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    • Figure 1. 

      Transformation and gene editing of G. uralensis mediated by A. rhizogenes. (a) Schematic of T-DNA regions for RUBY (top) and CRISPR/Cas9 (bottom) editing vectors. RUBY expression was driven by the Cauliflower mosaic virus (CaMV) 35S promoter. LB, left border; RB, right border; AtUbi10, Arabidopsis thaliana Ubiquitin 10 promoter; NOS, nopaline synthase terminator; AtU6, A. thaliana U6 spliceosomal RNA promoter. (b) Schematic of parallel cutting regeneration (upper branch) and transformation (lower branch). Leafy cuttings (5–8 cm long, 2–2.5 mm diameter) were harvested from the first to the third internodes with one to two leaves or buds retained. Control explants underwent rooting incubation and moist culture to produce control roots. For transformed cuttings, basal ends were inoculated with A. rhizogenes before identical culture procedures. RUBY-positive hairy roots with red fluorescence were visible at 14 d post-culture. (c) Hairy root transformation workflow of hypocotyls mediated by A. rhizogenes. (d) Comparison of untransformed and transgenic hairy roots in stem cuttings. Left: non-inoculated control with normal white roots; Right: stem cutting inoculated with A. rhizogenes, producing RUBY-positive red hairy roots. Scale bar = 1 cm. (e) Comparison of root phenotypes in hypocotyls. Left: non-inoculated control with normal white roots; Right: hypocotyl infected with A. rhizogenes produces RUBY-positive red hairy roots. Scale bar = 1 cm. (f) Transformation efficiency comparison of stem cutting and hypocotyl dip inoculation. *** Denotes significant difference at p < 0.001. (g) DNA sequencing results of a representative SRT2 gene editing in roots by CRISPR/Cas9. The sgRNA-target sequence is shown in green, the protospacer-adjacent motif is shown in red, and the mutations are shown as dashes (deletion) or orange letters (transition). 1–10: independent transgenic hairy roots with SRT2 gene editing. Ho: homozygous mutation; He: heterozygous mutation; Chi: weak chimerism. (h) qRT-PCR analysis of flavonoid biosynthesis-related gene expression in homozygous SRT2-edited hairy roots (lines X3 and X7). CK: non-edited wild-type hairy roots. Data are shown as mean ± SD. *** p < 0.001.