Studies On Regeneration System And Agrobacterium-mediated Transformation In Dendrobium Fimbriatum

Dendrobium fimbriatum.Hook, mainly distributed in the warm and monstrous areas of China, including Guangxi, Guizhou and Yunnan province, is one of the most important varieties of Chinese medical plants with significant medical values. It is major functions include nourishing yin cleaning heat & encouraging production of body fluids to extinguish thirst. It’s on the verge of extinction due to the excessive use of wild resources, together with its high demand of living environment, low breeding ratio and retarded growth. Therefore, breeding new variety, establishing rapid propagation system and modifying the plant through genetic engineering become the focus of its research. This thesis used the Dendrobium fimbriatum seeds as explanting material to establish regeneration system and agrobacterium-mediated genetic transformation system in hope of providing vital reference for future genetic engineering breeding of Dendrobium fimbriatum. The main results were as follows:1.Using Dendrobium fimbriatum seeds as the initial plants, research has been done on basic medium, plant growth regulator, additives which influence its protocorm induction, proliferation and bud differentiation of protocorm and seedling root. The results as follows:(1)The best protocorm induction medium for seed was:MS+6-BA 1.0 mg/L+NAA 0.2 mg/L+2,4-D 1.0 mg/L+sucrose 30 g/L+Agar 4.0 g/L, the protocorm induction rate of reached above 90% after 40 days culture; (2) The optimal proliferation culture medium formula:MS+6-BA 1.5 mg/L+NAA 0.8 mg/L+IBA 0.4 mg/L+sucrose 30 g/L+potato juice 15% +activated carbon 0.5g/L+Agar 4.0 g/L, the proliferation coefficient reached above 10; (3)The best formula for bud differentiation of medium:1/4 MS+6-B A 0.2 mg/L+NAA 1.0mg/L+sucrose 30 g/L+potato juice 15%+Agar 4.0 g/L, the bud differentiation rate reached 90%; (4)The best medium for strengthening seedling root:N6+0.1 mg/L KT+0.5 mg/L IAA+sucrose 30 g/L+potato juice 15%+activated carbon 0.5 g/L+Agar 4.0 g/L, the rooting rate reached 100%;2.Using Dendrobium fimbriatum protocom as recipient material, the author, after screening the working concentration of kanamycin and carbenicillin, optimized the transformation conditions of genetic transformation of agrobacterium-mediated.Dendrobium fimbriatum. The results as follows:(1) The concentration kanamycin of 200 mg/L used as the screening concentration can effectively inhibit the growth of the non-transformation materials; (2) The concentration carbenicillin of 200 mg/L used as the best concentration for genetic transformation & bacteria-elimination can completely inhibit the growth of agrobacterium; (3)The best condition for Agrobacterium-mediated transformation in Dendrobium fimbriatum is with immerged 20 min, OD600value, co culture time 5 d, moreover, the conversion rate reached the highest after adding AS 50 μmol/L. After the test of the protocorm which was screened for 2 months, using PCR identification showed that transformation positiveprotocorm had been got, and proved that the target gene had been taken into the genome of tassel Dendrobium fimbriatum.