Title of Invention	AGROBACTERIUM-MEDIATED TRANSFORMATION OF COTTON WITH NOVEL EXPLANTS
Abstract	A method is disclosed for producing a transgenic cotton plant comprising the steps of (a) obtaining cottonfibrous root explants, (b) culturing the fibrous root explants to induce callus formation, (c) exposing root callus to a culture of Agrobacterium tumefaciens that harbors a vector comprising an exogenous gene and a selectable marker, the Agrobacterium being capable of effecting the stable transfer of the exogenous gene and selection agent resistance gene to the genome of the cells of the explant, (d) culturing the callusiber the presence of the selection agent to which the selection agent resistance gene confers resistance so as to select for transformed cells, (producing somatic embryo formation in the selected callus culture, and (f) regenerating the induced somatic embryos into whole transgenic cotton plants.

Title of Invention

AGROBACTERIUM-MEDIATED TRANSFORMATION OF COTTON WITH NOVEL EXPLANTS

Abstract

A method is disclosed for producing a transgenic cotton plant comprising the steps of (a) obtaining cottonfibrous root explants, (b) culturing the fibrous root explants to induce callus formation, (c) exposing root callus to a culture of Agrobacterium tumefaciens that harbors a vector comprising an exogenous gene and a selectable marker, the Agrobacterium being capable of effecting the stable transfer of the exogenous gene and selection agent resistance gene to the genome of the cells of the explant, (d) culturing the callusiber the presence of the selection agent to which the selection agent resistance gene confers resistance so as to select for transformed cells, (producing somatic embryo formation in the selected callus culture, and (f) regenerating the induced somatic embryos into whole transgenic cotton plants.

