Approximately, 10 million non-coding RNAs (ncRNAs) from fiber tissue of the allotetraploid cotton (*G. hirsutum*) were sequenced 7 days after flowering (DAF), to identify 24 nt ncRNA as the dominant species, followed by 21 nt ncRNA, and 23 nt ncRNA. This study further screened ~560 miRNA gene loci and suggested the role of miRNAs in elongation and secondary cell wall synthesis stages of cotton fiber development [105].

#### **8. Functional genomics and genome-editing technologies in regulation of fiber genes**

Genome modification (GM) and genome-editing technologies (GETs) are invaluable in the discovery of genes of interest, and support functional genomics of many organisms, including cotton. Various GM and GETs have been developed to investigate regulation mechanisms of genes. One of the most commonly used GM approaches is RNA interference (*RNAi*) that drew its historic support from antisense technology in the discovery of gene structures and the functions of organisms. *RNAi* is a new emerging technique based on homology-dependent post transcriptional gene silencing, induced by double-stranded RNA (dsRNA). Associations of many important genes with fiber development were detected using *RNAi* [3]. Recently, considerable work has been done highlighting the suitability of this method in cotton improvement [106–108]. For example, modifying or regulating flowering time is arduous in a plant improvement program, but it is sometimes a critical step to produce novel varieties with high yield that are better adapted to a specific environment.

In a collaborative project, scientists from Uzbekistan and USA developed cotton plants with early flowering, higher yield, and improved fiber qualities when *RNAi* technology was applied to regulate phytochrome gene [108] (**Figure 1**). Considering the potential of this research, scientists have patented this technology. A number of novel RNAi cultivars were successfully fielded trialed in over 60,000 hectares in Uzbekistan [3]. This is the first time a major crop is developed through the new RNAi technology and has been planted in such a large area.

A majority of RNAi published studies have focused on the functional aspects of cotton fiberrelated genes [3, 109]. Later, Wang et al. [110] characterized dihydroflavanol 4-reductase (*DFR*) enzyme that mediates the biosynthesis of two polyphenols (anthocyanins and proanthocyanidins (PAs)) in Upland cotton. In order to silence *GhDFR1* in cotton, *DFR* gene was cloned from developing fibers, and used for virus-induced gene silencing. The results show a significant decrease in accumulation of anthocyanins and PAs when *GhDFR1* is silenced. In addition, a high decrease of two PA monomers, (-)-epicatachin and (-)-epigallocatachin, occurred in *GhDFR1*-silenced plant fibers while two new monomers, (-)-catachin and (-)-gallocatachin, were present compared to control plants infected with the empty vector. *GhDFR1* contribution in the biosynthesis of anthocyanins and PAs in cotton has thus been confirmed [110].

gravitropism like a mutant of the auxin efflux carrier PIN-FORMED2 (*PIN2*) gene. Expression of the auxin uptake carrier *AUXIN1* (*AUX1*) showed its upregulation, while *PIN2* was downregulated in the *GhTCP14*-expressing plants. *GhTCP14* showed transcription activity by binding to the promoters of the *PIN2*, *IAA3*, and *AUX1* genes; these are auxin response genes that use electrophoretic mobility shift assays. All results demonstrated the potential regulation of *GhTCP14* gene in auxin-mediated differentiation and elongation of cotton fiber cells [111]. Overexpression of the actin-bundling protein *GhFIM2* was functionally characterized in cotton. The abundance of actin bundles is accompanied with accelerated fiber growth at the fast-elongating stage, and it increased when the *GhFIM2* gene was overexpressed. Secondary cell wall biogenesis also showed activation when the *GhFIM2* gene was overexpressed. These results indicated the importance of *GhFIM2* gene in the development of cotton fiber cells [112]. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated (*Cas*) 9 protein system have emerged as a simple and efficient tool for genome editing in eukaryotic cells. Most of the commercially grown cotton is tetraploid, and it is much more

(H) matured bolls with fiber in RNAi Cocker 312 and control Cocker 312 cotton plants.

**Figure 1.** Different stages on the application of RNAi technology silencing *Phytochrome A* gene in *Gossypium hirsutum* grown at Uzbekistan (unpublished information collected from the reference 108). (A) Vector designed to introduce RNAi *PHYA1*; (B) RNAi mechanism in the cell; (C) *Agrobacterium tumefaciens* mediated cotton transformation with pHellsgate-8-*PHYA1* RNAi vector and plant regeneration using somatic embryogenesis through tissue culture system; (D) PCR-based marker confirmation of *PHYA1* RNAi regenerated cotton plant; (E) RNAiPhyA cotton line produces longer improved fiber compared to the control Cocker 312 plant; (F) RNAi plant showed rapid growth compared to the control at the young stage; (G) the cotton bolls showed different shape in control versus RNAiPhyA plant in the field;

Recent Developments in Fiber Genomics of Tetraploid Cotton Species

http://dx.doi.org/10.5772/intechopen.72922

139

Overexpression of an important gene in an alien/host genome can be useful to detect gene functions and structure. The cellular functions of the class I of TEOSINTE-BRANCHED1/ CYCLOIDEA/PCF (TCP) transcription factor *GhTCP14*, from Upland, cotton were characterized by Wang et al. [111]. According to their work, the main expression of *GhTCP14* gene was detected in fiber cells at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Overexpression of *GhTCP14* in *Arabidopsis thaliana* enhanced initiation and elongation of trichomes and root hairs. Moreover, it affected root Recent Developments in Fiber Genomics of Tetraploid Cotton Species http://dx.doi.org/10.5772/intechopen.72922 139

as the dominant species, followed by 21 nt ncRNA, and 23 nt ncRNA. This study further screened ~560 miRNA gene loci and suggested the role of miRNAs in elongation and second-

Genome modification (GM) and genome-editing technologies (GETs) are invaluable in the discovery of genes of interest, and support functional genomics of many organisms, including cotton. Various GM and GETs have been developed to investigate regulation mechanisms of genes. One of the most commonly used GM approaches is RNA interference (*RNAi*) that drew its historic support from antisense technology in the discovery of gene structures and the functions of organisms. *RNAi* is a new emerging technique based on homology-dependent post transcriptional gene silencing, induced by double-stranded RNA (dsRNA). Associations of many important genes with fiber development were detected using *RNAi* [3]. Recently, considerable work has been done highlighting the suitability of this method in cotton improvement [106–108]. For example, modifying or regulating flowering time is arduous in a plant improvement program, but it is sometimes a critical step to produce novel varieties with high

In a collaborative project, scientists from Uzbekistan and USA developed cotton plants with early flowering, higher yield, and improved fiber qualities when *RNAi* technology was applied to regulate phytochrome gene [108] (**Figure 1**). Considering the potential of this research, scientists have patented this technology. A number of novel RNAi cultivars were successfully fielded trialed in over 60,000 hectares in Uzbekistan [3]. This is the first time a major crop is developed through the new RNAi technology and has been planted in such a large area.

A majority of RNAi published studies have focused on the functional aspects of cotton fiberrelated genes [3, 109]. Later, Wang et al. [110] characterized dihydroflavanol 4-reductase (*DFR*) enzyme that mediates the biosynthesis of two polyphenols (anthocyanins and proanthocyanidins (PAs)) in Upland cotton. In order to silence *GhDFR1* in cotton, *DFR* gene was cloned from developing fibers, and used for virus-induced gene silencing. The results show a significant decrease in accumulation of anthocyanins and PAs when *GhDFR1* is silenced. In addition, a high decrease of two PA monomers, (-)-epicatachin and (-)-epigallocatachin, occurred in *GhDFR1*-silenced plant fibers while two new monomers, (-)-catachin and (-)-gallocatachin, were present compared to control plants infected with the empty vector. *GhDFR1* contribution

in the biosynthesis of anthocyanins and PAs in cotton has thus been confirmed [110].

Overexpression of an important gene in an alien/host genome can be useful to detect gene functions and structure. The cellular functions of the class I of TEOSINTE-BRANCHED1/ CYCLOIDEA/PCF (TCP) transcription factor *GhTCP14*, from Upland, cotton were characterized by Wang et al. [111]. According to their work, the main expression of *GhTCP14* gene was detected in fiber cells at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Overexpression of *GhTCP14* in *Arabidopsis thaliana* enhanced initiation and elongation of trichomes and root hairs. Moreover, it affected root

ary cell wall synthesis stages of cotton fiber development [105].

yield that are better adapted to a specific environment.

