): an assessment Critical Reviws Chakravarthy.pdf& Jayabalan, 2011), herbicide tolerance (Rajasekaran et al., 1996b), early maturity (Xanthopoulos & Kechagia, ... thuringiensis into

1

IntroductionThe genus Gossypium comprises of 51 species, out of them 46 species are diploid (2n = 2x = 26) while five species are tetraploid (2n = 2x = 52) (Fryxell, 1992). Gossypium arbo-reum L. (2n = 2x = 26), G. herbaceum L. (2n = 2x = 26), G. hirsutum L. (2n = 2x = 52) and G. barbadense L. (2n = 2x = 52) are the four cultivated species and others are wild species (Sun et al., 2006). G. hirsutum, widely known as the upland cotton—which is grown in both tropical and temperate latitudes—has dominated the world cot-ton commerce accounting for ~90% of the cotton produc-tion (Niles & Feaster, 1984; Wendel et al., 1992). Whereas, G. barbadense, commonly known as extra-long-staple cot-ton or Pima or Egyptian cotton, accounts for ~8% of total world production (Lee, 1984; Wendel et al., 1992). Cotton (G. hirsutum), the world’s premier source of natural fiber and seed oil, is cultivated in 70 nations with an annual contribution of $500 billion, and provides livelihood for

more than 180 million people (John, 1997; Rahman et al., 2012). Furthermore, cotton and its by-products are used for the manufacture of mattress padding, special paper, plastics, toothpaste, salad oil, fertilizers etc. (Wilkins et al., 2000). Extracts of cotton plants have been used medicinally for treating hypertension (Hasrat et al., 2004). Gossypol present in the cotton tissues has been found to show anticancer activity, besides its use as a male contra-ceptive (Coutinho, 2002; Moon et al., 2008).

The growth and productivity of cotton crop is often hampered by various biotic as well as abiotic stress factors. Significant amounts of crop productivity are jeopardized by the occurrence of various biotic stress factors such as insect pests (37%), weeds (36%), and pathogens (9%) (Oerke, 2006). Adoption of methods like conventional breeding, mutagenesis, and somaclonal variation have resulted in the improvement of characteristics such as heat tolerance (Garay & Barrow, 1988; Trolinder &

REVIEW ARTICLE

Current status of genetic engineering in cotton (Gossypium hirsutum L): an assessment

Vajhala S. K. Chakravarthy, Tummala Papi Reddy, Vudem Dashavantha Reddy, and Khareedu Venkateswara Rao

Centre for Plant Molecular Biology, Osmania University, Hyderabad, 500007, India

AbstractCotton is considered as the foremost commercially important fiber crop and is deemed as the backbone of the textile industry. The productivity of cotton crop, worldwide, is severely hampered by the occurrence of pests, weeds, pathogens apart from various environmental factors. Several beneficial agronomic traits, viz., early maturity, improved fiber quality, heat tolerance, etc. have been successfully incorporated into cotton varieties employing conventional hybridization and mutation breeding. Crop losses, due to biotic factors, are substantial and may be reduced through certain crop protection strategies. In recent years, pioneering success has been achieved through the adoption of modern biotechnological approaches. Genetically engineered cotton varieties, expressing Bacillus thuringiensis cry genes, proved to be highly successful in controlling the bollworm complex. Various other candidate genes responsible for resistance to insect pests and pathogens, tolerance to major abiotic stress factors such as temperature, drought and salinity, have been introduced into cotton via genetic engineering methods to enhance the agronomic performance of cotton cultivars. Furthermore, genes for improving the seed oil quality and fiber characteristics have been identified and introduced into cotton cultivars. This review provides a brief overview of the various advancements made in cotton through genetic engineering approaches.Keywords: Abiotic stress, cotton improvement, fiber quality, genetic transformation, insect resistance, oil quality, pathogen resistance

Address for Correspondence: Venkateswara Rao Khareedu, Centre for Plant Molecular Biology, Osmania University, Hyderabad, 500007, India. Email: [email protected]

(Received 26 05 2012; revised 04 October 2012; accepted 22 October 2012)

Critical Reviews in Biotechnology, 2012; Early Online: 1–18© 2012 Informa Healthcare USA, Inc.ISSN 0738-8551 print/ISSN 1549-7801 onlineDOI: 10.3109/07388551.2012.743502

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Genetic engineering in cotton

V. S. K. Chakravarthy et al.

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Shang, 1991), fungal resistance (Ganesan & Jayabalan, 2006), fiber quality (Zhang et al., 2001; Muthusamy & Jayabalan, 2011), herbicide tolerance (Rajasekaran et al., 1996b), early maturity (Xanthopoulos & Kechagia, 2001; Kandhro et al., 2002), photo-insensitivity (Raut et al., 1971) besides the development of cytoplasmic-male sterility (Ngematov et al., 1975) and gland-less mutants (McMichael, 1959). However, incorporation of desirable traits employing these approaches is constrained by various evolutionary bottlenecks (John, 1997; Wilkins et al., 2000). Moreover, several decades of intensive breeding employing a limited number of genotypes has narrowed the gene pool available for further improvement of cotton (Gingle et al., 2006).

About 30% of cotton production is lost to pests despite the measures for controlling insect pests by the applica-tion of chemical pesticides (Oerke, 2006). Furthermore, the prolonged and inappropriate usage of broad-spectrum insecticides has resulted in the development of resistance by the insects leading to pest outbreaks (Kerns & Gaylor, 1993; Kranthi et al., 2002). Growing awareness of the prob-lems associated with indiscriminate pesticide usage and lack of desired traits in cotton germplasm has heightened the interest to rely upon and adopt the transgenic technol-ogy. Worldwide, transgenic cotton fortified with single or stacked genes conferring insect resistance and herbicide tolerance were planted in ~25 mh occupying >15% of the total area (160 mh) under transgenic crops. Adoption of insect-resistant Bt cotton varieties has significantly con-tributed toward increased earnings of US$250 per hectare and has reduced the money spent for application of insec-ticides up to 50% (James, 2011). In India, insect-resistant transgenic Bt hybrids are widely cultivated in 10.6 mh by ~7 million farmers which accounts for 88% of the area under cotton cultivation. Furthermore, India occupies the foremost position in terms of transgenic cotton cultivation followed by USA (4.0 mh), China (3.9 mh), and Pakistan (2.6 mh) (James, 2011).

Earlier reviews on cotton biotechnology primarily dealt with the regeneration and genetic transformation with special emphasis on insect resistance, herbicide tol-erance and fiber quality (John, 1997; Pannetier et al., 1997; Wilkins et al., 2000; Kumria et al., 2003; Showalter et al., 2009). Recent reviews mainly described the application of somatic embryogenesis for production of transgenic cotton plants (Obembe et al., 2011) and biotechnological advances made thus far for genetic improvement of cot-ton (Zhu et al., 2011). The present communication pro-vides a comprehensive overview on the methods of gene delivery systems employed and progress achieved in the development of genetically engineered cotton tolerant to herbicide, biotic and abiotic factors, as well as the efforts made to enhance the quality of fiber and seed oil.

