Optimizing Regeneration Systerm, Assaying Physiological Changes During Regeneration And Establishing Agrobacterium-mediated Transformation System In Dendrobium Nobile Sw.

Dendrobium is the second largest family in Orchidaceae. D. noble, D. candium and D. huoshanness are valuable herbal medicines. One fouth or so of Dendrobium are important ornamentals, which are famous for their beautiful and attractive flowers and long florescences. So, it is great potential for study on Dendrobium nobile.In the present study, the changes of antioxidant enzymes during the differentiation of shoots and roots from PLBs, and changes of soluble proteins were studied. The other factors which influenced plant regeneration from PLBs were evaluated to optimize the plant regeneration system. At the same time, an efficient Agrobacterium-mediated transformation system for Dendrobium nobile was established. The primary results were summarized as follows:(1) Healthy green PLB were obtained from shoots and buds on the MS medium supplemented with 0.2 mg/L NAA. We explored deeply on the optimal proliferation medium, and ascertained the suitable concentration of BA (0.5 mg/L). We also compared the effects of seven organic additives on the increase of fresh weight and proliferation of PLB, indicating that both CW and peptone were the suitable additives, and 1.0 g/L peptone showed the most effective.(2) During the culture of shoots, axillary shoots were first induced to recover the growing vigor of weakly-growing shoots in vitro. We discovered that the quantity of extrinsic nitrogen regulated the proliferation and growth of buds.Through adjusting the concentrations of NAA, not only the buds could be induced to form multiple shoots, but also the proliferating rate of the buds was improved. Simultaneously it was found that the height of shoots was critical for rooting. Sucrose at suitable concentration (20 g/L) was also necessary for the normal growth of D. nobile.(3 ) In the process of proliferation of PLBs and weakly-growing shoots, F/P rate (growth rate of flesh weight / proliferation rate of PLBs) was a key parameter that could be used to distinguish whether the growth of PLBs was normal or not. When F/P rate was not less than 0.6, PLBs showed well growing and developed, otherwise weakly growing and showing greenwish to yellowish. And for shoots, the suitable F/P rate was around 1.14.(4) The results indicated that the activities of ascorbic peroxidase (APX), guaiacol peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT) and the contents of soluble proteins were decreased and then increased;and the activity of superoxide dismutase (SOD) was increased and then decreased. POD was the key antioxidant enzyme during rooting, which could be a physiological parameter for determing rooting or not. It was approved that the differentiation of shoots and roots from PLBs was aprocess under effects of proteins and antioxidant enzymes by the SDS-PAGE electrophoresis analysis of isozyme and soluble proteins, which provided an important reference for tissue culture of Dendrobium nobile.(5 )A system ofAgrobacterium-medi&ted transformation for Dendrobium nobile Sw. was established. GUS histochemical assay and PCR analysis showed that gus gene was integrated into D. nobile successfully.