Full Text	Agrobacterium-Mediated Transformation of Cotton With Novel Explants Technical Field The present invention relates to the general field of genetic engineering of plants, in particular to the introduction of exogenous genetic material into cotton by Agrojbacterium transformation of novel explants followed by somatic embryo regeneration. Background Cotton is the most extensively used natural fiber in the textile industry. Its annual production worldwide is over 100 million bales, valued at US$4 5_ billion^ Cotton lint or seed hair is_ a terminally differentiated single epidermal cell from 50 species of the genus Gossypium of the family Malvaceae. It is classified as a natural, cellulosic, monocellular and staple fiber. The cultivated cotton varieties, which have been cultivated for more than 5000 years, all come from two diploids (2n=2x=26) (G_. herbaceum and G. arboreum) and two allotetraploids (2n=4x=52) (G. hirsutum L., Upland; and G. barbadense L. , Sea Island). In 1997 the top five world cotton producers were the United States, China, India_, Pakistan and Uzbekstan, producing about 63 million bales. In the next century, most crops, including cereals, oil crops, fruits, vegetables and other economically important crops, will be genetically engineered with added or modified traits ranging from improvement of yield and quality, to herbicide resistance and pest resistance (Chappell, 1996; Fraley et al., 1986; Herrera-Estrella et al., 1983; Hoekema et al., 1983; Horsch et al., 1985; Jefferson, 1987; Ryals, 1996) . In cotton,, the new technology will be used to increase yield, improve fiber quality and^create. new varieties which are resistant to herbicides, pest insects, nematodes and diseases (John, 1996; John & Keller, 1996; John & Stewart, 1992; Murray et al., 1993; Rajasekaran et al., 1996; Schell, 1997; Stewart, 1992) . 1. Tissue Culture of Cotton: In 1935, Skovsted reported the first embryo culture of cotton. Beasley (1971) reported callus formation in cotton as an outgrowth from the micropylar end of fertilized ovules on MS medium. Somatic embryogenesis was achieved from a suspension culture of G. klotzschianum (Prive & Smith, 1979). In 1983, Davidonis & Hamilton first succeeded in efficient and repeatable regeneration of cotton (G. hirsutum L.) plants from callus after two-year cultivation. Cotton plants were since regenerated through somatic embryogenesis from different explants (Zhang & Feng, 1992; Zhang, 1994) including cotyledon (Davinonis et al., 1987; Davidonis & Hamilton, 1983; Finer, 1988; Firoozabady et al., 1987), hypocotyl (Cousins et al., 1991; Rangan & Zavala, 1984; Rangan & Rajasekaran, 1996; Trolinder & Goodin, 1988; Umbeck et al., 1987, 1989), stem (Altman et al., 1990; Finer & Smith, 1984), shoot apex (Bajaj et al., 1985; Gould et al., 1991; Turaev & Shamina, 1986), immature embryo (Beasley, 1971; Eid et al., 1973; Stewart & Hsu, 1977, 1978), petiole (Finer & Smith, 1984; Gawel et al., 1986; Gawel & Robacker, 1990), leaf (Finer & Smith, 1984; Gawel & Robacker, 1986), root (Chen & Xia, 1991; Kuo et al. , 1989), callus (Finer & McMullen, 1986; Trolinder et al., 1991) and protoplast (Chen et al., 1989). 2. Cotton Transformation: Explants (such as hypocotyl, cotyledon, callus generated from hypocotyl and cotyledon, as well as immature embryos) have been used for Agrobacterium-mediated transformation and particle bombardment (-de Framond et al., 1983; Finer & McMullen, 1990; Firoozabady et al., 1987; Perlak et al., 1990; Rangan & Rajasekaran, 1996; Rajasekaran et al., 1996; Trolinder et al., 1991; Umbeck et al. , 1987, 1989, 1992) . In addition, merjSternatic tissue of excised embryonic axes has also been used for cotton transformation by particle bombardment (Chlan et al., 1995; John, 1996; John & Keller, 1996; McCabe & Martinell, 1993). Zhou et_ al. (1983) transformed cotton by injecting DNA into the axile placenta one day after self-pollination. However, cotton transformation is highly dependent on genotype (Trolinder, 1985a, 1985b, 1986; Trolinder & Goodin, 1987, 1988a, 1988b). Apart from a few cultivars which are regeneratable and transformable, such as Gossypium hirsutum cv. Coker 312 and G. hirsutum Jin 7, most other important elite commercial cultivars, such as G. hirsutum cv. D&P 5415 and G. hirsutum. Zhongmian 12, are not regeneratable and transformable by these methods. Based on previous reports and the inventor's own experimental data, high efficiency of callus induction (60%) can be achieved using the hypocotyl as an explant. However, the transformation rate was only 20% (Firoozabady et al., 1987; limbeck et al., 1987). Several factors can lead to breakthrough of nontransformed calli, or to chimeric calli consisting of predominantly nontransformed cells: (1) low kanamycin levels (a high level of kanamycin is toxic to cotton explants and calli); (2) experience-dependent selection in later stages of callus proliferation; and (3) use of explants such as the hypocotyl which has only partial contact with the selective media (Firoozabady et al., 1987). When the cotyledon was used as an explant, although the transformation rate was higher than that with the hypocotyl, it was often difficult to eliminate Agrobacterium during subsequent culture (Jiao G.-L and Chen, Z.-X., personal communication; Umbeck et al., 1987, 1989). The transformation rate of meristemic tissue through particle bombardment was simply too low (0.02%-0.22%) compared to that of Agroibacterium mediated transformation. There thus remains aneed for methods of producing transgenic cotton plants that provide high rates of transformation along with high rates of transformants among regenerated somatic embryos. Summary of the Invention The present invention relates to a method for producing transgenic cotton plants, comprising the steps of (a) obtaining cotton fibrous root explants, (b) culturing the fibrous root explants to induce callus formation, (c) exposing root callus to a culture of Agrobacterium tumefaciens that harbors, a vector comprising an exogenous gene and a selectable marker, the Agrobacterium being capable of effecting the stable transfer of the exogenous gene and selection agent resistance gene to the genome of the cells of the explant, (d) culturing the callus in the presence of the selection agent to which the selection agent resistance gene confers resistance so as to select for transformed cells, (e) inducing somatic embryo formation in the selected callus culture, and (f) regenerating the induced somatic embryos into whole transgenic cotton plants. The present method provides for an improved rate of transformation when compared to previous methods that employ hypocotyl and cotyledon tissue. The method is believed to have wide applicability to a variety of cotton varieties. Brief description of the Figures Figure 1 shows the plasmid pBK9, containing a luciferase gene used to detect positive transformants obtained by the methods of the present invention. Figure 2 shows the plasmid pVIP96, the plasmid from which pBK9 was derived by insertion of the luciferase gene. Detailed Description In order to overcome the problems seen with prior art methods and increase the efficiency of transformation, fibrous root explants were used for Agrobacterium-mediated transformation of cotton. Although in Arabidopsis high efficiency of transformation was achieved in Agrobacterium-mediated transformation with fibrous root explants (Valvekens, et al., 1988), and the differentiation of young fibrous roots from cotton on MS medium containing 2.0 mg/L IAA, 0.02-0.04 mg/L IBA has been reported (Kuo, C.C., et al., 1989), there is no report in the literature about using fibrous roots as explants for cotton transformation. Fibrous root now have been successfully used as explants for Agrobacterium-mediated transformation and plant regeneration. In the process modified media for seedling culture, and regeneration and differentiation of embryogenic calli were used. Media used to culture seedlings to obtain explant material was designed to minimize browning of the roots (browning adversely effects the ability of explants to grow in culture and form callus), and to promote overall vigorous root growth. In a preferred embodiment MET (multi-effect triazole, a chemical agent used in agriculture to promote root growth) and NAA (a naphthalene acetic acid) are used together in the seedling culture medium to reduce the proportion of browned roots and increase callus initiation rate. MET is preferably used in concentrations ranging from about 0.05 mg/1 to about 0.2 mg/1, most preferably about 0.1 mg/1. NAA is preferably used in concentrations ranging from about 0.01 mg/1 to about 0.2 mg/1, most preferably about 0.05 mg/1. MET and NAA are also preferably used in the medium used to root transgenic seedlings regenerated from callus, in amounts similar to those described for the seedling culture medium. In a preferred embodiment of the callus-forming medium vitamin B5, 2,4-D ((2,4-dichlorophenoxy)acetic acid, MgCl and glucose are used, preferably about 0.05 mg/1 to about 0.15 mg/1 2,4-D, about 0.4 mg/1 to about 1.2 mg/1 MgCl, and about 1% to about 5% glucose, most preferably about 0.1 mg/L 2,4-D, 0.8 mg/L MgCl and 3% glucose. In an alternate preferred embodiment of the callus-forming medium my_o^inositol, vitamin B,, and dimethylallyl(amino)purine are used, a, preferably about 50 mg/1 to about 150 mg/1 myo-inositol, about 1 mg/1 to about 10 mg/1 vitamin B1, and about 0.1 mg/1 to about 7.5 mg/1 dimethylallyl(amino)purine, most preferably about 100 mg/1 myo-inositol, about 0.4 mg/1 vitamin B1 and about 5 mg/1 dimethylallyl(amino)purine. The same media used for callus induction can also be used during selection with antibiotics -- for example with 300-400 mg/L cefotaxime or 15-30 mg/L kanamycin. The presence of high concentrations (preferably about 1900 mg/1 to about 5700 mg/1, most preferably about 3800 mg/L) of nitrates (preferably NaNO3) was crucial for the observed effectiveness of the differentiation medium. With the fibrous roots as explants, although therate of callus-induction was lower compared with hypocotyl and cotyledon, a higher rate of transformation was achieved. Techniques for introducing exogenous genes into Agrobacterium such that they will be transferred stably to a plant or plant tissue exposed to the Agrobacterium are well-known in the art and do not form part of the present invention. It is advantagious to use a so- called "disarmed" strain of Agrobacterium or Ti plasmid, that is, a strain or plasmid wherein the genes responsible for the formation of the tumor characteristic of the crown gall disease caused by wild-type Agrobacteruim are removed or deactivated. Numerous examples of disarmed Agrobacterium strains are found in the literature (e.g., pAL4404, pEHAlOl and pEH 105 (Walkerpeach & Veltern, 1994)). It is further advantagious to use a so-called binary vector system, such as that described in U.S. Patent Nos. 