Methods of cotton transformationDevelopment of genetically engineered transgenic cot-ton plants depends on the optimization of a suitable

procedure for the transfer and stable integration of trans-genes into the genome. Currently, Agrobacterium and particle bombardment-mediated genetic transformation of target tissues followed by regeneration of plants—either through somatic embryogenesis or organogen-esis—are widely used for the development of transgenic cotton. Regeneration of plants from transformed tissues of cotton via embryogenesis is restricted to a limited num-ber of cultivars and efforts made to increase the range of cultivars that can produce somatic embryos, have met with limited success (Wilkins et al., 2004; Sakhanokho et al., 2005). Problems such as prolonged culture periods resulting in the appearance of somaclonal variation, high frequency of abnormal embryo development, low con-version rate of somatic embryos into plantlets and high genotype-dependency have further restricted the appli-cation of this technique for production of transgenics (Stelly et al., 1989; Mishra et al., 2003; Wilkins et al., 2004; Sun et al., 2006). Somaclonal variations resulting from prolonged culture periods are undesirable when produc-tion of true-to-type transgenic plant is the ultimate objec-tive. Methods for direct regeneration of transformed shoots have some advantages of being more genotype-independent than somatic embryogenesis besides allow-ing the speedy recovery of transgenic plants (Wilkins et al., 2000; Divya et al., 2008). However, constraints like low rooting efficiency of the shoots and low frequency of stable germline transformants need to be overcome (Luo & Gould, 1999; Gould et al., 1991; Hemphill et al., 1998; John, 1997). Different transformation techniques avail-able have merits as well as certain limitations (Showalter et al., 2009). Well established gene transfer systems like Agrobacterium and particle bombardment-mediated genetic transformations along with certain other meth-ods of transforming cotton are briefly described in this section. A comprehensive list of reports on the genetic transformation of cotton is provided in Table 1.

Agrobacterium tumefaciens-mediated transformationThe mechanism of gene transfer between Agrobacterium and the plant kingdom has facilitated the insertion of beneficial alien genes into diverse plant genomes (Barton et al., 1983; John, 1997). Agrobacterium-mediated transformation has been the most widely used and pre-ferred method of transferring genes into plants (Wilkins et al., 2000). For the first time, successful Agrobacterium-mediated transformation and regeneration of trans-genic cotton plants from hypocotyls and cotyledons via somatic embryogenesis were reported by Firoozabady et al. (1987) and Umbeck et al. (1987). Later, Perlak et al. (1990) developed insect-resistant transgenic plants by introducing cry1A (b) and cry1A (c) genes of Bacillus thuringiensis into the cotton cv. Coker 312 employing the Agrobacterium strain A208. Transgenic cotton plants resistant to H. armigera and S. litura were developed by introducing cry genes through Agrobacterium-mediated genetic transformation using different explants, such as hypocotyl, cotyledon and embryogenic calli (Singh et al.,

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Genetic engineering in cotton 3


Table 1. Genetic transformation of cotton by different gene-transfer methods.

Variety/explant used Transgenes PromotersTransformation method Conclusion Reference

Coker 312/hypocotyl tfdA and nptII CaMV35S and Nos Agrobacterium Transgenic plants exhibited increased tolerance to herbicide 2,4-D (>1.5 kg/h).

Bayley et al., 1992

Coker 315/cotyledon tfdA, gusA, and npt II CaMV35S and Nos Agrobacterium Transgenic plants showed increased (50–100-fold) tolerance to herbicide 2,4-d.

Lyon et al., 1993

Coker 312/hypocotyl CP4-EPSPS and nptII CaMV35S and CMoVb

Agrobacterium Transgenic plants revealed tolerance to herbicide glyphosate.

Nida et al., 1996

DP50/embryos phaB, phaC, and gusA E6, FbL2A and CaMV35S

Particle bombardment

Fibers of transgenic plants exhibited better insulating characteristics.

John and Keller, 1996

DP50, E1Dorado, Coker 312 and PimaS6/seed axes

bar and gusA CaMV35S Particle bombardment

Transgenic plants displayed tolerance to herbicide bialaphos.

Keller et al., 1997

Coker 315/cotyledons Go and nptII TobRB7 and CaMV35S

Agrobacterium Transgenic plants exhibited resistance against fungal pathogen, V. dahlia.

Murray et al., 1999

Ji 123/flower (direct injection of foreign DNA into embryos)

gfp CaMV35S Pollen-tube pathway Obtained transgenic plants and were con-firmed by GFP fluores-cence and Southern blot analyses.

Guocun et al., 1999

Coker 312/hypocotyl At-GR, Pea-APX and nptII

CaMV35S Agrobacterium Transgenic plants exhibited protection from chilling and high light intensity.

Kornyeyev et al., 2001

Coker 315/hypocotyl RNAiGh-SAD-1, Gh-FAD2-1 and nptII

Lec and Nos Agrobacterium Transgenic cottonseed oil showed increased levels of stearic acid (40%) and oleic acid (77%).

Liu et al., 2002

Coker 312/hypocotyl gusA and npt II Agp and Nos Agrobacterium Demonstrated developmental and tissue-specific expression of cotton α-globulin promoter using gusA gene.

Sunilkumar et al., 2002

Coker 312/hypocotyl Tv-ech and nptII CaMV35S Agrobacterium Transgenic plants exhib-ited resistance against two fungal pathogens, R. solani and A. alternata.

Emani et al., 2003

Coker 312/hypocotyl Pea-APX and nptII CaMV35S Agrobacterium Transgenic plants disclosed protection from chilling and high light intensity.

Kornyeyev et al., 2003

Coker 312/hypocotyl cdn1-C1 (antisense construct) and nptII

CaMV35S Agrobacterium Transgenic plants showed reduced levels of cadinane sesquiterpenoids in foliage and seeds.

Martin et al., 2003

Xin-Cai 1#, Lv 9902, 9903/Flower (direct injection of foreign DNA into embryos)

gafp and bar CaMV35S Pollen-tube pathway

Transgenic plants exhib-ited resistance against fungal pathogen, V. dahlia.

Wang et al., 2004

Coker 312/hypocotyl and cotyledon

GF14λ and nptII CaMV35S Agrobacterium Transgenic plants displayed increased water-stress tolerance and maintained higher pho-tosynthetic rates during drought.

Yan et al., 2004

(Continued)

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Coker 310 FR/embryo-genic calli

aphA-6 and npt II 16SrRNA Particle bombardment

Transgenic plants were obtained through plastid transformation and were confirmed by southern blot analyses.

Kumar et al., 2004

CRI 35/hypocotyl AroAM12 CaMV35S Agrobacterium Transgenic plants showed tolerance to herbicide glyphosate.

Xie et al., 2004

G007/Flower (fertilization with transformed pollen)

acsA, acsB, gusA and hpt

CaMV35S and Nos Pollen-tube pathway Fibers of transgenic plants exhibited increased length and strength.

Li et al., 2004

Coker 312/cotyledon and hypocotyl

D4E1 and nptII CaMV35S and Nos Agrobacterium Transgenic plants revealed resistance against fungal pathogen, T. basicola.

Rajasekaran et al., 2005

Coker 312/hypocotyl chi and nptII CaMV35S and Nos Agrobacterium Transgenic plants conveyed resistance against fungal pathogen, V. dahlia.

Tohidfar et al., 2005

F846/shoot apex AV2 and nptII CaMV35S Agrobacterium Transgenic plants conferred resistance to leaf curl disease.

Sanjaya et al., 2005

Coker 312/hypocotyl At-NHX1 and nptII Mas and Nos Agrobacterium Transgenic plants exhibited salinity-stress tolerance, improved photosynthetic performance and produced more fibre during stress.

He et al., 2005

Coker 312/hypocotyl RNAiCad1-C1 and nptII Agp and Nos Agrobacterium Transgenic cottonseed oil showed reduced gossypol level.

