Dihuang Regeneration System Of The Establishment Of Genetic Transformation System Research

Radix Rehmanniae, which is prepared from the fresh or dried rhizomes of fresh Rehmannia glutinosa. Libosch, is a famous traditional Chinese medicine with long history for its significant clinic effects and widely applications in the treatment of immune system, cardiovascular system, hematopoietic system, endocine system, cancer, sienium and so on.As a result of long-term vegetative propagation, degeneration comes to be one of the most intractable problems in the planting of Radix Rehmanniae. It was shown in many studies that the viruses are responsible for this phenomenon. According to our previous study, TMV and CMV are the main pathogenies from the plant. And we tried to explore antiviral gene transformation on the plant, but failed. The possible reasons might lie in two sides: the low regeneration frequency and the improper transforming condition. Hence this paper worked at optimizing the regeneration system of Rehmanniae glutinosa, and establishing the proper gene transformation system mediated by Agrobacterium.The main content and results of this study is described as followed:1．Two approaches of regeneration were experimented and optimized: callus tissue and direct differentiation of leaves.(1) Orthogonal design was used to find the optimal concentrations and combinations of plant-growth substance for inducting callus of Rehmannia glutinosa:The L₂₇ (3¹³ ) table was chosen, and five indicators-callus induction rates, the degree of callus, root redifferentiation ratio, shoot redifferentiation ratio and growth rates-were used to evaluate the effects of 2,4-D,NAA,6-BA and analyzed qualitatively and quantitatively. Based on the results, the plant-growth substances: 2,4-D,NAA,6-BA were carefully analyzed for the induction and regeneration of callus. The results indicated that the best medium for callus induction was MS+ 0．5mg/L NAA + 2．0 mg/L 6-BA.(2) Optimization of direct regeneration of leaves:To evaluate the effects of different Cytokinin / Auxin proportion on direct regeneration of leaves, shoot redifferentiation ratio and growth rates were examined and the results demonstrated that the best Cytokinin / Auxin proportion is 40．To study the effect of AgNO₃ on the regeneration capability of Rehmannia glutinosa .Libosch, shoot differentiation ratio and growth rates were calculated for various levels of AgNO₃．The statistics analysis showed that the environment of 5～10mg/L AgNO₃ can significantly improve the regeneration capability of Rehmannia glutinosa.To study the effect of CH on the regeneration capability of Rehmannia glutinosa. Libosch, shoot redifferentiation ratio and growth rates were calculated for various levels of CH. The statistics analysis showed that when the concentration of CH above 0．5mg/L is toxic to leaves and deleterious to regeneration.2．Exploration on Agrobacterium-mediated Transformation of Rehmannia glutinosa .LiboschLBA4404/pCAMBIA 1305．1 was transformated into the leaves of Rehmannia glutinosa, and the transient expression of GUS was shown. The elementary transformation system was established as followed:(1) The sensitivity of rooting capability of Rehmannia glutinosa to hygromycin was tested, and the minimum concentration of hygromycin was defined as 4mg/L.(2) The bacteriostasis experiment with 0～500mg/L carbenicillin showed that the minimum concentration of carbenicillin should be 200mg/L.(3) The appropriate infecting time length is 5～10 min.(4) Adding carbenicillin significantly impeded the occurring of shoots of the plant.(5) The existence of AgNO₃ can significantly improve the regeneration capability of Rehmannia glutinosa.(6) AS can improve the regeneration capability of Rehmannia glutinosa. Libosch.According to the above research, the in vitro regeneration system of Rehmannia glutinosa was optimized, and the role of different plant-growth substances was cleared up. And the elementary Agrobacterium-mediated transformation system of Rehmannia glutinosa was founded. These results established the foundation for target DNA transformation of the medicinal plant and for the further quality improvement.