Breeding for Disease Resistance in Plants PDF

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/381114607 DOC-20240514-WA0008. Chapter · June 2024 CITATIONS READS 0...

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/381114607 DOC-20240514-WA0008. Chapter · June 2024 CITATIONS READS 0 2,265 2 authors, including: Sambita Bhattacharyya G. B. Pant University of Agriculture and Technology, Pantnagar 7 PUBLICATIONS 2 CITATIONS SEE PROFILE All content following this page was uploaded by Sambita Bhattacharyya on 03 June 2024. The user has requested enhancement of the downloaded file.  GENETICS AND PLANT BREEDING VISTAS-EMERGING TRENDS IN GENETICS AND PLANT BREEDING M. Vennela graduated from Professor Jayashankar Telangana State Agricultural University, Hyderabad with a B.Sc. and an M.Sc. degree in Agriculture. She qualied for the ASRB NET in 2020 and is presently pursuing her Ph.D. in the discipline of Genetics and Plant Breeding at Dr. Rajendra Prasad Central Agricultural University, Bihar. Ms. Vennela GENETICS AND PLANT BREEDING VISTAS worked on sugarcane and rice crops for her research programmes. Her contributions to numerous publications include book chapters, conference papers, popular articles, short communications and research articles. EMERGING TRENDS IN Dr. D. Dinesh Varma completed his B.S.c., Agriculture from PJTSAU, Rajendranagar campus, Hyderabad and M.S.c., in Genetics and plant Breeding from Junagadh Agricultural university, Junagadh, Gujarat and Ph.D from Dr. Rajendra prasad central agricultural university, Pusa. He has published several research articles in many reputed national and GENETICS AND PLANT BREEDING international journals. He started working with seed companies from brief period of time. He has immnese experience in the elds of research. Dr. Sangeeta Kumari has completed her B.Sc. degree from All India Jaat Hero’s Memorial College, Rohtak, afliated with Maharishi Dayanand University, Rohtak, Haryana in 2010 and completed her M.Sc. (Botany) in 2012 from the Department of Botany in Maharishi Dayanand University, Rohtak, Haryana. She is qualied CSIR -UGC NET with all India rank 20th in 2013. Completed her Ph.D. (Botany) in 2022 from Maharishi Dayanand University, Rohtak, Haryana. She has published more than ve research papers in reputed journals. Currently she is working as Assistant Professor in RPS degree college, Mahenderghar, Haryana. Roshani Khetan completed her post-graduation in 2018 and graduation in 2016 from Banasthali University, Rajasthan in Biotechnology. She is currently pursuing a Ph.D. in Molecular Biology from SAGE University, Indore, in collaboration with ICMR NIREH Bhopal. She has extensive research experience in reproductive toxicity, mitochondrial biology, and epigenetic modications, with notable contributions to projects at ICMR- NIREH Bhopal. Roshani is also a prolic author, having published numerous research articles/paper & book chapters in reputable national and international journals. T. Deepthi completed her B.Sc., Horticulture from Sri Krishnadevaraya College of Horticultural Science (SKCHS), Anantapur and M.Sc., in Genetics and Plant Breeding from Annamalai University. She has published research articles in many reputed national and international journals. She shared her expertise to students by working as Assistant Professor in BEST Innovation University, Gorantla for a brief period of 1 year. She has immense experience in the eld of research. Editors : - M. Vennela Dr. Dinesh Varma Dr. Sangeeta Kumari Stella International Publication Roshani Khetan # 1781/3, Urban Estate, T. Deepthi Kurukshetra-136118 (Haryana) E-mail: stellapublica [email protected] MRP: T 990.00 Stella International Publication Kurukshetra  Genetics and Plant Breeding Vistas: Emerging Trends in Genetics and Plant Breeding i Genetics and Plant Breeding Vistas: Emerging Trends in Genetics and Plant Breeding Editors M.Vennela Dr. D Dinesh Varma Dr. Sangeeta Kumari Roshani Khetan T. Deepthi Stella International TM Publication ii Copyright © 2024, Stella International Publication TM All rights reserved. Neither this book nor any part may be reproduced or used in any form or by any means, electronic or mechanical, including photocopying, microfilming, recording or information storage and retrieval system, without the written permission of the publisher and author. Edition: 1st Publication Year: 2024 ISBN: 978-81-972019-5-0 Price: ₹ 990/- Published by: Stella International Publication 1781-3, U.E., Kurukshetra Haryana 136118 (India) Email: [email protected] Website: www.stellainternationalpublication.com iii Preface iv  Contents Preface iv 1. The Basis of Plant Genetics: An Overview 1 Ananthu Rajagopal and Aswini M S 2. CRISPER and Gene Editing in Plant Breeding 21 Yogesh Kashyap, Manisha Kumari, Patel Rushiprasad J. and Vinod Kumar 3. Climate Resilient Crops-A Genetic Approach 35 K Sri Anjanidevi 4. Advances in Quantitative Trait Loci (QTL) Mapping 54 Shirisha K. M. 5. Genetic Diversity and Crop Improvement 70 Mitali Srivastava and Manojkumar H G 6. Biofortification: Enhancing Nutritional Quality of Crops 94 Darshana A. S., Sruthi R., Parvathy and Narla Abigna 7. Genomics and Its Role in Modern Plant Breeding 113 Sachin, Yogender Kumar, Paras and Ashok 8. Hybridization Techniques in Plant Breeding 132 Narendra Deshwal, Ankita Yadav, Mahendra Kumar Ghasolia and Versha Sharma 9. Molecular Markers in Plant Breeding 148 Karuna and Navreet Kaur Rai 10. Transgenic Crops: Benefits and Controversies 174 Narendra Deshwal, Ankita Yadav, Versha Sharma and Mahendra Kumar Ghasolia v 11. Plant Tissue Culture and Its Role in Plant Breeding 188 Gangadhara Doggalli, Santhoshini Elango, C A Deepak and Shobha Immadi 12. Breeding for Disease Resistance in Plants 209 Gangadhara Doggalli, Abhishek V. Karadgi, Santhoshini Elango and Anusha T S 13. Role of MicroRNAs in Plant Genetics 240 Kanshouwa Modunshim Maring and Madhuri Arya 14. Conservation and Use of Genetic Resources in Breeding 259 Aswini M. S. and Ananthu Rajagopal 15. Genetic Engineering for Abiotic Stress Tolerance in Plants 273 Nikhil P. G., Niji M. S., Varsha P. Vengilat and Swathy V. 16. Advances in Root Breeding and Genetics 310 Sruthi R, Darshana A S, Parvathy and Narla Abigna 17. Genetically Modified Organisms (GMOs) in Agriculture 336 Soumya Patel 18. Plant Breeding for Organic Agriculture 354 Narla Abigna, Parvathy, Sruthi R. and Darshana A. S. 19. Next Generation Sequencing Technologies in Plant 378 Genetics Parvathy, Narla Abigna, Sruthi R and Darshana A S 20. The Ethics of Genetic Manipulation in Plant Breeding 399 Sourav Ranjan Nanda, Sambita Bhattacharyya, Shreya Singh and Samar Kumar Rath 21. Polyploidy and its Role in Plant Breeding 415 Swathy Sivan, Thouseem N and Revathi B S vi  CHAPTER: 1 THE BASIS OF PLANT GENETICS: AN OVERVIEW Ananthu Rajagopal1 and Aswini M S2 1 Ph.D. Scholar, Department of Genetics and Plant Breeding, UAS Bangalore 2 Ph.D. scholar, Department of Genetics and Plant Breeding, Kerala Agricultural University Abstract The study of genes, genetic variation, and heredity in plants is the focus of the field of plant genetics. Although it is frequently thought of as a subfield of botany and biology, it interacts with many other life sciences and has strong ties to the study of information systems. While there are many important distinctions between plant and animal genetics, there are also numerous commonalities. The field of genetics is credited with being pioneered by the late nineteenth-century scientist and Augustinian monk Gregor Mendel. His research revolved around "trait inheritance," examining the transmission of traits from parents to their progeny. It was discovered via Mendel's revolutionary research, mostly on pea plants, that organisms pass on traits through discrete "units of inheritance." Furthermore, the overview highlights the modern tools and techniques of plant genetics, including marker-assisted selection and genetic modification, which enable researchers and breeders to manipulate plant genomes for desired traits. By understanding these fundamentals, stakeholders in agriculture are equipped to address challenges related to crop improvement, biodiversity conservation, and global food security Keywords: DNA, Genes, Genetics, Inheritance and Markers 1 Introduction The field of plant genetics is concerned with the study of genes and genetic variation, and the study of heredity in plants specifically. Plant genetics is often seen as a branch of biology or botany, but it often interconnects with many other branches of life and is strongly related to information systems (Griffiths,2005). Although plant genetics is similar to animal genetics in many ways, there are some key differences between the two. Like all known organisms, plants use DNA to transfer their traits. In animal genetics, the main focus is on the parentage and lineage. However, this can sometimes be challenging in plant genetics, as plants can be self-fertile unlike most animals. Mendel, an Augustinian friar and scientist from the late 19th century, was one of the discoverers of genetics. He studied “trait inheritance” (mainly in pea plants) the process by which organisms inherited traits through discrete “units of inheritance”. The term “genes” is still used to refer to what is now considered a gene. A lot of Mendel’s work still forms the basis of modern plant genetics. Many plants have special genetic traits that make speciation simpler, including being well tuned for polyploidy. Plants are special because they can use chloroplasts to carry out the process of photosynthesis, which produces carbohydrates that are high in energy. identical to superficially identical mitochondria, chloroplasts are made of their own DNA. Thus, chloroplasts offer a greater genetic variety and gene storage in addition to an additional genetic layer of intricacy not present in animals. Plant genetics research has a significant economic influence because it has led to the genetic modification of several staple crops, which boost yields, impart resistance to pests and diseases, offer herbicide resistance, or improve nutritional value. History According to research, the earliest known proof of plant domestication dates back 11,000 years, to the time of primordial wheat. Farmers most certainly had a fundamental concept of heredity and inheritance, the basis of genetics, by 5,000 years ago, even though 2 selection probably happened accidentally at first. New crop species and varieties that form the foundation of the crops we cultivate, consume, and study today is the result of this selection over time. Gregor Johann Mendel, sometimes referred to as the "father of genetics," is credited with founding the study of plant genetics. He was born in Austria-Hungary on July 20, 1822, and was an Augustinian priest and scientist. He worked at the Abbey of St. Thomas in Brünn, which is now Brno in the Czech Republic. The pea plant was his go-to organism for researching characteristics and inheritance there. Mendel's research examined a wide range of phenotypic features of pea plants, including height, color of flowers, and qualities of seeds. Mendel demonstrated that these features are inherited according to two specific rules, which are later named after him. Published in 1866, his groundbreaking study on genetics, "Versuche über Pflanzen-Hybriden" (Experiments on Plant Hybrids), was largely ignored until 1900, when eminent UK botanists such as Sir Gavin de Beer realized its significance and republished an English translation. 