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## **Meet the editors**

Dr. Mehboob-ur-Rahman (Group Leader, Plant and Molecular Breeding Labs, NIBGE, Faisalabad, Pakistan) has been involved in exploring genomes of cotton and wheat—paving the way for the development of nine cotton and one wheat varieties. His group has taken the lead in introducing GM cotton (containing Cry1Ac gene, Mon531) varieties to the farming community.

These varieties uplifted the livelihood of 1.3 million cotton farmers (as land owners) and >7 million farm laborers. He published more than 10 chapters in foreign books, as well as over 60 publications in Journals including two in *Nature* as co-author. All these efforts were acknowledged by the International and National Agencies by bestowing him with several recognitions/ awards including Pride of Performance, ICAC Cotton Researcher of the Year 2014.

Dr. Yusuf Zafar, presently working as Chairman at the Pakistan Agricultural Research Council, has 35 years experience in Life Sciences/Biotechnology. He has been the founding team member of the NIBGE, Faisalabad Pakistan and NIGAB, PARC, Islamabad, Pakistan. He established Agricultural Biotechnology Division in NIBGE and developed several GM crops. He mentored over 15

PhD students and published over 200 research articles, including chapters. Dr. Zafar acted in high-profile positions including Director General, PAEC; Minister Technical in Vienna; Project Management Officer, Technical Cooperation–Asia & Pacific, IAEA, Vienna, etc. His services were recognized by the Government of Pakistan that awarded him with civil award in 2004 and the International Cotton Advisory Committee (Washington USA) that bestowed upon him the best Scientist of the Year award, 2012.

Contents

**Preface VII**

Harzallah

**Section 3 Genomics of Cotton Traits 63**

Chapter 5 **Genetic Mapping in Cotton 93**

**Strategies 65**

Ur-Rahman

**Section 1 Future Trends in Cotton Research 1**

**Cotton Production 3**

Mubarik and Aftab Ahmad

**Section 2 Cotton Fiber Types and Properties 43**

Chapter 1 **Introductory Chapter: Updates on Achieving Sustainable**

Chapter 2 **Targeted Genome Editing for Cotton Improvement 11**

Chapter 3 **Impact of the Bijective Relationship between Single and**

Chapter 4 **Temperature Extremes in Cotton Production and Mitigation**

Zulqurnain Khan, Sultan Habibullah Khan, Muhammad Salman

**Bundle Cotton Fiber's in Cotton Breeding Programs 45** Wafa Mahjoub, Jean-Paul Gourlot, Jean-Yves Drean and Omar

Syed Adeel Zafar, Mehmood Ali Noor, Muhammad Ahmed Waqas, Xiukang Wang, Tayyaba Shaheen, Mubashar Raza and Mehboob-

Adem Bardak, Khezir Hayat, Oktay Erdogan, Zahid Mahmood, Noor-ul-Islam Khan, Muhammad Atif Iqbal, Halil Tekerek, Mehboob-ur-Rahman and Hafiz Muhammad Imran

Mehboob-ur-Rahman and Yusuf Zafar

## Contents

**Preface XI**

#### Chapter 6 **Recent Developments in Fiber Genomics of Tetraploid Cotton Species 123**

Mirzakamol S. Ayubov, Ibrokhim Y. Abdurakhmonov, Venkateswara R. Sripathi, Sukumar Saha, Tokhir M. Norov, Zabardast T. Buriev, Shukhrat E. Shermatov, Khurshida A. Ubaydullaeva, Jack C. McCarty, Dewayne D. Deng and Johnie N. Jenkins

Preface

genic cotton as well as their biosafety issues.

cedures for sustaining cotton production, have been discussed.

tem. Its role for silencing undesirable traits in cotton is yet to be established.

fiber arrangement parameters, which ultimately determine the quality of the fabric.

The aim of editing this book was to share R&D efforts undertaken to study the properties of natural cotton fiber including color and quality, improving the genetics of naturally colored cotton, genomic insights for mitigating the impact of heat—one of the most devastating components depressing cotton production—genetic and genomics of different traits in cot‐ ton, genome editing for combating biotic and abiotic stresses, and the development of trans‐

In the first chapter, written by Drs Mehboob-ur-Rahman and Yusuf Zafar, the provoking approaches to sustain cotton yield are described. Till date, cotton varieties were developed for cultivation under high-performance environments to achieve the maximum yield. How‐ ever, the performance of these cotton varieties is much more vulnerable at resource poor farms. The changing climate would further exaggerate the situation by gradual escalation of temperature and depletion of fresh water resources and make cotton crop difficult to grow. In this chapter, possible solutions, including conventional as well as non-conventional pro‐

In the second chapter, genome editing—a way to tailor gene for silencing or enhancing its expression precisely, has been described. Conventionally, mutations were induced random‐ ly using various physical and chemical mutagens—making it difficult to target the gene of interest. Khan and colleagues described a number of approaches that can target the gene of interest very precisely. Among these, zinc finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs), and clustered regularly interspaced short palindromic re‐ peats (CRISPR), and RNA-guided nucleases (e.g., Cas9) were in common use to target the genome. The CRISPR/Cas has shown a great potential to edit genome of complex species including cotton. For example, lateral root formation was improved using CRISPR/Cas sys‐

In the second section, impact of the Bijective relationship between single and bundle cotton fiber was narrated by Mahjoub and colleagues. In this study, three different fiber types, chosen from a list of twelve cottons, were selected from a large panel of varieties with sub‐ stantial variations in the physical properties of fiber. These properties are elongation, single fibers and bundle tenacities, work of rupture, etc. They classified fiber based on length and their linear densities for improving precision and behavior of cotton fibers. This information is extremely important for breeders to know about the complex relationship among various

In the third section, a chapter was compiled for discussing the impact of heat—an emerging menace depression of cotton production worldwide—and various strategies to mitigate this stress. In this regard, Zafar and colleagues described that various physiological and bio‐ chemical changes occur in cotton plant upon exposure to extreme temperatures (high as

#### **Section 4 Transgenic Cotton and Biosafety Issues 153**

Chapter 7 **Transgenic Bt Cotton: Effects on Target and Non-Target Insect Diversity 155** Muhammad Arshad, Rashad Rasool Khan, Asad Aslam and Waseem Akbar

## Preface

Chapter 6 **Recent Developments in Fiber Genomics of Tetraploid**

Dewayne D. Deng and Johnie N. Jenkins

Chapter 7 **Transgenic Bt Cotton: Effects on Target and Non-Target Insect**

Muhammad Arshad, Rashad Rasool Khan, Asad Aslam and

**Section 4 Transgenic Cotton and Biosafety Issues 153**

Mirzakamol S. Ayubov, Ibrokhim Y. Abdurakhmonov, Venkateswara R. Sripathi, Sukumar Saha, Tokhir M. Norov, Zabardast T. Buriev, Shukhrat E. Shermatov, Khurshida A. Ubaydullaeva, Jack C. McCarty,

**Cotton Species 123**

**VI** Contents

**Diversity 155**

Waseem Akbar

The aim of editing this book was to share R&D efforts undertaken to study the properties of natural cotton fiber including color and quality, improving the genetics of naturally colored cotton, genomic insights for mitigating the impact of heat—one of the most devastating components depressing cotton production—genetic and genomics of different traits in cot‐ ton, genome editing for combating biotic and abiotic stresses, and the development of trans‐ genic cotton as well as their biosafety issues.

In the first chapter, written by Drs Mehboob-ur-Rahman and Yusuf Zafar, the provoking approaches to sustain cotton yield are described. Till date, cotton varieties were developed for cultivation under high-performance environments to achieve the maximum yield. How‐ ever, the performance of these cotton varieties is much more vulnerable at resource poor farms. The changing climate would further exaggerate the situation by gradual escalation of temperature and depletion of fresh water resources and make cotton crop difficult to grow. In this chapter, possible solutions, including conventional as well as non-conventional pro‐ cedures for sustaining cotton production, have been discussed.

In the second chapter, genome editing—a way to tailor gene for silencing or enhancing its expression precisely, has been described. Conventionally, mutations were induced random‐ ly using various physical and chemical mutagens—making it difficult to target the gene of interest. Khan and colleagues described a number of approaches that can target the gene of interest very precisely. Among these, zinc finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs), and clustered regularly interspaced short palindromic re‐ peats (CRISPR), and RNA-guided nucleases (e.g., Cas9) were in common use to target the genome. The CRISPR/Cas has shown a great potential to edit genome of complex species including cotton. For example, lateral root formation was improved using CRISPR/Cas sys‐ tem. Its role for silencing undesirable traits in cotton is yet to be established.

In the second section, impact of the Bijective relationship between single and bundle cotton fiber was narrated by Mahjoub and colleagues. In this study, three different fiber types, chosen from a list of twelve cottons, were selected from a large panel of varieties with sub‐ stantial variations in the physical properties of fiber. These properties are elongation, single fibers and bundle tenacities, work of rupture, etc. They classified fiber based on length and their linear densities for improving precision and behavior of cotton fibers. This information is extremely important for breeders to know about the complex relationship among various fiber arrangement parameters, which ultimately determine the quality of the fabric.

In the third section, a chapter was compiled for discussing the impact of heat—an emerging menace depression of cotton production worldwide—and various strategies to mitigate this stress. In this regard, Zafar and colleagues described that various physiological and bio‐ chemical changes occur in cotton plant upon exposure to extreme temperatures (high as well as low). In this chapter, various breeding approaches coupled with genomic strategies (marker-assisted as well as transgenic selection) were also described for developing resilient cotton varieties.

The second chapter of this section entitled "Genetic Mapping in Cotton" was written by Bar‐ dak and his colleagues. In this chapter, progress made toward understanding the extent of genetic diversity in cotton germplasm and identifying QTLs associated with resistance to various diseases including verticillium wilt, plant architecture, stomatal conductance, yield, and fiber qualities was discussed. Authors have also demonstrated the role of genome-wide association studies (GWAS) coupled with next-generation sequencing (NGS) in identifying DNA markers as well as cloning genes conferring various traits of interest. This information would help in initiating cotton breeding by design.

The third chapter of this section solely focused on updates on "fiber genomic studies." In this chapter, Ayubov and colleagues highlighted the importance of producing high-quality cotton to make the natural fiber more competitive than that of the synthetic fiber. Recent progress on sequencing the whole genome of cotton species together with parallel evolution of bioinformatic tools made it possible to identify closely linked DNA markers as well as genes responsible for fiber quality traits. Earlier, a number of DNA markers (SSRs, EST-SSRs, and SNPs) associated with fiber traits were developed, and few of them were used in marker-assisted selection (MAS). In this chapter, information about the new genomic re‐ sources, characterization of cotton genomes, discoveries of many novel genes, regulatory el‐ ements including small and micro-RNAs, and novel tools such as gene silencing or gene editing techniques for genome manipulation was provided comprehensively.

In the last section of the book, the impact of transgenic cotton containing Bt genes on nontargets has been reviewed comprehensively by Arshad and colleagues. It has been shown that transgenic crops may disturb the population structure of non-target insect unintention‐ ally. For instance, GM cotton containing Cry genes has been allowed for cultivation to con‐ trol lepidopterous pests; however, the introduction of GM cotton may support the survival of new insects and may attain the status of pests. The change in species population structure may influence IPM programs, which should be addressed by conducting some planned studies on target and non-target insect diversity. In this chapter, authors have compiled studies of various labs showing the impact of Bt cotton against major target and non-target insect pests in cotton agro-ecosystem. It was concluded that GM cotton is highly specific to the targeted pests and has no negative impact on non-target insect species. The decreased use of insecticides on Bt cotton demonstrated the positive impact on beneficial insect popu‐ lations and can also support the survival of rare species. It has been shown that the cultiva‐ tion of Bt cotton is safe and is an environment-friendly approach.

