ROSEA1-Based Visual Selection Reduces Plant Regeneration and Alters Developmental Regulator Expression

Anthocyanin-based visual reporters enable rapid, non-destructive identification of transgenic tissues, but their pigment output may not be physiologically neutral during organogenesis, raising concerns about their suitability as selection markers in transformation pipelines. To address this, we examined the regeneration performance of constitutive ROSEA1 expression across four eudicot species from three families: tomato, petunia, begonia, and marigold. Stable ROSEA1-overexpressing lines were evaluated in tomato and petunia, whereas transformation-stage assays were performed in begonia and marigold. Regeneration frequencies were quantified relative to controls, and transcript-level changes were assessed for anthocyanin biosynthetic genes and regeneration regulators; complementation assays with PLT5 co-expression and hormonal manipulations were also performed. In tomato and petunia, ROSEA1-overexpressing lines exhibited reduced regeneration frequencies and elevated anthocyanin accumulation, with two independent petunia lines showing that stronger pigment activation corresponded to more severe regeneration defects. In tomato, ROSEA1 coordinately upregulated CHI, F3H, F3'5'H, and DFR and was associated with reduced expression of PLT5, WUS, and LBD16 during early regeneration. PLT5 co-expression partially restored regeneration with modestly reduced pigmentation, indicating that pigment output alone does not fully explain the phenotype. The penalty extended to begonia and marigold, with genotype- and hormone-dependent severity in marigold. These findings indicate a context-dependent regeneration penalty associated with constitutive ROSEA1 expression and suggest that hormone optimization, genotype selection, and developmental support may help mitigate this limitation in some contexts.