Gentiana Macrophylla Pall Tissue, Cell Culture And The Secondary Metabolic Regulation Of Gentiopicrin

As an important Chinese traditional herb, large-leaved gentian has lots of medical functions including curing rheumatism, anti-inflammatory, abirritation and so on. Tissue culture of Gentiana macrophylla Pall is considered in this study to establish the optimal conditions of G.macrophylla. This work preliminarily researched the Gentiana suspension cell growth and enhanced the gentiopicrin content of the secondary metabolites of G. macrophylla by adding elicitors. This work was also to characterize the generation of nitric oxide (NO) in G. macrophylla cells induced by an elicitor and the signal role of NO in the elicitation of plant defense responses and secondary metabolite accumulation. Results are as follows.Tissue culture of G. macrophylla is considered in this study to establish the optimal conditions of G. macrophylla. The optimal medium of callus induction was the MS medium with0.5mg/L NAA and1.0mg/L6-BA added. The induced callus was transferred to the MS medium with0.3mg/L NAA and0.5mg/L6-BA added for its secondary culture. The callus from the secondary culture was transferred to the MS medium with0.3mg/L NAA and1.0mg/L6-BA added to differentiate. The buds differentiated from the callus were transplanted to the1/2MS medium with0.1mg/L IAA and then developed into many differentiation seedlings.The effects of elicitors on cell growth and gentiopicrin accumulation in shaken cell suspension cultures of G. macrophylla were investigated. The most effective elicitor, which stimulated the greatest increase in gentiopicrin accumulation (1.2-6.4times of non-elicited controls), regardless of its concentration, was yeast extract. Chitosan with50mg/L concentrations after48h increased the gentiopicrin accumulation. Salicylic acid and endophytic fungi for the Gentiana cell growth and secondary metabolites have no significant effect.The elicitor-induced fluorescence increase was effectively blocked by the NOS inhibitor and NO scavenger, proving that the fluorescence increase was mainly a result of NO production by the elicitor-treated cells. The elicitor treatment stimulated gentiopicrin production, increasing the gentiopicrin content of cell by about6.4fold. The NO donors SNP slightly but significantly stimulated the gentiopicrin production by itself and in combination with the yeast extract elicitor. The elicitor-induced gentiopicrin production was significantly depressed by both the NOS inhibitor L-Name and the NO scavenger PTIO, suggesting that NO plays a signal role in the elicitor-induced responses and secondary metabolism activities in the G. macrophylla cells.