Establishment Of Cell Mass Suspension Culture System And Study On Alkaloid Induction From Fritillaria Ussuriensis

Fritillaria ussuriensis Maxim belongs to Fritillaria genus, Lilium family, dry bulb as amedicine. There are a lot of therapeutic effects like relieving cough and asthma, moistening thelung and eliminating phlegm, protecting throat and beneficial to it. It is one of the basic rawmaterials of Chinese medicine products, widely used and largely marketing demand, belongingto the bulk of medicinal species. The wild resources have decreased year by year since its usageis bigger, and the artificial cultivation of sexual reproduction need a longer period, asexualreproduction often induce the germplasm degeneration. Therefore, this experiment based on thesolid culture, in order to improve the secondary metabolites of culture productivity, the first is tooptimize the study creatively the alkaloid induced cell mass production process parameters; thesecond is in the process of cultivation of precursor feeding material influence on the content ofsecondary metabolites change. Through this research, there is a benefit.to industrial andcommercial production of the active ingredients and avoiding disturbance of natural conditionsThe currently more advanced response surface methodology (Design Expert) was appliedin the screening of the hormone types, concentration of precursor feedings and data analysis.Other studies were conducted in single factor experiment design.The main experimental results as the followings：1. The optimal hormone formation of callus subculture is0.28mg/L2,4-D+0.36mg/L6-BA+0.74mg/L NAA.2. Establishing the optimal system of the cell mass suspension culture: MS liquid media;carbon source concentration is3%; the most suitable hormone concentration and combination is1.17mg/L2,4-D+1.21mg/L6-BA+0.33mg/L NAA; inoculation cell mass of29±2g. Underthese conditions, cell mass quantity of the fresh growth maximum value can reach68.87g, cellmass growth cycle of70days, and the cell mass grows most vigorous40thdays.3. The alkaloid content of is up to0.157%when phenylalanine precursor was fed into thesuspension culture cell mass media;0.203%when tyrosine precursor feeding;0.214%whentryptophan precursor feeding. It indicates that the effect of tryptophan precursor feeding is thebest; there are very significant differences between three kinds of additions (P＜0.01), and all ofthem are higher than control (0.132%), also reach to a very significant difference (P＜0.01).4. The three kinds of amino acids were combined, the total alkaloid content is up to0.301%when the optimal combination is phenylalanine10.95mg/L+tyrosine10.13mg/L+tryptophan 20.33mg/L. It is proved three kinds of N-containing amino acids have a good role in promotingthe synthesis of Fritillaria alkaloids.