Differential gene expression levels, chemical profiles, and biological activities of Pueraria candollei var. mirifica callus cultures at different growth stages

In vitro callus culture may provide a practical alternative for biomass production and medicinal compounds. The study of gene expression, chemical profile, and bioactivity at different growth stages of callus was conducted to determine the most suitable stage of callus based on the production requirements and provided important data for further study of the biosynthetic pathway. In this study, green (G), yellow (Y), orange (O), and brown (B) callus cultures of Pueraria candollei var. mirifica (PM) were collected at 30, 45, 60, and 75 days after subculture, respectively, and their gene expression levels, chemical profiles, and bioactivities were compared. The complexity and amount of constituents increased with culture time and increasing daidzein and genistein levels until the O callus stage, which was correlated with the constitutive expression of genes involved in the general isoflavonoid biosynthetic pathway at all growth stages. In contrast, the downstream biosynthetic genes encoding cytochrome P450 subfamily 81E and a putative prenyltransferase, which are responsible for diversifying the isoflavones to PM-specific secondary products, were significantly upregulated in the O callus. The observed color changes of the PM callus cultures were attributed to increases in daidzein and genistein levels. The callus cultures exhibited increased antioxidant activity with increasing age, reaching a highest half maximal inhibitory concentration of 0.30 mg/mL in the O callus. The cultures also exhibited cytotoxic activity, with the O and Y callus extracts (each at 3.13 μg/mL) showing significant activity against HepG2 cells (43.3% cell death) and MDA-MD-231 cells (40.1% cell death). These findings demonstrate that age significantly affects the chemical profiles and bioactivities of PM callus cultures.