| Echinacea purpurea,which belongs to Compositae familia, is a traditional North American perennial medicinal herb. It has many effective medicinal ingredients, such as polysaccharides, caffeoyl phenols, and alkylamides, so as to improve body immune system. Accordingly, it has become one of the most popular herbal products in North America and Europe as an immune promoter, and should have widely applied prospect in medicine. In recent years, we have successfully introduced this breed in China. However, as influenced by climate and soil condition, the content of the active components of Echinacea purpurea is too low and it is also difficult to ensure their medicine quality. As a result, what our subject concerns is just about how to utilize modern biotechniques to solve these problems. Our study will exploit a new way for mass production and secondary metabolites production of Chinese traditional medicine, which is of great significance for the development of Chinese pharmacy and its modernization. Based on these, we adopted techniques of plant physiology ,molecular biology,pharmaceutical analysis interactively, established a system to transform hairy roots of Echinacea purpurea and gained the secondary metabolites of Echinacea purpurea by the hairy roots culture. We also studied the content of their secondary metabolites, which would establish bedrock to obtain secondary metabolites of medicinal plants by hairy roots culture for industrialization. In this paper, we studied primarily three aspects as follows: (1) Establish a technical system for rapid propagation of test-tube plant , using mature seeds of the wild Canada Echinacea purpurea. Kelly callus was produced from the explants of Echinacea purpurea when cultured on MS+IBA(0.4 mg/L)+BA(0.1 mg/L). And then indefinite shoots were developed from the callus on MS+BA(0.3 mg/L)+IBA(0.4mg/L)+NAA(0.1mg/L) and MS+BA(0.3mg/L)+IBA(0.5 mg/L)+NAA (0.1 mg/L). Adventitious shoots took root and grew to regenerated plants on MS medium. (2) Establishment and identification of a system to transform hairy roots of Echinacea purpurea. Hairy roots were induced from the wounded brim of the explants, 25 days after inoculation of Echinacea purpurea explants with A.rhizogenes A4,R1601 or R1000. Hairy roots branched rapidly and grew upwards. The percentage of rooted leaf explants was 80% after A4 infection. Transformation of hairy roots was confirmed by PCR amplification of rolB genes of Ri plasmid in A.rhizogenes A4. (3) Studies on and the chemical constituents content determination of secondary metabolites in hairy roots of Echinacea purpurea, such as polysaccharide and phenolic. The transformed hairy roots grew rapidly after 60 days on a MS medium. By analyzing extraction of hairy roots, it was showed that the content of polysaccharides and phenolic compounds were 236 mg g-1 dw and 18.89 mg g-1 dw. |
