R E V I E W
An insight into cotton genetic engineering (Gossypium hirsutum L.):
current endeavors and prospects
Allah Bakhsh1•Emine Anayol2•Sancar Fatih O¨ zcan2•Tahira Hussain1• Muhammad Aasim3•Khalid Mahmood Khawar2•Sebahattin O¨ zcan2
Received: 10 December 2014 / Revised: 30 March 2015 / Accepted: 22 July 2015 / Published online: 4 August 2015 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2015
Abstract Cotton (Gossypium hirsutum L.) is the most significant cash crop and backbone of global textile industry. The importance of cotton can hardly be over emphasized in the economy of cotton-growing countries as cotton and cotton products contribute significantly to the foreign exchange earnings. Cotton breeders have continu-ously sought to improve cotton’s quality through conven-tional breeding in the past centuries; however, due to limited availability of germplasm with resistant to partic-ular insects, pests and diseases, further advancements in cotton breeding have been challenging. The progress in transformation systems in cotton paved the way for the genetic improvement by enabling the researchers to trans-fer specific genes among the species and to incorporate them in cotton genome. With the development of first genetically engineered cotton plant in 1987, several char-acteristics such as biotic (insects, viruses, bacteria and fungi) resistance, abiotic (drought, chilling, heat, salt), herbicide tolerance, manipulation of oil and fiber traits have been reported to date. Genetic engineering has emerged as a necessary tool in cotton breeding programs, strengthening classical strategies to improve yield and yield contributing factors. The current review highlights
the advances and endeavors in cotton genetic engineering achieved by researchers worldwide utilizing modern biotechnological approaches. Future prospects of the transgenic cotton are also discussed.
Keywords Binary vector Genetic transformation Diseases Resistance Genetic improvement Genetically modified (GM)
Introduction
The cultivation of genetically modified (GM) crops has gradually increased since commercialization of the first GM crop in 1996 and has reached more than 181 million hectares (James 2014). The global adaptation rates of biotech cotton have been 70 % as compared with con-ventional cotton (James 2013). Biotech cotton is widely accepted, cultivated and marketed crop after soybean and maize, and is planted on more than 24 million hectares in the world. Biotech cotton has made a significant contri-bution to the income of 16.5 million small resource-poor farmers in developing countries (James2014). Cotton is an important economical crop cultivated for food, fiber and feed. The most important product (lint) is a source of high quality natural fiber used in textile industry while cotton-seed is used for oil and cotton cake. The cotton cotton-seed meal is a protein enriched product that is a source of livestock feed (Keshamma et al. 2008; Bakhsh et al. 2009). The cotton breeders have improved it using conventional plant breeding methods (Agrawal et al. 1997). There are many problems with cotton plant that had been solved by breeding, yet diseases and insects continue to reduce yield (Poehlman 1987). More than 15 different insect species infest cotton; however, hemipteran and lepidopeteran are Communicated by A. K. Kononowicz.
& Allah Bakhsh
allah.bakhsh@nigde.edu.tr; abthebest@gmail.com
1 Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nig˘de University, 51240 Nig˘de, Turkey
2 Department of Field Crops, Faculty of Agriculture, University of Ankara, 06110 Dis¸kapi-Ankara, Turkey 3 Department of Biology, Karamanog˘lu Mehmetbey
University, 70200 Karaman, Turkey DOI 10.1007/s11738-015-1930-8
most devastating. To combat losses from these pests, highly toxic pesticides have been used in the past resulting in serious public health concern and also have been reported as unfriendly for environment (Bakhsh et al. 2012).
Weeds are sources of biotic stress in cotton resulting in reduced yield, increased cost of production, and hence income instability (Harvest Choice 2009). The herbicide-tolerant cotton became the need of the hour as weeds in cotton became unresponsive to herbicides used against them; size of the farm kept on increasing while number of farm workers decreased (King et al. 2001). The proper management of weeds has been considered a difficult task and time consuming. The development of glyphosate-tol-erant cotton has been a remarkable achievement as it has made weed management in cotton quite easy, comfortable and is compatible with environment.
The fungal pathogens cause 8–12 % yield losses annu-ally. Cotton fungal diseases such as Verticillium, Fusarium wilt and Alternaria lead to wilt and lesions on plant parts (Cui et al.2000). Cotton is also susceptible to attack by geminiviruses. Cotton leaf crumple and leaf curl viruses are major threats to cotton production in cotton-growing countries. The damage to cotton crop has been studied in Nigeria, Sudan and Tanzania where the disease caused a reduction in the number of bolls by 87.4 and 38.8 % in boll weight and 92.2 % in cotton seed yield (Singh et al.1999; Monga et al.2011; Mahmood-ur-Rahman et al.2012).
The development of cotton tolerant to various biotic stresses has been a challenge for the researchers. Drought stress is a critical factor in cotton adversely affecting fiber yield and quality of lint at the reproductive phase (Maqbool 2009). Salinity also affects cotton yield by hampering growth as a result of osmotic stress. Increased salt accu-mulation in transpiring leaf causes induction of leaf senescence (Munns2002; Pic et al.2002). Therefore, cot-ton breeding program focused mainly on tolerance to abi-otic stress.
The development of the synthetic fiber industry has stimulated the natural fiber industry to improve its fiber qualities. Hence cotton varieties with improved fiber characteristics can make a significant impact on the world economy (Li et al.2009a,b; Bajwa et al.2013).
