Importance and Potential Impact of Genetic Modification in Cotton

Cotton plants are particularly susceptible to a wide variety of insect pests and nematodes, and their cultivation has traditionally relied on the use of large amounts of highly toxic pesticides. Some estimates suggest that, prior to the widespread adoption of Bt cotton, nearly 25% of all insecticides used worldwide were needed for the production of cotton (Pannetier et al. 1997). Genetically modified cotton produced by incorporating the Bt gene was therefore a huge success in the United States following its introduction in 1996 (see also Chap. 11). Amongst the cotton-producing countries, India has the largest area under cotton cultivation and yet it has ranked third in terms of production until very recently. This was because the average yield of cotton in India was one of the lowest in the world. Several factors accounted for this low productivity including insect pests. The yield of lint+cotton-seed in this country averaged 561 kg ha-1 in 2001; however, it increased to 1019 kg ha-1 in 2007 (FAO 2008). In a recent publication from the International Food Policy Research Institute (IFPRI), Gruere et al. (2008) report that in the year 2007/ 08, India's cotton production exceeded that of the United States. Most of this rise in the production is attributed to an increasing use of Bt cotton varieties following their introduction in the year 2002 (Qaim and Zilberman 2003; James 2007; Gruere et al. 2008). It is not surprising then that, once approved by the respective regulatory agencies, cotton growers in many other countries have readily adopted GM cotton. The example of India illustrates the potential impact of biotechnology in enhancing global cotton production. Overall improvements in the production of cotton will be considerable once this technology is adopted by the rest of the cotton producing countries. Currently, Bt-mediated insect resistance and herbicide resistance are the only two transgenic traits available in cotton. When the traits that confer resistance to various other biotic and abiotic stresses become available, the combined impact of various transgenic traits on the total output will be much more substantial than what has been achieved thus far. In addition to its impact on the production, genetic engineering is likely to play a very important role in improving the quality of fiber as well as the seed.

15.3 Transformation of Cotton and its Improvement via Genetic Modification

There are some excellent reviews available on transgenic cotton (Murray et al. 1993; John 1997; Chlan et al. 2000; Wilkins et al. 2000; Rajasekaran et al. 2001; Kumria et al. 2003; Rathore et al. 2008). Table 15.1 provides a list of selected papers describing the key transformation methods and the introduction of certain useful traits via genetic engineering. General aspects of genetic transformation are discussed in Chap. 1.

15.3.1 Methods Used to Transform Cotton

The first two reports on successful cotton transformation were published by scientists at Agracetus (Umbeck et al. 1987) and Agrigenetics (Firoozabady et al. 1987). In both cases, tissue explants obtained from a young seedling were transformed via Agrobacterium tumefaciens. The transformed tissues growing on selection medium were cultured for several months before recovering the transgenic plants via somatic embryogenesis. This procedure is rather long and laborious, and is limited for use in only a few genotypes that are able to regenerate via somatic embryogenesis. However, it is a robust protocol and with some modifications, is widely used to obtain transgenic cotton plants in both academic and industrial laboratories (Table 15.1). A comprehensive investigation was undertaken in author's laboratory to understand both the transformation and regeneration processes (Sunilkumar and Rathore 2001; Rathore et al. 2006). This study made use of green fluorescent protein (GFP) gene as a reporter and showed clearly that the transfer of T-DNA per se, from Agrobacterium to the cotton cells at the wound site in a cotyledon, hypocotyl and cotyledonary petiole, is an efficient process. In addition, its integration into the cotton genome is also quite effective. It is the culture of transformed cells to obtain a friable, embryogenic callus capable of plant regeneration, that is a highly genotype-dependent process (Trolinder and Xhixian 1989). Even with the regenerable genotypes, a high degree of tissue culture skills are required to obtain transformed cotton plants. Bearing in mind the difficulties faced by many researchers in producing transgenic cotton, a simplified protocol describing various steps in detail has been published (Rathore et al. 2006).

