Establishing Taxus Media Cells Suspension System And The Effects Of Fungal Elicitor On Taxol Content Of Cells

Using the Taxus Media cell culture on a large scale is one of the most effective method to produce taxol,and adding elicitor can promote the synthesis of secondary metabolites,but using the Taxus Media cell culture to produce taxol has not realize the industrial production,the most important reason is the high cost,which make it a bottle-neck to realize it.In this study,ten-year-old Taxus Media,growing in Guanzhong,Shaanxi,was used as experiment material for the induction of callus.Screening explant and medium to induce callus,study the influence of different factors on the browning,such as Vc,establishment of stable cell suspension culture system.meanwhile,it do a discussion on the effect of fugal elicitor for the Taxol-production of Taxus Media suspension cell.Provide a theoretical basis for taxol production.The main results of the study are as follows:1.B5 and MS medium both can induce the callus from Taxus Media Successfully.Among them,B5 as the basic culture medium,induction rate of One-year-old stems(Φ≈2 mm)and old stems(Φ≈2 mm)are higher,MS as the basic culture medium,induction rate of detached leaves are higher.Two kinds of culture medium do not have a significant effect on the time of formation of callus.The inducing rate of different explants callus have a obvious difference.For example,B5 as the basic medium,the callus rate of old stems(Φ≈2 mm),one-year-old stems(Φ≈2 mm),one-year-old stems(Φ≈1mm),old leaves,one-year-old leaves are 61.5%,86.7%,23.0%,15.0%and 10.0%.Disinfecting way can effect the fugal rate of different explant callus.For one-year-old stems(Φ≈2 mm)about spring-summer,we should use the 75%C2H5OH disinfecting 30s,0.1%HgCl2 disinfecting 14 min and 75%C2H5OH disinfecting 1 min,0.1%HgCl2 disinfecting 20 min for autumn-winter explants.2.Vc、LH and PVP three anti-browning agents have a obvious benefit on Taxus Media callus growth,Add amount of Vc、LH and PVP were 5 mg·L-1、1.0 g·L-1 and 1.00 mg·L-1.3.The inoculum of Taxus Media cell suspension cultivating system is about7.5 g·L-1,for the whole suspension process,the synthesis period is 30 d,among them,0～9 d is adjust phase,9～18 d is logarithmic phase,18～30 d is stable phase and after 30 d is decline phase.During the 0～12 d of cell cultivating,the production of the Taxol is 0,after 12 d,the cell start to produce taxol gradually.In the 24 d,the production of the taxol is the highest,the accumulation is 17.04 mg·L-1.in the 30 d,the accumulation of Taxol is 18.84 mg·L-1,the highest.We observe find that the status of the cell present rounded or oval in the microscope.4.If use the fugal culture solution as elicitor,the best experiment condition is the 14 d of cell suspension cultivating,then add a fugal culture solution which has been cultivated 7 d,the quantity is 9%,here,the taxol production of suspension cell is highest,reach the number 45.32 mg·L-1,is 2.31 time than control group.If use the fugal mycelia extract as elicitor,the result show that when we add the mycelia extract which has been fermentation for 6 d in the 16 d of cell suspension,the quantity is 9%(v/v),the Taxol production of suspension cell is 47.64 mg·L-1,the highest,2.74 times than control group.