4,940,838 and 5,464,763 (Schilperoort, et al.) and Hoekema et al., 1983. A binary vector system allows for manipulation in E. coli of the plasmid carrying the exogenous gene to be introduced into the plant, making the process of vector construction much easier to carry out. Similarly, vector construction, including the construction of chimeric genes comprising the exogenous gene that one desires to introduce into the plant, can be carried out using techniques well-known in the art and does not form part of the present invention. Chimeric genes should comprise promoters that have activity in the host in which expression is desired. For example, it is advantageous to have a series of selectable markers for selection of transformed cells at various stages in the transformation process. A selectable marker (for example a gene conferring resistance to an antibiotic such as kanamycin, cefotaxime or streptomycin) linked to a promoter active in bacteria would permit selection of bacteria containing the marker (i.e., transformants). Another selectable marker linked to a plant-active promoter, such as the CaMV 35S promoter or a T-DNA promoter such as the NPT II NOS promoter, would allow selection of transformed plant cells. The exogenous gene that is desired to be introduced into the plant cell should comprise a plant-active promoter in functional relation to the coding sequence, so that the promoter drives expression of the gene in the transformed plant. Again, plant-active promoters, such as the CaMV 35S, the NPT II NOS promoter or any of a number of tissue- specific promoters, are well-known in the art and selection of an appropriate promoter is well within the ordinary skill in the art. The present method can be used to produce transgenic plants expressing any number of exogenous genes, and is not limited by the choice of such a gene. The selection of the desired exogenous gene depends on the goal of the researcher, and numerous examples of desirable genes that could be used with the present invention are known in the art (e.g., the family of Bacillus thuringiensis toxin genes, herbicide resistance genes such as shikimate synthase genes that confer glyphosate resistance, U.S. Patent No. 5,188,642, or a 2,4-D monooxygenase gene that confers 2,4-D resistance, Bayley et al., Theoretical and Applied Genetics, vol. 82, pp. 645-49, male sterility genes such as the antisense genes of U.S. Patent No. 5,741,684 (Fabijanski, et al.), or even the elaborate crop protection systems described in U.S. Patent No. 5,123,765 (Oliver, et al.)). Agrobacteriurn-mediated cotton transformation is considered in the art to be heavily variety-dependant. The Coker series of cotton varieties have been shown to be relatively easy to transform. However, DP 5412, Zhongmain 12 and many other varieties still have difficulties associated with transformation. The situation is the same for G. barbadense and other diploid species. Particle bombardment, DNA injection and infection of meristem tissue with Agrobacterium are some alternative methods, which can be used to transform, in theory, all the cotton varieties. The problems associated with these methods are: low efficiency of transformation and unstable/unreliable results. It is believed that the present method has broad applicability to transformation of cotton varieties, as it overcomes or minimizes several of the problems associated with previous work relating to cotton transformation (such as breakthrough of non- transformed callus, poor explant growth and low transformation rate, poor somatic regeneration) through the use of fibrous root explants. The following abbreviations are used to designate culture media useful in connection with the present invention: LB medium (lOg bacto-tryptone + 5g bacto-yeast extract + lOg NaCl); B5 medium (Gamborg et al., 1968; Sigma, Cat. No. G-5768) ; MS medium (Murashige et al., 1962; Sigma, Cat. No. M-5524) ; SH medium (Stewart & Hsu, 1977. Planta 137, 113-117); CB-1.1 (½ MS + ½ B5 Vitamin + 0.1mg/L NAA); CB-1.2 (½ B5 medium); CB-2.1 (MS macro + B5 micro + 0.05mg/L 2,4-D + 0.1mg/L kinetin + 3% glucose + 2g/L gellan gum (PhytaGel™, Sigma) + 0.93mg/L MgCl2-6H20, pH5.8); CB-2.2 (MS macro + 100mg/L myo-inositol + 0.4mg/L vitamin Bl + 5mg/L 2iP (6 - (??- dimethyially(amino)purine)+ 0.2mg/L NAA + 3% glucose + 2g/L gellan gum (PhytaGel™, Sigma) + 0.93mg/L MgCl2-6H20, pH5.8); CB-3.1 (CB-2.1+ 500 mg/L cefotaxime + 50mg/L kanamycin); CB-3.2 (CB-2.2 + 500mg/L cefotaxime + 50mg/L kanamycin); CB-4 (Modified CB-3.1 or CB-3.2 by adding double amount of KN03 and removing NH4N03 with 250 mg/L cefotaxime and 20mg/L kanamycin); CB-5 (SH +1.5% Sucrose + 2g/L gellan gum (PhytaGel™, Sigma) + 0.93g/L MgCl2, pH7.0). The following Examples are intended to illustrate the present invention, and not in any way to limit its scope, which is solely defined by the claims. EXAMPLE 1: Regeneration of Cotton Plants from Root Tissue Culture Preparation of Root Explants: Cotton seeds were sterilized in 70% ethanol for 10-15 min., and then treated with 10% H20, for 30-120 mins. Treated seeds were rinsed in sterile water for 24 hrs at 28°C and germinated on either CB-1.1 medium or CB-1.2 medium at 28°C - 30°C, 16h light (60-90 uE rrT2 s-1)- Seven to ten days sterile seedlings thus grown were used to prepare explants. It was found that plentiful healthy roots (longer and thicker) with white color were obtained using CB-1.1 medium, whereas shorter and thinner roots with grey to brown color were obtained using the CB-1.2 medium. Therefor, CB-1.1 was chosen for further work. Induction of calli: Fibrous roots were cut from seedlings and cultured on CB-2.1 medium, or CB-2.2 medium at 28°C - 30°C for three days, 16h light (60-90 pE m-2 s-1) . The optimum size for root explants was 5-7 mm. A few small calli initiated on the cut sites of root segments in as little as 3 days. In general, transformed hypocotyl or cotyledon explants started to initiate callus on inducing medium after 3 days. However, previous to the present invention, transformed root explants were generally found to initiate callus only after 10 days of cultivation. The color of the root explants was white. One week later, small calli were also initiated from other parts of the root segments. The color of the root explants changed to grey or even brown. At the end of 2 weeks of cultivation, calli initiated from the whole root explants and grew well. Of the two inducing media, CB- 2.2 was found to induce good callus formation, while CB-2.1 did not. On CB-2.2 medium, root explants grew well, and the microcallus initiated on the cut sites of the explant. About 10% of root explants initiated callus after only 3 days on the medium. On the other hand, while CB-2.1 medium supported the growth of the root explants well, there was no callus initiation on the cut sites. The efficiency of callus-induction with root explants on the CB-2.2 medium was 10%, which was lower than that with hypocotyl or cotyledon explants (20-30%). A summary of results showing callus induction and transformation efficiency appears in Table 1, below. Regeneration of root calli: After one month, the calli were transferred to new medium for subculture on either CB-2.1 medium or CB-2.2 medium. After 2 months of subculture, the mature calli were transferred to CB- 4 (without antibiotic) for induction of somatic embryos. Glutamine and L-asparagine were added in amounts of 0.5 mg/L and 0.2 mg/L, respectively, to promote embryogenesis. Primary somatic embryos were formed on the embryogenic calli after 2 months of cultivation, with 2 subcultures in between on the same media. Primary somatic embryos were subcultured on the same media for another month before mature somatic embryos were formed. Some of the somatic embryos developed to plantlets. These small plantlets were transferred to CB-5 medium for root induction. When the plantlets had made roots on the CB-5 medium (4-6 weeks), they were transferred to soil and maintained in an incubator under high humidity for 3-4 weeks at 28°C, 16h light (60-90 µE m-2 s-1) , and then transferred to large pots with soil in a green house. EXAMPLE 2: Agrobacterium Transformation and Culture The plasmid pBK9 (35S:LUC) (see Fig. 1) was generated by cloning the luc coding sequence from the BamHI/Stul fragment of plasmid pGEMluc into the blunt-ended Stul site of the plasmid pVIP96 (see Fig. 2). Prepared competent cells (400 microliter) in Eppendorf tube from -80°C were put on ice to thaw. Plasmid DNA was added in the cells. After gentle mixing, the mixture was incubated on ice for 4 5 minutes. The Eppendorf tube containing the mixture was put into liquid nitrogen for 1 minute and afterwards in a water bath (37°C) for 3 minutes. After the incubation, 800 microliter LB medium (without antibiotics) was added into the mixture and the tube with the mixture was incubated at 28°C for 3 hours. After a brief centrifugation at 12,000 rmp, 800 microliter supernatant was removed. The rest of the medium was mixed well with the cell pellet and the mixture was plated onto LB plates containing 100 mg/L kanamycin and 100 mg/L streptomycin. Successful transformed LBA4404 cells formed colonies on the plates in about 48 hours at 28°C. Agrobacterium strain LBA4404 harboring the plasmid pBK9 (35S:LUC) was initiated on LB plate with kanamycin (50 mg/L), streptomycin (50 mg/L) and refamycin (50 mg/L). A single colony was inoculated into LB liquid medium without antibiotics and grown overnight for about 18 h at 28°C on a gyratory shaker. The optical density (A600) value was adjusted to 0.1 - 0.4 in liquid LB medium prior to use. EXAMPLE 3: Transformation of Root Explant Tissue and Regeneration of Transgenic Cotton Plants Root explants were obtained by cultivating sterile- cotton seeds as described in Example 1, above, on C3- 1.1 medium. Fibrous roots were cut from seedlings and cultured on CB-2.2 for two days, 16h light (60-90 µE m- 2s-1) . The fibrous roots were then cut into small segments (5-10 mm) and incubated with the cell suspension culture of Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pBK9 (35S:LUC) (A600 = 0.1-0.6) of Example 2 for 15 min. After drainage of the bacterial solution, the root explants were cultured at 28°C, 16h light (60-90 µE m-2s-1) for an additional two days. The optimum concentration of the Agrobacterium strain LBA4404 for root explants was lower (A600=0.1-0.4) than that for hypocotyl and cotyledon explants (A600=0.3-0.6). Optimal bacterial concentrations did not affect the growth of the root explants and the subsequent callus induction. Co-cultured explants were washed twice with sterile distilled water and transferred to CB-3.1 medium or CB-3.2 medium for cultivation at 28°C, 16h light (60-90 µE m-2 s-1). After four weeks, kanamycin-resistant calli were selected and subcultured on the same media for the second selection. At the same time, some of the calli were selected to detect the LUC expression with the luciferase luminescence image system (see Example 4, below). The process of inducing callus took about 2 months. The efficiency of callus induction from root explant was lower compared with that from hypocotyl and cotyledon explants. Kanamycin-resistant calli were transferred to CB-4 medium to induce embryogenic calli and somatic embryos. After 4-6 weeks of cultivation, with one subculture, mature somatic embryos appeared on the calli. Plantlets developed afterwards from some of the embryos. The green plantlets were then transferred to rooting medium (CB-5) for root induction. When plantlets had made roots, they were transferred to soil and maintained in an incubator under high humidity for 3-4 weeks at 28°C, 16h light (60-90 µE nf2 s"1) , and then transferred to large pots with soil in a green house. EXAMPLE 4: Detection of Luciferase Activity Plant materials (such as callus, leaf and whole plantlet) were sprayed with a solution containing 0.5 mM potassium luciferin and 0.01% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20) and left for 30 min. The luciferase luminescence from these plant materials was visualized using an image-intensifying camera and photon-counting image processors purchased from Prinston Instruments Inc., 3660 Quakerbridge Road, Trenton, NJ 08619. The exposure time was 6 min. The electronic images were converted to Microsoft Powerpoint TIFF files and printed out from a standard color printer. 