> **Mehboob-Ur-Rahman** National Institute for Biotechnology & Genetic Engineering Faisalabad, Pakistan

> > **Yusuf Zafar** Pakistan Agricultural Research Council Islamabad, Pakistan

**Section 1**

**Future Trends in Cotton Research**

**Future Trends in Cotton Research**

well as low). In this chapter, various breeding approaches coupled with genomic strategies (marker-assisted as well as transgenic selection) were also described for developing resilient

The second chapter of this section entitled "Genetic Mapping in Cotton" was written by Bar‐ dak and his colleagues. In this chapter, progress made toward understanding the extent of genetic diversity in cotton germplasm and identifying QTLs associated with resistance to various diseases including verticillium wilt, plant architecture, stomatal conductance, yield, and fiber qualities was discussed. Authors have also demonstrated the role of genome-wide association studies (GWAS) coupled with next-generation sequencing (NGS) in identifying DNA markers as well as cloning genes conferring various traits of interest. This information

The third chapter of this section solely focused on updates on "fiber genomic studies." In this chapter, Ayubov and colleagues highlighted the importance of producing high-quality cotton to make the natural fiber more competitive than that of the synthetic fiber. Recent progress on sequencing the whole genome of cotton species together with parallel evolution of bioinformatic tools made it possible to identify closely linked DNA markers as well as genes responsible for fiber quality traits. Earlier, a number of DNA markers (SSRs, EST-SSRs, and SNPs) associated with fiber traits were developed, and few of them were used in marker-assisted selection (MAS). In this chapter, information about the new genomic re‐ sources, characterization of cotton genomes, discoveries of many novel genes, regulatory el‐ ements including small and micro-RNAs, and novel tools such as gene silencing or gene

In the last section of the book, the impact of transgenic cotton containing Bt genes on nontargets has been reviewed comprehensively by Arshad and colleagues. It has been shown that transgenic crops may disturb the population structure of non-target insect unintention‐ ally. For instance, GM cotton containing Cry genes has been allowed for cultivation to con‐ trol lepidopterous pests; however, the introduction of GM cotton may support the survival of new insects and may attain the status of pests. The change in species population structure may influence IPM programs, which should be addressed by conducting some planned studies on target and non-target insect diversity. In this chapter, authors have compiled studies of various labs showing the impact of Bt cotton against major target and non-target insect pests in cotton agro-ecosystem. It was concluded that GM cotton is highly specific to the targeted pests and has no negative impact on non-target insect species. The decreased use of insecticides on Bt cotton demonstrated the positive impact on beneficial insect popu‐ lations and can also support the survival of rare species. It has been shown that the cultiva‐

**Mehboob-Ur-Rahman**

Faisalabad, Pakistan

Islamabad, Pakistan

Pakistan Agricultural Research Council

**Yusuf Zafar**

National Institute for Biotechnology & Genetic Engineering

editing techniques for genome manipulation was provided comprehensively.

tion of Bt cotton is safe and is an environment-friendly approach.

cotton varieties.

VIII Preface

would help in initiating cotton breeding by design.

**Chapter 1**

Provisional chapter

**Introductory Chapter: Updates on Achieving**

DOI: 10.5772/intechopen.74410

Cotton, a major source of natural fiber and vegetable oil [1], belongs to the genus Gossypium. The word cotton derived from "quotn", an Arabic word [2], and Gossypium derived from "goz", an Arabic word [3]. The genus Gossypium is composed of ~50 species; out of these five are tetraploids (2n = 4x = 52) which evolved about 1–2 million years ago [4–6] through hybridization of two diploid species containing genomes much similar like "A" (A2 (G. arboreum) and A1 (G. herbaceum)) and "D" (G. raimondii). Among these, G. hirsutum (AD1) and G. barbadense (AD2) are cultivated, while three species including G. tomentosum (AD3), G. mustelinum (AD4), and G. darwinii (AD5) are wild which are endemic to Hawaii, Brazil, and Galapagos Islands. In total, 45 are diploids containing one genome each of the total eight different genomes (A–G and K) [4].

Significant progress toward increasing lint yield and improving lint quality has been made during the last seven decades through using conventional and nonconventional breeding approaches. Transgenic cotton conferring resistance to chewing insect pests as well as to glyphosate not only reduces the cost of production but also increases the yield per unit area in developing countries. Cultivation of transgenic cotton helped IPM programs as well as reduced the toxic impact of pesticide in the environment. Also, the cultivation of Bt cotton was found safe for the nontargets. At the moment, cotton is cultivated on >30 million hectares in 80 different countries of the world [7, 8]. World cotton average yield is fluctuating over the last 3 years, and significant reduction (~9% in 2015–2016 than that of 2014–2015) in yield was observed [9]. There are a number of factors including changing climate, resistance development in target insect pests and weeds, increased heat and drought stress, excessive rains and water logging, evolution of new strains of diseases, etc. contributed toward the decline in yield [10, 11]. Another menace is the high demand of inputs for harvesting acceptable yield from Bt cotton. Also, the synthetic fiber is competing out the natural cotton fiber owing to its reduced

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Introductory Chapter: Updates on Achieving

**Sustainable Cotton Production**

Sustainable Cotton Production

Mehboob-ur-Rahman and Yusuf Zafar

Mehboob-ur-Rahman and Yusuf Zafar

http://dx.doi.org/10.5772/intechopen.74410

1. Introduction

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Introductory Chapter: Updates on Achieving Sustainable Cotton Production** Introductory Chapter: Updates on Achieving Sustainable Cotton Production

DOI: 10.5772/intechopen.74410

 Mehboob-ur-Rahman and Yusuf Zafar Mehboob-ur-Rahman and Yusuf Zafar

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74410

#### 1. Introduction

Cotton, a major source of natural fiber and vegetable oil [1], belongs to the genus Gossypium. The word cotton derived from "quotn", an Arabic word [2], and Gossypium derived from "goz", an Arabic word [3]. The genus Gossypium is composed of ~50 species; out of these five are tetraploids (2n = 4x = 52) which evolved about 1–2 million years ago [4–6] through hybridization of two diploid species containing genomes much similar like "A" (A2 (G. arboreum) and A1 (G. herbaceum)) and "D" (G. raimondii). Among these, G. hirsutum (AD1) and G. barbadense (AD2) are cultivated, while three species including G. tomentosum (AD3), G. mustelinum (AD4), and G. darwinii (AD5) are wild which are endemic to Hawaii, Brazil, and Galapagos Islands. In total, 45 are diploids containing one genome each of the total eight different genomes (A–G and K) [4].

Significant progress toward increasing lint yield and improving lint quality has been made during the last seven decades through using conventional and nonconventional breeding approaches. Transgenic cotton conferring resistance to chewing insect pests as well as to glyphosate not only reduces the cost of production but also increases the yield per unit area in developing countries. Cultivation of transgenic cotton helped IPM programs as well as reduced the toxic impact of pesticide in the environment. Also, the cultivation of Bt cotton was found safe for the nontargets. At the moment, cotton is cultivated on >30 million hectares in 80 different countries of the world [7, 8]. World cotton average yield is fluctuating over the last 3 years, and significant reduction (~9% in 2015–2016 than that of 2014–2015) in yield was observed [9]. There are a number of factors including changing climate, resistance development in target insect pests and weeds, increased heat and drought stress, excessive rains and water logging, evolution of new strains of diseases, etc. contributed toward the decline in yield [10, 11]. Another menace is the high demand of inputs for harvesting acceptable yield from Bt cotton. Also, the synthetic fiber is competing out the natural cotton fiber owing to its reduced

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cost in the international market [3, 10, 11]. Thus, extraordinary efforts are needed to sustain cotton production by 2050.

using some other forward and reverse genetic approaches—VIGS and CRISPR-Cas system

Introductory Chapter: Updates on Achieving Sustainable Cotton Production

http://dx.doi.org/10.5772/intechopen.74410

5

Another important genetic resource in the form of genetic maps construed using several mapping populations developed through interspecific (G. hirsutum/G. barbadense) and intraspecific (G. hirsutum/G. hirsutum) crosses is available. Initially, this information may help in developing cotton cultivars by design. Unlike rice, deployment of DNA markers in routine cotton breeding is limited to simple traits; however, selection for complex traits is yet to be realized even in advanced countries. For example, ~1000 QTLs for ~40 different traits have been reported. In total, over 49 genetic maps and about 25,000 genetic markers have been reported in different research articles. This information can be used to breed "designer cotton

In developing countries, cotton breeders are not using the diagnostic markers linked with simple traits because of lack of resources and trained manpower. It is extremely important to identify DNA markers close enough to the complex traits in spite of the fact that genetic base of the parent genotypes is narrow. In this regard, collaborative efforts among few labs for searching new DNA markers have been initiated by deploying new assays like nestedassociation mapping and association mapping approaches. Such coordinated efforts can be successful if the goal is common, for example, breeding for resistance to lepidopteron insect pests, drought, and salinity. However, there are few research issues typical to a specific region. For example, cotton leaf curl disease infects cotton in Pakistan and is a potential threat to all other cotton-growing countries where whitefly is prevalent. For addressing this issue, collaborative and coordinated research programs including sharing of cotton germplasm for screening in the hotspot regions would help in tackling such issues. USDA screened >5000 accessions of cotton in hotspot regions of Pakistan, and more than a dozen of asymptomatic cotton genotypes were identified [11, 13, 19]. This information and genetic material are useful for Pakistan and also for the whole cotton-growing community as the threat is spreading to other countries (reported in India and China). Thus, cotton production would be sustained. There are 53,000–63,946 cotton germplasm accessions that are preserved in gene banks of different cotton-growing countries. Sharing these resources is extremely important to characterize the extent of phenotypic and genotypic diversity present in the genus Gossypium. A highthroughput phenotyping platform is required to study traits precisely in a large number of genotypes/accessions in the shortest possible time [16]. The use of such automated technologies will expedite the progress toward initiating marker-assisted selection as well as elucidating various genetic circuits of simple and complex traits. Duplicated accessions can be discarded to avoid redundancy. Selected genotypes/accessions reflecting the maximum phenotypic diversity can be explored using genomic tools including SSR-based characterization, genotyping by sequencing, and re-sequencing, together with the application of association mapping analysis including nested-association mapping (NAM) and genome-wide association

Another important genetic resource is the cultivated G. arboreum and G. herbaceum species. These have been evolved in drought-prone regions and are still under plow in marginal lands of Indo-Pak regions [21, 22]. These two species have relatively greater root biomass than that of

may have a role in the future for assigning functions to different genes [12, 17].

varieties" [18].

study (GWAS) methods [20].

#### 2. Possibilities toward achieving sustainable cotton production

Bringing sustainability in cotton production is a major challenge for the resource-poor farming communities—dominantly living in developing countries. Other than releasing cotton varieties expressing high yield potential by growing in high input environments—progressive farmers can afford, there is a need to open R&D fronts for developing cotton varieties which can withstand the impact of changing climate and produce sustainable yields in low input or in optimum input environments [11]. Deployment of high-tech genomic tools in breeding is one of the approaches to initiate breeding by design aiming at the development of resilient cotton cultivars [12, 13].

Efforts for achieving maximum yield potential under optimum input environments are not successful largely because of lack of genetic diversity among the genetic resource use to develop new cotton varieties. Narrow genetic base does not only limit the future breeding progress but also makes the crop vulnerable to insect pests and diseases and also to the negative impact of changing climate. For enhancing the genetic diversity of newly developed cultivars, underutilized genetic resources, land races, obsolete varieties, old accessions, etc. are the golden assets for cotton breeders to cultivate genes conferring novel traits [11]. Usually, cotton breeders avoid using the genetic resource from undomesticated germplasm as it hinders the breeding progress because of the linkage drag of some undesirable traits present in the wild germplasm. For having success using conventional breeding approach, selection of diverse parent genotypes, appropriate population size, and accurate testing of newly developed lines in a given ecosystem are the prerequisites to develop cotton varieties with improved genetics [14].

After sequencing of Arabidopsis genome, a number of crop species including cultivated cotton species have been sequenced. Also, the progenitor species, i.e., A-genome and D-genome, of the tetraploid-cultivated cotton species have been sequenced. The next question is to understand the extent of genetic diversity residing in genomes, and associating the diversity in gene spellings with phenotypic diversity or important traits (quality, productivity, resistance to biotic and abiotic stresses, etc.) would remain a major challenge [15]. In this regard, massive re-sequencing of thousands of cotton genotypes/accessions of the same species as well as different species would help in elucidating gene function of important traits [16] as well as the footprint of selection [15]. In the present scenario, the function of identical genes can also be deduced by comparing with the similar genes present in Arabidopsis thaliana—shared common ancestry with cotton ~83–86 million years ago [15]. Thus, many genes which are analogous to genes present in Arabidopsis can be characterized with varying degree of success. However, genes conferring traits typical to cotton will have to characterize by deploying various molecular assays including finding DNA markers associated with the traits using nested-association mapping (NAM) populations, genome-wide association study (GWAS) analysis, etc. or by using some other forward and reverse genetic approaches—VIGS and CRISPR-Cas system may have a role in the future for assigning functions to different genes [12, 17].

cost in the international market [3, 10, 11]. Thus, extraordinary efforts are needed to sustain

Bringing sustainability in cotton production is a major challenge for the resource-poor farming communities—dominantly living in developing countries. Other than releasing cotton varieties expressing high yield potential by growing in high input environments—progressive farmers can afford, there is a need to open R&D fronts for developing cotton varieties which can withstand the impact of changing climate and produce sustainable yields in low input or in optimum input environments [11]. Deployment of high-tech genomic tools in breeding is one of the approaches to initiate breeding by design aiming at the development of resilient

Efforts for achieving maximum yield potential under optimum input environments are not successful largely because of lack of genetic diversity among the genetic resource use to develop new cotton varieties. Narrow genetic base does not only limit the future breeding progress but also makes the crop vulnerable to insect pests and diseases and also to the negative impact of changing climate. For enhancing the genetic diversity of newly developed cultivars, underutilized genetic resources, land races, obsolete varieties, old accessions, etc. are the golden assets for cotton breeders to cultivate genes conferring novel traits [11]. Usually, cotton breeders avoid using the genetic resource from undomesticated germplasm as it hinders the breeding progress because of the linkage drag of some undesirable traits present in the wild germplasm. For having success using conventional breeding approach, selection of diverse parent genotypes, appropriate population size, and accurate testing of newly developed lines in a given ecosystem are the prerequisites to develop cotton varieties with improved

After sequencing of Arabidopsis genome, a number of crop species including cultivated cotton species have been sequenced. Also, the progenitor species, i.e., A-genome and D-genome, of the tetraploid-cultivated cotton species have been sequenced. The next question is to understand the extent of genetic diversity residing in genomes, and associating the diversity in gene spellings with phenotypic diversity or important traits (quality, productivity, resistance to biotic and abiotic stresses, etc.) would remain a major challenge [15]. In this regard, massive re-sequencing of thousands of cotton genotypes/accessions of the same species as well as different species would help in elucidating gene function of important traits [16] as well as the footprint of selection [15]. In the present scenario, the function of identical genes can also be deduced by comparing with the similar genes present in Arabidopsis thaliana—shared common ancestry with cotton ~83–86 million years ago [15]. Thus, many genes which are analogous to genes present in Arabidopsis can be characterized with varying degree of success. However, genes conferring traits typical to cotton will have to characterize by deploying various molecular assays including finding DNA markers associated with the traits using nested-association mapping (NAM) populations, genome-wide association study (GWAS) analysis, etc. or by

2. Possibilities toward achieving sustainable cotton production

cotton production by 2050.