Sunilkumar et al., 2006

Zhongmian 35/hypocotyl

aroA-M1 CaMV35S Agrobacterium Transgenic plants exhibited tolerance to herbicide glyphosate.

Zhao et al., 2006

Luyuan 890/shoot apex

betA and als CaMV35S Agrobacterium Transgenic plants disclosed water and polyethylene glycol (PEG) stress tolerance and maintained higher photosynthetic rates during drought.

Lv et al., 2007

Coker 310 FR/cotyledon

ALSdm, gusA and nptII CaMV35S and Nos

Agrobacterium Transgenic plants disclosed tolerance to herbicide imazethapyr.

Rawat et al., 2008

CC 71/seedling apical meristem (in planta)

gusA and nptII CaMV35S and Nos

Agrobacterium Transgenic plants exhibited GUS activity.

Keshamma et al., 2008

Coker 312/embryogenic calli

AVP1, gusA and nptII CaMV35S and Nos

Silicon carbide whiskers

Transgenic plants showed salinity-stress tolerance.

Asad et al., 2008

SVPR 2/shoot apex chi, bar and hpt Ubi1 and CaMV35S

Agrobacterium Transgenic plants conveyed resistance against two fungal pathogens, F. oxysporum and A. macrospora.

Ganesan et al., 2009


Ntosm and nptII CaMV35S Agrobacterium Transgenic plants exhib-ited water and PEG stress tolerance and improved growth during drought.

Parkhi et al., 2009

(Continued)

Table 1. (Continued).


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Lumianyan 19 and 21/shoot apex

TsVP and als CaMV35S Agrobacterium Transgenic plants showed water and PEG stress tol-erance and improved root and shoot growth during drought.

Lv et al., 2009

Simian 3/flower (pistil-dip method)

bar CaMV35S Agrobacterium Transgenic plants exhib-ited tolerance to herbicide glufosinate.

Tian et al., 2010

YZ 1/hypocotyl RNAiGh-aGP4 and nptII CaMV35S Agrobacterium Gene silencing in transgenic plants caused suppressed fiber growth, elongation and fiber quality.

Li et al., 2010

Z 35/hypocotyl hpa1xoo

and nptII CaMV35S and Nos Agrobacterium Transgenic plants exhib-ited resistance against fungal pathogen, V. dahlia.

Miao et al., 2010


At-NPR1 and npt II CaMV35S Agrobacterium Transgenic plants con-veyed resistance against three fungal pathogens, viz., F. oxysporum, V. dahliae, R. solani, A. alternata and a reniform nematode, R. reniformis.

Parkhi et al., 2010a


At-NPR1 and npt II CaMV35S Agrobacterium Transgenic plants exhibited resistance against fungal pathogen, V. dahlia.

Parkhi et al., 2010b

Durga/hypocotyl AnnBj1 and nptII CaMV35S Agrobacterium Transgenic plants showed salinity, mannitol and PEG stress tolerance and produced more biomass during drought.

Divya et al., 2010

CIM 496/mature embryo

Ghsp26, gusA and hpt CaMV35S Agrobacterium Transgenic plants exhib-ited water-stress tolerance.

Maqbool et al., 2010

Coker 312/hypocotyl tAC1 and nptII CaMV35S and Nos

Agrobacterium Transgenic plants con-ferred resistance to leaf curl disease.

Hashmi et al., 2011

HS 6/embryo ACP (antisense construct) and nptII

CaMV35S and Nos

Agrobacterium Transgenic plants con-veyed resistance to leaf curl disease.

Amudha et al., 2011

ND94-7/shoot apex phyA and nptII Pyk10 and Nos Particle bombardment Transgenic plants exhibited increased (>3.1-fold) extracellular phytase activity and efficiently used organic phosphorous and phytate.

Liu et al., 2011a

ND94-7/shoot apex phyA and nptII Pyk10 and Nos Agrobacterium Transgenic plants showed increased (4.2–6.3-fold) extracellular phytase activity and efficiently used organic phosphorous and phytate.

Liu et al., 2011b


AVP1 and nptII CaMV35S Agrobacterium Transgenic plants exhibited water and salinity-stress tolerance and produced more fibre (>20%) during drought.

Pasapula et al., 2011

(Continued)

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2004; Wu et al., 2005; Kumar et al., 2009). Recently, Mao et al. (2011) obtained insect-resistant transgenic cotton plants expressing dsCYP6AE14 using Agrobacterium-mediated genetic transformation of hypocotyl and cotyledon explants. Although certain cotton varieties have been transformed through Agrobacterium and plants have been subsequently regenerated through embryogenesis, commercially important cultivars proved recalcitrant to regeneration due to their inability to develop embryogenic tissues. Regeneration of trans-genic cotton plants from shoot apex explants infected with Agrobacterium harboring β-glucuronidase (gusA) and neomycin phosphotransferase (nptII) genes were reported by Gould and Cedeno, (1998) and Zapata et al., (1999). Direct regeneration of plants from the infected explants facilitated the development of trans-genic plants in genotype-independent manner within a short time span. Subsequently, transgenic cotton plants have been produced in diverse cotton varieties through Agrobacterium-mediated transformation of apical meri-stems (Lv et al., 2008, 2009; Nandeshwar et al., 2009; Liu et al., 2011b).

Particle bombardment-mediated transformationBiolistic transformation, utilizing high velocity metal particles to deliver biologically active DNA into the plant cells, emerged as an alternative approach to bypass the problems posed by the genotype-dependent transforma-tion system. Finer and McMullen (1990) were the first to obtain transgenic cotton plants by particle bombard-ment of suspension cultures derived from embryogenic calli. Also, successful transfer of gusA, nptII, amino glycoside phosphotransferase (apha-6), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) genes into cotton were achieved after bombardment of embryogenic cultures and embryo

axes (McCabe & Martinell, 1993; John & Keller, 1996; Rajasekaran et al., 2000; Kumar et al., 2004). Recently, Liu et al. (2011a) produced transgenic cotton plants express-ing Aspergillus ficuum phytase gene (phyA) for enhanced utilization of phosphorous using shoot apex explants employing biolistic transformation. However, a number of drawbacks, such as low transformation frequency of explants, occurrence of chimeras, high frequency of epidermal transformants, insertion of fragmented cop-ies of transgenes, co-suppression due to multiple copies were found associated with the particle bombardment-mediated gene delivery system (DeBlock, 1993; Depicker & Montagu, 1997; Wilkins et al., 2000).

Other methods employed for cotton transformationBesides widely used techniques, attempts were also made for genetic transformation of cotton using vari-ous alternative approaches. The process of producing transgenic cotton plants by Agrobacterium-mediated transformation, direct introduction of exogenous DNA into cotton embryos and pollinating the flowers with pre-transformed pollen via pollen-tube pathway in a tis-sue culture independent manner were reported. Initially, introduction of exogenous DNA into embryos of self-pollinated cotton flowers through the pollen-tube path-way was reported by Zhou et al. (1983). Adopting similar approaches, Huang et al. (1999) and Lu et al. (2002) pro-duced transgenic cotton plants expressing green fluo-rescent protein gene (gfp) and cellulose synthesizing genes (acsA, acsB, acsC, and acsD) of Acetobacter xylinum. Wang et al. (2004) produced Verticillium wilt resistant transgenic cotton plants by applying 0.1–0.2 μg of DNA of Gastrodia antifungal protein (gafp) and bialaphos resistance (bar) genes on the floral stigmatic region. Herbicide-tolerant transgenic cotton plants were produced by direct inoculation of Agrobacterium



Jimian 14/hypocotyl Gh-CKX, gusA and nptII CaMV35S and Nos Agrobacterium Transgenic plants revealed reduced levels of ovular cytokinins and fiber initiation.