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Skovsted, A. 1935. Cytological studies in cotton. III. A hybrid between Gossypium davidsonii Kell, and G. sturrtii F. Muell. J. Genet. 30, 397405. Stewart, J. McD. 1992. Biotechnology of cotton: achievements and perspectives. ICAC Review Articles on Cotton Production Research No. 3, 1-50. Stewart, J. McD and Hsu, C. L. 1978. Hybridization of deploid and tetraploid cottons through in-ovulo embryo culture. J. Heridity 69, 404-8. Srewart, J. McD. and Hsu, C. L., 1977. In ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.). Planta 137, 113-7. Thomas, J. C, Adams, D. G., Keppene, V. D. , Wasmann, C. C, Brown, J. K. , Kanost, M. R. and Bohnert, H. J. 1995. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cel, Rep. 14, 758-62. Trolinder, N. L. 1985a. Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). A Dissertation in Biology (Dec, 1985). Trolinder, N. L. 1985b. Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Reports 8, 133-6. Trolinder, N. L. 1986. Somatic embryogenesis and plant regeneration in Gossypium hirsutum L. Dissertations Abstracts International 47, 2550B-2551B. Trolinder, N. L. and Goodin, J. E. 1987. Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Reports 6, 231-4. Trolinder, N. L. and Goodin, J. E. 1988a. Somatic embryogenesis and regeneration in cotton. I. Effects of source of explant and hormone regime. Plant Cell Tissue Organ Culture 12, 31-42. Trolinder, N. L. and Goodin, J. E. 1988b. Somatic embryogenesis and regeneration in cotton. II. Requirements for embryo development and plant regeneration. Plant Cell Tissue Organ Culture 12, 43-53. Trolinder, N. L., Quisenberry, J., Bayley, C, Ray, C, and Ow, D. 1991. 2,4-D resistant transgenic cotton. Proc. Beltwide Cotton Prod. Res. Conf., 840. Turaev, A. M. and Shamina, Z. B. 1986. Soniec Plant Physiol. 33, 439. Umbeck, P. F. , 1991. Genetic engineering of cotton plants and lines. US Patent No. 5,004,863. Umbeck, P. F. 1992. Genetic engineering of cotton plants and lines. US Patent No. 5,159,135 (continued from US Patent 5,004,863, 1991). Umbeck, P. F, Johnson, G., Barton, K. and Swain, W. 1987. Genetically transformed cotton (Gossypium hirsutum L.) plants. Bio/technol. 5, 263-6. Umbeck, P. F., Swain, W. and Yang, N.-S., 1989. Inheritance and expression of genes for kanamycin and chloramphenical resistance in transgenic cotton plants. Crop. Sci. 29, 196-201. Valvekens, D., Van Montagu, M., Van Lijsebettens, M. 1988. Agrobacterium tumefaciens-mediated transformation of Arabidopsis root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536-40. Walkerpeach, C.R. and Veltern, J. 1994. Agrojbacterium- mediated gene transfer to plant cells: cointegrate and binary vector systems. Plant Mol. Biol. Mannuel Bl, 1-19. Zhang, H.-B. 1994. The tissue culture of cotton (II). Plant Physiol. Commun. 30, 386-91. Zhang, H.-B. and Feng, R. 1992. The tissue culture of cotton (I). Plant Physiol. Commun. 30, 386-91. Zhou, G.-Y., Weng, J., Zeng, Y.-S., Huang, J.-G., Qian, S.-Y. and Liu, G.-L. 1983. Introduction of exogenous DNA into cotton embryos. Methods in Enzymology 101,433-81. We claim: 1. A method for producing a transgenic cotton plant comprising the steps of: (a)obtaining cotton fibrous root explants from seedlings cultured in medium containing multi effect triazole, (b) culturinq the fibrous root explants in medium containing a plant hormone to induce callus formation, (c) exposing root callus to a culture of Agrobacterium tumefaciens that harbors a vector comprising an exogenous gene and a selectable marker, the Agrobacterium being capable of effecting the stable transfer of the exogenous gene and selection agent resistance gene to the genome of the cells of the callus, (d) culturing the callus in the presence of the selection agent to which the selection agent resistance gene confers resistance so as to select for transformed cells, (e) inducing somatic embryo formation in the selected -callus culture, and (f) regenerating the induced somatic embryos into whole transgenic cotton plants. 2. The method as claimed in claim 1, wherein the multi-effect triazole is in a concentration of about 0.05 mg/l to about 0.2 mg/1. 3. The method as claimed in claim 2, wherein the multi-effect triazole is in a concentration of about 0.1 mg/i. 4. The method as claimed in claim 1, wherein the cotton seedlings are grown in the presence of a naphthalene acetic acid. 5. The method as claimed in claim 4, wherein the a naphthalene acetic acid is in a concentration of about 0.01 mg/1 to about 0.2 mg/1. 6 The method as claimed in claim 5, wherein the a naphthalene acetic acid is in a concentration of about 0.05 mg/1. 7. The method as claimed in claim 1, wherein the step of regenerating the somatic embryos is carried out in the presence of multi-effect triazole. 8. The method as claimed in claim 7, wherein the multi-effect triazole is in a concentration of about 0.05 mg/1 to about 0.2 mg/1. 9. The method as claimed in claim 8, wherein the multi-effect triazole is in a concentration of about 0.1 mg/1. io. The method as claimed in claim 7, wherein the step of regenerating the somatic embryos is carried out in the additional presence of a naphthalene asetic acid. II The method as claimed in claim 10, wherein the a naphtalene acetic and is in a concentration of about 0.01 mg/1 to about 0.2 mg/1. 13. The method as claimed in claim 11, wherein the a naphthalene acetic acid is in a concentration of about 0.05 mg/1. 13 The method as claimed in claim 1, wherein the step of inducing callus formation is carried out in a callus inducing culture medium comprising myo-inositol, vitamin B1, and a dimethylallyl (amino)purine. 11. The method as claimed in claim 1, wherein the step of inducing somatic embryo formation is carried out in a somatic embryo inducing culture medium comprising myo-inositol, vitamin B1, and a dimethylallyl(amino)purine. 15. The method as claimed in claim 13, wherein the callus inducing culture medium comprises myo-inositol in an amount from 50 mg/L to 150 mg/L, vitamin B2 in an amount from 0.2 to 10 mg/L and a dimethylallyl(amino)purine in an amount from 0.1 to 7.5 mg/L. 16. The method as claimed in claim 15, wherein the callus inducing culture medium comprises 100 mg/L myo-inositol, 0.4 mg/L vitamin B1 and 5 mg/L dimethylallyl(amino)purine. 17. The method as claimed in claim 14, wherein the somatic embryo inducing culture medium comprises myo-inositol in an amount from 50 to 100 mg/L, vitamin B, in an dimethyla in an amount from 0.01 to 0.5 mg/w 18. The method as claimed in plasning wherein the somatic emoryo inducing medium comprises 100 mg/L myo-inositol, 0.4 mg/L vitamin Bi and 5 mg/L dimethylallyl(amino)purine. 19. The method as claimed in claim 1, wherein the step of inducing callus formation is carried out in a callus inducing culture medium comprising vitamin B5, (2, 4-dichlorophenoxy)acetic acid, MgCl2 and glucose. 20. The method as claimed in claim 1, wherein the step of inducing somatic embryo formation is carried out in a somatic embryo inducing culture medium comprising vitamin B5, (2, 4-dichlorophenoxy) acetic acid, MgCl2 and glucose. 21. The method as claimed in claim 19, wherein the callus inducing culture medium comprises vitamin B5 in an amount from 0.2 mg/L to 10 mg/L, (2,4- dichlorophenoxy)acetic acid in an amount from 0.05 mg/L to 0.15 mg/L, MgCl2 in an amount from 0.4 mg/L to 1.2 mg/L and glucose in an amount from 1% to 5%. 22. The method as claimed in claim 21, wherein the callus inducing culture medium comprises 0.4 mg/L vitamin B5, 0.1 mg/L (2,4-dichlorophenoxy)acetic acid, 0.8 mg/L MgCl and 3% glucose. 23. The method as claimed in claim 20, wherein the somatic embryo inducing culture medium comprises vitamin B5 in an amount from 0.2 mg/L to 10 mg/L, (2,4- dichlorophenoxy) acetic acid in an amount from 0.05 mg/L to 0.15 mg/L, MgCl in an amount from 0.4 mg/L to 1.2 mg/L and glucose in an amount from 1% to 5%. 24. The method as claimed in claim 23, wherein the somatic embryo inducing medium comprises 0.4 mg/L vitamin B5, 0.1 mg/L (2,4-dichlorophenoxy)acetic acid, 0.8 mg/L MgCl and 3% glucose. 25. A method as claimed in any of claims 13-24, wherein the medium------ comprises gellan gum. 26. A method as claimed in claim 25, wherein the gellan gum is present in an amount from 1.0 g/L to 3.0 g/L. 27. The method as claimed in claim 1, wherein the step of inducing somatic embryo culture is carried out in a somatic embryo-inducing medium comprising a nitrate in an amount from 1900 mg/L to 5700 mg/L. 28. The method as claimed in claim 27, wherein the somatic embryo- inducing medium comprises 3800 mg/L nitrate. 39. A method as claimed in either claim 27 or 28, wherein the nitrate is NaNO3. A method is disclosed for producing a transgenic cotton plant comprising the steps of (a) obtaining cottonfibrous root explants, (b) culturing the fibrous root explants to induce callus formation, (c) exposing root callus to a culture of Agrobacterium tumefaciens that harbors a vector comprising an exogenous gene and a selectable marker, the Agrobacterium being capable of effecting the stable transfer of the exogenous gene and selection agent resistance gene to the genome of the cells of the explant, (d) culturing the callusiber the presence of the selection agent to which the selection agent resistance gene confers resistance so as to select for transformed cells, (producing somatic embryo formation in the selected callus culture, and (f) regenerating the induced somatic embryos into whole transgenic cotton plants.

