The Study On Establishment Of Salvia Miltiorrhiza Bunge Hairy Root Culture System And Elicitors's Effect

Salvia miltiorrhiza Bunge is a kind of Chinese traditional herb medicine. Recently, there are many researches about it, and the hairy root of the Salvia miltiorrhiza Bunge is the hotspot, as it has its own features. In this research, the Agrobacterium Rhizogenes ATCC15834 and leaves of Salvia miltiorrhiza Bunge were used as materials. The leaves of Salvia miltiorrhiza Bunge were infected by the agrobacterium, then the hairy root was produced. After co-culture, sterilization and expand training, the dynamic changes of the hairy root would be detected. Then, six elicitors, including yeast extract, Aspergillus niger, ultrasonic waves, Zn2+, Ca2+ and Mg2+, were used to influence the hairy root culture system. After that, the growth percentage of hairy roots and three kinds of tanshinone contents (TanshinoneⅠ,DihydrotanshinoneⅠ, TanshinoneⅡA), as well as the influence of the protective enzyme (SOD,POD,CAT) were detected during experiments. And this provides the research basic to the hairy roots.1.The Agrobacterium Rhizogenes ATCC15834 was used to infect the Salvia miltiorrhiza Bunge leaves, In this experiment, the infection time (30min), co-culture time (2-3h), sterilization method (40℃) and the type of culture medium(MS-N) were established. According to the observation of SEM, there are a lot of growing points on the surface of the hairy root. After growth, they became empty tubes. Hairy root was composed by many empty tubes, and this made hairy roots to easy be cultured in the liquid medium.2.According to experiments, the hairy root which was cultured in the liquid medium has several features: (1) the growth percentage of hairy roots and contents of the carbohydrate in culture media have inverse relationship; (2) the growth of hairy root has delaied periods (0-3d), rapid growth periods (3-21d) and moderate growth periods(21-24d); (3) the accumulation of tanshinone had a quick increase after the 9th day of culture, and there is also a second rapid growth of the tanshinone which occurred after the 15th day of culture, and it decreased after the 21th day; (4) according to the growth and the tanshinone synthesis of hairy roots, the proper time to add elicitors should be the 18th day.3.Two biotic elicitors (yeast extract and Aspergillus niger) and three abiotic elicitors (Ca2+, Mg2+ and Zn2+) can promote the growth percentage of hairy roots, while the ultrasonic waves can inhibit the growth of them. Six elicitors all inhibit the activity of SOD. The two biotic elicitors can promote the activity of POD, but inhibit the activity of CAT. Mg2+ and Ca2+all can inhibit the activity of POD and CAT, but they have the contrary effect. After treatment of ultrasonic waves, effects were contrary with the biotic elicitors. When the hairy roots had a high growth rate, the activities of SOD, POD and CAT are all relatively high.4. Different elicitors have the different effects for the growth and the tanshinone synthesis of hairy roots. The results are as follows: (1) the two biotic elicitors can promote the production of tanshinone with time, while the Zn2+, Mg2+ and Ca2+ did not have this effects. The ultrasonic can inhibit the synthesis of tanshinone. (2) After treatment of elicitors, three protective enzymes all increased, and they all became stabilized with time.