A RUBY-based in vivo hairy root induction system for rapid assessment of transformant and genome editing efficiency...

Hairy root transformation systems are increasingly recognized as effective tools for functional genomics and gene editing studies in various crop species. In this study, we employed the RUBY reporter gene as a visible marker to establish a rapid, simple, and efficient in vivo system for potato hairy root induction and transformation. Explants were wounded by transverse cuts and gently scraped across the surface of a culture plate containing Rhizobium rhizogenes harboring the desired construct, then directly planted into pots containing moist, sterile vermiculite. The 2-3-week-old shoots regenerated from tubers of the Atlantic potato cultivar were identified as sufficient explants for Rhizobium rhizogenes-mediated transformation, achieving transformation efficiencies of up to 100%. This procedure was further adapted for stem nodes and leaf explants and achieved high transformation efficiencies of 80% and 93%, respectively. In addition, the in vivo hairy root transformation system was successfully utilized to validate CRISPR/Cas9 activities by inducing targeted mutations of the StDL1 gene in potato. Heteroduplex analysis on PAGE revealed a high editing frequency (96%), and Sanger sequencing of selected lines confirmed diverse deletions at the target site. Overall, this in vivo hairy root transformation method enables rapid and robust assessment of genome editing efficiency and transgene expression, providing a valuable tool for functional genomics and genetic improvement in potato, with potential applications in other Solanaceae crops.