**regulation of fiber genes**

138 Past, Present and Future Trends in Cotton Breeding

**8. Functional genomics and genome-editing technologies in**

**Figure 1.** Different stages on the application of RNAi technology silencing *Phytochrome A* gene in *Gossypium hirsutum* grown at Uzbekistan (unpublished information collected from the reference 108). (A) Vector designed to introduce RNAi *PHYA1*; (B) RNAi mechanism in the cell; (C) *Agrobacterium tumefaciens* mediated cotton transformation with pHellsgate-8-*PHYA1* RNAi vector and plant regeneration using somatic embryogenesis through tissue culture system; (D) PCR-based marker confirmation of *PHYA1* RNAi regenerated cotton plant; (E) RNAiPhyA cotton line produces longer improved fiber compared to the control Cocker 312 plant; (F) RNAi plant showed rapid growth compared to the control at the young stage; (G) the cotton bolls showed different shape in control versus RNAiPhyA plant in the field; (H) matured bolls with fiber in RNAi Cocker 312 and control Cocker 312 cotton plants.

gravitropism like a mutant of the auxin efflux carrier PIN-FORMED2 (*PIN2*) gene. Expression of the auxin uptake carrier *AUXIN1* (*AUX1*) showed its upregulation, while *PIN2* was downregulated in the *GhTCP14*-expressing plants. *GhTCP14* showed transcription activity by binding to the promoters of the *PIN2*, *IAA3*, and *AUX1* genes; these are auxin response genes that use electrophoretic mobility shift assays. All results demonstrated the potential regulation of *GhTCP14* gene in auxin-mediated differentiation and elongation of cotton fiber cells [111]. Overexpression of the actin-bundling protein *GhFIM2* was functionally characterized in cotton. The abundance of actin bundles is accompanied with accelerated fiber growth at the fast-elongating stage, and it increased when the *GhFIM2* gene was overexpressed. Secondary cell wall biogenesis also showed activation when the *GhFIM2* gene was overexpressed. These results indicated the importance of *GhFIM2* gene in the development of cotton fiber cells [112].

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated (*Cas*) 9 protein system have emerged as a simple and efficient tool for genome editing in eukaryotic cells. Most of the commercially grown cotton is tetraploid, and it is much more difficult to target both sets of homologous alleles. In an initial effort to standardize CRISPR/ Cas9 in in the tetraploid cotton, a single copy gene, green fluorescent protein (GFP), has been utilized to determine the efficacy of the system in generating targeted mutations (indels) using three independent sgRNAs [113]. Literature analysis showed that application of novel generation GETs for cotton in general and fiber trait improvements in specific are in their very early stages and require more future attentions, coordinated efforts, and continuous investments.

**Author details**

Sukumar Saha3

Uzbekistan

**References**

Mirzakamol S. Ayubov1

Khurshida A. Ubaydullaeva1

\*, Tokhir M. Norov1

Alabama A and M University, Alabama, USA

10.1073/pnas.0605389103

10.3389/fpls.2016.00202

rer/2006/p73.pdf

\*Address all correspondence to<EMAIL_ADDRESS>
, Ibrokhim Y. Abdurakhmonov1

, Jack C. McCarty3

, Zabardast T. Buriev1

1 Center of Genomics and Bioinformatics, Academy Sciences of Uzbekistan, Tashkent,

2 Center for Molecular Biology, Department of Biological and Environmental Sciences,

[1] Bi C, Paterson AH, Wang X, Xu Y, Wu D, Qu Y, Jiang A, Ye Q, Ye N. Analysis of the complete mitochondrial genome sequence of the diploid cotton *Gossypium raimondii* by сomparative genomics approaches. BioMed Research International. 2016;**2016**:5040598

[2] Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proceedings of the National Academy of Sciences USA. 2006;**103**:18054-18059. DOI:

[3] Abdurakhmonov IY, Ayubov MS, Ubaydullaeva KA, Buriev ZT, Shermatov SE, Ruziboev HS, Shapulatov UM, et al. RNA interference for functional genomics and improvement of cotton (*Gossypium* sp.). Frontiers in Plant Science. 2016;**7**:202. DOI:

[4] Stable Global Stocks in 2017/18 [Internet]. 2017. Available from: https://www.icac.org/

[5] Arpat AB, Waugh M, Sullivan JP, Gonzales M, Frisch D, Main D, Wood T, Leslie A, Wing RA, Wilkins TA. Functional genomics of cell elongation in developing cotton fibers. Plant

[6] Desalegn ZN, Ratanadilok N, Kaveeta R. Correlation and heritability for yield and fiber quality parameters of Ethiopian cotton (*Gossypium hirsutum* L.) estimated from 15 (dial-

[7] Chee PW, Shen X, Lubbers EL, Paterson AH. Fine mapping for fiber length on chromosome 1 in cotton [Internet]. 2006. Available from: http://www.ugacotton.com/vault/

[8] Wendel JF. New world cottons contain old world cytoplasm. Proceedings of the National

Press-Release/2017/PR-30-17-Stable-Global-Stocks-in-2017-18

lel) crosses. Kasetsart Journal (Natural Science). 2009;**43**:1-11

Molecular Biology. 2004;**54**:911-929. DOI: 10.1007/s11103-004-0392-y

Academy of Sciences. 1989;**86**:4132-4136. DOI: 10.1073/pnas.86.11.4132

3 Genetics and Sustainable Agriculture Research, USDA-ARS, Mississippi, USA

, Dewayne D. Deng3

, Venkateswara R. Sripathi2

Recent Developments in Fiber Genomics of Tetraploid Cotton Species

, Shukhrat E. Shermatov1

,

141

,

and Johnie N. Jenkins3

http://dx.doi.org/10.5772/intechopen.72922

#### **9. Conclusion**

For all crops—cotton in particular—where limited genetic diversity exists among the agriculturally elite types, genetic improvements will depend on innovative exploitation of genetic resources, and efficient strategies that effectively utilize both conventional and advanced molecular technologies. Comprehensive information is needed from independent and diverse research to understand molecular and genetic mechanisms associated with fiber development and additional agronomic traits. A cotton fiber provides a single cell crucible to understand the mechanisms of primary and secondary cell wall synthesis. It facilitates not only the study of cotton fibers, but also helps for further understanding of how all plant cell walls grow in relation to cell division and cell elongation. The elongating fibers also exemplify a scheme for production, and utilization of complex biochemical substances are first synthesized and then transported beyond the metabolic confines of cell membrane in the so-called "outer-space." A cotton fiber cell can be studied in detail relatively away from the noise of metabolic activity within the "inner-space" where thousands (and soon to be tens of thousands) of functionally characterized genes participate in a spatial and temporal interplay of plant growth. These genes first promote the regulated cell growth in organ-specific manner, and later march the specialized fiber cells toward senescence and apoptosis. To study the complex but very interesting process of fiber development, the cotton research community has extensively applied the genetic tools from mapping to genome modification technologies through characterization of key genes and development of molecular markers. This enabled researchers to tag complex fiber QTLs, clone and characterize genes and breed novel superior fiber cultivars using MAS and GE technologies. However, functional genomics of fiber is still behind other crops in the utilization of new generation native GETs due to complexity and multi-allelic nature of fiber-related genes, which require well-planned cooperative research activities and larger investments.

#### **Acknowledgements**

We thank Academy of Sciences of Uzbekistan, and Committee for Coordination Science and Technology Development of Uzbekistan for basic science (FA-F5-T030), and several applied (FA-A6-T081, FA-A6-T085 and YF5-FА-Т-005) research grants. We greatly acknowledge the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA) – Agricultural Research Service (ARS) and U.S. Civilian Research and Development Foundation (CRDF) for international cooperative grants P121, P121B, and UZB-TA-2992, which were devoted to cotton microRNA research. We also thank Dr. Sharma for his efforts to review this manuscript.

#### **Author details**

difficult to target both sets of homologous alleles. In an initial effort to standardize CRISPR/ Cas9 in in the tetraploid cotton, a single copy gene, green fluorescent protein (GFP), has been utilized to determine the efficacy of the system in generating targeted mutations (indels) using three independent sgRNAs [113]. Literature analysis showed that application of novel generation GETs for cotton in general and fiber trait improvements in specific are in their very early stages and require more future attentions, coordinated efforts, and continuous investments.

