Optimization Of Agrobacterium-Mediated Transformation Of Cotton And The Development Of Mutants

Cotton is an important cash crop for its natural fiber. Many excellent cotton cultivars have been developed through genetic engineering since 1987, which play a important role in further improving yield and quality of fiber, feed, and oil and in increasing the production efficiency and reducing both the monetary and environmental cost of production. However, many hinders still exisist in cotton biotechnology. The main reason is that cultivars of transgenic cotton, which requires transformation of appropriate tissue followed by regeneration, remains extremely difficult in somatic embryogenesis and regeneration. Recalcitrant cotton cultivars, long tissue culture duration, the unpredictability of tissue culture, and a high degree of genotype dependence are more troublesome in regeneration of cotton. The length of time in cotton transformation can result in a higher rate of somaclonal variation. We have optimized some factors that can significantly affect the efficiency of regeneration and transformation in cotton. Using this high efficiency transgenic procedure, an promoter-trapping system has been introduced into cotton genome, which laid a solid foundation for further research of functional genomics in cotton. And the result is as follows:1. Thirty elite genotypes were selected for comparison of their regeneability using IBA+KT and 2,4-D+KT hormone combination. The results showed that two genotypes, namely, 'Y668' and 'YZ-1' have predominant ability of somatic embryogenesis, which got high ratio of somatic embrogenesis within two months. The identification of these two elite genotypes will significantly improve the transgenic efficiency and save time consumed in the somatic embryogenesis.2. In this study, three techniques, namely, flow cytometric analysis, chromosome number counting and RAPD analysis were adopted for somaclonal variation detection of plants regenerated from somatic embryogenes. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were all stable in regenerated plants, however, Eleven RAPD primers from 187 tested revealed 16 polymorphic bands derived from 50.0 % of the regenerated plants (7 out of 14), which meant that cytological changes was not correlated with the frequency of RAPD polymorphisms. Somaclonal variation in embryogenic calli induced by IBA+KT and 2,4-D+KT hormone combination were evaluated by RAPD and SSR for the first time, the...