Studies On Agrobacterium Tumefaciens-mediated Transformation System Of Fuyu Persimmon

In this study, an efficient transformation system had been established by Agrobacterium tumefaciens-mediated transformation technology on the base of high-frequency regeneration systems. Factors that affect the efficiency of genetic transformation of Fuyu persimmon were studied and optimized. It was originally proved by the PCR examination that the ACS gene had been transferred into Fuyu persimmon. The major results as following:1. Effects of Sp and Cef on shoot differentiation of leaf explants and rooting of in vitro shoots were investigated. The results indicated that Fuyu persimmon was highly sensitive to Sp. As its concentration increased to 30 mg/L in shoot differentiation medium, adventitious buds differentiation was completely inhibited. Selection pressures were determined that: 20 mg/L Sp for rooting of shoots. It was suitable that 300 mg/L Cef can controlled the propagation of Agrobacterium tumefaciens, and the frequency of adventitious shoot induction can reach 46.3%.2. Conditions for efficient T-DNA transformation were studied through selecting the best of pre-cultured time, the concentration of Agrobacterium tumefaciens the infection time, the co-cultured time and so on. The results indicated that Leaves pre-cultured in darkness for three days were dipped into Agrobacterium suspension for seven minutes, then co-cultured in darkness for four day on the medium of 1/2MS + ZT 1.0mg/L + TDZ 0.5 mg/L + AS 150μmol/L, the best delayed selection time was 4d, could increase the frequency of transformation. The Frequency of resistant shoots could reach 13.64%.3. In this study, we obtained 246 resistant shoots, only 4 resistant shoots were Fuyu persimmon transgenetic plants carrying ACS gene which identified by PCR.4. Effects of concentration of sucrose, dakness treatment, buttle-closed exercise and light intensity on Fuyu persimmon were studied for cultivating vigorous and healthy rooted plants. The results showed that 1/2MS medium with 20 g/L sucrose was the best basal medium, darkness cultured 8d, buttle-closed exercise should have been for 21 d in the light intensity of 30 000 Ix. In this way, the plantlet growed very well, which had many thick and long roots.5. In order to enhance survival rate of transplanting, factors of temperature, humidity, substratum was studied on trandplants environment of Fuyu persimmon. Result shows that, the Pearlite combined with Vermiculite by1:1 was the best material. The best temperature is between 25～30℃, containing the humidity with 90%～100%, could have better survival rate of-transplanting.