For all crops—cotton in particular—where limited genetic diversity exists among the agriculturally elite types, genetic improvements will depend on innovative exploitation of genetic resources, and efficient strategies that effectively utilize both conventional and advanced molecular technologies. Comprehensive information is needed from independent and diverse research to understand molecular and genetic mechanisms associated with fiber development and additional agronomic traits. A cotton fiber provides a single cell crucible to understand the mechanisms of primary and secondary cell wall synthesis. It facilitates not only the study of cotton fibers, but also helps for further understanding of how all plant cell walls grow in relation to cell division and cell elongation. The elongating fibers also exemplify a scheme for production, and utilization of complex biochemical substances are first synthesized and then transported beyond the metabolic confines of cell membrane in the so-called "outer-space." A cotton fiber cell can be studied in detail relatively away from the noise of metabolic activity within the "inner-space" where thousands (and soon to be tens of thousands) of functionally characterized genes participate in a spatial and temporal interplay of plant growth. These genes first promote the regulated cell growth in organ-specific manner, and later march the specialized fiber cells toward senescence and apoptosis. To study the complex but very interesting process of fiber development, the cotton research community has extensively applied the genetic tools from mapping to genome modification technologies through characterization of key genes and development of molecular markers. This enabled researchers to tag complex fiber QTLs, clone and characterize genes and breed novel superior fiber cultivars using MAS and GE technologies. However, functional genomics of fiber is still behind other crops in the utilization of new generation native GETs due to complexity and multi-allelic nature of fiber-related genes, which

require well-planned cooperative research activities and larger investments.

We thank Academy of Sciences of Uzbekistan, and Committee for Coordination Science and Technology Development of Uzbekistan for basic science (FA-F5-T030), and several applied (FA-A6-T081, FA-A6-T085 and YF5-FА-Т-005) research grants. We greatly acknowledge the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA) – Agricultural Research Service (ARS) and U.S. Civilian Research and Development Foundation (CRDF) for international cooperative grants P121, P121B, and UZB-TA-2992, which were devoted to cotton microRNA research. We also thank Dr. Sharma

**9. Conclusion**

140 Past, Present and Future Trends in Cotton Breeding

**Acknowledgements**

for his efforts to review this manuscript.

Mirzakamol S. Ayubov1 , Ibrokhim Y. Abdurakhmonov1 , Venkateswara R. Sripathi2 , Sukumar Saha3 \*, Tokhir M. Norov1 , Zabardast T. Buriev1 , Shukhrat E. Shermatov1 , Khurshida A. Ubaydullaeva1 , Jack C. McCarty3 , Dewayne D. Deng3 and Johnie N. Jenkins3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Center of Genomics and Bioinformatics, Academy Sciences of Uzbekistan, Tashkent, Uzbekistan

2 Center for Molecular Biology, Department of Biological and Environmental Sciences, Alabama A and M University, Alabama, USA

3 Genetics and Sustainable Agriculture Research, USDA-ARS, Mississippi, USA

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s10681-017-1853-0

jxb/ers278

[126] Hu H, Wang M, Ding Y, Zhu S, Zhao G, Tu L, Zhang X. Transcriptomic repertoires depict the initiation of lint and fuzz fibers in cotton (*Gossypium hirsutum* L.). Plant Biotechnology Journal. 2017:1467-7652. DOI: 10.1111/pbi.12844

**Section 4**

**Transgenic Cotton and Biosafety Issues**

**Transgenic Cotton and Biosafety Issues**

[126] Hu H, Wang M, Ding Y, Zhu S, Zhao G, Tu L, Zhang X. Transcriptomic repertoires depict the initiation of lint and fuzz fibers in cotton (*Gossypium hirsutum* L.). Plant

[127] Thyssen GN, Fang DD, Turley RB, Florane CB, Li P, Mattison CP, Naoumkina M. A Gly65Val substitution in an actin, GhACT\_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants. Plant Journal. 2017;**90**:111-121. DOI:

[128] Naoumkina M, Bechere E, Fang DD, Thyssen GN, Florane CB. Genome-wide analysis of gene expression of EMS-induced short fiber mutant Ligon lintless-y (li y) in cotton (*Gossypium hirsutum* L.). Genomics. 2017;**109**:320-329. DOI: 10.1016/j.ygeno.2017.05.007

[129] Ma Q, Wu M, Pei W, Wang X, Zhai H, Wang W, et al. RNA-Seq-mediated transcriptome analysis of a fiberless mutant cotton and its possible origin based on SNP markers.

[130] Hinchliffe DJ, Condon BD, Thyssen G, et al. The GhTT2\_A07 gene is linked to the brown colour and natural flame retardancy phenotypes of Lc1 cotton (*Gossypium hirsutum* L.) fibres. Journal of Experimental Botany. 2016;**67**:5461-5471. DOI: 10.1093/jxb/erw312 [131] Zou C, Wang Q, Lu C, Yang W, Zhang Y, Cheng H, Feng X, Prosper MA, Song G. Transcriptome analysis reveals long noncoding RNAs involved in fiber development in cotton (*Gossypium arboreum*). Science China Life Sciences. 2016;**59**:164-171. DOI:

Biotechnology Journal. 2017:1467-7652. DOI: 10.1111/pbi.12844

PLoS One. 2016;**11**:e0151994. DOI: 10.1371/journal.pone.0151994

10.1111/tpj.13477

152 Past, Present and Future Trends in Cotton Breeding

10.1007/s11427-016-5000-2

**Chapter 7**

**Provisional chapter**

**Transgenic Bt Cotton: Effects on Target and Non-Target**

Occurrence of diversity in ecosystem sustains particular characteristic of a biological community and also ensures stability of the community. Transgenic crops may affect insect biodiversity by unintended impacts on non-target arthropod population. For example, transgenic GM cotton specific to target lepidopterous pests can change the cotton pest spectrum and may induce the growth of new harmful pest species having no pest status. The change in species composition may influence IPM approach in cotton crop. The results of authors' research studies as well as global impact indicate that GM cotton is highly specific to target pests and has no unintended impact on non-target insect population. GM cotton provides significant season-long field control of target pests (*Helicoverpa armigera*, *Earias* spp. and *Pectinophora gossypiella*), with no significant control of *Spodoptera* species. The decreased insecticide use in GM cotton has a positive impact on beneficial insect populations and can increase the stability of rare species.  As GM cotton has no adverse effects on the non-target insect population and can reduce the use of broad-spectrum insecticides, it can become an important tool of IPM program in cotton agro-ecosystem

**Transgenic Bt Cotton: Effects on Target and Non-Target**

DOI: 10.5772/intechopen.73182

© 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,

© 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.

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

Cotton plant has been genetically modified to incorporate gene conferring insecticidal protein (Cry1Ac) derived from the naturally occurring soil bacterium *Bacillus thuringiensis* (Bt) var.

**Keywords:** target insects, non-target insects, diversity, GM cotton, Pakistan

**Insect Diversity**

and Waseem Akbar

**Abstract**

of Pakistan.

**1. Introduction**

**1.1. Transgenic Bt cotton**

**Insect Diversity**

Muhammad Arshad, Rashad Rasool Khan,

Muhammad Arshad, Rashad Rasool Khan, Asad Aslam

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Asad Aslam and Waseem Akbar

http://dx.doi.org/10.5772/intechopen.73182

**Provisional chapter**

#### **Transgenic Bt Cotton: Effects on Target and Non-Target Insect Diversity Insect Diversity**

**Transgenic Bt Cotton: Effects on Target and Non-Target**

DOI: 10.5772/intechopen.73182

Muhammad Arshad, Rashad Rasool Khan, Asad Aslam and Waseem Akbar and Waseem Akbar Additional information is available at the end of the chapter

Muhammad Arshad, Rashad Rasool Khan, Asad Aslam

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73182

#### **Abstract**

Occurrence of diversity in ecosystem sustains particular characteristic of a biological community and also ensures stability of the community. Transgenic crops may affect insect biodiversity by unintended impacts on non-target arthropod population. For example, transgenic GM cotton specific to target lepidopterous pests can change the cotton pest spectrum and may induce the growth of new harmful pest species having no pest status. The change in species composition may influence IPM approach in cotton crop. The results of authors' research studies as well as global impact indicate that GM cotton is highly specific to target pests and has no unintended impact on non-target insect population. GM cotton provides significant season-long field control of target pests (*Helicoverpa armigera*, *Earias* spp. and *Pectinophora gossypiella*), with no significant control of *Spodoptera* species. The decreased insecticide use in GM cotton has a positive impact on beneficial insect populations and can increase the stability of rare species.  As GM cotton has no adverse effects on the non-target insect population and can reduce the use of broad-spectrum insecticides, it can become an important tool of IPM program in cotton agro-ecosystem of Pakistan.

**Keywords:** target insects, non-target insects, diversity, GM cotton, Pakistan

#### **1. Introduction**

#### **1.1. Transgenic Bt cotton**

Cotton plant has been genetically modified to incorporate gene conferring insecticidal protein (Cry1Ac) derived from the naturally occurring soil bacterium *Bacillus thuringiensis* (Bt) var.

