RUBY-Mediated Visual Selection Facilitates Transgenic Screening and Red Floral Pigmentation in Petunia × hybrida

Visual transgenic marker systems enabling rapid and non-destructive transformant detection are crucial for efficient plant genetic transformation and selection. <i>RUBY</i> is a highly effective reporter system based on betalain biosynthesis; however, its application to species outside of the order Caryophyllales (i.e., species lacking betalains) has not been established. In this study, we performed the first systematic evaluation of the <i>RUBY</i> system using <i>Petunia</i> × <i>hybrida</i> lines obtained via <i>Agrobacterium</i>-mediated gene transformation. Stable <i>RUBY</i> transgenic plants were obtained from an optimized transformation and organogenesis system. The transgenic lines displayed a gradient of betalain accumulation, with pigment intensity positively correlated with <i>RUBY</i> expression levels and metabolite contents. In a morphological analysis, there was a negative correlation between <i>RUBY</i> expression and corolla opening, suggesting that <i>RUBY</i> pigment overaccumulation is associated with altered floral development and morphology. <i>RUBY</i> overexpression significantly reduced expression levels of gibberellin biosynthetic genes (<i>PhGA20ox1</i> and <i>PhGA3ox1</i>) and flowering- and senescence-related regulators (<i>PhNF-YC2</i> and <i>PhOBF1</i>). These findings indicate that high-level betalain accumulation is associated with changes in floral development and gene expression, highlighting both the utility of the <i>RUBY</i> system as a visual reporter and the importance of carefully evaluating potential developmental effects under strong expression conditions.