Establishment Of Tribulus Terrestris L. Callus Culture System And Determination Of Total Flavonoids Content

Tribulus terrestris L. is a kind of Zygophyllaceae, Tribulus,whose dry and mature fruit were seleted as medicine. It has the ability to grow in many kinds of environment, such as wild,grasses near the river, hillside and roadside. However,its growth places are widely distributed in the north of the Yangtze River and the most of the northern regions are genuine producing areas.Tribulus terrestris L. has the ability to adopt different growth stituationa,though it can adopt different kinds of soil, loosen and rich sandy loam soil is the best suitable for it. The flavonoid component it contained is 1.5 times of its saponin, which has a significant effect on cardiovascular, such as increasing blood flow, blocking platelet aggregation. What’s more, it has the ability of anti-hypoxia.In the state of natural, dry and mature seeds of Tribulus terrestris L. were selected in this study. Aseptic seedlings were successfully obtained through investigating types of disinfectant,duration of action and the methods of treating seeds. Healthy green aseptic seedlings were used as experimental materials to induce Tribulus terrestris L. callus to investigate the influence of different explants, explants age, inoculation methods, culture mediums, culture temperature,light conditions and hormone ratios on callus induction in order to determine the best induction conditions of the callus of Tribulus terrestris L: lay the aseptic seedling stems of 28 d on the culture medium surface of the MS + 0.50 mg/L 2, 4-D + 0.10 mg/L 6-BA; 25 ℃ culture temperature; 24 h/d dark culture.Callus proliferation conditions of Tribulus terrestris L. were optimized on the basis of the above, the best conditions were obtained after investigating the effect of light time, pH value,carbon source and its content, subculture cycle and hormone ratio on the growth of callus: MS +1.00 mg/L 2, 4-D + 0.20 mg/L 6-BA + ZT 1.00 mg/L; 12 h/d light culture; pH 6.2; 25 g/L saccharose as the carbon source; 35 d subculture cycle.After establishing a stable callus culture system of Tribulus terrestris L., this study adjusted the hormone ratio, at the same time, given 16 h/d light culture on the callus. Finally it promoted the redifferentiation of the callus and urged them to form complete plants. Suitable medium for regrowth of Tribulus terrestris L. to take root is MS + 0.4 mg/L IBA + 0.3 mg/L 6-BA; suitable medium for Tribulus terrestris L.’s regrowth germination is MS + 0.1 mg/L IBA + 0.3 mg/L6-BA.Finally, HPLC chromatographic conditions to measure the total flavonoids contents ofTribulus terrestris L. were optimized in this study. And the total flavonoids content of the whole herb, seeds and callus were measured and compared. The total flavonoids content of its seeds was 1.46%; the total flavonoids content of the whole herb was 1.37 times more than that of its seeds, the total flavonoids content of the whole herb was 2.01%; the total flavonoids content in its callus culture system was 5.22%, which was the highest and 3.57 times higher than that of its seeds. According to this study, fast secondary metabolite accumulation of Tribulus terrestris L.could be completed through establishing callus culture system and enlarging culture system.