Diterpenoid production in hairy root cultures of Salvia miltiorrhiza

Transformed root cultures of Salvia miltiorrhiza have been established by infecting pieces of sterile grown plants with Agrobacterium rhizogenes strains LBA 9402, ATCC 15834, TR 105, R 1601 and A 4 1027. These strains showed different abilities to induce hairy roots on this plant. A promoting effect of acetosyringone on the Agrobacterium-mediated hairy root initiation in Salvia miltiorrhiza was also observed. All transformed root cultures were assayed for opines by TLC, and were positive for agropine and mannopine. A sensitive quantitative HPLC procedure was developed for the simultaneous quantification of tanshinone I, tanshinone II A, tanshinone II B, tanshinone V, dihydrotanshinone I, cryptotanshinone, tanshinone VI, and the colourless diterpene ferruginol. These seven major tanshinones and ferruginol were found not only in the roots, but also in the liquid medium. Apparently, the hairy roots released secondary metabolises into the culture medium. The main products in the hairy root cultures were the tanshinones which are the active principles of this Plant species. The effects of culture conditions on hairy root growth and the diterpene production have also been studied. The most apparent effect was seen when ammonium nitrate levels of the medium were modified.