Genetic Engineering of Crop Plants for Food and Health Security: Volume 2 2

This edited volume presents recent advancements in genetic transformation and genome editing, offering a comprehensive understanding of their applications for creating more sustainable crops. These innovations are revolutionizing crop development, enhancing yields, and mitigating environmental challenges. The demand for sustainable crop production, driven by a growing global population and mounting environmental challenges, has never been more pressing. Genetic transformation and genome editing techniques offer precise and targeted ways to enhance crop traits, increase yields, and reduce the need for chemical inputs. The book traces the evolution of these technologies, from the early days of genetic modification to the current era of CRISPR-based genome editing. It covers cutting-edge technologies, from CRISPR-Cas9 to advanced gene editing techniques, while discussing the current scenario and future prospects of GM crops developed either by transgenic or genome editing approaches. The book explores the ethical and regulatory aspects surrounding genetic transformation, providing a complete perspective on this evolving domain. It is an essential read for researchers, students, and professionals in the fields of agriculture, plant sciences, biotechnology, agronomy, as well as policymakers. Preface Acknowledgments Contents Editors and Contributors About the Editors Contributors 1: Pros and Cons of Plant Genetic Engineering Technologies 1.1 Introduction 1.2 Pros and Cons of Genetic Engineering 1.3 Pros of Genetic Engineering 1.3.1 Productivity of GM Crops 1.3.2 Herbicide Resistance 1.3.3 Insect Resistance 1.3.4 Nutrition Content 1.3.5 Abiotic Stress Resistance 1.3.6 Effect on the Environment 1.4 Cons of Genetic Engineering 1.4.1 Herbicide Tolerance and Insect Resistance 1.4.2 Horizontal Gene Transfer 1.4.3 Risk to Biodiversity 1.4.4 Antibiotic Resistance 1.4.5 Ethical Concern 1.5 Conclusion and Future Perspective References 2: Regulatory Requirement for Genetically Modified (GM) Crops in India and GM Detection Approaches 2.1 Introduction 2.2 Approval of GM Crops in India 2.3 Need for GM Detection 2.4 Regulation of Import of GM Plant Material into India 2.5 Route for GM Detection 2.6 Publicly Available GMO Databases 2.6.1 GM Approval Database (https://www.isaaa.org/gmapprovaldatabase/default.asp Isaaa (n.d.-b)) 2.6.1.1 European Union Database of Reference Methods for GMO Analysis (http://gmocrl.jrc.ec.europa.eu/gmomethods/ Gmocrl (n.d.)) 2.6.1.2 United Nations Cartagena Protocol Biosafety Clearing House (http://bch.cbd.int/database/lmo-registry/ Bch (n.d.)) 2.6.1.3 The European GMO Database (https://www.euginius.eu/euginius/pages/home.jsf Euginius (n.d.)) 2.6.1.4 OECD Biotrack by Organisation for Economic co-Operation and Development (OECD) (http://www.oecd.org Oecd (n.d.)) 2.6.1.5 Patent Search Databases 2.7 GMO Detection Methods 2.7.1 Strategy for DNA-Based GMO Detection 2.7.2 GMO Matrix: A Decision Support System 2.7.3 Polymerase Chain Reaction (PCR) 2.7.4 Real-Time PCR (qPCR) 2.7.5 Digital PCR (dPCR) 2.7.6 Loop-Mediated Isothermal Amplification (LAMP) 2.7.7 Next-Generation Sequencing (NGS) 2.7.8 GMO Detection Methods Developed/Validated at ICAR-NBPGR 2.8 Conclusion References 3: Molecular Confirmation of Transgenic Events in Plants 3.1 Introduction 3.2 Molecular Characterization 3.2.1 Confirmation of Transgene Integration 3.2.2 Copy Number Analysis 3.2.3 Zygosity Analysis 3.2.4 Analysis of Site of Integration 3.2.5 Analysis of Transgene Expression 3.3 Molecular Confirmation in Genome-Edited Plants 3.4 Conclusion References 4: Genetic Engineering in Crop Plants Using Tissue Culture and Tissue Culture-Free Environment 4.1 Introduction 4.2 Genetic Engineering 4.3 Tissue Culture-Mediated Genetic Transformation 4.4 Tissue Culture-Mediated Genetic Transformation: Techniques 4.4.1 Microprojectile Bombardment 4.4.2 Microinjection 4.4.3 Silicon Carbide-Mediated Transformation 4.4.4 Pollen Tube-Mediated Transformation 4.4.5 Sonication 4.4.6 Electroporation 4.4.7 Liposome-Mediated Transformation 4.5 Advantages of Tissue Culture-Mediated Genetic Transformation 4.6 Disadvantages of Tissue Culture-Mediated Genetic Transformation 4.7 Tissue Culture-Free Genetic Transformation 4.8 Tissue Culture-Free Genetic Transformation: Techniques 4.8.1 In Planta Particle Bombardment (iPB) 4.8.2 Tissue Culture-Free Transformation Mediated Through Agrobacterium tumefaciens 4.8.3 Floral Dip 4.8.4 Infiltration Methodologies 4.9 Advantages of Tissue Culture-Free Genetic Transformation 4.10 Disadvantages of Tissue Culture-Free Genetic Transformation 4.11 Genome Editing: An Alternate to Transgenic Mediated Genetic Engineering 4.11.1 Genetic Transformation Via Meristem Induction 4.11.2 Heritable Genetic Transformation 4.11.3 Nanotechnology-Based Genetic Transformation 4.12 Genome Editing: Future Perspective 4.13 Conclusion References 5: Genetic Engineering of Plants for Vaccine, Recombinant Protein, and Drugs Production for Health Security 5.1 Introduction 5.2 Plant Cell Cultures: Current State and Future Perspectives 5.2.1 Recent Advances in Plant Cell Cultures in Bioreactors 5.2.2 Currently Used Bioreactor Types 5.3 Other Hosts Derived from Plants for Recombinant Protein Production 5.3.1 Plant Cell Packs 5.3.2 Cell Lysates 5.3.3 Hairy Root Cultures 5.3.4 Whole Plants 5.3.4.1 Transgenic Plants 5.3.4.2 Transplastomic Plants 5.3.4.3 Transient Expression 5.4 Molecular Tools Applied for Recombinant Protein Production in Plants 5.4.1 Vector Design in Molecular Farming 5.4.1.1 Protein Modifications to Improve Yield 5.4.1.1.1 Codon Usage 5.4.1.1.2 Resistance to Digestion 5.4.1.1.3 Signal Peptides 5.4.1.1.4 Promoters 5.4.1.2 DNA Assembly Methods for Complex DNA Constructs 5.5 Conclusion References 6: Genetic Modification of Tropical Root and Tuber Crops: Prospects and Perspectives 6.1 Introduction 6.2 Tropical Root and Tuber Crops: Problems and Prospects for Genetic Modification 6.2.1 Cassava 6.2.2 Sweet Potato 6.2.3 Yams 6.2.3.1 Greater Yam 6.2.3.2 Lesser Yam 6.2.3.3 White Yam 6.2.4 Aroids 6.2.4.1 Taro 6.2.4.2 Tannia 6.2.4.3 Elephant Foot Yam 6.2.5 Minor Tuber Crops 6.3 Applications of Genetic Modification in Tropical Tuber Crops 6.3.1 Tuber Yield and Size 6.3.2 Nutritional Quality 6.3.2.1 Starch Quality 6.3.2.2 Biofortification 6.3.2.2.1 Micronutrients 6.3.2.2.2 Vitamin A 6.3.2.2.3 Tuber Storage Proteins 6.3.3 Antinutrient Factors 6.3.3.1 Cyanogenic Glycosides 6.3.3.2 Alkaloids and Saponins 6.3.3.3 Phytates and Oxalates 6.3.4 Nutrient-Use Efficient Genotypes in Tuber Crops 6.3.5 Sterility and Poor Seed Setting 6.3.6 Biotic Stress Tolerance 6.3.7 Abiotic Stress Tolerance 6.3.8 Herbicide Tolerance 6.3.9 Post-Harvest Losses and Storage Quality 6.4 Regulatory Process 6.5 Conclusion References 7: Insights into the Genetic Improvement of Tomato 7.1 Introduction 7.2 Morphology 7.3 Reproductive Biology 7.3.1 Floral Biology 7.4 Gene Bank Repository 7.5 Conventional Breeding 7.5.1 Breeding Efforts 7.5.2 Breeding Objectives and Methodology 7.5.3 Tomato Breeding for Therapeutic/Nutraceutical Formulations 7.5.4 Marker-Assisted Selection 7.6 The Tomato Genome and Pan-Genomes 7.7 Tomato Leaf Curl Disease (ToLCD) 7.8 Genome Editing Tools for Tomato Improvement 7.8.1 Genome Editing