Zeng et al., 2012

Z 35/shoot apex At-LOS5 and hpt CaMV35S Agrobacterium Transgenic plants exhibited tolerance to water stress and increased fresh weight during drought.

Yue et al., 2012

Luyuan 890/shoot apex

betA and als CaMV35S Agrobacterium Transgenic plants showed tolerance to cold stress and improved photosynthesis during stress.

Zhang et al., 2012

Coker 315/cotyledon Sus and nptII S7 and S1 Agrobacterium Transgenic plants contained more fibre and seed.

Xu et al., 2012

W0/cotyledon and hypocotyl

Gh-SusA1 and nptII CaMV35S and Nos Agrobacterium Transgenic plants produced more fibre and biomass.

Jiang et al., 2012

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culture on the pistils of pollinated flowers (Tian et al., 2010). Transformation of cotton was also reported by manual pollination of flowers using transformed pol-len. Li et al. (2004) reported cotton transformation by fertilizing the flowers using the pollen co-cultivated with Agrobacterium harbouring acsA, acsB, gusA and hygromycin phosphotransferase (hpt) genes. Gounaris et al. (2005) reported the generation of transgenic cotton plants after fertilization of flowers employing the pollen bombarded with Arabidopsis thaliana 3′-hydroxylmethyl glutaryl coenzyme A reductase (hmgr) gene, along with the nptII gene.

Chemical-induced cotton transformation was carried out by exposing cotyledon-derived embryogenic suspen-sion cultures in MS medium containing spermidine and polybrene along with the DNA carrying hpt and gusA genes (Sawahel, 2001). Beringer et al. (2004) obtained herbicide-tolerant transgenic cotton plants by agitating cotyledon-derived embryogenic calli in a solution con-taining mannitol and sorbitol together with needle-like silicon carbide microfibers (WHISKERS™) coated with DNA containing the bar gene. Using a similar approach, Asad et al. (2008) produced salt tolerant transgenic cotton plants expressing Arabidopsis vacuolar pyrophospha-tase proton pump (AVP1) gene. Keshamma et al. (2008) reported in planta genetic transformation of cotton by pricking the meristematic regions and dipping cotton seedlings in the Agrobacterium culture. However, these techniques were not repeatable and involved screen-ing of a large number of plants for identifying the stable transformants. Furthermore, plants selected as transgen-ics failed to transfer the transgene into subsequent gen-erations (Showalter et al., 2009; Obembe et al., 2011).

Genetic engineering of cotton deploying agronomically useful genesGenetic engineering has enabled the incorporation of potential candidate genes for several beneficial traits thereby surpassing the limitations normally associated with the conventional methods of crop improvement. This technology allowed the precise transfer of alien genes into the cotton genome for resistance to insect pests (Wu et al., 2011), fungal pathogens (Parkhi et al., 2010a,b), tolerance to herbicides (Nida et al., 1996), drought (Yan et al., 2004), soil salinity (Liu et al., 2012) and silencing of undesirable genes (Sunilkumar et al., 2006) as well as improvement in nutrient acquisition (Liu et al., 2011a,b). Results pertaining to cotton bio-technology after introducing alien genes for tolerance to herbicides, abiotic stress tolerance, improvement of oil and fiber characteristics, as well as resistance to various pathogens and insect pests are dealt with in the following sections.

Herbicide toleranceWeed management has been an integral part of cot-ton cultivation as weeds compete for water, nutrients,

sunlight and increase the trash content of the harvested cotton fiber (Nida et al., 1996). Several herbicides with different modes of activity, toxicity, and environmental effects have been used for weed control in diverse crops (Kishore et al., 1992). Application of various herbicides often showed adverse effects on the plants owing to their inability to distinguish between crop plants and weeds (Lyon et al., 1993). To overcome the debilitating effects associated with nonspecific killing of plants, new crop protection strategies have been evolved by introducing herbicide tolerance genes derived from different sources, such as bacteria, fungi, and plants. In this section, the transgenic cottons exhibiting tolerance against various herbicides has been described.

Glyphosate-tolerant transgenic cotton plants were generated by introducing 5-enolpyruvilshikimate-3-phosphate synthase (CP4-EPSPS) gene derived from A. tumefaciens strain CP4 through Agrobacterium-mediated genetic transformation (Nida et al., 1996). The first commercialized glyphosate-tolerant Roundup Ready® transgenic cotton, expressing CP4-EPSPS under the control of figwort mosaic virus (FMV) 35S promoter, could effectively sustain glyphosate applications of >0.84 kg a.e. (acid-equivalent) h–1 till the four leaf stage of the plants (May et al., 2004). However, beyond this stage, the application of glyphosate caused damage to the reproductive organs resulting in substantial yield losses, owing to suboptimal levels of EPSPS in the devel-oping pollen and tapetum. This problem was successfully overcome through the development of Roundup Ready Flex® cotton by introducing CP4-EPSPS gene driven by FMV35S promoter along with an additional CP4-EPSPS driven by the elongation factor-1α (P-Ef1α) promoter of A. thaliana. These transgenic cotton lines exhibited ample tolerance against glyphosate applications of >1.68 kg a.e. h–1 even after four leaf stage with ~97% of pollen viability. Whereas, pollen from transformants expressing CP4-EPSPS alone under the control of FMV35S promoter were aborted (Chen et al., 2006)

Different cotton varieties with tolerance to herbicide glyphosate were successfully produced after the commer-cialization of EPSPS-containing Roundup Ready® and Roundup Ready Flex® cottons. Zhao et al. (2006) reported the development of transgenic cotton plants expressing chimeric aroA-M1 gene fused to the chloroplast transit peptide of A. thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) gene against glyphosate tolerance. In 2006, Bayer CropScience developed glyphosate-tolerant trans-genic cotton (Gly TolTM) by introducing a double-mutated version of Zea mays EPSPS (2mepsps) (Wallace et al., 2011). Although, transgenic glyphosate-tolerant cotton plants were successful, yet the continuous application of herbicide for weed control in transgenic cotton fields resulted in the emergence of 12 weed species tolerant to glyphosate (Dill et al., 2008).

The bar gene isolated from Streptomyces hygroscopi-cus has been widely used as the selection marker of choice along with candidate genes in the production of

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transgenic cotton plants (Guo et al., 2007; Ganesan et al., 2009; Li et al., 2009). Initially, Keller et al. (1997) used the bar gene to produce transgenic cotton plants tolerant to the herbicide bialaphos. The commercially developed transgenic cotton tolerant to gluphosinate by Bayer CropScience has been named as Liberty Link®. The num-ber of weed species controlled by gluphosinate is low as compared to that of glyphosate. However, the advantage of gluphosinate was that no cases of emergence of weed species tolerant to gluphosinate are reported. Transgenic cotton plants tolerant to herbicide bromoxynil were developed by expressing nitrilase gene of Klebisella ozaene. As the nitrilase enzyme converts bromoxynil into non-herbicidal 3, 5-dibromo-4-hydroxy benzoic acid (DBHA), the herbicide could not show any phytotoxic effects on the transgenic plants (Stalker et al., 1988). The commercially grown transgenic cotton tolerant to bro-moxynil has been designated as BXN® cotton. However, there are a number of weed species that have inherent tol-erance to bromoxynil, restricting its application for con-trolling major weeds. Transgenic cotton engineered with the modified 2, 4-D mono-oxygenase (tfdA) gene isolated from Alcaligenes eutrophus provided considerable pro-tection toward 2,4,-dichlorephenoxyacetic acid (2, 4-D) herbicide (Bayley et al., 1992; Lyon et al., 1993). Further, transgenic cotton plants produced with the mutated forms of acetohydroxyacid synthase (AHAS) gene of cot-ton, exhibited tolerance to herbicides such as imidazoli-none and sulfonylurea (Rajasekaran et al., 1996a).

