ROSEA1-based visual selection reduces plant regeneration and alters developmental regulator expression

ROSEA1-based visual selection reduces plant regeneration and alters developmental regulator expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ROSEA1-based visual selection reduces plant regeneration and alters developmental regulator expression Tao Jiang, Sameena Ejaz Tanwir, Fangchen Liu, Wisnu Handoyo Ardi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9298244/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. ROSEA1 Anthocyanin biosynthesis Regeneration capacity Metabolic flux Visual markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message ROSEA1 -based visual selection is not developmentally neutral, reduces regeneration, and can be partly mitigated by hormone optimization or genotype selection. Introduction Visual reporters that produce pigmentation in plant cells provide a convenient means to identify transgenic events without destructive assays or specialized equipment. Among these, the R2R3-MYB transcription factor ROSEA1 from Antirrhinum majus has become a widely adopted marker owing to its ability to induce robust anthocyanin accumulation across diverse plant species (Butelli et al., 2008 ; Bedoya et al., 2012 ). Unlike conventional reporters such as β-glucuronidase (GUS), which require destructive histochemical assays and overnight incubation (Jefferson et al., 1987 ), or green fluorescent protein (GFP), which necessitates fluorescence microscopy and UV illumination (Baulcombe et al., 1995 ; Stewart, 2001 ), ROSEA1 , together with related anthocyanin-inducing transcription factors, enables real-time, non-destructive tracking of transgenic tissues through distinctive purple coloration visible to the unaided eye. This class of visual marker has been incorporated into transformation vectors for applications ranging from tracking viral infections to visual selection in rubber tree and hairy root systems (Bedoya et al., 2012 ; Majer et al., 2013 ; Huang et al., 2021 ). Despite this widespread adoption, the effects of ROSEA1 expression on plant regeneration capacity, a critical determinant of transformation success, remain poorly characterized. ROSEA1 , a member of the R2R3-MYB transcription factor family, activates anthocyanin biosynthesis by forming a regulatory complex with endogenous basic helix-loop-helix (bHLH) and WD40 proteins, collectively known as the MYB-bHLH-WD40 (MBW) complex (Schwinn et al., 2006 ; Koes et al., 2005 ). This highly conserved regulatory module coordinates the expression of structural genes throughout the flavonoid pathway, including chalcone synthase ( CHS ), chalcone isomerase ( CHI ), flavanone 3-hydroxylase ( F3H ), dihydroflavonol 4-reductase ( DFR ), and anthocyanidin synthase ( ANS ) (Holton and Cornish, 1995 ; Tanaka et al., 2008 ). The broad conservation of endogenous bHLH partners across angiosperms enables ROSEA1 expression alone to induce visible pigmentation in most plant species (Naing et al., 2018 ; Albert et al., 2014 ). Landmark work demonstrated that co-expression of ROSEA1 and its bHLH partner, DELILA , produced intensely purple tomato fruits with anthocyanin levels comparable to those of blueberries, thereby establishing the metabolic engineering potential of these transcription factors (Butelli et al., 2008 ). However, constitutive activation of flavonoid biosynthesis may also impose developmental and metabolic consequences beyond pigmentation itself. Anthocyanin production requires substantial investment of carbon skeletons derived from phenylalanine and malonyl-CoA, potentially diverting metabolic flux from primary pathways and other phenylpropanoid branches, including lignin biosynthesis and protein synthesis (Winkel-Shirley, 2001 ; Gould, 2004 ; Pourcel et al., 2010 ). The synthesis and vacuolar sequestration of these pigments involve multiple biosynthetic enzymes and downstream tailoring reactions, including multiple glycosylation steps, representing considerable cellular resource commitment (Shi and Xie, 2014 ; Winkel-Shirley, 2001 ). Trade-offs among anthocyanin accumulation, vegetative growth, reproductive output, and stress tolerance have been documented across diverse species (Chalker-Scott, 1999 ; Gould, 2004 ). In maize, ectopic/inducible activation of the anthocyanin regulators C1 and R is sufficient to activate flavonoid pathway genes and drive strong anthocyanin accumulation, highlighting the scale of transcriptional and metabolic reprogramming required for high pigment output. In contrast, anthocyanin-rich tomato fruits exhibit altered ripening dynamics (Zhang et al., 2013 ). These observations suggest that strong anthocyanin-promoting programs could interfere with the regenerative capacity of cultured explants, in which cellular resources must support both pigment biosynthesis and the energetically demanding process of shoot organogenesis. The potential impact of anthocyanin accumulation on regeneration is particularly significant, as regeneration capacity is the primary bottleneck limiting the efficiency of plant transformation in economically important species. Genetic transformation underpins modern plant biotechnology, enabling functional genomics, precision breeding, and CRISPR/Cas-based genome editing (Altpeter et al., 2016 ; Zhang et al., 2019 ). The success of any transformation pipeline depends on two sequential processes: delivery of foreign DNA into plant cells and subsequent regeneration of whole plants from transformed tissues (Altpeter et al., 2016 ; Lee and Wang, 2023 ). While DNA delivery methods have been substantially optimized, regeneration recalcitrance, the inability of plant tissues to form organized structures such as shoots or somatic embryos under in vitro conditions, whether during tissue culture or after genetic transformation, continues to restrict the scope of genetic engineering across legumes, cereals, woody species, and many elite cultivars (Somers et al., 2003 ; Gordon-Kamm et al., 2019 ; Birch, 1997 ). Moreover, the genotype-specificity of regeneration responses means that protocols optimized for one cultivar frequently fail when applied to related genotypes, and genome-editing tools can be applied only to the narrow subset of genotypes amenable to regeneration (Bregitzer et al., 1998 ; Debernardi et al., 2020 ). De novo shoot organogenesis proceeds through a highly coordinated developmental sequence governed by precisely coordinated hormonal and transcriptional programs. The foundational work of Skoog and Miller established that the balance between auxin and cytokinin determines cell fate: high cytokinin-to-auxin ratios promote shoot formation, while high auxin-to-cytokinin ratios favor root development (Skoog and Miller, 1957 ). This paradigm has guided the development of callus-induction media (CIM) and shoot-induction media (SIM), which are central to most transformation protocols (Valvekens et al., 1988 ; Ikeuchi et al., 2019 ). At the molecular level, key transcription factors orchestrate regeneration, including WUSCHEL ( WUS ), which serves as the master regulator of shoot apical meristem maintenance (Gallois et al., 2004 ; Gordon et al., 2007 ), the PLETHORA genes ( PLT3 , PLT5 , PLT7 ) that promote pluripotency establishment (Kareem et al., 2015 ), and LATERAL ORGAN BOUNDARIES DOMAIN factors ( LBD16 , LBD29 ), which contribute to competence acquisition (Sugimoto et al., 2010 ; Liu et al., 2018 ). Perturbations to the expression of these regulators are associated with impaired regeneration outcomes. Notably, several lines of evidence suggest potential mechanistic links between anthocyanin biosynthesis and regeneration pathways: cytokinin has been shown to stimulate both shoot organogenesis and anthocyanin accumulation (Deikman and Hammer, 1995 ; Das et al., 2012 ), while the phenylpropanoid pathway shares metabolic precursors with auxin biosynthesis (Brown et al., 2001 ). Consequently, constitutive ROSEA1 expression may perturb regeneration through metabolic competition, hormonal crosstalk, or transcriptional interference with key developmental networks. Despite the growing deployment of ROSEA1 and related anthocyanin-inducing MYB factors as visible reporters in transformation systems, their effects on regeneration have not been systematically evaluated (Kortstee et al., 2011 ; Bedoya et al., 2012 ; Fatihah et al., 2019 ). Existing evidence suggests context-dependent outcomes. In cassava, anthocyanin induction via the R2R3-MYB HbAN1 did not significantly affect regeneration (Zhen et al., 2024 ). In several dicots, anthocyanin-based markers such as MYB10 have been reported to be compatible with organogenesis during transformation, including in apple, strawberry, and potato (Kortstee et al., 2011 ). By comparison, maize anthocyanin regulators were first established as visible transformation markers in maize with the Lc gene (Ludwig et al., 1990 ), and maize C1/B-peru regulators were later shown to induce pigmentation in transformed wheat tissues (Chawla et al., 1999 ), while related anthocyanin-based visible-marker applications have also been reported in sugarcane (Bower et al., 1996 ), although the developmental consequences for regeneration were not systematically compared across systems. However, no study has quantified the magnitude of any regeneration penalty across multiple species, profiled underlying transcriptional changes, or tested whether such effects are conserved across phylogenetically diverse lineages. This gap creates uncertainty when selecting visual markers and may contribute to suboptimal transformation outcomes, particularly in regeneration-sensitive genotypes. If such a penalty exists, defining its magnitude and mechanistic basis would enable mitigation strategies, such as hormone optimization, genotype selection, or developmental support, to retain the advantages of visual selection while minimizing regeneration deficits. Here, we show that ROSEA1 expression substantially impairs regeneration capacity across four plant species representing three eudicot families, both in stable transgenic lines and during plant transformation. These findings indicate that anthocyanin-based visual selection can impose a regeneration cost associated with disrupted developmental competence. We further identify hormone optimization, genotype selection, and developmental support as practical strategies to mitigate this trade-off. Material and Methods Plant material and growth conditions Seeds of tomato ( Solanum lycopersicum cv. 