Preliminary Study On The Identification, Cultivation And The Regulation Of Secondary Metabolites Of Tripterygium Wilfordii Hook.f Hairy Root

Tripterygium wilfordii Hook.f.(Celastraceae), woody climber, has important agriculcuralactivity and medical value. The main active ingredients are terpenoids and alkaloids. While，because of the dramatically increased demands and long-time excessive excavation in recentyears for metabolites extraction，the survival of T. wilfordii are seriously threated and the wildresources are reduceed sharply. In addition, with the long growth period of T. wilfordii and thedifficulty of chemical synthesis for its active substance, it’s far from the satisfication forhuman demands. Therefore, it has increasing significance in tissue cultures to settle thelimitation of T. wilfordii natural resources. The hairy root culture has huge potential in theproduction of plant secondary metabolites, for its characteristics of rapid growth, high contentof secondary substances with stable synthetic process. Based on the previous reseach, thisstudy focused on the PCR identification, the establishment of liquid culture system, the effectsof precucor and elicitor on the contents of secondary metabolites in T. wilfordii hairy roots.The main results were as following:1The identification of T. wilfordii hairy roots. The PCR-based analysis was performed tospecifically amplify rolB(506bp), rolC(487bp), aux1(815bp) genes in the T-DNA of T.wilfordii hairy roots induced previously. We got the. The results showed the T-DNAfragments from the Ri plasmid of Agrobacterium rhizogenes strain A4had been integratedinto T. wilfordii tissues.Therefore, these roots were testified to be fairy roots.2Solid medium for the growth of T. wilfordii hairy roots. The results showed that whensupplemented with50ml/l coconut milk and1mg/L IBA to the1/2MS basic medium, thehairy roots grew the best, The subculture (100ml flask) of T. wilfordii hairy roots wasconducted to by transferring the roots to this suitable medium with5.37g and0.5g to thissuitable medium with5.37g and0.5g fresh weight and dry weight respectively after50dcultivation.3Liquid mediums were screened for better culture incubation of T. wilfordii hairy roots.The T. wilfordii hairy roots on1/2MS liquid medium were cultivated statically for30d, andthen the roots were transferred into1/2MS liquid medium shaking on an incubator at120rpm.The medium was supplemented with50ml/l coconut milk and1mg/L IBA as mentionedabove. After2~3subculture, the growth tended to be stable and consistent. 4Preliminary tests were performed to investigate the effects of precusors and elicitors onthe contents of triptolide and wilforine in T. wilfordii hairy roots. The results indicated that theprecusors and elicitors had different impacts on the production of secondary metabolites in T.wilfordii hairy roots.(1) MeJA could significantly promote the accumulation of triptolide andwilforine, whose contents were1448.0and3851.4μg/flask(1.9and6.7folds, respectively,more than the control). In addition, the secretion contents of triptolide were significantlyincreased in the culture medium. It reached1.1folds than the control after MeJA treated for15days.(2) Nicotinic acid and salicylic acid enhanced the total amount of wilforine, as well.(3) When treated by nicotinic acid, isoleucine, aspartic acid and salicylic, the production oftriptolide was decreased to varying degrees, and the same phenomenon was observed forwilforine when treated by isoleucine and aspartic acid...