Precursor feeding and elicitation improve mitragynine production in Mitragyna speciosa shoot culture

Mitragyna speciosa (Roxb.) Korth. [Rubiaceae] is a unique source of mitragynine, an opioid agonist and possesses an analgesic effect by non-selective binding to opioid receptors. The present study shoot culture from axillary buds were established and cultured in the liquid McCown woody plant medium (WPM) supplemented with 2 mg/L thidiazuron, 1 mg/L benzyladenine and 20 g/L sucrose. To improve mitragynine production in the shoot culture, feeding of precursors and elicitors was investigated. Tryptamine, loganin and tryptamine/loganin (final concentration of 0.2, 0.4 mM) were fed on the day of subculture and harvested at day 14 and 21 of culture. The results showed that feeding of tryptamine affected the mitragynine content rather than loganin. However, a combination of tryptamine and loganin (at 0.4 mM of each) enhanced mitragynine production to 0.364 ± 0.009 mg/g DW (after 14 days; P< 0.01) and 0.273 ± 0.015 mg/g DW (after 21 days; P< 0.05) when compared to control (0.169 ± 0.019 mg/g DW). Plant hormone-like elicitors including jasmonic acid (0 – 100µM), abscisic acid (0 – 100µM) and salicylic acid (0 – 500µM) were selected. The optimal concentration and optimal time exposure of each elicitor were designed using the response surface methodology (RSM) and central composite design (CCD). The equations, obtained from RSM and CCD were used for calculation of optimal conditions in each elicitor. At optimal conditions, the elicited shoot culture with jasmonic acid (100µM; 48h) increased mitragynine production (0.482 ± 0.021 mg/g DW) about 2.4 times higher than the control (0.188 ± 0.030 mg/g DW). In addition, abscisic acid (50µM; 24h) and salicylic acid (500µM; 48h) increased the mitragynine production to 0.273 ± 0.007 mg/g DW and 0.279 ± 0.016 mg/g DW, respectively when compared to the control (0.116 ± 0.008 mg/g DW).