> 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

Kurstaki. Genes that express the delta-endotoxins are called "cry genes [1]. In lepidopterans, the chewing mouthparts promote the ingestion of Bt toxins and the crystals are solubilized in the midgut having alkaline environment (pH 9 to 12). The crucial step in the activation of crystal proteins is the cleavage of toxins which may vary in different insect species [2]. Larvae stop feeding after Bt toxin ingestion due to the onset of paralysis in midgut, altered permeability and disintegration of the epithelium that leads starvation to death of the insect within 2–3 days after exposure. The larval death may vary depending on insect species, larval age and the amount of toxin ingested [3].

**1.3. Status of GM cotton in Pakistan**

Bt cotton seed (**Table 1**).

Adoption of Bt-cotton in Pakistan was not fast than that of the other major cotton growing countries. The cultivation of Bt-cotton in Pakistan started upon the release of Bt-cotton candidate lines (IR-NIBGE-2, IR-FH-901, IR-CIM-443 and IR-CIM-448, developed by NIBGE Faisalabad) in 2003–2004 for testing their performance in various localities of Pakistan. Later on these varieties started capturing area each year. In 2005–2006, area under these varieties was 0.20 million ha, of which 0.093 million ha was in the cotton belt of Punjab Province [45]. In 2009, Ministry of Food and Agriculture made a positive development for the introduction of Bt cotton varieties in the country to maximize cotton production and for this purpose a letter of intent was signed with Monsanto company, but process was delayed. During the meantime, these cotton varieties including IR-NIBGE-2 (approved as IR-NIBGE-1524), IR-FH-901 (approved as IR-NIBGE-901), IR-CIM-448 (approved as IR-NIBGE-3701) and Bt-121 acquired >40% of the total cultivated area of cotton in both the province (Sindh and Punjab). Later on these cotton varieties along with some new varieties were approved by the Punjab Seed Council (PSC) on March 31, 2010 to counteract the cultivation of adulterated and unapproved

Transgenic Bt Cotton: Effects on Target and Non-Target Insect Diversity

http://dx.doi.org/10.5772/intechopen.73182

157

Later on some more Bt cotton varieties were approved for commercialization but all these varieties contain a single Cry1Ac toxic gene. In 2014, Bt cotton was grown an area of 2.9 million ha indicating an adoption rate of 88% in the country. Of the approved 32 Bt cotton varieties, half were developed by private seed companies and half by public sector research institutes. It was estimated that about 700,000 resource poor and small farmers were benefited from Bt cotton cultivation. The economic benefits achieved from Bt cotton cultivation was US\$1615 million for 2010–2013 [46]. However, the productivity of cotton in Pakistan is low (0.5 tons/ha) as compared to other Bt cotton growing countries. The agricultural productivity can be enhanced by increased adoption of Bt cotton, which would considerably

**Sr. # Variety/lines Center of release Year of cultivation and approval** 1 IR-NIBGE-3701 NIBGE Faisalabad, Pakistan Released for testing at farmer fields in

2 IR-NIBGE-901 NIBGE Faisalabad, Pakistan Released for testing at farmer fields in

3 NS-121 Neelum Seed, Multan, Pakistan Released in 2006, approved in 2010

4 MNH-886 Cotton Research Institute, Multan,

5 FH-142 Cotton Research Institute, AARI

6 IUB-2013 Islamia University Bahawalpur,

Pakistan

Pakistan

Faisalabad, Pakistan

**Table 1.** The most popular Bt-cotton varieties (covered at least 10% area in any province) of Pakistan.

2003–2004 but approved in 2010 for Punjab, and in 2011 for Sindh

2003–2004 but approved in 2011 for

Sindh

Approved in 2012

Approved in 2013

Approved in 2014

Monsanto developed and commercialized the first insect-resistant transgenic GM cotton expressing Cry1Ac gene (Bollgard® I) in 1996 [4]. GM cotton, the first transgenic non-food crop, has provided a specific, safe and effective tool for the control of lepidopterous pests [5–8] as compared to insecticides (pyrethroids and carbamates) that adversely affect nontarget arthropods and other invertebrates [9].

Transgenic Bt cotton has provided an important tool for developing an integrated pest management (IPM) strategy [10, 11], especially for lepidopterous larvae in cotton [12–15]. GM cotton expressing Cry genes is cultivated on 33.1 million ha in different cotton growing countries including United States [16, 17], China [18–20], India [21–26], South Africa [27–29], Mexico [30], Argentina [31, 32] and Pakistan [33–43] and experienced many benefits like reduced use of broad-spectrum insecticides, improved control of target pests, reduced production cost, increased yield and better opportunity for biological control.

The targeted pests have developed the resistance against Bollgard I in most of the countries. To overcome this issue, Monsanto has released Bt cotton containing two Bt genes Cry1Ac and Cry2Ab (Bollgard II). However, there are some other alternative means to minimize the development of resistance in target pests including: a) planting of refuge crop that does not contain Bt based product for susceptible target insect pests, b) consistent and high level of expression of Bt proteins in all plant structures, c) monitoring for shift in baseline susceptibility of target pests to Bt based products, d) use of other IPM control strategies (sowing time, new chemistry insecticides etc.) [43].

#### **1.2. Global status of GM cotton**

It is estimated that there is a rapid adoption of GM crops globally (up to 30 countries), and almost 18 million farmers have been grown these crops on more than 2 billion ha. GM crops have reflected substantial economic, health, environmental and social benefits to farmers by increasing crop productivity and conserving biodiversity [44].

GM cotton is being planted in USA since 1996 and it is estimated that 93% of total cotton area (3.98 million ha) is under cultivation of Biotech cotton. Biotech cotton is the third most important GM crop in Brazil and estimated to occupy 1.01 million ha in 2016/17. In India, farmers increased the cotton productivity by planting GM cotton on 11.2 million ha representing 96% of cotton area. Paraguay approved GM Cotton in 2011 for commercial production, and keeping in view the benefit of this technology, about 12,000 ha was planted up to 2015–2016. In Pakistan GM cotton is being cultivated on 2.9 million ha (97%) of the total 3 million ha of cotton area [44].

#### **1.3. Status of GM cotton in Pakistan**

Kurstaki. Genes that express the delta-endotoxins are called "cry genes [1]. In lepidopterans, the chewing mouthparts promote the ingestion of Bt toxins and the crystals are solubilized in the midgut having alkaline environment (pH 9 to 12). The crucial step in the activation of crystal proteins is the cleavage of toxins which may vary in different insect species [2]. Larvae stop feeding after Bt toxin ingestion due to the onset of paralysis in midgut, altered permeability and disintegration of the epithelium that leads starvation to death of the insect within 2–3 days after exposure. The larval death may vary depending on insect species, larval age

Monsanto developed and commercialized the first insect-resistant transgenic GM cotton expressing Cry1Ac gene (Bollgard® I) in 1996 [4]. GM cotton, the first transgenic non-food crop, has provided a specific, safe and effective tool for the control of lepidopterous pests [5–8] as compared to insecticides (pyrethroids and carbamates) that adversely affect non-

Transgenic Bt cotton has provided an important tool for developing an integrated pest management (IPM) strategy [10, 11], especially for lepidopterous larvae in cotton [12–15]. GM cotton expressing Cry genes is cultivated on 33.1 million ha in different cotton growing countries including United States [16, 17], China [18–20], India [21–26], South Africa [27–29], Mexico [30], Argentina [31, 32] and Pakistan [33–43] and experienced many benefits like reduced use of broad-spectrum insecticides, improved control of target pests, reduced production cost,

The targeted pests have developed the resistance against Bollgard I in most of the countries. To overcome this issue, Monsanto has released Bt cotton containing two Bt genes Cry1Ac and Cry2Ab (Bollgard II). However, there are some other alternative means to minimize the development of resistance in target pests including: a) planting of refuge crop that does not contain Bt based product for susceptible target insect pests, b) consistent and high level of expression of Bt proteins in all plant structures, c) monitoring for shift in baseline susceptibility of target pests to Bt based products, d) use of other IPM control strategies (sowing time, new chemistry

It is estimated that there is a rapid adoption of GM crops globally (up to 30 countries), and almost 18 million farmers have been grown these crops on more than 2 billion ha. GM crops have reflected substantial economic, health, environmental and social benefits to farmers by

GM cotton is being planted in USA since 1996 and it is estimated that 93% of total cotton area (3.98 million ha) is under cultivation of Biotech cotton. Biotech cotton is the third most important GM crop in Brazil and estimated to occupy 1.01 million ha in 2016/17. In India, farmers increased the cotton productivity by planting GM cotton on 11.2 million ha representing 96% of cotton area. Paraguay approved GM Cotton in 2011 for commercial production, and keeping in view the benefit of this technology, about 12,000 ha was planted up to 2015–2016. In Pakistan GM cotton is being cultivated on 2.9 million ha (97%) of the total 3 million ha of cotton area [44].

and the amount of toxin ingested [3].