for Yield and Fruit Quality Improvement in Tomato 7.8.1.1 Fruit Quality Improvement 7.8.1.2 Abiotic Stress Resistance Breeding in Tomato 7.8.1.3 Biotic Stress Resistance Breeding in Tomato 7.9 Conclusion References 8: Insights into the Genetic Improvement of Carrot (Daucus carota L.) 8.1 Introduction 8.2 Colorful Carrots Bring Healthy Colors to Our Life 8.2.1 Major Pigments and Their Significance in the Nutritive Content of Carrots 8.2.1.1 Carotenes 8.2.1.2 Lutein 8.2.1.3 Lycopene 8.2.1.4 Anthocyanin 8.3 Phytonutrient Profile of Carrots 8.3.1 Phenolics 8.3.2 Polyacetylenes 8.3.3 Ascorbic Acid 8.3.4 Vitamins 8.3.5 Minerals 8.3.6 Dietary Fibers 8.4 Genomic Description of Daucus carota L. 8.5 Genetic Manipulation to Improve Potential Metabolic Efficiency in Carrot 8.5.1 Engineered Nucleases: Efficient Tool for Site-Directed Mutagenesis 8.5.2 SSR Markers for Carrots 8.5.3 MicroRNA Profiling and Expression 8.5.4 Use of Vector and Nonvector Methods 8.5.5 Microprojectile Bombardment Method 8.5.6 Genome Editing in Carrots via CRISPR/Cas9 System 8.6 Conclusion 8.7 Future Prospects References 9: Insights into the Genetic Improvement of Canola 9.1 Introduction 9.2 Marker-Assisted Breeding 9.2.1 Marker-Aided Selection 9.2.2 Selection by Genome 9.2.3 Signal-Aided Breeding Focused on Enhancing Crop Performance in Response to Abiotic Limitations 9.3 Change Over to Canola from Rapeseed 9.4 Canola Varieties Improvement and Innovation 9.4.1 Rapeseed Genome with Enhanced Resolution 9.4.2 Pan-Genome 9.5 Abiotic and Biotic Stress Resistance 9.6 Canola’s Salt Stress Molecular Response 9.7 Technique of Salinity Resistance in Canola at the Molecular Level 9.8 Proline Synthesis 9.9 ROS (Reactive Oxygen Species) Scavenging System 9.10 QTL Response to Drought in Brassica Species 9.11 Screening of Elite Germplasm by Molecular Markers 9.11.1 Arabidopsis thaliana-Based Genetic Modifications of Brassica napus 9.11.2 Genomic Modulation of Canola Seed Storage Proteins 9.11.3 Canola Seed Storage Proteins: A Non-Genetic Approach 9.11.4 Oil Production from Transgenic Plants 9.12 Genetically Modified Plants’ Impact on Oilseed Crop Production 9.13 Relevance of Plant Gene Transfer 9.14 Conclusion References 10: Genetic Improvement of Mustard 10.1 Introduction 10.2 India’s Current Goal for Improving Rapeseed and Mustard Crops 10.3 Identifying Rapeseed-Mustard Germplasm with Desirable Characteristics for Crop Improvement 10.4 Strategic Studies to Improve Crop Improvement Programmes: Use of Genetic Resources 10.4.1 Creating Core and Mini Core Groups 10.5 Induced Mutations for Rapeseed Mustard Improvement 10.5.1 Mutations for Morphological Traits 10.5.2 Chlorophyll Mutations 10.5.3 Dwarf Mutations 10.5.4 Flower Mutations 10.5.5 Siliqua Characteristics 10.5.6 Seed Coat Colour 10.5.7 Root Morphology 10.5.8 Insect Pest-Resistant Mutation 10.6 Modifications to Increase Seed Yield and Related Characteristics 10.7 Double Haploids and Mutagenesis 10.8 Molecular Basis of Mutations 10.9 Transgenic Approaches for Enhancement of the Species Brassica 10.10 CRISPR/Cas9 to Enhance Brassica’s Agronomic Characteristics 10.11 miRNAs for Brassica Improvement 10.12 Nutritional Enhancement in Brassicaceae 10.13 Amino Acid Enhancement 10.14 Improving Antioxidants 10.15 Brassica Transgenic Methods for Abiotic Stress Tolerance 10.16 Utilising Molecular Methods for Genetic Enhancement of Oil Quality in Brassica juncea 10.16.1 Fatty Acid 10.16.2 Oleic Acid 10.16.3 Glucosinolates 10.16.4 Tocopherols 10.17 Conclusion References 11: Genetic Improvement of Foxtail Millet (Setaria italica L.) 11.1 Introduction 11.2 Taxonomy 11.3 Floral Biology 11.4 Nutritional Importance of