Genetic engineering technologies allow the researchers to transfer one or more genes to plant genome. The widely used method is Agrobacterium-mediated transformation. Agrobacterium tumefaciens naturally infects the wound sites in dicotyledonous plants and causes crown gall tumor formation. The first evidence indicating this bacterium as the causative agent of the crown galls was reported by Smith and Towsend (1907) while Zaenen et al. (1974) reported that T-DNA transferred from Ti plasmid in bac-terium to plant cells is the cause of crown gall disease
(Horsch et al. 1985). The transfer and integration of T-DNA to the plant genome depend on many bacterial and plant specific factors which include plant genotype, selec-tion of binary vector, bacterial strain and explant, plant culture media and use of antibiotics to suppress the growth of agrobacterium following cocultivation (Alt-morbe et al. 1989; Bidney et al. 1992; Komari et al. 1996; Hiei et al. 1994; Nauerby et al. 1997; Klee 2000).
In 1987, the first genetically altered cotton plant was reported (Firozabady et al. 1987; Umbeck et al.1987) by two different research groups. The 12 days old aseptically germinated seedlings and hypocotyl were used as explants. The phosphotransferase (npt II) was transferred to these transgenic plants and later its integration was confirmed by molecular analysis. These studies triggered research in cotton transformation with different genes of economic interest (Haq2004).
Cotton regeneration via callus has been challenging except Coker genotypes that have been reported as responsive for gene transfer. Therefore, the genes of interest were transferred to Coker at first and then back-crossed into other cotton varieties using conventional method (Satyavathi et al.2002). However, the development of tissue culture protocols to induce efficient proliferation in a genotype-independent manner continued to transfer desirable traits in cotton. In this context, a method of rapid genotype-independent transformation and regeneration of cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobac-terium-mediated transformation (Gould and Magallanes-Cedeno1998; Zapata et al.1999; Keshamma et al.2008; Khan et al. 2011; Rao et al. 2011; Bakhsh et al. 2012; Bajwa et al.2013).
Insect-resistant cotton
Numerous attempts have been made to generate insect-resistant cotton by transferring genes from unrelated sources (Lycett and Grierson1990; Dhaliwal et al. 1998). Genes from Bacillus thuringiensis (Bt) which are toxic to lepidopterans (Hofte and Whitely1989; Cohen et al.2000), coleopterans (Krieg et al.1983; Herrnstadt et al.1986) and dipterans (Andrews et al.1987) were introduced in cotton. Presently, the area under GM cotton (insect and herbicide resistant) is more than 100 million hectares worldwide (Fig.1) and is expected to be increased in coming years.
An important achievement was reported by Monsanto USA by the modification of Bt genes (cry1Ab and cry1Ac) for better expression in plant cells (Perlak et al. 1991). Expression of these modified genes in cotton (cry1Ac) and potato (cry3Aa) resulted in significant protection against lepidopteran and coleopteran pests, respectively. The Bt
gene was inherited as a single dominant Mendelian trait in the progeny and plants were phenotypically normal. Field tests of these transgenic plants showed good protection against cotton pink bollworm (Pectinophora zea) (Perlak et al.1990). Cotton cultivars transformed with insecticidal gene cry1Ab have been reported by various researchers using Agrobacterium-mediated transformation (Majeed et al.2000; Tohidfar et al.2008; Khan et al. 2011,2013) (Table1). These transgenic lines have shown significant resistance against targeted insect pests.
A less laborious and simple protocol for production of cotton transgenics by co-cultivating embryogenic calli with A. tumefaciens harboring cry1Ia5 gene was reported by Leelavathi et al. (2004). Wu et al. (2005) reported the generation of 12 insect-resistant cotton plants expressing cry1Ac and API-B. The transgenic cotton plants showed high Heliothis armigera larval mortality in bioassays. Katageri et al. (2007) reported insect-resistant Indian cot-ton genotype (Bikaneri Nerma) harboring cry1Ac gene. Insect bioassays and field tests of the promising lines (T2 and T3 generations) ensured potential of the transgenic cotton for resistance against cotton bollworm. Hussain et al. (2007) transformed a Pakistani cotton cultivar with snow drop Galanthus nivalis (GNA) and cry1Ac gene to encode resistance against sucking as well as chewing insect pests. Transgenic cotton lines expressing cry1C, cry2A and cry9C showed enhanced resistance against Heliothis armigera (Guo et al.2007). The efficacy of cry1F insec-ticidal protein was deemed satisfactory for the control of
fall armyworm (Spodoptera frugiperda) in cotton by Sie-bert et al. (2008) under field conditions.
Rashid et al. (2008) transformed a Pakistani local cotton variety CIM-482 with two insecticidal genes (cry1Ac and cry2A) using Agrobacterium-mediated transformation to engineer durable resistance in cotton against targeted insect pests. Transgenic lines showed mortality of 75–100 % of second instar of Heliothis armigera compared with 0 % for the control. Later on, the same transgenic lines were sub-jected to extensive field trail to evaluate their efficacy against targeted insect pest in further generations (Bakhsh et al. 2009). The results showed significant increase in yield of cotton lines in multilocational trials. Many researchers have evaluated the resistant level of transgenic cotton lines against targeted insect pest by leaf biotoxicity and artificial field infestation assays (Fig.2) (Rashid et al. 2008; Bakhsh et al.2009; Khan et al.2011,2013). Bakhsh (2010) reported the genetic transformation of cotton culti-var NIAB-846 transformed with insecticidal genes driven by green tissue-specific (RbcS) and 35S CaMV promoters. Furthermore, the insecticidal gene expression was com-pared in transgenic cotton plant under these two different promoters (Bakhsh et al. 2012). These transgenic lines consistently expressed cry1Ac protein under control of tissue-specific promoter compared with CaMV 35S promoter.