4 Past, Present and Future Trends in Cotton Breeding

cotton cultivars [12, 13].

genetics [14].

Another important genetic resource in the form of genetic maps construed using several mapping populations developed through interspecific (G. hirsutum/G. barbadense) and intraspecific (G. hirsutum/G. hirsutum) crosses is available. Initially, this information may help in developing cotton cultivars by design. Unlike rice, deployment of DNA markers in routine cotton breeding is limited to simple traits; however, selection for complex traits is yet to be realized even in advanced countries. For example, ~1000 QTLs for ~40 different traits have been reported. In total, over 49 genetic maps and about 25,000 genetic markers have been reported in different research articles. This information can be used to breed "designer cotton varieties" [18].

In developing countries, cotton breeders are not using the diagnostic markers linked with simple traits because of lack of resources and trained manpower. It is extremely important to identify DNA markers close enough to the complex traits in spite of the fact that genetic base of the parent genotypes is narrow. In this regard, collaborative efforts among few labs for searching new DNA markers have been initiated by deploying new assays like nestedassociation mapping and association mapping approaches. Such coordinated efforts can be successful if the goal is common, for example, breeding for resistance to lepidopteron insect pests, drought, and salinity. However, there are few research issues typical to a specific region. For example, cotton leaf curl disease infects cotton in Pakistan and is a potential threat to all other cotton-growing countries where whitefly is prevalent. For addressing this issue, collaborative and coordinated research programs including sharing of cotton germplasm for screening in the hotspot regions would help in tackling such issues. USDA screened >5000 accessions of cotton in hotspot regions of Pakistan, and more than a dozen of asymptomatic cotton genotypes were identified [11, 13, 19]. This information and genetic material are useful for Pakistan and also for the whole cotton-growing community as the threat is spreading to other countries (reported in India and China). Thus, cotton production would be sustained. There are 53,000–63,946 cotton germplasm accessions that are preserved in gene banks of different cotton-growing countries. Sharing these resources is extremely important to characterize the extent of phenotypic and genotypic diversity present in the genus Gossypium. A highthroughput phenotyping platform is required to study traits precisely in a large number of genotypes/accessions in the shortest possible time [16]. The use of such automated technologies will expedite the progress toward initiating marker-assisted selection as well as elucidating various genetic circuits of simple and complex traits. Duplicated accessions can be discarded to avoid redundancy. Selected genotypes/accessions reflecting the maximum phenotypic diversity can be explored using genomic tools including SSR-based characterization, genotyping by sequencing, and re-sequencing, together with the application of association mapping analysis including nested-association mapping (NAM) and genome-wide association study (GWAS) methods [20].

Another important genetic resource is the cultivated G. arboreum and G. herbaceum species. These have been evolved in drought-prone regions and are still under plow in marginal lands of Indo-Pak regions [21, 22]. These two species have relatively greater root biomass than that of the cultivated G. hirsutum species. Thus, these species harbor important genes for high root biomass including deep root system and scavenge water from deep layers of soils. These two species are also resistant to cotton leaf curl disease and other biotic stresses. In spite of the fact that the genomes of G. arboreum and G. herbaceum have been sequenced, a limited number of genetic maps using diploid interspecific populations have been reported. Identifying QTLs associated with traits typical to these diploid species would be another priority research area for initiating DNA-based selection procedures—previously not possible. The negative linkage drags can be avoided in desirable plants in segregating and/or backcross populations by monitoring the introgression of desirable alleles/genes using the associated DNA markers. Most times, backcross breeding scheme is deployed to recover the genome of recurrent genotype while retaining the desirable alleles of the donor genotype. In the whole scheme, extensive backcrossing followed by identifying plants containing the desirable alleles using DNA markers would be an important breeding procedure for developing improved cotton germplasm.

grow varieties producing more cottonseed yield per hectare rather than more lint yield per hectare. A significant fluctuation in lint percentage (35–50%) in the cotton germplasm has been found. Thus, emphasis should be made to breed for >45% lint recovery which would add a

Introductory Chapter: Updates on Achieving Sustainable Cotton Production

http://dx.doi.org/10.5772/intechopen.74410

7

Another breeding objective is to improve the lint quality—always remained a major challenge for breeders as the trait is controlled by multiple thousands of genes. Through conventional breeding, limited success has been achieved; however, further improvement is handicapped due to lack of compatible genetic resources. The success of resolving the complex traits has been demonstrated in model plant species using modern genomic tools. Currently, efforts are underway to clone QTLs involved in conferring these complex traits in cotton. Genome sequence information of the cultivated species also unraveled the genetics of fiber initiation relatively simpler than that of the fiber elongation traits. Once the key genes involved in defining lint characters are identified and these can be used to engineer the pathways of the diploid cotton species like G. arboreum and others, thus fiber production can be sustained in low input environments. Another immediate thought is to initiate projects for "re-sequencing" the representative genotypes of the closely related cotton species using next-generation sequencing approaches which would be a way forward for associating genetic variations with the traits using bioinformatics tools—would add synergy for sustaining cotton production. These studies would also help in identifying new tissue-specific promoters, unlike the consti-

Protection to insect pests and diseases has been engineered in different crop species including cotton using different novel genes (Cry genes) excised from a soil bacterium. Success of Bt cotton in protecting cotton crop from bollworms has been demonstrated since its release in 1996. Now, resistance conferred by the Cry1Ac gene has been weakened. Also, pink bollworm infestation on GM-cotton in India has been reported (scientific evidences are lacking). Potential of minor pests for emerging as major pests is another threat to cotton sustainability. For example, before the cultivation of Bt cotton in Pakistan, mealy bug and dusky bug have never been problematic to cotton as indirectly controlled by the application of insecticides applied to kill lepidopteron insect pests, but on Bt cotton these two insects infested in the recent past. This scenario may also arise in other countries. The situation can be mitigated through educating cotton farmers for monitoring and controlling the new emerging pests by taking measures including the applications of insecticides. Till now, GMcotton containing few genes (largely of Cry series and genes conferring resistance to glyphosate) has been commercialized [23, 24]. Thus, new genes and/or their transcription factors conferring tolerance to biotic and abiotic stresses from other plant sources including wild species can be characterized followed by the introduction in cotton. These genes would have high chances of acceptance by the end user. Improvement in expression of transgene can be made by designing effective gene cassette with efficient promoters, followed by identifica-

Identification of new marker genes (e.g., genes conferring fluorescent proteins) instead of using conventional marker genes (e.g., antibiotic-resistant gene) would be useful for making the use of GM foods more safe. These genes should be tested for an extended time

couple of million bales toward the total cotton production.

tutive promoters, which may save plant energy.

tion of the best event with high gene expression [17].

The use of mutagens for breaking the negatively linked traits has been used to develop new cotton varieties. Earlier radiations (gamma rays) have been extensively used during the early 1960s—induced changes in structure of chromosomes randomly. Usually, soaked seed and/or pollen grains are exposed to physical mutagens. These mutagens can be used to expose F1/F2 seed developed through crossing two different species, and the best mutant plants can be identified by surveying with DNA markers originating from the adapted species. This technique was not largely found worth working because of induction of several deleterious mutations and unwanted linkages in the newly developed mutated genotypes.

The use of chemical mutagens is another approach for inducing mutations by exposing the cottonseed with known mutagens; that is, EMS would not only be helpful in assigning functions to different genes but would also be instrumental in adding new alleles through adding or deleting or replacing nucleotides in the genes which would enhance the genetic diversity—a potential buffer to the epidemic of insect pests and diseases.

Hybrid vigor, increased growth over the parent genotypes, has been explored in corn that resulted in multifold increase in production worldwide. Such efforts were also translated in vegetable and arable crops including cotton but could not gain the popularity like corn. For example, in few parts of the world, cultivation of hybrid cotton has shown increase in lint production. The hybrid cotton can be exploited if the hybrid seed surpasses the yield by 30% over the open-pollinated variety (OPV); resultantly, farmers may get rewards of their investments made to procure costly hybrid seed. In this regard, extensive studies are needed to identify the best combiners. Hybrid cotton breeding is also handicapped due to the nonavailability of reliable genetic as well as mechanical means for getting rid of anthers/pollens. Farmers may produce hybrid seed sufficient to meet their own demands (farmers in few provinces of China do this practice) for sowing cotton hybrids instead of OPVs. Thus, farmers can save money meant for buying the hybrid seed. Trainings for the production of hybrid cottonseed may be given to farmers by the public sector organizations [11, 22].

In most developing countries, for example, in Pakistan, the cost of cottonseed is paid based on total weight of the cottonseed irrespective of the lint potential which encourages farmers to grow varieties producing more cottonseed yield per hectare rather than more lint yield per hectare. A significant fluctuation in lint percentage (35–50%) in the cotton germplasm has been found. Thus, emphasis should be made to breed for >45% lint recovery which would add a couple of million bales toward the total cotton production.

the cultivated G. hirsutum species. Thus, these species harbor important genes for high root biomass including deep root system and scavenge water from deep layers of soils. These two species are also resistant to cotton leaf curl disease and other biotic stresses. In spite of the fact that the genomes of G. arboreum and G. herbaceum have been sequenced, a limited number of genetic maps using diploid interspecific populations have been reported. Identifying QTLs associated with traits typical to these diploid species would be another priority research area for initiating DNA-based selection procedures—previously not possible. The negative linkage drags can be avoided in desirable plants in segregating and/or backcross populations by monitoring the introgression of desirable alleles/genes using the associated DNA markers. Most times, backcross breeding scheme is deployed to recover the genome of recurrent genotype while retaining the desirable alleles of the donor genotype. In the whole scheme, extensive backcrossing followed by identifying plants containing the desirable alleles using DNA markers would be an important breeding procedure for developing improved

The use of mutagens for breaking the negatively linked traits has been used to develop new cotton varieties. Earlier radiations (gamma rays) have been extensively used during the early 1960s—induced changes in structure of chromosomes randomly. Usually, soaked seed and/or pollen grains are exposed to physical mutagens. These mutagens can be used to expose F1/F2 seed developed through crossing two different species, and the best mutant plants can be identified by surveying with DNA markers originating from the adapted species. This technique was not largely found worth working because of induction of several deleterious muta-

The use of chemical mutagens is another approach for inducing mutations by exposing the cottonseed with known mutagens; that is, EMS would not only be helpful in assigning functions to different genes but would also be instrumental in adding new alleles through adding or deleting or replacing nucleotides in the genes which would enhance the genetic diversity—a

Hybrid vigor, increased growth over the parent genotypes, has been explored in corn that resulted in multifold increase in production worldwide. Such efforts were also translated in vegetable and arable crops including cotton but could not gain the popularity like corn. For example, in few parts of the world, cultivation of hybrid cotton has shown increase in lint production. The hybrid cotton can be exploited if the hybrid seed surpasses the yield by 30% over the open-pollinated variety (OPV); resultantly, farmers may get rewards of their investments made to procure costly hybrid seed. In this regard, extensive studies are needed to identify the best combiners. Hybrid cotton breeding is also handicapped due to the nonavailability of reliable genetic as well as mechanical means for getting rid of anthers/pollens. Farmers may produce hybrid seed sufficient to meet their own demands (farmers in few provinces of China do this practice) for sowing cotton hybrids instead of OPVs. Thus, farmers can save money meant for buying the hybrid seed. Trainings for the production of hybrid cottonseed may be given to

In most developing countries, for example, in Pakistan, the cost of cottonseed is paid based on total weight of the cottonseed irrespective of the lint potential which encourages farmers to

tions and unwanted linkages in the newly developed mutated genotypes.

potential buffer to the epidemic of insect pests and diseases.

farmers by the public sector organizations [11, 22].

cotton germplasm.

6 Past, Present and Future Trends in Cotton Breeding

Another breeding objective is to improve the lint quality—always remained a major challenge for breeders as the trait is controlled by multiple thousands of genes. Through conventional breeding, limited success has been achieved; however, further improvement is handicapped due to lack of compatible genetic resources. The success of resolving the complex traits has been demonstrated in model plant species using modern genomic tools. Currently, efforts are underway to clone QTLs involved in conferring these complex traits in cotton. Genome sequence information of the cultivated species also unraveled the genetics of fiber initiation relatively simpler than that of the fiber elongation traits. Once the key genes involved in defining lint characters are identified and these can be used to engineer the pathways of the diploid cotton species like G. arboreum and others, thus fiber production can be sustained in low input environments. Another immediate thought is to initiate projects for "re-sequencing" the representative genotypes of the closely related cotton species using next-generation sequencing approaches which would be a way forward for associating genetic variations with the traits using bioinformatics tools—would add synergy for sustaining cotton production. These studies would also help in identifying new tissue-specific promoters, unlike the constitutive promoters, which may save plant energy.

