Studies On Accumulation Of Taxol And Biosynthetic Intermediate In The Callus Of Taxus Cuspidate Sieb.et Zucc.var

In this study, the young shoots of Taxus cuspidata Sieb.et Zucc.var. were sampled forcallus induction, and then factors including culture base ingredients, culture conditions, aminoacids and elicitors were tested for their effects on callus growth, and secondary metoblites andimportant intermediate compound synthesis and accumulation, and as well, severalparameters affecting callus induction, growth and compounds accumulation hereby wereoptimized, which aimed to provide important data for large-scale production of raw materialof taxol sources by means of cellular engineering in the future. The results are as follows:1. The formula of B5+2,4-D0.5mg·L^-1+NAA1.5mg·L^-1was proven to be the mostsuitable culture media for callus induction at24℃under darkness, and B5+NAA1.0mg·L^-1+6-BA0.2mg·L^-1+2,4-D1.5mg·L^-1at24℃under darkness for callus proliferation. Thecallus growth period was about22～24d, and showed an “S”type.2. The most suitable culture base for taxol accumulation in callus was B5+NAA1.0mg·L^-1+6-BA0.1mg·L^-1+2,4-D2.0mg·L^-1, containing30g·L^-1sucrose, mixed nitrogen(thetotal concentration was32mmol·L^-1) a30:2ratio of NO₃^-to NH₄⁺,1.25mmol·L^-1NaH₂PO₄,1.02mmol·L^-1CaCl₂,24℃, In the late stage of culture lumination （4600lx）,12h·d^-1. For10-deacetylbaccatin Ⅲ（10-DAB） synthesis and accumulation, it was: B5+NAA1.5mg·L^-1+6-BA0.2mg·L^-1+2,4-D2.0mg·L^-1, containing50g·L^-1sucrose, a24:8ratio of NO₃^-to NH₄⁺,NaH₂PO₄1.25mmol·L^-1,0.51mmol·L^-1CaCl₂,24℃, illumination （1900lx）,4h·d^-1in thelate stage of culture.3. The effects of various amino acids on callus growth, taxol and10-DAB accumulation.The results showed that the content and yield of taxol reached a maximum of193μg·g^-1,3.4412mg·L^-1，and increased2.02fold,2.44fold in the presence of1mmol·L^-1glutamic acidcompared with the control; in addition, the content and yield of10-DAB reached a maximumof290μg·g^-1,6.1023mg·L^-1, and increased1.4fold,2.7fold in the presence of1mmol·L^-1ofphenylalanine in the culture media.4. The effects of several elicitors on callus growth and taxol and10-DAB accumulationwere investigated. The results showed that365nm UV irradiation for30min treatment taxol content and production reached a maximum of129μg·g^-1,1.6838mg·L^-1, and increased1.35fold,1.19fold, UV irradiation for10min treatment10-DAB content and production reached amaximum of199.4μg·g^-1,2.4684mg·L^-1, and increased1.34fold,1.13fold; treatment ofoxalic acid affected both taxol and10-DAB synthesis and accumulation, that’s that0.1mmol·L^-1of oxalic acid treatment taxol content and production reached a maximum of153μg·g^-1,2.3199mg·L^-1, respectively, and increased1.60fold,1.64fold; After addition of0.05mmol·L^-1, the content and yield of10-DAB reached a maximum of209μg·g^-1,3.4044mg·L^-1,and increased1.41fold and1.55fold. By contrary, treatment of salicylic acid causedinhibition on both the growth of cell and accumulation of10-DAB, however, it was useful forthe accumulation of taxol; Treatment of0.05mmol·L^-1hydrogen peroxide, the maximumvalue of the content of taxol and callus10-DAB reached1.9115mg·L^-1,3.3492mg·L^-1,respectively, and increased1.35fold and1.53fold.5. The additions of carbon, ascorbic acid （vitamin C） and polyethylene pyrrole into thecultures could relieve the browning of callus to some extent. And the addition of50mg·L^-1ascorbic acid（vitamin C） exhibited significant inhibition on the browning of callus camparedwith the other two agents.