1884 saw Mendel's death. Mendel's segregation ratios were studied by statisticians and botanists in the early 1900s. The basis of population genetics was established by W.E. Castle's discovery that, although individual qualities may segregate and vary over time due to selection, the genetic ratio ceases to change and reaches a state of stability when selection is halted and environmental variables are taken into consideration. G. H. Hardy and W. Weinberg separately found this, which ultimately led to the publication of the Hardy–Weinberg equilibrium notion in 1908. DNA The genetic instructions required for the formation and operation of all known living things as well as certain viruses are found in deoxyribonucleic acid (DNA), a type of nucleic acid. Long-term preservation of data is the primary function of DNA molecules. Since DNA carries the instructions needed to make other components of cells, such proteins and RNA molecules, it is frequently compared to a collection of blueprints, a recipe, or a code. The genetic information is carried by DNA segments called genes, and the locations of these genes 3 within the genome are called genetic loci. Other DNA sequences, on the other hand, serve structural functions or control how this genetic information is used (Figure 1). Figure1.The structure of part of a DNA double helix This sequence of DNA is used by geneticists (including plant geneticists) to identify and elucidate the function of various genes within a genome. Through plant research and breeding, various techniques can be used to manipulate various plant genes (genotypes) and loci (genotypes) encoded by the plant chromosome DNA sequence to create different or desirable genotypes that lead to different or desirable phenotypes. Chromosomes A chromosome is a DNA packet containing all or most of an organism's genetic material. Most chromosomes have extremely long, thin DNA fibers covered in nucleosome-forming packing proteins, the most significant of which are histones in eukaryotic cells (Hammond et al., 2017). To preserve the integrity of the DNA molecule, these proteins attach to it and compress it with the help of chaperone proteins. The intricate three-dimensional structure of these chromosomes is important for the control of transcription (Wilson, J., & Hunt, T. 2002). 4  Its only during the metaphase stage, the chromosomes are usually visible under the light microscope. Prior to this, each chromosome goes through a S phase of duplication, in which two copies are connected by a centromere. This can produce an X-shaped structure (Figure2.) if the centromere is placed equatorially, or a two-arm configuration if it is located distally. Figure 2. Structure of Chromosome Sister chromatids are the new term for the linked copies.A metaphase chromosome is an X-shaped structure that is highly condensed during metaphase and is therefore the easiest to identify and examine. Chromosomes in animal cells undergo chromosome segregation during anaphase, when they achieve their maximum compaction level. A large portion of genetic variability is attributed to chromosomal recombination that occurs during meiosis and following sexual reproduction. The cell may experience a mitotic catastrophe if these structures are improperly altered through procedures known as chromosomal instability and translocation. 5 DNA packaging a) Prokaryotes: Prokaryotes lack nuclear structure. Instead, the DNA is reorganized into a nucleoid (Thanbichler et al., 2005). The bacterial cell's nucleoid is a unique structure that takes up a specific area. But this structure is dynamic, and a variety of histone-like proteins that attach to the bacterial chromosome work to preserve and modify it. Chromosomes in archaea have even more ordered DNA, contained in structures resembling eukaryotic nucleosomes. Other extrachromosomal DNA, such as plasmids, are also present in some bacteria. These are cytoplasmic circular structures that aid in horizontal gene transfer and hold cellular DNA. Bacterial chromosomes are typically attached to the bacteria's plasma membrane (Antonin, W., & Neumann, H. (2016). This enables its separation from plasmid DNA in applications related to molecular biology through the centrifugation of lysed bacteria and pelleting of the membranes (together with the associated DNA). Similar to eukaryotic DNA, prokaryotic chromosomes and plasmids are typically supercoiled. To make the DNA accessible for transcription, regulation, and replication, it must first be released into its relaxed state. b) Eukaryotes: A dense complex of proteins and DNA known as chromatin is formed by the association of a long linear DNA molecule with proteins on each eukaryotic chromosome. Much of an organism's DNA is contained in its chromosomes, (Figure 3) but a tiny bit that is inherited from mothers can be found in the mitochondria. With just a few such red blood cells, it is found in the majority of cells. The nucleosome, the earliest and most fundamental unit of chromosomal organization, is made possible by histones. Numerous enormous linear chromosomes are housed in the nucleus of eukaryotes, or cells with nuclei, like those found in plants, fungi, and mammals. Every chromosome consists of a centromere and one or two arms that extend from it; however, these arms are typically invisible. 6  Figure 3. DNA packaging of chromosome Meiosis The DNA genome of plants experiences double-strand breaks following meiosis. Such breaks can be repaired through a type of recombination process that uses the gene products RAD51 and DMC1, which are homologous to recombinases used by eukaryotes in general. The production of double-strand breaks, homologous recombinational repair, and homologous chromosomal pairing are essential components of eukaryotic meiosis. These mechanisms seem to be tailored to repair germline DNA damage (Mirzaghaderi et al., 2016) Plant Specific Genetics Similar to every other known living thing, plants use DNA to pass on their traits. However, the presence of chloroplasts sets plants apart from other living things. Chloroplasts are made of DNA, just like mitochondria. Similar to animals, plants also undergo somatic mutations on a regular basis; however, since flowers arise at the tips of branches made of somatic cells, these changes can readily influence the germ line. This has been known for millennia, and the term "sports" refers to the mutant branches. It is possible to obtain a new cultivar if the fruit on the sport is profitable. 7  Certain plant species have the ability to fertilize themselves, and others are almost completely self-fertile. This implies that, unlike mammals, plants can act as both a mother and a father to their progeny. When doing plant crosses, scientists and amateurs need to take extra precautions to keep the plants from fertilizing themselves (Caime, S. 2018). Hybrids between plant species are produced in plant breeding for both decorative and commercial purposes. For instance, the development and spread of hybrid corn types has contributed to an almost five-fold rise in corn output during the previous century. The combination of plants that will likely result in a hybrid with hybrid vigor can be predicted using plant genetics, or on the other hand, many genetic breakthroughs have arisen from researching the consequences of hybridization. In general, polyploid plants are better equipped to endure and even thrive. There are more than two sets of homologous chromosomes in polyploid species. A normal human will have two copies of each of the 23 distinct chromosomes, for a total of 46, since humans, for instance, have two sets of homologous chromosomes. Conversely, wheat is a hexaploid plant with six copies of each of its seven different chromosomes, making it a total of 42. In hereditary germline polyploidy, animals are less likely to experience it, and spontaneous chromosomal expansions might not even last through fertilization. This is less of an issue with plants, though. Numerous procedures can result in the creation of polyploid individuals, but once they are, they typically are unable to revert to their original form. Self-fertile polyploid individuals have the potential to create a new, genetically unique lineage, perhaps leading to the emergence of a new species. We call this "instant speciation" a lot. Numerous crops used for human consumption, such as wheat, maize, potatoes, peanuts, strawberries, and tobacco, are either inadvertently or purposefully produced as polyploids due to their larger fruit, which is a characteristic that is commercially advantageous. Model Organism a) Arabidopsis thaliana 8  The model organism for the study of plant genetics has been Arabidopsis thaliana, commonly referred to as thale cress (Figure 4). A. thaliana has contributed as much to our understanding of plant genetics as the fruit fly species Drosophila did to that of early genetics. In the year 2000, it became the first plant whose genome had ever been sequenced. Because of its tiny genome, the initial sequencing is more doable. With a 125 Mbp genome, it can encode roughly 25,000 different genes. A database known as The Arabidopsis Information Resource (TAIR) has been built as a repository for various data sets and information on the species due to the extraordinary quantity of research that has been carried out on the plant. Figure 4. Arabidopsis thaliana There are numerous naturally occurring inbred accessions of A. thaliana (sometimes called "ecotypes") that have been helpful in genetic studies. Utilizing this natural variation, loci crucial for biotic and abiotic stress resistance have been found. b) Brachypodium distachyon Brachypodium distachyon (Figure 5) is a species of grass endemic to southern Europe, northern Africa, southwestern Asia, and eastern India. It is also known by the popular name like purple false brome and stiff brome. It shares relationship main species of cereal grains, including millet, rye, sorghum, wheat, barley, and oats. 9  It is an ideal model organism for functional genomics studies in temperate grasses, cereals, and speciality biofuel crops like switchgrass because of its various attributes. These characteristics include a tiny physical appearance, self-fertility, short lifespan, simple growing needs, an effective transformation system, a small genome (~270 Mbp) diploid accessions, and a number of polyploid accessions. Figure 5. Brachypodium distachyon Other model plants Additional models comprise the C4 grass Setaria viridis, the alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the clover Medicago truncatula, the antirrhinum majus (snapdragon), and maize (corn). Genetic linkages and recombination The large-scale arrangement of genetic material into chromosomes results in genetic linkage; in the absence of recombination, all genes located on the same chromosome would segregate as a unit and exhibit absolute genetic linkage. Maintaining