Full Text

Agrobacterium-Mediated Transformation of
Cotton With Novel Explants
Technical Field
The present invention relates to the general field
of genetic engineering of plants, in particular to the
introduction of exogenous genetic material into cotton
by Agrojbacterium transformation of novel explants
followed by somatic embryo regeneration.
Background
Cotton is the most extensively used natural fiber
in the textile industry. Its annual production
worldwide is over 100 million bales, valued at US$4 5_
billion^ Cotton lint or seed hair is_ a terminally
differentiated single epidermal cell from 50 species of
the genus Gossypium of the family Malvaceae. It is
classified as a natural, cellulosic, monocellular and
staple fiber. The cultivated cotton varieties, which
have been cultivated for more than 5000 years, all come
from two diploids (2n=2x=26) (G_. herbaceum and G.
arboreum) and two allotetraploids (2n=4x=52) (G.
hirsutum L., Upland; and G. barbadense L. , Sea Island).
In 1997 the top five world cotton producers were the
United States, China, India_, Pakistan and Uzbekstan,
producing about 63 million bales.
In the next century, most crops, including
cereals, oil crops, fruits, vegetables and other
economically important crops, will be genetically
engineered with added or modified traits ranging from
improvement of yield and quality, to herbicide
resistance and pest resistance (Chappell, 1996; Fraley
et al., 1986; Herrera-Estrella et al., 1983; Hoekema et
al., 1983; Horsch et al., 1985; Jefferson, 1987; Ryals,
1996) . In cotton,, the new technology will be used to
increase yield, improve fiber quality and^create. new
varieties which are resistant to herbicides, pest
insects, nematodes and diseases (John, 1996; John &
Keller, 1996; John & Stewart, 1992; Murray et al.,
1993; Rajasekaran et al., 1996; Schell, 1997; Stewart,
1992) .
1. Tissue Culture of Cotton: In 1935, Skovsted
reported the first embryo culture of cotton. Beasley
(1971) reported callus formation in cotton as an
outgrowth from the micropylar end of fertilized ovules
on MS medium. Somatic embryogenesis was achieved from
a suspension culture of G. klotzschianum (Prive &
Smith, 1979). In 1983, Davidonis & Hamilton first
succeeded in efficient and repeatable regeneration of
cotton (G. hirsutum L.) plants from callus after
two-year cultivation. Cotton plants were since
regenerated through somatic embryogenesis from
different explants (Zhang & Feng, 1992; Zhang, 1994)
including cotyledon (Davinonis et al., 1987; Davidonis
& Hamilton, 1983; Finer, 1988; Firoozabady et al.,
1987), hypocotyl (Cousins et al., 1991; Rangan &
Zavala, 1984; Rangan & Rajasekaran, 1996; Trolinder &
Goodin, 1988; Umbeck et al., 1987, 1989), stem (Altman
et al., 1990; Finer & Smith, 1984), shoot apex (Bajaj
et al., 1985; Gould et al., 1991; Turaev & Shamina,
1986), immature embryo (Beasley, 1971; Eid et al.,
1973; Stewart & Hsu, 1977, 1978), petiole (Finer &
Smith, 1984; Gawel et al., 1986; Gawel & Robacker,
1990), leaf (Finer & Smith, 1984; Gawel & Robacker,
1986), root (Chen & Xia, 1991; Kuo et al. , 1989),
callus (Finer & McMullen, 1986; Trolinder et al., 1991)
and protoplast (Chen et al., 1989).
2. Cotton Transformation: Explants (such as
hypocotyl, cotyledon, callus generated from hypocotyl
and cotyledon, as well as immature embryos) have been
used for Agrobacterium-mediated transformation and
particle bombardment (-de Framond et al., 1983; Finer &
McMullen, 1990; Firoozabady et al., 1987; Perlak et
al., 1990; Rangan & Rajasekaran, 1996; Rajasekaran et
al., 1996; Trolinder et al., 1991; Umbeck et al. , 1987,
1989, 1992) . In addition, merjSternatic tissue of
excised embryonic axes has also been used for cotton
transformation by particle bombardment (Chlan et al.,
1995; John, 1996; John & Keller, 1996; McCabe &
Martinell, 1993). Zhou et_ al. (1983) transformed
cotton by injecting DNA into the axile placenta one day
after self-pollination. However, cotton transformation
is highly dependent on genotype (Trolinder, 1985a,
1985b, 1986; Trolinder & Goodin, 1987, 1988a, 1988b).
Apart from a few cultivars which are regeneratable and
transformable, such as Gossypium hirsutum cv. Coker 312
and G. hirsutum Jin 7, most other important elite
commercial cultivars, such as G. hirsutum cv. D&P 5415
and G. hirsutum. Zhongmian 12, are not regeneratable
and transformable by these methods.
Based on previous reports and the inventor's own
experimental data, high efficiency of callus induction
(60%) can be achieved using the hypocotyl as an
explant. However, the transformation rate was only 20%
(Firoozabady et al., 1987; limbeck et al., 1987).
Several factors can lead to breakthrough of
nontransformed calli, or to chimeric calli consisting
of predominantly nontransformed cells: (1) low
kanamycin levels (a high level of kanamycin is toxic to
cotton explants and calli); (2) experience-dependent
selection in later stages of callus proliferation; and
(3) use of explants such as the hypocotyl which has
only partial contact with the selective media
(Firoozabady et al., 1987). When the cotyledon was
used as an explant, although the transformation rate
was higher than that with the hypocotyl, it was often
difficult to eliminate Agrobacterium during subsequent
culture (Jiao G.-L and Chen, Z.-X., personal
communication; Umbeck et al., 1987, 1989). The
transformation rate of meristemic tissue through
particle bombardment was simply too low (0.02%-0.22%)
compared to that of Agroibacterium mediated
transformation.
There thus remains aneed for methods of producing
transgenic cotton plants that provide high rates of
transformation along with high rates of transformants
among regenerated somatic embryos.
Summary of the Invention
The present invention relates to a method for
producing transgenic cotton plants, comprising the
steps of (a) obtaining cotton fibrous root explants,
(b) culturing the fibrous root explants to induce
callus formation, (c) exposing root callus to a culture
of Agrobacterium tumefaciens that harbors, a vector
comprising an exogenous gene and a selectable marker,
the Agrobacterium being capable of effecting the stable
transfer of the exogenous gene and selection agent
resistance gene to the genome of the cells of the
explant, (d) culturing the callus in the presence of
the selection agent to which the selection agent
resistance gene confers resistance so as to select for
transformed cells, (e) inducing somatic embryo
formation in the selected callus culture, and (f)
regenerating the induced somatic embryos into whole
transgenic cotton plants.
The present method provides for an improved rate
of transformation when compared to previous methods
that employ hypocotyl and cotyledon tissue. The method
is believed to have wide applicability to a variety of
cotton varieties.
Brief description of the Figures
Figure 1 shows the plasmid pBK9, containing a
luciferase gene used to detect positive transformants
obtained by the methods of the present invention.
Figure 2 shows the plasmid pVIP96, the plasmid
from which pBK9 was derived by insertion of the
luciferase gene.
Detailed Description
In order to overcome the problems seen with prior
art methods and increase the efficiency of
transformation, fibrous root explants were used for
Agrobacterium-mediated transformation of cotton.
Although in Arabidopsis high efficiency of
transformation was achieved in Agrobacterium-mediated
transformation with fibrous root explants (Valvekens,
et al., 1988), and the differentiation of young fibrous
roots from cotton on MS medium containing 2.0 mg/L IAA,
0.02-0.04 mg/L IBA has been reported (Kuo, C.C., et
al., 1989), there is no report in the literature about
using fibrous roots as explants for cotton
transformation.
Fibrous root now have been successfully used as
explants for Agrobacterium-mediated transformation and
plant regeneration. In the process modified media for
seedling culture, and regeneration and differentiation
of embryogenic calli were used.
Media used to culture seedlings to obtain explant
material was designed to minimize browning of the roots
(browning adversely effects the ability of explants to
grow in culture and form callus), and to promote
overall vigorous root growth. In a preferred
embodiment MET (multi-effect triazole, a chemical agent
used in agriculture to promote root growth) and NAA (a
naphthalene acetic acid) are used together in the
seedling culture medium to reduce the proportion of
browned roots and increase callus initiation rate. MET
is preferably used in concentrations ranging from about
0.05 mg/1 to about 0.2 mg/1, most preferably about 0.1
mg/1. NAA is preferably used in concentrations ranging
from about 0.01 mg/1 to about 0.2 mg/1, most preferably
about 0.05 mg/1. MET and NAA are also preferably used
in the medium used to root transgenic seedlings
regenerated from callus, in amounts similar to those
described for the seedling culture medium. In a
preferred embodiment of the callus-forming medium
vitamin B5, 2,4-D ((2,4-dichlorophenoxy)acetic acid,
MgCl and glucose are used, preferably about 0.05 mg/1
to about 0.15 mg/1 2,4-D, about 0.4 mg/1 to about 1.2
mg/1 MgCl, and about 1% to about 5% glucose, most
preferably about 0.1 mg/L 2,4-D, 0.8 mg/L MgCl and 3%
glucose. In an alternate preferred embodiment of the
callus-forming medium my_o^inositol, vitamin B,, and
dimethylallyl(amino)purine are used, a, preferably
about 50 mg/1 to about 150 mg/1 myo-inositol, about 1
mg/1 to about 10 mg/1 vitamin B1, and about 0.1 mg/1 to
about 7.5 mg/1 dimethylallyl(amino)purine, most
preferably about 100 mg/1 myo-inositol, about 0.4 mg/1
vitamin B1 and about 5 mg/1 dimethylallyl(amino)purine.
The same media used for callus induction can also be
used during selection with antibiotics -- for example
with 300-400 mg/L cefotaxime or 15-30 mg/L kanamycin.
