Establishment of an efficient hairy root transformation system and functional analysis of GsCGTa in Grona styracifolia

Abstract Grona styracifolia is a medicinal herb rich in bioactive compounds, including schaftoside, from the Lingnan region. However, functional genomic analyses of Grona styracifolia have been limited by the lack of a stable and highly efficient genetic transformation system. In this study, key parameters affecting Rhizobium rhizogenes -mediated hairy root transformation, including the explant type and seedling age, inoculation conditions, duration of cocultivation, stage of acetosyringone (AS) application, and cefotaxime (Cef) concentration, were systematically optimized. Optimal transformation was achieved by inoculating 10-d-old cotyledonary nodes as explants for 20 min with an MMA (MES‑MgCl 2 ‑AS) resuspension solution (OD 600 = 0.6), followed by a 3‑d co‑cultivation period and subsequent culture on 1/2 MS solid medium supplemented with 300 mg/L Cef. Through the integrated optimization, the transformation efficiency significantly increased from 35.56% to 71.11%. On the basis of this system, GsCGTa , a key structural gene in the schaftoside biosynthetic pathway, was functionally characterized. A transcriptome analysis revealed that C -glycosyltransferase a ( GsCGTa ) overexpression induced selective transcriptional reprogramming of pathway-related structural genes. In addition, we observeddistinct growth and metabolic responses when transgenic hairy roots expressing GsCGTa were subjected to different light qualities: green light promoted hairy root induction and elongation, whereas blue light was more conducive to the accumulation of schaftoside-related metabolites. In summary, the highly efficient hairy root transformation system established in this study provides a reliable technical platform for gene research in G. styracifolia and offers essential technical support for molecular breeding and quality improvement of this medicinal plant.