blocks of advantageous alleles as a unit has clear benefits for an organism. This benefit must be weighed against the need for genetic variety that evolution requires in order to produce novel allelic arrays through crossing-over, a process that 10 results in recombinant chromosomes. Recombination events can give rise to either reciprocal recombination or similar events. Genetic information is exchanged between two nonsister chromatids to cause reciprocal recombination. The nonreciprocal transfer of DNA sequences from one nonsister chromatid to another is known as gene conversion. Mechanistic models that account for both gene conversion and reciprocal recombination have been sought after since they seem to follow a similar pattern. Genetic variation Genetic variation refers to the variations in DNA between individuals or communities within the same species. Genetic recombination and mutation are two of the several sources of genetic diversity. Genetic variety ultimately originates from mutations, although it is also influenced by other processes like genetic drift. Genetic variety ultimately originates from random mutations. Most mutations are neutral or harmful, and mutations are probably uncommon. However, natural selection may occasionally favor the new alleles. One type of chromosomal mutation is polyploidy. There are various ways to identify genetic variation. Studies of phenotypic variation in either discrete (traits that fall into discrete categories and are coded for one or a few genes, such as the white, pink, or red petal color in certain flowers) or quantitative (traits that vary continuously and are coded for by many genes) traits can be used to identify genetic variation (Darwin,1889) When an organism has three or more sets of genetic variation, it is said to be polyploid (3n or more). New alleles or allele combinations can be produced during meiosis as a result of crossing over (genetic recombination) and random segregation. Moreover, variation is also influenced by random fertilization. LINEs, SINEs, endogenous retroviruses, transposable genomic elements, and other entities can all promote variation and recombination Genetic diversity for a given multicellular organism's genome can be inherited through the germline or acquired in somatic cells. 11 Inbreeding and Hybdrization Both plants and animals experience the phenomena known as inbreeding depression, which is the decreased survival and fertility of offspring of related individuals. This indicates that diversity for heritable fitness qualities occurs within populations. Different animals experience inbreeding depression at different rates. The distinction between plants classified as outbreeders (species with reproductive mechanisms promoting cross-pollination, which typically exhibit inbreeding depression and tend to be intolerant of inbreeding, e.g., maize and alfalfa) and inbreeders (species with reproductive mechanisms promoting self-pollination in which inbreeding depression is minimal, who tolerate many generations of inbreeding, e.g., wheat and oat) was done for the first time by Charles Darwin, a British naturalist best known for his theories of evolution and natural selection. Hybridization is the process of marrying or crossing two plants or lines with different genotypes. Pollen grains from one genotype i.e., the male parent i.e., are placed on the stigma of flowers from the other genotype i.e., the female parent i.e., to cross plants. Preventing accidental cross-pollination and self-pollination in the flowers of the female parent used for crossing is crucial. Hybrid or F1 refers to the seeds and offspring produced by hybridization. The offspring of F1 that result from selfing or crossing F1 plants are referred to as segregating generations, as are the following generations. The term "Cross" is frequently used to refer to the hybridization products, such as F1 and segregating generations. Genetically modified crops Foods labeled as genetically modified (GM) are created from organisms whose DNA has been altered through genetic engineering techniques. Compared to earlier techniques like selective breeding and mutation breeding, genetic engineering techniques facilitate the development of new traits as well as more control over existing traits. In 2017, 89% of maize, 94% of soybeans, and 91% of cotton grown in the US came from genetically modified strains, demonstrating the economic significance of plant genetic modification. Yields have increased by 22% and farmer profits, particularly in poor nations, have soared by 68% since 12 the introduction of genetically modified crops. The reduction in the amount of land needed for GM crops has been a significant adverse effect. 1994 saw the introduction of genetically modified foods onto the commercial market with Calgene's failed Flavr Savr delayed-ripening tomato. The majority of food alterations have mostly targeted cash crops that farmers are in high demand for, like cotton, soybean, corn, and canola. Better nutritional content and resistance to diseases and herbicides are two features of genetically modified crops. Additional crops of this type include the commercially significant genetically modified papaya, which is immune to the highly damaging papaya ringspot virus, and the nutritionally enhanced golden rice, which is currently under research.The scientific community is in agreement that food made from genetically modified crops is now available and does not pose an increased risk to human health than traditional food, but every GM food product should be evaluated individually before being introduced. However, the general population is far less likely than scientists to believe that genetically modified foods are safe. Each country has a different legal and regulatory position regarding genetically modified foods; some forbid or limit them, while others allow them with wildly varying levels of control. Public worries about food safety, regulations, labeling, environmental effects, research methodologies, and the fact that some genetically modified seeds are protected by corporate intellectual property rights are still present. Modern ways to genetically modify plants Numerous studies in contemporary plant genetics have been prompted by genetic manipulation, which has also resulted in the sequencing of numerous plant genomes. These days, there are two main methods for changing an organism's genes: The Agrobacterium method the "Gene gun" method. "Gene gun" method Another name for the gene gun approach is "biolistics" (ballistics with biological components). This method has been particularly helpful for in vivo (within a living organism) transformation of monocot species, 13 such as rice and corn. This method essentially injects genes into the chloroplasts and plant cells. Small gold or tungsten particles with a diameter of around two micrometers are coated with DNA. The plant tissue to be modified is positioned beneath the vacuum chamber containing the particles. A brief burst of high-pressure helium gas is used to push the particles at a high speed (Figure 6.) They strike a fine mesh baffle positioned above the tissue as the DNA coating penetrates every tissue or cell. Figure 6. Gene Gun Agrobacterium method Dicots, like tomatoes and soybeans, have long benefited from the successful application of the Agrobacterium transformation process. This technique has recently been modified and shown to work well with monocots like rice and corn, which are grasses. Because the Agrobacterium method may generate a higher frequency of foreign DNA single-site insertions, making monitoring easier, it is frequently chosen over the gene gun method. Using this technique, the desired gene plus a marker are substituted for the tumor-inducing (Ti) region that was previously removed from the transfer DNA, or T-DNA (Figure 7). The creature is subsequently given this altered T-DNA. The procedure could entail either directly injecting the tissue with a culture of altered Agrobacterium or injecting it following tissue-damaging micro-projectile 14 bombardment. The damaged tissues force the plant to release phenolic compounds which induces the tissue invasion by Agrobacterium. Micro- projectile bombardment often enhances the efficiency of Agrobacterium infection. When an organism successfully incorporates the intended gene, it is identified by the marker. The organism's tissues are then transferred, depending on the marker employed, to a medium that either contains an antibiotic or a herbicide. The antibiotic eliminates Agrobacterium from the environment. Only tissues that display the marker will endure and contain the desired gene. The next phases entail using these surviving plants to produce complete plants, which are cultivated in tissue culture under carefully regulated environmental conditions. This involves a number of media, all of which have hormones and nutrients in them. Figure 7. Agrobacterium induced gene transfer The genetics of sex determination in plants, and plant sex chromosomes Plants' sex chromosomes and sex inheritance are remarkably comparable to those of animals. Most plants under study have heterozygous males, or male heterogamety (XY males, XX females) when the chromosomes are clearly distinct (perhaps half of plants with separate sexes, see Westergaard, 1958). Males of many dioecious plants are 15 "inconstant," meaning they only sometimes bear fruit (Lloyd, 1975b; Lloyd and Bawa, 1984). Genetic evidence indicating heterozygous males has been offered by self-fertilization of these plants in multiple species. The male genotype needs to contain a dominant suppressor of femaleness (SuF), as will be discussed later. If SuF / SuF is viable, then a 3:1 male to female ratio should result from selfing; if the Y chromosome is genetically degraded and this genotype is inviable, should result in 2:1 ratio. It has been possible to find each of these ratios (Westergaard, 1958; Testolin et al, 1995). As a result, several plant Y chromosomes have at least some genetic degeneracies. There are multiple types of evidence that point to the participation of two loci in determining sex. A portion of the data originate from hybrids between related monoecious or hermaphrodite species and dioecious plants (Westergaard, 1958). Cytological studies have provided direct evidence of Y chromosomal deletions in Silene dioica and latifolia. The SuF region and two areas containing factors regulating early and late anther development are the three functionally distinct Y chromosomal regions (Figure 8) (Westergaard, 1958; Grant et al, 1994; Farbos et al, 1999; Lardon et al, 1999). In these species, recombination is missing for the majority of the Y chromosome and X and Y pairing in male meiosis is limited to the tips (Westergaard, 1958; Parker, 1990; Lardon et al, 1999) Marker assisted selection A trait of interest is chosen indirectly by a procedure known as marker assisted selection, or marker aided selection (MAS), in which the trait itself is not taken into consideration but rather a marker (morphological, biochemical or DNA/RNA variation) associated with the trait (such as productivity, disease resistance, abiotic stress tolerance, and quality). This method has been thoroughly studied and suggested for the breeding of plants and animals. For instance, rather than focusing on the degree of disease resistance, MAS is used to select individuals who have disease resistance by identifying a marker allele that is associated with disease resistance. It is assumed that the marker's high frequency of association with the gene or quantitative trait locus (QTL) of interest 16 results from genetic linkage, which is the close chromosomal proximity between the marker locus and the locus that determines disease resistance. When selecting for qualities that are expensive or difficult to quantify, have little heredity, or manifest late in development, MAS might be helpful. The specimens are inspected at specific stages of the breeding process to make sure they exhibit the desired characteristic. Figure 8 (Charlesworth, D. (2002) Marker types These days, DNA-based markers are used in most MAS research. Morphological markers were the first, nevertheless, to enable indirect selection of an interest characteristic. Karl Sax noticed segregation of seed size related with segregation for a seed coat colour marker in beans (Phaseolus vulgaris L.) in 1923, which led him to publish the relationship of a simply inherited genetic marker with a quantitative feature in plants for the first time. J. Rasmusson showed how a simple hereditary gene for flower colour may be linked to the quantitative feature of flowering time in peas in 1935. 