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

Chinese cabbage (Brassica rapa ssp. pekinensis) is a globally important leafy vegetable, but its recalcitrance to Agrobacterium-mediated genetic transformation has severely limited functional genomics research. Here we demonstrate that both Agrobacterium infection and antibiotic selection impose significant inhibition on cotyledonary petiole regeneration, representing one principal bottleneck to high-throughput transformation. Infection with different Agrobacterium strains suppressed regenerated shoot per explant by 30.98-69.16%. Supplying the salicylic-acid-signalling 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 reporters eYGFPuv or RUBY eliminated phytotoxicity, reduced false-positive shoots, and further elevated genetic transformation efficiency to 19.33-20.00% (versus 2.67–6.67% under antibiotic selection). The integrated protocol yielded stable RUBY over-expressing lines whose biomass 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.