Studies On In Vitro Culture And Active Constituents Of Ri Plasmidtransformed Roots Of Trichosanthes Kirilowii Maxim, Cassia Obtusifolia L., Ipomoea Batatas(L.)Lam

The technique of Ri plasmid transformation is an important research area of plant genetic engineering. At present, Ri plasmid transformation has been combined with phytochemistry, The integration of two fields has provided newfashioned technique for exploitation of natural products.Ri plasmid has been shown to have reliable efficiency in transferring genes into many plant species, The transformation of Ri plasmid is based on T-DNA transfer. Hairy-root is the result of the transformation of T-DNA into plant genome. Such hairy-root can grow rapidly and demonstrates genetic stability.On the one hand, hairy root can produce the same secondary metabolite as the original plant; on the other it can produce compounds, which are lacked by original plant. These special characteristics made hairy root become a medium for large-scale production of natural products and plant genetic engineering.In this experiment, we succeeded in plantlets regeneration of sweet potato, snake gourd and sickle senna in vitro. Hairy roots were successfully induced from above-mentioned three plants using Agrobacterium rhizogenes A4 , explants are leaf disc^ stem axis and leaf disc respectively. The fact that transferred DNA (T-DNA) of Ri plasmid was successfully transferred into plant cells and integrated into genomes of the plants was identified by opine assay.In order to optimize the speed of growth and ultimate biomass of hairy root, Many parameters have already been considered. Through comparing the effect ofdifferent medium types and pH values of medium on hairy root growth and biomass, the results indicated that MS medium (pH 5.6) was the optimum for sweet potato hairy root growth ; 1/5MS medium (pH 5.8-6.0) is fit for the hairy root growth of snake gourd; 1/10MS medium (pH 5.8-6.0) is fit for hairy root growth, development of branch root and accumulation of ultimate biomaas of sickle senna.Light is another important factor which affect the process of secondary metabolism of hairy root. The Hairy root of sweet potato is apt to accumulate secondary metabolites in faint light. Light is also favorable for rapid growth of hairy roots of sickle senna which are transferred from solid medium to liquid medium, but it is unfavorable for the growth of hairy root of sickle senna which are consistently subcultured on suspension medium.The effects of carbon source on the growth and biomass of snake gourd hairy root were investigated, Saccharose was found favorable for hairy root growth comparing with glucose at same concentration in medium.The aim of hairy root culture is to obtain useful metabolite. So we need to understand the regularity of hairy root growth and identify the harvest time, the time-course curves of hairy-roots of three plants in liquid medium were determined. From the curves, we found the exponential phase of sweet potato and sickle senna is 7-20 days. As for snakegourd, the exponential phase is unconspicuous.Through the pharmacological experiment, we have pursued the active part (SP2. 4) from hairy root of sweet potato, this part was demonstrated to be effective in resisting K562 cell clone and the mechanism of resistance is cytotoxic. The detail structure of this active part has not been identified for lack of sample, but we elementarily confirmed that typical alkaloid reaction appeared in the process of assay of this active part.Betulinic acid was isolated from the hairy root of sickle senna and original plant for the first time. Apart from Betulinic acid, we also compared the content and types of anthraquinone between hairy root of sickle senna and original plant, HPLC analyzing results show that there are two new types of anthraquinone appeared in hairy root.Polyacrylamide gel electrophoresis was employed to compare trichosanthin isolated from hairy-root of snake gourd and that from cultivated snake gourd. Both of them have one kind protein with 29000 molecular weight. Another protein with 31000molecular weight has also been isolated from medium of snakegourd hairy root. The content of coarse trichosanthin in hairy root is 0.6%, this value is five times as high as that of cultivated snake gourd.The conservation and large-scale culture of hairy root were studied in this paper. Hairy root clone can be kept in liquid medium or solid medium; the optimal explant is stem apex.The research of plant bioreactor is at primary stage. Judging from the growth and biomaas of hairy root cultured in bioreactor, we can say bioreactor is superior to suspension culture method. Bioreactor can supply more hairy root products in short time and expense is less than suspension culture.The change of pH value in bioreactor can reflect the change of metabolism, so the identification of relationship between two aspects will lay a foundation for industrial production of hairy root.