A RUBY-Assisted Hairy Root Transformation Platform for Metabolomic Profiling and Preliminary Functional Analysis of HpRHL76 in Herpetospermum pedunculosum

Herpetospermum pedunculosum is an endangered cucurbitaceous medicinal plant whose functional genomic and metabolic studies are constrained by the lack of efficient aseptic seedling and genetic transformation systems. Here, we established a rapid aseptic seedling production method and a RUBY-assisted hairy root transformation platform integrated with metabolomic evaluation. The cotton-supported liquid culture method provided a favorable balance between germination performance and experimental operability. Microsyringe-mediated stem-base inoculation with Rhizobium rhizogenes K599 carrying 35S::RUBY enabled rapid visual screening of RUBY-positive hairy roots, with a maximum transformation efficiency of 93.33%. Widely targeted metabolomics identified 242 metabolites and revealed major differences in amino acid/nitrogen and sugar metabolism, lipid-related metabolism, specialized metabolite biosynthesis, hormone-related pathways, and ABC transporters among wild-type, vector-free K599-induced, and RUBY-expressing roots. In addition, 80 HpRHL-like bHLH genes were identified. As a proof of concept, RT-qPCR confirmed HpRHL76 overexpression, which was associated with an increase in mean root hair length from approximately 0.4 to 1.1 mm. Together, this integrated framework provides a practical basis for preliminary candidate gene assessment, functional genomic studies, and future metabolic engineering of medicinally relevant compounds in H. pedunculosum, and may serve as a methodological reference for other difficult-to-transform medicinal plants.