Protection to insect pests and diseases has been engineered in different crop species including cotton using different novel genes (Cry genes) excised from a soil bacterium. Success of Bt cotton in protecting cotton crop from bollworms has been demonstrated since its release in 1996. Now, resistance conferred by the Cry1Ac gene has been weakened. Also, pink bollworm infestation on GM-cotton in India has been reported (scientific evidences are lacking). Potential of minor pests for emerging as major pests is another threat to cotton sustainability. For example, before the cultivation of Bt cotton in Pakistan, mealy bug and dusky bug have never been problematic to cotton as indirectly controlled by the application of insecticides applied to kill lepidopteron insect pests, but on Bt cotton these two insects infested in the recent past. This scenario may also arise in other countries. The situation can be mitigated through educating cotton farmers for monitoring and controlling the new emerging pests by taking measures including the applications of insecticides. Till now, GMcotton containing few genes (largely of Cry series and genes conferring resistance to glyphosate) has been commercialized [23, 24]. Thus, new genes and/or their transcription factors conferring tolerance to biotic and abiotic stresses from other plant sources including wild species can be characterized followed by the introduction in cotton. These genes would have high chances of acceptance by the end user. Improvement in expression of transgene can be made by designing effective gene cassette with efficient promoters, followed by identification of the best event with high gene expression [17].

Identification of new marker genes (e.g., genes conferring fluorescent proteins) instead of using conventional marker genes (e.g., antibiotic-resistant gene) would be useful for making the use of GM foods more safe. These genes should be tested for an extended time

period on the number of different model organisms for making the conclusions acceptable to the public in effective manner. All these new genes from alien background may be supplemented, or resistance can be delayed by adopting some other strategies including development of short-duration varieties and characters offering defense umbrella (small leaves, pubescence, light green leaves, etc.) for avoiding any significant damage by the insect pests and diseases. Secondly, research on other aspects such as chemical ecology would add synergy in managing eco-friendly insect pests and diseases and thus can enhance yield.

Author details

References

pp. 7-22

426 p

Mehboob-ur-Rahman<sup>1</sup>

\* and Yusuf Zafar2

1 National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

[1] Abdurakhmonov IY. Role of genomic studies in boosting yield. In: Proceedings of International Cotton Advisory Board (ICAC); 20 September–4 October 2013; Cartagena. 2013.

Introductory Chapter: Updates on Achieving Sustainable Cotton Production

http://dx.doi.org/10.5772/intechopen.74410

9

[2] Lee JA, Fang DD. Cotton as a world crop: Origin, history, and current status. In: Fang D, Percy R, editors. Cotton. 2nd ed. Madison: American Society of Agronomy; 2015. pp. 1-24.

[3] Gledhill D. The Names of Plants. 4th ed. Cambridge: Cambridge University Press; 2008.

[4] Cronn RC, Small RL, Haselkorn T, Wendel JF. Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes.

[5] Adams KL, Cronn R, Percifield R, Wendel JF. Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:

[6] Adams KL, Wendel JF. Allele-specific bidirectional silencing of an alcohol dehydrogenase gene in different organs of interspecific diploid cotton hybrids. Genetics. 2005;171:2139-2142.

[7] Abdurakhmonov IY, Buriev ZT, Shermatov SS, Abdullaev AA, Urmonov K, Kushanov F, Egamberdiev S, Shapulatov U, Abdukarimov A, Saha S, Jenkins J, Kohel RJ, Yu JZ, Pepper AP, Kumpatla SP, Ulloa U. Genetic diversity in Gossypium genus. In: Caliskan M, editor. Genetic Diversity in Plants. Rijeka: InTech. pp. 331-338. DOI: 10.5772/35384 [8] Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS. Engineering cotton seed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proceedings of the National Academy of Sciences of the United States of America. 2006;

\*Address all correspondence to<EMAIL_ADDRESS>
2 Pakistan Agricultural Research Council, Islamabad, Pakistan

DOI: 10.2134/agronmonogr57.2013.0019

American Journal of Botany. 2002;89:707-725

4649-4654. DOI: 10.1073/pnas.0630618100

103:18054-18059. DOI: 10.1073/pnas.0605389103

DOI: 10.1534/genetics.105.047357

Genome editing through CRISPR-Cas is an emerging tool which can be used to edit the genes to improve or silent their expression. For example, gossypol-free cottonseed can be produced by silencing the genes conferring gossypols in seed. Major advantage of this assay is that the function of gene can be characterized and new cultivars can be evolved without introgressing foreign gene; hence, the technology will be acceptable to countries having skeptical views about the GM technology [11, 12, 15, 17]. Thus, it is summarized that the adoption of hightech management practices, utilization of untapped genetic resources in breeding, cultivation of cotton varieties with excellent genetics, monitoring of risk and efficacy of transgene in ecosystem, and continued search for new genetic resources would help in sustaining cotton production [11, 12].

#### 3. Conclusions

Cotton breeding, largely based on recombination genetics, has paved the way to develop cotton varieties which were heat tolerant, early maturing, and high yielding with improved fiber traits. Historically, the breeding subject of cotton revolves around the evolution of new cultivars, maintenance of varieties, and their seed production. Presently, phenotypic-based selection procedures have been changed to DNA-based selection systems (marker-assisted selection)—made possible for evolving cotton varieties with brilliant genetics in the shortest possible time. Finding the enormous number of variations has been greatly facilitated by the advent of next-generation sequencing tools, and these variations were assigned functions using advanced bioinformatic tools. All these discoveries and effectiveness of the technologies have made it possible to initiate breeding by design using DNA markers as well as by precise genome editing. Unlike conventional breeding practices, genes (e.g., Cry1Ac, etc.) have been transferred from alien sources for improving resilience to chewing insect pests and herbicides. In the present book, research efforts representing a wide range of research endeavors, being undertaken in different parts of the world, were comprehensively discussed. Both the editors (Drs. Mehboob-ur-Rahman and Yusuf Zafar) ensured the compilation of high-quality research, opinions, and progress made toward enhancing and understanding the cotton genome and its application for developing resilient cotton varieties—a way to sustain cotton production beyond 2050. In the end, editors acknowledge efforts and hard work of the authors in compiling their respective chapters.

#### Author details

period on the number of different model organisms for making the conclusions acceptable to the public in effective manner. All these new genes from alien background may be supplemented, or resistance can be delayed by adopting some other strategies including development of short-duration varieties and characters offering defense umbrella (small leaves, pubescence, light green leaves, etc.) for avoiding any significant damage by the insect pests and diseases. Secondly, research on other aspects such as chemical ecology would add synergy in managing eco-friendly insect pests and diseases and thus can

Genome editing through CRISPR-Cas is an emerging tool which can be used to edit the genes to improve or silent their expression. For example, gossypol-free cottonseed can be produced by silencing the genes conferring gossypols in seed. Major advantage of this assay is that the function of gene can be characterized and new cultivars can be evolved without introgressing foreign gene; hence, the technology will be acceptable to countries having skeptical views about the GM technology [11, 12, 15, 17]. Thus, it is summarized that the adoption of hightech management practices, utilization of untapped genetic resources in breeding, cultivation of cotton varieties with excellent genetics, monitoring of risk and efficacy of transgene in ecosystem, and continued search for new genetic resources would help in sustaining cotton

Cotton breeding, largely based on recombination genetics, has paved the way to develop cotton varieties which were heat tolerant, early maturing, and high yielding with improved fiber traits. Historically, the breeding subject of cotton revolves around the evolution of new cultivars, maintenance of varieties, and their seed production. Presently, phenotypic-based selection procedures have been changed to DNA-based selection systems (marker-assisted selection)—made possible for evolving cotton varieties with brilliant genetics in the shortest possible time. Finding the enormous number of variations has been greatly facilitated by the advent of next-generation sequencing tools, and these variations were assigned functions using advanced bioinformatic tools. All these discoveries and effectiveness of the technologies have made it possible to initiate breeding by design using DNA markers as well as by precise genome editing. Unlike conventional breeding practices, genes (e.g., Cry1Ac, etc.) have been transferred from alien sources for improving resilience to chewing insect pests and herbicides. In the present book, research efforts representing a wide range of research endeavors, being undertaken in different parts of the world, were comprehensively discussed. Both the editors (Drs. Mehboob-ur-Rahman and Yusuf Zafar) ensured the compilation of high-quality research, opinions, and progress made toward enhancing and understanding the cotton genome and its application for developing resilient cotton varieties—a way to sustain cotton production beyond 2050. In the end, editors acknowledge efforts and hard work of the authors in compil-

enhance yield.

8 Past, Present and Future Trends in Cotton Breeding

production [11, 12].

3. Conclusions

ing their respective chapters.

Mehboob-ur-Rahman<sup>1</sup> \* and Yusuf Zafar2

#### References

[9] Better yields to boost cotton production in 2016/17. International Cotton Advisory Committee (ICAC) press release. 2016. Available at: https://www.icac.org/Press-Release/2016/ PR-14-Better-Yields-to-Boost-Cotton-Production-in

**Chapter 2**

**Provisional chapter**

**Targeted Genome Editing for Cotton Improvement**

Conventional tools induce mutations randomly throughout the cotton genome—making breeding difficult and challenging. During the last decade, progress has been made to edit the gene of interest in a very precise manner. Targeted genome engineering with engineered nucleases (ENs) specifically zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases (e.g., Cas9) has been described as a "game-changing technology" for diverse fields as human genetics and plant biotechnology. In eukaryotic systems, ENs create double-strand breaks (DSBs) at the targeted DNA sequence which are repaired by nonhomologous end joining (NHEJ) or homologydirected recombination (HDR) mechanisms. ENs have been used successfully for targeted mutagenesis, gene knockout, and multisite genome editing (GenEd) in model plants and crop plants such as cotton, rice, and wheat. Recently, cotton genome has also been edited for targeted mutagenesis through CRISPR/Cas for improved lateral root formation. In addition, an efficient and fast method has been developed to evaluate guide RNAs transiently in cotton. The targeted disruption of undesirable genes or metabolic pathway can be achieved to increase quality of cotton. Undesirable metabolites like gossypol in cottonseed can be targeted efficiently using ENs for seed-specific low-gossypol cotton. Moreover, ENs are also helpful in gene stacking for herbicide resistance, insect resistance,

**Targeted Genome Editing for Cotton Improvement**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Cotton is an important source of natural fiber and has been playing a major role in economy and social structure of several countries. In addition, cotton serves as cash crop for more than

DOI: 10.5772/intechopen.73600

Zulqurnain Khan, Sultan Habibullah Khan,

Zulqurnain Khan, Sultan Habibullah Khan,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73600

and abiotic stress tolerance.

**1. Introduction**

**Keywords:** TALENs, CRISPR/Cas, DSBs, NHEJ, HDR, cotton

**Abstract**

Muhammad Salman Mubarik and Aftab Ahmad

Muhammad Salman Mubarik and Aftab Ahmad

#### **Targeted Genome Editing for Cotton Improvement Targeted Genome Editing for Cotton Improvement**

DOI: 10.5772/intechopen.73600

Zulqurnain Khan, Sultan Habibullah Khan, Muhammad Salman Mubarik and Aftab Ahmad Zulqurnain Khan, Sultan Habibullah Khan, Muhammad Salman Mubarik and Aftab Ahmad

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73600

#### **Abstract**

[9] Better yields to boost cotton production in 2016/17. International Cotton Advisory Committee (ICAC) press release. 2016. Available at: https://www.icac.org/Press-Release/2016/

[10] Hake K. Climate disruptions to fiber yield growth [Internet]. 2012. Available at: http://

[11] Rahman M. Cotton improvement for environmentally stressed economies. The ICAC

[12] Abdurakhmonov IY, Ayubov MS, Ubaydullaeva KA, Buriev ZT, Shermatov SE, Ruziboev HS, et al. RNA interference for functional genomics and improvement of cotton (Gossypium spp.). Frontiers in Plant Science. 2016;7:202. DOI: 10.3389/fpls.2016.00202 [13] Rahman M, Khan AQ, Rahmat Z, Iqbal MA, Zafar Y. Genetics and genomics of cotton leaf curl disease, its viral causal agents and whitefly vector: A way forward to sustain world cotton fiber security. Frontiers in Plant Science. 2017;8:1157. DOI: 10.3389/fpls.2017.01157

[14] Constable GA. Integration of all research disciplines for future production systems. The

[16] Abdurakhmonov IY. Some new directions and priority tasks for worldwide cotton genetics, breeding, genomics and biotechnology research. The ICAC Recorder. 2016;XXXIV(1)

[15] Paterson AH. New directions in cotton research. The ICAC Recorder. 2016;XXXIV(1)

[18] Karanthi KR. New directions in cotton research. The ICAC Recorder. 2016;XXXIV(1)

[19] Zafar Y. Declining rate of cotton production in Pakistan. New directions in cotton research.