The presence of high concentrations (preferably about
1900 mg/1 to about 5700 mg/1, most preferably about
3800 mg/L) of nitrates (preferably NaNO3) was crucial
for the observed effectiveness of the differentiation
medium. With the fibrous roots as explants, although
therate of callus-induction was lower compared with
hypocotyl and cotyledon, a higher rate of
transformation was achieved.
Techniques for introducing exogenous genes into
Agrobacterium such that they will be transferred stably
to a plant or plant tissue exposed to the Agrobacterium
are well-known in the art and do not form part of the
present invention. It is advantagious to use a so-
called "disarmed" strain of Agrobacterium or Ti
plasmid, that is, a strain or plasmid wherein the genes
responsible for the formation of the tumor
characteristic of the crown gall disease caused by
wild-type Agrobacteruim are removed or deactivated.
Numerous examples of disarmed Agrobacterium strains are
found in the literature (e.g., pAL4404, pEHAlOl and pEH
105 (Walkerpeach & Veltern, 1994)). It is further
advantagious to use a so-called binary vector system,
such as that described in U.S. Patent Nos. 4,940,838
and 5,464,763 (Schilperoort, et al.) and Hoekema et
al., 1983. A binary vector system allows for
manipulation in E. coli of the plasmid carrying the
exogenous gene to be introduced into the plant, making
the process of vector construction much easier to carry
out.
Similarly, vector construction, including the
construction of chimeric genes comprising the exogenous
gene that one desires to introduce into the plant, can
be carried out using techniques well-known in the art
and does not form part of the present invention.
Chimeric genes should comprise promoters that have
activity in the host in which expression is desired.
For example, it is advantageous to have a series of
selectable markers for selection of transformed cells
at various stages in the transformation process. A
selectable marker (for example a gene conferring
resistance to an antibiotic such as kanamycin,
cefotaxime or streptomycin) linked to a promoter active
in bacteria would permit selection of bacteria
containing the marker (i.e., transformants). Another
selectable marker linked to a plant-active promoter,
such as the CaMV 35S promoter or a T-DNA promoter such
as the NPT II NOS promoter, would allow selection of
transformed plant cells. The exogenous gene that is
desired to be introduced into the plant cell should
comprise a plant-active promoter in functional relation
to the coding sequence, so that the promoter drives
expression of the gene in the transformed plant.
Again, plant-active promoters, such as the CaMV 35S,
the NPT II NOS promoter or any of a number of tissue-
specific promoters, are well-known in the art and
selection of an appropriate promoter is well within the
ordinary skill in the art.
The present method can be used to produce
transgenic plants expressing any number of exogenous
genes, and is not limited by the choice of such a gene.
The selection of the desired exogenous gene depends on
the goal of the researcher, and numerous examples of
desirable genes that could be used with the present
invention are known in the art (e.g., the family of
Bacillus thuringiensis toxin genes, herbicide
resistance genes such as shikimate synthase genes that
confer glyphosate resistance, U.S. Patent No.
5,188,642, or a 2,4-D monooxygenase gene that confers
2,4-D resistance, Bayley et al., Theoretical and
Applied Genetics, vol. 82, pp. 645-49, male sterility
genes such as the antisense genes of U.S. Patent No.
5,741,684 (Fabijanski, et al.), or even the elaborate
crop protection systems described in U.S. Patent No.
5,123,765 (Oliver, et al.)).
Agrobacteriurn-mediated cotton transformation is
considered in the art to be heavily variety-dependant.
The Coker series of cotton varieties have been shown to
be relatively easy to transform. However, DP 5412,
Zhongmain 12 and many other varieties still have
difficulties associated with transformation. The
situation is the same for G. barbadense and other
diploid species. Particle bombardment, DNA injection
and infection of meristem tissue with Agrobacterium are
some alternative methods, which can be used to
transform, in theory, all the cotton varieties. The
problems associated with these methods are: low
efficiency of transformation and unstable/unreliable
results. It is believed that the present method has
broad applicability to transformation of cotton
varieties, as it overcomes or minimizes several of the
problems associated with previous work relating to
cotton transformation (such as breakthrough of non-
transformed callus, poor explant growth and low
transformation rate, poor somatic regeneration) through
the use of fibrous root explants.
The following abbreviations are used to designate
culture media useful in connection with the present
invention:
LB medium (lOg bacto-tryptone + 5g bacto-yeast
extract + lOg NaCl);
B5 medium (Gamborg et al., 1968; Sigma, Cat. No.
G-5768) ;
MS medium (Murashige et al., 1962; Sigma, Cat. No.
M-5524) ;
SH medium (Stewart & Hsu, 1977. Planta 137,
113-117);
CB-1.1 (½ MS + ½ B5 Vitamin + 0.1mg/L NAA);
CB-1.2 (½ B5 medium);
CB-2.1 (MS macro + B5 micro + 0.05mg/L 2,4-D +
0.1mg/L kinetin + 3% glucose + 2g/L gellan gum
(PhytaGel™, Sigma) + 0.93mg/L MgCl2-6H20, pH5.8);
CB-2.2 (MS macro + 100mg/L myo-inositol + 0.4mg/L
vitamin Bl + 5mg/L 2iP (6 - (??-
dimethyially(amino)purine)+ 0.2mg/L NAA + 3%
glucose + 2g/L gellan gum (PhytaGel™, Sigma) +
0.93mg/L MgCl2-6H20, pH5.8);
CB-3.1 (CB-2.1+ 500 mg/L cefotaxime + 50mg/L
kanamycin);
CB-3.2 (CB-2.2 + 500mg/L cefotaxime + 50mg/L
kanamycin);
CB-4 (Modified CB-3.1 or CB-3.2 by adding double
amount of KN03 and removing NH4N03 with 250 mg/L
cefotaxime and 20mg/L kanamycin);
CB-5 (SH +1.5% Sucrose + 2g/L gellan gum
(PhytaGel™, Sigma) + 0.93g/L MgCl2, pH7.0).
The following Examples are intended to illustrate
the present invention, and not in any way to limit its
scope, which is solely defined by the claims.
EXAMPLE 1: Regeneration of Cotton Plants from Root
Tissue Culture
Preparation of Root Explants: Cotton seeds were
sterilized in 70% ethanol for 10-15 min., and then
treated with 10% H20, for 30-120 mins. Treated seeds
were rinsed in sterile water for 24 hrs at 28°C and
germinated on either CB-1.1 medium or CB-1.2 medium at
28°C - 30°C, 16h light (60-90 uE rrT2 s-1)- Seven to ten
days sterile seedlings thus grown were used to prepare
explants. It was found that plentiful healthy roots
(longer and thicker) with white color were obtained
using CB-1.1 medium, whereas shorter and thinner roots
with grey to brown color were obtained using the CB-1.2
medium. Therefor, CB-1.1 was chosen for further work.
Induction of calli: Fibrous roots were cut from
seedlings and cultured on CB-2.1 medium, or CB-2.2
medium at 28°C - 30°C for three days, 16h light (60-90
pE m-2 s-1) . The optimum size for root explants was 5-7
mm. A few small calli initiated on the cut sites of
root segments in as little as 3 days. In general,
transformed hypocotyl or cotyledon explants started to
initiate callus on inducing medium after 3 days.
However, previous to the present invention, transformed
root explants were generally found to initiate callus
only after 10 days of cultivation. The color of the
root explants was white. One week later, small calli
were also initiated from other parts of the root
segments. The color of the root explants changed to
grey or even brown. At the end of 2 weeks of
cultivation, calli initiated from the whole root
explants and grew well. Of the two inducing media, CB-
2.2 was found to induce good callus formation, while
CB-2.1 did not. On CB-2.2 medium, root explants grew
well, and the microcallus initiated on the cut sites of
the explant. About 10% of root explants initiated
callus after only 3 days on the medium. On the other
hand, while CB-2.1 medium supported the growth of the
root explants well, there was no callus initiation on
the cut sites. The efficiency of callus-induction with
root explants on the CB-2.2 medium was 10%, which was
lower than that with hypocotyl or cotyledon explants
(20-30%). A summary of results showing callus
induction and transformation efficiency appears in
Table 1, below.
Regeneration of root calli: After one month, the
calli were transferred to new medium for subculture on
either CB-2.1 medium or CB-2.2 medium. After 2 months
of subculture, the mature calli were transferred to CB-
4 (without antibiotic) for induction of somatic
embryos. Glutamine and L-asparagine were added in
amounts of 0.5 mg/L and 0.2 mg/L, respectively, to
promote embryogenesis. Primary somatic embryos were
formed on the embryogenic calli after 2 months of
cultivation, with 2 subcultures in between on the same
media. Primary somatic embryos were subcultured on the
same media for another month before mature somatic
embryos were formed. Some of the somatic embryos
developed to plantlets. These small plantlets were
transferred to CB-5 medium for root induction. When
the plantlets had made roots on the CB-5 medium (4-6
weeks), they were transferred to soil and maintained in
an incubator under high humidity for 3-4 weeks at 28°C,
16h light (60-90 µE m-2 s-1) , and then transferred to
large pots with soil in a green house.
EXAMPLE 2: Agrobacterium Transformation and Culture
The plasmid pBK9 (35S:LUC) (see Fig. 1) was
generated by cloning the luc coding sequence from the
BamHI/Stul fragment of plasmid pGEMluc into the
blunt-ended Stul site of the plasmid pVIP96 (see Fig.
2).
Prepared competent cells (400 microliter) in
Eppendorf tube from -80°C were put on ice to thaw.
Plasmid DNA was added in the cells. After gentle
mixing, the mixture was incubated on ice for 4 5
minutes. The Eppendorf tube containing the mixture was
put into liquid nitrogen for 1 minute and afterwards in
