Effects Of Elicitors On Biomass And Metabolites Accumulation In Hairy Root Culture Of Tripterygium Wilfordii Hook.f.

Tripterygium wilfordii Hook.f.is a perennial vine of the genus Tipterygium in the family euonymaceae.It has a long use in Chinese medicine and for pest control as an important natural resource.T.wilfordii contains diterpenoids,triterpenoids,sesquiterpene alkaloids and other secondary metabolites with different biological activity.Wilforgine and wilforine of sesquiterpene alkaloids have insecticidal activity.T.wilfordii hairy roots have high content of alkaloids,fast growth rate and stable content.It has the potential to replace natural resources to produce sesquiterpene alkaloids.The adding of elicitors is an effective way to improve the content of bioactive compents in vitro culture.According to our knowledge,it is few reports about the elicitors on production of secondary metabolites in T.wilfordii in vitro culture.The medium salt strength is optimized for biomass accumulation and secondary metabolites production in T.wilfordii hairy roots.Five different elicitos and the combination of elicitors were explored for the growth and secondary metabolites accumulation in hairy roots following the determination of hairy root growth curve.The main research results are as follows:1.The screening of the salt concentration of MS liquid medium showed that the four kinds of medium with different salt concentrations,including 1/4 MS,1/2 MS,3/4 MS and MS,had different effects on the growth of hair roots and secondary metabolites.According to the result,the hair roots grew well in the medium of 1/2 MS and MS,with the biomass of 0.86 g/flask ease of medium salt concentration,and the contents of both alkaloids and alkaloids reached the maximum value in MS medium.Therefore,MS medium was selected as the basic medium for further studies.2.The growth curve of T.wilfordii hairy roots showed that the hairy roots grew slowly from 0 to 14 days,which was in a growth delay period.Hairy roots grew rapidly and passed into the logarithmic growth period at days 14 to 28 and into stable growth period at days 28 to 35.After 35 d,the dry weight of the hairy roots decreased and the content of sesquiterpene alkaloids also decreased.The contents of the internal sesquiterpene alkaloids in 21-28 d increased rapidly.However,the content of wilforgine and wilforine in 28-35 d was basically stable.Considering a variety of factors,the 28 d was selected to add elicitors.3.Five kinds of elicitors had different influences on the hairy root growth and secondary metabolites.Methyl jasmonate had little effect on the growth of hairy roots.However,it could significantly increase the total production of sesquiterpene alkaloids.The total production of wilforine was 5.06 times as much as the control after the treatment of 100 ?mol/L.Low concentration of salicylic acid promoted the growth of the hairy roots.The total production of wilforgine and wilforine reached 1777.09 and 592.16 g/flask when the concentration was 100 ?mol/L.High concentration of silver nitrate inhibited the growth of hairy roots,but significantly increased the yield of sesquiterpene alkaloids.The total production of wilforgine and wilforine reached the highest after silver nitrate treatment at 50 mg/L,which was 3.85 and 2.26 times of the control,respectively.10 g/L sorbitol promoted the growth of hairy roots and increased the production of wilforgine and wilforine.Chitosan of different concentrations had no obvious effect on the growth of hairy roots.And the the optimum concentration of chitosan was 100 mg/L.The elicitors showed selectivity to the growth of T.wilfordii hairy roots and the accumulation of wilforgine and wilforine.Methyl jasmonate and silver nitrate were the two inducers with better induction effect.4.The combination of methyl jasmonate,salicylic acid and silver nitrate had different influence on the hairy root growth and sesquiterpene alkaloids accumulation.The combination of methyl jasmonate(100 ?mol/L)and salicylic acid(100 ?mol/L)was optimum.The total production of wilforgine and wilforine was 5.33 and 8.56 times of that of control,both of which were larger than the maximum production of two kinds of alkaloid after a single inducer.