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To study the effects of the extract of fungal elicitor, AgNO3, MeJA and yeast on the growth and content of secondary metabolites of adventitious roots in Tripterygium wilfordii. The above elicitors were supplemented to the medium, the growth and the content of secondary metabolites were measured. When the medium was supplemented with the elicitor Glomerella cingulata or Collectotrichum gloeosporioides, the content of triptolide was increased by 2.24 and 1.93-fold, the alkaloids content was increased by 2.02 and 2.07-fold, respectively. The optimal concentration of G. cingulata was 50 μg/mL for accumulation of triptolide, alkaloids and for the growth of adventitious roots. AgNO3 inhibited the growth of adventitious roots and the accumulation of the alkaloids, whereas it (at 25 μmol/L) increased the accumulation of triptolide by 1.71-fold compared to the control. The growth of adventitious roots, the contents of triptolide and alkaloids were increased 1.04, 1.64 and 2.12-folds, respectively when MeJA was at 50 μmol/L. When the concentration of yeast reached 2 g/L, the content of triptolide increased 1.48-folds. This research demonstrated that supplementation of AgNO3 and yeast enhanced the biosynthesis of triptolide in adventitious roots and the synergism of G. cingulata and MeJA could promote the biosynthesis of both triptolide and alkaloids.

Objective: To get the information of resources, cultivation, commodity circulation and other aspects of Tripterygium wilfordii and Tripterygium hypoglaucum., Methods: Collect samples in 13 locations of Tripterygium wilfordii and Tripterygium hypoglaucum, compare their plant morphological characteristics and growth habit, and investigate their wild resources conditions, planting information, easy-confused varieties and different commodity features., Results: (1) Tripterygium wilfordii and Tripterygium hypoglaucum were mainly collected under woods or on the edge of woods,and light and moisture attributed to their distribution to some extent. (2) Wild resources of Tripterygium wilfordii and Tripterygium hypoglaucum were shrinking, and both of their cultivation history were relatively short and their cultivation technique were still in a low level. (3) Due to lack of harvesting and processing standards, decoction pieces, varying from roots, rhizomes to stems of plants, were all sold as commercial medicines., Conclusion: Wild resources of Tripterygium wilfordii and Tripterygium hypoglaucum are shrinking,and the standardized research on cultivation-harvest processing and commercial medicines remains to be further carried out.

Using MS as basic medium, supplemented with 1.0 mg · L(-1) IBA, the adventitious roots of Tripterygium wilfordii were induced, and the good adventitious root culture system was established by leaves or callus induced by leaves as explants. The adventitious roots were also induced with 2.0-4.0 mg · L(-1) NAA and the good adventitious root culture system established by using suspension cells from callus as materials to induce adventitious root. The content of triptolide of three adventitious roots culture system were exceeded in the natural root bark. The content of triptolide of AR3 adventitious roots was the highest about 5.3 times as that in the natural root bark. By using 5 L stirred fermentor during pilot enlarge cultivation, compared with 250 mL flask cultivation, the adventitious roots increment and secondary metabolites content per liter medium showed no significant difference. The accomplishment of this analysis laid a foundation by tissue culture production of the secondary metabolites of T. wilfordii.

In order to solve the shortage of natural Tripterygium wilfordii Hook. f. plant resource for the production of the important secondary metabolites triptolide and wilforine, hairy roots were induced from its root calli by Agrobacterium rhizogenes. Induced hairy roots not only could be maintained and grown well in hormone-free half-strength Murashige and Skoog medium but also could produce sufficient amounts of both triptolide and wilforine. Although hairy roots produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots, they could grow fast and could be a suitable system for producing both secondary metabolites compared with other tissues. Addition of 50 micrometer methyl jasmonate (MeJA) could slightly affect hairy root growth, but dramatically stimulated the production of both triptolide and wilforine, whereas 50 micrometer salicylic acid had no apparent effect on hairy root growth with slightly stimulatory effects on the production of both secondary metabolites. Addition of precursor nicotinic acid, isoleucine, or aspartic acid at the concentration of 500 micrometer had varying effects on hairy root growth, but none of them had stimulatory effects on triptolide production, and only the former two had slightly beneficial effects on wilforine production. The majority of triptolide produced was secreted into the medium, whereas most of the produced wilforine was retained inside of hairy roots. Our studies provide a promising way to produce triptolide and wilforine in T. wilfordii hairy root cultures combined with MeJA treatment.

