Study On Somatic Embryogenesis Mechanism Of Cotton And Production Of P53Gene Transgenic Plants

In this study, the plant regeneration systems of xinhai20, xinhai24(Xinjiang Sea Island cotton) and xinluzao1hao, zhongmiansuo44hao(upland cotton) have been established through the induction of somatic embryogenesis. We revealed the variation of the physiology, biochemistry and cell ultrastructure of cotton somatic embryogenesis. By using two different approaches, Agrobacterium-mediated embryogenic callus and the floral spray, the p53gene was introduced into Sea Island cotton and upland cotton, it has been proved that transgenic plants can be obtained through the two approaches by PCR amplification and Southern bolt hybridization. The main conclusions of this study are as follows:At the stage of Sea Island cotton callus induction,0.1mg/L2,4-D+0.1mg/L KT hormone combination can effectively induce callus. After subculture twice, they were inoculated with0.1mg/L NAA+0.1mg/L KT callus induction medium to produce a large number of adventitious roots for the differentiation of good preparation. In the upland cotton callus inductional phase, the optimal hormone combination is0.05mg/L2,4-D+0.1mg/L KT.At the stage of differentiation and somatic embryogenesis, reducing the amount of NH4NO3, redoubling the amount of KNO3is more conducive to the growth of callus and somatic embryogenesis. Adding0.1mg/L NAA into the medium can effectively improve somatic embryo survival rate.Ultrastructural studies have shown that cotton somatic embryogenesis, embryogenic callus cells are linked closely together; At the Ultrastructural level, large central vacuole disappeared, they exist many organelles and a large number of mitochondria, the nucleus is located in the cell central, and have prominent nucleoli structure. Non-embryogenic callus cells arranged in irregular, with a large central vacuole, have a little number of mitochondria, nucleus tends to one end of the cell.In the process of cotton somatic embryogenesis, callus contained the lowest content of soluble sugar, the soluble protein content of the embryogenic callus were highest, SOD, POD and CAT activities were higher than non-embryogenic callus. The activities of three kinds of antioxidant enzyme of Embryonic calli play an extremely important role in stability of intracellular environment and differentiation.In the process of formation of embryogenic callus, the number of mitochondria in the process will be significantly increased. From the number of mitochondria and cell area to consider, it has been proved the area of mitochondria in embryogenic callus cells is larger than in non-embryogenic callus by using the MATLAB software. The number of mitochondria in embryogenic is significantly more than in non-embryogenic callus.Agrobacterium-mediated embryogenic callus exist a lot of problems such as a long cycle, easily be restricted by genotype, consumption of human and financial. The rate of Agrobacterium mediated callus transformation is higher, transgenic progeny were more single copy inserted, and has a relatively stable background of genetic transformation. Floral spray has a lot of advantages such as genetically modified way is simple, time-consuming is short. But this way was easily affected by seasonal restrictive, and has a higher copy, so floral spray is at the disadvantage of genetic stability.