'Micro-Tom'), petunia ( Petunia hybrida cv. 'Mitchell'), and marigold ( Tagetes erecta cv. 'Marvel II Yellow' and Tagetes patula cv. 'Bonanza Yellow') were purchased from Ball Horticultural Company (West Chicago, IL, USA). Begonia 'UF183-11', an advanced breeding line developed at the University of Florida, served as plant material. Seeds were surface-sterilized by immersion in 75% (v/v) ethanol for 1 min, followed by 15% (v/v) commercial bleach (6% sodium hypochlorite) for 10 min with gentle agitation, and then rinsed six times with sterile distilled water. Sterilized seeds were germinated on Murashige and Skoog (MS) basal medium supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar, pH adjusted to 5.8. Cultures were maintained at 25°C under a 16-h light/8-h dark photoperiod (100 µmol m⁻² s⁻¹) in a controlled environment chamber. For begonia, mature leaves from greenhouse-grown stock plants were washed under running tap water for 30 min, then surface-sterilized in a laminar flow hood with 70% (v/v) ethanol for 1 min, followed by 1.5% (v/v) sodium hypochlorite for 10 min, and rinsed three to five times with sterile distilled water. For marigold, seeds were pretreated for 2 h in a solution containing Contrex AP detergent (4.0 mg L⁻¹) and Dithane M-45 fungicide (4 mg L⁻¹), rinsed with sterile distilled water, and then surface sterilized in a laminar flow hood with 20% (v/v) Clorox (7.5% sodium hypochlorite) plus two drops of Tween 20 for 20 min, followed by 10% (v/v) Clorox for 10 min. After five rinses with sterile distilled water, seeds were germinated on hormone-free MS medium, and cotyledons from 4- to 5-day-old seedlings were used as explants for regeneration experiments. Vector construction The ROSEA1 coding sequence from Antirrhinum majus was cloned into the pOX135 binary vector backbone under the control of the CaMV 35S promoter for constitutive expression (Jiang et al., 2025 a). For transformation experiments comparing ROSEA1 -expressing (ROSox) and control (CK) treatments, the empty vector lacking the ROSEA1 insert served as the control. To generate stable overexpression lines (ROSox), the same 35S:: ROSEA1 construct was used. All constructs carried nptII and a GFP fusion cassette driven by the CsVMV promoter, with nptII conferring kanamycin resistance. Construct integrity was verified by Sanger sequencing before transformation into Agrobacterium tumefaciens strain EHA105. Agrobacterium -mediated transformation Agrobacterium tumefaciens strain EHA105 harboring the binary vectors was cultured in 25 mL LB broth supplemented with spectinomycin (100 mg L⁻¹) and rifampicin (50 mg L⁻¹) at 28°C with shaking at 180 rpm overnight. A secondary culture was initiated by transferring 1 mL of the overnight culture into 10 mL of fresh LB broth containing the same antibiotics and growing it until the optical density at 600 nm (OD₆₀₀) reached 0.3–0.5, except for marigold, for which cultures were grown to OD₆₀₀ = 0.6–1.0. Bacterial cells were pelleted by centrifugation and resuspended in liquid MS medium supplemented with 100 µM acetosyringone for plant infection (Jiang et al., 2026a ). For tomato transformation, cotyledon explants excised from 7- to 10-day-old seedlings were immersed in the Agrobacterium suspension for 10 min with gentle agitation. Inoculated explants were blotted on sterile filter paper and co-cultivated on MS medium (MS salts and vitamins, 30 g L⁻¹ sucrose, 2.5 g L⁻¹ Phytagel, pH 5.8) in the dark at 25°C for 2 days. Explants were then transferred to callus induction medium (CIM) consisting of MS medium supplemented with 2 mg L⁻¹ zeatin, 0.2 mg L⁻¹ NAA, 100 mg L⁻¹ kanamycin, and 100 mg L⁻¹ timentin. Cultures were maintained for 2–5 weeks with subculturing onto fresh CIM every 2 weeks. Regenerating shoots were subsequently transferred to shoot induction medium (SIM) composed of MS medium with 1 mg L⁻¹ zeatin, 100 mg L⁻¹ kanamycin, and 100 mg L⁻¹ timentin, followed by rooting on hormone-free MS medium containing 100 mg L⁻¹ kanamycin and 100 mg L⁻¹ timentin. Regenerated shoots exhibiting purple pigmentation, indicative of anthocyanin accumulation, were selected and transferred to a rooting medium. Rooted plantlets were acclimatized in a growth chamber and transferred to soil. T₀ transgenic plants were confirmed by PCR amplification of the GFP transgene. Seeds from T₀ plants were germinated on kanamycin-containing medium to select T₁ progeny for subsequent regeneration assays. For petunia transformation, cotyledon explants from 7- to 10-day-old seedlings were inoculated as described above and co-cultivated on MS medium in the dark at 25°C for 2 days. Explants were then transferred to CIM comprising MS medium supplemented with 1 mg L⁻¹ BAP, 0.1 mg L⁻¹ NAA, 100 mg L⁻¹ kanamycin, and 100 mg L⁻¹ timentin. Because shoot regeneration occurred directly on CIM without a distinct induction phase, explants were maintained on the same medium for 2–4 weeks with subculturing every 2 weeks until regenerated shoots were sufficiently developed for transfer to hormone-free MS medium containing 100 mg L⁻¹ kanamycin and 100 mg L⁻¹ timentin for elongation and rooting (Jiang et al., 2025 ). For Begonia transformation, leaf segments (approximately 0.5 × 0.5 cm) from sterile in vitro culture were inoculated with the Agrobacterium suspension and co-cultivated on regeneration medium (REM), consisting of MS medium supplemented with 1.5 mg L⁻¹ TDZ and 0.375 mg L⁻¹ NAA, in the dark at 25°C for 2 days. The explants were then transferred to REM supplemented with 75 mg L⁻¹ kanamycin and 100 mg L⁻¹ timentin. Cultures were subcultured every 2 weeks. After 4–9 weeks, regenerating explants were transferred to hormone-free MS medium with 75 mg L⁻¹ kanamycin and 100 mg L⁻¹ timentin to promote shoot elongation and rooting (Jiang et al., 2026b ). For marigold transformation, cotyledon explants from 4- to 5-day-old seedlings were vacuum-infiltrated with the Agrobacterium suspension for 5–10 min to enhance T-DNA delivery, then co-cultivated on MS medium in the dark at 25°C for 2 days. To evaluate the effect of cytokinin concentration on regeneration under ROSEA1 expression, three CIM formulations differing only in BAP level were tested: CIM1 (MS medium + 0.5 mg L⁻¹ NAA + 0.75 mg L⁻¹ BAP), CIM2 (MS medium + 0.5 mg L⁻¹ NAA + 2.0 mg L⁻¹ BAP), and CIM3 (MS medium + 0.5 mg L⁻¹ NAA + 2.5 mg L⁻¹ BAP), all supplemented with 100 mg L⁻¹ kanamycin and 100 mg L⁻¹ timentin. After callus formation, explants from each CIM treatment were transferred to a corresponding shoot induction medium (SIM: MS + NAA 0.1 mg L⁻¹ + BAP 1 mg L⁻¹) at the respective BAP concentration to maintain hormonal continuity during regeneration. Regenerated shoots were subsequently transferred to hormone-free MS medium containing 200 mg L⁻¹ kanamycin and 100 mg L⁻¹ timentin for elongation and rooting. Unless stated otherwise, all basal media consisted of MS salts and vitamins, 30 g L⁻¹ sucrose, and 7 g L⁻¹ agar (pH 5.8). Regeneration assays Two categories of regeneration assay were conducted throughout this study: transformation-stage assays, in which freshly inoculated explants were tracked during Agrobacterium -mediated transformation, and stable-line assays, in which explants derived from confirmed ROSox transgenic plants were compared with non-transgenic wild-type controls (CK) under identical culture conditions. Because all quantitative comparisons of regeneration capacity reported in this work originate from these assays, the scoring criteria and experimental design are described in detail below. In transformation-stage assays, explants were cultured on the species-specific CIM formulations described above. Regeneration was scored at discrete time points rather than across continuous intervals: at weeks 2, 3, and 4 for tomato; at weeks 1, 2, and 3 for petunia; at weeks 3, 5, 7, and 9 for begonia; and at weeks 2, 4, 6, and 8 for marigold. In stable-line assays, leaf or cotyledon explants excised from greenhouse-grown ROSox and CK plants were placed on the corresponding regeneration medium and cultured under the same light, temperature, and subculture regime used for transformation-stage experiments (Jiang et al., 2026b ). Regeneration capacity was evaluated using two complementary metrics. Regeneration frequency was defined as the percentage of explants that produced at least one visible shoot ≥ 5 mm in length by a given scoring date. Shoots per explant were calculated as the total number of shoots (≥ 5 mm) divided by the total number of explants in that treatment. In addition, explant area was measured from calibrated overhead photographs at each time point using ImageJ (NIH, Bethesda, MD) to provide a quantitative record of tissue expansion independent of shoot formation. Each treatment included a minimum of 15 explants per