This study investigates the effects of artificial in vitro polyploidisation of Astragalus membranaceus, focusing specifically on gene expression and metabolic pathway associated with the biosynthesis of calycosin and calycosin-7-O-β-D-glucoside. Using oryzalin as an antimitotic agent, we have systematically investigated different genotypic lines, under both in vitro and ex vitro conditions. Amid cases of reduced gene expression in certain lines, results showed a significant upregulation in specific lines, particularly in genotypes 16, 54, and 74. Genotype 54 showed an exceptional response, manifesting a statistically significant upregulation in all investigated genes studied under in vitro conditions (i.e. AmPAL, AmC4H and AmI3′H). Interestingly, even under ex vitro conditions after two years of greenhouse cultivation, certain lines showed significant variations in gene expression. The genotype 16, although no longer tetraploid, exhibited the highest expression levels among the examined genotypes, with statistically significant upregulation of both the AmPAL and AmC4H genes. In addition, the induced autotetraploid genotype 74 showed a significant upregulation of the AmI3′H gene and a concomitant downregulation of the AmC4H gene. These results highlight the complex regulatory mechanisms affected by the polyploidisation of A. membranaceus and provide promising avenues for manipulating gene expression to enhance the production of pharmacologically significant compounds.Key messageThis study investigates the effects of artificial polyploidisation on gene expression of Astragalus membranaceus, suggesting diverse regulatory influences for potential plant bioactive compound production.