Inducement Of Callus And Establishing Cell Suspension Culture System Of Epimedium Acuminatum

The callus induction, subculture and suspension culture of E.acuminatum young leaves as the explants material, were studied in order to provide the technical support for the high efficient production of barewort flavonoids at a large scale. The key results are as follows:1. The callus induction of E. acuminatum young leaves: The factors including different sterilization agents, mediums, hormone combinations and illumination conditions which affect the callus induction of E. acuminatum were studied. The content of flavonoids in the calli was measured by means of the ultraviolet spectrophotometric analysis. The results indicate that the callus culture of E. acuminatum leaves can be effectively induced under the following conditions: disinfection of 2% Na Cl O for 14 mins, the medium MS + 2,4-D 2.0mg /L + 6-BA 0.5mg /L + IBA 0.5mg /L + NAA 1mg /L, at 24 ℃ 1000 lx illumination conditions. Total flavonoid content of induced embryonic calli is up to 5.38%, higher than that of pharmacopoeia standard(5.0%).2. The subculture: The factors including different medium, hormone combinations and its dense which affect the subculture of callus were studied. The result showed that the best subculture condition of E. acuminatum callus is as follows: MS + 2,4-D 2.0mg/L + 6-BA 0.5mg/L + IBA 0.5mg/L+ NAA 1mg/L. The dark conditions were favorable for the subculture and preservation.3. The suspension culture: The factors including inoculum size, shaking speed, concentration of sugar and hormone combinations were studied forthe system of efficient cell suspension culture of E. acuminatum. The results indicated that the most suitable conditions are as follows: the medium MS without agar, 25 ml liquid medium per 2g callus in a 100 ml flask, 30g/L sugar with speed of 120 rpm, the most suitable medium is MS + 2,4-D 2mg/L + 6-BA0.5mg/L+NAA0.5mg/L,The original p H is 5.8.Under the above environment,the cells suspension culture of E. acuminatum show the results that the growth cycle is 24 d,and after 9d it entered into the logarithmic phase with rapid growth of cell biomass,while reached its maximum rate of growth at the 24 d,that was 2.35 times, Total flavonoid content at this time also reached the highest value in the cell cycle,was 5.68%,while glycoside of E. acuminatum also reached a maximum of 0.748% during cells logarithmic growth period, after that it entered the decline phase,there is no stable phase. Suspension cell growth curve of E. acuminatum shows substantially "S" type,similar with the accumulation curve of glycoside of E. acuminatum and total flavonoids content, there is first increased and then decreased.