Development and Optimization of a Non-Tissue Culture Hairy Root Transformation System in Camellia oleifera Mediated...

Camellia oleifera is an important woody oil crop species endemic to China. However, gene functional analysis and molecular breeding in oiltea are greatly constrained by the low efficiency of conventional Agrobacterium-mediated transformation systems. In this study, a rapid and efficient Agrobacterium rhizogenes-mediated hairy root transformation system was established using the elite cultivar 'Xianglin 210'. A vector carrying the RUBY visual reporter gene was introduced into leaf and hypocotyl explants, and the transformation efficiencies of three Agrobacterium strains (GV3101, MSU440, and K599) were systematically evaluated. Key transformation parameters, including bacterial suspension density and vacuum infiltration duration, were further optimized. The results demonstrated that leaf explants exhibited significantly higher transformation efficiency than hypocotyl explants. Among the tested strains, K599 showed the highest hairy root induction efficiency. The optimal transformation conditions were obtained using a bacterial suspension at OD₆₀₀ = 0.8 combined with vacuum infiltration for 40 min. Under these conditions, the hairy root induction rate reached 69.00%, and the proportion of RUBY-positive hairy roots reached 58.00%. Notably, the entire transformation process was achieved without callus induction or plant regeneration, and stable transgenic hairy roots were obtained within 60 days. In conclusion, this study demonstrates a rapid and tissue culture-independent hairy root transformation protocol for C. oleifera. While further functional studies are needed, this platform provides a practical tool with strong potential for future candidate gene validation and functional genomics in oil tea crops.