[20] Rahman M, Shaheen T, Tabbasam N, Iqbal MA, Ashraf M, Zafar Y, Paterson AH. Genetic resources in cotton and their improvement. Agronomy for Sustainable Development.

[21] Rahman M, Yasmin T, Tabassum N, Ullah I, Asif M, Zafar Y. Studying the extent of genetic diversity among Gossypium arboreum L. genotypes/cultivars using DNA finger-

[22] Iqbal MA, Rahman M. Identification of marker-trait associations for lint traits in cotton.

[23] Rahman M. 2015. Role of genetic and genetic engineering in optimizing input use. Enhancing the mechanism of input interaction in cotton production. Proceedings of 73rd

[24] Rahman M, Zaman M, Shaheen T, Irem S, Zafar Y. Safe use of cry genes in genetically

[17] Saha S. New directions in cotton research. The ICAC Recorder. 2016;XXXIV(1)

PR-14-Better-Yields-to-Boost-Cotton-Production-in

www.cicr.org.in.isci-image/3.pdf

ICAC Recorder. 2016;XXXIV(1)

The ICAC Recorder. 2016;XXXIV(1)

2012;32:419-432. DOI: 10.1007/s13593-011-0051-z

ICAC Meeting; 2014; Thessaloniki, Greece. pp. 4-6

printing. Genetic Resources and Crop Evolution. 2008;55:331-339

Frontiers in Plant Science. 2017;8:86. DOI: 10.3389/fpls.2017.00086

modified crops. Environmental Chemistry Letters. 2015;13:239-249

Recorder. 2016;XXXIV(1)

10 Past, Present and Future Trends in Cotton Breeding

Conventional tools induce mutations randomly throughout the cotton genome—making breeding difficult and challenging. During the last decade, progress has been made to edit the gene of interest in a very precise manner. Targeted genome engineering with engineered nucleases (ENs) specifically zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases (e.g., Cas9) has been described as a "game-changing technology" for diverse fields as human genetics and plant biotechnology. In eukaryotic systems, ENs create double-strand breaks (DSBs) at the targeted DNA sequence which are repaired by nonhomologous end joining (NHEJ) or homologydirected recombination (HDR) mechanisms. ENs have been used successfully for targeted mutagenesis, gene knockout, and multisite genome editing (GenEd) in model plants and crop plants such as cotton, rice, and wheat. Recently, cotton genome has also been edited for targeted mutagenesis through CRISPR/Cas for improved lateral root formation. In addition, an efficient and fast method has been developed to evaluate guide RNAs transiently in cotton. The targeted disruption of undesirable genes or metabolic pathway can be achieved to increase quality of cotton. Undesirable metabolites like gossypol in cottonseed can be targeted efficiently using ENs for seed-specific low-gossypol cotton. Moreover, ENs are also helpful in gene stacking for herbicide resistance, insect resistance, and abiotic stress tolerance.

**Keywords:** TALENs, CRISPR/Cas, DSBs, NHEJ, HDR, cotton

#### **1. Introduction**

Cotton is an important source of natural fiber and has been playing a major role in economy and social structure of several countries. In addition, cotton serves as cash crop for more than

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

20 million farmers in Asia and Africa. Despite the availability of synthetic alternatives, cotton remains an important source of fiber because of the advantages related to cost of production and unique features offered by cotton lint. Consumption of cotton products in the world is increasing day by day with a lot of paces, but world cotton production is stagnant because of biotic and abiotic stresses. To meet the demands of the masses, production of cotton needs to be very high with good quality. Cotton is also affected by diseases, causing significant losses to industry. The most damaging diseases are Texas root rot, bacterial blight, blue disease, cotton leaf curl disease (CLCuD), and some strains of *Verticillium* and *Fusarium* wilt. Abiotic factors (heat, drought, salinity, and waterlogging) affect cotton yield, especially during early stages of plant development. Along with conventional breeding and genetic engineering, other novel techniques such as GenEd could be helpful for resistance development in cotton against biotic and abiotic stresses. GenEd tools have also been used for growth, quality, and yield enhancement in other crop plants. So, translation of this marvelous technology for improvement of fiber, quality, and yield of cotton would definitely have long-lasting benefits. In this chapter, we provide a picture of the use of GenEd tools for genetic improvement of cotton and other crop plants.

DNA template or donor DNA, the DSBs are repaired without errors in HDR fashion. HDR is

Targeted Genome Editing for Cotton Improvement http://dx.doi.org/10.5772/intechopen.73600 13

Reprograming and redesigning of artificial DNA-binding proteins and ENs have made GenEd quite an easy job. Most of the softwares are freely available online for the designing and cloning of ENs. Apart from ZFNs, TALENs, and CRISPR, other ENs such as homing endonucleases or meganucleases (DADGILAGLI) have also been used for targeted GenEd

The first targeted induction of DSBs was achieved using the natural meganuclease I-SceI, which has an 18-bp recognition site [30]. Experiments performed in tobacco using I-SceI to introduce chromosome breaks at integrated, defective reporter genes which, upon correction by homologous recombination, confer a selectable phenotype [30, 31]. Zinc fingers were fused with FokI nuclease to create artificial endonuclease for targeting predetermined DNA sites [32]. Zinc-finger nuclease-assisted gene targeting was first implemented in animal systems [33]. In the late 1990, ZFNs were designed and used for the first time to target genes of *Drosophila melanogaster* [34]. In case of ZFs, three DNA bases are targeted with one monomer. ZF monomers have been deciphered, and a table was built with possible combinations of three DNA bases to design ZFs against a DNA sequence (**Figure 1a**). Two efficient ZFN assembly platforms are available for successful designing and cloning of ZFNs: oligomerized pool engineering (OPEN) [35] and context dependent assembly (CoDA) [36]. Previously, modular assembly method was used to assemble multi-finger ZFN arrays, but the efficiency was reported low owing to inefficiency for context-depen-

Using two ZFN monomers results in DSB formation by a functional nuclease dimer, as initially shown for FokI endonuclease coupled to three ZFs636 recognizing 9-bp-binding sites [32, 37]. Induction of ZFN expression in *Arabidopsis* by heat shock during seedling development resulted in mutations at the ZFN recognition sequence. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFN-induced mutations [38]. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene, integrated at various chromosomal sites in ten different transgenic tobacco lines [39]. ZFN-mediated gene targeting at endogenous plant genes of tobacco acetolactate synthase genes (ALS SuRA and SuRB) was observed with high frequency exceeding 2% of transformed cells. Targeting of SuR loci resulted in resistance to

Co-expression of ZFNs with heterologous donor molecule led to precise targeted addition of an herbicide tolerance gene at the intended locus in maize. Mutant maize plants also transmitted genetic changes to further generation [41]. HDR-based gene replacement has been achieved successfully by replacing a 7-kb fragment flanked by two ZFN cutting sites with a 4-kb donor cassette, which integrates genes of kanamycin resistance and red fluorescent protein (RFP) [42]. In the last decade, artificial zinc-finger proteins (AZPs) have been used against begomoviruses (beet severe curly top virus (BSCTV) and tomato yellow leaf curl

imidazolinone and sulfonylurea herbicides with allelic mutations [40].

[29], but their applicability is very low compared to the above-mentioned nucleases.

an efficient pathway to make targeted insertions and/or gene corrections.

**2.1. Zinc-finger nucleases**

dent activity.

#### **2. GenEd tools for targeted genome modification**

Mutagenesis at target sites was a long-standing goal in the field of genome engineering and biotechnology. Along with chemical mutagens, transposons, recombinases, and TILLING technologies have been used historically to mutate certain genes for functional genomics and reverse genetic studies. The last decade has observed a revolution in the field of targeted genome modifications. GenEd has been found successful with equal efficiency in both plants and animals. Targeted genome modifications have modernized the field of genome engineering and biotechnology by GenEd from unicellular to multicellular and from prokaryotic to eukaryotic organisms.

GenEd tools like zinc-finger nucleases, transcription activator-like effectors, and CRISPR/Cas have been used massively for targeted genome modification. These GenEd reagents have the ability to search and bind specific DNA sequence and, hence, can be programmed to target any DNA sequence of choice. All of the ENs mentioned above have a catalytic ability to create double-strand breaks (DSBs) at the target DNA sequence. Zinc fingers and TALEs are fused with FokI nuclease domain to induce DSBs on dimerization, while CRISPR/Cas9 has its own catalytic activity with two nuclease domains: RuVC and HNH. DSBs at a predefined DNA sequence can be utilized efficiently for targeted genome modifications. DSBs in the DNA are repaired through cell endogenous repair systems: nonhomologous end joining (NHEJ) and homology-directed recombination. NHEJ is an error-prone repair mechanism in which DSBs are repaired with some insertions and/or deletions (Indels). On the provision of a homologous DNA template or donor DNA, the DSBs are repaired without errors in HDR fashion. HDR is an efficient pathway to make targeted insertions and/or gene corrections.

Reprograming and redesigning of artificial DNA-binding proteins and ENs have made GenEd quite an easy job. Most of the softwares are freely available online for the designing and cloning of ENs. Apart from ZFNs, TALENs, and CRISPR, other ENs such as homing endonucleases or meganucleases (DADGILAGLI) have also been used for targeted GenEd [29], but their applicability is very low compared to the above-mentioned nucleases.

#### **2.1. Zinc-finger nucleases**

20 million farmers in Asia and Africa. Despite the availability of synthetic alternatives, cotton remains an important source of fiber because of the advantages related to cost of production and unique features offered by cotton lint. Consumption of cotton products in the world is increasing day by day with a lot of paces, but world cotton production is stagnant because of biotic and abiotic stresses. To meet the demands of the masses, production of cotton needs to be very high with good quality. Cotton is also affected by diseases, causing significant losses to industry. The most damaging diseases are Texas root rot, bacterial blight, blue disease, cotton leaf curl disease (CLCuD), and some strains of *Verticillium* and *Fusarium* wilt. Abiotic factors (heat, drought, salinity, and waterlogging) affect cotton yield, especially during early stages of plant development. Along with conventional breeding and genetic engineering, other novel techniques such as GenEd could be helpful for resistance development in cotton against biotic and abiotic stresses. GenEd tools have also been used for growth, quality, and yield enhancement in other crop plants. So, translation of this marvelous technology for improvement of fiber, quality, and yield of cotton would definitely have long-lasting benefits. In this chapter, we provide a picture

of the use of GenEd tools for genetic improvement of cotton and other crop plants.

Mutagenesis at target sites was a long-standing goal in the field of genome engineering and biotechnology. Along with chemical mutagens, transposons, recombinases, and TILLING technologies have been used historically to mutate certain genes for functional genomics and reverse genetic studies. The last decade has observed a revolution in the field of targeted genome modifications. GenEd has been found successful with equal efficiency in both plants and animals. Targeted genome modifications have modernized the field of genome engineering and biotechnology by GenEd from unicellular to multicellular and from prokaryotic to eukaryotic organisms.  These GenEd reagents have the ability to search and bind specific DNA sequence and, hence, can be programmed to target any DNA sequence of choice. All of the ENs mentioned above have a catalytic ability to create double-strand breaks (DSBs) at the target DNA sequence. Zinc fingers and TALEs are fused with FokI nuclease domain to induce DSBs on dimerization, while CRISPR/Cas9 has its own catalytic activity with two nuclease domains: RuVC and HNH. DSBs at a predefined DNA sequence can be utilized efficiently for targeted genome modifications. DSBs in the DNA are repaired through cell endogenous repair systems: nonhomologous end joining (NHEJ) and homology-directed recombination. NHEJ is an error-prone repair mechanism in which DSBs are repaired with some insertions and/or deletions (Indels). On the provision of a homologous

**2. GenEd tools for targeted genome modification**

12 Past, Present and Future Trends in Cotton Breeding

cessfully with engineered proteins and nucleases.

The first targeted induction of DSBs was achieved using the natural meganuclease I-SceI, which has an 18-bp recognition site [30]. Experiments performed in tobacco using I-SceI to introduce chromosome breaks at integrated, defective reporter genes which, upon correction by homologous recombination, confer a selectable phenotype [30, 31]. Zinc fingers were fused with FokI nuclease to create artificial endonuclease for targeting predetermined DNA sites [32]. Zinc-finger nuclease-assisted gene targeting was first implemented in animal systems [33]. In the late 1990, ZFNs were designed and used for the first time to target genes of *Drosophila melanogaster* [34]. In case of ZFs, three DNA bases are targeted with one monomer. ZF monomers have been deciphered, and a table was built with possible combinations of three DNA bases to design ZFs against a DNA sequence (**Figure 1a**). Two efficient ZFN assembly platforms are available for successful designing and cloning of ZFNs: oligomerized pool engineering (OPEN) [35] and context dependent assembly (CoDA) [36]. Previously, modular assembly method was used to assemble multi-finger ZFN arrays, but the efficiency was reported low owing to inefficiency for context-dependent activity.