a water bath (37°C) for 3 minutes. After the
incubation, 800 microliter LB medium (without
antibiotics) was added into the mixture and the tube
with the mixture was incubated at 28°C for 3 hours.
After a brief centrifugation at 12,000 rmp, 800
microliter supernatant was removed. The rest of the
medium was mixed well with the cell pellet and the
mixture was plated onto LB plates containing 100 mg/L
kanamycin and 100 mg/L streptomycin. Successful
transformed LBA4404 cells formed colonies on the
plates in about 48 hours at 28°C. Agrobacterium strain
LBA4404 harboring the plasmid pBK9 (35S:LUC) was
initiated on LB plate with kanamycin (50 mg/L),
streptomycin (50 mg/L) and refamycin (50 mg/L). A
single colony was inoculated into LB liquid medium
without antibiotics and grown overnight for about 18 h
at 28°C on a gyratory shaker. The optical density (A600)
value was adjusted to 0.1 - 0.4 in liquid LB medium
prior to use.
EXAMPLE 3: Transformation of Root Explant Tissue
and Regeneration of Transgenic Cotton
Plants
Root explants were obtained by cultivating sterile-
cotton seeds as described in Example 1, above, on C3-
1.1 medium. Fibrous roots were cut from seedlings and
cultured on CB-2.2 for two days, 16h light (60-90 µE m-
2s-1) . The fibrous roots were then cut into small
segments (5-10 mm) and incubated with the cell
suspension culture of Agrobacterium tumefaciens strain
LBA4404 harboring the plasmid pBK9 (35S:LUC) (A600 =
0.1-0.6) of Example 2 for 15 min. After drainage of
the bacterial solution, the root explants were cultured
at 28°C, 16h light (60-90 µE m-2s-1) for an additional
two days. The optimum concentration of the
Agrobacterium strain LBA4404 for root explants was
lower (A600=0.1-0.4) than that for hypocotyl and
cotyledon explants (A600=0.3-0.6). Optimal bacterial
concentrations did not affect the growth of the root
explants and the subsequent callus induction.
Co-cultured explants were washed twice with
sterile distilled water and transferred to CB-3.1
medium or CB-3.2 medium for cultivation at 28°C, 16h
light (60-90 µE m-2 s-1).
After four weeks, kanamycin-resistant calli were
selected and subcultured on the same media for the
second selection. At the same time, some of the calli
were selected to detect the LUC expression with the
luciferase luminescence image system (see Example 4,
below). The process of inducing callus took about 2
months. The efficiency of callus induction from root
explant was lower compared with that from hypocotyl and
cotyledon explants.
Kanamycin-resistant calli were transferred to CB-4
medium to induce embryogenic calli and somatic embryos.
After 4-6 weeks of cultivation, with one subculture,
mature somatic embryos appeared on the calli.
Plantlets developed afterwards from some of the
embryos. The green plantlets were then transferred to
rooting medium (CB-5) for root induction. When
plantlets had made roots, they were transferred to soil
and maintained in an incubator under high humidity for
3-4 weeks at 28°C, 16h light (60-90 µE nf2 s"1) , and then
transferred to large pots with soil in a green house.
EXAMPLE 4: Detection of Luciferase Activity
Plant materials (such as callus, leaf and whole
plantlet) were sprayed with a solution containing 0.5
mM potassium luciferin and 0.01% (w/v)
polyoxyethylenesorbitan monolaurate (Tween-20) and left
for 30 min. The luciferase luminescence from these
plant materials was visualized using an
image-intensifying camera and photon-counting image
processors purchased from Prinston Instruments Inc.,
3660 Quakerbridge Road, Trenton, NJ 08619. The
exposure time was 6 min. The electronic images were
converted to Microsoft Powerpoint TIFF files and
printed out from a standard color printer.
Callus growing on selected medium for one month
was selected to test LUC expression with the video
image system. The positive transformed callus had
white spots whereas untransformed callus did not. Out
of the 139 pieces of kanamycin resistant calli, 49
pieces were positive with LUC activity. The successful
transformation rate was therefor 35%, which was much
higher than that seen using cotyledon or hypocotyl as
explant (20%).
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We claim:
1. A method for producing a transgenic cotton plant
comprising the steps of:
(a)obtaining cotton fibrous root explants from seedlings
cultured in medium containing multi effect triazole,
(b) culturinq the fibrous root explants in medium
containing a plant hormone to induce callus formation,
(c) exposing root callus to a culture of
Agrobacterium tumefaciens that harbors a
vector comprising an exogenous gene and a
selectable marker, the Agrobacterium being
capable of effecting the stable transfer of
the exogenous gene and selection agent
resistance gene to the genome of the cells of
the callus,
(d) culturing the callus in the presence of
the selection agent to which the selection
agent resistance gene confers resistance so
as to select for transformed cells,
(e) inducing somatic embryo formation in the
selected -callus culture, and
(f) regenerating the induced somatic embryos
into whole transgenic cotton plants.
2. The method as claimed in claim 1, wherein the multi-effect
triazole is in a concentration of about 0.05 mg/l
to about 0.2 mg/1.
3. The method as claimed in claim 2, wherein the multi-effect
triazole is in a concentration of about 0.1 mg/i.
4. The method as claimed in claim 1, wherein the cotton seedlings
are grown in the presence of a
naphthalene acetic acid.
5. The method as claimed in claim 4, wherein the a naphthalene
acetic acid is in a concentration of about 0.01
mg/1 to about 0.2 mg/1.
6 The method as claimed in claim 5, wherein the a naphthalene
acetic acid is in a concentration of about 0.05
mg/1.
7. The method as claimed in claim 1, wherein the step of
regenerating the somatic embryos is carried out in
the presence of multi-effect triazole.
8. The method as claimed in claim 7, wherein the multi-effect
triazole is in a concentration of about 0.05 mg/1
to about 0.2 mg/1.
9. The method as claimed in claim 8, wherein the multi-effect
triazole is in a concentration of about 0.1 mg/1.
io. The method as claimed in claim 7, wherein the step of
regenerating the somatic embryos is carried out in
the additional presence of a naphthalene asetic
acid.
II The method as claimed in claim 10, wherein the a naphtalene
acetic and is in a concentration of about 0.01
mg/1 to about 0.2 mg/1.
13. The method as claimed in claim 11, wherein the a naphthalene
acetic acid is in a concentration of about 0.05
mg/1.
13 The method as claimed in claim 1, wherein the step of inducing
callus formation is carried out in a callus
inducing culture medium comprising myo-inositol,
vitamin B1, and a dimethylallyl (amino)purine.
11. The method as claimed in claim 1, wherein the step of inducing
somatic embryo formation is carried out in a
somatic embryo inducing culture medium comprising
myo-inositol, vitamin B1, and a
dimethylallyl(amino)purine.
15. The method as claimed in claim 13, wherein the callus inducing
culture medium comprises myo-inositol in an amount
from 50 mg/L to 150 mg/L, vitamin B2 in an amount
from 0.2 to 10 mg/L and a
dimethylallyl(amino)purine in an amount from 0.1
to 7.5 mg/L.
16. The method as claimed in claim 15, wherein the callus inducing
culture medium comprises 100 mg/L myo-inositol,
0.4 mg/L vitamin B1 and 5 mg/L
dimethylallyl(amino)purine.
17. The method as claimed in claim 14, wherein the somatic embryo
inducing culture medium comprises myo-inositol in
an amount from 50 to 100 mg/L, vitamin B, in an
dimethyla in an amount from 0.01
to 0.5 mg/w
18. The method as claimed in plasning wherein the somatic emoryo
inducing medium comprises 100 mg/L myo-inositol,
0.4 mg/L vitamin Bi and 5 mg/L
dimethylallyl(amino)purine.
19. The method as claimed in claim 1, wherein the step of inducing
callus formation is carried out in a callus
inducing culture medium comprising vitamin B5,
(2, 4-dichlorophenoxy)acetic acid, MgCl2 and
glucose.
20. The method as claimed in claim 1, wherein the step of inducing
somatic embryo formation is carried out in a
somatic embryo inducing culture medium comprising
vitamin B5, (2, 4-dichlorophenoxy) acetic acid, MgCl2
and glucose.
21. The method as claimed in claim 19, wherein the callus inducing
culture medium comprises vitamin B5 in an amount
from 0.2 mg/L to 10 mg/L, (2,4-
dichlorophenoxy)acetic acid in an amount from 0.05
mg/L to 0.15 mg/L, MgCl2 in an amount from 0.4 mg/L
to 1.2 mg/L and glucose in an amount from 1% to
5%.
22. The method as claimed in claim 21, wherein the callus inducing
culture medium comprises 0.4 mg/L vitamin B5, 0.1
mg/L (2,4-dichlorophenoxy)acetic acid, 0.8 mg/L
MgCl and 3% glucose.
23. The method as claimed in claim 20, wherein the somatic embryo
inducing culture medium comprises vitamin B5 in an
amount from 0.2 mg/L to 10 mg/L, (2,4-
dichlorophenoxy) acetic acid in an amount from 0.05
mg/L to 0.15 mg/L, MgCl in an amount from 0.4 mg/L
to 1.2 mg/L and glucose in an amount from 1% to
5%.
24. The method as claimed in claim 23, wherein the somatic embryo
inducing medium comprises 0.4 mg/L vitamin B5, 0.1
mg/L (2,4-dichlorophenoxy)acetic acid, 0.8 mg/L
MgCl and 3% glucose.
25. A method as claimed in any of claims 13-24, wherein
the medium------ comprises gellan gum.
26. A method as claimed in claim 25, wherein the gellan
gum is present in an amount from 1.0 g/L to 3.0
g/L.
27. The method as claimed in claim 1, wherein the step of inducing
somatic embryo culture is carried out in a somatic
embryo-inducing medium comprising a nitrate in an
amount from 1900 mg/L to 5700 mg/L.
28. The method as claimed in claim 27, wherein the somatic embryo-
inducing medium comprises 3800 mg/L nitrate.
39. A method as claimed in either claim 27 or 28,
wherein the nitrate is NaNO3.

