Establishment of an efficient genetic transformation system based on Agrobacterium rhizogenes and the RUBY marker in Brassica juncea

Brassica juncea is an important and widely cultivated vegetable. Current genetic transformation techniques for this crop are limited by low efficiency. To establish a stable and efficient genetic transformation system for B. juncea, this study first combined Agrobacterium rhizogenes-mediated transformation with the novel visual marker gene RUBY. Rootless seedlings from two B. juncea genotypes, as explants, were infected by the A. rhizogenes strain K599 carrying a RUBY overexpression vector, successfully inducing red hairy roots. Subsequently, through exogenous hormone induction, a complete regeneration pathway was achieved, where transgenic positive hairy roots differentiated via callus to form regenerated shoots, establishing an efficient hairy roots–callus–regenerated shoots regeneration system. Ultimately, transgenic B. juncea plants carrying the RUBY gene, exhibiting distinctly red leaves throughout the plant, were obtained. Quantitative real-time polymerase chain reaction confirmed the high expression levels of the RUBY gene in these transgenic plants. It was worth noting that overexpression of RUBY gene may lead to growth abnormalities such as leaf wrinkling in some plants. This system using the visible marker of the RUBY gene significantly enhanced the efficiency and accuracy of screening for positive transformant plants and provided a new and efficient technical platform for the precise improvement of target traits and the functional analysis of genes in B. juncea.