A visual reporter system for transgene screening and monitoring genome editing in tomato

The stable expression of integrated transgenes remains a major restriction in plant transformation, as different vector backbones and host contexts often lead to variable or silenced expression. Systematic evaluations of the binary vector architecture and broadly applicable visual markers for rapid line selection are still lacking. In this study, we developed a streamlined 2A-linked unit of CYP76AD1, DODA, and Glucosyltransferase (RUBY)-based chromogenic reporter system and validated its robustness in the transformation in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum). A comparative analysis of two binary vector backbones revealed strong structural effects on the stability of expression, with one backbone enabling consistently high pigmentation in tomato. RUBY's intensity provides an immediate readout of transgene activity and allows the rapid identification of single-copy, stably expressing lines through seedling segregation. RUBY expression does not influence plants' growth or epidermal development, demonstrating excellent biocompatibility. We also established an inducible pXVE::RUBY module and a CRISPR-associated protein 9 (Cas9)-porcine teschovirus-1 2A peptide (P2A)-RUBY system for real-time monitoring of transgene induction and visualizing genome editing events. This platform offers an efficient and readily implementable strategy to overcome the disconnect between transgenes' integration and expression, while substantially improving the overall performance of plant transformation and genome editing pipelines.