The identification and quantitative determination of rosmarinic acid and salvianolic acid B in hairy root cultures of Dracocephalum forrestii W.W. Smith

• Induction and establishment of D. forrestii transformed roots.
• Genetic characterization of hairy roots.
• Phytochemical analysis of phenolic compounds.

Dracocephalum forrestii, an important plant in Tibetan medicine, was transformed with Agrobacterium rhizogenes strain A4 to obtain hairy root culture. A maximum transformation frequency of 82% after seven weeks was obtained by inoculation of internodal stem segments. Six lines of hairy roots were obtained and cultured in darkness in Woody Plant (WPM) liquid medium. The transgenic nature of the hairy roots was investigated by PCR and RT-PCR. Three main compounds: rosmarinic acid (RA), salvianolic acid B (SAB) and caffeic acid (CA) were identified in extracts of D. forrestii hairy roots by UPLC-PDA-ESI-MS analysis. Quantitative HPLC analysis showed that RA levels were greatly increased in hairy roots (max. 19.97 mg/g DW) compared with the roots of one-year-old field grown plants (4.7 mg/g DW), but SAB levels were significantly lower in transformed roots (max. 11.7 mg/g DW) than in untransformed roots (16.4 mg/g DW). Caffeic acid was detected only in transformed roots. A time course experiment with the most productive hairy root line showed that a culture period of 30 days and a medium supplemented with 3% of sucrose was optimal for biomass and production of analysed phenolic compounds. Production remained stable over at least 3.5 years of continuous subculturing of the hairy roots.