Insect resistanceIndeterminate growth characteristics of cotton crop offer food and shelter to a wide range of insect pests. About 130 species of insects are known to infest and cause damage to the cotton crop. Different candidate genes of bacterial, insect and plant origins have been identified and were successfully introduced into cotton to combat against major insect pests (Perlak et al., 1990; Thomas et al., 1995; Wu et al., 2006). Diverse cotton transgenics developed using different insecticidal genes are listed in Table 2.

Bacterial genes employed for insect pest resistanceGenes encoding crystalline (Cry) proteins of B. thuringi-ensis have been classified as CryI, CryII, CryIII, CryIV, CryV, and CryVI based on their insecticidal activities (Crickmore et al., 1998; Wilkins et al., 2000). Of these, Cry δ-endotoxins, derived from subspecies of B. thuringien-sis, viz., B. kurstaki and B. aizawai, have been extensively analyzed and are the first candidate genes introduced into the cotton to control the cotton bollworm complex.

Different transgenic cotton varieties expressing native or modified versions of Cry proteins either singly (Cry1Ac) or stacked (Cry1Ac+Cry2Ab and Cry1Ac+Cry1F) have provided resistance against lepidopteran pests like boll-worm (Helicoverpa zea), tobacco budworm (Heliothis virescens), pink bollworm (Pectinophora gossypiella), and fall armyworm (Spodoptera furgiperda) (Tabashnik et al.,

2002; Greenplate et al., 2003; Siebert et al., 2008). The expression of synthetic Cry1EC protein in cotton under the control of chimeric CaMV35S(r) promoter when fused to a tobacco pathogenesis related promoter (PR-1a), not only enabled constitutive expression of the insecticidal protein, but also promoted enhanced expression during insect bite and salicylic acid treatment. Furthermore, these transgenics effectively targeted S. litura larvae with 100% mortality (Kumar et al., 2009).

In general, B. thuringiensis δ-endotoxins were highly toxic to different lepidopteran pests. However, insects such as H. viscerens, P. gossypiella, H. armigera, and H. zea were found to develop resistance against CryIAc protein expressed in transgenic plants (Gahan et al., 2001; Morin et al., 2003; Gunning et al., 2005; Anilkumar et al., 2008; Bravo & Soberón, 2008). Moreover, certain economically important pests were found to show a low level of sensitivity toward Cry proteins (Estruch et al., 1996; Wu et al., 2011). To develop an effective pest control strategy, genes encoding vegetative insecticidal proteins (Vips), derived from certain Bacillus species, were employed for production of insect-resistant trans-genic plants. Unlike B. thuringiensis δ-endotoxins, which expressed during sporulation, Vip3A is expressed dur-ing vegetative growth phase starting at mid-log phase as well as during sporulation (Estruch et al., 1996). The Vip1 and Vip2 originated from B. cereus revealed potent insecticidal activity against corn rootworm (Diabrotica virgifera), whereas Vip3A of B. thuringiensis proved toxic to a wide spectrum of lepidopteran insects, including black cutworm (Agrotis ipsilon), fall armyworm (S. fur-giperda), beet armyworm (S. exigua), Swinhoe (Chino partellus), budworm (H. punctigera), and sugarcane borer (Diatracea saccharalis) (Estruch et al., 1996; Zhu et al., 2006a). Furthermore, Vip3A did not share sequence homology with known δ-endotoxins, bind to the specific receptors other than those recognized by Cry proteins and exhibit wide insecticidal activity against various lepidopteran and coleopteran insects (Yu et al., 1997; Lee et al., 2003; Wu et al., 2011).

Transgenic cotton plants were produced by introduc-ing two different versions of Vips such as synthetic vip3A and tvip3A fused to chloroplast transit peptide, to achieve the accumulation of transgene product in chloroplasts (Wu et al., 2011). The expression level of Vip3A was three times more in the transgenics containing tvip3A gene in comparison with the transgenics carrying vip3A alone. Moreover, transgenic cotton plants expressing vip3A and tvip3A genes exhibited resistance to three lepi-dopteran pests of cotton, viz., S. furgiperda, S. exigua, and H. zea. Also, transgenic cotton plants expressing Vip3A and CryIAb proteins exhibited high entomotoxic effect against a wide range of lepidopteran pests including H. virescens, H. zea, and P. gossypiella (Kurtz et al., 2007).

Insect genes employed for resistance to insect pestsApart from cry genes, various candidate genes derived from insects have been introduced into cotton to confer

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Table 2. Different genes introduced into cotton for insect resistance.


Coker 312/hypocotyl cryAc, cry1Ab, and nptII

CaMV35S and Nos Agrobacterium Transgenic plants exhibited resistance against lepidopteran pests, H. zea and S. exigua.

Perlak et al., 1990

Pima S 6, Sea Island, Delta Pine 90 and 50/embryonic axes

cry2Ab and gusA CaMV35S Particle bombardment

Transgenic plants exhib-ited resistance against lepidopteran pests, H. viscerens, H. zea, and S. furgiperda.

McCabe and Martinell, 1993

Coker 312/cotyledon Manduca sexta protease inhibitors and nptII

CaMV35S, Ppc-1 and Nos

Agrobacterium Transgenic plants displayed antimetabolic effect against homopteran pest B. tabaci.

Thomas et al., 1995

CIM 443/embryo cry1Ab and nptII CaMV35S and Nos Particle bombardment and Agrobacterium

Transgenic plants exhibited resistance against lepidopteran pest, H. armigera.

Majeed et al., 2000

Coker 312/hypocotyl cry1EC and nptII CaMV35S and Nos Agrobacterium Transgenic plants conveyed resistance against lepidopteran pest, S. litura.

Singh et al., 2004

Coker 312/embryogenic calli

cry1Ia5 and nptII CaMV35S and Nos Agrobacterium Obtained transgenic plants and were confirmed by Southern blot analyses.

Leelavathi et al., 2004

Ekang 9 and Jihe 321/embryogenic calli

cry1Ac, api-B and nptIICaMV35S and Nos Agrobacterium Transgenic plants exhibited resistance against lepidopteran pest, H. armigera.

Wu et al., 2005

Zhongmiansuo 35/hypocotyl

ACA and nptII CoYMV and Nos Agrobacterium Transgenic plants disclosed resistance against homopteran pest, A. gossypii.

Wu et al., 2006

Coker 201, YZ 1, EK 10 and EM 22/hypocotyl

cry1C, cry2A, cry9C and bar

Ubi and CaMV35S Agrobacterium Transgenic plants showed resistance against lepidopteran pest, H. armigera.

Guo et al., 2007

Bikaneri Nerma/ apical meristem

crylC and nptII CaMV35S and Nos Agrobacterium Transgenic plants conveyed resistance against lepidopteran pest, H. armigera.

Katageri et al., 2007

CIM 482/embryo cry1Ac, cry2A and nptII

CaMV35S Sonication and Agrobacterium

Transgenic plants conferred resistance against lepidopteran pest, H. armigera.

