Study On The Factors Effect On Stable Production Of Taxol In Taxus Cells

Cell culture of Taxus is one of the effective ways to solve the shortage resources of taxol. According to previous studies, the synthesis capacity of taxol in Taxus cells can be improved by changing the cultivating method and metabolizing regulation. But now, industrial production by cell culture still not implement, the capital reason is the instability of taxol production during Taxus cell culture. In this study, we concerned the factors which influence the stability of Taxus cell, including cells homogenicity, temperature, medium, addition of demethylation reagent and elicitor, and established a method to detect DNA methylation by HPLC. The main results are as follows:1. Established a new method for inducing callus in high frequency from Taxus explant. The optima conditions for inducing callus are: spring, explant cut for about 1cm stem with leafstalk, MS medium with 0.25mg/L 2,4-D and 1.0 mg/L NAA. In those high production cell lines of Taxus media, taxol production can be reached to 400μg/g.2. The technology of stably subculture in Taxus cells by low temperature (4℃) was established in this study: Taxol production is quickly decreased after subcultured from preservatived in low temperature And it can be effectively inhibited with 5-7 days treatment of second low temperature. Adding elicitor after low temperature and heat-shock, taxol production can be increased to 132.2μg/g, 6.19 times higher than that just induced.3. The effects of different culture conditions on Taxus media cell were systematic studied. The results showed: phylohormone has larger effect on the stability of Taxus cells than medium types and subculture period. The culture method has little effect on the stability of Taxus cells. Taxol production could reach to maximum if high producing cells are about 40% in inhomogeneos culture system.4. Study on the influence of DNA methylation level on taxol production. The results showed that 1 mg/L 5-Aza-CdR for 12 days did not extremely affect the cell growth, but effectively increased taxol production. Demethylation reagent would be more useful to Taxus chinensis both in biomass and taxol production. Induced by dihydro methyl jasmonate after adding 5-Aza-CdR into Taxus media cells, taxol production can be reached to 362.2μg/g, raising 6.19 times, compared of 8.16 times increased in Taxus chinensis.5. A method to detect DNA methylation by HPLC in Taxus cells was established, and it was confirmed to have a satisfactory stability and reproducibility after repeated experiments. The level of methylation in Taxus chinensis is generally higer than in Taxus media which detected by this method. The HPLC results showed that there indeed have some relationship between the increase of taxol production and the decrease of DNA methylation by using the demethylation reagent 5-Aza-CdR.