A method is disclosed for producing a transgenic cotton plant comprising the steps of (a) obtaining cottonfibrous root explants, (b)
culturing the fibrous root explants to induce callus formation, (c) exposing root callus to a culture of Agrobacterium tumefaciens that harbors
a vector comprising an exogenous gene and a selectable marker, the Agrobacterium being capable of effecting the stable transfer of the
exogenous gene and selection agent resistance gene to the genome of the cells of the explant, (d) culturing the callusiber the presence of
the selection agent to which the selection agent resistance gene confers resistance so as to select for transformed cells, (producing somatic
embryo formation in the selected callus culture, and (f) regenerating the induced somatic embryos into whole transgenic cotton plants.

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Patent Number

249095

Indian Patent Application Number

IN/PCT/2001/1016/KOL

PG Journal Number

40/2011

Publication Date

07-Oct-2011

Grant Date

29-Sep-2011

Date of Filing

01-Oct-2001

Name of Patentee

TEMASEK LIFE SCIENCES LABORATORY LIMITED

Applicant Address

1 RESEARCH LINK, THE NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE 117604

Inventors:

#	Inventor's Name	Inventor's Address
1	JIAO GAI-LI	BLOCK 322, CLEMENTI AVENUE 5, #03-237, SINGAPORE, 120322
2	LIU JIAN WEI	13 TOH YI DRIVE, #07-05, SINGAPORE 590013

PCT International Classification Number

C12N 15/82

PCT International Application Number

PCT/SG1999/00016

PCT International Filing date

1999-03-10

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA