Enhanced Agrobacterium-Mediated Transformation in Chinese Cabbage via Tenoxicam, Phytohormone Optimization, and Visual Reporters

Chinese cabbage (<i>Brassica rapa</i> ssp. <i>pekinensis</i>) is a globally important leafy vegetable, but functional genomics research on its recalcitrance to <i>Agrobacterium</i>-mediated genetic transformation is severely limited. In this study, we demonstrate that both <i>Agrobacterium</i> infection and antibiotic selection significantly inhibit cotyledonary petiole regeneration, representing one principal bottleneck to high-throughput transformation. Infection with different <i>Agrobacterium</i> strains suppressed the regenerated shoot per explant by 30.98-69.16%. Supplying the salicylic acid signaling inhibitor tenoxicam in the seed germination medium raised post-infection regeneration by up to 37.90%. Compared with non-infected controls, the optimal NAA concentration for explant regeneration after infection was higher, and 0.5 mg/L increased post-infection regeneration by 27.66%. Replacing antibiotic selectable markers with the visual reporter eYGFPuv or RUBY eliminated phytotoxicity, reduced false-positive shoots, and further elevated transformation efficiency to 19.33-20.00% (versus 2.67-6.67% under antibiotic selection). The integrated protocol yielded stable RUBY overexpressing lines, the biomass of which declined with rising transcript levels. Restricting RUBY expression to the inner head leaves generated a novel germplasm with less yield penalty. This work provides a high-efficiency transformation method that will accelerate gene discovery and genome editing in Chinese cabbage.