Establishment Of Regeneration System And Studies On Genetic Transformation In Kolkwitzia Amabilis

In this experiment, we choose endangered plants Kolkwitzia amabilis as the research object. The study includes experiment on accelerating germination, establishing the regeneration system and researching the genetic transformation.The main results are as follows:We used the seeds of Kolkwitzia amabilis as our experimental material. The seeds were soaked with distilled water. Then concentrated sulfuric acid were used to deal with the seed coats different time respectively, and then combined with gibberellin(GA) soaking treatments and alternating temperature. While the seeds were put into petri dishes and made them to sprout. The result showed that germination rate, germinability of Kolkwitzia amabilis seeds were effectively promoted by being immersed 48 h with water after using 48 h distilled water to breaking hard seeds or using concentrated sulfuric acid for 20 min, and then 24 h with 100mg/L gibberellin(GA3). Alternating temperature with 6 hours period for 20 cycles, can substitute using gibberellin(GA3).Chosing Aseptic leaves as explants, Kolkwitzia amabilis regeneration system is established with the two-step method. Preparing for genetic transformation. The result shows that the optimal medium for callus induction was MS+6-BA 0.3 mg/L+NAA 2.0 mg/L. In order to efficient bud differentiation, we first put the callus in the high concentration of cytokinin medium(MS+6-BA 4.0 mg/L+TDZ 0.5 mg/L+IBA 0.1 mg/L)training a week, then changing to MS medium without adding hormone for a week at low temperature, finally switching to low cytokinin bud differentiation medium(MS+6-BA 1.0 mg/L+TDZ 0.5mg/L+IBA 0.1 mg/L). And the optimum rooting medium is MS+NAA 0.2 mg/L.Based on the system of regeneration, the genetic transformation system of Kolkwitzia amabilis was established. Using tissue culture method of seedlings, report gene was detected by GUS staining. The results showed that the best pre-cultured period is 4 days, the concentration of bacilli is OD600=0.6 adding 100 ?mol/L AS, the best infection time is 40 minutes, and the co-culture period is 3 days. 50 mg/L Kan and 400 mg/L Cef can get good screening effect. Results of GUS staining proved that the GUS gene was integrated into the explants.The research establish a stable regeneration system of Kolkwitzia amabilis and lay the foundation for genetic transformation, in order to provide a new way for the cultivation of new varieties of Kolkwitzia amabilis.