Foxtail Millet 11.5 Genomic Resources for Millet Improvements 11.6 Crop Improvement 11.7 Objective of Breeding 11.8 Breeding Methods for Quality and Character Improvements in Foxtail Millet 11.8.1 Conventional Breeding Approach 11.8.2 Molecular Breeding Approach 11.8.2.1 Marker-Assisted Selection (MAS) Approach for Foxtail Millet Improvement 11.8.2.2 Implementation of Genetic Markers in Plant Breeding for Millet Quality Improvement 11.9 Transcriptomic Approaches (Stresses Affecting Productivity) 11.9.1 Improvement for Abiotic Stress 11.9.2 Improvement for Biotic Stress 11.10 Conclusion References 12: Genetic Improvement of Groundnut 12.1 Introduction 12.1.1 Genetics 12.2 Genetic Resources 12.3 Genetic Analysis in Bambara Groundnut 12.3.1 The Significance of Traditional Varieties and Landraces 12.3.2 Opportunity for Bambara Groundnut Breeding 12.4 Development of Transgenic Plant: Fundamental Idea and Process 12.4.1 Establishing a Culture for a Desired Organ, Tissue, or Cell 12.5 Techniques for Inserting Genes 12.5.1 Transformation Mediated by Agrobacterium 12.6 Expression of a Target Gene in Transgenic Plants 12.7 Successful Character Enhancement in Groundnut Using a Transgenic Approach 12.8 Biotic Stress Resistance 12.9 Ability to Resist Fungal Illness 12.10 Resistance to Insect Pests 12.11 Absorbance of Abiotic Stress 12.11.1 Adaptability to Hydration Stress 12.11.2 Resistance to Salinity 12.12 Traditional Methods for Groundnut Improvement 12.12.1 Genetics and Genetic Diversification 12.13 Enhancing Groundnuts with Genomic Tools 12.14 The Molecular Underpinnings of High-Oleic (HO) Peanut Genetics 12.14.1 High-Oleic (HO) Trait Genetics and the Development of Cultivars 12.14.2 Gene Profiling for HO Content 12.15 Molecular Enhancement Through Primary Gene Pool Resources 12.15.1 Conventional Breeding 12.16 Conclusion References 13: Genetic Improvement of Jute: An Alternative for Plastic and Future Food 13.1 Introduction 13.2 Versatile Nature of Jute 13.2.1 A Fiber Crop 13.2.2 A Viable Substitute for Plastic 13.2.3 A Source of Food 13.2.4 A Nutritious Fiber Crop 13.2.5 A Potential Therapeutic Crop 13.2.6 A Raw Material for Paper Pulp and Other Items 13.3 Breeding and Biotechnology for Jute Improvement 13.3.1 Breeding and Wide Hybridization in Jute 13.3.2 Mutation in Jute Crop 13.3.3 Genetic Transformation of Jute 13.3.4 RNA Interference (RNAi) Technology 13.4 Toward Improvement of Jute 13.4.1 Insertional Mutagenesis 13.4.2 CRISPR/Cas9 13.5 Conclusion References 14: Genome Editing: A Safe Alternative to Genetic Engineering of Crops 14.1 Introduction 14.2 Principles of Genome Editing 14.3 History of Genome Editing 14.4 Genome-Editing Tools 14.4.1 Meganucleases (MNs) 14.4.2 Zinc Finger Nucleases (ZFNs) 14.4.3 Transcription Activator-Like Effector Nucleases (TALENs) 14.4.4 CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein Cas9) RNA-Guided Nucleases 14.4.4.1 CRISPR/Cas System in Bacterial Adaptive Immunity 14.4.4.2 Classification of CRISPR/Cas System 14.4.4.3 The Type II CRISPR/Cas System 14.4.4.4 Improvements in CRISPR/Cas9 Genome Editing 14.4.4.4.1 Cas9-NG 14.4.4.4.2 CjCas9 14.4.4.4.3 xCas9 14.4.4.4.4 Cas12a (Cpf1) 14.4.4.4.5 Cas13 14.4.4.4.6 Cas14a 14.4.4.4.7 Cas9 Nickase 14.4.4.4.8 dCas9 14.4.4.4.9 Base Editing 14.4.4.4.10 Prime Editing 14.4.4.4.11 DNA-Free Editing 14.4.4.4.12 Multiplex Genome Editing 14.4.4.4.13 Epigenome Editing 14.4.4.4.14 PAM-Free Nuclease 14.4.4.4.15 Dimeric RNA-Guided FokI Nucleases (RFNs) 14.4.4.4.16 CRISPR/Cas9 and λ-Red Recombinase-Based MAGE Technology (CRMAGE) 14.4.4.4.17 Other Improvements 14.5 Comparison of Various Genome-Editing Tools 14.6 Strategy for Genome Editing in