17 Markers can be: Morphological: These markers loci were the first to be discovered that clearly affect a plant's morphology. Many times, one may identify these indicators just by looking at them. The existence or absence of an awn, the colour of the leaf sheath, height, rice grain colour, aroma, and other characteristics are examples of this kind of marker. Numerous chromosome positions have been assigned to several hundreds of genes that influence morphological features in well-characterized crops such as maize, tomato, pea, barley, or wheat. Biochemical: A protein that is observable and extractable, such as storage proteins and isozymes. Cytological: Chromosome characteristics that can be seen by microscopy are known as cytological markers. These often assume the shape of chromatin areas impregnated with certain dyes used in cytology, known as chromosomal bands. A chromosomal band's presence or absence might be associated with a certain trait, meaning that the trait's locus is either inside or close to (tightly linked) to the banded region. The foundation of early genetic studies in crops like wheat and maize was made up of morphological and cytological markers. Gene vs Marker Markers are genetically associated with the gene of interest, whereas the gene of interest directly leads to the the production of protein(s) or RNA that generate a desired characteristic or phenotype. Due to their proximity on the same chromosome and the consequent reduction in recombination (chromosome crossing events) involving the marker and the gene of interest, the gene of interest and the marker tend to migrate together during gamete segregation. For certain features, the desired alleles can be directly and confidently tested for in the presence of the identified gene. Conclusion In conclusion, the basics of plant genetics provide a fundamental understanding of how traits are inherited, varied, and expressed in plants. Through the principles of Mendelian genetics, the processes of cross- 18 pollination, inbreeding, and hybridization, and the advancements in genetic modification techniques, researchers and breeders can manipulate plant genomes to develop new varieties with improved traits. From Mendelian principles to modern genetic engineering techniques, this knowledge empowers researchers and breeders to manipulate plant genomes, improving crop yield, resilience, and nutritional quality. By harnessing genetic diversity through strategies like hybridization and managing the risks associated with inbreeding, we pave the way for sustainable agriculture, biodiversity conservation, and food security in an ever-changing world. References 1. Griffiths, A. J. (2005). An introduction to genetic analysis. Macmillan. 2. Mirzaghaderi, G., & Hörandl, E. (2016). The evolution of meiotic sex and its alternatives. Proceedings of the Royal Society B: Biological Sciences, 283(1838), 20161221. 3. Caime, S. (2018). Present-Day Influencers on Consumer Understanding and Acceptance of Genetically Modified Foods. 4. Westergaard, M. (1958). The mechanism of sex determination in dioecious flowering plants. Advances in genetics, 9, 217-281. 5. Lloyd, D. G. (1976). The transmission of genes via pollen and ovules in gynodioecious angiosperms. Theoretical population biology, 9(3), 299-316. 6. Lloyd, D. G. (1984). Modification of the gender of seed plants in varying conditions. Evol. Biol., 17, 255-338. 7. Westergaard, M. (1958). The mechanism of sex determination in dioecious flowering plants. Advances in genetics, 9, 217-281. 8. Testolin, R., Cipriani, G., & Costa, G. (1995). Sex segregation ratio and gender expression in the genus Actinidia. Sexual Plant Reproduction, 8, 129-132. 9. Charlesworth, D. (2002). Plant sex determination and sex chromosomes. Heredity, 88(2), 94-101. 10. Lardon, A., Georgiev, S., Aghmir, A., Le Merrer, G., & Negrutiu, I. (1999). Sexual dimorphism in white campion: complex control 19  of carpel number is revealed by Y chromosome deletions. Genetics, 151(3), 1173-1185. 11. Parker, J. S. (1990). Sex chromosome and sex differentiation in flowering plants. Chromosome Today, 10, 187-198. 12. Grant, S., Houben, A., Vyskot, B., Siroky, J., Pan, W. H., Macas, J., & Saedler, H. (1994). Genetics of sex determination in flowering plants. Developmental Genetics, 15(3), 214-230. 13. Farbos, I., Veuskens, J., Vyskot, B., Oliveira, M., Hinnisdaels, S., Aghmir, A.,... & Negrutiu, I. (1999). Sexual dimorphism in white campion: deletion on the Y chromosome results in a floral asexual phenotype. Genetics, 151(3), 1187-1196. 14. Hammond, C. M., Strømme, C. B., Huang, H., Patel, D. J., & Groth, A. (2017). Histone chaperone networks shaping chromatin function. Nature reviews Molecular cell biology, 18(3), 141-158. 15. Wilson, J., & Hunt, T. (2002). Molecular biology of the cell: a problems approach. In Molecular biology of the cell: a problems approach (pp. 4th-ed). 16. Thanbichler, M., Wang, S. C., & Shapiro, L. (2005). The bacterial nucleoid: a highly organized and dynamic structure. Journal of cellular biochemistry, 96(3), 506-521. 17. Antonin, W., & Neumann, H. (2016). Chromosome condensation and decondensation during mitosis. Current opinion in cell biology, 40, 15-22. 18. Darwin, C. (1889). Journal of Researches Into the Natural History and Geology of the Countries Visited During the Voyage of HMS" Beagle" Round the World: Under the Command of Capt. Fitz Roy, RN (No. 1). Ward, Lock and Company. 20  CHAPTER: 2 CRISPER AND GENE EDITING IN PLANT BREEDING Yogesh Kashyap1, Manisha Kumari2, Patel Rushiprasad J.1 and Vinod Kumar3 1 Ph.D. Scholar, Department of Genetics and Plant Breeding, N.M. College of Agriculture, Navsari Agricultural University, Navsari, Gujrat 396450. 2 Ph.D. Scholar, Department of Genetics and Plant Breeding, C. P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Dantiwada, Gujarat, India, 385506. 3 Department of Horticulture (Vegetable Science), Vikram University, Ujjain, Madhya Pradesh. Abstract Genome editing refers to the precise alteration of an organism's DNA, allowing for the modification of specific genes or genomic elements. This transformative field has seen significant advancements, with various technologies emerging to enable targeted changes in the genetic code. One of the most revolutionary and widely adopted genome editing tools is the CRISPR/Cas9 system. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a natural defence mechanism found in bacteria against invading viruses. Scientists harnessed this system and coupled it with the Cas9 enzyme to create a powerful genome editing tool. The RNA- guided endonuclease, known as CRISPR/Cas9, enables researchers to precisely cut DNA at specific locations. Once the DNA is cut, the cell's natural repair mechanisms come into play, allowing for the introduction of desired genetic modifications. The CRISPR/Cas9 system, an RNA-guided endonuclease (RGEN), has brought about a revolutionary transformation in genome engineering. Its simplicity in constructing customized vectors targeting specific genomic loci makes it significantly easier to introduce compared 21 to ZFNs or TALENs. This groundbreaking technology has rapidly become a standard tool for targeted gene modification within just a few years. This chapter provides insights into the emergence, establishment, improvement, and applications of CRISPR/Cas9, as well as its anticipated evolution. The strategies involving CRISPR/Cas9-mediated genome editing is poised to continually expedite studies on functional genomics for the foreseeable future. Furthermore, the utilization of nuclease- inactivated Cas9 (dCas9) with various functional domains is expected to unlock the technology's full potential, surpassing other platforms like ZF and TALE-based approaches. Beyond its impact on genetic engineering, CRISPR/Cas9 is poised to reshape not only the varieties of genetic research but also various domains within the life sciences. As genome editing technologies continue to evolve, including advancements such as base editing and prime editing, the ability to manipulate DNA with increased precision and efficiency is expanding. The field holds tremendous promise for advancing our understanding of genetics, treating genetic diseases, and shaping the future of various industries. Keywords: Genome editing, ZFNs, TALENs, CRISPR/Cas9, RNA- guided endonuclease What is Genome Editing? Genome editing or gene editing, represents a form of genetic engineering wherein modifications such as insertion, deletion, modification, or replacement of DNA occur within the genome of a living organism. In contrast to earlier genetic engineering methods that randomly integrated genetic material into a host genome, genome editing specifically directs insertions to predetermined, site-specific locations. The fundamental mechanism underlying genetic manipulations utilizing programmable nucleases involves the identification of target genomic loci. This is followed by the binding of an effector DNA-binding domain (DBD) and the induction of double-strand breaks (DSBs) at the targeted DNA site, facilitated by restriction endonucleases such as FokI and Cas. The subsequent repair of DSBs takes place through either homology- directed recombination (HDR) or non-homologous end joining (NHEJ). 