure and/or kinship in the panel; and (v) testing the associations between genotypes and phenotypes using the appropriate statistical approach (Xu et al., 2017). We chose to articulate the description of GWAS methodology around these five steps (Fig. 4), starting with the building of a sample panel.

3.1 GWAS Panel Composition The objective of association mapping is to exploit the genetic diversity of a designed population to resolve complex trait variation and potentially identify QTLs, single genes or even individual nucleotides involved in this variation (Zhu et al., 2008). GWAS in crops present the advantage of using a permanent resource: a population of diverse (and preferably homozygous) accessions which needs to be genotyped once while being phenotyped for many traits. The diversity panel must ideally represent most of the genetic diversity available in the “species” and most of the phenotypic diversity for a given trait. Furthermore, each partition of this diversity must be carried by enough accessions to avoid spurious associations. Consequently, several questions arise when building a diversity panel (Fig. 4a). First, what should be the size of the panel? Then, how this panel should be composed? At which geographical scale should the accessions be sampled, local or worldwide? Do we consider a single species, or do we extend the genotype origins to wild relatives? Do we focus on elite lines, cultivars, heirloom varieties or wild accessions? The success of GWAS is determined by the mapping resolution, marker density, statistical methods and mapping power. Since these aspects are sensitive to genetic diversity, genome-wide LD extent and relatedness among the individuals composing the panel, the choice of germplasm is critical (Zhu et al., 2008). Furthermore, most of these questions stem from the fact a source of false positives in GWAS is population stratification (Rafalski, 2010). Indeed, through domestication, crop species were usually subjected to severe bottleneck, a genetic erosion and differentiation which was continued through breeder’s selection leading to complex population structure. Differences are also expected between germplasms originating from different regions of the world. Examples of factors to take into account include the

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Fig. 4  General concept and main parameters impacting GWAS. GWAS is based on the study of a diversity panel (a) described genetically (b) and phenotypically for a trait of interest (c). The ultimate goal of GWAS is to determine whether there is a statistical association between allelic variation for a given genetic variant and phenotypic variation. These results are usually presented as Manhattan plot (d) which has to be analysed to determine which QTL and genes are the most likely candidates involved in the phenotypic variation. All these steps rely on specific hypotheses and present limits which are presented in this section

division of maize germplasm into heterotic groups (Reif et al., 2005), a severe post-­ domestication bottleneck associated with popularisation of soybean in North America (Hyten et  al., 2006) or fruit size and historical patterns of breeding in tomato (van Berloo et al., 2008; Robbins et al., 2011). It is possible to classify plant populations used for association studies into five groups (Yu et  al., 2006; Yu &

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Buckler, 2006): (i) ideal panels with low population structure or kinship, (ii) panels composed of several groups, (iii) panels with population structure, (iv) panels with both population structure and kinship and (v) panels with strong differentiation caused by population structure and kinship. Because of local adaptation, selection and breeding history in many plant species, many diversity panels used for GWAS would fall into category four (Zhu et al., 2008). 3.1.1 Panel Size The first crop GWAS studies used a limited number of markers and small-sized population size. Since then, high-throughput genotyping and phenotyping techniques allowed the study of larger association panels (Gupta et al., 2019). Population size remains an important issue. Larger panels are expected to provide higher power, and in practice, between 100 and 500 individuals are needed (Kumar et al., 2012). Rice and maize are the two major models for crop GWAS, and panels of several hundreds of genotyped inbreds have been phenotyped (Huang & Han, 2014). For instance, in rice, 1083 cultivated O. sativa spp. indica and O. sativa spp. japonica varieties and 446 wild rice accessions (Oryza rufipogon) were collected and sequenced with low genome coverage (Huang et al., 2012b). It is also true for less studied crops, as a panel of 917 worldwide diverse accessions of sorghum being, for example, used for GWAS (Morris et al., 2013a). The number or individuals, the distribution of allele frequencies and the precision of phenotyping present a complex interaction (Purcell et al., 2003; Yu et al., 2008). A larger population size should lead to an increased power to detect meaningful associations with larger effect, sufficiently high frequency, overcoming rare variants (Alqudah et al., 2020). Indeed, standard GWAS have low power for rare alleles, although they represent a substantial proportion of natural variation (Huang & Han, 2014). In rice, for instance, around 44% of the SNPs are of low frequency (minor allele frequency (MAF)