Agrobacterium-mediated Genetic Transformation And Production Of Transgenic Plants With PhyA Gene In Cotton

This study used the upland cotton cv. CCRI521, Jiwu2031 and Nongda94-7 as target materials and transformed the expression vector phyA gene into the hypocotyls of these varieties via Agrobactetium-mediated, and optimized transformation conditions by orthogonal design and obtained a large amount of kanamycin-resistant callus of cotton; The transgenic plants of Coker312 and Coker201 were also obtained via Agrobactetium-mediated gene transfer of embryogenic callus; The transgenic plants of Coker312, CCRI521 and Nongda94-7 were obtained by transforming the phyA gene into the shoot meristems, and T₂ generation were obtained after kanamycin selection culture, PCR, genome-Southern analysis and were identified by genetic analysis and activity analysis of roots excreted phytase further. The objective of this study is to increase new content and new germplasm for cotton molecular breeding and provide the effective method for solving the problem of using the phosphorus in soil. The results were as follows:1. The effects of Agrobacterium concentration, infection time, the time and the temperature of co-cultute on the induction rate of kanamycin-resistant calli were studied in Agrobacterium-mediated transformation using the hypocotyls as explants. Four factors were optimized by orthogonal design. The result showed that the transformation ratio was highest on the condition of 0.3 OD of bacteria concentration, 15 min of infection time, 24℃of co-culture temperature and 24 h of co-cultivation time. This paper studied the effect of different concentration of kanamycin on the response of induction and growth of callus for three varieties of upland cotton via CCRI521, Jiwu2031 and Nongda94-7. The hypocotyls were cultured in callus induction medium supplemented with different concentration of kanamycin, the effects of kanamycin on the frequency of callus formation and proliferation of callus were studied. The results indicated that the sensitivity of different varieties was different and the optimal kanamycin concentration was 100 mg/L for the callus of Jiwu2031 and Nongda94-7, and 150 mg/L for CCRI521.2. The naked apical meristems of cotton plantlets coming from sterilized seeds were used as the receptor of Agrobacterim-mediated transformation. Integration of the phyA gene was confirmed by Southern hybridization analysis. Six T₁ indivudal plants and twelve T₂ indivudal plants with southern blot hybridization signal were choosen arbitrarily and and analyzed the activity of phytase secreted from roots after culturing for 20 days. The result revealed the activity of phytase secreted from roots T₁ indivudal plants and T₂ indivudal plants were increased 1.32～7.76 times.3. Ernbryogenic callus of Coker312 and Coker201 were used as the receptor of Agrobacterim-mediated transformation. PCR analysis using primer sets of the interest gene phyA indicated that plants which came from different cell lines produced the wanted 1.4 kb fragments. No wanted fragments were observed in the control plants.