To counteract resistance development in insects, various strategies have been proposed: the expression of multiple Bt toxins at high doses (Gryspeirt and Gre´goire 2012), Fig. 1 Area by country under GM cotton (insect and herbicide
resistant). Countries like Costa Rica, Colombia and South Africa have planted \0.1 million hectare of GM cotton. The data have been
collected and reproduced from sources, James (2013) and GMO Compass; retrieved on December 09, 2014
fusing Bt toxins together (Naimov et al.2003), or Bt toxin tagged with any non-Bt gene having lectin capability to boost the binding domains of Bt in insect midgut (Mehlo et al.2005). The research suggests that the targeted larvae of commercial crops, including cotton, are likely to develop partial resistance against single Bt toxin. In addi-tion to gene pyramiding and refugia strategy, chloroplast transformation has been suggested as a means of localized expression of transgenes in chloroplasts (Lo¨ssl and Waheed 2011; Kiani et al. 2013).
The vegetative insecticidal protein genes (vip) from Bacillus species (Bacillus thuriengenesis and Bacillus cereus) have been used to transform crop plants. Vip1 and Vip2 genes induce significant insecticidal activity against western corn rootworm (Diabrotica virgifera), but do not show any activity against lepidopteran insects while Vip3 toxins group which does not have any similarity to Vip1 and Vip2 showed remarkable insecticidal activity against lepidopterans of maize and cotton (Estruch et al. 1996; Fang et al. 2007).
Table 1 Salient selected examples of insect, herbicide, fungus and abiotic stress-tolerant cotton. The introduced genes with encoded traits have been compiled
Character Gene introduced Encoded trait
(resistance/tolerance against)
References
Insect resistance cry1Ac?cry1Ab Helicoverpa zea and Spodoptera exigua
Perlak et al. (1990)
cry1Ab Helicoverpa armigera Majeed et al. (2000) cry1Ia5 Heliothis armigera Leelavathi et al. (2004)
cry1Ac Heliothis armigera Wu et al. (2005)
cry1Ac Helicoverpa armigera Katageri et al. (2007)
cry1Ac Heliothis armigera Hussain et al. (2007)
cry1C, cry2A, cry9C Helicoverpa armigera Guo et al. (2007) cry1F Spodoptera frugiperda Siebert et al. (2008) cry1Ac?cry2A Heliothis armigera Rashid et al. (2008)
cry1Ab Heliothis armigera Tohidfar et al. (2008)
cry1Ab Heliothis armigera Khan et al. (2011)
cry1Ac Heliothis armigera Bakhsh et al. (2012)
cry1Ac Heliothis armigera Kiani et al. (2013)
Herbicide tolerance EPSPS Glyphosate Padgette et al. (1996)
aroA-M1 Glyphosate Zhao et al. (2006)
Pat Glufosinate Daud et al. (2009)
Mutated EPSPS Glyphosate Tong et al. (2010)
Wilt resistant cotton Gastrodianin Verticillum dahliae Wang et al. (2004) Chitinase Verticillum dahliae Tohidfar et al. (2005) D4E1 (antimicrobial
peptide)
Verticillum dahliae and Fusarium verticillioides
Rajasekaran et al. (2005)
Arabidopsis NPR1 Verticillum dahliae Parkhi et al. (2010a,b) P35 (antiapoptotic) Verticillum dahliae Tian et al. (2010)
Hpa1Xoo Verticillum dahliae Miao et al. (2010)
Lipid transfer protein Verticillum dahliae Munis et al. (2010)
GbVe Verticillum dahliae Zhang et al. (2012a,b)
Abiotic stress tolerance LOS5 Drought tolerance Yue et al. (2012)
GHSP26 Drought tolerance Maqbool et al. (2010)
IPT Salt tolerance Liu et al. (2012)
betA Drought, heat and chilling Chen and Murata (2002),
Park et al. (2004), Yang and Lu (2005), Zhang et al. (2008a,b), Yang et al. (2008), Fitzgerald et al. (2009), Zhang et al. (2009), Duan et al. (2009), He et al. (2010), Bao et al. (2011) and Zhang et al. (2012a,b)
Besides cotton bollworms, another class of insect pests affecting cotton productivity is sap-sucking pests from hemiptera order mainly jassids, whiteflies and aphids (Amudha et al.2011). These pests are multiplying fast and have different feeding behaviors compared with others insects from lepidoptera. This makes them more difficult to control by conventional insecticides. The genes from Bacillus thuringiensis are not effective against sap-sucking pests. The plants naturally defend themselves against these insects by the synthesis of different proteins, i.e., lectins and protease inhibitors (Yarasi et al. 2008). The mecha-nism of actions of plant lectins and protease inhibitors has been extensively studied (Vijayan and Chandra 1999; Vasconcelos and Oliveira 2004). Genes for plant lectins have been transferred to cotton successfully (Rao et al. 1998; Yao et al.2003; Wu et al.2006; Yarasi et al.2008)
and their efficacy is well documented (Hussain 2002; Vajhalal et al. 2013).
