Tanshinone production in ti-transformed Salvia miltiorrhiza cell suspension cultures

Abstract

Transformed cell cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium tumefaciens strain C58. The transformed cells in suspension formed macroscopic clumps of cell aggregates up to 2-3 cm in size rather than homogeneous cell suspensions. These transformed cells grew well in hormone-free media. It was found that the B5 medium supported the best growth while the 6,7-V medium promoted tanshinone production in the transformed cell suspension cultures. The effect of initial sucrose concentrations on cell growth was also studied. The best growth was observed when cells were cultivated in the B5 medium containing 30 g l-1 sucrose. Although low levels of tanshinones were produced in fast growing cell cultures, there existed a rapid increase in tanshinone production when the cell aggregates were transferred to the fresh yeast-extract-containing medium. By this two-stage culture method, about 22 mg tanshinones were produced in 1 liter of medium. Green cell aggregates were formed when cells were cultured under illumination. Light was found to have an inhibitory effect on tanshinone biosynthesis. A sensitive high-performance liquid chromatographic method was developed for the measurement of tanshinones. Cryptotanshinone, tanshinone I and tanshinone IIA were identified from the transformed cultures. | Transformed cell cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium tumefaciens strain C58. The transformed cells in suspension formed macroscopic clumps of cell aggregates up to 2-3 cm in size rather than homogeneous cell suspensions. These transformed cells grew well in hormone-free media. It was found that the B5 medium supported the best growth while the 6,7-V medium promoted tanshinone production in the transformed cell suspension cultures. The effect of initial sucrose concentrations on cell growth was also studied. The best growth was observed when cells were cultivated in the B5 medium containing 30 g l-1 sucrose. Although low levels of tanshinones were produced in fast growing cell cultures, there existed a rapid increase in tanshinone production when the cell aggregates were transferred to the fresh yeast-extract-containing medium. By this two-stage culture method, about 22 mg tanshinones were produced in 1 liter of medium. Green cell aggregates were formed when cells were cultured under illumination. Light was found to have an inhibitory effect on tanshinone biosynthesis. A sensitive high-performance liquid chromatographic method was developed for the measurement of tanshinones. Cryptotanshinone, tanshinone I and tanshinone IIA were identified from the transformed cultures.