Thus, genotype-dependence, in terms of regeneration via somatic embryogenesis, does remain a limitation in introducing a transgenic trait directly into commercial varieties. The same constraints also apply to methods that utilize particle bombardment-mediated transformation of cultured cells. These limitations have served as an impetus to find alternative methods to produce transgenic cotton. Since regeneration of plants from shoot apical meristem is genotype-independent, relatively rapid, and a rather straightforward process, many laboratories have targeted the cells within this explant for transformation. The research involving

Table 15.1 Summary of selected studies on transgenic cotton. C Cotyledon, ESC embryogenic cell suspension, H hypocotyls, P cotyledonary petiole, SAM shoot apical meristem

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Analysis and Comments

Reference

Agrobacterium Agrobacterium Gene gun

Agrobacterium Agrobacterium

Agrobacterium

Gene gun

Gene gun

Gene gun

Coker 310, 312,

5110 Coker 201

Coker 310

Coker 312 Coker 312

Coker 315

Delta Pine 50, Delta Pine 90, Sea Island, Pima S-6 Coker 312, Delta Pine 50, Sea Island

Delta Pine 50

H/somatic embryogenesis C/somatic embryogenesis ESC/somatic embryogenesis

H/somatic embryogenesis H/somatic embryogenesis

C/somatic embryogenesis

SAM from mature seed/shoot regeneration in culture SAM from mature seed/shoot regeneration in culture SAM from mature seed/ Shoot regeneration in culture cat and nptll nptll and OCS

CrylAc, CrylAb and nptll nptll and tfdA

nptll, gusA and tfdA gusA

Fiber-specific,

FbL2A promoter driving phaB and phaC, and gusA Fiber-specific, E6 or FbL2A promoter driving phaB and phaC, and gusA

Enzyme assays and Southern for confirmation Immunoblot and Southern for confirmation Southern for confirmation

Western and bioassay for confirmation 2,4-D monooxygenase activity, PCR, and 2,4-D resistance for confirmation Southern, GUS enzyme assay and 2,4-D resistance for confirmation GUS histochemical analysis and Southern for confirmation

GUS assay, Southern, Western, and biochemical analyses for confirmation

GUS assay, Southern, Northern, microscopic, and biochemical analyses for confirmation; fiber's thermal properties were altered

Umbeck et al.

  • 1987) Firoozabady et al. (1987) Finer and McMullen (1990) Perlak et al.
  • 1990) Bay ley et al.

Lyon et al.

McCabe and Martineil (1993)

Rinehart et al. (1996)

John and Keller (1996), Chowdhury and John (1998)

Agrobacterium Coker315and C, H, ESC/somatic and gene gun Acala varieties embryogenesis

Agrobacterium

Agrobacterium

Agrobacterium

Coker 312 CUBQHRPIS Coker 315

Agrobacterium Coker 312

Agrobacterium Coker 312

Agrobacterium Coker 312 Agrobacterium Coker 312

H/somatic embryogenesis

SAM from seedling/ shoot regeneration in culture

H/somatic embryogenesis

H/somatic embryogenesis

H/somatic embryogenesis

H/somatic embryogenesis

H/somatic embryogenesis

Agrobacterium

Coker 315

C/somatic embryogenesis nptll, mutant native AHAS genes nptll, FMV 35S promoter diving CP4-EPSPS nptll, gusA

Tobacco basic chitinase, glucose oxidase, nptll nptll, gusA

Cotton a-globulin promoter driving gusA, nptll CaMV 35S promoter driving GFP gene, nptll

Seed-specific RNAi of Cotton SAD-1

Southern and resistance to herbicides, imidazolinone and sulfonylurea, for confirmation Southern, ELIS A, and resistance to herbicide, glyphosate for confirmation Resistance to kanamycin and Southern for confirmation

Rajasekaran et al. (1996)

Nida et al.

