Studies On The Genetic Transformation And Evaluation For The Performance Of Transgenic Cotton

Cotton, Gossypium spp., is an economically important crop that is grown throughout the world. Traditional breeding and genetic engineering plays a major role in further improving the yield and quality of fiber. Thus it is very important in crop production to increase the production efficiency and reduce both the monetary and environmental cost of production. One is the use of genetic transformation to incorporate valuable alien genes into the cotton genome. The in-planta transformation methods avoid tissue culture steps and direct transformation of genes into in-vivo plants. The first transgenic upland cotton, expressing CryIAc insecticidal protein, was released into commercial production in 1996, and we are interested to study the agronomic performance between Bt and non-Bt cotton cultivars. Thus we have planned several experiments on cotton with the following objectives: To optimize and develop new methods of direct transformation mediated via the pollen tube pathway; To compare the different transformation methods for cotton; To study the performance of the transgenic Bt cotton cultivars. The main experimental results are presented as follows:(i) Optimization of modified Agrobacterium transfected pollen via pollen tube pathway method for genetic transformation of Gossypium hirsutum L.A new method for the genetic transformation of cotton pollen by means of Agrobacterium co-cultivation and pollen tube pathway (PTP)-mediated transformation is reported. The bar gene, which is involved in herbicide tolerance, was transferred into pollen grains of upland cotton. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by Agrobacterium strain LBA4404 harboring a vector pCB4, which contains the gene of interest, bar driven by the CaMV 35S promoter and NOS (nopaline synthase) terminator sequences. The integration and expression of bar in the genome of transgenic plants were analyzed with kanamycin and basta bioassay tests, PCR and Southern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double fertilization and that normal cottonseeds were produced in the cotton ovary. The experiment was repeated threetimes and data was averaged. Of 1245 seeds obtained from 334 bolls pollinated with transfected pollen grains; 14 were positive for basta? selection; and 11 of these were transgenic plants integrated with bar, yielding a 0.88% transformation rate. Progeny analysis of these plants showed a classical Mendelian pattern of inheritance. None of the transgenic plants obtained were positive for the Agrobacterium contamination test. (ii) Comparison of transformation methods for genetic transformation in Gossypium hirsutum L.Protocols for consistent production of transgenic herbicide tolerant upland cotton were established and compared the different transformation methods. Agrobacterium strain LBA4404 harboring a vector pCB4 carrying the bar gene was utilized to transform into upland cotton cultivar 'Coker312'. Ovarian injection of plasmid DNA (method-I), pollination by using pollen co-cultivated with Agrobacterium (method-II), Agrobacterium drop/dip (method-Ill) and apical meristem explants (method-IV) methods were standardized and compared. The herbicide basta leaf sensitivity test, polymerase chain reaction (PCR), and southern hybridization confirmed that the bar gene was integrated with the plant genome. The progeny test proved that all the transgenics obtained in our experiment followed Mendelian laws of inheritance. Though, we obtained transgenic plants in all methods, but they were differed with respect to transformation efficiency, time taken, reproducibility and simplicity of methods. The method-II found to be economically and technically viable compared with other three methods. The time taken by this method-II was on par with method-I and reproducibility was on par with method-IV. In addition this method-II used minimum facilities than tissue mediated method-IV. The transformation efficiency was higher in metho...