| Summary: | 碩士 === 朝陽科技大學 === 應用化學系生化科技碩博士班 === 102 === Salvia miltiorrhiza Bunge, a perennial plant belongs to Lamiaceae Family. Its roots contain tanshinones and phenolic acids. In the recent years, roots of S. miltiorrhiza have been used for the treatment of cardiovascular diseases. In the present study, hairy roots were induced in leaves of S. miltiorrhiza by infecting with Agrobacterium rhizogenes. In a liquid culture system, these hairy roots were cultured for the production of Salvianolic acid B (SAB). Three basal salt media (MS, B5, and WPM) in half (½x) and full (1x) strengths were evaluated for hairy root growth and SAB accumulation. Among these three basal salt media, B5 was selected because here nitrogen source in form of NO3- and NH4+ are separate and can be controlled to investigate their influence on production of hairy root biomass and SAB. Thus, different concentrations of six medium components viz. sucrose, KNO3, (NH4)2SO4, CuSO4, CoCl2 and NaH2PO4 were investigated for hairy roots biomass and SAB content. At 4 weeks of culture, results showed that ½ strength of MS basal medium gave the least hairy root biomass and was not suitable for the hairy roots culture of S. miltiorrhiza. In case of WPM (1x and ½x), production of the maximum root biomass (6.36 g dw/L) and SAB content (33.6 mg/g) was obtained after 3 weeks of culture. Thereafter, there was a decreasing trend in SAB content in the hairy roots. In the B5 medium (1x and ½x), the maximum production of root biomass (7.87 g dw/L) was obtained at 8 weeks of culture. Thereafter, there was a decreasing trend in the SAB content. It was found that there was no significant difference in biomass when hairy roots were cultured in the B5 basal medium containing different concentrations of (NH4)2SO4 from 0 to 2.0 mM for 4 weeks. However, with the increasing concentrations of (NH4)2SO4, there was a decreasing trend in the SAB content. The SAB content was 3.1-fold higher in the medium without (NH4)2SO4 compared to the medium with 1.0 mM (NH4)2SO4 indicating the influence of (NH4)2SO4 on SAB production. Results showed that growth of hairy roots varied with concentration of CuSO4 (0-75 µg/L) in the medium. B5 medium containing 50 µg/L CuSO4 produced 1.5-fold higher root biomass compared to medium with 25 µg/L CuSO4 demonstrating its influence on root biomass of S. miltiorrhiza. Thus, the present study demonstrates that concentrations of NH4+ and Cu+ in the culture medium effect hairy root production and its SAB content. To verify the effect of NH4+ ions on growth of hairy roots, a further experiment was carried out. The hairy roots were grown in the B5 medium without (NH4)2SO4 for 3 weeks, before adding different concentrations of (NH4)2SO4 in the medium. Results showed that production of biomass was directly proportional to concentration of (NH4)2SO4 (2 mM > 1 mM > 0 mM > CK) at the 4th week of evaluation. However, further culture of hairy roots beyond 4 weeks retarded the biomass production.
In conclusion, this study demonstrates that the type of basal medium, concentrations of Cu+ ions and (NH4)2SO4 in the culture medium significantly influence the biomass and SAB production in hairy root culture of S. miltiorrhiza, thus, needing culture medium optimization for the optimum results.
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