Some protection against McFadden et al.

verticillium wilt with each gene (2000)

PCR and Southern for confirmation; a step-wise and comprehensive account of transgenic cotton production

Enzymatic assays confirmed overexpression; chilling-induced photoinhibition of photosystem II reduced Histochemical and biochemical analyses; seed-specificity of promoter demonstrated Fluorescence microscopy analysis; developmental- and tissue-specific activity of promoter demonstrated Southern, Northern and biochemical analyses; seed oil

Sunilkumar and Rathore (2001), Rathore et al. (2006) Kornyeyev et al. (2001, 2003a, b)

Sunilkumar et al. (2002a)

Sunilkumar et al. (2002b)

(continued)

Table 15.1 (continued)

Transformation Cultivar method

Target tissue/mode of transformant recovery

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Gene gun

Agrobacterium

Agrobacterium

MCU5, DCH32, Coker 310FR

Coker 312

Coker 312

Coker 315 Coker 310FR Coker 312 Coker 312

Shoot tip from seedling/shoot regeneration in culture C, H/ somatic embryogenesis

H/somatic embryogenesis

C/somatic embryogenesis

H-derived friable callus/somatic embryogenesis

H/somatic embryogenesis

H/somatic embryogenesis

Transgenes

Analysis and Comments

Reference and Cotton FAD2-1, nptll gusA, nptll

Cotton P-tubulin promoter driving gusA, nptll

Endochitinase gene from

Trichoderma virens, nptll

Sense and antisense suppression of sucrose synthase, nptll

Chloroplast- specific expression of aphA-6 and nptll

Cotton ghCTL2 promoter driving gusA, nptll

Seed-specific antisense of cotton FAD-2, nptll with substantially higher stearic acid or oleic acid levels Histochemical, PCR and Southern analyses and resistance of progeny to kanamycin for confirmation Histochemical analyses;

preferential activity in fiber and root tip observed Southern, Northern and biochemical analyses; protection against Rhizoctonia solani and Alternaria alternata observed Southern, immunolocalization, electron microscopy and biochemical analyses; fiber development inhibited PCR and Southern to confirm plastid genome transformation; strict maternal inheritance of Kanamycin-resistance Preferential activity in different cell types during secondary wall deposition including lint fibers

Biochemical analysis; seed oil with higher oleic acid level

Satyavathi et al. (2002)

Sunilkumar et al. (2005)

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Coker 315

Coker 312

Coker 312

Coker 312

Zhongmiansuo 35 Coker 312

Coker 312

C/somatic embryogenesis

C, H/ somatic embryogenesis

H/somatic embryogenesis

C, H/somatic embryogenesis

H/somatic embryogenesis

H, P/somatic embryogenesis

H/somatic embryogenesis

Soybean lectin promoter or CaMV 35S promoter driving antisense cdnl-C4, nptll Synthetic antimicrobial peptide D4E1, nptll Arabidopsis NHX1, nptll

Cotton ACTIN1 promoter driving gusA, RNAi of ghACTl, nptll Phloem-specific promoter driving ACA gene, nptll Seed-specific RNAi of cotton 5-cadinene synthase, nptll

CaMV 35S promoter driving spinach SPS gene, nptll

Southern, Northern, and Western analyses; no reduction in gossypol levels; induction of the target gene by bacterial blight was blocked

Southern, PCR and RT-PCR analyses; transgenic plants resistant to several fungal pathogens PCR, Northern, and Western analyses; more biomass and more fiber produced under salt stress conditions Histochemical analyses;

preferential activity observed in the fiber; RNAi-inhibition of fiber elongation Southern and Western for confirmation; resistance to cotton aphid observed Southern, RT-PCR, Northern and biochemical analyses for confirmation; over 98% reduction in the seed gossypol level obtained Southern, RT-PCR, Northern, Western and biochemical analyses for confirmation; improved fiber quality

Townsend et al. (2005)

Rajasekaran et al. (2005)

Sunilkumar et al. (2006)