The adventitious root of Tripterygium wilfordii was used as experiment material to study effects of various concentration of aspartic acid, isoleucine, cysteine and arginine in MS medium on the growth and triptolide, wilforgine, wilforine contents of the adventitious roots. The results showed that compared with the control, supplemented with 0.25 mmol x L(-1) aspartic acid at 3rd week, the growth of the adventitious roots only accounted for 80%, but the content of triptolide of the adventitious roots and the medium was 1.36, 1.30 times, the content of wilforgine was 1.16, 1.37 times, the content of wilforine was 1.22, 1.63 times, respectively. At 3rd week 0.05 mmol x L(-1) isoleucine, the growth of adventitious roots was 97.3%, wilforgine of adventitious roots and medium 1.02, 1.27 times, wilforine 1.36 times and 1.15 times. At 1st week 0.25 mmol x L(-1) cysteine, the growth of the adventitious roots comprised 77.5% of the control, while content of triptolide of adventitious roots reached 1.87 times. At 2nd week 1.00 mmol x L(-1) cysteine, the growth of adventitious roots was 44.6% of the control, the content of wilforine in medium was 2.97 times. At 3rd week 0.50 mmol x L(-1) arginine, the growth of adventitious roots was 124.2%, the content of wilforgine and wilforine was 1.3, 1.4 times, respectively.

A total of twenty-three endophytic bacteria were isolated from Tripterygium wilfordii, among which three strains were selected for further studying based on their capabilities of growth-promotion and wheat germination. All three isolated strains could produce phytohormone and siderophore, and LG3 and LJ10 strains were capable of dissolving phosphorus. Additionally, LG3 and LY4 strains were both proved to have nitrogen-fixing function and 1-aminocyclopropane-1carboxy-late deaminase activity. Bacterial 16S rDNA sequence identification and homology analysis suggested that LG3 and LY4 strains belonged to the Enterobacter and LJ10 belonged to the Pantoea. The results of reinoculation experiment demonstrated that three endophytic bacteria could not only promote the growth of Tripterygium wilfordii, but also improve the triptolide contents of different organs significantly.

Objective: To study the genetic relationship and diversity of 3 species which belong to Tripterygium., Method: Samples were collected and divided into 4 types: typical T. wilfordii, typical T. hypoglaucum and their middle type according to morphological characters, and T. regelii. RAPD markers were used to measure the genetic relationship and diversity of 110 individuals from 22 natural populations in China., Result and Conclusion: 10 primers were selected from 100 ones screened. A total of 128 bands were scored and 123 of them were polymorphic. Cluster analysis indicated that all the samples could be divided into 3 parts: 5 individuals from T. regelii gathered closely and were separated from other population and formed single branch. All 4 populations from T. hypoglaucum gathered directly. The middle type showed a nearer relationship with T. wilfordii than with T. hypoglaucum, although there was genetic differentiation between populations from middle type and those from T. wilfordii. The middle type has more diversity than T. wilfordii. The distance between different middle type populations and T. wilfordii was different. The existence of middle type populations between T. wilfordii and T. hypoglaucum suggested that the 2 species should be combined into one species. Considering all populations except T. regeli, the generic differentiation among populations was significant and the genetic diversity existed mainly among different populations. So samples of Tripterygii should be collected from different ppopulations for the reservation of the genetic diversity.

Objective: To study the content variation of triptolide in medicinal material of Tripterygium and provide theoretical basis for the hereditary improvement, the gathering and process, the quality evaluation and the provenance division in medicinal material of Tripterygium., Method: HPLC method was used to determine the content of triptolide., Result: The relations between triptolide and germplasm, growth year, gathering season were found out basically., Conclusion: The triptolide contents in xylem are affected by hereditary factors remarkably. While the triptolide contents in phloem are not affected obviously. The accumulation of triptolide needs the certain growth years. However when growth is beyond certain years, the triptolide content decreases with the disintegration of secondary metabolism in xylem. The triptolide in xylem is highest in winter and decreasing in growing season. The triptolide in phloem is less affected by the season.

The experiment of cutting propagation of Tripterygium wilfordii Hook. f. was carried out. It showed a better result when stems were instantly planted after being moistened or soaked by 500 - 1000 ppm IBA or NAA. A remarkably higher survival rate was achieved while plasticfilm and shade were accompanied. September and October or rainy June are prime moments for operation. It' s recommendable to choose those newly corking twigs for cuttage.

Objective: To establish a method for the determination of triptophenolide in Chinese herb Leigongteng (Tripterygium wilfordii Hook. f.)., Method: TLCS was used. The sample was purified with a column and developed upwardly with CHCl3-EtOEt(2:1), sprayed with Keddle testing solution and scanned. The content was calculated with external two-point method., Result: The linear range was 2.1 micrograms-10.5 micrograms, r = 0.998., Conclusion: The method is proper for the quality control of Leigongteng.