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. RUBY 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 RUBY system using Petunia × hybrida lines obtained via Agrobacterium-mediated gene transformation. Stable RUBY 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 RUBY expression levels and metabolite contents. In a morphological analysis, there was a negative correlation between RUBY expression and corolla opening, suggesting that RUBY pigment overaccumulation is associated with altered floral development and morphology. RUBY overexpression significantly reduced expression levels of gibberellin biosynthetic genes (PhGA20ox1 and PhGA3ox1) and flowering- and senescence-related regulators (PhNF-YC2 and PhOBF1). These findings indicate that high-level betalain accumulation is associated with changes in floral development and gene expression, highlighting both the utility of the RUBY system as a visual reporter and the importance of carefully evaluating potential developmental effects under strong expression conditions.