Haigler et al. (2007)

particle bombardment of isolated shoot apical meristems followed by the recovery of plants has provided unambiguous evidence for the transgenic status of the regenerants and proved the feasibility of this approach (McCabe and Martinell 1993; McCabe et al. 1998). The gene gun-based microprojectile bombardment is a direct, physical method that can deliver the genes into the epidermal cells of the L1 layer or the germline progenitor cells of L2/L3 layer within the apical meristem. As expected, the progeny plants from L1 transformants did not inherit the transgene. In contrast, the germline transformants resulting from the transformation of L2/L3 cells passed on the transgenic trait to subsequent generations. However, the primary transformants recovered from these shoot apices are chimeric and the efficiencies of recovering germline transformation events are very low. As this method is highly labor- and resource-intensive, it has not been used by others. There are reports from three laboratories on Agrobacterium-mediated transformation of shoot apical meristem to obtain transgenic cotton plants (Zapata et al. 1999; Satyavathi et al. 2002; Uceer and Koc 2006). The ability to tolerate kanamycin was used as a major criterion to identify the putative transformants and each report provided some molecular evidence. However, these reports did not provide any information on the type of cells that were transformed within the shoot apical meristem. The transformation efficiencies reported in these papers differ drastically, thus raising questions about the criteria used to assign transgenic status to the regenerated plants. Additional, convincing evidence that includes phenotypic analysis, molecular proof that discounts the possibility of Agrobacterium contamination of the plant tissue, and genetic analysis in several generations will be needed to confirm the reliability, efficiency, and robustness of this method. If an unambiguous proof of Agrobacterium-mediated transformation of shoot apical meristem is provided, it will ensure a widespread adoption of this technique by other researchers who are interested in a genotype-independent method to transform cotton.

  1. 3.2 Selectable Markers and Reporter Genes used for Cotton Transformation
  2. 3 generally discusses the use of marker genes in transgenic plants. Neomycin phosphotransferase II (nptII) gene in combination with kanamycin as the selection agent was used in the first two investigations reporting successful cotton transformation (Firoozabady et al. 1987; Umbeck et al. 1987). The papers listed in Table 15.1 suggest that this gene continues to be used widely to obtain transgenic cotton. Its wide popularity stems from the fact that kanamycin is relatively inexpensive and does not adversely affect regeneration from cultured cotton tissues. Hygromycin phosphotransferase (hpt) gene is also suitable for producing transgenic cotton and has been used as a selectable marker in some studies (Finer and McMullen 1990). Cotton has been transformed with the bialaphos resistance (bar) gene; however, the initial selection of transgenic tissue was based on the expression of a linked nptII

gene in these studies (Keller et al. 1997). Bialaphos-tolerant cotton has been also developed by Bayer CropScience and is marketed by FiberMax under the name LibertyLink (Perkins 2004). The list provided in Table 15.1 shows that b-glucuronidase (gusA) remains the gene of choice to evaluate different transformation methods as well as for the characterization of promoter activities in various tissues in cotton. This is because GUS activity assays are relatively simple and the enzyme activity can be quantitated (Jefferson et al. 1987). The utility and versatility of GFP reporter gene (allowing non-invasive monitoring of its expression) was demonstrated by observing the tissue- and development-specific activity of CaMV 35S promoter in cotton (Sunilkumar et al. 2002b).