Using two ZFN monomers results in DSB formation by a functional nuclease dimer, as initially shown for FokI endonuclease coupled to three ZFs636 recognizing 9-bp-binding sites [32, 37]. Induction of ZFN expression in *Arabidopsis* by heat shock during seedling development resulted in mutations at the ZFN recognition sequence. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFN-induced mutations [38]. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene, integrated at various chromosomal sites in ten different transgenic tobacco lines [39]. ZFN-mediated gene targeting at endogenous plant genes of tobacco acetolactate synthase genes (ALS SuRA and SuRB) was observed with high frequency exceeding 2% of transformed cells. Targeting of SuR loci resulted in resistance to imidazolinone and sulfonylurea herbicides with allelic mutations [40].

Co-expression of ZFNs with heterologous donor molecule led to precise targeted addition of an herbicide tolerance gene at the intended locus in maize. Mutant maize plants also transmitted genetic changes to further generation [41]. HDR-based gene replacement has been achieved successfully by replacing a 7-kb fragment flanked by two ZFN cutting sites with a 4-kb donor cassette, which integrates genes of kanamycin resistance and red fluorescent protein (RFP) [42]. In the last decade, artificial zinc-finger proteins (AZPs) have been used against begomoviruses (beet severe curly top virus (BSCTV) and tomato yellow leaf curl

**2.2. TALEs and TALENs**

**Table 1.** ZFN-mediated genome modifications in plants.

**Table 2.** Genome editing in plants through TALEN technology.

TALE proteins are bacterial proteins (plant pathogens: genus *Xanthomonas*) and produced to bind DNA in the infected plant to hijack the expression system in a way that attenuate the disease process. Natural TALEs have a binding domain and an effector domain which binds DNA sequence and alter expression system of host, respectively. The binding domain consists of variable number of amino acid repeats in which each repeat contains

**Organism Gene Editing Reference** *Arabidopsis ADH1* NHEJ [1] Tobacco *EBE* of *Hax3* NHEJ [2] Rice *EBE* (*AvrXa7* and *PthXo3*) NHEJ [3] Potato *Vlnv* NHEJ [23] Wheat *MLO* NHEJ [26] *N. benthamiana FucT*, *XylT* NHEJ [54] Rice *OsSD1*, *OsBADH2* NHEJ [139] *Brachypodium BdABA1*, *BdSPL* NHEJ [139] Tobacco *SuRA*, *SuRB* NHEJ, HDR [140] Barley *PAPhy\_A* NHEJ [141] *Brassica oleracea FRIGIDA* NHEJ [142] Soybean *FAD2–1A*, *FAD2–1B* NHEJ [109] Barley *PAPHY-A* NHEJ [143] Rice *OsMST8*, *OsMST7*, *OsEPSPS* NHEJ [144] Maize *Glossy2 locus* NHEJ [145] *Arabidopsis CLV3* NHEJ [146]

**Sr. # Plant species Gene Gene modification Reference** Maize *IPK1* NHEJ [35] Tobacco *SuRA*, *SuRB* NHEJ [40] *Arabidopsis ADH1*, *TT4* NHEJ [41] *Petunia mGUS* NHEJ [132] Soybean DCL NHEJ [24] Tobacco *Kan*, *RFP* HDR [42]

Targeted Genome Editing for Cotton Improvement http://dx.doi.org/10.5772/intechopen.73600 15

**Figure 1.** Genome editing tools: (a) a pair of ZFNs with zinc-finger monomers and a pair of FokI nuclease to cut a dsDNA, (b) a pair of TALENs with TALE-binding repeats and FokI nuclease domains, and (c) CRISPR/Cas9 targeting dsDNA along with sgRNA and nuclease domains to create DSBs.

virus (TYLCV), respectively) [43, 44]. This strategy can be used for suppression of begomoviruses infecting cotton plants [45]. Moreover, ZFNs and AZPs can be useful for gene insertion, deletion, replacement, and functional genomics studies in cotton. Selected reports of ZFNmediated genome modification are given in **Table 1**.

**Table 1.** ZFN-mediated genome modifications in plants.

#### **2.2. TALEs and TALENs**

TALE proteins are bacterial proteins (plant pathogens: genus *Xanthomonas*) and produced to bind DNA in the infected plant to hijack the expression system in a way that attenuate the disease process. Natural TALEs have a binding domain and an effector domain which binds DNA sequence and alter expression system of host, respectively. The binding domain consists of variable number of amino acid repeats in which each repeat contains

**Table 2.** Genome editing in plants through TALEN technology.

virus (TYLCV), respectively) [43, 44]. This strategy can be used for suppression of begomoviruses infecting cotton plants [45]. Moreover, ZFNs and AZPs can be useful for gene insertion, deletion, replacement, and functional genomics studies in cotton. Selected reports of ZFN-

**Figure 1.** Genome editing tools: (a) a pair of ZFNs with zinc-finger monomers and a pair of FokI nuclease to cut a dsDNA, (b) a pair of TALENs with TALE-binding repeats and FokI nuclease domains, and (c) CRISPR/Cas9 targeting

mediated genome modification are given in **Table 1**.

dsDNA along with sgRNA and nuclease domains to create DSBs.

14 Past, Present and Future Trends in Cotton Breeding

33–35 amino acids and recognizes a DNA base pair. The DNA recognition is specifically modulated by two hypervariable amino acid residues (also called as repeat variable diresidues (RVDs)) at positions 12 and 13 in each repeat. Therefore, TALE repeats can be engineered by varying the RVDs to create a TALE protein that can bind a specific sequence in the genome (**Figure 1b**).

In case of TALEs and TALENs, the designing and assembly can be done with more ease and comfort. Owing to the single base-pair specificity of the TALE RVDs, modular assembly has been used frequently. Golden gate assembly of Cermak et al. has advantage of being fast, simple, and cost-effective [1]. Many free online softwares are available to design TALEs and TALENs [45]. The assembly of TALENs has also been offered on commercial basis by different companies, and many kits are available to construct TALENs against target sequence [45]. These TALE domains can be linked with a designed effector domain (nuclease like Fok1, repressor like KRAB, or activator like VP64) to create a chimeric protein capable of targeted genome manipulation. Successful genome modifications have been achieved using TALENs in different plant species (**Table 2**).

#### **2.3. CRISPR/Cas RNA-guided system**

CRISPR/Cas is an RNA-guided endonuclease (RGEN) system. RGENs are the easiest and simplest to design and clone. Cas9-gRNA is based on simple Watson and Crick base pairing of RNA-DNA, and 20-bp guide RNA is designed to target a DNA sequence of interest (**Figure 1c**). The efficiency of RNA-guided Cas9 system is remarkable to rewrite genomic sequence for genetic improvement of crops against different threats of multiple origins. Due to the ease of designing, simplicity in cloning, and cost-effectiveness, CRISPR/Cas is the most widely used EN.

cotton line with single copy of GFP gene incorporated previously [53]. Multiplexing ability of CRISPR/Cas system has given a distinction to this system. Multiplexed, targeted gene editing has been achieved in *Nicotiana benthamiana* for glycol engineering and monoclonal antibody production [54]. CRISPR/Cas system has been used efficiently for GenEd in plants (**Table 3**).

**Table 3.** Genome editing in plants through CRISPR/Cas9.

**Plant species Targeted gene Modification References** *Arabidopsis PDS3*, *FLS2*, *RACK1b*, *RACK1c* NHEJ [66] Barley, cabbage *HvPM19*, *BoIC.GA4.a* NHEJ [147] *Camelina FAD2* NHEJ [148, 149] *C. reinhardtii CpFTSY*, *ZEP* NHEJ [150] Cotton *GFP* (transgene), *CLA1*, *VP* NHEJ [28, 53] Dandelion *1-FFT* NHEJ [151] Flax *EPSPS*, *BFP (transgene)* NHEJ, HDR [152] Grape *IdnDH* NHEJ [22] Lettuce, *N. attenuata BIN2*, *AOC* NHEJ [128] Liverwort *ARF1* NHEJ [153] *Lotus japonicus SYMRK*, *LjLb1*, *LjLb2*, *LjLb3* NHEJ [154] Maize *IPK* NHEJ [25]

Targeted Genome Editing for Cotton Improvement http://dx.doi.org/10.5772/intechopen.73600 17

Specific DNA-binding proteins such as zinc fingers, TALEs, and dCas9 can be fused with different effector domains like activators, repressors, and epigenome modifiers to modulate gene expression (**Figure 2**). DSB created by ENs/RGEN can be used for different purposes (**Figure 3**). Controlled and tuneable expression of genes can be tremendously used for genetic improvement of plants. Modification of epigenetic marks can be further saved from regulation

**Figure 2.** Functional domain engineering of zinc fingers, TALEs and dCAs9, for different purposes. ZFs/TALE/dCas9 proteins can be engineered and fused with different effector/functional domains for targeted genome modifications. In this figure, different functional domains have been shown which can be fused with ZFs/TALE/dCas9 for creation of DSBs, gene insertion, gene activation, gene mutation, gene repression, gene stacking, and epigenetic modifications.

CRISPR/Cas has emerged as a new tool for targeting DNA using single-guide RNA (sgRNA), enabling genetic editing of any region in the genome [46, 47]. This single RNA-single protein CRISPR system is derived from a natural microbial adaptive immune system that uses RNAguided nuclease to recognize and cleave foreign DNA elements. This system consists of two components, a chimeric sgRNA and a CRISPR-associated protein (Cas9), which specifically unwinds and cleaves the target DNA, with the cleavage site dictated solely by complementarity to the sgRNA [48]. The only restriction in this system to target a DNA sequence is the presence of protospacer adjacent motif (PAM) region. CRISPR system has been proven to be incredibly valuable for site-specific genome engineering. Recently, in bacterial and human cells, nuclease's deactivated version of Cas9 protein called as dCas9 was created for programmable RNA-dependent DNA-binding protein [49]. Targeting nuclease-inactive Cas9 protein (dCas9) to coding region of a gene can block the binding and elongation of RNA polymerase, leading to dramatic suppression of transcription. Moreover, it has also been reported that dCas9 can also be modulated to recruit different protein effectors (activators or repressors) to DNA in a highly specific manner [50] to activate (CRISPRa) or suppress (CRISPRi) a gene. More recently, fusing dCas9 with Krüppel-associated box (KRAB) repressor domain resulted in an efficient transcriptional interference [50, 51]. In addition, CRISPRi was also used for multiplexed control of endogenous genes [52] and stable repression of genes with silencing efficiency typically achieved by RNAi while minimally impacting transcription of nontargeted genes. CRISPR/Cas9 has the efficiency to target the green fluorescent protein (GFP) gene within the genome of transgenic

**Table 3.** Genome editing in plants through CRISPR/Cas9.

33–35 amino acids and recognizes a DNA base pair. The DNA recognition is specifically modulated by two hypervariable amino acid residues (also called as repeat variable diresidues (RVDs)) at positions 12 and 13 in each repeat. Therefore, TALE repeats can be engineered by varying the RVDs to create a TALE protein that can bind a specific sequence

In case of TALEs and TALENs, the designing and assembly can be done with more ease and comfort. Owing to the single base-pair specificity of the TALE RVDs, modular assembly has been used frequently. Golden gate assembly of Cermak et al. has advantage of being fast, simple, and cost-effective [1]. Many free online softwares are available to design TALEs and TALENs [45]. The assembly of TALENs has also been offered on commercial basis by different companies, and many kits are available to construct TALENs against target sequence [45]. These TALE domains can be linked with a designed effector domain (nuclease like Fok1, repressor like KRAB, or activator like VP64) to create a chimeric protein capable of targeted genome manipulation. Successful genome modifications have been achieved using TALENs

CRISPR/Cas is an RNA-guided endonuclease (RGEN) system. RGENs are the easiest and simplest to design and clone. Cas9-gRNA is based on simple Watson and Crick base pairing of RNA-DNA, and 20-bp guide RNA is designed to target a DNA sequence of interest (**Figure 1c**). The efficiency of RNA-guided Cas9 system is remarkable to rewrite genomic sequence for genetic improvement of crops against different threats of multiple origins. Due to the ease of designing, simplicity in cloning, and cost-effectiveness, CRISPR/Cas is the most

CRISPR/Cas has emerged as a new tool for targeting DNA using single-guide RNA (sgRNA), enabling genetic editing of any region in the genome [46, 47]. This single RNA-single protein CRISPR system is derived from a natural microbial adaptive immune system that uses RNAguided nuclease to recognize and cleave foreign DNA elements. This system consists of two components, a chimeric sgRNA and a CRISPR-associated protein (Cas9), which specifically unwinds and cleaves the target DNA, with the cleavage site dictated solely by complementarity to the sgRNA [48]. The only restriction in this system to target a DNA sequence is the presence of protospacer adjacent motif (PAM) region. CRISPR system has been proven to be incredibly valuable for site-specific genome engineering. Recently, in bacterial and human cells, nuclease's deactivated version of Cas9 protein called as dCas9 was created for programmable RNA-dependent DNA-binding protein [49]. Targeting nuclease-inactive Cas9 protein (dCas9) to coding region of a gene can block the binding and elongation of RNA polymerase, leading to dramatic suppression of transcription. Moreover, it has also been reported that dCas9 can also be modulated to recruit different protein effectors (activators or repressors) to DNA in a highly specific manner [50] to activate (CRISPRa) or suppress (CRISPRi) a gene. More recently, fusing dCas9 with Krüppel-associated box (KRAB) repressor domain resulted in an efficient transcriptional interference [50, 51]. In addition, CRISPRi was also used for multiplexed control of endogenous genes [52] and stable repression of genes with silencing efficiency typically achieved by RNAi while minimally impacting transcription of nontargeted genes. CRISPR/Cas9 has the efficiency to target the green fluorescent protein (GFP) gene within the genome of transgenic

in the genome (**Figure 1b**).