In addition to the common approaches of achieving insect resistance, plant-mediated RNAi technology has emerged to combat insects, especially to address resistance development in targeted insect pests (Price and Gatehouse 2008). RNAi initially characterized in Caenorhabditis elegans (Fire et al.1998) has emerged as an efficient gene-silencing approach in various organisms (Hannon 2002). The gene of different insects has been knock downed via orally fed dsRNA including insects from Hymenoptera (Lynch and Desplan 2006), Coleoptera (Tomoyasu et al. 2008), Diptera (Dzitoyeva et al. 2001), Lepidoptera (Terenius et al. 2011). However, results from reports of Mao et al. (2011), Zhu et al. (2012) and Mao and Zeng (2014) are more encouraging who knock downed Fig. 2 The efficacy of insecticidal genes in transgenic cotton
evaluated by leaf biotoxicity and artificial insect infestation of plants. The fresh leaves from transgenic plant are taken and placed on a wet filter paper in petri dish with Helicoverpa larvae which are pre-fasted for 4–6 h. Mortality rates of Helicoverpa are recorded. a Alive larvae
feeding on a non transgenic leaf, b dead larvae after ingesting tissue of transgenic leaf. c and d A glass vial containing Helicoverpa larvae is tied to the main stem of the plant and opened to allow the insects to feed on the plant. Plant health and number of the bolls are recorded before and after infestation (Bakhsh et al.2009)
cytochrome P450 gene (CYP6AE14) in cotton, ecdysone receptor (EcR) and hunchback (hb) gene in tobacco to encode resistance against Helicoverpa armigera, Spo-doptera exigua and Myzus persicae, respectively. How-ever, the technology is at its early phase and being investigated by different research groups worldwide.
GM crops have the potential to contribute significantly to global food security and poverty reduction. The impact studies of insect-resistant cotton show that this technology is highly beneficial for farmers and consumers economi-cally, as well as it has positive effects on the environment and human health (Qaim2009) (Table2). Biotech cotton in China generated economic benefits valued at over $15 billion between 1996 and 2012, with $2.2 billion gained during the past year. India enhanced farm income by use of Bt cotton by US$5.1 billion in the period 2002–2008 and US$1.8 billion in 2008 alone (Brookes and Barfoot2010) while US$1.7 billion was reported from Pakistan recently (Kouser and Qaim 2012). The adoption of transgenic insect-resistant cotton has resulted in the reduction of use of insecticides worldwide, Mexico (77 %), China (65 %), Argentina (47 %), India (41 %), and South Africa (33 %), respectively, which ultimately led to significant increase in farm yield (Qaim2009).
Herbicide-tolerant cotton
The introduction of herbicide-tolerant crops especially cotton, corn, soybean and canola has made weed man-agement easy since their commercialization. The better weed management ultimately has resulted in improved crop yield and also use of such crops has been friendly (Green2012). The EPSPS gene isolated from CP4 strain of
Agrobacterium has shown tolerance against glyphosate herbicide and was successfully incorporated in cotton and other crops (Padgette et al.1996) (Table1). The first her-bicide-resistant cotton was adopted in 1997 under Mon-santo trait designation (MON 1445/1698) containing CP4 EPSPS as primary gene trait encoding resistance to gly-phosate. Since its introduction, herbicide-resistant cotton has been adopted at a very rapid pace. Later on, glufosi-nate-resistant cotton was introduced in 2009 (Table3). The mechanism of glyphosate action has been described by Duke et al. (2003).
Zhao et al. (2006) reported the development of herbi-cide-tolerant Chinese cotton cultivar (Zhongmian 35). In this work, glyphosate tolerance encoding gene (aroA-M1) fused with chloroplast transit peptide under the control of 35S promoter was introduced in cotton. The efficacy of introduced gene in T0 and T1progeny showed improved tolerance for glyphosate. The glufosinate tolerance was introduced in transgenic cotton germplasm (BR001) and its background germplasm (Coker 312) using Agrobac-terium-mediated transformation and Pollen tube pathway approaches (Daud et al. 2009). Later on, Tong et al. (2010) reported a mutated cotton cultivar tolerant to gly-phosate. After repeated field evaluations, it was concluded that glyphosate-tolerant transgenic line has a great potential to combat weeds in cotton field. To address the inefficiency of EPSPS gene in cotton male reproductive organs, multiple constructs were transformed under dif-ferent promoters (Chen et al. 2006). After 8 years of field test, MON 88913 event was found the best expressing EPSPS in vegetative as well as in reproductive organs (Cerny et al. 2010) that was later commercialized as Roundup Ready Flex Cotton (Monsanto Co., St. Louis, MO, USA).
Table 2 Economic impact of insect-resistant cotton in major cotton-growing countries
India China South Africa Argentina Mexico USA Average
Reduction in insecticide use (%) 41 65 33 47 77 36 49.8
Increase in yield (%) 37 24 22 33 9 10 22.5
Increase in profit (US $/ha) 135 470 91 23 295 58 178.7
Data showed significant economic increase in farm yield as well as reduction in insecticide use (Qaim
2009; Sadashivappa and Qaim2009)
Table 3 Commercialized herbicide-resistant cotton currently being cultivated
Herbicide-resistant cotton Introduced gene(s) Trait designation Commercialized year
Glyphosate CP4 EPSPS MON1445/1698 1997
Two CP4 EPSPS MON88913 2006
ZM-2MEPSPS GHB614 2009
Glufosinate PAT A2704-12 2009
EPSPS gene has been isolated from agrobacterium tumefaciens strain CP4 while PAT gene has been isolated from Streptomyces hygroscopicus
The herbicide-tolerant crops were planted on 126 mil-lion hectares area out of total 160 milmil-lion hectares. Among these 126 million hectares, herbicide-resistant crops cov-ered an area of 94 million hectare as a single trait while as a stacked trait (insect and herbicide resistance), these crops covered an area of 42 million hectares (James2011; Green 2012). The worldwide adoption of herbicide-tolerant crops has gradually increased (about 10 % per year). The critics of herbicide-tolerant crops claim that this technology is only beneficial for large-scale crop production while higher yields have been reported from small-scale farmers who benefit from advanced weed control methods by planting such crops (Green 2012). It is clear that the herbicide resistance trait does not give any agronomic advantage or disadvantage to the crops. Some critics often object the technology as harmful for public health, environment and also a source of generation of superweeds. However, many researchers have concluded that herbicide-resistant crops are relatively safe, quite efficient in weed control and environmentally friendly (Entine2006).