22 This precise and targeted approach in genome editing offers a more controlled and predictable method for altering genetic material in living organisms. Genome-Editing Technologies Over the last decade, a new class of technologies, commonly known as genome-editing technologies, has emerged. These cutting-edge tools rely on engineered endonucleases (EENs) designed to cleave DNA in a highly specific manner. This precision is achieved through the incorporation of a sequence-specific DNA-binding domain or RNA sequence (Carroll D.,2014 & Gaj T. et al., 2013). By recognizing the unique DNA sequence, these nucleases efficiently and accurately cleave the targeted genes. The induced double-strand breaks (DSBs) in the DNA subsequently trigger cellular DNA repair mechanisms, including homology-directed repair (HDR) and error-prone non-homologous end joining (NHEJ) (Wyman C et al., 2006). These repair processes ultimately lead to gene modifications occurring precisely at the designated target sites. This breakthrough in genome editing technology has significantly advanced our ability to manipulate genetic material with unprecedented precision and efficiency. 1. Zinc finger nucleases (ZFNs) Zinc fingers were first discovered in African clawed toad (Xenopus laevis) in 1985. Zinc-finger nucleases (ZFNs) are highly specific genomic scissors which consist of two functional domain a DNA binding domain and a DNA cleaving domain. The engineered zinc finger domains have the capability to target specific DNA sequences, providing zinc-finger nucleases with the ability to focus on particular sequences within intricate genomes. Leveraging the natural DNA repair mechanisms present in organisms, these tools can be effectively utilized to accurately modify the genetic material of higher organisms. Each Zinc Finger (ZF) protein has the capacity to identify and recognize three consecutive nucleotide bases within the DNA substrate. Illustrated in Figure 1, a standard Zinc Finger Nuclease (ZFN) monomer 23 is formed by the fusion of two functionally distinct domains: an artificially engineered domain at the N-terminal and a non-specific DNA cleavage domain from the Fok I DNA restriction enzyme at the C-terminal. The essential enzymatic activity of the Fok I domain relies on its dimerization. Hence, a ZFN dimer, comprising two 3- or 4-ZF domains, is capable of recognizing an 18- or 24-base target sequence. ZFN target sites consist of two ZF binding sites separated by 5 to 7 bp spacer sequence and this spacer is recognized by the Fok1 cleavage domain. This statistically unique sequence is present in the genomes of most organisms. ZFN can be introduced into a organism by various ways like in the form of recombinant protein, agrobacterium mediated gene transfer and through various methods of direct DNA transfer. ZFN stimulate cell`s natural DNA-repair process i.e. homologous recombination and non-homologous end joining (NHEJ) for DNA repair. Zinc Finger Nucleases (ZFNs) have demonstrated successful applications in gene modification, primarily within animal systems such as human cells, zebrafish, and plants including Arabidopsis, tobacco, and maize. However, the creation of functional ZFNs demands an exhaustive and time-intensive screening process. Figure 1: Schematic illustration of the ZFN structure and the principle of ZFN-mediated genomic modifications Gaj et al., (2013) & Moore et al. (2012) 24  ZNFs technology have various uses like it can be use for repairing mutations, insertion of gene or DNA fragment at a specific site, disabling an allele, allele editing, repair or replace of aberrant genes, gene therapy and treatment of HIV. However, obtaining functional ZFNs requires an extensive and time-consuming screening process. (Hsu PD et al.,2012) Further, ZFNs have other limitations, such as off-target effect (Cathomen T. et al.,2008) or even toxic to the host cells. These shortcomings limit the application of ZFNs in plant genome editing. Until now, there have been no reports on ZFN applications in horticultural crops. 2. Transcription Activator-Like Effector Nucleases (TALENs) Transcription activator-like effector nucleases (TALENs), was named as methods of the year in 2011. TALEs are naturally occurring proteins from the plant pathogenic bacteria Xanthomonas, these bacteria secrete effector protein (TALEs) to increase its susceptibility to the pathogens (Boch J et al.,2010). These effectors are capable of DNA binding and activating the expression of their genes by mimicking the eukaryotic transcription factors. After been pumped into host cells, the TAL effectors enter the nucleus and bind to effector-specific sequences in the host gene promoters and activate transcription. (Bogdanove AJ et al., 2010). AvrBs3 was the first investigated TAL effector protein found in 1989 (Bonas U et al., 1989). TALENs are restriction enzyme engineered to cut specific sequences of DNA, they are made of DNA binding domain (TAL effector) at the N-terminal and DNA cleavage domain (Fok 1) at the C-terminal (Figure 2). The DNA recognition, property of the TAL effectors is mediated by tandem amino acid repeats (34 residues in length). Two hypervariable amino acids known as repeat-variable di-residues (RVDs) located at the 12th and 13th position in each repeat determine the binding specificity of the TAL effectors (Moscou MJ & Boch J et al., 2009). HD, NG, NI, and NN are the four most common RVDs, accounting for each of the four nucleotides C, T, A, G, respectively. Simultaneous bindings of the left and right TALE enable dimerization of the Fok I cleavage domain, resulting in DSBs of the target DNA. Induced DSBs of the target DNA are repaired either by NHEJ or HDR resulting in gene 25 mutations that include nucleotide insertion, deletion, or substitution around the cleavage site. Due to easier manipulation, the genes modified by TALENs have been successfully used in both animal and plant species within three years of deciphering their function. TALENs technology have various uses like it can be use for gene therapy, to efficiently modify plant genome, to correct the genetic error that underlie disease, to harness the immune system to fight cancer and TALEN- mediated targeting can generate T cells that are resistant to chemotherapeutic drugs and show anti-tumor activity. Figure.2 Schematic illustration of the TALENs structure and the principle of TALENs mediated genomic modifications Gaj et al., (2013) & Moore et al. (2012) 26 3. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPER) An RNA-guided endonuclease (RGEN), known as CRISPR/Cas9, has been dramatically changing the field of genome engineering. Because CRISPR/Cas9 is much easier to introduce than ZFNs or TALENs because of its simple construction of customized vectors targeting particular genomic loci, this epoch- making technology has rapidly become a standard tool for targeted gene modification within a time span of just a few years. 3.1 CRISPR/Cas System in Prokaryotic Adaptive Immunity CRISPER is a part of the adaptive immune system of bacteria and archaea, protecting their genome against invading nucleic acid such as virus by cleaving the foreign DNA (Ishino 1987 & Mojica et al., 2000). CRISPER consists of repetitive sequences and interspersed regions, where the repetitive segments share identical sequences, and the intervening regions consist of distinct sequences originating from foreign DNA (Mojica 2005 & Pourcel et al., 2005). The CRISPR locus operates with CRISPR-associated (Cas) proteins to serve as an adaptive immune system defending against the introduction of foreign DNA (Wiedenheft 2012 & Westra et al., 2014). In this system, the invading DNA becomes integrated into the spacer region within the CRISPR locus, undergoing transcription as an long pre-crRNA (CRISPR RNA) encompassing numerous repeats and spacers. Following this, in the type II CRISPR/Cas system, the pre-crRNA undergoes processing to form crRNA containing a single spacer sequence that is complementary with the foreign DNA. This is facilitated by another short RNA molecule, trans-crRNA (tracrRNA), transcribed from a distinct locus. The resulting crRNA-tracrRNA heteroduplex serves as a guidance molecule, targeting exogenous DNA bearing an identical sequence to the crRNA. This induce a DNA double-strand break (DSB) at the specified locus, association with Cas protein (Bhaya 2011; Reeks 2013; Barrangou & Marraffini 2014). 27 3.2 Application of CRISPR/Cas9 in Genome Editing In the process of genome editing using the CRISPR/Cas system, only two essential components are required: a chimeric guide RNA (gRNA) that emulates the crRNA-tracrRNA complex, and a Cas9 protein possessing nuclease activity (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013). Although targeting specificity primarily relies on the sequence of the guide RNA (gRNA), Cas9 also require specific bases, referred to as a protospacer adjacent motif (PAM) (Bolotin et al. 2005). The PAM sequences vary among species. For example, Streptococcus thermophilus Cas9 (StCas9) requires 5′-NNAGAAW-3′ (Cong et al. 2013; Esvelt et al., 2013) while Streptococcus pyogenes Cas9 (SpCas9) requires 5′-NGG-3′ (Jinek et al., 2012). Currently, SpCas9 is the most widely used for genetic engineering (Hsu et al., 2014). The structure of guide RNA (gRNA) plays a crucial role in CRISPR/Cas9-based genome editing. While crRNA and tracrRNA can be transcribed independently, similar to the natural CRISPR/Cas system, a fused chimeric gRNA structure is a simpler design that frequently results in high activity (Hsu et al., 2013). A chimeric gRNA is composed of a 5′ end region derived from crRNA and a 3′ end region derived from tracrRNA. Essentially, the DNA-recognition sequence within the crRNA region spans 20 base pairs long. However, it has been reported that enhancing specificity can be achieved by either adding or truncating a few bases (Cho et al.,2014; Fu et al., 2014). The 3′ end of the crRNA region and the 5′ end of the tracrRNA region are generally linked with four nucleotides (5′-GAAA-3′) to form a major stem loop, known as a tetraloop (Kim et al.,2014). On the 3′ side, the tracrRNA region incorporates additional minor loops, and these sequences are recognized as crucial for achieving high gRNA expression (Hsu et al., 2013). 