Tissue culture of Salvia miltiorrhiza adventitious roots (II) effects of carbon, nitrogen, and phosphate sources on culture of Salvia miltiorrhiza adventitious roots

Objective: To study the effects of carbon, nitrogen, and phosphate sources on the growth of Salvia miltiorrhiza adventitious roots and the contents of tanshinone II_A and protocatechuic aldehyde. Methods: The adventitious roots were obtained through tissue culture by manipulation of carbon, nitrogen, and phosphate sources and the contents of tanshinone II_A and protocatechuic aldehyde were determined by HPLC. Results: Carbon, nitrogen, and phosphate sources were necessary for the culture of S. miltiorrhiza adventitious roots. The highest times of root multiplication were achieved at sucrose level of 30 g/L after 20 d culture, 60 g/L sucrose and low level sucrose were favorable for biosyntheses of tanshinone II_A and protocatechuic aldehyde, respectively. The highest root yield and tanshinone II _A content on day 25 were obtained by intermittent sugar adding during cultivation, and the production of adventitious roots and tanshinone II_A were 2.3- and 2.4-fold compared with those of control, respectively. The maximum root growth rate, contents of tanshinone II_A and protocatechuic aldehyde were achieved while NH₄⁺-NO₃^- was 1:4, 1:4, and 1:1, respectively when concentration of total nitrogen source was kept at 60 mmol/L. To compare with the control group, changing of KH₂PO₄ concentration could favor for the adventilious root growth, but high KH₂PO₄ concentration inhibited tanshinone II_A biosynthesis. Conclusion: The results show that various carbon, nitrogen, and phosphate sources have the significant effects on adventitious root culture of S. miltiorrhiza. The best carbon source and its concentration, nitrogen and phosphate sources for the growth of S. miltiorrhiza adventitious root and the synthesis of secondary metabolite are confirmed.