15.3.3 Genetically Engineered Traits in Cotton

Bt was the first commercially useful gene introduced into cotton (Perlak et al. 1990). This cotton was later developed and marketed under the trade name Bollgard by Monsanto/Delta & Pine Land (Jones et al. 1996; Jenkins et al. 1997). These plants expressed a truncated, codon-modified CrylAc gene from Bacillus thurin-giensis (Bt) encoding a 8-endotoxin that is toxic to tobacco budworm and American bollworm (Jenkins et al. 1997). These Bt cottons were readily accepted by farmers in the United States and other countries that had allowed their cultivation. Bollgard II, introduced in 2003, contains Cry2Ab in addition to CryIAc (Micinski et al. 2006; Robinson 2006). This second Bt gene broadens the resistance to include fall armyworm, beet armyworm, cabbage looper, and soybean looper (Perlak et al. 2001). Syngenta has developed VIP-Cotton containing a different gene from B. thuringiensis that encodes a vegetative insecticidal protein (VIP; Estruch et al. 1996). The VIP is structurally, biochemically, and functionally different from the Bt 8-endotoxins and exhibits insecticidal activity against a variety of lepidopterans (McCaffery et al. 2006). Another type of insect-resistant cotton has been developed by Dow AgroSciences by combining CryIF and CryIAc genes. This product, WideStrike cotton, also confers resistance to several Lepidopteran pests (Bacheler et al. 2006; Micinski et al. 2006). Thus, a choice of more than one insect resistance genes with different modes of action, especially if they are stacked, will help broaden the spectrum of insects that can be controlled by the genetically modified plants and also help counter the development of resistance in the target insects.

Roundup Ready cotton that is resistant to glyphosate-based herbicide (see also Chap. 10 for references) was introduced in 1997 by Monsanto (Nida et al. 1996). This trait was engineered by expressing a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (derived from Agrobacterium sp. strain CP4) under the control of FMV 35S promoter. In 2006, Roundup Ready Flex cotton became available that allows safe application of the herbicide well beyond the five-leaf stage (Chen et al. 2006). Glyphosate-tolerant cottons help in the effective management of weeds and were also readily adopted by the United States cotton growers. Glufosinate- or bialaphos-tolerant cotton, developed by Bayer CropScience and marketed by FiberMax under the name LibertyLink, is also available commercially (Perkins 2004).

As is the case with most other crop plants, no commercial, transgenic products are yet available in cotton that address the problems of biotic or abiotic stresses. However, there are some published reports describing transgene-mediated resistance to various fungal diseases in cotton (Murray et al. 1999; McFadden et al. 2000; Emani et al. 2003; Wang et al. 2004b; Rajasekaran et al. 2005). Although some of these studies appear promising, in each case, the transgene conferred protection to only a limited spectrum of pathogens. Similarly, there are a number of reports describing attempts to engineer cotton to tolerate abiotic stresses, including freezing (Kornyeyev et al. 2001, 2003a, b; Payton et al. 2001), water-logging (Ellis et al. 2000), salt stress (He et al. 2005) and drought (Yan et al. 2004).

Since cotton is grown mainly for its fiber, it is an obvious target for improvement via genetic engineering. In addition to the usual desirable properties that include strength, fineness, length, and uniformity, cotton fiber can benefit from characteristics such as better dye binding, wrinkle resistance, and shrinkage resistance. Improvements in these last three categories will help cotton fiber compete more effectively against synthetic fibers. The number of genes involved in controlling some of these traits is likely to be large and the mechanism controlling these characteristics is expected to be complex. Several laboratories are involved in identifying and isolating genes that are involved in fiber initiation, elongation, and development. As these genes become available and are characterized, their coding and regulatory sequences will be used to engineer the cotton plant to address issues related to fiber quality improvement. Nevertheless, some interesting work to modify cotton fiber has been already conducted by scientists at Agracetus and elsewhere. An early example of such research involved the synthesis of novel biological materials in the fiber. Expression of some genes derived from Alcaligenes eutrophus in the developing cotton fibers resulted in the deposition of poly-D-(-)-3-hydroxy-butyrate (PHB) in their lumens (John and Keller 1996; Rinehart et al. 1996). The modified fiber exhibited altered thermal properties resulting in improved insulating characteristics (Chowdhury and John 1998). Although this product was not developed further, the results demonstrated the feasibility of improving cotton fiber in a manner that is impossible to achieve by traditional breeding methods. Two recent studies have examined the effects of manipulating endogenous gene expression in cotton fiber cells (Ruan et al. 2003; Li et al. 2005). Although each of these studies involved suppression of a cotton gene that adversely affected fiber growth/development, the results indicate the feasibility of altering fiber properties. In a more recent study, Haigler et al. (2007) showed that constitutive overexpression of spinach sucrose phosphate synthase gene in cotton resulted in the improvements in fiber quality when the cotton plants were grown under controlled environmental conditions. Chapman et al. (2008) reported an interesting and unexpected outcome of manipulating the oil composition by overexpressing a non-functional rapeseed FAD-2 gene in cottonseed. They showed that, while the seeds from transformed lines were smaller, of poor quality and had lower oil content, the lint produced was significantly increased, suggesting a redirection of carbon reserves. These reports on transgenic manipulation of cotton fiber are promising. However, considering the importance of this agricultural product, the progress in improving its characteristics and yield through biotechnology has been rather slow. As more fiber-specific genes and their regulatory sequences become available, transgenic technology is expected to make a significant impact on the quality and yield of this most important product of the cotton plant (Li et al. 2002; Wang et al. 2004a).