Plants 14.7 Applications of Genome Editing in Crop Improvement 14.8 Advantages of Genome Editing 14.9 Challenges and Concerns of Genome Editing 14.10 Regulatory Considerations 14.11 Future Prospects 14.12 Conclusion References 15: Transgenic and Genome-Edited Maize: Status and Prospect 15.1 Introduction 15.2 Transgenesis for Maize Improvement 15.3 Genome Editing for Maize Improvement 15.3.1 Zinc Finger Nucleases 15.3.2 TALENS 15.3.3 CRISPR/Cas 15.3.4 Base Editing 15.3.5 Prime Editing 15.4 Conclusion References 16: Genome Editing for Trait Improvement in Potato (Solanum tuberosum L.) 16.1 Introduction 16.2 Genome Editing with Engineered Nuclease (GEEN) Technologies 16.2.1 Meganucleases (MNs) 16.2.2 Zinc Finger Nucleases (ZFNs) 16.2.3 Transcription Activator-Like Effector Nucleases (TALENs) 16.2.4 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein (Cas) 16.3 Expansion of CRISPR/Cas9 Toolkit 16.4 Applications of CRISPR/Cas9 in Potato Trait Improvement 16.4.1 Potato with Improved Resistance to Biotic Stress and Herbicide 16.4.2 Potato with Reduced Toxicity 16.4.3 Potato with Self-Compatibility 16.4.4 Potato with Reduced Enzymatic Browning 16.4.5 Potato Phytoene Desaturase Mutants 16.4.6 Potato with Better Starch Quality 16.5 Challenges with Potato Genome Editing 16.6 Conclusion References 17: Genetic Improvement of Chickpea Using CRISPR-Based Genome Editing Strategy 17.1 Introduction 17.2 Genetic Improvement of Chickpea Using Conventional Methods and Marker-Assisted Selection 17.2.1 Breeding for Biotic Stress Tolerance 17.2.2 Breeding for Abiotic Stress Tolerance 17.3 Genome Editing Technologies 17.3.1 Zinc Finger Nucleases (ZFNs) 17.3.2 Transcription Activator-Like Effector Nuclease (TALEN) 17.3.3 CRISPR/Cas System 17.4 Applications of CRISPR/Cas9 System in Chickpea and Other Legumes 17.5 Genetic Transformation in Chickpea 17.6 Regulatory Barriers and Genome Editing 17.7 Conclusion and Future Perspectives References 18: Future Perspective on CRISPR-Cas9-Based Targeted Genome Editing in Date Palms 18.1 Introduction 18.2 Limitations in the Functional Genomic Studies of Date Palms 18.3 Steps of CRISPR/Cas9-Mediated Editing 18.3.1 Target and Off-Target Selection 18.3.2 Design of the gRNA Cassette 18.3.3 Selection of the CRISPR/Cas Nuclease 18.3.4 Delivery of the CRISPR/Cas9 Construct and Selection of Transformed and Mutated Date Palm Plants 18.4 Multiplex Genome Editing Using CRISPR and Its Potential Use in Date Palms 18.5 Relaxing the Off-Target Effects of Cas9 in Date Palms 18.6 Applications of CRISPR/Cas9 Genome Editing to Date Palms 18.6.1 Abiotic and Biotic Stress Tolerance 18.6.2 Nutritional Improvement 18.6.3 Improvement of Other Traits 18.7 Limitations of CRISPR/Cas9-Based Genome Editing in Date Palms 18.8 Conclusion References 19: Updates on Global Status of Transgenic and Genome-Edited Crops 19.1 Introduction 19.2 Overview of Biotech/Transgenic Crops 19.2.1 Current Status of Biotech Crops 19.2.2 Global Acreage of Transgenic Crops 19.2.3 Status of Approved Events of GM Crops 19.3 Global Production of GM Crops 19.3.1 Africa: Embracing GM Crops for Agricultural Transformation 19.3.2 Asia: GM Crops and Agricultural Sustainability 19.3.3 Europe: Striving Toward Crop Improvement 19.3.4 North America (USA and Canada): A Stronghold on GMO Crop Production 19.3.5 Australia: Growing with the Change 19.4 Regulation on GM Crops 19.5 Significance of GM Crops 19.5.1 Herbicide-Tolerant Crops 19.5.2 Insect/Pest-Resistant Crops 19.5.3 Biofortification for Nutritional Enhancement 19.6 Risk Assessment 19.7 Challenges Associated with GM Crops 19.8 The GMO Register 19.9 Genome-Edited Crops 19.10 Conclusion References