16 Past, Present and Future Trends in Cotton Breeding

in different plant species (**Table 2**).

widely used EN.

**2.3. CRISPR/Cas RNA-guided system**

cotton line with single copy of GFP gene incorporated previously [53]. Multiplexing ability of CRISPR/Cas system has given a distinction to this system. Multiplexed, targeted gene editing has been achieved in *Nicotiana benthamiana* for glycol engineering and monoclonal antibody production [54]. CRISPR/Cas system has been used efficiently for GenEd in plants (**Table 3**).

Specific DNA-binding proteins such as zinc fingers, TALEs, and dCas9 can be fused with different effector domains like activators, repressors, and epigenome modifiers to modulate gene expression (**Figure 2**). DSB created by ENs/RGEN can be used for different purposes (**Figure 3**). Controlled and tuneable expression of genes can be tremendously used for genetic improvement of plants. Modification of epigenetic marks can be further saved from regulation

**Figure 2.** Functional domain engineering of zinc fingers, TALEs and dCAs9, for different purposes. ZFs/TALE/dCas9 proteins can be engineered and fused with different effector/functional domains for targeted genome modifications. In this figure, different functional domains have been shown which can be fused with ZFs/TALE/dCas9 for creation of DSBs, gene insertion, gene activation, gene mutation, gene repression, gene stacking, and epigenetic modifications.

to abiotic stress [55–57]. In response to abiotic stress, sometimes, plants may undergo wholegenome duplication events, and functional redundancy in multigene families may also be observed. Single-gene knockout often produces undesirable results/phenotypes making difficult to unravel the exact function. A comprehensive understanding of molecular basis of abiotic stresses (including drought, salinity, and heat) and their tolerance mechanisms have

Targeted Genome Editing for Cotton Improvement http://dx.doi.org/10.5772/intechopen.73600 19

A VIGS-mediated gene silencing of sucrose non-fermenting-1-related protein kinase 2 (GhSnRK2) mitigated drought tolerance in cotton plants, indicating that GhSnRPK2 positively conditions drought stress and low-temperature tolerance [58]. Moreover, RNAi of cotton PHYA1 genes improved drought, salt, and heat tolerance in transgenic plants, due to increased photosynthesis and better developed root systems [59]. This kind of genes can also be targeted for deletion with pair of ENs or RGENs. Moreover, ZFs, TALEs, and dCas9 can be

To increase the tolerance in cotton against drought stress, transcription factors are excellent candidates for the plant scientists. Various transcription factors (such as MYB, WRKY, ERF, NAC, bZIP) are involved in normal development as well as in drought stress response. These transcription factors have been cloned and proven useful for stress tolerance in cotton and/or in other plants. The genetic engineering of transcription factor genes could activate drought tolerance pathways and enhance drought tolerance in cotton. Recently, a bZIP transcription factor gene, GhABF2, has been reported in the drought and salt tolerance in *Arabidopsis* and cotton. The transcriptomic analysis revealed that GhABF2 regulates genes related to ABA. Overexpressing GhABF2 in cotton increased SOD and CAT activities as compared to wild-type plants. Moreover, overexpressed plants showed better results in the field, and meanwhile its yield was recorded higher than wild-type plants [60]. Stacking of these gene/transcription factors in best-growing cotton varieties with strong promoters could produce more resistant varieties. In another case, overexpressing GbMYB5 positively involved in response to drought stress in cotton and tobacco by reduced water loss from stomata and

Mitogen-activated protein kinases (MAPKs) are important signaling molecules that respond to drought stress. In a study, SlMAPK3 was induced by drought stress, and CRISPR/Cas9 system was utilized to generate SIMAPK3 mutants [62]. Field tests of transgenic maize plants with reduced ethylene biosynthesis by silencing 1-aminocyclopropane-1-carboxylic acid synthase 6 significantly improved grain yield under drought stress conditions [63]. Similarly, decreasing the sensitivity of maize to ethylene also resulted in higher yield [64]. Overexpression of ARGOS genes and negative regulators of the ethylene response enhances drought tolerance

Due to its simple design and efficient cloning of single or multiple gRNAs, CRISPR/Cas9 system using multiplex genome editing represents a promising and very powerful tool to specifically modulate the expression and activity of genes involved in abiotic stress responses. Multiplexing through CRISPR/Cas9 has been used successfully in model and crop plants [19, 66, 67]. Multiplex genome editing may also be useful for studying functions of gene families as well as an interaction between multiple genes. Multiple genes involved in stress regulatory network, signal transduction, and metabolite production may be simultaneously targeted

been one of the major goals of plant researchers to engineer stress tolerance in plants.

used for suppression of such genes at the transcriptional level.

showed hypersensitivity to ABA [61].

in transgenic maize plants [64, 65].

**Figure 3.** Targeted genome modifications through inducing DSBs. (a) Induction of DSB at the target site using one pair of ENs or one RGEN. DSB is further used to targeted mutations through NHEJ or gene insertion/correction by providing donor DNA. (b) Creation of DSBs using two ENs for targeted mutation, gene replacement/deletion, and chromosome inversion. (c) The use of paired nickases for gene correction and decreasing off-targeting. (d) Multiplexed Cas9 can produce DSBs at different sites for targeted mutagenesis, or induction of DSBs at two different chromosomes may lead to multiple gene disruption or chromosome translocation.

as GMOs. ZFNs, TALEs, and TALENs and Cas9, dCas9, and multiplexed Cas9 can be used efficiently for genetic improvement of cotton through gene deletion, insertion, replacement, correction, and modulation of expression.

#### **3. Use of GenEd tools against abiotic stresses in cotton**

Abiotic stress is a multigenic and complex trait. A substantial interaction between several components of signaling, regulatory, and metabolic pathways leads to response/adaptation to abiotic stress [55–57]. In response to abiotic stress, sometimes, plants may undergo wholegenome duplication events, and functional redundancy in multigene families may also be observed. Single-gene knockout often produces undesirable results/phenotypes making difficult to unravel the exact function. A comprehensive understanding of molecular basis of abiotic stresses (including drought, salinity, and heat) and their tolerance mechanisms have been one of the major goals of plant researchers to engineer stress tolerance in plants.

A VIGS-mediated gene silencing of sucrose non-fermenting-1-related protein kinase 2 (GhSnRK2) mitigated drought tolerance in cotton plants, indicating that GhSnRPK2 positively conditions drought stress and low-temperature tolerance [58]. Moreover, RNAi of cotton PHYA1 genes improved drought, salt, and heat tolerance in transgenic plants, due to increased photosynthesis and better developed root systems [59]. This kind of genes can also be targeted for deletion with pair of ENs or RGENs. Moreover, ZFs, TALEs, and dCas9 can be used for suppression of such genes at the transcriptional level.

To increase the tolerance in cotton against drought stress, transcription factors are excellent candidates for the plant scientists. Various transcription factors (such as MYB, WRKY, ERF, NAC, bZIP) are involved in normal development as well as in drought stress response. These transcription factors have been cloned and proven useful for stress tolerance in cotton and/or in other plants. The genetic engineering of transcription factor genes could activate drought tolerance pathways and enhance drought tolerance in cotton. Recently, a bZIP transcription factor gene, GhABF2, has been reported in the drought and salt tolerance in *Arabidopsis* and cotton. The transcriptomic analysis revealed that GhABF2 regulates genes related to ABA. Overexpressing GhABF2 in cotton increased SOD and CAT activities as compared to wild-type plants. Moreover, overexpressed plants showed better results in the field, and meanwhile its yield was recorded higher than wild-type plants [60]. Stacking of these gene/transcription factors in best-growing cotton varieties with strong promoters could produce more resistant varieties. In another case, overexpressing GbMYB5 positively involved in response to drought stress in cotton and tobacco by reduced water loss from stomata and showed hypersensitivity to ABA [61].

Mitogen-activated protein kinases (MAPKs) are important signaling molecules that respond to drought stress. In a study, SlMAPK3 was induced by drought stress, and CRISPR/Cas9 system was utilized to generate SIMAPK3 mutants [62]. Field tests of transgenic maize plants with reduced ethylene biosynthesis by silencing 1-aminocyclopropane-1-carboxylic acid synthase 6 significantly improved grain yield under drought stress conditions [63]. Similarly, decreasing the sensitivity of maize to ethylene also resulted in higher yield [64]. Overexpression of ARGOS genes and negative regulators of the ethylene response enhances drought tolerance in transgenic maize plants [64, 65].

as GMOs. ZFNs, TALEs, and TALENs and Cas9, dCas9, and multiplexed Cas9 can be used efficiently for genetic improvement of cotton through gene deletion, insertion, replacement,

**Figure 3.** Targeted genome modifications through inducing DSBs. (a) Induction of DSB at the target site using one pair of ENs or one RGEN. DSB is further used to targeted mutations through NHEJ or gene insertion/correction by providing donor DNA. (b) Creation of DSBs using two ENs for targeted mutation, gene replacement/deletion, and chromosome inversion. (c) The use of paired nickases for gene correction and decreasing off-targeting. (d) Multiplexed Cas9 can produce DSBs at different sites for targeted mutagenesis, or induction of DSBs at two different chromosomes may lead

Abiotic stress is a multigenic and complex trait. A substantial interaction between several components of signaling, regulatory, and metabolic pathways leads to response/adaptation

correction, and modulation of expression.

to multiple gene disruption or chromosome translocation.

18 Past, Present and Future Trends in Cotton Breeding

**3. Use of GenEd tools against abiotic stresses in cotton**

Due to its simple design and efficient cloning of single or multiple gRNAs, CRISPR/Cas9 system using multiplex genome editing represents a promising and very powerful tool to specifically modulate the expression and activity of genes involved in abiotic stress responses. Multiplexing through CRISPR/Cas9 has been used successfully in model and crop plants [19, 66, 67]. Multiplex genome editing may also be useful for studying functions of gene families as well as an interaction between multiple genes. Multiple genes involved in stress regulatory network, signal transduction, and metabolite production may be simultaneously targeted via CRISPR/Cas9 technologies for engineering stress tolerance in crop plants. An additional strategy could be pyramiding/stacking of multiple stress regulatory genes through HDRmediated gene targeting.

for controlling BeYDV [88], BSCTV [13], and TYLCV [12] with very few off-target activities, and these successful reports highlight the enormous potential of CRISPR/Cas system against geminiviruses. Due to inexpensive, simple, and rapid mechanism for triggering site-specific genome modifications, the programmable Cas9-gRNA system is potentially transforming next-generation genome-scale studies. The efficiency of RGEN system is remarkably high for crop improvement against potential threats of multiple origins (viral and bacterial diseases)

Targeted Genome Editing for Cotton Improvement http://dx.doi.org/10.5772/intechopen.73600 21

The strategy of targeting rep gene or rep protein-binding sites to occupy or disrupt the binding sites could be very fascinating using TALE and TALEN approach with high specificity. Recently, it has been demonstrated [89] that artificial TALE proteins could be a platform for broad-spectrum resistance against begomoviruses. Targeting viral DNA or host factors associated with pathogenesis of viral disease for disruption could be the possible strategies for virus suppression and disease resistance. There is a great possibility and progress in the idea of using TALEN and TALE repressors for antiviral gene therapy as well, to suppress potent viruses that cause global mortality and morbidity like HIV [90]. So far, different regions of viral genomes have been targeted to inhibit replication and to suppress viruses. As a result, decrease in titer of the virus by using ENs has been achieved by many researchers [13, 91].

DNA methylation is generally defined as an epigenetic mark of transcriptional gene silencing. Epigenetic regulation is although mysterious but can be modulated for a desirable change in the genome. Gene regulation without any change in DNA remained a challenge for years, but now factors have been deciphered which are responsible for epigenetic suppression or activation of genes. So, it has become possible with the help of engineered proteins to modulate gene expression of a gene epigenetically as well. So far, ZFs, TALEs, and CRISPR/Cas were dominantly used for this purpose [92–95], but recently TALEs and dCas9 have become available for this purpose. These proteins fused with different effector domains like 10–11 translocation methylcytosine dioxygenase 1 (TET1) [96], lysine-specific demethylase 1A (LSD1) [97], and methyltransferase which have been used as potential epigenome editors. ZFs fused with TET1 (ZF-TET1) were successfully used for demethylation purpose [96]. In addition, TET1 was used in demethylation of cytocine at CpG sites, and LSD1 has been used for demethyl-

DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Repeat regions, promoters, enhancers, and gene body are the main sites for DNA methylation in the genome. Epigenetic regulation also contributes in splicing. Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato [98]. It was revealed that DNA demethylation is required for tomato fruit ripening through both activation of induced genes and inhibition of ripening-repressed genes. DNA methylation controls many aspects of plant

**5. GenEd tools for epigenetic modifications in cotton**

ation of H3K4me1/2 and deacetylation of H3K27.

especially CLCuD.