biological replicate, and at least three independent biological replicates were performed for every experiment unless otherwise noted. The exact number of explants (n) and replicates for each dataset is reported in the corresponding figure legends. Anthocyanin extraction and quantification Anthocyanins were extracted following the acidified methanol protocol with modifications. Fresh tissue (50–100 mg) from regenerating explants or callus was weighed, flash-frozen in liquid nitrogen, and homogenized to a fine powder. Samples were extracted in 300 µL of acidified methanol [1% (v/v) HCl in methanol] by vortexing and incubating overnight at room temperature in the dark. Following extraction, 200 µL of Milli-Q water and 500 µL of chloroform were added to facilitate phase separation. Samples were vortexed vigorously and centrifuged (12,000 × g, 5 min, 4°C). A 350-µL aliquot of the upper aqueous-methanolic phase was transferred to a fresh tube and diluted to a final volume of 1.05 mL with 415.8 µL methanol, 4.2 µL concentrated HCl, and 280 µL Milli-Q water. Absorbance was measured at 530 nm (anthocyanin peak) and 657 nm (chlorophyll interference) using a UV-visible spectrophotometer (NanoDrop 2000; Bio Tek). Corrected anthocyanin absorbance was calculated as Acorr = A₅₃₀ − 0.25 × A₆₅₇. Anthocyanin content was expressed as milligrams of cyanidin-3-glucoside equivalents per gram fresh weight (mg g⁻¹ FW) using the molar extinction coefficient ε = 33,000 l mol⁻¹ cm⁻¹ and molecular weight 449.2 g mol⁻¹. Blank extractions processed without tissue were included to correct for background absorbance. RNA isolation and quantitative real-time PCR Total RNA was extracted from 80–100 mg of flash-frozen regenerating tissue using RNAzol RT reagent (Molecular Research Center) following the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer; samples with A₂₆₀/A₂₈₀ ratios between 1.9 and 2.1 were used for cDNA synthesis. Genomic DNA contamination was eliminated by on-column DNase I digestion using the RNase-Free DNase Set (QIAGEN). First-strand cDNA was synthesized from 1 µg of DNase-treated RNA using the QuantiTect Reverse Transcription Kit (QIAGEN). The resulting cDNA was diluted 20- to 25-fold for use as qPCR template. Quantitative real-time PCR (qRT-PCR) was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). Each 10-µL reaction contained 5 µL of 2× SYBR Green master mix, 4 µL of diluted cDNA template, and 0.5 µL of a 10 µM forward and reverse primer mixture. Gene-specific primers were designed using Primer-BLAST (NCBI) to span exon-exon junctions where possible and to generate amplicons of 100–150 bp with melting temperatures of 58–62°C. Target genes included ROSEA1 (transgene expression), anthocyanin biosynthetic genes ( CHI , chalcone isomerase; F3H , flavanone 3-hydroxylase; F3 ' 5 ' H , flavonoid 3′,5′-hydroxylase; DFR , dihydroflavonol 4-reductase), and regeneration-associated transcription factors ( PLT5 , PLETHORA5; WUS , WUSCHEL; LBD16 , LATERAL ORGAN BOUNDARIES DOMAIN 16). These regeneration-associated regulators were selected based on their established roles in pluripotency acquisition and shoot meristem identity during de novo organogenesis. Primer sequences are provided in Supplementary Table 1. Thermal cycling conditions consisted of initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Melt curve analysis was performed to verify primer specificity. Relative gene expression was calculated using the 2 ⁻ΔΔCt method with ACTIN2 as the internal reference gene. Three biological replicates, each comprising three technical replicates, were analyzed per genotype and time point. Imaging and microscopy Macroscopic images of regenerating explants and culture plates were captured using a digital camera under standardized lighting conditions. To confirm transgene expression and T-DNA integration, explants were visualized using a fluorescence stereomicroscope equipped with appropriate filter sets. Purple pigmentation indicative of anthocyanin accumulation was documented at each time point. Anthocyanin extracts were also photographed to illustrate differences in color intensity between genotypes. Statistical analysis All data visualization and statistical analyses were performed using GraphPad Prism 10 (version 10.4.2). Biochemical and gene expression analyses were conducted using at least three independent biological replicates, with three technical replicates per biological sample for qRT-PCR. Morphological traits were quantified from a minimum of ten biological replicates per genotype. Data are presented as mean ± s.d. Statistical significance was assessed using two-tailed Student's t-tests for pairwise comparisons; P values < 0.05 were considered statistically significant. Exact P-values and sample sizes are indicated in the corresponding figure legends. Results ROSEA1 expression compromises regeneration during tomato transformation and in stable transgenic lines The R2R3-MYB transcription factor ROSEA1 from Antirrhinum majus has been widely adopted as a visual reporter for plant transformation due to its ability to induce anthocyanin accumulation, resulting in a distinctive purple pigmentation that facilitates the identification of transgenic events (Butelli et al., 2008 ; Schwinn et al., 2006 ). To determine whether ROSEA1 -based visual selection influences regeneration during the transformation process itself, tomato explants were subjected to Agrobacterium -mediated transformation with a ROSEA1 -containing construct and compared with empty vector controls transformed in parallel under identical culture conditions. ROSox tissues developed purple pigmentation in regenerating regions, visible as discrete purple foci emerging from the wound sites, whereas control (CK) explants remained green throughout the culture period (Fig. 1 a). This contrast confirmed strong ROSEA1 activity and validated its utility as a visual selection marker. However, ROSox explants produced fewer visible regenerative structures than CK explants, as evidenced by both magnified views and plate-level observations. The proportion of regenerating explants was reduced by 44.4% in ROSox compared with CK (Fig. 1 b), indicating a substantial decline in regeneration initiation. Shoot yield was similarly affected, with shoots per explant reduced by 55.5% in ROSox (Fig. 1 c). Together, these results demonstrate that the visual marker function of ROSEA1 is accompanied by a measurable reduction in regeneration efficiency during transformation. To determine whether the regeneration deficit persisted beyond the transformation stage and reflected a stable phenotype associated with constitutive ROSEA1 expression, stable transgenic tomato lines overexpressing ROSEA1 (ROSox) were generated and subjected to regeneration assays. ROSox lines exhibited sustained purple pigmentation throughout three weeks of culture on regeneration medium, with coloration intensifying over time (Fig. 2 a). Shoot emergence in ROSox lagged behind CK from the earliest time point examined, and this difference widened from week 1 to week 3 (Fig. 2 a). Although the pigment signal remained robust in stable lines, it was associated with reduced developmental output rather than serving solely as a neutral marker of transgenic tissue. Quantification analysis of regeneration parameters revealed substantial differences between genotypes. Regeneration frequency at week 2 was reduced by 60.0% in ROSox relative to CK, representing the maximum divergence (Fig. 2 b). By week 3, regeneration frequency in ROSox had partially recovered but remained 43.5% lower than CK (Fig. 2 b), indicating a sustained penalty despite overall progression of regeneration. Shoot production per explant was significantly reduced, declining by 52.4% at week 2 and by 42.6% at week 3 in ROSox relative to CK (Fig. 2 c). To confirm that the pigmentation phenotype reflected elevated anthocyanin biosynthesis, pigments were extracted from regenerated tissues and quantified. Anthocyanin extracts from ROSox were intensely colored compared with the pale solutions obtained from CK (Fig. 2 d). Spectrophotometric quantification confirmed that anthocyanin levels were 20.7-fold and 7.6-fold higher in ROSox than CK at weeks 2 and 3, respectively (Fig. 2 e). To test whether the regeneration penalty was specific to ROSEA1 or a general consequence of anthocyanin pathway activation, stable transgenic tomato lines overexpressing the bHLH transcription factor DELILA (DELox) were evaluated in parallel (Fig. S1 ). Notably, no regeneration penalty was observed in DELox lines (Fig. S1 a). At week 3, DELox tissues accumulated 73.7% more anthocyanin than CK (Fig. S1 b), confirming that DELILA also enhanced anthocyanin biosynthesis, albeit to a lower level than ROSEA1 . In contrast to ROSox, however, DELox did not exhibit reduced regenerative capacity; shoot production was 13.3% higher than in CK at the same stage, although this difference was not statistically significant (Fig. S1 c). These results indicate that anthocyanin accumulation per se is insufficient to impair regeneration, and that the inhibitory effect observed in ROSox likely depends on the specific regulatory context and/or the magnitude of pathway activation. ROSEA1 rewires gene expression during regeneration, coupling activation of anthocyanin synthesis with suppression of regeneration-associated regulators Quantitative real-time PCR (qRT-PCR) was performed on ROSox and CK tissues harvested at weeks 2 and 3 to characterize the transcriptional changes underlying the regeneration deficit. These time points were selected because week 2 corresponded to the period of maximum divergence in