3.3 Targeting Specificity of CRISPR/Cas9 As mentioned earlier, the targeting specificity of CRISPR/Cas9 is determined by the approximately 20-base pair gRNA sequence and the 3- base pair PAM sequence of SpCas9. Yet, the level of base recognition stringency varies among these sequences. Concerning the PAM sequence, SpCas9 can interact with 5′-NGA-3′ (Zhang et al., 2014), 5′-NAG-3′ (Hsu et al., 2013; Jiang et al., 2013), and 5′-NGG-3′ sites. Concerning the 28 gRNA targeting sequence, the specificity diminishes as the distance from the PAM site increases. The sequence extending up to 12 base pairs adjacent to the PAM site is termed the seed sequence and exhibits relatively high targeting specificity (Jinek et al. 2012; Cong et al. 2013). Source: Vecterbiolabs.com 3.4 Double-Nicking and Dimeric FokI-dCas9 Strategies for Highly- Specific Genome Editing Due to significant concerns regarding off-target mutations, numerous advanced strategies have been developed to achieve highly specific CRISPR/Cas9-mediated genome editing. The primary challenge in achieving specificity with CRISPR/Cas9 lies in its monomeric architecture, unlike the dimeric structure found in ZFNs and TALENs. In 29 a conventional CRISPR/Cas9 genome editing system, a single gRNA is paired with a Cas9 nuclease. As the Cas9 nuclease exhibits cleavage activity for both DNA strands, the site of induced double-strand breaks (DSB) is determined by the single gRNA. Now the Cas9 nuclease cut the DNA strand at this specific site. After cleavage repair id done by homologous recombination and non - homologous end joining (NHEJ) which is a error prone mechanism. Applications of CRISPR-Cas9 system Gene disruption (without donor template DNA) Gene knock-out (with a reporter knock-in) Non-protein coding gene disruption Specific mutations o Desired SNP introduction or correction o Desired insertions/deletions Promoter Study o Luciferase replaced the 5’ exon Conditional knockout o For essential genes or tissue-specific study inserting LoxP sites o Around the exon to be knocked-out Large chromosomal deletions o Using two signature Cas9 RNAs to induce double stranded breaks at sites that flank the region of interest Exogenous gene Insertion o Adeno-associated virus integration site 1 (AAVS1) in human genome is a safe harbor for transgene integration o A controlled Gene Knock-in e.g. controlled copy number and location CRISPR interference and activation of transcription Importance of CRISPR High potency and specificity Broad applicability in vitro and in vivo Potential one-time curative treatment Ability to edit out diseases Ability to address any site in the genome or foreign genome Ability to target multiple DNA sites simultaneously Multi-functional programmability – Delete, insert, repair 30 Conclusion genome editing represents a groundbreaking frontier in scientific advancement with the potential to revolutionize various fields, from medicine to agriculture. The ability to precisely modify genetic material offers unprecedented opportunities to treat genetic disorders, enhance crop resilience, and even address pressing environmental concerns. However, as with any powerful technology, genome editing also raises ethical and safety considerations that must be carefully navigated. Striking a balance between innovation and responsibility is crucial to harness the full potential of genome editing while ensuring its ethical and safe application. Continued research, open dialogue, and robust regulatory frameworks will play pivotal roles in shaping the future of genome editing, ensuring that it becomes a force for positive change without compromising fundamental values and principles. As we move forward, it is imperative for the scientific community, policymakers, and society as a whole to collaborate in a transparent and informed manner, fostering a responsible and ethical approach to genome editing that benefits humanity as a whole. References 1. Barrangou, R., & Marraffini, L. A. (2014). CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Molecular cell, 54(2), 234-244. 2. Bhaya, D., Davison, M., & Barrangou, R. (2011). CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual review of genetics, 45, 273-297. 3. Boch, J., & Bonas, U. (2010). Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annual review of phytopathology, 48, 419-436. 4. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S.,... & Bonas, U. (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326(5959), 1509- 1512. 31 5. Bogdanove, A. J., Schornack, S., & Lahaye, T. (2010). TAL effectors: finding plant genes for disease and defense. Current opinion in plant biology, 13(4), 394-401. 6. Bolotin, A., Quinquis, B., Sorokin, A., & Ehrlich, S. D. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 151(8), 2551-2561. 7. Bonas, U., Stall, R. E., & Staskawicz, B. (1989). Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Molecular and General Genetics MGG, 218, 127-136. 8. Carroll, D. (2014). Genome engineering with targetable nucleases. Annual review of biochemistry, 83, 409-439. 9. Cathomen, T., & Joung, J. K. (2008). Zinc-finger nucleases: the next generation emerges. Molecular Therapy, 16(7), 1200-1207. 10. Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., & Kim, J. S. (2014). Analysis of off-target effects of CRISPR/Cas- derived RNA-guided endonucleases and nickases. Genome research, 24(1), 132-141. 11. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N.,... & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823. 12. Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu, J. K., & Yan, N. (2012). Structural basis for sequence-specific recognition of DNA by TAL effectors. Science, 335(6069), 720-723. 13. Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J., & Church, G. M. (2013). Orthogonal Cas9 proteins for RNA- guided gene regulation and editing. Nature methods, 10(11), 1116-1121. 14. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature biotechnology, 32(3), 279-284. 15. Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology, 31(7), 397-405. 32 16. Hsu, P. D., & Zhang, F. (2012). Dissecting neural function using targeted genome engineering technologies. ACS chemical neuroscience, 3(8), 603-610. 17. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of bacteriology, 169(12), 5429-5433. 18. Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR- Cas systems. Nature biotechnology, 31(3), 233-239. 19. Mak, A. N. S., Bradley, P., Cernadas, R. A., Bogdanove, A. J., & Stoddard, B. L. (2012). The crystal structure of TAL effector PthXo1 bound to its DNA target. Science, 335(6069), 716-719. 20. Mali, P., Esvelt, K. M., & Church, G. M. (2013). Cas9 as a versatile tool for engineering biology. Nature methods, 10(10), 957-963. 21. Mojica, F. J., Díez‐Villaseñor, C., Soria, E., & Juez, G. (2000). Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Molecular microbiology, 36(1), 244-246. 22. Moore, F. E., Reyon, D., Sander, J. D., Martinez, S. A., Blackburn, J. S., Khayter, C., & Langenau, D. M. (2012). Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs). PloS one, 7(5), e37877. 23. Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501-1501. 24. Pourcel, C., Salvignol, G., & Vergnaud, G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology, 151(3), 653-663. 33 25. Reeks, J., Naismith, J. H., & White, M. F. (2013). CRISPR interference: a structural perspective. Biochemical Journal, 453(2), 155-166. 26. Westra, E. R., Buckling, A., & Fineran, P. C. (2014). CRISPR– Cas systems: beyond adaptive immunity. Nature Reviews Microbiology, 12(5), 317-326. 27. Wiedenheft, B., Sternberg, S. H., & Doudna, J. A. (2012). RNA- guided genetic silencing systems in bacteria and archaea. Nature, 482(7385), 331-338. 28. Wyman, C., & Kanaar, R. (2006). DNA double-strand break repair: all's well that ends well. Annu. Rev. Genet., 40, 363-383. 34  CHAPTER: 3 CLIMATE RESILIENT CROPS-A GENETIC APPROACH K Sri Anjanidevi Ph.D. Scholar, Department of Plantation, Spices Medicinal and Aromatic Plants, Dr. YSRHU, COH-Venkataramannagudem, Andhra Pradesh- 534101 Abstract Climate-resilient crops development represents a critical frontier in agricultural innovation, aiming to safeguard global food security amidst the escalating challenges posed by climate change. At its core, this endeavor revolves around identifying and harnessing genetic traits within crops that confer resilience to the evolving environmental conditions driven by shifting climatic patterns. Scientists and breeders employ a multifaceted approach, drawing upon a diverse array of breeding techniques, genetic resources, and cutting-edge technologies to develop crops capable of thriving in the face of droughts, floods, heatwaves, and emerging pests and diseases. One pivotal aspect of developing climate- resilient crops involves meticulous trait identification, where researchers pinpoint specific genetic characteristics crucial for adaptation to climatic stressors. These traits encompass a broad spectrum, including enhanced drought tolerance, heat resilience, resistance to pathogens, improved nutrient utilization efficiency, and tolerance to saline soils. Through extensive exploration of genetic diversity within crop species and related wild relatives, breeders uncover hidden reservoirs of resilience, laying the groundwork for breeding programs aimed at fortifying crops against the impacts of climate change. Keywords: Climate resilience, Crop Sustainability, Breeding approaches. 35 Introduction Climate change poses significant challenges to global food security, but the cultivation of climate-resilient crops offers a promising solution. By harnessing the power of plant breeding and biotechnology, we can develop crops with the traits needed to thrive in a changing climate while ensuring sustainable food production and livelihoods for future generations. Conventional approaches such as selective breeding, hybridization, and crossbreeding are complemented by advanced techniques like marker-assisted selection (MAS) and genetic engineering. MAS enables breeders to expedite the identification of desirable traits by pinpointing specific genetic markers associated with climate resilience, while genetic engineering offers the precision to introduce or modify genes responsible for conferring resilience traits directly into crop genomes. Field trials serve as a crucial proving ground, allowing breeders to assess the performance of candidate varieties under real-world environmental conditions. Rigorous evaluations across diverse agroecological zones provide insights into the adaptability, agronomic performance, and stress tolerance of promising breeding lines. Iterative cycles of selection and refinement further hone the resilience of crop varieties, ensuring they meet the needs of farmers facing increasingly unpredictable growing conditions. Climate Resilience Climate resilience is the ability to anticipate, prepare for, and respond to hazardous events, trends, or disturbances related to climate. Improving climate resilience involves assessing how climate change will create new, or alter current, climate-related risks, and taking steps to better cope with these risks. Genetic Approach Hybridization Hybridization is a fundamental technique in developing climate- resilient crops, integrating genetic diversity to confer adaptive traits. In this process, plants with desirable traits are crossbred to create offspring with a combination of beneficial characteristics. With climate change 36 increasingly challenging crop productivity, hybridization serves as a pivotal strategy to breed crops capable of withstanding various environmental stresses. To initiate hybridization for climate resilience, scientists meticulously select parent plants with traits suited to specific stressors. These may include drought tolerance, heat resistance, pest and disease resilience, or efficient nutrient utilization. By crossing these parents, breeders aim to combine favourable traits in the progeny, creating hybrids with enhanced resilience to climatic fluctuations. The success of hybridization lies in the careful selection of parent plants. Genetic diversity is crucial, as it increases the likelihood of capturing desirable traits and enables the exploration of adaptive mechanisms across different gene pools. Through extensive screening and evaluation, breeders identify superior hybrids demonstrating resilience under simulated stress conditions in controlled environments. Field trials play a crucial role in validating the performance of hybrid progeny under real-world conditions. These trials assess not only the resilience of hybrids to prevailing climatic stresses but also their agronomic performance, yield potential, and overall adaptability across diverse agroecological zones. Data gathered from field trials inform breeding decisions, guiding the selection and advancement of promising hybrids. Furthermore, hybridization is a dynamic process, often requiring iterative cycles of selection and refinement. Breeders continuously evaluate hybrid populations, selecting individuals exhibiting the most desirable traits for further breeding. This iterative approach allows for the gradual improvement of hybrids, fine-tuning their resilience to evolving climate challenges over successive generations. Ultimately, hybridization holds immense promise in the development of climate-resilient crops, offering a pathway to address the complex and multifaceted challenges posed by climate change. By harnessing the power of genetic diversity through hybridization, breeders can create novel crop varieties capable of thriving in increasingly unpredictable environments, ensuring food security and sustainability for future generations. 