Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding

Ag (+) was employed as an abiotic elicitor to stimulate the secondary metabolite production in hairy root culture of Salvia miltiorrhiza, a valuable herbal plant. The addition of Ag (2)S (2)O (3) at 15 - 40 microM to the culture between 12 and 22 days post inoculation resulted in more than 2-fold increase in the yields of the three diterpenoid tanshinones, tanshinone I, tanshinone IIA and cryptotanshinone. This stimulating effect was dependent on the Ag (+) dose, the day of Ag (+) addition to the culture and the tanshinone species, while the total yield of the three tanshinones (TT) was mainly dependent on the Ag (+) dose, with the highest being attained at 30 microM. The Ag (+) treatment also caused a dose-dependent inhibition of hairy root growth. Sucrose feeding or medium renewal before the addition of Ag (+) to the culture effectively prevented the growth inhibition, and significantly increased the biomass concentration and volumetric tanshinone yield. With combined medium renewal and Ag (+) treatment, in particular, the TT yield was increased 6.6-fold relative to that of the control (55.7 mg/L versus 7.3 mg/L). The elicitor function of Ag (+) in the hairy root culture was validated by its ability to induce the characteristic elicitor responses of plants, the increase in cross-cell membrane ion fluxes and the production of reactive oxygen species.