Rashid et al., 2008


AaHIT and nptII CaMV35S and Nos Agrobacterium Transgenic plants exhibited resistance against lepidopteran pest, H. armigera.

Wu et al., 2008

Coker 100/cotyledon cry1Ab and nptII CaMV35S and Nos Agrobacterium Transgenic plants showed resistance against lepidopteran pest, H. armigera.

Yazdanpanah et al., 2009


cry1EC and hptII CaMV35S and Nos Agrobacterium Transgenic plants con-ferred resistance against lepidopteran pest, S. litura.

Kumar et al., 2009

RG 8/shoot tip cry1Ac and nptII CaMV35S Agrobacterium Obtained transgenic plants and were confirmed by Southern blot and ELISA analyses.

Nandeshwar et al., 2009

(Continued)

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protection against lepidopteran pests. The expression of a synthetic Androctonus australis hector insect toxin (AaHIT) in transgenic cotton plants conferred resis-tance to cotton bollworm as evidenced by the bioassays performed on detached leaves of transgenic plants (Wu et al., 2008). Likewise, the expression of insect (Manduca sexta)-derived protease inhibitors, viz., anti-trypsin, anti-chymotrypsin, and anti-elastase protease inhibitor genes in transgenic cotton caused delayed fecundity in the whitefly Bemesia tabaci (Thomas et al., 1995). Recently, Mao et al., (2011) demonstrated the suppression of P450 mono-oxygenase (CYP6AE14) gene of cotton bollworm through RNA interference mechanism by introducing the double-stranded RNA of CYP6AE14 into cotton plants. Although, the suppression of CYP6AE14 expression was not lethal to H. armigera, yet it has significantly retarded the larval growth and reduced the extent of damage in transgenic plants as compared to the untransformed control plants. The main advantage of this approach has been that it did not affect the non target insects owing to the high specificity of dsRNA toward a particular gene present in the target pest.

Plant genes employed for resistance to insect pestsTwo groups of plant-derived insecticidal proteins, viz., proteases and lectins, have been used to confer resis-tance to various lepidopteran and homopteran insect pests of cotton. Transgenic cotton plants expressing cow-pea (Vigna unguiculata)-derived trypsin inhibitors were shown to protect plants against the cotton bollworm (Li et al., 1998). Another group of insecticidal proteins are lectins which are carbohydrate-binding proteins that spe-cifically recognize glycans of glycoproteins, glycolipids or polysaccharides with high affinity (Goldstein & Hayes, 1978; Chrispeels & Raikhel, 1991). Lectins derived from diverse plant species, viz., Galanthus nivalis, Pinellia ternata, Amaranthus caudatus, Allium sativum, etc., have been found to provide effective protection against several insect pests when expressed in transgenic plants

(Rao et al., 1998; Yao et al., 2003; Wu et al., 2006; Yarasi et al., 2008). Transgenic cotton plants expressing Bt toxins are successful in providing substantial resistance against lepidopteran pests. However, these transgenics failed to convey resistance to cotton plants against homopteran sucking pests (Lawo et al., 2009). Further, whiteflies and aphids act as vectors for transmitting major viral dis-eases of cotton such as leaf curl and cotton blue diseases (Distéfano et al., 2010; Amudha et al., 2011). In cotton, two reports dealing with the introduction of plant lec-tins to combat against sucking insect pests are available. Transgenic cotton plants, expressing A. caudatus agglu-tinin (aca) gene under the control of Commelina Yellow Mottle Virus (CoYMV) phloem-specific promoter and A. sativum agglutinin (asa) gene under the regulation of CaMV35S promoter, were produced (Wu et al., 2006; Balogun et al., 2011). As such, no transgenic cotton vari-ety resistant to sucking pests is available.

Fungal and viral resistanceIn cotton, diverse microbial and viral pathogens cause ~11% yield loss and use of fungicide sprays costs about $5 per acre (Wilkins et al., 2000; Oerke & Dehne, 2004). Genes encoding various antifungal proteins, such as chi-tinases (Emani et al., 2003), glucose oxidases (Murray et al., 1999), as well as components of signalling pathways involved in the defense response (Parkhi et al., 2010a,b), have been exploited to generate transgenic cotton plants resistant to various fungal pathogens. Transgenic cot-ton plants overexpressing Gastrodia elata antifungal protein (gafp) and baculovirus apoptosis inhibitor genes (p35 and op-iap) exhibited resistance to Verticillum wilt (Wang et al., 2004; Tian et al., 2010). Rajasekaran et al. (2005) have introduced a synthetic antimicrobial pep-tide (D4E1) into Coker 312, and in vitro analysis of the crude leaf extracts of transgenic plants inhibited the growth of Fusarium verticillioides and Verticillium dahlia fungal strains. Transgenic plants expressing D4E1 pep-tide, when infected with Thielaviopsis basicola, showed

Table 2. Different genes introduced into cotton for insect resistance.


MNH 93/embryo cry1Ab and nptII CaMV35S and Nos Particle bombardment and Agrobacterium

Transgenic plants exhibited resistance against lepidopteran pest, H. armigera.

Khan et al., 2011

Coker 310 FR/cotyledon

cry1Ac and nptII CaMV35S, MMV, FMV and Nos

Agrobacterium Obtained transgenic plants and were confirmed by molecular analyses.

Rawat et al., 2011


vip3A, tvip3A and nptII

CaMV35S and Nos Agrobacterium Transgenic plants exhibited resistance against lepidopteran pests, S. frugiperda, S. exigua and H. armigera.

Wu et al., 2011

R 15/hypocotyl and cotyledon

DsCYP6AE14 and nptII

CaMV35S and Nos Agrobacterium Transgenic plants showed resistance against lepidopteran pest, H. armigera.

Mao et al., 2011

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marked reduction in the disease symptoms while seeds exhibited decreased colonization of Aspergillus flavus in comparison with the control plants. Genes encoding various fungal and plant chitinases, namely Trichoderma virens chitinase (Tv-ech) (Emani et al., 2003) and rice chitinase (chi II) (Ganesan et al., 2009) were expressed in cotton to confer resistance against various fungal patho-gens, such as Rhizoctonia solani, Alternaria alternata, F. oxysporum, and A. macrospora. Furthermore, the expression of Talaromyces flavus glucose oxidase (GO) gene in cotton with a root-specific promoter conveyed resistance to the root pathogen V. dahliae (Murray et al., 1999). However, a high-level expression of GO in cotton roots resulted in reduced plant height, low seed set, decreased seedling germination and lateral root formation. Miao et al. (2010) reported Agrobacterium-mediated genetic transformation of the cotton using a harpin-encoding gene (hpa1

Xoo) from Xanthomonas

oryzae pv. oryzae for obtaining resistance against various pathogens through a priming mechanism. Verticillum wilt resistance of these transformants was analyzed by exposing the transgenic plants to the spores of two dif-ferent strains of V. dahliae. After ten days, control plants treated with fungal spores developed severe disease symptoms, whereas low-levels of fungal damage were observed on transgenic plants. Parkhi et al. (2010a,b) pro-duced transgenic cotton plants expressing AtNPR1 gene to bestow resistance against different fungal pathogens including a non-defoliating (TS-2) isolate of V. dahliae, F. oxysporum, R. solani, and A. alternata besides a reniform nematode (Rotylenchulus reniformis).

