A developmental regulator-based approach for highly efficient and genotype-independent transformation of flowering Chinese cabbage

• The AtGRF5-AtGIF1 chimera greatly enhances transformation efficiency (up to 17.69 %) in flowering Chinese cabbage. • The established system significantly reduces genotype-dependent transformation barriers. • The combination of A. rhizogenes K599 and SC explants is optimal for transformation. Flowering Chinese cabbage ( Brassica rapa ssp. chinensis var. parachinensis ), a highly nutritious and economically significant vegetable, is extensively cultivated across East Asia. However, its genetic improvement through biotechnology has been significantly constrained by the low transformation efficiency and strong genotype dependency associated with conventional Agrobacterium -mediated transformation. To overcome these limitations, we developed an optimized genetic transformation system using several developmental regulators (DRs). Five DR constructs, including AtWUS-AtBBM, AtGRF5-AtGIF1, AtWIND1, BaPLT5 , and BaWOX5 , were introduced into flowering Chinese cabbage via Agrobacterium -mediated transformation. The visual reporter gene RUBY was employed to facilitate efficient detection of transformation events. Among these constructs, A. rhizogenes strain K599 carrying the AtGRF5-AtGIF1 chimera demonstrated the most significant enhancement, yielding red callus induction (20.17%) and positive transformation (17.69%) rates, respectively. Notably, this construct significantly mitigated genotype-dependent responses, supporting high-efficiency transformation across all three tested cultivars. Further optimization demonstrated that seedling cutoff root (SC) explants exhibited significantly higher transformation efficiency than hypocotyl with cotyledon (HC) explants, and among the tested Agrobacterium strains, A. rhizogenes K599 proved to be the most effective, particularly when delivering the AtGRF5-AtGIF1 construct. Collectively, our work establishes a robust protocol for high-efficiency transformation of flowering Chinese cabbage, holding broad promise for functional genomics research and CRISPR-based molecular breeding in this crop.