Wilt-resistant cotton
Genetic engineering has resulted in the development of cotton with increased resistance against fungi. Cotton is prone to a few fungal diseases, but Fusarium and Verticil-lium wilt are the most important diseases under certain environmental conditions. Fusarium wilt is caused by soil-inhabiting fungus Fusarium oxysporum while the causative agent for Verticillium wilt is Verticillium dahlia. The majority of the cotton cultivars (upland) show susceptibility to V. dahliae and no successful breeding of cotton varieties with the resistance against Verticillium wilt has been reported yet. V. dahliae remains in soil for a long time in the form of microsclerotia (Miao et al.2010; Klosterman et al. 2009). Symptoms may be observed on relatively young plants. However, the expression is greater after flowering. Foliar symptoms consist of interveinal chlorosis or necrosis. As the disease progresses, severe stunting and prema-ture defoliation can occur. Discoloration of the vascu-lar system can be observed on infected plants. Younger bolls may abscise or become malformed. Symptoms of the Verticillium can be confused with Fusarium wilt, and may require laboratory diagnosis. The researchers have made untiring efforts to obtain transgenic cotton resistant to V. dahliae. Genes used for these studies included GbVe (Zhang et al.2012a,b), Arabidopsis NPR1 (Parkhi et al. 2010a,b), anti-apoptotic gene p35 (Tian et al.2010), HR-induced Hpa1Xoo (Miao et al.2010) and some antifungal genes, including chitinase (Tohidfar et al. 2005), D4E1 (Rajasekaran et al. 2005), lipid transfer proteins (Munis et al. 2010) and gastrodianin (Wang et al. 2004). The
transgenic seedlings showed varying levels of resistance from complete control of Verticillium growth in plant extracts engineered (Tohidfar et al.2005; Rajasekaran et al. 2005), to increased resistance against disease under field trials (Zhang et al.2011; Parkhi et al. 2010a; Miao et al. 2010; Wang et al. 2004) or enhanced resistance response against weak pathogen varieties only (Parkhi et al.2010b). The transgenic cotton, showing improved tolerance to Verticillium dahliae, has been developed by Tohidfar et al. (2005). The crude leaf extracts from transgenic cotton expressing a chitinase gene inhibited V. dahlia. Transgenic cotton plants showing resistant to Fusarium and Alternaria have also been reported by Ganesan et al. (2009). The hairpin protein Hpa1Xoo from Xanthomonas oryzae pv. oryzae causes the hypersensitive cell death in plants. Miao et al. (2010) transformed a susceptible cotton cultivar with hpa1Xoo to encode resistance against Verticillium dahliae (Fig.3). Zhang et al. (2012a, b) reported the transgenic cotton cultivar harboring a tomato homologous gene, Gbve1 with improved resistance against Verticillium wilt.
Virus-resistant cotton
Cotton leaf curl disease (CLCuD) is one of the major problems in cotton production and has emerged as a serious disease. CLCuD is transmitted by the whitefly, Bemisia tabaci and is associated with members of genus Bego-movirus (family Geminiviridae) collectively referred to as cotton leaf curl virus (CLCuV). Geminiviruses occur mainly in tropical and warm climatic areas, where the viruses have unleashed important diseases and significant agricultural losses in dicotyledonous hosts (Mahmood-ur-Rahman et al. 2012). The genome of geminiviruses is either monopartite or bipartite, and DNA b of monopartite viruses is gener-ally required for viral infection (Mansoor et al. 1993;
Fig. 3 Resistance phenotypes of hpa1Xoo-transformed T-34 and untransformed Z35 to Verticillium wilt in the nursery as reported by Miao et al. (2010) with permission
Mahmood-ur-Rahman et al.2012). CLCuV-infected cotton plants contain CLCuV DNA A and a single-stranded satel-lite DNA molecule called DNA b (1350 nt) which together induce symptoms typical of CLCuV, both in cotton and tobacco (Briddon and Markham 2000). DNA b requires CLCuV DNA A for replication and encapsidation, and encodes putative proteins that share no homology with the DNA b of other begomoviruses.
The development of virus-resistant transgenic cotton is environmentally safe for the management of viral disease (Beachy 1997). The virus-resistant transgenic cotton has been produced using two different approaches. Either gene used for encoding resistance against particular virus is a part of the virus itself, or taken from another source. The pathogen-derived resistance (former approach) utilizes a fragment or complete viral gene to be transformed in plant that further meddles viral replication. The mechanism behind the viral resistance in plants has been clearly explained. It had been initially proposed that these small RNAs might be involved in the production of the double-stranded RNA (dsRNA) to serve as a target for PTGS, since they were shown to be complementary to targeted mRNAs. The dsRNA degradation results in the small RNAs, and their role would be to act as a silencing signal (Hammond et al. 2001). In case of begomoviruses, expression of viral coat protein (Neves-Borges et al.2001), replicase (Yang et al. 2004) and movement proteins has proved more promising. The viral CP gene was the first and one of the most widely used genes to confer pathogen-derived resistance against plant viruses (Prins2003). CP-mediated resistance has been successfully applied to numerous crop species (Beachy1997; Miller and Hemen-way1998; Pang et al.2000).
Geminivirus replicates inside the host nucleus, where sense mRNAs (translatable RNA) are transcribed, as anti-sense transcripts are also produced in the host nucleus, so that there are chances for duplex formation between the sense and the antisense mRNA. dsRNA duplex is prone to degradation. Therefore, it is possible that there would be greater likelihood of success in using antisense RNA technology for engineering virus-resistant plants against geminiviruses (Prins2003; Waterhouse et al.1998). Anti-sense RNA expression against the CLCuV DNA A-specific genes could provide tolerance and/or resistance to CLCuV in elite cultivars of cotton.