Apart from fungal diseases, cotton plants also suffer from certain viral diseases. The leaf curl disease (CLCuD) of cotton, caused by cotton leaf curl virus (CLCuV), is one of the major viral diseases and is becoming a seri-ous threat especially in India and Pakistan (Amudha et al., 2011; Hashmi et al., 2011). Transgenic cotton plants, expressing antisense movement protein (AV2) and antisense coat protein (ACP) genes of CLCuV, were produced to provide resistance to CLCuD (Sanjaya et al., 2005; Amudha et al., 2011). Recently, Hashmi et al. (2011) produced transgenic cotton plants by Agrobacterium-mediated genetic transformation employing truncated forms of cotton leaf curl Khokhran virus replicase (tAC1) gene. The transgenic plants expressing tAC1 gene exhib-ited decreased symptoms of CLCuV infection along with lower CLCuV titre when compared to the untransformed plants.

Abiotic stress toleranceNonspecific and erratic occurrence of various abiotic stress factors like salinity, drought, water-logging, strong or insufficient light, extreme temperatures and mineral imbalance have been found to affect growth, develop-ment and yield of crop plants (Bohnert & Jensen, 1996; Chen & Murata, 2002; Gong et al., 2012). Genes involved in signaling and regulatory pathways, functional and structural protectants and stress-conferring proteins can

be used as potent candidate genes to impart tolerance against various abiotic stresses (Wang et al., 2003). In transgenic cotton, expression of superoxide dismutase, glutathione reductase and ascorbate peroxidase genes have been found to furnish abiotic stress tolerance against chilling and intense light conditions. Transgenic plants, expressing Mn superoxide dismutase (Mn-SOD) from Nicotiana plumbaginifolia fused with chloroplast transit peptide of ribulose 1,5-bisphosphate carboxylase or ascorbate peroxidase (APX) from Pisum sativum and glutathione reductase (GR) from A. thaliana, provided significant protection against chilling and high light intensities (Payton et al., 2001; Kornyeyev et al., 2001, 2003). Cotton plants, overexpressing choline dehydro-genase (betA) gene from E. coli (Lv et al., 2007; Zhang et al., 2011) and choline mono-oxygenase (Ah-CMO) gene from Atriplex hortensis (Zhang et al., 2009), disclosed elevated levels of glycine betaine and were found tolerant to cold and drought stress conditions.

Cotton plants, expressing vacuolar sodium antiporter gene (At-NHX1) from A. thaliana and annexin gene (AnnBj1) from Brassica juncea, showed improved photo-synthetic ability, increased plant biomass and fibers under different stress treatments (He et al., 2005; Divya et al., 2010). In cotton, expression of Arabidopsis 14-3-3 protein (GF14λ), tobacco osmotin (Nt-Osm), and G. arboreum heat shock-protein (Ghsp26) genes resulted in increased water-stress tolerance and reduced leaf-wilting under drought stress (Yan et al., 2004; Parkhi et al., 2009; Maqbool et al., 2010). Transgenic cotton, expressing H+-pyrophosphatase (H+-PPase) genes of Thellungiella halophila (Ts-VP) and A. thaliana (AVP1), disclosed increased salinity tolerance upto 250 mM NaCl as well as improved photosynthetic ability under drought, and transgenic plants could pro-duce ~40% enhanced seed yield than that of untrans-formed control plants (Lv et al., 2008, 2009; Pasapula et al., 2011). Similarly, the expression of A. thaliana drought tol-erant (AtLOS5) gene in cotton plants conferred increased drought tolerance. Furthermore, these transgenic plants could accumulate more proline (20%) and abscisic acid (25%), and have produced enhanced biomass after expo-sure to drought conditions (Yue et al., 2012). The overex-pression of A. tumefaciens isopentenyl transferase (ipt) gene under the control of G. hirsutum cysteine proteinase (Ghcysp) promoter not only increased the accumulation of cytokinins and chlorophyll, but also resulted in improved fiber quality of transgenic cotton plants under saline conditions (Liu et al., 2012). Attempts were also made to improve cotton plant survival under water-logging con-ditions by the constitutive expression of cotton alcohol dehydrogenase (Adh2) and rice pyruvate decarboxylase (Pdc1) genes (Ellis et al., 2000).

Transgenic cotton for quality traitsAlterations in the seed oilApart from fiber, cotton seed also serves as an important source of edible oil. Cotton is graded as the fifth best oil

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producing plant in the world and the second best poten-tial source of plant seed proteins (Benbouza et al., 2010). Refined cotton-seed oil is consumed extensively in many parts of the world including USA, China, Uzbekistan, India, Middle East, etc. The fatty acid profile of cotton-seed comprises ~55% polyunsaturated fatty acids, ~17% monounsaturated fatty acids and ~26% saturated fatty acids (Lukonge et al., 2007). Despite its desirable charac-teristics for human consumption, use of cotton-seed oil has been limited because of the presence of cardio- and hepato-toxic terpenoid substances like gossypol (Risco & Chase, 1997; Sunilkumar et al., 2006).

The glandless cotton varieties developed through conventional breeding produced gossypol-free cotton-seed oil suitable for feeding monogastric animals and for human consumption (Lusas & Jividen, 1987; Sunilkumar et al., 2006). However, the glandless cotton which lack natural immunity could not gain commercial success owing to their high susceptibility to a range of insect pests and pathogens (Sunilkumar et al., 2006). Later, Romano and Scheffler, (2008) reported tissue-specific reduction of gossypol in seeds (>50%), whereas retaining the gos-sypol in different vegetative plant parts through conven-tional hybridization using varieties with different gland densities and their distribution. Strategies like antisense technology and RNAi-mediated silencing of the target genes are more advantageous to achieve alterations in the fatty acid composition and reduction in the gossypol content. Cotton plants engineered with an antisense G. arboreum δ-(+) cadinene synthase (cdn1-Cl) gene under the control of CaMV35S promoter resulted in the reduc-tion of seed-gossypol content upto ~70% and reduced the accumulation of foliar-gossypol to the extent of 92.4% (Martin et al., 2003). This kind of undesirable down regu-lation of gossypol in nontarget tissues can be success-fully overcome through the adoption of RNAi approach using tissue-specific promoters (Sunilkumar et al., 2006). Hairpin RNA-mediated silencing of fatty acid desaturase genes, viz., stearoyl-acyl Δ9-desaturase (ghSAD-1) and oleoyl-phosphatidylcholine ω6-desaturase (ghFAD2-1) under the control of seed specific soybean lectin pro-moter, resulted in increased stearic acid (~40%) and oleic acid contents (~77%) in cotton seed oil (Liu et al., 2002). Sunilkumar et al. (2006) produced transgenic cotton plants with low (0.1 µg/mg) levels of seed-gossypol by RNAi-mediated disruption of Δ-cadinene synthase gene expression during seed development.