156 Past, Present and Future Trends in Cotton Breeding

insecticides etc.) [43].

**1.2. Global status of GM cotton**

target arthropods and other invertebrates [9].

increased yield and better opportunity for biological control.

increasing crop productivity and conserving biodiversity [44].

Adoption of Bt-cotton in Pakistan was not fast than that of the other major cotton growing countries. The cultivation of Bt-cotton in Pakistan started upon the release of Bt-cotton candidate lines (IR-NIBGE-2, IR-FH-901, IR-CIM-443 and IR-CIM-448, developed by NIBGE Faisalabad) in 2003–2004 for testing their performance in various localities of Pakistan. Later on these varieties started capturing area each year. In 2005–2006, area under these varieties was 0.20 million ha, of which 0.093 million ha was in the cotton belt of Punjab Province [45]. In 2009, Ministry of Food and Agriculture made a positive development for the introduction of Bt cotton varieties in the country to maximize cotton production and for this purpose a letter of intent was signed with Monsanto company, but process was delayed. During the meantime, these cotton varieties including IR-NIBGE-2 (approved as IR-NIBGE-1524), IR-FH-901 (approved as IR-NIBGE-901), IR-CIM-448 (approved as IR-NIBGE-3701) and Bt-121 acquired >40% of the total cultivated area of cotton in both the province (Sindh and Punjab). Later on these cotton varieties along with some new varieties were approved by the Punjab Seed Council (PSC) on March 31, 2010 to counteract the cultivation of adulterated and unapproved Bt cotton seed (**Table 1**).

Later on some more Bt cotton varieties were approved for commercialization but all these varieties contain a single Cry1Ac toxic gene. In 2014, Bt cotton was grown an area of 2.9 million ha indicating an adoption rate of 88% in the country. Of the approved 32 Bt cotton varieties, half were developed by private seed companies and half by public sector research institutes. It was estimated that about 700,000 resource poor and small farmers were benefited from Bt cotton cultivation. The economic benefits achieved from Bt cotton cultivation was US\$1615 million for 2010–2013 [46]. However, the productivity of cotton in Pakistan is low (0.5 tons/ha) as compared to other Bt cotton growing countries. The agricultural productivity can be enhanced by increased adoption of Bt cotton, which would considerably

**Table 1.** The most popular Bt-cotton varieties (covered at least 10% area in any province) of Pakistan.

reduce insecticide applications, better quality of cotton, increased farm income, less exposure of insecticides to farmers and farm laborers and ultimate impact on food security efforts in the country.

*2.1.1. Cotton bollworm (Helicoverpa Armigera)*

between Bt and non-Bt cotton for oviposition [76].

*2.1.2. Pink bollworm (Pectinophora gossypiella)*

*2.1.3. Spotted bollworm (Earias spp.)*

Commonly known as cotton bollworm (CBW) is one of the damaging pests of cotton and many other field crops worldwide [8, 71–73]. In India, this pest causes an estimate crop loss of US \$350 million annually and farmers have to spray 15–20 times. Farmers in Pakistan also rely heavily on the use of chemical to control this pest and this indiscriminate use of insecticide particularly pyrethroids has developed resistance in this pest against insecticides [74, 75].

Transgenic Bt Cotton: Effects on Target and Non-Target Insect Diversity

http://dx.doi.org/10.5772/intechopen.73182

159

Our research studies have shown that transgenic Bt cotton offers great potential to significantly reduce the pesticide application for the control of major lepidopterous pest, *H. armigera* in Pakistan. The bollworm larval densities in Bt cotton remained below the threshold level; hence, no insecticide application is needed in Bt cotton. The results have shown no ovipositional differences between Bt and conventional cotton, as female moths cannot differentiate

Transgenic Bt cotton varieties have lethal effect against *H. armigera* [77–81] and proved to be very effective in controlling this pest, causing 80–90% mortality in Australia [70], more than 90% in China [82] and 40–50% in India [83]. However, some studies have showed inadequate control of *H. armigera* with Bt cotton [84]. Some studies have showed no oviposition difference of *H. armigera* between transgenic Bt and non-Bt cotton [85, 86]. While, other reported greater number of eggs in Bt cotton than conventional cotton because of better leaf canopy due to lower damage [48]. It is also observed that there is a variation in Bt cotton resistance throughout the growing season and has shown the higher resistance to *H. armigera* at the last 10 days of May (94.5%) and July (83.3%), which decreased in August (22.7%) [84]. Similarly, some other field research studies conducted in Pakistan [87] and somewhere else [88–91] have showed significantly lower population of *H. armigera* in Bt cotton as compared to non-Bt cotton.

It is the most important pest throughout the world, wherever the cotton is grown [92, 93] and almost difficult to control this pest because of its cryptic feeding habit. Bt cotton containing Cry1Ac can effectively control this pest [94–96]. Our research results indicated a lower density of rosette flowers and larvae in Bt cotton as compared to conventional cotton [97]. The study indicated that some larvae survived in Bt cotton, late in the season (end of September and October). It may be due to the decreased Bt toxin expression at lateral stage of plant [98]. However, it is admired that Bt cotton effectively suppressed the larval density in early season to an extent that pest could not cause an economic damage in the late season. Our results and those of other investigators support the efficacy of Bt cotton for pink bollworm control [99–101].

It is an important pest of cotton in Indo-Pak subcontinent and cause damage to fruiting bodies and shedding of squares, flowers and bolls [102, 103]. Although, the primary target of transgenic Bt cotton is to control cotton bollworm, *H. armigera* but it also has a significant impact on other bollworm species, including *Earias insulana* & *E. vittella*. It occurs as an early

#### **1.4. GM cotton and insecticide use**

Farmers rely heavily on the use of insecticides to control insect pests in cotton crop [47, 48]. This dependence on insecticides escalated the production cost. GM cotton containing Bt genes resulted in reduced application of pesticides for controlling the insect pests [8, 21, 49–51]. Insecticide application in Bt cotton has reduced up to 14 applications in China [52], 5-6 in Australia [53], 7 in South Africa [54] and 2.5 in India [55]. The introduction of Bt cotton in Southeast Asia significantly reduced the insecticide applications by 72%, increased yield of 11.4% and an estimated profit of US \$126.02/ha [56]. The reduced insecticide use may increase the predator abundance and can affect the arthropod communities overall in Bt cotton field [57–61].

#### **2. Diversity of insects on cotton crop**

Cotton crop hosts a rich diversity of insect pests, predators and parasitoids. About 145 insect and mite pests have been reported in the cotton crop in Pakistan [97]. Cotton insect pests cause 35–40% yield loss [62]. The insect pest complex on cotton is divided into two groups: chewing insect pests and sucking insect pests. Among the chewing insect pests, cotton bollworm complex (*Helicoverpa armigera* (Hubner), *Pectinophora gossypiella* (Saunders) and *Earias Spp.*) are the most destructive ones in Pakistan and causes 30–40% yield reduction [63], because of damage to flowers, squares and bolls [64, 65].

Farmers consider insecticides as a main sole to manage the insect pests in cotton crop. Most of the insecticides used, are broad-spectrum, which disturb the insect biodiversity, damage the beneficial insect fauna, hazardous to human health and environment, as well as leading to insect pests resurgence and outbreaks of secondary pests [68]. The insecticide application to cotton crop is the most intensive and the crop is to be considered as the largest insecticide consumer throughout the world [69]. It is estimated that in Pakistan, farmers spend US\$300 million on pesticides annually, of which more than 80% is used on cotton, especially for bollworms.

#### **2.1. Impact of GM cotton on target insect pests**

Among the target insect pests of Bt cotton *Helicoverpa armigera* Hubner, *Earias* spp., *Spodoptera* spp. (Lepidoptera: Noctuidae), *Pectinophora gossypiella* Saunder (Lepidoptera: Gelechidae) and *Spodoptera* spp. are more serious pests of cotton in Pakistan. They damage the cotton plant by feeding on squares, flowers and bolls and in severe damage caused significant yield reduction [70].

