Elicitation of solid callus cultures of Salvia miltiorrhiza Bunge with salicylic acid and a synthetic auxin (1-naphthaleneacetic acid)

The presented study analyses the influence of salicylic acid (SA) and the synthetic auxin 1-naphthaleneacetic acid (NAA) on total tanshinone level and on dihydrotanshinone (DHT), cryptotanshinone (CT), tanshinine I (TI) and tanshinone IIA (TIIA) level in Salvia miltiorrhiza callus cultures growing on solid Murashige and Skoog (MS) medium. The influence of SA and NAA was evaluated at 10-day intervals throughout a 80-day treatment period. SA was applied at 0.1, 0.2 and 0.4 mM, and NAA at 2.69, 13.43, 26.85 and 40.28 μM. DHT, CT, TI and TIIA concentrations were measured using HPLC. NAA did not increase the concentration of any tanshinone. SA increased content in a concentration- and time-dependent manner; however, the yields were relatively low, possibly due to the metabolic specificity of S. miltiorrhiza cultivars in Poland. Total tanshinone concentration reached 226.38 ± 37.33 μg g−1 DW after 50 days of 0.4 mM SA elicitation. After 50 days of SA elicitation, the following maximum tanshinone concentrations were observed for 0.4 mM SA: DHT (71.58 ± 12.72 μg g−1 DW), CT (108.54 ± 18.29 μg g−1 DW), TI (29.50 ± 4.13 μg g−1 DW) and TIIA (16.75 ± 2.74 μg g−1 DW). To account for these observed differences in tanshinone biosynthesis, the distribution of SA and auxin responsive cis-active motif in the proximal promoters of the mevalonic acid, methylerythritol-4-phosphate and tanshinone-precursor biosynthesis pathway genes was evaluated in A. thaliana and S. miltiorrhiza. Our findings indicate that the SA-responsive cis-active elements have a much broader distribution than those recognized by auxin-responsive transcription factors. Tanshinone concentration in S. miltiorrhiza callus growing on solid, modified MS medium may by increased by salicylic acid elicitation.