Improved fiber qualityWorldwide, the fiber of cotton suffers from competition with synthetic fibers used in the manufacture of yarns and textiles, because of its poor characteristics (John, 1997; Karaca et al., 2002). Improvement of traits such as fiber length, strength and uniformity employing various candidate genes driven by fiber-specific promoters would enhance the competitiveness of cotton vis-à-vis synthetic fibers (John & Keller, 1996; John, 1997). John and Keller (1996) developed transgenic cotton plants synthesizing

a thermoplastic polymer, poly-d-(-)-3-hydroxybutyrate (PHB), by fiber-specific expression of phaB and phaC genes from A. eutrophus. Fiber from these transgenic plants exhibited improved thermal and insulating prop-erties. In the brown cotton, Li et al. (2004) reported ~15% increase in fiber length and strength by expressing acsA and acsB cellulose synthesis genes from A. xylinum. Furthermore, Li et al. (2009) achieved increases in length (28.36%) and strength (8.32%) of cotton fiber by express-ing silkworm fibroin (fbn) gene. Attempts to improve cotton fiber quality were also made by introducing plant-derived genes such as expansins (Zhu et al., 2006b) and sucrose synthases (Jiang et al., 2012). Overexpression of potato sucrose synthase (Sus) gene in cotton under the control of subterranean clover stunt virus constitutive segment promoter (S7) resulted in increased accumula-tion of hexose sugars in transgenic fiber. Moreover, the transgenic plants could yield more fiber (30%) and seed (23%) compared to the control plants (Xu et al., 2012). Similarly, Jiang et al., (2012) also reported increase in length and strength of cotton fiber produced from trans-genic plants expressing G. hirsutum sucrose synthase (Gh-SusA1) gene.

Biosafety aspects and regulatory mechanismCultivation of transgenic crops expressing Cry proteins derived from Bt has increased substantially worldwide since their introduction in 1996 (James, 2011). Various organisms in the environment are directly or indirectly exposed to insecticidal proteins expressed in the trans-genic plants. Conservation of natural enemies is impor-tant to avoid the outbreaks of secondary pests which are insensitive to the insect control proteins (Kos et al. 2009). It is also equally important to minimize the harmful effects to useful organisms in addition to effective resis-tance management. Continuous expression of transgene in the plant creates a significant opportunity for nontar-get herbivores to acquire and bio-accumulate the toxin for higher trophic levels (Torres & Ruberson, 2008). In this section, results pertaining to the investigations made to evaluate the ecological impact of transgenic crops are summarized.

Detailed analysis of potential effects of transgenic crops on various organisms is essential before their release into the environment. Since the introduction of Bt crops more than fifteen years ago, numerous studies have addressed the potential effects of Cry proteins on nontarget organisms in both laboratory and field condi-tions. Several field studies have indicated that predator and parasitoid abundance are similar in Bt and non-Bt crops (Romeis et al. 2006). The study covering a period of 6 years in the Bt cotton fields on the abundance of nontarget arthropod natural enemies indicated that the exposure to the Bt toxin over multiple generations did not cause any chronic long-term effects (Naranjo, 2005). The possible direct effects of transgenic maize expressing Cry3Bb1 insecticidal protein was analyzed by feeding the

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nontarget predatory spider (Theridion impressum) and the study revealed no differences in mortality, weight development and offspring production of spider (Meissle & Romeis, 2009). Recently, Zhang et al. (2012) conducted experiments to study the long-term effects of Bt cotton on the nontarget A. gossypii fed on transgenic and non-transgenic controls. Results indicated that although trace amounts of Cry protein was detected in the aphids fed on Bt plants, it did not cause any negative effects on the A. gossypii. Ecological risk assessment of maize MIR162 expressing Vip3Aa20 on nontarget aquatic invertebrate (Daphnia magna) revealed that the toxin did not affect the survival, but have resulted in a negligible develop-mental delay when compared to unexposed controls (Raybould & Vlachos, 2011). Indirect feeding studies to investigate the movement of Cry1Ac toxin from trans-genic cotton plants were performed by continuous lifetime predation of Podisus maculiventris on a Cry1Ac toxin ingested herbivorous prey S. exigua. Results sug-gested that the Cry1Ac toxin present in the herbivorous prey did not cause any negative effects on the predator (Torres & Ruberson, 2008). Lawo et al. (2009) assessed the impact of Bt cotton expressing Cry1Ac on aphid (A. gossypii) populations and the results clearly showed that none of the aphid samples contained Bt protein, indicating that natural enemies fed on aphids are not affected either directly or indirectly.

A consecutive 3-year study conducted on two dif-ferent cotton fields cultivated with Cry1Ac- and CpT1-expressing transgenics disclosed that long-term cultivation of insect-resistant transgenic cotton did not pose significant effects on soil microorganisms (Li et al. 2011). In a study, Bt corn (MON 863) expressing Cry3Bb1 protein and its near-isogenic non-Bt variety DKC 46-26 were evaluated for the persistence of Cry3Bb1 protein in the soil using western blot and ELISA analyses. This study showed that the Cry3Bb1 protein does not persist or accumulate in the soil and is rapidly degraded, indi-cating that this protein probably pose little ecological or environmental risk (Icoz & Stotzky, 2008).The overall studies collectively indicate that cultivation of transgenic plants poses negligible risk to nontarget organisms and these crops are unlikely to adversely affect environment.

Globally, genetically modified organisms are sub-jected to different regulations. The regulatory approval processes of transgenic crops are not uniform and dif-fered among various countries. Despite the fact that transgenic Bt crops are friendlier to the environment, natural enemies and nontarget pests, compared to con-ventional pest control practices, there are still concerns about their potential impacts (Romeis et al. 2006; Zhang et al. 2012). For countries that are signatories to the Cartagena Protocol, environmental risk assessment is required for the regulatory approval of genetically modi-fied organisms (Hilbeck et al. 2011). In India, genetically modified organisms and products are regulated accord-ing to the “Rules for the manufacture, use, import, export and storage of hazardous microorganisms, genetically

engineered organisms or cells, 1989” (commonly referred as Rules, 1989) notified by the Ministry of Environment and Forests (MoEF), Government of India under the Environment (Protection) Act (1986).

Conclusions and perspectivesBy the year 2050, the human population is expected to reach 9 billion and as such require sustainable agricul-tural production to meet the demands of food and fiber. Cotton plants being the major source of fiber and seed oil, play a significant role in meeting the future needs of an ever increasing population. The commercially avail-able insect-resistant and herbicide-tolerant transgenic cotton cultivars, expressing Bt and herbicide-tolerant genes, have substantially minimized the production costs and contributed to higher yields. During the past decade, a variety of novel candidate genes, which added value to the quality of cotton products, have been identi-fied. Transgenic cotton plants tolerant to several abiotic stresses, as well as resistance to various pests and patho-gens have been produced. Moreover, attempts have been made to improve the fiber quality and nutritional value of the cotton-seed oil through genetic engineering approaches. Identification and isolation of new candi-date genes, spatial and temporal regulation of trans-genes, refinement of in vitro regeneration protocols and gene transfer techniques for transforming elite cultivars are expected to contribute for genetic enhancement of cotton. Furthermore, concerns like polarization regard-ing apprehensions of environmental risks, development of insect resistance, nontarget effects and gene-flow from transgenic plants to nontransgenic varieties need to be addressed satisfactorily. Adoption of plastid transfor-mation technology using stacked genes with different mechanisms of action may be employed for achieving broad-based and durable resistance/tolerance against different factors besides transgene containment so as to gain regulatory approvals. Development and an under-standing of the specificity of RNAi gene knockdown facilitate the expression of a cocktail of dsRNAs effective against various target traits for genetic enhancement of cotton.

Declaration of interestThe work on development of transgenic cotton in our lab-oratory is supported by grants from the Swarna Bharathi Biotech Pvt., Ltd., Hyderabad, India. V.S.K.C. is thankful to the University Grants Commission, Government of India, New Delhi, for the award of Research Fellowship. The authors report no declaration of interest.

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): an assessment Critical Reviws Chakravarthy.pdf& Jayabalan, 2011), herbicide tolerance (Rajasekaran et al., 1996b), early maturity (Xanthopoulos & Kechagia, ... thuringiensis into