Cotton plant produces about 1.6 times more seed than fiber. Cottonseed contains ^21% oil and a substantial portion of the global production is used to obtain edible oil. Since cottonseed oil is rather low in monounsaturated fatty acid, gene-suppression technologies have been used to improve its fatty acid composition in favor of higher oleic acid. Use of antisense technology to suppress A-12 desaturase gene resulted in doubling of oleic acid from a wild-type level of ^15% to ^30% and a reduction in linoleic acid level from ^55% to ^35% (Sunilkumar et al. 2005). Interestingly, RNAi-mediated suppression of the same target gene resulted in a fivefold increase in oleic acid level and a concomitant reduction in the linoleic acid (Liu et al. 2002). In a separate set of transformants, RNAi-mediated downregulation of the SAD-1 gene resulted in a > 10-fold increase in stearic acid level in cottonseed oil. Importantly, it was possible to stack the two traits by intercrossing (Liu et al. 2002). These results demonstrate that transgenic technology can be used to modify fatty acid biosynthetic pathway in a tissue-specific manner to improve storage and cooking properties of the cottonseed oil. In addition to the oil, cottonseed also contains ^23% protein that is of relatively high quality. Global cottonseed output of ^44 MMT year1 can potentially meet the basic protein requirements of 500 million people. However, the ability to utilize this abundant, protein-rich resource for food is hampered by the presence of toxic gossypol. This cardio- and hepatotoxic terpenoid, present in cottonseed glands, renders the seed unsafe for human and monogastric animal consumption. Glands containing gossypol and related terpenoids are present in most parts of a cotton plant. The terpenoids are believed to play a protective role in defending the cotton plant against various insect pests and diseases (Hedin et al. 1992; Townsend et al. 2005). To avoid the weakening of defensive capability of the cotton plant, the elimination of gossypol must be strictly limited to the seed. Since traditional breeding methods have failed to achieve this goal, biotechnological approaches were tested in many laboratories around the world to solve the problem of cottonseed toxicity. Most of these attempts over the past decade have been unsuccessful (see Townsend et al. 2005 and references therein). However, in a relatively recent breakthrough, the feat of selective and significant reduction of gossypol in cottonseed was achieved by disrupting its biosynthesis through RNAi-mediated suppression of 8-cadinene synthase activity in the developing seed (Sunilkumar et al. 2006). Some of the RNAi lines obtained showed a 98% reduction in the concentrations of gossypol in the seed. Importantly, these transformants maintained normal levels of gossy-pol and related terpenoids in all other parts of the plant. These studies involving alteration of oil composition and gossypol reduction suggest that a genetically modified cotton plant, in addition to meeting the clothing requirements, can also play an important role in fulfilling the nutritional needs of the growing human population. More details on different engineered traits may be found in Sect. C of this volume.

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