Thidiazuron-induced efficient propagation of Salvia miltiorrhiza through in vitro organogenesis and medicinal constituents of regenerated plants

Abstract

An efficient system for micropropagation using leaf explants was established in a high-grade Chinese medicinal herb, Salvia miltiorrhiza Bunge. A high morphogenetic plasticity was found in the leaf explants, and it showed four types of in vitro morphogenesis, including direct rhizogenesis, indirect rhizogenesis, direct shoot formation and indirect shoot formation. In direct organogenesis, a significantly higher number of shoots was obtained in the presence of 0.1 or 0.5 mg/L TDZ and a significantly higher number of roots was obtained in the plant growth regulator-free (PGR-free) treatment, respectively. In indirect organogenesis, the ratio of NAA to BA showed a significant effect and it followed the regular pattern on in vitro organogenesis that higher ratios promote rooting and lower ratios promote shoot formation from the callus. Generally, in S. miltiorrhiza, the application of TDZ could induce effective responses including induction of direct and indirect shoot formation and also callogenesis when it combined with 2,4-D. In callogenesis, 16 callus lines were induced and maintained, and significant differences of proliferation rate and redifferentiation capacity were found between several lines of the calli. Therefore, it could promote efficiency of regeneration pathway by selecting appropriate callus lines. According to these results, a two-step protocol via direct shoot formation and another four-step protocol via indirectly shoot formation were developed for obtaining regenerated plantlets. The requirements and characteristics of these two regeneration protocols were compared thoroughly, and either has its advantages and applications. Eventually, the regenerated plants had a 100 % survival rate after 45 days of acclimatization. The chemical contents of the regenerated plants were proved by high-performance liquid chromatography, and the extract of the root contained all the major medicinal constituents, including salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA. The in vitro culture system established in this study has various aspects of applications including mass propagation and production of tanshinones. It may provide an efficient model for further investigating on the mechanism of organogenesis in S. miltiorrhiza, especially direct rhizogenesis from leaf explants without the supplement of exogenous PGRs.