37 Mutation Breeding Mutation breeding is a powerful technique utilized in the development of climate-resilient crops, offering a means to introduce genetic variation and novel traits that enhance the ability of plants to withstand environmental stresses associated with climate change. This method involves the induction of random mutations in the plant's DNA using physical or chemical agents, resulting in the creation of mutant populations with diverse genetic backgrounds. The process of mutation breeding begins with the selection of appropriate parent plants or germplasm with desirable traits or genetic variability. Seeds or plant tissues are then treated with mutagenic agents, such as gamma rays, X- rays, neutron radiation, ethyl methane sulfonate (EMS), or sodium azide, to induce random mutations in the DNA. Mutagenized populations are subsequently generated, and large numbers of mutant plants are grown and screened for the expression of target traits under controlled environmental conditions. Screening and selection of mutants with desired traits are crucial steps in mutation breeding programs. High-throughput screening methods, including physiological assays, biochemical analyses, and phenotypic evaluations, are employed to identify individuals exhibiting enhanced resilience to specific stresses or improved agronomic performance. Promising mutants are further evaluated through field trials to assess their performance under real-world conditions and to confirm the stability and heritability of the desired traits across generations. Once, validated selected mutants are crossed with elite cultivars or breeding lines to introgress the beneficial traits into commercially viable genetic backgrounds. This process involves traditional breeding techniques, such as backcrossing and pedigree selection, to develop stable breeding lines or varieties with improved climate resilience while retaining desirable agronomic traits and quality characteristics. Advanced breeding lines derived from mutation breeding are then released as new climate-resilient crop varieties, contributing to sustainable agriculture and food security in a changing climate. Mutation breeding offers several advantages over other breeding methods, including the induction of genetic variation without the need for 38 specific knowledge of the underlying genes or molecular mechanisms. Additionally, mutation breeding can complement other breeding strategies, such as conventional breeding and biotechnological approaches, by expanding the genetic diversity available for crop improvement. Overall, mutation breeding plays a critical role in the development of climate-resilient crops, providing breeders with a valuable tool to address the challenges posed by climate change and ensure global food security. Marker-assisted selection Marker-assisted selection (MAS) is a pivotal technique in the development of climate-resilient crops, providing breeders with a powerful tool to expedite the breeding process and select for specific traits associated with adaptation to changing environmental conditions. MAS involves the use of molecular markers linked to genes or genomic regions controlling target traits of interest. By precisely identifying and selecting plants carrying desired genetic variants associated with climate resilience, MAS enables breeders to improve the efficiency and precision of crop breeding programs. In the context of climate resilience, MAS offers several advantages. Firstly, it allows breeders to indirectly select for complex traits that are difficult or time-consuming to phenotype directly, such as drought tolerance, heat tolerance, disease resistance, or nutrient use efficiency. By targeting molecular markers associated with these traits, breeders can accurately identify plants with the desired genetic background and prioritize them for further evaluation and breeding efforts. Furthermore, MAS facilitates the introgression of specific genes or genomic regions conferring climate resilience from wild or exotic germplasm into elite breeding lines or commercial cultivars. This is particularly valuable in crop improvement programs where genetic diversity for target traits may be limited in cultivated germplasm. Through MAS, breeders can efficiently transfer beneficial alleles or gene variants associated with climate resilience, thereby broadening the genetic base and enhancing the adaptive potential of crop varieties. The integration of MAS into climate-resilient crop development also enables the 39 implementation of precision breeding strategies. By fine-tuning selection decisions based on molecular information, breeders can accelerate the development of new varieties with improved stress tolerance and agronomic performance. MAS-guided selection enables breeders to focus resources on the most promising breeding lines, reducing the time and resources required for traditional phenotypic evaluations and field trials. DNA markers [Single Nucleotide Polymorphisms (SNPs), Simple Sequence Repeats (SSRs), Sequence Characterized Amplified Regions (SCAR), Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), etc.]. DNA markers are PCR (Polymerase Chain Reaction) or non-PCR based. Various molecular markers and their application for QTL mapping in agronomic crops were reviewed by Younis et al. (2020). MAS facilitates the pyramiding of multiple genes or genomic regions associated with different aspects of climate resilience into a single genotype. This allows breeders to develop multi-trait climate-resilient varieties with enhanced adaptability and stability across diverse environmental conditions. DNA-based MAS is an effective method for saving time in breeding as it is growth-stage independent, unaffected by environmental conditions, effective to use in early generations, and is efficient when field evaluation is very slow or expensive. By combining complementary traits through MAS, breeders can create crop varieties that are better equipped to withstand the complex challenges posed by climate change, ensuring greater productivity and sustainability. Overall, marker-assisted selection is a valuable tool in the development of climate-resilient crops, offering breeders a means to accelerate breeding progress, enhance precision, and broaden the genetic base of crop varieties. By harnessing the power of molecular markers, breeders can expedite the development of new crop varieties with improved stress tolerance and resilience to environmental fluctuations, ultimately contributing to global food security and crop sustainability in the face of climate change. 40 Gene overexpression Gene overexpression, a technique within genetic engineering, plays a crucial role in the development of climate-resilient crops by enhancing the expression of specific genes associated with stress tolerance and adaptive traits. This approach involves introducing extra copies of target genes or regulatory elements into the plant genome, resulting in increased production of proteins or metabolites that confer resilience to environmental stresses. By modulating gene expression levels, researchers can enhance the plant's capacity to withstand adverse conditions and maintain productivity under challenging environmental scenarios. One of the key applications of gene overexpression in climate- resilient crop development is the enhancement of drought tolerance. By overexpressing genes involved in stress signaling pathways, osmotic regulation, water uptake, or water use efficiency, researchers can improve the plant's ability to maintain cellular hydration and cope with water scarcity. For instance, overexpression of genes encoding aquaporins, which regulate water transport across cell membranes, can enhance water uptake and retention in plant tissues, enabling crops to endure periods of drought stress without significant yield losses. Similarly, gene overexpression can be used to enhance heat tolerance in crops by upregulating genes involved in heat shock responses, antioxidant defense mechanisms, or thermotolerance pathways. By increasing the expression of heat shock proteins (HSPs) or enzymes involved in reactive oxygen species (ROS) scavenging, researchers can mitigate the damaging effects of high temperatures on cellular structures and biochemical processes, thereby improving the plant's resilience to heat stress. Additionally, gene overexpression can confer resistance to biotic stresses such as pest infestations and pathogen infections, which can exacerbate the impacts of climate change on crop production. By overexpressing genes encoding antimicrobial peptides, detoxification enzymes, or defence-related proteins, researchers can enhance the plant's ability to recognize and defend against invading pathogens or insect pests, reducing yield losses and improving crop resilience in agroecosystems under pressure from pest outbreaks. Moreover, gene overexpression can be utilized to improve nutrient use efficiency and nutrient uptake in crops, 41 enhancing their ability to thrive in nutrient-deficient soils or under suboptimal fertilization regimes. By overexpressing genes involved in nutrient acquisition, assimilation, or remobilization, researchers can optimize nutrient utilization and allocation within the plant, improving overall nutrient uptake efficiency and crop productivity under nutrient- limiting conditions. In conclusion, gene overexpression is a powerful strategy in the development of climate-resilient crops, offering a targeted approach to enhance stress tolerance, pest resistance, and nutrient use efficiency. By modulating gene expression levels, researchers can engineer crops with improved adaptive traits, enabling them to thrive in the face of climate change and contribute to global food security and sustainability. However, careful consideration of potential environmental and regulatory implications is essential to ensure the safe and responsible deployment of genetically modified crops in agricultural systems. RNA interference (RNAi) RNA interference (RNAi) is a transformative tool in the development of climate-resilient crop varieties, offering a