Establishment of an efficient Agrobacterium rhizogenes-mediated hairy root transformation system for functional analysis in tomato

Abstract

Tomato is an important model for functional genomics in Solanaceae, and elucidating its gene functions provides valuable insights into conserved genetic regulatory networks across dicotyledonous plants. Hairy root transformation is a key technology for functional studies in tomato. However, systematic optimization of its parameters remains limited. Here, we established an efficient Agrobacterium rhizogenes-mediated hairy root transformation system for tomato cv. Micro-Tom. Using the RUBY visual reporter gene, we systematically evaluated multiple parameters, including A. rhizogenes strain, bacterial optical density (OD600), inoculation duration, acetosyringone (AS) concentration, and the type and concentration of antibiotics for Agrobacterium eradication. ArQual was identified as the most suitable strain. Optimization revealed that an OD600 of 0.7, inoculation duration of 10 min, and AS concentration of 200 μM, these optimized conditions yielded the highest transformation efficiency of 66.01%. To validate the utility of this system, we first demonstrated that overexpression of SlMYB28 promotes anthocyanin accumulation in stable transgenic lines. We then applied the optimized hairy root transformation system to functionally characterize SlMYB28, achieving 59.33% transformation efficiency and distinct purple pigmentation in transgenic roots. These results support the positive regulatory role of SlMYB28 in anthocyanin biosynthesis and demonstrate the utility of this system for rapid visual validation of stress-responsive genes. This rapid, efficient, and visually tractable transformation platform provides a useful tool for rapid functional screening and preliminary characterization in tomato.