Promising strategy for improving calycosin-7-O-β-D-glucoside production in Astragalus membranaceus adventitious root cultures

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Culture duration, elicitation, and precursor feeding can significantly affect secondary metabolite production in adventitious root cultures. Hydrogen peroxide (H2O2) can act as a diffusible signal to induce the production of secondary metabolites. L-phenylalanine (L-Phe) is the precursor in the biosynthesis of the bioactive compound calycosin-7-O-β-D-glucoside (CG). To improve the accumulation of CG in Astragalus membranaceus adventitious roots (AMARs), the present study investigated the effects of culture duration and H2O2 and L-Phe treatment on the accumulation of CG in AMARs. The CG content fluctuated during AMAR culture, with the highest productivity observed after 35 d of suspension culture. Furthermore, the H2O2 treatment efficiently enhanced CG content in a concentration- and time-dependent manner, and under 20 μM H2O2, the CG reached its maximum level after 12 h of treatment. However, the addition of L-Phe to H2O2 treatment remarkably improved the CG accumulation, with a maximum CG level observed after 120 h of treatment with 200 μM H2O2 and 300 μM L-Phe, which was 8.6-fold greater than the cultures treated with 200 μM H2O2 alone. The effectiveness of these parameters was confirmed by measuring the expression of four major CG-biosynthesis genes, and both the chalcone reductase and isoflavone synthase genes were identified as key regulatory nodes in the CG biosynthetic pathway. The improvement strategy was also applied using a large-scale bioreactor system. Both the CG content and antioxidant activity of the H2O2-elicited and L-Phe-fed bioreactor culture were greater than those of the untreated bioreactor culture and field-grown plants. Thus, the simultaneous application of H2O2 elicitation and L-Phe-feeding in 35-d-old AMAR cultures is a promising strategy to economically improve the industrial production of CG.