Development of a hairy root transformation platform for enhanced cyclopeptide production and gene function analysis...

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.