precise mechanism to regulate gene expression and enhance stress tolerance in plants. This technology leverages small RNA molecules to silence specific target genes involved in stress responses, pathogen defense, or other regulatory pathways, thereby modulating plant physiology and improving resilience to environmental challenges associated with climate change. Targeting genes involved in drought-responsive signaling pathways or water transport mechanisms, researchers can attenuate the detrimental effects of water scarcity on crop productivity. For example, silencing genes encoding negative regulators of drought tolerance or aquaporins, which control water movement across cell membranes, can enhance water retention and stress tolerance in plants, enabling them to better withstand periods of water deficit. Similarly, RNAi can be employed to enhance heat tolerance in crops by targeting genes involved in heat stress responses, protein denaturation, or oxidative damage repair mechanisms. By silencing the expression of heat-sensitive genes or upregulating heat shock proteins (HSPs) and other protective molecules, researchers can mitigate the adverse effects of high temperatures on cellular integrity and 42 biochemical processes, thereby improving the plant's resilience to heat stress and reducing yield losses. Additionally, RNAi-mediated gene silencing can confer resistance to biotic stresses such as pest infestations and pathogen infections, which pose significant threats to crop production in the context of climate change. By targeting genes essential for pest or pathogen virulence, researchers can disrupt key physiological processes in invading organisms, rendering them less harmful or susceptible to control measures. This approach offers a sustainable and environmentally friendly alternative to conventional pesticide applications, reducing the ecological footprint of agriculture while safeguarding crop yields. RNAi can be utilized to optimize nutrient use efficiency and nutrient uptake in crops, enhancing their ability to thrive in nutrient-deficient soils or under suboptimal fertilization regimes. By targeting genes involved in nutrient uptake, assimilation, or allocation, researchers can fine-tune nutrient utilization pathways in plants, ensuring optimal growth and productivity under limiting nutrient conditions. This strategy contributes to the development of climate-resilient crop varieties capable of maximizing resource use efficiency and adapting to changing environmental conditions. Genome editing has emerged as a groundbreaking technique in the development of climate-resilient crop varieties, offering unprecedented precision and efficiency in targeted genetic modification. This technology enables researchers to precisely alter specific DNA sequences within the plant genome, facilitating the introduction of beneficial traits associated with stress tolerance, pest resistance, and nutrient use efficiency. By leveraging genome editing tools such as ZFNs, TALENs and CRISPR-Cas9, researchers can engineer crops with enhanced resilience to environmental challenges posed by climate change. gene editing techniques have the added advantage that once the genome has been modified in the transformed plants, the inserted transgene (like Cas) can be removed in subsequent generations through segregation. 43 Zinc Finger Nucleases (ZFNs): Zinc Finger Nucleases (ZFNs) are engineered DNA-binding proteins that have revolutionized the field of genome editing. They are designed to precisely target and modify specific DNA sequences within the genome. ZFNs consist of two main components: zinc finger proteins (ZFPs) and a nuclease domain derived from the FokI restriction enzyme. Zinc finger proteins are naturally occurring DNA-binding domains found in many eukaryotic transcription factors. Each zinc finger domain typically recognizes and binds to a specific three-base-pair DNA sequence, enabling precise targeting of desired genomic loci. By combining multiple zinc finger domains, researchers can engineer custom ZFPs capable of recognizing longer DNA sequences with high specificity. The nuclease domain of ZFNs is derived from the FokI restriction enzyme, which cleaves DNA at specific recognition sites. The nuclease domain is engineered to function only when two ZFNs bind adjacent target sites on opposite DNA strands, bringing the nuclease domains into close proximity and facilitating double-stranded DNA cleavage. This requirement for dimerization ensures that ZFNs induce targeted DNA cleavage only at specific genomic loci where both ZFNs bind, minimizing off-target effects and enhancing the precision of genome editing. To use ZFNs for genome editing, pairs of ZFNs are designed to target specific DNA sequences flanking the desired editing site. When introduced into cells, ZFNs bind to their target sites in the genome, leading to the formation of a double- strand break (DSB) at the target locus. The cell's natural DNA repair machinery then repairs the DSB through either non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. In the NHEJ pathway, the broken DNA ends are directly ligated together, often resulting in small insertions or deletions (indels) at the cleavage site due to imprecise repair. This process can be exploited to disrupt target genes by introducing frame-shift mutations or premature stop codons, leading to gene knockout or loss-of-function phenotypes. In the HDR pathway, exogenous DNA templates containing desired sequence modifications can be introduced along with the ZFNs. The cell uses these templates as a repair template, incorporating the desired modifications into the genome during the repair process. This 44 allows for precise gene editing, including gene insertion, replacement, or correction of disease-causing mutations. Overall, Zinc Finger Nucleases offer a powerful and versatile tool for precise genome editing, enabling targeted modifications of specific DNA sequences in a wide range of organisms. While ZFNs have been widely used in research applications, the development of newer genome editing technologies such as CRISPR-Cas9 has largely superseded their use due to their simpler design, higher efficiency, and broader applicability. However, ZFNs remain valuable tools in certain contexts where specific design requirements or regulatory considerations may favor their use. Transcription activator-like effector nucleases (TALENs) TALENs are a type of genome editing technology that allows researchers to precisely modify DNA sequences within the genome of an organism. TALENs are engineered proteins composed of a DNA-binding domain derived from transcription activator-like effectors (TALENs) and a DNA-cleaving domain derived from the FokI endonuclease. The DNA- binding domain of TALENs consists of a series of repetitive amino acid motifs, each of which recognizes and binds to a specific nucleotide in the target DNA sequence through a code known as the TALE code. By arranging these motifs in a specific order, researchers can design TALENs with customized DNA-binding specificities, enabling them to target virtually any desired sequence within the genome with high precision. The DNA-cleaving domain of TALENs is derived from the FokI endonuclease, which is capable of inducing double-strand breaks (DSBs) in the target DNA sequence. TALENs are typically used as pairs, with each TALEN targeting one strand of the DNA duplex. When the two TALENs bind to their target sites in close proximity, the FokI domains dimerize and induce a DSB between the binding sites. Once a DSB is generated, the cell's natural DNA repair mechanisms are activated to repair the break. Two main pathways are involved in DNA repair: non- homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ often results in small insertions or deletions (indels) at the site of the DSB, leading to gene disruption or knockout. HDR, on the other hand, 45 can be harnessed to introduce precise sequence changes at the target site by providing a DNA repair template with the desired sequence alterations. TALENs offer several advantages as a genome editing tool. They have a high degree of specificity, allowing for precise targeting of desired DNA sequences while minimizing off-target effects. Additionally, TALENs can be easily customized to target different genomic loci by simply changing the DNA-binding domain, making them highly versatile for a wide range of applications in basic research, biotechnology, and medicine. Overall, TALENs represent a powerful tool for genome editing, enabling researchers to make precise modifications to the DNA sequence of an organism and investigate the function of specific genes, develop novel therapies for genetic diseases, and improve crops for agriculture and food security. Table 1: Representative examples of CRISPR/Cas9 mediated improvement in crops for abiotic and biotic stress tolerance Plant Target Target Type of Transform Refere Speci Gene Trait modifica ation nces es tion method Toma SILBD40 Drought Knockou Agrobacteri Liu et to t um al.,2020 tumefaciens Rice OsERA1 Drought Knockou Agrobacteri Ogata et t um al., tumefaciens 2020 Toma SINPR1 Drought Knockou Agrobacteri Li et al., to t um 2019 b tumefaciens Maiz ZmSRL5 Drought Knockou Agrobacteri Pan et e t um al.,2020 tumefaciens Rice OsRR22 Salinity Knockou Agrobacteri Zhang t um et tumefaciens al.,2019 46 Toma HyPRP1do Salinity Knockou Agrobacteri Tran et to main t um al., tumefaciens 2020 Rice OsNAC00 Multiple Knockou Agrobacteri Bo et 6 t um al.,2019 tumefaciens Soyb GmNAC06 Salinity Knockou A.rhizogene Li et ean t s al.,2021 Rice OsDST Multiple Knockou Agrobacteri Santosh t um kumar tumefaciens et al., 2020 Rice Xa 13 Bacterial Knockou Agrobacteri Li et blight t um al.,2020 tumefaciens a Rice AvrXa7 Bacterial Knockou Agrobacteri Zafar et blight t um al.,2020 tumefaciens Rice Os8N3 Xanthom Knockou Agrobacteri Kim et onas t um al., oryzae tumefaciens 2019 Rice OsSWEET Xanthom Knockou Agrobacteri Zeng et 14 onas t um al.,2020 oryzae tumefaciens Toma SIJAZ2 Bacterial Knockou Agrobacteri Ortigos to speck t um a et al., tumefaciens 2019 Cassa AC2, AC3 African Interfere Agrobacteri Mehta va cassava nce um et al., mosaic tumefaciens 2019 virus Toma PMR4 Powdery Knockou Agrobacteri Martine to mildew t um z et tumefaciens al.,2020 (Razzaq et al., 2021) 47 CRISPR/Cas9 CRISPR/Cas9 is a revolutionary genome editing technology that has transformed the field of molecular biology and biotechnology. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR-associated protein 9) together form a precise and efficient system for editing DNA sequences within the genome of organisms. The CRISPR/Cas9 system was originally discovered as a bacterial immune system that provides defense against viral infections. It consists of two main components: the CRISPR RNA (crRNA) and the Cas9 protein. The crRNA contains a short RNA sequence that is complementary to a specific target sequence within the genome, known as the protospacer sequence. The crRNA forms a complex with another RNA molecule called the trans-activating CRISPR RNA (tracrRNA) to guide the Cas9 protein to the target DNA sequence. Once the CRISPR/Cas9 complex binds to the target DNA sequence, th