Metabolomic profiling reveals synchronized accumulation of biomass and bioactive flavonoids during in vitro development of Anoectochilus roxburghii

Background Anoectochilus roxburghii is a perennial orchid valued for its bioactive constituents. Traditionally, the harvest of in vitro cultured plants has relied primarily on plant height or approximate cultivation duration; however, identifying the optimal harvest window requires a precise understanding of the temporal dynamics of metabolite accumulation. Results In this study, we combined morphological phenotyping with widely targeted metabolomics to characterize the developmental trace of in vitro cultured A. roxburghii across five stages (120, 180, 240, 300, and 360 days after inoculation, DAI). Morphological characteristics generally showed a rapid growth from 120 DAI to 180 DAI and then to a stable plateau at 300–360 DAI. Time-series metabolomics demonstrated that lipids, terpenoids, and alkaloids were enriched in the early stages (120–180 DAI), whereas flavonoids and kinsenoside predominantly accumulated in the late stages (300–360 DAI). Weighted gene co-expression network analysis (WGCNA) identified distinct metabolite modules, revealing synchronized accumulation of biomass and flavonoids, alongside an inverse relationship between lipids and sugars and amino acids. These patterns indicate a metabolic transition from primary to secondary metabolism during development. Conclusion Our findings suggest that 300–360 DAI represents the optimal harvest window for balancing yield and quality, providing a scientific basis for precision in vitro cultivation.