Study On Tissue Culture And Genetic Transformation Of Betula Luminifera

Using orthogonal experiment design and complete combination design, a systematic research of tissue culture and efficient micropropagation system of Betula luminifera leaf-explants and stem explants was studied. In order to establish Agrobacterium tumefaciens-mediated efficient Betula luminifera H. winkl. sterile leaf-fragments transfor-mation system, we surveyed the5factors influencing transformation frequency in4levels, including pre-culture time, the concentration of agrobacterium liquid, acetosyringone(AS), infection time and co-culture time with transformation rate scored by GUS transient expres-sion using orthogonal design and studied the fit concentration of Carbenicillin and Hyg in transformation system.In order to obtain high efficiency genetic transformation, efficient and stable system of micropropagation was established. Screening the optimal basic medium, optimal medium for shoot differentiation and root differentiation using B. luminifera leaf-explants and stem explants."One step into buds"(stem and leaf produced adventitious buds directly), without the bud proliferation phase, average number of buds can reach5.43and12, the differentiation rate was99.85%and80%, improving the efficiency of experiment. The regeneration system of B. luminifera leaf-explants was established:1. The buds of B. luminifera leaf-explants and stem-explants grew well, dark green on MS medium. The optimal basic medium of the regeneration system of B. luminifera was MS medium.2. In this experiment, use B. luminifera stems as explants, the adventitious bud differentiation medium containing TDZ,6-BA and TDZ,6-BA and NAA two kinds of hormone combinations. The results showed that B. luminifera stems in the bud differentiation stage, just add the cytokinin6-BA, TDZ, can promote the induction of bud differentiation, auxin NAA had no significant effect on bud differentiation rate.3. The appropriate medium for differentiation of B. luminifera stem-explants was MS+0.50mg-L-16-BA+0.10mg-L-1TDZ+30g·L-1sugar+5.50g·L-1agar, on which the inducing rate of the adventitious shoot was99.85%and the average buds was5.43.4. The optimal medium for differentiation of B. luminifera leaf-explants was MS+TDZ1.0mg·L-1+NAA0.05mg·L-1+30g·L-1sugar+5.50g·L-1agar, on which the inducing rate of the adventitious shoot was80.00%and the average buds was12.5. In B. luminifera rooting stage, use1/2MS medium as basic medium under the premise of hormones, IBA and NAA comparison test, finally selected:IBA was more suitable for the rooting culture of B. luminifera than NAA.6. The inducing rate of the root was100%, the average roots14.5, the length10-11cm, the root stout, base without callus, and produced a lot of hair roots when the adventitious shoots were cultured on medium of1/2MS+IBA1.0mg·L-1+Suc.20g·L-1+Agar5.5g·L-1.7. The B. luminifera leaf-explants has high Hygromycin resistance background. The inducing rate of the root was80%and buds grew well when the optimal differentiation medium was without Hygromycin. The buds died when the differentiation medium containing hygromycin20mg·L-1. The Hygromycin resistance level of the leaf was20mg·L-1.8. Agrobacterium of EHA105could not grow and buds cound grow well when Carb. concentration was400mg·L-1.9. The results were as follows, the sterile leaf-fragmengts of tisssue culture pre-cultured for7day, were dipped in agrobacterium liquid whose concentration was OD600=1.0for20min with AS200μmol·L-1and co-cultived for5days, the GUS gene instantaneous expression was98%.