Visualizing application of RUBY-MAT system in maize genetic transformation and gene expression pattern detection

RUBY-MAT system, integrating the RUBY reporter and MAT module, was innovatively applied to Agrobacterium-mediated maize transformation, and precise spatiotemporal gene expression analysis. Maize genetic improvement relies on efficient genetic transformation and gene expression monitoring technologies. In this study, we provided RUBY-MAT system with RUBY reporter and MAT (morphogene-assisted transformation), and realized its visual application in Agrobacterium-mediated genetic transformation of maize immature embryos and gene expression patterns. Experimental results demonstrated that RUBY-MAT system can be successfully applied in Agrobacterium-mediated genetic transformation of maize immature embryos, and RUBY reporter enables non-invasive monitoring of positive transformation events, as early as 7 days after culture, through the clearly distinguishable betalain of positive callus. Dynamic expression monitoring further revealed that RUBY reporter can reflect the spatiotemporal expression patterns of specific promoters in real time: in constitutively expressed RUBY plants, high activity was observed in tissues including tassels, anthers, and stem nodes; in ZmH1A promoter-driven RUBY lines, betalain accumulation clearly indicated gene expression in vascular bundles and stomatal guard cells; while in lines harboring the putative young leaf-specific ZmGLP1 promoter, betalain specifically accumulated in young leaves. Quantitative analysis confirmed a strong linear correlation between betalain absorbance and promoter activity, establishing the RUBY reporter as a reliable quantitative tool for monitoring transformation efficiency and gene expression at tissue and cellular levels. Collectively, the RUBY-MAT system offers robust technical support for maize genetic transformation and functional gene analysis, demonstrating broad application potential in both field and laboratory studies.