#### **4. Use of GenEd tools against biotic stresses in cotton**

Conventional methods have been used for integrated pest management (IPM). Physical, chemical, and biological methods have been used for pest and disease management since domestication of crops. For insect resistance, the most widely used technology is *Bacillus thuringiensis* (Bt) technology. Through expression of Bt genes, *Cry* toxin, many insect-resistant crops have been developed [68]. Bt crops helped in decreasing insect attack and the use of pesticides and, hence, had done a good job for decreasing pollution as well. But unfortunately, resistance against Bt has been observed in certain parts of the world like resistance in pink bollworm in India. Apart from Bt technology, RNAi technology has also been used for insect resistance in crop plants. The first report of RNAi technology for cotton bollworm resistance was developed [69] by expression of dsDNA of insect-derived cytochrome P450 monooxygenase gene (dsCYP6AE14). Stacking of dsCYP6AE14 and plant cysteine proteases, such as GhCP1 from cotton (*Gossypium hirsutum*) and AtCP2 from *Arabidopsis*, can increase insect resistance in plants against cotton bollworms. In addition, stacking of new genes with old transgenic cotton varieties will further produce durable resistance against insects. Bt alternate transgenic approaches have also been used at the laboratory scale to develop new strategies of insect resistance in plants.

To counter the insect resistance against Bt crops, alternate strategies include expression of other toxins [70], engineering with proteases [71], proteinase inhibitors [72], receptor proteins [73, 74], and double-stranded RNA [75]. Among all these, dsRNA has been proposed as a method of choice and next-generation insecticide [75]. Moreover, expression of small dsRNA of CYP450 genes in transgenic plants to target vital bollworm functions has been reported as alternative to Bt applications [69]. Most recently, CRISPR/Cas9 was used to knock down a male-determining factor gene, *Nix*, in *Aedes aegypti* mosquitoes, leading to partial sex-change phenotypes [76]. The demonstration of using CRISPR/Cas for inhibition of mosquito-borne disease suggests that GenEd tools can also be translated for inhibition of other insect-borne diseases like whitefly that acts as vector for CLCV transmission to cause CLCuD.

Viral diseases are generally controlled by eliminating the vector population which transmits them. Scientists have been using conventional breeding [77, 78], pathogen-derived resistance [79–81], and nonpathogen-derived resistance [82, 83] to control the diseases. Most efforts were focused on silencing gene(s) of helper virus, but genes on satellite molecules were ignored. Such efforts proved effective but for a short period of time, and then virus relapsed because of multiple infections, synergistic effects, and evolution. A variety of multiplex genome engineering models in plants and animals are available either with expressing multiple gRNAs under single RNA Pol-III promoter [84, 85] or under different promoters at the same time [86, 87]. The CRISPR/Cas9 system has been successfully used for controlling BeYDV [88], BSCTV [13], and TYLCV [12] with very few off-target activities, and these successful reports highlight the enormous potential of CRISPR/Cas system against geminiviruses. Due to inexpensive, simple, and rapid mechanism for triggering site-specific genome modifications, the programmable Cas9-gRNA system is potentially transforming next-generation genome-scale studies. The efficiency of RGEN system is remarkably high for crop improvement against potential threats of multiple origins (viral and bacterial diseases) especially CLCuD.

The strategy of targeting rep gene or rep protein-binding sites to occupy or disrupt the binding sites could be very fascinating using TALE and TALEN approach with high specificity. Recently, it has been demonstrated [89] that artificial TALE proteins could be a platform for broad-spectrum resistance against begomoviruses. Targeting viral DNA or host factors associated with pathogenesis of viral disease for disruption could be the possible strategies for virus suppression and disease resistance. There is a great possibility and progress in the idea of using TALEN and TALE repressors for antiviral gene therapy as well, to suppress potent viruses that cause global mortality and morbidity like HIV [90]. So far, different regions of viral genomes have been targeted to inhibit replication and to suppress viruses. As a result, decrease in titer of the virus by using ENs has been achieved by many researchers [13, 91].

#### **5. GenEd tools for epigenetic modifications in cotton**

via CRISPR/Cas9 technologies for engineering stress tolerance in crop plants. An additional strategy could be pyramiding/stacking of multiple stress regulatory genes through HDR-

Conventional methods have been used for integrated pest management (IPM). Physical, chemical, and biological methods have been used for pest and disease management since domestication of crops. For insect resistance, the most widely used technology is *Bacillus thuringiensis* (Bt) technology. Through expression of Bt genes, *Cry* toxin, many insect-resistant crops have been developed [68]. Bt crops helped in decreasing insect attack and the use of pesticides and, hence, had done a good job for decreasing pollution as well. But unfortunately, resistance against Bt has been observed in certain parts of the world like resistance in pink bollworm in India. Apart from Bt technology, RNAi technology has also been used for insect resistance in crop plants. The first report of RNAi technology for cotton bollworm resistance was developed [69] by expression of dsDNA of insect-derived cytochrome P450 monooxygenase gene (dsCYP6AE14). Stacking of dsCYP6AE14 and plant cysteine proteases, such as GhCP1 from cotton (*Gossypium hirsutum*) and AtCP2 from *Arabidopsis*, can increase insect resistance in plants against cotton bollworms. In addition, stacking of new genes with old transgenic cotton varieties will further produce durable resistance against insects. Bt alternate transgenic approaches have also been used at the laboratory scale to develop new strategies

To counter the insect resistance against Bt crops, alternate strategies include expression of other toxins [70], engineering with proteases [71], proteinase inhibitors [72], receptor proteins [73, 74], and double-stranded RNA [75]. Among all these, dsRNA has been proposed as a method of choice and next-generation insecticide [75]. Moreover, expression of small dsRNA of CYP450 genes in transgenic plants to target vital bollworm functions has been reported as alternative to Bt applications [69]. Most recently, CRISPR/Cas9 was used to knock down a male-determining factor gene, *Nix*, in *Aedes aegypti* mosquitoes, leading to partial sex-change phenotypes [76]. The demonstration of using CRISPR/Cas for inhibition of mosquito-borne disease suggests that GenEd tools can also be translated for inhibition of other insect-borne

Viral diseases are generally controlled by eliminating the vector population which transmits them. Scientists have been using conventional breeding [77, 78], pathogen-derived resistance [79–81], and nonpathogen-derived resistance [82, 83] to control the diseases. Most efforts were focused on silencing gene(s) of helper virus, but genes on satellite molecules were ignored. Such efforts proved effective but for a short period of time, and then virus relapsed because of multiple infections, synergistic effects, and evolution. A variety of multiplex genome engineering models in plants and animals are available either with expressing multiple gRNAs under single RNA Pol-III promoter [84, 85] or under different promoters at the same time [86, 87]. The CRISPR/Cas9 system has been successfully used

diseases like whitefly that acts as vector for CLCV transmission to cause CLCuD.

**4. Use of GenEd tools against biotic stresses in cotton**

mediated gene targeting.

20 Past, Present and Future Trends in Cotton Breeding

of insect resistance in plants.

DNA methylation is generally defined as an epigenetic mark of transcriptional gene silencing. Epigenetic regulation is although mysterious but can be modulated for a desirable change in the genome. Gene regulation without any change in DNA remained a challenge for years, but now factors have been deciphered which are responsible for epigenetic suppression or activation of genes. So, it has become possible with the help of engineered proteins to modulate gene expression of a gene epigenetically as well. So far, ZFs, TALEs, and CRISPR/Cas were dominantly used for this purpose [92–95], but recently TALEs and dCas9 have become available for this purpose. These proteins fused with different effector domains like 10–11 translocation methylcytosine dioxygenase 1 (TET1) [96], lysine-specific demethylase 1A (LSD1) [97], and methyltransferase which have been used as potential epigenome editors. ZFs fused with TET1 (ZF-TET1) were successfully used for demethylation purpose [96]. In addition, TET1 was used in demethylation of cytocine at CpG sites, and LSD1 has been used for demethylation of H3K4me1/2 and deacetylation of H3K27.

DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Repeat regions, promoters, enhancers, and gene body are the main sites for DNA methylation in the genome. Epigenetic regulation also contributes in splicing. Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato [98]. It was revealed that DNA demethylation is required for tomato fruit ripening through both activation of induced genes and inhibition of ripening-repressed genes. DNA methylation controls many aspects of plant growth and development. TALE-LSD1 was used to modify methylation pattern of different sites, confirmed through chromatin immunoprecipitation [99]. Gao et al. have confirmed that for epigenome modifications, TALEs are more effective than dCas9 in mammalian cells. Moreover, they have evaluated TALE and dCas9, for gene activation and repression purpose, and highlight the use of designed transcription factors for epigenome modifications.

life and oxidative stability along with decrease in polyunsaturated fats. RNAi-based silencing of two key fatty acid desaturase genes, GhSAD-1 and GhFAD2-1, in cottonseeds significantly increased stearic and oleic acid contents in transgenic lines. In addition, palmitic acid contents were significantly low in both high-stearic and high-oleic transgenic cotton lines. These results provide an opportunity for nutritional improvement of cottonseed oil through genetic engineering [110]. Engineering of cotton in same manner through CRISPR/Cas9 or TALENs will improve

Targeted Genome Editing for Cotton Improvement http://dx.doi.org/10.5772/intechopen.73600 23

Cottonseeds contain high-quality protein and oil so it is also an important source of nutrient-rich food crop and edible oil. For every kilogram of fiber collected, about 1.65 kg of seeds are produced. Therefore, cotton can potentially provide the protein requirements of half a billion people if it could be used directly as food. However, cottonseeds are toxic for humans and other monogastric animals because of the presence of gossypol in the seed glands. Gossypol is a toxic terpenoid compound that causes heart and liver damage in human beings. Gossypol-free cottonseed may enhance the overall value of cottonseed and may generate a new market for cottonseed. Therefore, gossypol-free cottonseeds could provide protein requirement to poultry, aquaculture, and millions of humans worldwide. Gossypol is not only mainly localized in cottonseeds but also presents in other parts of cotton plant. In leaves and reproductive tissues of plant, gossypol and other related terpenoids play a protective role against insects, provoking infertility in insects. RNAi has been used successfully to reduce gossypol contents in cottonseeds by silencing (+)-δ-cadinene synthase which catalyzes the very first reaction involving the cyclization of farnesyl diphosphate to (+)-δ-cadinene. However, RNAi has several disadvantages like off-targets and reproducibility [111]. Thus, the promise of cottonseed to ensure food security and protein requirement of the developing countries like Pakistan remained unfulfilled. Recently, Ma et al. [112] have mapped a gene (*GoPGF*), acting as a positive regulator of formation of pigment glandular trichomes, storage organs of gossypol. Tissue-specific silencing of this gene will result in gossypol-free seeds while maintaining the level of secondary metabolites in the other parts of the plant [106]. Targeting dCas9 to regulatory region of a gene can block the binding and elongation of RNA polymerase, leading to dramatic suppression of transcription. Moreover, it has also been reported that dCas9 can also be modulated to recruit different protein effectors (activators or repressors) to DNA in a highly specific manner [50] to activate (CRISPRa) or suppress (CRISPRi) a gene. More recently, fusing TALEs and dCas9 with KRAB repressor domain resulted in an efficient transcriptional interference [51, 113, 114]. In addition, CRISPRi was also used for multiplexed control of endogenous genes [114] and stable repression of genes with silencing efficiency typically achieved by RNAi while minimally impact-

Flowering is a very critical developmental stage in cotton. All of the production depends on flowering. From emergence to drying up or falling off, it takes just 5–7 days. Flowering depends largely on temperature, availability of water, and other environmental conditions. Growth and development stages in cotton, from planting to emergence, from emergence to square, from square to flowering, and from flowering to boll development, are water sensitive. SELF-PRUNING 5G (SP5G) is a repressor of flowering in tomato and drives loss of day length sensitivity in flowering. CRISPR/Cas9-based mutation in SP5G resulted in

cottonseeds valuable for farmers and oilseed industry.

ing transcription of nontargeted genes.

Epigenetic modifications of chromatin at the DNA or histone level are considered to be one of the major forces that influence gene expression [100, 101]. Genome-wide changes in methylation patterns have been linked with physiological and developmental responses. Genetic imprinting in *Arabidopsis* endosperm and embryo was also driven by extensive demethylation of whole genome coupled with hypermethylation of non-CG residues especially CHH sites on transposable elements [102, 103]. In plants, genes, transposons, and repetitive sequences were found to be methylated in different densities at various developmental stages, which suggested that the transcription of certain genes is controlled epigenetically [104, 105]. Indeed, promoter DNA hypermethylation was related to target gene repression in undifferentiated *Arabidopsis* cells [106]. Jin et al. [107] reported that annual pattern of cytosine methylation drives fiber growth in cotton and moreover also studied the degree of CHH DNA methylation in the promoter regions of the growth-regulating genes SUR4, KCS13, and ERF6 on yearly basis.

However, potential application of TALEs for targeting DNA or histone for epigenome editing has been demonstrated, but more research is needed for development and validation of epigenetically modified crops/organism (EMO). About 500 genes have been identified that are epigenetically modified between wild cotton varieties and domesticated cotton, some of which are known to relate to agronomic and domestication traits. By selectively turning gene expression on and off, breeders could create new varieties of cotton without altering the genes.