The Two-step Cultivation And Metabolism Regulation Of Tripterygium Wilfordii Hook. F. Adventitious Roots

Tripterygium wilfordii Hook.f. is Celastraceae Tripterygium woody perennial vine as well as traditional Chinese medicinal plants. It contains a variety of active substances. In agriculture, we found it had activity in toxic action, development inhibition and antifeedant activity to many pests. The adventitious roots lines was established using the adventitious roots after inducing from tender leaves. In this research, we studied the growth ratio of adventitious roots and the content of triptolide, wilforgine and wilforine; the effects of elictors on the contents of adventitious roots growth and secondary metabolites were studied. We researched the effect of different culture medium and two-step cultivation on the growth ratio and content of triptolide, wilforgine and wilforine in adventitious roots. Different T. wilfordii cultures insecticidal activity was explored. The main results and conclusions were given as follows:1. The growth curve of adventitious roots and the compounded rule of secondary metabolites:In the 0~15 d, adventitious roots was in lag phase; in 15~20 d, adventitious roots was in log phase; In the 20~30 d, adventitious roots was in stationary phase. A lot of secondary metabolites compounded in the period. After 30 d, adventitious roots entered decline phase. The weight of adventitious roots and the content of secondary metabolites in adventitious roots started down.2. The selection of NO and H202:Through regulating different concentration of H202 and NO to enhance the growth and secondary metabolite content of adventitious roots in the medium,we access to two better training conditions: In MS+4.0 mg/L NAA+0.5 mg/L KT+1 μmol/L H202 cultured conditions,the content of the triptolide in adventitious root was 81.62 μg/g, 1.2 times as control; the content of the triptolide in the culture medium was 444.02 μg/bottle; 1.4 times as control; In MS+4.0 mg/L NAA+0.5 mg/LKT+ 70 μmol/L NO cultured conditions,the content of the triptolide in adventitious root was 59.18 μg/g, the content of the triptolide in the culture medium was 398.91μg/bottle; 1.49 times as the control.3. Effect of different culture mediums and two-step cultivation on the growth ratio and content of triptolide, wilforgine and wilforine in adventitious roots of T.wilfordii:Training with the adventitious roots in the MS、B5、NT、H、6,7-V、1/2MS、1/4MS、White eight mediums, we found that MS medium was the most suitable for adventitious roots growth;White medium was the most suitable for accumulation of secondary metabolites. We access to MS medium as growth medium and H medium as production medium. We choose different time for the second step training.We access to better training conditions- conducting the second culture stage at the 20 th day using H medium as growth medium and white medium as production medium. The content of the triptolide in adventitious and the culture medium was 476.19 μg/flask, wilforgine was 207 μg/flask, Wilforine was 739.03μg/flask。4. Different T. wilfordii cultures insecticidal activity:Insecticidal activity of T. wilfordii adventitious roots cultivated in different mediumes extracts on plutella xylostella larvae was evident. The extrats from that cultivated in H culture medium is the most toxic one with LC50 of 7.9603 mg/mL, followed by that cultivated in NT culture medium with LC50 of 8.7416 mg/mL.In conclusion, this research optimized the T. wilfordii adventitious roots culture conditions, improved the content of secondary metabolites. This provided basis for metabolic engineering and theoretical foundation for industrial production.