This study aimed to investigate the elicitation of volatile organic compounds (E)-2-hexenal and 2,3-butanediol on bioactive metabolites in Astragalus membranaceus var. mongholicus adventitious root cultures by adding them into the medium. The experiment was performed for 72 h and the roots were dynamically sampled for quantification of representative astragaloside IV, calycosin-7-O-β-D-glucoside (CG), ononin, and the gene expression. Compared with the controls, the combination of 2,3-butanediol and (E)-2-hexenal advanced the peak accumulation of astragaloside IV and was the most effective, but their individual application delayed it. Meanwhile, 2,3-butanediol and (E)-2-hexenal had no obviously promoting effect on the production of CG and ononin but chronologically changed their accumulation patterns. The underlying mechanism was uncovered by the correlation analysis between the metabolites and the gene expression, as did the identification of the target genes. Collectively, 2,3-butanediol and (E)-2hexenal were important cues shaping the production of bioactive products in the herbal plant.

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