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.

Genetic transformation of citrus is essential for functional genomics and trait improvement, yet remains limited by prolonged regeneration cycles, genotype-dependent responses, and inefficient screening of transformed tissues. Here, we established an Agrobacterium-mediated indirect organogenesis-based transformation system for Carrizo citrange and evaluated two visible reporter systems, the anthocyanin regulator ROSEA1 and the betalain biosynthetic cassette RUBY, for monitoring transformed tissues throughout regeneration. The regeneration protocol enabled visible callus initiation within 7 days following a 5-day pre-culture treatment and recovery of PCR-confirmed transgenic plantlets within 5 months. Both visible reporters were compatible with callus development and shoot regeneration throughout the indirect organogenesis workflow. During callus proliferation, visible pigmentation closely corresponded with GFP fluorescence, enabling rapid, non-destructive identification of transformed tissues. However, reporter performance diverged during shoot regeneration: RUBY maintained stable pigmentation throughout regeneration, whereas ROSEA1-associated pigmentation progressively declined despite continued GFP fluorescence. Consequently, RUBY showed an approximately 4.7-fold higher pigment-positive shoot fraction than ROSEA1 at week 17, and the RUBY-positive fraction was similar to the GFP-positive fraction in the same treatment. Putative transgenic events were confirmed by PCR in independent lines of both constructs, together with GFP fluorescence in leaves and root tips. Together, these findings describe a practical indirect organogenesis-based transformation platform for citrus and demonstrate that both ROSEA1 and RUBY are effective visual reporters during callus proliferation, whereas RUBY provides more reliable visual identification during shoot regeneration. RUBY is therefore recommended as the visible reporter of choice for citrus transformation extending beyond the callus stage. The workflow was established in Carrizo citrange, among the most readily transformed citrus genotypes, and extension to other varieties remains to be demonstrated.

Plant tissue culture is essential for clonal propagation, germplasm conservation, genetic transformation, and genome editing in woody plants, yet these species remain highly challenging in vitro due to developmental recalcitrance, phenolic oxidation, endogenous contamination, seasonal effects, hyperhydricity, rooting difficulty, and strong genotype dependence. Medium formulation is therefore often a decisive factor rather than a routine technical step. This review examines the development, composition, and application of tissue culture media for woody species, emphasizing how specialized formulations emerged to overcome limitations of general-purpose media such as Murashige and Skoog medium. Major woody-targeted media, including Woody Plant Medium, Quoirin and Lepoivre medium, Anderson’s medium, Driver and Kuniyuki Walnut medium, Rugini Olive Medium, Litvay medium, and DCR medium, are discussed in relation to the physiological needs of their target species. Key medium components are reviewed, including mineral nutrients, carbon sources, vitamins, plant growth regulators, organic additives, gelling agents, and pH, with attention to nitrogen form, ionic strength, calcium supply, iron availability, and pathway-dependent plant growth regulator selection. The review further highlights applications in conservation, micropropagation, transformation, and genome editing, and discusses emerging approaches such as statistical optimization, machine learning, nanoparticles, temporary immersion systems, biostimulants, and morphogenic regulators.