Chitosan promoting formononetin and calycosin accumulation in Astragalus membranaceus hairy root cultures via mitogen-activated protein kinase signaling cascades.

Chitosan, behaving as a potent biotic elicitor, can induce plant defense response with the consequent enhancement in phytoalexin accumulation. Accordingly, chitosan elicitation was conducted to promote the production of two phytoalexins, i.e. formononetin and calycosin (also known as health-promoting isoflavones), in Astragalus membranaceus hairy root cultures (AMHRCs). Compared with control, 12.45- and 6.17-fold increases in the yields of formononetin (764.19 ± 50.81 μg/g DW) and calycosin (611.53 ± 42.22 μg/g DW) were obtained in 34 day-old AMHRCs treated by 100 mg/L of chitosan for 24 h, respectively. Moreover, chitosan elicitation could cause oxidative burst that would induce the expression of genes (MPK3 and MPK6) related to mitogen-activated protein kinase signaling (MAPK) cascades, which contributed to the transcriptional activation of pathogenesis-related genes (β-1,3-glucanase, Chitinase, and PR-1) and eight biosynthesis genes involved in the calycosin and formononetin pathway. Overall, the findings in this work not only highlight a feasible chitosan elicitation practice to enhance the in vitro production of two bioactive isoflavones for nutraceutical and food applications, but also contribute to understanding the phytoalexin biosynthesis in response to chitosan elicitation.