Establishment Of Regeneration System And Technology For Genetic Transformation With RolB Gene Of Populus Tomentosa Carr.

In order to solve the problem of difficult in rooting in cuttage of P. tomentosa Carr., rolB gene, which has good effects on inducing roots of plants, had been introduced into P. tomentosa Carr. by the Agrobaterium-mediated method in the base of establishment of high regeneration system of P. tomentosa Carr. by plant tissue culture techniques. The main results as follows:1. Suckers of P. tomentosa Carr. with 3 to 6 leaves were the optimal explants, and the optimal method of sterilization was that suckers were dipped in 75 % alcohol about 30s and then in 1% "84" disinfectors about 5 min. As a result of such sterilization, the survival rate of explants was 100%. The inducing medium was MS medium supplemented with 0.5 mg/L 6-BA, 0.1mg/L NAA, sugar 3%, agar 0.52%, pH 6.0.The subculture medium was MS medium supplemented with 0.3 mg/L 6-BA, 0.1mg/L NAA, sugar 3%, agar 0.52%, pH 6.0. The rooting culture medium was MS containing sugar 3%, agar 0.52%, pH 6.0.2. The optimal recipe for adventitious buds regeneration from P. tomentosa Carr. was screened, which was MS containing 6-BA (2.0 mg/L) and NAA (0.1 mg/L), sugar 3 %, agar 0.52 %, pH 6.0. For adventitious buds regeneration of P. tomentosa Carr., the optimal ratio of hormone concentration (6-BA: NAA) should be in the range of 2-5:1. The frequency of regeneration greatly differed from in various clones among the clones , G3 and G2 were thebest in the regeneration frequency. Regeneration frequency was found to increase with 2 days culture in dark. Moreover, frequency of regeneration also varied in different types of vessels.3. Toxicity of Kanamycim (Kan) and Ceftomine (Cef) to adventitious buds regeneration, shoots multiplication and rooting were estimated. The result showed that the Kanamycim sensitive critical concentration to adventitious buds regeneration, shoots multiplication and rooting was 30mg/L and 50mg/L, respectively. The suitable Cef concentration to control the propagation of Agrobaterium was between 200 mg/L and 400 mg/L.4. The influencing factors of the transformation efficiency were studied. The optimum method and process of genetic transformation of P. tomentosa Carr. were obtained. The optimized condition for transformation of P. tomentosa Carr. were advanced culture for 2-3 days and infected 10minutes with OD600=0.4 concentration of. Agrobacterium. It was found that the presence of acetosyringone and late selection of 2 days increased transformation efficiency.5. The transgenic plants were screened in the differentiation culture media, subculture media containing antibiotics and identified through PCR. The results showed that rolB gene had been integrated into the chromosome of transgenic P. tomentosa Carr. Finally, the transgenic plants were successfully transplanted in the greenhouse.