Amudha et al. (2011) developed cotton transgenics resistant to CLCuD using antisense coat protein (ACP). A binary vector carrying the ACP gene along with the nptII (neomycin phosphotransferase II) gene driven by CaMV-35S promoter and nos (nopaline synthase) terminator was used for the transformation. The transgenics were raised in the greenhouse individually and screened for virus resis-tance by inoculating with viruliferous whiteflies. Following
the infection with the viruliferous whiteflies, transgenic plants remained symptomless (Fig.4).
Discovery of RNA interference has opened up entirely new arena for its therapeutic potential in controlling viral diseases. The phenomenon has been reported as evolu-tionary conserved in plants, animals and fungi and is known to protect cells from invading viruses and trans-posons. Chakrabarty et al. (2010) utilized RNAi-mediated approach successfully to target CLCuV in cotton.
Abiotic stress-tolerant cotton
Abiotic stresses in plants are limiting factors in crop pro-ductivity. Drought hampers crop productivity and quality significantly (Boyer 1982; Araus et al. 2002; Hong-Bo et al. 2005). The researchers have elucidated the stress signaling and regulatory pathways using advanced molec-ular approaches (Fig.5). Genes encoding tolerance to drought, salinity and chilling stresses are being introduced in crop plants as single gene or pyramided genes strategy (Lata et al. 2011; reviewed in Bakhsh2014).
The conventional plant breeding has focused on the development of cotton with enhanced drought tolerance; however, the pace for developing new cultivars has been relatively slow (Bakhsh 2014). Drought stress induces a cascade of physiological and biochemical responses in plants. The various organic osmolytes, metabolites (abscisic acid), late embryogenesis abundant proteins (LEA), regu-latory proteins are induced under stress conditions (Shi-nozaki and Yamaguchi-Shi(Shi-nozaki2007). The various genes involved in ABA synthesis are attempted to introduce in crop plants (Xiong and Zhu2003; Taylor et al.2005).
LOS/ABS locus encodes a molybdenum co-factor that is essential for activating aldehyde oxidase that further Fig. 4 Plants screened for cotton leaf curl disease (CLCuD) after being inoculated with viruliferous whitefly (24 h after acquisition period), adapted from Amudha et al. (2011) with permission
catalyzes abscisic aldehyde to abscisic acid in last step of ABA synthesis. A Chinese cotton cultivar overexpressing LOS5/ABA3 showed enhanced drought tolerance as com-pared with control. Due to reduced transpiration, more ABA and proline accumulation resulted in improved drought tolerance in transgenic plants; increased antioxi-dants were also reported (Yue et al. 2012). Soybean overexpressing LOS5/ABA3 has also been reported to be tolerant to drought stress in field conditions (Li et al.2013). Recently, Mittal et al. (2014) reported transgenic cotton (Gossypium hirsutum) expressing AtRAV1/2 (one of the gene families of Basic 3-DNA-binding domain transcrip-tion factors) and/or AtABI5 (one of basic leucine zipper transcription factors) showed resistance to drought stress under field and green house conditions. The improved photosynthesis and water use efficiency (associated with absorption through larger root system and greater leaf area) in plants were reported. Transgenic plants expressing stacked AtRAV1/2 and AtABI5 were superior.
The heat shock proteins are induced in plants when plants undergo water deficiency (Joshi and Nguyen 1996; Vierling1991). The role of heat shock proteins has been well described by Zhu et al. (1993). Maqbool et al. (2007, 2010) developed a cotton cultivar overexpressing GHSP26; a small heat shock protein gene. The transgenic plants accumulated GHSP26 transcripts when water was with-drawn for a specified period. Expression of a small heat
shock protein was reported many folds higher in transgenic cotton plants as compared with control variety. The role of universal stress proteins against abiotic stress has also been established in cotton (Shamim et al.2013).
Salinity is considered a threat to the agriculture world-wide (Cha-um et al. 2006). The salinity affects approxi-mately 20 % cultivated area worldwide (Zhu2001). Cotton is a salt-tolerant crop; however, cotton seedlings are more susceptible to saline condition during germination (Ashraf 2002). This environmental challenge causes substantial crop losses and hence reduces yield (Ashraf and Harris 2004). Many factors such as low rainfall and higher surface evaporation, use of salty water for irrigation, contamination of freshwater with sea water and improper agricultural practices are mainly responsible for saline soils (Foolad 2004).
Salt stress leads to alterations in physiological responses (Nawaz et al. 2010), integrity of cellular membranes and activity of various enzymes are unjustified (Khan 2003) and also impaired nutrient uptake (Chaves et al. 2009). Photosynthesis is one of the major physiological processes which affects adversely and reduces plant growth and yield due to restricted functioning of stomata or non-stomatal limitations and/or combination of both (Saeed et al.2009). Different plant developmental stages such as seed germi-nation, root enlargement and expansion, leaf area, shoot development and seed production are adversely affected
ABIOTIC STRESSES
Drought, Salt, Cold, Heat
Signal Percepon
ABA-Dependent ABA-Independent
NAC/ZF-HD AREB/ABF (bZIP)
MYC/MYB DREB2/CBF Drought-Salt DREB
Cold-DREB/CBF
MYCR/MYBR
ABRE NACR/HDZFR DRE/CRT
Acvaon/Expression of Target Stress Inducible Genes
STRESS TOLERANCE Fig. 5 An illustration of signal
transduction pathways during abiotic stress, as previously described by Lata et al. (2011). Dehydration results in induction of ABA biosynthesis that further activates two regulatory ABA-dependent gene
expressions; bZIP/ABRE and the MYC/MYB. DREBs are the members of the ERF family of transcription factors and follow ABA-independent signal transduction pathway. DRE drought responsive element, ABRE abscisic acid responsive binding element, MYBRS MYB recognition site, MYCRS MYC recognition site, bZIP basic-domain leucine zipper (reviewed in Bakhsh2014)
due to salt stress which results in significant decrease in biomass production and yield (Ahmad et al.2002).
