Establishment Of Bletilla Striata (Thunb.) Rchb.f Cell Suspension Culture System And Determination Of Secondary Metabolites

Bletilla striata (Thunb.) Rchb. f is a Perennial herb belonging to Orchidaceae, Inour country it is a valuable medicinal plant, which has bleeding myogenic effect,swelling detoxification, B. striata is not only used for medicinal, but also a highornamental value. In recent years, it’s very popular in the flower market at home andabroad. The cultivation of B. striata is mainly using common division propagation,but on account of its long growth cycle, low Propagation coefficient, It is not suitablefor large-scale commercial production. With the development of plant tissue culturetechnology and cell suspension culture technology, test-tube reproduction of the B.striata has become possible.In this study, the Bletilla striata polysaccharide (BSPS)was obtained from the suspension-cells cultured in the established cell suspensionculture system with the calli induced from the seed and Pseudobulb of B. striata.onthe study of influence of different combinations of hormone, nitrogen source,phosphorus source, carbon source, inostol and the cell inculated amount on theB. striata’ cell growth and synthesization of the BSPS and Rheochrysidin，thedynamic curve was established in order to the highest of the BSPS andRheochrysidin though cell suspension of B. striata.The research results are asfollows:1. Plenty of more uniformly and quickly growing callus was harvested from thehalf of MS medium with2.0mg/L2,4-D,0.2mg/L NAA,1.8mg/L6-BA.2. After optimized culture condition for cell suspension culture, the resultsshowed that the best culture condition was the modified the half of MS medium with340mg/L KH2PO4,3300mg/L NH4NO3,100mg/L inositol,30g/L sugar and0.2mg/L NAA,1.0mg/L6-BA,2.0mg/L2,4-D and added0.0038g(DW)/L cell in20mlliquid medium. 3. The detection system of the BSPS and Rheochrysidin: Glucose as a standard,we adopt Anthrone-sulfuric acid method to determine total sugar; and use3,5-dinitrosalicylic acid method for determination of reducing sugar; Polysaccharideis equal to the total sugar minus reducing sugar. At about48days, the content of theBSPS was3.72mg(DW)/L. The content of Rheochrysidin was detected by HighPerformance Liquid Chromatography (HPLC).When cell suspension cultures wasabout30d,8.12μg Rheochrysidin can be extracted from1g dry weight cells.4. Under the optimal culture condition, the dynamic curves between the Bletillastriata (Thunb.) Rchb. f.’s cell growth and biosynthesis of the BSPS showed thatthe Bletilla striata (Thunb.) Rchb. f.’s cell growth reached to the top at the27thday, and the biomass was2.79g (FW)/L. At the same time the content of the BSPSand Rheochrysidin reached to the top at the48th and30th with3.72mg (DW)/L，8.12μg/g, respectively. Then, the biomass of B. striata. and the content of BSPS andRheochrysidin began to decline.