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. Here, we show that constitutive ROSEA1 expression reduces shoot regeneration across four eudicot species from three families. In tomato and petunia, stable ROSEA1 -overexpressing lines displayed markedly lower regeneration frequencies than controls, together with increased anthocyanin accumulation. In petunia, comparison of two independent lines with contrasting pigment intensities further showed that stronger activation of the anthocyanin program was associated with a more severe regeneration defect. In tomato, transcript analysis showed that ROSEA1 coordinately activated anthocyanin biosynthetic genes, including CHI, F3H, F3′5′H, and DFR , while downregulating the regeneration regulators PLT5, WUS, and LBD16 during the early regeneration phase. Co-expression of PLT5 with ROSEA1 partially alleviated the regeneration defect while modestly reducing anthocyanin accumulation, supporting the conclusion that the phenotype cannot be explained by pigment output alone. The regeneration penalty also extended to begonia and marigold, although its magnitude varied by species. In marigold, the effect was genotype-dependent, and altered hormonal conditions changed the severity of the penalty, highlighting context dependence. These results indicate that strong ectopic activation of the ROSEA1 -dependent anthocyanin program compromises developmental competence during regeneration, likely through suppression of developmental regulators rather than anthocyanin accumulation alone. These findings identify an important limitation of anthocyanin-based reporter systems and suggest that hormone optimization, genotype selection, and developmental support may help mitigate this trade-off in plant transformation pipelines across diverse species.