Salinity affects growth of the cotton plant as a result of osmotic stress and also results in senescence of leaves with accumulated salts (Munns 2002; Pic et al. 2002). The studies revealed that these processes are connected to the hormonal signals that are produced during salt stress con-dition. Ghanem et al. (2008) reported the reduction of cytokinin contents gradually in salt-stressed tomato. Hence, it was believed that salinity tolerance in crop plants can be improved if senescence of leaves (salt induced) is delayed. The researchers believe that the introduction of Agrobacterium tumefaciens isopentenyl transferase (IPT) gene is capable of not only delaying leaf senescence but also enhancing salt stress tolerance. Recently, delayed leaf senescence in transgenic cotton plants expressing isopen-tenyl transferase was reported by Liu et al. (2012). These transgenic cotton plants showed improved salt stress under salt stress of 200 mM NaCl.
The glycine betaine is believed to have a crucial role in many plants during abiotic stress (drought, salinity and chilling). The synthesis of glycine betaine from choline comprises two steps, i.e., choline is converted to betaine aldehyde which in turns converts to glycine betaine. The first step is catalyzed by choline oxygenase while second step is catalyzed by betaine aldehyde dehydrogenase (Rhodes and Hanson1993). The studies are evident for the accumulation of glycine betaine in normal cotton plants (Blunden et al.2001; Gorham1996); however, these gly-cine betaine contents are varying among genotypes. More accumulation of endogenous gene leads to more stress responsive cotton genotype (Meek and Oosterhuis 2000; Naidu et al.1998). The overexpression of genes encoding glycine betaine led to the induction of abiotic stress tol-erance in cotton (Lv et al.2007).
The overexpression of choline oxygenase in cotton resulted in more accumulation of glycine betaine and hence more salt stress tolerance in cotton seedling (Zhang et al. 2009). Glycine betaine as an osmoprotectant has been studied widely to induce tolerance against drought, salinity, chilling and high temperature stresses (Chen and Murata 2002; Park et al. 2004; Yang and Lu 2005; Zhang et al. 2008a,b,2009,2012a,b; Yang et al.2008; Fitzgerald et al. 2009; ; Duan et al.2009; He et al.2010; Bao et al.2011). Chilling stress is another yield limiting stress factor in crops. The annual losses incurred because of chilling stress have been estimated at 100 billion dollar (Allen and Ort 2001; Deng and Jian2001). Chilling stress results in severe damage of cellular membranes and negative ROS (reactive oxygen species) (Kratsch and Wise2000; Xing and Raja-shekar2001). The ROS activity is responsible for oxidative damage and scrambles cellular functions negatively. Nat-urally, cotton is sensitive to chilling stress and can hardly
withstand if temperature is between 0 and 10°C, especially during early growth stage (Zeng et al. 2012; Zhang et al. 2012a,b). Hence it is evident that temperature below 10°C may limit crop productivity (Bradow 1991; DeRidder and Crafts-Brandner 2008).
Cotton with improved fiber characteristics
Cotton being a natural fiber occupies its important role in agricultural economy of the countries. The cotton fiber characteristics and their quality are the sole of textile industry which includes length, strengthen, maturity and fineness of fiber (Chee and Campbell 2009). As genetic engineering is advancing, the researchers are focusing new ways and means to improve quality of cotton by incorpo-rating exogenous genes.
Cotton fiber is polygenic trait controlled by number of genes. The molecular genetics of multigenic families pro-vide new insights into transcription and expression signa-tures to better understand cotton fiber growth and development. The role of genes in any specific stage of development depends on their function and depends on other related genes with similar functions. The major and minor isoforms of gene family members are expressed at different levels depending on the specific developmental stage of the fiber cell. The expression of expansin genes upregulates fiber cell expansion (Vogler et al.2003; Zhang et al. 2008a, b). Genes contributing to the synthesis of fiber-related proteins, waxes, lignin and polysaccharides are characterized (Ma et al. 1995; John 1996; Song and Allen1997; Orford and Timmis1998; Liu et al.2000). The genes have a specific roles in fiber synthesis have been used as an ideal candidate for the improvement of fiber quality and traits in cotton (Hovav et al.2005; Rapp et al. 2010).
Transgenic cotton engineered with fiber-related genes has been reported to enhance length, strength, color and other fiber-related properties (Richter 1998; May and Wofford 2000; Zhang et al. 2004; Li et al. 2004; Shang-guan et al. 2007; Qin et al. 2007). The Gram-negative bacterium Acetobacter xylinum harbors acsA and acsB genes which contribute to cellulose biosynthesis. Using these genes, Li et al. (2004) reported transgenic cotton lines with improved fiber quality. The silkworm protein fibroin has also been utilized for the fiber improvement. The specific crystalline structure brings it high tensile breaking strength and soft texture (Shen and Wang1990; Chen et al. 2003). The fibroin gene was incorporated in cotton successfully to improve fiber characteristics (Li et al. 2009a,b). A fiber-related gene from a different source was expressed in cotton by Bajwa et al. (2013). They intro-duced fiber-specific gene from Calotropis procera into a
local cotton cultivar and reported increased fiber fineness and strength as compared to the control variety.
