Development of a hairy root transformation platform for enhanced cyclopeptide production and gene function analysis in Pseudostellaria heterophylla

Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm. is a widely used traditional Chinese medicinal herb in which cyclopeptides are the major bioactive constituents. However, naturally low cyclopeptide contents and a lack of an efficient genetic transformation system have hindered functional characterization of cyclopeptide biosynthetic genes. We establish a rapid and efficient Agrobacterium rhizogenes -mediated hairy root induction and transformation system for P. heterophylla . By combining in vitro embryo culture to overcome seed dormancy using a filter paper sandwich co-cultivation method, we achieve a hairy root induction rate of 73.43% at 18 d, and a transformation efficiency of 48.94% using A. rhizogenes strain K599. The system is validated by stable expression of DsRed2 and RUBY reporter genes. Metabolite profiling reveals hairy roots accumulate significantly higher levels of heterophyllins A and B, but lower levels of polysaccharides and saponins compared with normal roots, indicating selective activation of cyclopeptide biosynthesis. We also clone and functionally characterize the precursor gene PhPreHA —the overexpression of which in hairy roots leads to a marked increase in heterophyllin A accumulation. This study pioneers a hairy root-based genetic transformation system in P. heterophylla and provides the first functional validation of a cyclopeptide precursor gene. In doing so, it offers a robust platform for pathway dissection and metabolic engineering of medicinal plants. • First rapid and efficient A. rhizogenes -mediated hairy root transformation system established for P. heterophylla (73.43% induction rate, 48.94% transformation efficiency). • Hairy roots of P. heterophylla selectively accumulate higher cyclopeptides of heterophyllins A/B vs. normal roots. • Functional validation of cyclopeptide precursor gene PhPreHA : its overexpression boosts heterophyllin A accumulation in hairy roots.