Enhanced production of calycosin-7-O-β-D-glucoside and astragaloside Ⅳ from adventitious root cultures of Astragalus membranaceus var. mongholicus by green leaf volatiles

•Green leaf volatiles stimulated the production of bioactive compounds in A. mongholicus adventitious roots.•Hexanol was competent and endurable as a chemical elicitor;•Promoting mechanism of hexanol on isoflavonoids was time-dependent. Green leaf volatiles (GLVs) serve as signaling molecules in plant chemical defense. However, it remains unclear whether they are applicable in medicinal plant tissue cultures for reducing the demands for natural sources and industrialization purpose. In this study, their elicitation was explored in adventitious roots of Astragalus membranaceus var. mongholicus (A. mongholicus) using astragaloside Ⅳ and calycosin-7-O-β-D-glucoside (CG) as indicators. The results showed that exposure to GLVs, namely hexanal, hexanol, and E-2-hexenal promoted the CG production in a time-dependent manner. Amongst, hexanol simultaneously increased the production of the two analytes and another isoflavonoid ononin, so did the root yields. Compared with the others, hexanol induced differential transcriptome profiles, and more enzyme genes involved in the CG biosynthesis at the 14-day time point than that at the 7-day point. Real-time quantitative PCR analysis denoted that hexanol significantly suppressed the expression of the genes involved in the biosynthesis of the scaffolds of CG but stimulated these associated with the conjugation such as UCGT2 and CGMT within two weeks. Quantification of the synthetic precursors formononetin and calycosin confirmed its short-term inhibition on the biosynthesis of the scaffolds. Hexanol also exerted long-term promotion on the CG biosynthesis via upregulating the transcription of IFS1.1 and subsequent biosynthesis of formononetin even after five to six weeks of the treatment. In conclusion, hexanol was a competent elicitor in A. mongholicus adventitious roots and the application of GLVs is feasible in in vitro propagation strategy of medicinal and economical plants.