Studies On Alkaloids Accumulation And Optimization Of Liquild Culture System Of Protocorm-like Bodies Of D.huoshanense

Dendrobium huoshanense is a kind of precious traditional Chinese medicine in Anhui Province,and is protected by government for its medicinal value.It can diminish inflammation,clear the sight and improve the immunity of human body because of its affluent amylose and alkaloids.Currently,the wild resources of D.huoshanense has endangered,and the preservation of its germplasm resources primarily through the in vitro propagation technology.Because of its low alkaloids content,and has been unable to meet the needs of the market,but the liquid culture can be achieved in a relatively short period to produce alkaloids.The experiments were made with the stem section of D.huoshanense as explantlet,which acquired the protocorm like-bodies.In order to provide technical parameters for the establishment of large-scale liquid culture,producing a higher content alkaloids,and provides a theoretical basis for the synthesis of alkaloids,the research was studied on the effects of external factors to the growth and alkaloids accumulation in PLBs of D.huoshanense and also included the optimization of liquid culture system.The external factors included inoculum size,rocking speed,basic medium,carbon sources,hormone and natural extracts.The main results were as followes:1.The optimum medium for PLBs induction is:Knudson+2,4-D 0.1 mg/L+KT 0.05 mg/L; the optimum subculture time on solid medium is 55d and its growth curve was similar as "S" type;2.Through the observation of PLBs by paraffin slice technique,we found that the formation of PLBs is by the route of somatic embryogenesis;3.The fresh weight and the alkaloids production of unit medium reached peak,which were 8.7 g/flask and 0.153 g/DW·flask when the inoculum size was 7.0 g/flask,and the PLBs is full without brown and differentiation.And when the rocking speed was 120 r/m,the fresh weight and the alkaloids production of unit medium were also reached peak,which were 12.47 g/flask and 0.144 g/DW·flask,and the growth status was the same as above;4.The optimum concentration combination for PLBs proliferation was determined as1/2MS+glucose 40g/L+NAA 0.5 mg/L+water chestnut juice 150g/L.The medium which suit for the alkaloids production of unit medium accumulation was 1/2MS+ sucrose 30g/L+NAA 2.0 mg/L+2.0 mg/L KT+water chestnut juice 150g/L.5.The growth curve was similar as "S" type in the 48 days of culture.It reached peak and declined after 40 days of culture.And the fresh weight reached peak in the 40th day, which is 21.57 g·FW/flask;6.The alkaloids accumulation curve of PLBs in liquid medium was also similar as "S" growth type in the 48 days of culture.And the alkaloids production of unit medium reached peak in the 44th day,which was 0.28 g/DW·flask.The accumulation of alkaloids of PLBs was basically synchronous,but the accumulation of alkaloids to be slightly delayed;...