Improved cotton oil devoid of gossypol
Naturally cotton plant contains several sesquiterpenes including gossypol in arial plant parts and in the root epi-dermal cells (Bell1986). The presence of sesquiterpenes (gossypol) in arial plant parts is considered as cotton genetic defense umbrella against various insect pests. But the existence of high levels of gossypol in cottonseed makes it low value by-product for feed purpose. Many efforts have been made to address the biochemical syn-thesis of gossypol and other related terpenoids. Benedict et al. (1995) and later on Liu et al. (1999) established that d-cadinene is the first step in the biosynthesis of sesquiterpenoids. d-cadinene results from the catalytic activity of a cyclase enzyme (cadinene synthase) from farnesyl diphosphate. Furthermore, (?)-d-cadinene is hydrolyzed to 8-hydroxy cadinene that is catalyzed by a cadinene-8-hydroxylase, P450 cytochrome mono-oxyge-nase (Luo et al.2001). The following step in the biosyn-thesis involves hydroxylation of (?)-d-cadinene to 8-hydroxy-cadinene that is catalyzed by (?)-d-cadinene-8-hydroxylase, a cytochrome P450 mono-oxyge-nase (Luo et al. 2001). The further biochemcial steps leading to the formation of hemigossypol, gossypol and heliocides are described by Wang et al. (2003) and Bene-dict et al. (2004).
As (?)-d-Cadinene is a critical intermediate in gossypol biosynthesis; therefore, the gene (?)-d-cadinene synthase was targeted using RNAi approaches to reduce gossypol in cotton seed. The objective was to reduce gossypol level in cottonseed while maintaining the level of gossypol same as in other plant parts. The attempts to reduce gossypol levels using anti sense (?)-d-cadinene synthase mechanism remained unfruitful in the start (Martin et al. 2003; Townsend et al. 2005). Either unexpected results were obtained or very less amount of gossypol was reduced. However, later on, a highly seed-specific a-globulin pro-moter was utilized to drive a hairpin transcripts targeting (?)-d-cadinene synthase gene(s) in cotton. Several trans-genic cotton lines expressing ihpRNA transcripts showed significantly reduced levels of gossypol (Sunilkumar et al. 2006). As seed-specific promoters were used in the study, therefore, the levels of gossypol and related terpenoids involved in defense against insects and diseases were not lowered in arial plant parts and roots of transgenic plants as compared to controls. Later on based on 3 years of field trials of these transgenic cotton lines, same group reported that the major difference between the ultra-low gossypol cottonseed (ULGCS) and wild-type cottonseeds was in
terms of their gossypol levels; other seed composition and features remained the same (Palle et al. 2013). The study also demonstrated the potential of RNAi-based constructs for commercial use.
Conclusion and prospects
Cotton is considered as the foremost commercially important fiber crop in all parts of the world and is deemed as the backbone of the textile industry. The productivity of cotton is severely hampered by the occurrence of pests, weeds, pathogens apart from various environmental fac-tors. Using traditional plant breeding approaches, plant breeders introduced many improvements, however, due to limited availability in germplasm; many questions were addressed with the advent of new genetic engineering approaches. The researchers have put forth their efforts to improve various traits in cotton, viz., resistance against insect pests (chewing as well as sucking), herbicide resis-tance, tolerance to abiotic stresses and improved fiber quality, etc. Crop losses, due to biotic factors, are sub-stantial 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. A significant improvement in cotton economy has been reported in countries like USA, China, India and Pakistan (major cotton-growing countries) after the introduction of transgenic Bt cotton cultivars in the agricultural system. Likewise, commercialized herbicide resistance cotton is another successful example of the adoption of new tech-nology. The various gene(s), responsible for tolerance against major abiotic stress factors such as drought and salinity, have been introduced into cotton cultivars. Fur-thermore, genes for improving the seed oil quality and fiber characteristics have also been introduced into cotton cul-tivars. The economic benefits of commercially released insect and herbicides resistance cotton have revolutionized modern agriculture worldwide as highlighted in the review. Transgenic cotton with resistant to fungus and virus has been reported but we have not witnessed any large-scale field trials. Likewise, abiotic stress-tolerant cotton has successfully been developed; however, it is pertinent to note that most of these studies have been conducted in laboratory or in green house under controlled stress con-ditions. Recently, Mittal et al. (2014) reported the perfor-mance of stress-tolerant transgenic cotton expressing AtRAV1/2 and AtABI5 under green house and field con-ditions. The commercialization of stress-tolerant cotton can have a huge impact on sustainable agriculture. The pilot
scale field testing of other stress-tolerant transgenic crops (soybean, peanut, corn and rice) has also been reported from various researchers. The need of the hour is to expand these field evaluations at larger scale to determine the potential of these transgenic crops against abiotic stresses. The cultivation of first drought-tolerant maize in USA in an area of 50,000 hectares in drought-prone belt is a signifi-cant step towards the commercialization of abiotic stress-tolerant crops. Commercializing drought-stress-tolerant cotton will be a remarkable achievement for cotton productivity worldwide as losses from abiotic stresses are quite preva-lent. The economic benefits of insect and herbicides resistance crops have revolutionized agriculture produc-tivity; hence, a further boom in cotton production is being expected by the development of new generation biotech cotton.
Author contribution statement Emine Anayol, Sancer Fatih O¨ zcan and Tahira Hussain collected literature in chronological order. Allah Bakhsh compiled, composed and wrote the main manuscript. Muhammad Aasim, Khalid Mahmood Khawar and Sebahattin O¨ zcan read and criti-cally analyzed the manuscript, and gave valuable sugges-tions that helped to present the article in its current form.
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