#### *2.1.1. Cotton bollworm (Helicoverpa Armigera)*

reduce insecticide applications, better quality of cotton, increased farm income, less exposure of insecticides to farmers and farm laborers and ultimate impact on food security efforts in

Farmers rely heavily on the use of insecticides to control insect pests in cotton crop [47, 48]. This dependence on insecticides escalated the production cost. GM cotton containing Bt genes resulted in reduced application of pesticides for controlling the insect pests [8, 21, 49–51]. Insecticide application in Bt cotton has reduced up to 14 applications in China [52], 5-6 in Australia [53], 7 in South Africa [54] and 2.5 in India [55]. The introduction of Bt cotton in Southeast Asia significantly reduced the insecticide applications by 72%, increased yield of 11.4% and an estimated profit of US \$126.02/ha [56]. The reduced insecticide use may increase the predator abundance and can affect the arthropod communities overall in

Cotton crop hosts a rich diversity of insect pests, predators and parasitoids. About 145 insect and mite pests have been reported in the cotton crop in Pakistan [97]. Cotton insect pests cause 35–40% yield loss [62]. The insect pest complex on cotton is divided into two groups: chewing insect pests and sucking insect pests. Among the chewing insect pests, cotton bollworm complex (*Helicoverpa armigera* (Hubner), *Pectinophora gossypiella* (Saunders) and *Earias Spp.*) are the most destructive ones in Pakistan and causes 30–40% yield reduction [63], because of damage to flowers, squares and bolls [64, 65]. Among sucking insect pests i.e., whitefly, jassid, thrips,

Farmers consider insecticides as a main sole to manage the insect pests in cotton crop. Most of the insecticides used, are broad-spectrum, which disturb the insect biodiversity, damage the beneficial insect fauna, hazardous to human health and environment, as well as leading to insect pests resurgence and outbreaks of secondary pests [68]. The insecticide application to cotton crop is the most intensive and the crop is to be considered as the largest insecticide consumer throughout the world [69]. It is estimated that in Pakistan, farmers spend US\$300 million on pesticides annually, of which more than 80% is used on cotton, especially

Among the target insect pests of Bt cotton *Helicoverpa armigera* Hubner, *Earias* spp., *Spodoptera* spp. (Lepidoptera: Noctuidae), *Pectinophora gossypiella* Saunder (Lepidoptera: Gelechidae) and *Spodoptera* spp. are more serious pests of cotton in Pakistan. They damage the cotton plant by feeding on squares, flowers and bolls and in severe damage caused significant yield

the country.

**1.4. GM cotton and insecticide use**

158 Past, Present and Future Trends in Cotton Breeding

**2. Diversity of insects on cotton crop**

aphid and cotton mealy bug are important [66, 67].

**2.1. Impact of GM cotton on target insect pests**

Bt cotton field [57–61].

for bollworms.

reduction [70].

Commonly known as cotton bollworm (CBW) is one of the damaging pests of cotton and many other field crops worldwide [8, 71–73]. In India, this pest causes an estimate crop loss of US \$350 million annually and farmers have to spray 15–20 times. Farmers in Pakistan also rely heavily on the use of chemical to control this pest and this indiscriminate use of insecticide particularly pyrethroids has developed resistance in this pest against insecticides [74, 75].

Our research studies have shown that transgenic Bt cotton offers great potential to significantly reduce the pesticide application for the control of major lepidopterous pest, *H. armigera* in Pakistan. The bollworm larval densities in Bt cotton remained below the threshold level; hence, no insecticide application is needed in Bt cotton. The results have shown no ovipositional differences between Bt and conventional cotton, as female moths cannot differentiate between Bt and non-Bt cotton for oviposition [76].

Transgenic Bt cotton varieties have lethal effect against *H. armigera* [77–81] and proved to be very effective in controlling this pest, causing 80–90% mortality in Australia [70], more than 90% in China [82] and 40–50% in India [83]. However, some studies have showed inadequate control of *H. armigera* with Bt cotton [84]. Some studies have showed no oviposition difference of *H. armigera* between transgenic Bt and non-Bt cotton [85, 86]. While, other reported greater number of eggs in Bt cotton than conventional cotton because of better leaf canopy due to lower damage [48]. It is also observed that there is a variation in Bt cotton resistance throughout the growing season and has shown the higher resistance to *H. armigera* at the last 10 days of May (94.5%) and July (83.3%), which decreased in August (22.7%) [84]. Similarly, some other field research studies conducted in Pakistan [87] and somewhere else [88–91] have showed significantly lower population of *H. armigera* in Bt cotton as compared to non-Bt cotton.

#### *2.1.2. Pink bollworm (Pectinophora gossypiella)*

It is the most important pest throughout the world, wherever the cotton is grown [92, 93] and almost difficult to control this pest because of its cryptic feeding habit. Bt cotton containing Cry1Ac can effectively control this pest [94–96]. Our research results indicated a lower density of rosette flowers and larvae in Bt cotton as compared to conventional cotton [97]. The study indicated that some larvae survived in Bt cotton, late in the season (end of September and October). It may be due to the decreased Bt toxin expression at lateral stage of plant [98]. However, it is admired that Bt cotton effectively suppressed the larval density in early season to an extent that pest could not cause an economic damage in the late season. Our results and those of other investigators support the efficacy of Bt cotton for pink bollworm control [99–101].

#### *2.1.3. Spotted bollworm (Earias spp.)*

It is an important pest of cotton in Indo-Pak subcontinent and cause damage to fruiting bodies and shedding of squares, flowers and bolls [102, 103]. Although, the primary target of transgenic Bt cotton is to control cotton bollworm, *H. armigera* but it also has a significant impact on other bollworm species, including *Earias insulana* & *E. vittella*. It occurs as an early to mid-season pest in cotton and hence transgenic Bt cotton can effectively control this pest during early-mid phase of the crop, when toxin expression is high. Baseline susceptibility data has shown that Cry1Ac was highly toxic to spotted bollworm with LC50 ranged from 0.006 to 0.105 μg/ml of diet and 0.88 ng/cm2 for leaf-dip bioassays [104].

pest complex [90]. Most of the research studies have showed the higher population of sucking insect pests mainly; jassid, whitefly, aphid and thrips in transgenic Bt cotton [85]. Some other research studies conducted in Pakistan [63] and India [48] have found no significant differences in sucking insect pests; whitefly, jassid and thrips population among transgenic Bt and non-Bt cotton. As Bt cotton has no resistance against sucking insect pests and requires continuous use of pesticides and other control tactics for effective management of

Transgenic Bt Cotton: Effects on Target and Non-Target Insect Diversity

http://dx.doi.org/10.5772/intechopen.73182

161

Seed treatment provided the better protection against early-season sucking pests in transgenic cotton. As, there is no direct impact of Bt toxin on the non-target insect species but the ingestion of Bt toxin may prolong the development time during which herbivores are more exposed to parasitoids and predators [129]. It is suggested that Bt cotton along with pesticide applications could provide protection against target and non-target insect pests. But for the long term implementation of Bt cotton as a component of IPM, it is important that such varieties should be transformed with Bt genes that have also the resistance against non-target suck-

Cotton crop hosts a rich diversity of insect predators and parasitoids, which have the significant role in regulating the pest population [130, 131]. Most of the field studies have shown no significant effects of Bt crops on natural enemies [40, 42, 60, 124, 129, 132–134]. Some reported the reduced activity of parasitoids in Bt cotton due to the absence of hosts or direct toxic

Bt cotton may act as a refuge for insect predators and spiders in large scale cotton production, where non-Bt cotton may be sprayed with insecticides [58]. Although Bt cotton is effective against target pests and have no direct influence on natural enemies [80] but there are the options that natural enemy population may be indirectly influenced by the behavioral change of non-target organisms or by the removal of their prey/hosts [124, 126, 137]. Some laboratory studies have reported indirect effects on natural enemies' population through unhealthy prey/hosts but at the same time population may be increased because of increased parasitism

Bt cotton can affect natural enemies in field by the removal of eggs, larvae and pupae of lepidopterous pests that serves as food sources [91]. Some studies showed the adverse effects of Bt toxin on the survival and development of some predators [109]. It may be due to the ingestion of Bt toxin during feeding on lepidopterous larvae or may be due to the consumption of intoxicated non-target prey that may pick up the Bt toxin [141]. While, most of the studies experienced no effect of Bt toxin on a main predator, *Chrysoperla carnea* [142] and reported no significant difference in abundance of insect predators between unsprayed Bt and non-Bt cotton fields [143]. The reduced insecticide use in Bt cotton can increase the predaceous arthropod population [144]. Some other field studies reported no significant difference of natural enemy populations between Bt and non-Bt cotton fields and where the differences were present, natural enemy populations were significantly higher in Bt than non-Bt cotton,

these insect pests [84, 105, 128].

effects of Bt toxin [86, 135, 136].

ing pests to reduce the number of pesticide applications.

