Establishment of a high-efficiency hairy root transformation system for Forsythia suspensa and enhancement of transformation efficiency by developmental regulators

Forsythia suspensa (Thunb.) Vahl is an economically important woody medicinal plant, yet the lack of efficient genetic transformation protocols hinders genomic and molecular breeding studies. Conventional Agrobacterium tumefaciens -mediated transformation faces low efficiency and long culture cycles, while Agrobacterium rhizogenes -mediated hairy root transformation offers a faster, simpler alternative. We systematically optimized key parameters for hairy root transformation in Forsythia suspensa , including culture medium, Agrobacterium rhizogenes strain, seedling age, and explant type. The optimal protocol consisted of infection with strain K599 and Hoagland‑moistened vermiculite as the substrate, achieving successful hairy root induction from seedlings, stems, and leaves. Among nine developmental regulators (DRs) tested, co‑transformation of FsMP elevated leaf transformation efficiency to 95%. Furthermore, combining the RUBY reporter with CRISPR/Cas9 enabled rapid, efficient gene editing in hairy roots. We established an efficient, rapid, and cost‑effective Agrobacterium rhizogenes -mediated hairy root transformation and gene editing platform for Forsythia suspensa that bypasses conventional tissue culture, providing valuable tools for gene function validation and molecular breeding in this and other woody species.