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

Piotr Szymczyk,Grażyna Szymańska,Ewa Kochan,Renata Grąbkowska,Janusz Szemraj

doi:10.1007/s11240-021-02141-z

Piotr Szymczyk, Grażyna Szymańska + Show 3 more

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https://doi.org/10.1007/s11240-021-02141-z

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Abstract

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.

Highlights

Salvia miltiorrhiza, known as Danshen/Tanshen, is a medicinal plant that has been used in Chinese medicine since ancient times (Su et al 2015)
The induction and proliferation of S. miltiorrhiza callus was performed according to Wu et al (2003) with modifications: callus induction was performed with 10 × 10 mm leaf pieces from the S. miltiorrhiza plants, compared to 5 × 5 mm described in the original method, and 100 mm glass Petri dishes were used instead of the original 22 × 160 mm glass tubes
Our findings show generally lower tanshinone accumulation dynamics; this may be related to the fact they were grown on solid rather than liquid medium, or the generally lower initial tanshinone productivity demonstrated by plants growing in Poland (Buchwald et al 2006; Cheng et al 2013)

Summary

Introduction

Known as Danshen/Tanshen, is a medicinal plant that has been used in Chinese medicine since ancient times (Su et al 2015). The diterpene tanshinones are synthesized from two five-carbon precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), mainly by the plastidial methylerythritol-4-phosphate (MEP) pathway. The IDP and DMADP moiety is used for the stepwise biosynthesis of a 20-carbon geranylgeranyl diphosphate (GGPP), which serves as a substrate for copalyl diphosphate synthase 1 and 2, producing (+)-copalyl diphosphate. The (+)-copalyl diphosphate is transformed into miltiradiene, the first committed tanshinone biosynthesis pathway intermediate, by the kaurene-like synthase 1 (Bai et al 2018; Guo and Ma 2019; Szymczyk et al 2016)

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