*2.2.2. Impact of GM cotton on non-target natural enemies*

of unhealthy prey/host due to Bt toxin [124, 137–140].

mainly due to lower insecticide use in Bt cotton fields [145].

Bt cotton containing Cry1Ac proved to be effective against this pest and significantly control the larval population [78, 105, 106]. Another research study conducted in Pakistan investigated the infestation trend of spotted bollworm in different plant parts of transgenic Bt and conventional cotton cultivars and reported minimum infestation of 3.36% in transgenic variety, "IR-FH-901" as compared to conventional variety, "FH-900" with 10.5% infestation [65].

#### *2.1.4. Armyworm (Spodoptera spp.)*

Commonly known as beet armyworm and fall armyworm is a multivoltine, polyphagous pest and can cause significant damage to cotton crop in case of severe infestation. Bt cotton with Cry1Ac proved not to be effective against armyworm, *Spodoptera* spp. [65, 105, 107–110]; hence, no significant differences in larval density between Bt and non-Bt conventional cotton [111, 112] and insecticide applications are needed to control this pest in Bt cotton. In Pakistan Bt cotton varieties proved to be less affective against armyworm and less mortality (13.3–53.3%) noted on different Bt cotton varieties containing CriAc. Some other field studies have shown that there were no significant differences in larval density among Bt and non-Bt cotton [112, 113]. As Bt cotton varieties expressing single toxin gene (Cry1Ac) have no resistance against armyworm, *Spodoptera* species, to overcome this problem a Bollgard® II cotton was developed that contain Cry1Ac and Cry2Ab, which provide the adequate control of armyworm and cotton bollworms [114–123].

#### **2.2. Impact of GM cotton on non-target insect pests**

The potential impact of GM crops on non- target organisms is a strategic concern among farmers, policy makers and scientist working on the development of GM crops as an ideal pest control tactic. Non-target organisms include all organisms except for the pest to be controlled. Examples of non-target organisms would be birds, reptiles, mammals, fish and other insects. A number of studies have shown that Bt toxin is highly selective and has no adverse effects on non-target insect fauna in cotton [124–127].

#### *2.2.1. Impact of GM cotton on non-target major sucking insect pests*

Among the non-target, sucking insect pests of GM cotton, whitefly, jassid, thrips, aphid and cotton mealy bug are the most important in Pakistan. These are very destructive pests during seedling and vegetative phase of cotton as they suck the sap of the plant, make it weak and in case of severe infestation wilting and shedding of leaves occur.

The field research study indicated that transgenic Bt cotton proved to be very effective against certain chewing lepidopterous pests and reduced the insecticide applications [37].  Most of the research studies have showed the higher population of sucking insect pests mainly; jassid, whitefly, aphid and thrips in transgenic Bt cotton [85]. Some other research studies conducted in Pakistan [63] and India [48] have found no significant differences in sucking insect pests; whitefly, jassid and thrips population among transgenic Bt and non-Bt cotton. As Bt cotton has no resistance against sucking insect pests and requires continuous use of pesticides and other control tactics for effective management of these insect pests [84, 105, 128].

Seed treatment provided the better protection against early-season sucking pests in transgenic cotton. As, there is no direct impact of Bt toxin on the non-target insect species but the ingestion of Bt toxin may prolong the development time during which herbivores are more exposed to parasitoids and predators [129]. It is suggested that Bt cotton along with pesticide applications could provide protection against target and non-target insect pests. But for the long term implementation of Bt cotton as a component of IPM, it is important that such varieties should be transformed with Bt genes that have also the resistance against non-target sucking pests to reduce the number of pesticide applications.

#### *2.2.2. Impact of GM cotton on non-target natural enemies*

to mid-season pest in cotton and hence transgenic Bt cotton can effectively control this pest during early-mid phase of the crop, when toxin expression is high. Baseline susceptibility data has shown that Cry1Ac was highly toxic to spotted bollworm with LC50 ranged from 0.006 to

Bt cotton containing Cry1Ac proved to be effective against this pest and significantly control the larval population [78, 105, 106]. Another research study conducted in Pakistan investigated the infestation trend of spotted bollworm in different plant parts of transgenic Bt and conventional cotton cultivars and reported minimum infestation of 3.36% in transgenic variety, "IR-FH-901" as compared to conventional variety, "FH-900" with 10.5% infestation [65].

Commonly known as beet armyworm and fall armyworm is a multivoltine, polyphagous pest and can cause significant damage to cotton crop in case of severe infestation. Bt cotton with Cry1Ac proved not to be effective against armyworm, *Spodoptera* spp. [65, 105, 107–110]; hence, no significant differences in larval density between Bt and non-Bt conventional cotton [111, 112] and insecticide applications are needed to control this pest in Bt cotton. In Pakistan Bt cotton varieties proved to be less affective against armyworm and less mortality (13.3–53.3%) noted on different Bt cotton varieties containing CriAc. Some other field studies have shown that there were no significant differences in larval density among Bt and non-Bt cotton [112, 113]. As Bt cotton varieties expressing single toxin gene (Cry1Ac) have no resistance against armyworm, *Spodoptera* species, to overcome this problem a Bollgard® II cotton was developed that contain Cry1Ac and Cry2Ab, which provide the adequate control of armyworm and cotton bollworms

The potential impact of GM crops on non- target organisms is a strategic concern among farmers, policy makers and scientist working on the development of GM crops as an ideal pest control tactic. Non-target organisms include all organisms except for the pest to be controlled. Examples of non-target organisms would be birds, reptiles, mammals, fish and other insects. A number of studies have shown that Bt toxin is highly selective and has no adverse

Among the non-target, sucking insect pests of GM cotton, whitefly, jassid, thrips, aphid and cotton mealy bug are the most important in Pakistan. These are very destructive pests during seedling and vegetative phase of cotton as they suck the sap of the plant, make it weak and in

The field research study indicated that transgenic Bt cotton proved to be very effective against certain chewing lepidopterous pests and reduced the insecticide applications [37]. But at the same time, non-target sucking insect pests may become the significant insect pests, because the reduced use of insecticides in Bt cotton can increase the sucking insect

for leaf-dip bioassays [104].

0.105 μg/ml of diet and 0.88 ng/cm2

160 Past, Present and Future Trends in Cotton Breeding

*2.1.4. Armyworm (Spodoptera spp.)*

**2.2. Impact of GM cotton on non-target insect pests**

effects on non-target insect fauna in cotton [124–127].

*2.2.1. Impact of GM cotton on non-target major sucking insect pests*

case of severe infestation wilting and shedding of leaves occur.

[114–123].

Cotton crop hosts a rich diversity of insect predators and parasitoids, which have the significant role in regulating the pest population [130, 131]. Most of the field studies have shown no significant effects of Bt crops on natural enemies [40, 42, 60, 124, 129, 132–134]. Some reported the reduced activity of parasitoids in Bt cotton due to the absence of hosts or direct toxic effects of Bt toxin [86, 135, 136].

Bt cotton may act as a refuge for insect predators and spiders in large scale cotton production, where non-Bt cotton may be sprayed with insecticides [58]. Although Bt cotton is effective against target pests and have no direct influence on natural enemies [80] but there are the options that natural enemy population may be indirectly influenced by the behavioral change of non-target organisms or by the removal of their prey/hosts [124, 126, 137]. Some laboratory studies have reported indirect effects on natural enemies' population through unhealthy prey/hosts but at the same time population may be increased because of increased parasitism of unhealthy prey/host due to Bt toxin [124, 137–140].

Bt cotton can affect natural enemies in field by the removal of eggs, larvae and pupae of lepidopterous pests that serves as food sources [91]. Some studies showed the adverse effects of Bt toxin on the survival and development of some predators [109]. It may be due to the ingestion of Bt toxin during feeding on lepidopterous larvae or may be due to the consumption of intoxicated non-target prey that may pick up the Bt toxin [141]. While, most of the studies experienced no effect of Bt toxin on a main predator, *Chrysoperla carnea* [142] and reported no significant difference in abundance of insect predators between unsprayed Bt and non-Bt cotton fields [143]. The reduced insecticide use in Bt cotton can increase the predaceous arthropod population [144]. Some other field studies reported no significant difference of natural enemy populations between Bt and non-Bt cotton fields and where the differences were present, natural enemy populations were significantly higher in Bt than non-Bt cotton, mainly due to lower insecticide use in Bt cotton fields [145].

