Alkaloid Content Determination Of Cultured Coptis Cells By Two-stage Suspension-culture

As medicine, coptis is the dry roots of the Coptis chinensis Franch. Coptis deltoidea C.Y. Cheng et Hsiao,and Coptis teeta Wall in Ranuculaceae. Coptis roots contains a lot of alkaloids, and there is remarkable curative effect on various diseases. already widely used in clinical Growth of coptis dry roots is extremely slow, pharmaceutical is very lack. Plus coptis belong to yin born plants, artificially cultivated is difficult.To Production secondary metabolites by Plant cell culture can overcome the chemical synthesis shortage and the difficult of Artificially cultivated.Due to the process of training in plant cells.there is contradiction between cell growth and the synthesis of secondary metabolites. And different medium to promote cells to grow and secondary metabolite synthesis has different effect.So In the previous research foundation, this experiment deep-explor the buttercup family plant coptis by two-step culture method. The basic medium were change for set up for 6,7-V1 and 6,7-V2.And set different concentrations of hormone for the two kinds of culture media, with the cell growth and Berberine content determination, expecting to get the best hormone concentration combination. The results are as follows:1,Explant disinfection:coptis leaves as selected explant, fisrt prepare the experiment on the disinfection methods of rhizome coptis blade callus induction training with three years borned.optimal disinfection method and time is:first use soft-brush brush dust, Soak fluffy coptis blade about 5min with soap, And remove soap. Then flush water 2h or so. After washing, use the disinfected paper absorb surface moisture, soaking in 75% ethanol for 30s, At last. remove it and timely use HgCl2 added 1～2 drops 0.1% of polysorbate-80 soaking 12min, and flushed 6 times by sterile water.2,Primary training:the optimal hormone concentration ratio in inducing coptidis blade callus is 6,7-V+2.4-D 1.0 mg/L +KT 0.2 mg/L. In this hormone concentration, coptis blade callus is started in the 32 day,and the callus induction rate is 89.5%.3,Subculture:subculture on the solid medium when callus appeared about 50d.and transfer once about every 15day.This is to prevent the impact of browning, and materials leak too much fluid.4,Callus state regulation:hoar, waterlogging shape callus can be improved in suculturing 4 times within ZT, or match with 6-BA and NAA.For light yellow, yellow, light green, green callus, state also can be better improved in suculturing 2 or 3 times alone within ZT 0.05 mg/L+6-BA 0.5 mg/L. or match with casein acids hydrolysate (CH500).5,Two-step method culture cell:Best hormone concentration ratio is 6.7-V1+2.4-D 1.5 mg/L+KT 0.2 mg/L in suspending of one-step method.dry weight of harvest cell is 21.58 g/L.fresh weight is 182.92 g/L,and the content of total alkaloid is 8.3%; Best hormone concentration ratio is 6,7-V:+NAA 1.5+KT 0.2 in suspending of two-step method.dry weight of harvest cell is 17.89 g/L.fresh weight is 129.01 g/L.and the content of total alkaloid is 13.8%.;In concerned of cell growth, cell growth index determinated by one-step method is higher than two-step method. And in concerned of alkaloid content, alkaloid content is obviously higher than determinated by one-step method, although each alkaloid content is not proportional increase. Keywords:Coptis, Callus, Cell Suspension Culture, Alkaloid...