#### *2.2.3. Impact of GM cotton on the overall abundance and insect diversity*

Bt cotton can alters the insect diversity especially predators and parasitoids by reducing the abundance of *Helicoverpa* spp. and some other lepidopterous species [146–148]. A little numerical difference was found in the overall abundance and diversity of insect community in unsprayed Bt and conventional cotton fields [149], but another field study showed that species richness and total abundance reduced by 2.4–16.3 and 71.0–78.3%, respectively in Bt than non-Bt cotton fields [150]. Similarly, a three-year field studies have revealed no significant differences in species richness, evenness and diversity between unsprayed Bt and non Bt cotton, but plots receiving insecticides have slightly higher evenness.

to increased beneficial abundance and the change of pest status. The biotechnological efforts, in developing the transgenic Bt cotton varieties, should also focus on the sustainable temporal

, Asad Aslam1

and Waseem Akbar2

Transgenic Bt Cotton: Effects on Target and Non-Target Insect Diversity

http://dx.doi.org/10.5772/intechopen.73182

163

and intra-plant expression of Cry1Ac toxin in all plant parts.

\*, Rashad Rasool Khan1

2014;**5**:241-243. DOI: 10.1080/21645698.2014.997119

toxins. Annual Review of Entomology. 1992;**37**:615-636

1 Department of Entomology, University of Agriculture Faisalabad, Pakistan

2 Plant Protection Division, Nuclear Institute of Agriculture, Tando Jam, Hyderabad,

[1] Naglaa AA. The story behind Bt cotton: Where does Sudan stand? GM Crops & Food.

[2] Perlak FJ, Oppenhuizen M, Gustafson K, Voth R, Sivasupramaniam S, Heering D, Carey B, Ihrig RA, Roberts JK. Development and commercial use of Bollgard® cotton in the

[3] Gill SS, Cowles EA, Pietrantonio PV. The mode of action of *Bacillus thuringiensis* endo-

[4] Purcell JP, Perlak FJ. Global impact of insect-resistant (Bt) cotton. AgBioforum. 2004;

[5] Betz FS, Hammond BG, Fuchs RL. Safety and advantages of *Bacillus thuringiensis* protected plants to control insect pests. Regulatory Toxicology and Pharmacology. 2000;**32**:

[6] Shelton AM, Zhao JZ, Roush RT. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annual Review of Entomology.

[7] Mendelsohn M, Kough J, Vaituzis Z, Matthews K. Are Bt crops safe? Nature Bio-

[8] KM W, Guo YY. The evolution of cotton pest management practices in China. Annual

[9] KM W, Guo YY. Changes in susceptibility to conventional insecticides of a Cry1Acselected population of *Helicoverpa armigera* (Hubner) (Lepidoptera: Noctuidae). Pest

USA - early promises versus today's reality. The Plant Journal. 2001;**27**:489-501

\*Address all correspondence to<EMAIL_ADDRESS>
**Author details**

Pakistan

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Muhammad Arshad1

The reduced insecticide use in Bt cotton may increase the minor insect pests' community, which are suppressed under intense insecticide applications [92]. The mirid bugs, which were minor insect pests in northern China, now have attained the status of main pests and population has increased 12-folds mainly due to the Bt cotton cultivation on large scale [151].

However, Shannon's index for total arthropod community and the neutral arthropod guild found significantly higher in Bt cotton fields than those in non-Bt cotton [152]. A comparison of Shannon-Weaver diversity indices in Bt and non-Bt cotton under sprayed and unsprayed conditions revealed that Bt cotton increased the diversity of arthropod communities and pest subcommunities; however, it decreased the diversity of natural enemy sub-communities [153]. A comparison of canopy and ground dwelling arthropod community revealed no significant difference in the abundance of total insect community between unsprayed Bt and non-Bt cotton [134]. In addition, the relative greater abundance of honey bees; *Apis mellifera*, *A. cerana*, *A. dorsata* and other pollinators in Bt than non-Bt cotton, indicate that Bt cotton may be a good source of nectar and pollen for insect pollinators [152]. Similarly, some other field studies have revealed that Bt cotton increased the stability of insect community, pest and natural enemy sub-communities and found no significant effects on the non-target insect diversity [154, 155].

#### **3. Conclusions**

A plenty of insects inhabit the cotton crop, including the target and non-target insects. Transgenic Bt cotton has resistance against major target insect pests; *H. armigera, Earias* spp. & *P. gossypiella* and significantly reduce the insecticide applications. This reduction in pesticide use has a positive impact on natural enemies and increased the stability of beneficial rare species. Bt cotton varieties with Cry1Ac toxin are ineffective against armyworm, *Spodoptera* spp. However, some inhibitory effects of Bt toxin on the growth of armyworm larvae are observed but there is a chance that this pest may become the major and alarming pest in Bt cotton field in Pakistan. Bt cotton has no resistance against sucking insect pests; jassid, whitefly, thrips, aphid & mealybug and insecticides are used to control these pests. To increase the stability of Bt based products as an important tool of IPM in cotton, it is crucial that such varieties should be transformed with *Bt* toxin genes, which also have other resistance traits against non-target insect pests to reduce the number of insecticide applications. There is also need to re-determine the economic threshold levels for sucking pests and bollworms in Bt cotton due to increased beneficial abundance and the change of pest status. The biotechnological efforts, in developing the transgenic Bt cotton varieties, should also focus on the sustainable temporal and intra-plant expression of Cry1Ac toxin in all plant parts.

#### **Author details**

*2.2.3. Impact of GM cotton on the overall abundance and insect diversity*

162 Past, Present and Future Trends in Cotton Breeding

but plots receiving insecticides have slightly higher evenness.

**3. Conclusions**

Bt cotton can alters the insect diversity especially predators and parasitoids by reducing the abundance of *Helicoverpa* spp. and some other lepidopterous species [146–148]. A little numerical difference was found in the overall abundance and diversity of insect community in unsprayed Bt and conventional cotton fields [149], but another field study showed that species richness and total abundance reduced by 2.4–16.3 and 71.0–78.3%, respectively in Bt than non-Bt cotton fields [150]. Similarly, a three-year field studies have revealed no significant differences in species richness, evenness and diversity between unsprayed Bt and non Bt cotton,

The reduced insecticide use in Bt cotton may increase the minor insect pests' community, which are suppressed under intense insecticide applications [92]. The mirid bugs, which were minor insect pests in northern China, now have attained the status of main pests and population has increased 12-folds mainly due to the Bt cotton cultivation on large scale [151].

However, Shannon's index for total arthropod community and the neutral arthropod guild found significantly higher in Bt cotton fields than those in non-Bt cotton [152]. A comparison of Shannon-Weaver diversity indices in Bt and non-Bt cotton under sprayed and unsprayed conditions revealed that Bt cotton increased the diversity of arthropod communities and pest subcommunities; however, it decreased the diversity of natural enemy sub-communities [153]. A comparison of canopy and ground dwelling arthropod community revealed no significant difference in the abundance of total insect community between unsprayed Bt and non-Bt cotton [134]. In addition, the relative greater abundance of honey bees; *Apis mellifera*, *A. cerana*, *A. dorsata* and other pollinators in Bt than non-Bt cotton, indicate that Bt cotton may be a good source of nectar and pollen for insect pollinators [152]. Similarly, some other field studies have revealed that Bt cotton increased the stability of insect community, pest and natural enemy sub-communities and found no significant effects on the non-target insect diversity [154, 155].

A plenty of insects inhabit the cotton crop, including the target and non-target insects. Transgenic Bt cotton has resistance against major target insect pests; *H. armigera, Earias* spp. & *P. gossypiella* and significantly reduce the insecticide applications. This reduction in pesticide use has a positive impact on natural enemies and increased the stability of beneficial rare species. Bt cotton varieties with Cry1Ac toxin are ineffective against armyworm, *Spodoptera* spp. However, some inhibitory effects of Bt toxin on the growth of armyworm larvae are observed but there is a chance that this pest may become the major and alarming pest in Bt cotton field in Pakistan. Bt cotton has no resistance against sucking insect pests; jassid, whitefly, thrips, aphid & mealybug and insecticides are used to control these pests. To increase the stability of Bt based products as an important tool of IPM in cotton, it is crucial that such varieties should be transformed with *Bt* toxin genes, which also have other resistance traits against non-target insect pests to reduce the number of insecticide applications. There is also need to re-determine the economic threshold levels for sucking pests and bollworms in Bt cotton due Muhammad Arshad1 \*, Rashad Rasool Khan1 , Asad Aslam1 and Waseem Akbar2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Entomology, University of Agriculture Faisalabad, Pakistan

2 Plant Protection Division, Nuclear Institute of Agriculture, Tando Jam, Hyderabad, Pakistan

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