regeneration frequency between genotypes, whereas week 3 captured the partial recovery phase. ROSEA1 transcript abundance in ROSox was readily detected at both time points and remained substantially higher than in CK (Fig. 3 a), indicating sustained transgene expression throughout the culture period. Anthocyanin pathway genes were coordinately activated in ROSox tissues. CHI (chalcone isomerase), which catalyzes an early step in flavonoid biosynthesis, increased 117.5-fold at week 2 and 108.9-fold at week 3 relative to CK. DFR (dihydroflavonol 4-reductase), which directs the metabolic flux toward anthocyanin production rather than alternative flavonoid branches, increased 9.1-fold at week 2 and 23.8-fold at week 3 (Fig. 3 c). F3H (flavanone 3-hydroxylase) exhibited a more gradual response, increasing 1.8-fold at week 2 and 5.6-fold at week 3. F3′5′H (HF1), encoding a cytochrome P450 flavonoid 3′,5′-hydroxylase typically regulated downstream of MYB-bHLH-WD40 complexes controlling late anthocyanin biosynthesis, increased 75.3-fold at week 2 and 39.2-fold at week 3 (Fig. 3 c). The induction of multiple genes spanning both early and late steps of the pathway, rather than a single gene, is consistent with the role of ROSEA1 as a transcriptional activator of the anthocyanin biosynthetic module (Schwinn et al., 2006 ). Given the reduced regeneration capacity observed in ROSox lines, the expression of genes encoding key regulators of shoot regeneration was examined. PLT5 (PLETHORA5), WUS (WUSCHEL), and LBD16 (LATERAL ORGAN BOUNDARIES DOMAIN 16) were selected for analysis because these transcription factors play central roles in establishing pluripotency and shoot meristem identity during de novo organogenesis (Kareem et al., 2015 ; Sugimoto et al., 2010 ). PLT5 promotes the acquisition of regenerative competence by facilitating the establishment of pluripotency during the early phase of callus formation (Kareem et al., 2015 ). WUS functions as master regulator of shoot apical meristem identity and is required for de novo shoot formation (Zhang et al., 2017 ). LBD16 operates within the auxin-mediated lateral organ development pathway and contributes to the acquisition of cellular competence preceding regeneration (Liu et al., 2018 ). In contrast to anthocyanin genes, regeneration-associated regulators were markedly repressed at the early time point. At week 2, PLT5 expression was reduced by 89.3%, WUS by 71.1%, and LBD16 by 82.8% relative to CK (Fig. 3 b). This repression coincided with the stage at which developmental competence is normally established during the regeneration process (Kareem et al., 2015 ; Ikeuchi et al., 2019 ). By week 3, PLT5 rebounded to 2.4-fold above CK levels, WUS to 1.4-fold above, whereas LBD16 remained 16.0% lower than CK (Fig. 3 b). This temporal pattern, characterized by early suppression followed by partial recovery, mirrored the regeneration phenotype (Fig. 2 b), with maximal transcriptional repression at week 2 corresponding to the greatest reduction in regeneration capacity. Sustained ROSEA1 activity was associated with persistent anthocyanin pathway induction and a dynamic perturbation of developmental regulators, characterized by early-stage suppression during the critical window of competence establishment. To test whether reinforcement of the regeneration program could counteract the inhibitory effect of ROSEA1 , stable tomato lines co-expressing PLT5 and ROSEA1 (P+ROSox) were evaluated alongside ROSox lines (Fig. 4 ). Relative to ROSox, P+ROSox lines showed a 73.7% increase in shoots per explant and a 41.9% increase in regeneration frequency, indicating that PLT5 co-expression substantially alleviated the regeneration defect associated with ROSEA1 overexpression (Fig. 4 d, e). In contrast, anthocyanin accumulation in P+ROSox tissues was reduced by 18.8% relative to ROSox (Fig. 4 b, c), demonstrating that restoration of regeneration was achieved with only a modest reduction in pigment output. These results indicate that regeneration inhibition in ROSox is not solely due to anthocyanin accumulation but is more likely driven by the suppression of regeneration-promoting developmental pathways. Importantly, this effect can be at least partially mitigated through PLT5 -mediated reinforcement of the regeneration program. The regeneration penalty associated with ROSEA1 is conserved across diverse plant species To test whether the regeneration deficit observed in tomato reflects a broader consequence of ROSEA1 expression, stable petunia lines overexpressing ROSEA1 (ROSox) were examined. In an initial screening experiment, a ROSox line (later designated ROSox-2) accumulated visible purple pigmentation throughout culture, whereas control (CK) explants remained green (Fig. S2a, b). Plate-level observations revealed that ROSox-2 cultures consistently produced fewer regenerants and exhibited delayed shoot progression relative to CK over a 1- to 3-week culture window (Fig. S2a, b). Quantification of explant expansion supported this macroscopic phenotype: explant area in ROSox-2 lagged behind CK at all time points, with the deficit increasing over time, being reduced by 18.3% at week 1, 32.2% at week 2, and 41.6% at week 3 relative to CK (Fig. S2c). Regeneration frequency was reduced by 47.2% at week 2 and remained 40.0% lower at week 3 (Fig. S2d). Anthocyanin extracts from week-3 tissues were visibly pink in ROSox-2 but colorless in CK, with pigment levels 21.0-fold higher than CK (Fig. S2e, f). Motivated by this reproducible phenotype, we next tested whether the regeneration penalty scales with ROSEA1 -driven pigment output by comparing two independent ROSox lines with distinct anthocyanin intensities (ROSox-1, moderate; ROSox-2, strong) in a time-course assay extending to week 4 (Fig. 5 a, b). Both lines maintained purple pigmentation throughout culture and produced fewer regenerants with delayed shoot progression at the plate level relative to CK (Fig. 5 a, b). Quantification of explant expansion confirmed that reduced tissue growth emerged early and persisted: compared with CK, mean explant area was reduced by 27.6% in ROSox-1 and 29.2% in ROSox-2 at week 1, by 46.2% in ROSox-1 and 42.1% in ROSox-2 at week 2, and by 42.8% in ROSox-1 and 47.1% in ROSox-2 at week 3 (Fig. 5 c). This growth deficit was accompanied by impaired regeneration performance: at week 3, mean regeneration frequency was reduced by 51.0% in ROSox-1 and 59.8% in ROSox-2 relative to CK; by week 4, ROSox-1 remained 29.3% lower than CK, whereas ROSox-2 remained 64.1% lower (Fig. 5 d). Likewise, regenerated shoots per explant at week 4 were reduced by 63.2% in ROSox-2, with a milder effect in ROSox-1 (Fig. 5 e). Biochemical analysis confirmed the intended pigment gradient: anthocyanin content at week 4 was 4.3-fold higher in ROSox-1 and 21.7-fold higher in ROSox-2 relative to CK (Fig. 5 f, g). These results demonstrate that, as in tomato, strong ROSEA1 -driven anthocyanin accumulation in petunia is associated with reduced regeneration capacity and tissue expansion, and the severity of the penalty scales with pigment output across independent transgenic lines. Begonia, phylogenetically distant from Solanaceae and representing the order Cucurbitales, was included to broaden the taxonomic scope of the analysis and test whether the ROSEA1 -associated penalty extends beyond the Solanaceae family. Agrobacterium -mediated transformation introduced ROSEA1 into begonia leaf explants, and the success of transformation was confirmed by co-expression of a fluorescent reporter. Fluorescence signals and developing structures were observed in ROSox explants at weeks 3 and 6 (Fig. 6 a), confirming successful T-DNA integration and ruling out failed transformation as an explanation for any subsequent differences in regeneration. At week 9, when regeneration scoring was performed, CK cultures had produced visibly more regenerants than ROSox cultures at the plate level (Fig. 6 b). Quantification supported this observation: regeneration frequency was reduced by 57.4% in ROSox relative to CK (Fig. 6 c), while shoots per explant were reduced by 70.1% (Fig. 6 d). The ROSEA1 -associated penalty thus extends beyond Solanaceae into a phylogenetically distinct eudicot clade. To further evaluate context dependence, marigold ( Tagetes erecta ), a member of the Asteraceae family, was examined under varying cytokinin regimes. Cytokinins are key determinants of regeneration efficiency in many species, and manipulating hormone levels represents a potential strategy to mitigate regeneration deficits (Lowe et al., 2016 ). Agrobacterium -mediated transformation of marigold 'Marvel II Yellow' was carried out under two callus-induction media differing in cytokinin content: CIM1 (0.5 mg L⁻¹ NAA + 0.75 mg L⁻¹ BAP), representing a lower cytokinin condition, and CIM2 (0.5 mg L⁻¹ NAA + 2.0 mg L⁻¹ BAP), representing an intermediate cytokinin condition. ROSox explants exhibited reduced regenerative progression relative to CK on both formulations at the plate level (Fig. 6 e). Reporter fluorescence in regenerating tissues verified that these outcomes arose from successfully transformed cells (Fig. 6 f). On CIM1, regeneration frequency was reduced by 33.1% in ROSox relative to CK, whereas on CIM2, regeneration frequency was reduced by 30.9% (Fig. 6 g). Shoot output followed a similar trend, decreasing by 35.2% on CIM1 and by 16.2% on CIM2 (Fig. 6 h). Across both hormone regimes, ROSEA1 expression was associated with reduced regeneration, although the apparent penalty was less pronounced under higher cytokinin conditions. The attenuation of the regeneration penalty at elevated cytokinin levels prompted us to ask whether further increases could fully offset the ROSEA1 effect. To test this, ‘Marvel II Yellow’ explants were cultured on CIM3 (0.5 mg L⁻¹ NAA + 2.5 mg L⁻¹ BAP), which supported substantially higher baseline regeneration than CIM1 or CIM2. Under CIM3 conditions, CK and ROSox cultures appeared macroscopically similar throughout the culture period (Fig. S3a). No significant differences were detected between treatments for either regeneration frequency (Fig. S3b) or shoots per explant (Fig. S3c). Collectively, these results suggest that the ROSEA1 -associated regeneration penalty becomes progressively less apparent with increasing cytokinin and is not detectable under strongly cytokinin-dominant conditions. The ROSEA1 -associated penalty also appeared to vary with genotype. In ‘Bonanza Yellow’ cultured on CIM1, baseline regeneration was low for both CK and ROSox treatments, and plate phenotypes appeared similar (Fig. S3d). Neither regeneration frequency nor shoot output differed significantly between treatments (Fig. S3e,f); ROSox values for regeneration showed a slight, non-significant increase relative to CK (Fig. S3e,f). The inherently low regeneration capacity of this genotype may have masked additional ROSEA1 -associated effects, as floor effects can obscure modest reductions in already low-performing systems. Together, these results suggest that the impact of ROSEA1 on regeneration in marigold depends on both hormone regime and genotype-specific regeneration capacity and further indicate that mitigation strategies based on cytokinin optimization and genotype selection could influence transformation outcomes. Discussion Visual reporters that enable non-destructive identification of transgenic events are valuable tools in plant transformation; however, their developmental neutrality has rarely been systematically evaluated. In this study, constitutive ROSEA1 expression was consistently associated with reduced regeneration across multiple eudicot species, although the magnitude of this effect varied with species, genotype, and hormone regime (Figs. 1 , 2 , 5 , 6 ). In tomato, this phenotype coincided with activation of anthocyanin biosynthetic genes and reduced expression of key regeneration-associated regulators during an early regeneration window (Fig. 3 ), providing a mechanistic basis for the observed trade-off. Together, these findings demonstrate that ROSEA1 -based visual selection is not inherently developmentally neutral and that marker systems capable of reprogramming secondary metabolism may significantly influence developmental plasticity during tissue culture. Mechanistic basis of the ROSEA1 –regeneration trade-off The data presented here argue against a simple model in which anthocyanin accumulation alone directly suppresses regeneration. This distinction is most clearly illustrated by the DELILA comparison: although DELox tomato lines accumulated 73.7% more anthocyanin than the control at week 3, they did not exhibit reduced regenerative capacity (Fig. S1 ). By contrast, ROSox lines showed substantially greater anthocyanin induction, reaching 20.7-fold above the control at week 2 and 7.6-fold at week 3, accompanied by a marked reduction in regeneration (Fig. 2 ). A similar dosage-dependent pattern was observed in petunia, where the more intensely pigmented ROSox-2 line exhibited a stronger regeneration defect than the moderately pigmented ROSox-1 (Fig. 5 ). Together, these comparisons indicate that the severity of the phenotype correlates more closely with the extent of ROSEA1 -driven pathway activation than with pigment accumulation per se. This interpretation is consistent with the biology of ROSEA1 in Antirrhinum , where it functions as an R2R3-MYB regulator of floral pigmentation intensity and patterning rather than as a canonical developmental regulator (Schwinn et al., 2006 ), and with previous reports that ectopic ROSEA1 expression can alter cellular programs beyond pigmentation, including abiotic stress responses (Naing et al., 2018 ). A key line of support comes from the transcriptional data. During the competence acquisition phase, ROSEA1 overexpression was associated with reduced expression of PLT5, WUS , and LBD16 (Fig. 3 b), coinciding with the developmental stage at which the divergence in regeneration between ROSox and the control was maximal (Figs. 2 , 3 ). This temporal overlap is notable because these genes occupy central positions in regeneration: PLT factors promote the acquisition of pluripotency (Kareem et al., 2015 ), WUS is required for shoot formation (Gallois et al., 2004 ), and LBD16 contributes to the establishment of organogenic competence upstream of WUS (Liu et al., 2018 ; Ikeuchi et al., 2019 ). Consistent with this framework, lines co-expressing PLT5 and ROSEA1 (P+ROSox) exhibited a 73.7% increase in shoots per explant and a 41.9% increase in regeneration frequency relative to ROSox, whereas anthocyanin levels declined by only 18.8% (Fig. 4 ). The disproportionate recovery of regeneration relative to pigment reduction supports the conclusion that the ROSEA1 -associated penalty is not driven solely by anthocyanin accumulation but instead reflects suppression of regeneration-promoting developmental pathways that can be partially restored by PLT5. Importantly, these data do not indicate that ROSEA1 itself directly regulates regeneration; rather, ectopic ROSEA1 activity likely perturbs the transcriptional environment required for shoot induction, thereby indirectly suppressing the regeneration program. One plausible mechanistic layer underlying this effect is metabolic burden. Anthocyanin biosynthesis requires a substantial investment of phenylalanine-derived carbon skeletons and malonyl-CoA, potentially diverting metabolic flux from pathways that support cell proliferation and differentiation (Gould, 2004 ). Anthocyanin accumulation is closely linked to growth, energy allocation, and environmental responsiveness rather than representing a metabolically neutral output (Shi et al., 2023 ) and engineered high-anthocyanin tomato genotypes have been reported to show reduced vegetative growth and yield (Cerqueira et al., 2023 ). A similar principle may extend beyond anthocyanin-based systems. In ornamental eggplant, constitutive overexpression of the betalain marker cassette RUBY caused profound developmental alterations, including complete reproductive sterility due to defects in both male and female organs (Tanwir et al., 2026 , in revision). Although that system involves betalain rather than anthocyanin biosynthesis, it similarly demonstrates that strong constitutive activation of visible pigment pathways may not be developmentally neutral and can impose substantial physiological trade-offs. These studies focused on whole-plant growth rather than regeneration-stage tissues, but they are broadly consistent with our observation that the regeneration penalty became most apparent when anthocyanin induction was especially pronounced (Figs. 2 , 5 , S1), compatible with a threshold-like effect. However, the partial rescue observed in P+ROSox lines, in which regeneration improved markedly despite only modest reductions in pigment output (Fig. 4 ), suggests that metabolic cost alone may not fully account for the phenotype. A complementary explanation is broader regulatory pleiotropy. As a transcription factor, ROSEA1 may influence pathways beyond anthocyanin biosynthesis. Indeed, transcriptomic and metabolomic studies of tomato expressing snapdragon anthocyanin regulators have revealed widespread reprogramming across flavonoid and phenylpropanoid networks rather than a confined increase in pigment accumulation (Tohge et al., 2015 ). The coordinate suppression of PLT5, WUS , and LBD16 (Fig. 3 b) may therefore reflect either a shared upstream perturbation or parallel interference across multiple developmental pathways. Supporting this view, callus cultures of Nicotiana benthamiana expressing ROSEA1 and DELILA under a strong constitutive promoter exhibited enhanced pigmentation but reduced shoot formation compared with those driven by a weaker, tissue-specific promoter (Fatihah et al., 2019 ; Zheng et al., 2007). Although regeneration was not quantitatively assessed in that study, the qualitative trend aligns with the present findings, in which stronger regulatory activation is associated with a more pronounced regeneration penalty (Figs. 2 , 5 , S1). A further layer likely involves altered hormone signaling. While there is currently no evidence that ROSEA1 directly represses PLT5, WUS , or LBD16 , it is plausible that intermediate effects on auxin distribution, cytokinin responsiveness, or other signaling pathways contribute to the phenotype. Nevertheless, the biological plausibility of such a link is considerable because anthocyanin-associated regulation is closely integrated with sugar signaling and multiple phytohormone pathways (Das et al., 2012 ; LaFountain and Yuan, 2021 ; Shi et al., 2023 ). Notably, elevated cytokinin largely alleviated the regeneration penalty in marigold (Fig. 6 e–h; Fig. S3), indicating that exogenous hormone supply can buffer at least part of the ROSEA1 -associated inhibition, consistent with the broader principle that regeneration barriers can often be mitigated through hormonal optimization (Valvekens et al., 1988 ; Ikeuchi et al., 2019 ). Taken together, the mechanistic evidence supports a hierarchical model in which ectopic ROSEA1 expression drives extensive activation of the anthocyanin network, accompanied by broader metabolic and transcriptional reprogramming that compromises regeneration competence, at least in part through suppression of PLT5, WUS , and LBD16 (Fig. 3 ). Within this framework, metabolic burden, transcription-factor pleiotropy, and altered hormone signaling should be viewed not as competing explanations but as interconnected components of a broader mechanistic framework. Conservation of the regeneration penalty across species and genotypes The regeneration penalty associated with ROSEA1 extended across three eudicot families: Solanaceae (tomato and petunia; Figs. 1 , 2 , 5 ), Begoniaceae (begonia; Fig. 6 a–d), and Asteraceae (marigold; Fig. 6 e–h), indicating that the phenotype reflects broadly conserved developmental constraints rather than a species-specific anomaly. This phylogenetic breadth is consistent with the deep conservation of both the MYB-bHLH-WD40 module, which controls anthocyanin biosynthesis, and the WUS-, PLT-, and LBD-associated pathways that govern regeneration (Albert et al., 2014 ; Lloyd et al., 2017 ; Ikeuchi et al., 2019 ). However, because ROSEA1 is a transcriptional regulator rather than a metabolic endpoint, these findings are best generalized to ROSEA1 -based visual selection rather than to anthocyanin markers as a class. This distinction is reinforced by the DELox comparison, which demonstrates that moderate anthocyanin enhancement does not necessarily compromise organogenesis (Fig. S1 ). These results support a regulator- and context-dependent model in which the developmental cost is determined by regulator identity, expression level, and endogenous partner context. The magnitude of the regeneration penalty appeared to vary across species, ranging from approximately 30% in marigold under moderate cytokinin conditions to nearly 70% in begonia (Fig. 6 ). This variation may reflect differences in baseline regenerative capacity, the endogenous availability of bHLH and WD40 partners that may be needed to assemble functional MBW complexes with ROSEA1 (Naing et al., 2018 ), and species-specific differences in phenylpropanoid metabolism. Genotype-specific responses within marigold further suggest this complexity. In ‘Bonanza Yellow’, which showed relatively low baseline regeneration, no significant penalty was detected (Fig. S3), possibly because of a floor effect. In contrast, genotypes with higher regenerative capacity tended to show a clearer ROSEA1 -associated reduction in regeneration (Fig. 6 e–h). This pattern is consistent with the well-established genotype dependence of regeneration capacity (Bregitzer et al., 1998 ; Gordon-Kamm et al., 2019 ) and may also suggest genotype-by-marker-system interactions, with the developmental burden associated with ROSEA1 potentially becoming more evident in genotypes with moderate to high regenerative potential. Practical implications for transformation pipeline design The distinctive purple pigmentation induced by ROSEA1 enables rapid, non-destructive identification of transformed sectors under ambient lighting, offering clear advantages over β-glucuronidase (GUS), which requires destructive histochemical assays, and green fluorescent protein (GFP), which often necessitates specialized fluorescence imaging equipment (Jefferson et al., 1987 ; Stewart, 2001 ; Leclercq et al., 2010 ; Bedoya et al., 2012 ). Our findings do not diminish these advantages but indicate that they can be offset by reduced regeneration efficiency, particularly in systems with moderate regenerative competence (Figs. 1 , 2 , 5 , 6 ). Importantly, this cost is not an inherent consequence of anthocyanin production per se. The DELox comparison demonstrates that increased pigment output does not necessarily impair regeneration (Fig. S1 ), indicating that regulator identity and expression context are key determinants of whether a developmental penalty arises. Several mitigation strategies emerge from these findings. Hormone optimization, particularly increased cytokinin, can partially or fully compensate for the phenotype, as demonstrated in marigold (Fig. 6 e–h; Fig. S3), consistent with the broader principle that regeneration barriers can often be alleviated by adjusting the hormonal environment (Valvekens et al., 1988 ; Ikeuchi et al., 2019 ). Co-expression of PLT5 partially rescued the ROSox regeneration defect while only modestly reducing anthocyanin accumulation (Fig. 4 ), suggesting that reinforcement of developmental competence is an effective complementary strategy. Inducible or tissue-specific expression systems, such as the XVE promoter (Zuo et al., 2000 ), could delay ROSEA1 activation until after regeneration is established. Alternative reporters, such as RUBY , which drives betalain rather than anthocyanin pigmentation, may impose a distinct developmental burden (He et al., 2020 ). Finally, genotype pre-screening is advisable because the ROSEA1 -associated penalty is most pronounced in genotypes with intermediate regeneration capacity; selecting highly regenerable backgrounds can help maintain both visual selection efficiency and acceptable transformation frequencies. More broadly, these findings highlight a critical interference between secondary metabolism and developmental plasticity. The coordinated reduction in PLT5, WUS , and LBD16 expression (Fig. 3 ) may arise from direct transcriptional effects, indirect metabolic perturbation, or a combination of both. Dissecting these mechanisms will be essential for designing next-generation visual reporters that retain strong pigmentation while minimizing developmental cost. Comparative analysis using different anthocyanin regulators under matched expression systems will be particularly informative for distinguishing pigment-associated effects from regulator-specific pleiotropy. In conclusion, ROSEA1 -based visual selection can impose a regeneration cost associated with reduced expression of key developmental regulators during competence acquisition (Fig. 3 ). This effect extends across phylogenetically diverse species (Figs. 1 , 2 , 5 , 6 ) but can be mitigated through hormone optimization, co-expression of developmental regulators, and genotype selection. The findings provide a mechanistic framework for understanding how a secondary-metabolism regulator can influence regeneration and offer practical guidance for the informed deployment of ROSEA1 in transformation pipelines. Declarations Acknowledgements We thank Jaideep Chandranshu Cherukula for plant and facility management, and Keila Emily Rodriguez for the previously generated transgenic lines used in this project. Author contributions T.J. conceived and designed the study. S.E.T. performed plant regeneration experiments in tomato. F.L. performed plant regeneration experiments in petunia. S.E.T. conducted the qRT-PCR analysis. Y.N. performed the plant regeneration experiments in marigold, and W.H.A. performed those in begonia. F.H. and S. Z. maintained plant growth in the greenhouse. T.J. conducted data analysis and visualization. T.J. wrote the manuscript with input from all authors. H.H. and T.J. revised the manuscript. Funding This work was supported by USDA-NIFA (grant number 2019-67013-29236) and the USDA HATCH program (grant number FLA-MFC-006387) to H.H. Competing interests The authors declare no competing interests. Ethics declarations Not applicable. Data availability All data supporting the findings of this study are available from the corresponding author upon reasonable request. Code availability No custom code was developed for this study. All analyses were performed using publicly available software as described in the Methods section. References Albert, N.W., Davies, K.M., Lewis, D.H., Zhang, H., Montefiori, M., Brendolise, C., Boase, M.R., Ngo, H., Jameson, P.E. and Schwinn, K.E., 2014. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. The Plant Cell , 26(3), pp.962–980. Altpeter, F., Springer, N.M., Bartley, L.E., Blechl, A.E., Brutnell, T.P., Citovsky, V., Conrad, L.J., Gelvin, S.B., Jackson, D.P., Kausch, A.P., Lemaux, P.G., Medford, J.I., Orozco-Cárdenas, M.L., Tricoli, D.M., Van Eck, J., Voytas, D.F., Walbot, V., Wang, K., Zhang, Z.J. and Stewart, C.N., 2016. Advancing crop transformation in the era of genome editing. The Plant Cell , 28(7), pp.1510–1520. Baulcombe, D.C., Chapman, S. and Santa Cruz, S., 1995. Jellyfish green fluorescent protein as a reporter for virus infections. The Plant Journal , 7(6), pp.1045–1053. Bedoya, L.C., Martínez, F., Orzáez, D. and Daròs, J.-A., 2012. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiology , 158(3), pp.1130–1138. Birch, R.G., 1997. Plant transformation: problems and strategies for practical application. Annual Review of Plant Physiology and Plant Molecular Biology , 48, pp.297–326. Bower, R., Elliott, A.R., Potier, B.A.M. and Birch, R.G., 1996. High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Molecular Breeding , 2(3), pp.239–249. Bregitzer, P., Dahleen, L.S. and Campbell, R.D., 1998. Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars. Plant Cell Reports , 17(12), pp.941–945. Brown, D.E., Rashotte, A.M., Murphy, A.S., Normanly, J., Tague, B.W., Peer, W.A., Taiz, L. and Muday, G.K., 2001. Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis . Plant Physiology , 126(2), pp.524–535. Butelli, E., Titta, L., Giorgio, M., Mock, H.-P., Matros, A., Peterek, S., Schijlen, E.G.W.M., Hall, R.D., Bovy, A.G., Luo, J. and Martin, C., 2008. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology , 26(11), pp.1301–1308. Cerqueira, J.V.A., Zhu, F., Mendes, K., Nunes-Nesi, A., Martins, S.C.V., Benedito, V., Fernie, A.R. and Zsögön, A., 2023. Promoter replacement of ANT1 induces anthocyanin accumulation and triggers the shade avoidance response through developmental, physiological and metabolic reprogramming in tomato. Horticulture Research , 10(2), uhac254. Chalker-Scott, L., 1999. Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology , 70(1), pp.1–9. Chawla, H.S., Cass, L.A. and Simmonds, J.A., 1999. Developmental and environmental regulation of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes. In Vitro Cellular & Developmental Biology-Plant , 35 (5), pp.403–408. Das, P.K., Shin, D.-H., Choi, S.-B. and Park, Y.-I., 2012. Sugar-hormone cross-talk in anthocyanin biosynthesis. Molecules and Cells , 34(6), pp.501–507. Debernardi, J.M., Tricoli, D.M., Ercoli, M.F., Hayta, S., Ronald, P., Palatnik, J.F. and Dubcovsky, J., 2020. A GRF–GIF chimera boosts regeneration and transformation in wheat. Nature Biotechnology , 38(12), pp.1274–1279. Deikman, J. and Hammer, P.E., 1995. Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana . Plant Physiology , 108(1), pp.47–57. Fatihah, H.N.N., Moñino López, D., van Arkel, G.,"; Schaart, J.G., Visser, R.G.F. and Krens, F.A., 2019. The ROSEA1 and DELILA transcription factors control anthocyanin biosynthesis in Nicotiana benthamiana and Lilium flowers. Scientia Horticulturae , 243, pp.327–337. Gallois, J.-L., Nora, F.R., Mizukami, Y. and Sablowski, R., 2004. WUSCHEL induces shoot stem cell activity and developmental plasticity in the root meristem. Genes & Development , 18(4), pp.375–380. Gordon, S.P., Heisler, M.G., Reddy, G.V., Ohno, C., Das, P. and Meyerowitz, E.M., 2007. Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development , 134(19), pp.3539–3548. Gordon-Kamm, B., Sardesai, N., Arling, M., Lowe, K., Hoerster, G. and Betts, S., 2019. Using morphogenic genes to improve recovery and regeneration of transgenic plants. Plants , 8(2), 38. Gould, K.S., 2004. Nature's Swiss army knife: the diverse protective roles of anthocyanins in leaves. Journal of Biomedicine and Biotechnology , 2004(5), pp.314–320. He, Y., Zhang, T., Sun, H., Zhan, H. and Zhao, Y., 2020. A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research , 7, 152. Holton, T.A. and Cornish, E.C., 1995. Genetics and biochemistry of anthocyanin biosynthesis. The Plant Cell , 7(7), pp.1071–1083. Huang, T., Xin, S., Fang, Y., Chen, T., Chang, J., Ko, N.C.K., Huang, H. and Hua, Y., 2021. Use of a novel R2R3-MYB transcriptional activator of anthocyanin biosynthesis as visual selection marker for rubber tree (Hevea brasiliensis) transformation. Industrial Crops and Products , 174 , p.114225. Ikeuchi, M., Favero, D.S., Sakamoto, Y., Iwase, A., Coleman, D., Rymen, B. and Sugimoto, K., 2019. Molecular mechanisms of plant regeneration. Annual Review of Plant Biology , 70, pp.377–406. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W., 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal , 6(13), pp.3901–3907. Jiang, T., Lian, Z., Rodriguez, K., Liu, F., Karn, A., Handoyo, W., Kyum, M., Tanwir, S.E., Van Deynze, A. Bradford, K., and Huo, H., 2026b. Synergistic developmental regulators enable efficient plant regeneration and transformation across species. Jiang, T., Liu, F., Tanwir, S.E., Shaheen, N., Wang, G., Copsey, L., Lian, Z. and Huo, H., 2026a. Identification and functional characterization of AmbHLH002 as a conserved bHLH regulator of anthocyanin biosynthesis in Antirrhinum majus. Horticultural Plant Journal , 12 (3), pp. 704–719. Jiang, T., Rodriguez, K., Tanwir, S.E., Liu, F., Hussain, F., Cherukula, J.C. and Huo, H., 2025. Regulatory role of MicroRNA164 in heat and salinity stress responses via candidate target genes during seed germination in petunia. Horticulture Advances , 3 (1), p.15. Kareem, A., Durgaprasad, K., Sugimoto, K., Du, Y., Pulianmackal, A.J., Trivedi, Z.B., Abhayadev, P.V., Pinon, V., Meyerowitz, E.M., Scheres, B. and Prasad, K., 2015. PLETHORA genes control regeneration by a two-step mechanism. Current Biology , 25(8), pp.1017–1030. Koes, R., Verweij, W. and Quattrocchio, F., 2005. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends in Plant Science , 10(5), pp.236–242. Kortstee, A.J., Khan, S.A., Helderman, C., Trindade, L.M., Wu, Y., Visser, R.G.F., Brendolise, C., Allan, A., Schouten, H.J. and Jacobsen, E., 2011. Anthocyanin production as a potential visual selection marker during plant transformation. Transgenic Research , 20(6), pp.1253–1264. LaFountain, A.M. and Yuan, Y.-W., 2021. Repressors of anthocyanin biosynthesis. New Phytologist , 231(3), pp.933–949. Leclercq, J., Lardet, L., Martin, F., Chapuset, T., Oliver, G. and Montoro, P., 2010. The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens -mediated transformation in Hevea brasiliensis (Müll. Arg.). Plant Cell Reports , 29(5), pp.513–522. Lee, K. and Wang, K., 2023. Strategies for genotype-flexible plant transformation. Current Opinion in Biotechnology , 79, 102848. Liu, J., Hu, X., Qin, P., Prasad, K., Hu, Y. and Xu, L., 2018. The WOX11–LBD16 pathway promotes pluripotency acquisition in callus cells during de novo shoot regeneration in tissue culture. Plant and Cell Physiology , 59(4), pp.739–748. Lloyd, A., Brockman, A., Aguirre, L., Campbell, A., Bean, A., Cantero, A. and Gonzalez, A., 2017. Advances in the MYB–bHLH–WD repeat (MBW) pigment regulatory model: addition of a WRKY factor and co-option of an anthocyanin MYB for betalain regulation. Plant and Cell Physiology , 58(9), pp.1431–1441. Lowe, K., Wu, E., Wang, N., Hoerster, G., Hastings, C., Cho, M.-J., Scelonge, C., Lenderts, B., Chamberlin, M., Cushatt, J., Wang, L., Ryan, L., Khan, T., Chow-Yiu, J., Hua, W., Yu, M., Banh, J., Bao, Z., Brink, K., Igo, E., Rudrappa, B., Shamseer, P.M., Bruce, W., Newman, L., Shen, B., Zheng, P., Bidney, D., Falco, C., Register, J., Zhao, Z.-Y., Xu, D., Jones, T. and Gordon-Kamm, W., 2016. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. The Plant Cell , 28(9), pp.1998–2015. Ludwig, S.R., Bowen, B., Beach, L. and Wessler, S.R., 1990. A regulatory gene as a novel visible marker for maize transformation. Science , 247(4941), pp.449–450. Majer, E., Daròs, J.-A. and Zwart, M.P., 2013. Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome. Viruses , 5(9), pp.2153–2168. Naing, A.H., Ai, T.N., Lim, K.B., Lee, I.J. and Kim, C.K., 2018. Overexpression of Rosea1 from snapdragon enhances anthocyanin accumulation and abiotic stress tolerance in transgenic tobacco. Frontiers in Plant Science , 9, 1070. Pourcel, L., Irani, N.G., Lu, Y., Riedl, K., Schwartz, S. and Grotewold, E., 2010. The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Molecular plant , 3 (1), pp.78–90. Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E., Oyama, R., Bailey, P., Davies, K. and Martin, C., 2006. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum . The Plant Cell , 18(4), pp.831–851. Shi, L., Li, X., Fu, Y. and Li, C., 2023. Environmental stimuli and phytohormones in anthocyanin biosynthesis: a comprehensive review. International Journal of Molecular Sciences , 24(22), 16415. Shi, M.-Z. and Xie, D.-Y., 2014. Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana . Recent Patents on Biotechnology , 8(1), pp.47–60. Skoog, F. and Miller, C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symposia of the Society for Experimental Biology , 11, pp.118–130. Somers, D.A., Samac, D.A. and Olhoft, P.M., 2003. Recent advances in legume transformation. Plant Physiology , 131(3), pp.892–899. Stewart, C.N., Jr., 2001. The utility of green fluorescent protein in transgenic plants. Plant Cell Reports , 20(5), pp.376–382. Sugimoto, K., Jiao, Y. and Meyerowitz, E.M., 2010. Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Developmental Cell , 18(3), pp.463–471. Tanaka, Y., Sasaki, N. and Ohmiya, A., 2008. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. The Plant Journal , 54(4), pp.733–749. Tanwir, S.E., Jiang, T., Creech, M.R., Kim, J., Cherukula, J.C., Wang, S. and Huo, H. (2026) Engineering betalain biosynthesis via RUBY enhances ornamental aesthetics but alters fertility in Solanum aethiopicum . Molecular Horticulture , Manuscript in revision. Tohge, T., Zhang, Y., Peterek, S., Matros, A., Rallapalli, G., Tandrón, Y.A., Butelli, E., Kallam, K., Hertkorn, N., Mock, H.-P., Martin, C. and Fernie, A.R., 2015. Ectopic expression of snapdragon transcription factors facilitates the identification of genes encoding enzymes of anthocyanin decoration in tomato. The Plant Journal , 83(4), pp.686–704. Valvekens, D., Van Montagu, M. and Van Lijsebettens, M., 1988. Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proceedings of the National Academy of Sciences of the United States of America , 85(15), pp.5536–5540. Vandenbussche, M., Zethof, J., Souer, E., Koes, R., Tornielli, G.B., Pezzotti, M., Ferrario, S., Angenent, G.C. and Gerats, T., 2003. Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia. The Plant Cell , 15 (11), pp.2680–2693. Winkel-Shirley, B., 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology , 126(2), pp.485–493. Zhang, T.-Q., Lian, H., Zhou, C.-M., Xu, L., Jiao, Y. and Wang, J.-W., 2017. A two-step model for de novo activation of WUSCHEL during plant shoot regeneration. The Plant Cell , 29(5), pp.1073–1087. Zhang, Y., Butelli, E. and Martin, C., 2013. Engineering anthocyanin biosynthesis in plants. Current Opinion in Plant Biology , 16(2), pp.218–223. Zhang, Y., Malzahn, A.A., Sretenovic, S. and Qi, Y., 2019. The emerging and uncultivated potential of CRISPR technology in plant science. Nature Plants , 5(8), pp.778–794. Zhen, X.H., Pan, R.R., Lu, X.H., Ge, Y.J., Li, R.M., Liu, J., Wang, Y.J., Yi, K.X., Li, C.X., Guo, J.C. and Yao, Y., 2024. An anthocyanin-based visual reporter system for genetic transformation and genome editing in cassava. International Journal of Molecular Sciences , 25 (21), p.11808. Zuo, J., Niu, Q.-W. and Chua, N.-H., 2000. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. The Plant Journal , 24(2), pp.265–273. Additional Declarations No competing interests reported. Supplementary Files SupplementaryinformationLegends.docx Supplementaryfile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9298244","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619575107,"identity":"bcf47a0f-b867-4f8c-914c-1d5ae2847cb3","order_by":0,"name":"Tao Jiang","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Jiang","suffix":""},{"id":619575109,"identity":"6f75bdaf-a208-4b78-905f-01a3ee69f9c0","order_by":1,"name":"Sameena Ejaz Tanwir","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Sameena","middleName":"Ejaz","lastName":"Tanwir","suffix":""},{"id":619575111,"identity":"155a5970-637d-4474-9aa6-ff29d787369b","order_by":2,"name":"Fangchen Liu","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Fangchen","middleName":"","lastName":"Liu","suffix":""},{"id":619575113,"identity":"aa78a115-dd53-4494-9f24-ceb336388e82","order_by":3,"name":"Wisnu Handoyo Ardi","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Wisnu","middleName":"Handoyo","lastName":"Ardi","suffix":""},{"id":619575115,"identity":"04f594c6-5aa6-4e3c-a779-2dc66a9d1c21","order_by":4,"name":"Yeyen Novitasari","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Yeyen","middleName":"","lastName":"Novitasari","suffix":""},{"id":619575117,"identity":"531c970d-deb4-4e3f-b266-d8878b58ed10","order_by":5,"name":"Sandy Zammar","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Sandy","middleName":"","lastName":"Zammar","suffix":""},{"id":619575120,"identity":"8925a9f4-5c02-4c8c-86ca-7068b08296e1","order_by":6,"name":"Fida Hussain","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Fida","middleName":"","lastName":"Hussain","suffix":""},{"id":619575121,"identity":"f1e0491d-4390-4d69-98eb-7d8ed7a6f40f","order_by":7,"name":"Heqiang Huo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAkElEQVRIiWNgGAWjYBACAwkg8YFBTgbEJl4L4wwGYx7StDDzkKTFXLrH8LFtmwEPA3vzNgmitFjOOWNsnAvSwnOsjDgtBjdyzKRz2/7wMEjkmJGgxRJki/wbUrQwgrRI8BCtJa3YsOecAQ8bT1qxBZFakjc++FFmIMfPfnjjDaK0wAEbacpHwSgYBaNgFOAFAK2JJCgPtvQoAAAAAElFTkSuQmCC","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"Heqiang","middleName":"","lastName":"Huo","suffix":""}],"badges":[],"createdAt":"2026-04-02 05:09:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9298244/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9298244/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106560484,"identity":"bbbfa75d-0e00-48eb-ab8f-267c2d36b72f","added_by":"auto","created_at":"2026-04-09 22:26:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROSEA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ereduces plant regeneration during transformation in tomato. a, \u003c/strong\u003eRepresentative phenotypes of tomato explants during Agrobacterium-mediated transformation under control (CK) and \u003cem\u003eROSEA1\u003c/em\u003e (ROSox) treatments. CK explants regenerated green shoots without visible anthocyanin accumulation, whereas ROSox explants developed purple anthocyanin pigmentation and exhibited reduced regeneration. Left and middle panels show close-up views at week 2 and week 4, respectively; right panels show representative Petri-dish overviews at week 4. \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, Regeneration frequency (%) (b) and shoots per explant (c) in CK (green) and ROSox (purple) treatments. Data are presented as mean ± s.d. (n = 4 biological replicates; 20 explants per replicate). Statistical significance was assessed using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-tests; \u003cem\u003eP\u003c/em\u003e-values are indicated. Scale bars, 5 mm (close-ups in a) and 1 cm (Petri dishes in a).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/af841c2a58bcd9213f6c2734.png"},{"id":106560486,"identity":"4f878c6c-d9fb-4ee6-ba70-b3d8eced2fc6","added_by":"auto","created_at":"2026-04-09 22:26:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1744880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROSEA1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpression reduces regeneration in stable transgenic tomato lines. a, \u003c/strong\u003eRepresentative regeneration phenotypes of stable transgenic tomato lines overexpressing \u003cem\u003eROSEA1\u003c/em\u003e(ROSox; anthocyanin-pigmented tissues) and control lines (CK) from week 1 to week 3 after regeneration induction. Scale bars, 1 cm. \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, Regeneration frequency (%) (b) and shoots per explant (c) in CK (green) and ROSox (purple) lines at week 2 and week 3. Data are presented as mean ± s.d. (n = 4 biological replicates; 20 explants per replicate). \u003cstrong\u003ed\u003c/strong\u003e, Representative anthocyanin extracts from CK and ROSox tissues collected at week 2 and week 3. \u003cstrong\u003ee\u003c/strong\u003e, Anthocyanin content (mg g⁻¹ FW) in CK and ROSox tissues at week 2 and week 3 (n = 4 biological replicates; three technical replicates per biological replicate). Statistical significance was assessed using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-tests; \u003cem\u003eP\u003c/em\u003e-values are indicated.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/8c4f7e00851d7fbb0d37a5d2.png"},{"id":106725144,"identity":"fd0de2ce-f52a-4a75-955d-b97cf54ac068","added_by":"auto","created_at":"2026-04-12 18:31:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1099763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROSEA1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eactivates anthocyanin-biosynthesis genes and represses regeneration-associated genes in tomato. a, \u003c/strong\u003eqRT–PCR analysis of \u003cem\u003eROSEA1\u003c/em\u003eexpression level in control (CK; green) and \u003cem\u003eROSEA1\u003c/em\u003e overexpression (ROSox; purple) tomato lines at week 2 and week 3. \u003cstrong\u003eb\u003c/strong\u003e, qRT–PCR analysis of regeneration-associated genes (for example, \u003cem\u003ePLT5\u003c/em\u003e, \u003cem\u003eWUS\u003c/em\u003e and \u003cem\u003eLBD16\u003c/em\u003e) in CK and ROSox lines at week 2 and week 3. \u003cstrong\u003ec\u003c/strong\u003e, qRT–PCR analysis of anthocyanin-biosynthesis genes (for example, \u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, \u003cem\u003eF3′5′H\u003c/em\u003eand \u003cem\u003eDFR\u003c/em\u003e) in CK and ROSox lines at week 2 and week 3. Expression values were normalized to the reference gene \u003cem\u003eACTIN2\u003c/em\u003e. Data are presented as mean ± s.d. (n = 3 biological replicates; three technical replicates per biological replicate). Statistical significance was assessed using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-tests; \u003cem\u003eP\u003c/em\u003e-values are indicated.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/39c5a38013edf05fff1657b1.png"},{"id":106725669,"identity":"be107846-1e86-4e72-b204-9b801917f3bf","added_by":"auto","created_at":"2026-04-12 18:33:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":324821,"visible":true,"origin":"","legend":"\u003cp\u003eCo-expression of \u003cem\u003ePLT5\u003c/em\u003e alleviates the regeneration defect of stable transgenic tomato lines overexpressing \u003cem\u003eROSEA1\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Representative regeneration phenotypes of stable transgenic tomato lines overexpressing \u003cem\u003eROSEA1\u003c/em\u003e alone (ROSox; anthocyanin-pigmented tissues) or co-expressing \u003cem\u003ePLT5\u003c/em\u003e and \u003cem\u003eROSEA1\u003c/em\u003e (P+ROSox) at week 2 and week 3 after regeneration induction. Scale bars, 1 cm. \u003cstrong\u003eb\u003c/strong\u003e, Representative anthocyanin extracts from ROSox and P+ROSox tissues. \u003cstrong\u003ec\u003c/strong\u003e, Anthocyanin content (mg g⁻¹ FW) in ROSox and P+ROSox tissues. \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, Shoots per explant (d) and regeneration frequency (%) (e) in ROSox and P+ROSox lines. Data are presented as mean ± s.d. (n = 4 biological replicates, 20 explants per replicate). Statistical significance was assessed using two-tailed Student’s t-tests; \u003cem\u003eP\u003c/em\u003e-values are indicated.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/afa1ae9475b2ad6a916d1617.png"},{"id":106727477,"identity":"4b77a928-83b4-48cd-bf54-88abd106c27d","added_by":"auto","created_at":"2026-04-12 18:39:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2082556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROSEA1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpression reduces plant regeneration in stable transgenic petunia lines.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003ea\u003c/strong\u003e, Time-course of regeneration from week 1 to week 4 in petunia explants for control (CK, top) and two independent \u003cem\u003eROSEA1\u003c/em\u003e overexpression lines (ROSox-1, middle; ROSox-2, bottom), showing reduced plantlet formation in ROSox. \u003cstrong\u003eb\u003c/strong\u003e, Representative close-up views of regenerating tissues from week 1 to week 4 for CK, ROSox-1, and ROSox-2. \u003cstrong\u003ec\u003c/strong\u003e, Explant area quantified from week 1 to week 3 (CK, green; ROSox-1, light purple; ROSox-2, dark purple). Data are presented as mean ± s.d. (n = 20 biological replicates). \u003cstrong\u003ed\u003c/strong\u003e, Regeneration frequency (%) at week 3 and week 4 in CK, ROSox-1 and ROSox-2 (n = 6 biological replicates; 20 explants per replicate). \u003cstrong\u003ee\u003c/strong\u003e, Regenerated shoots per explant at week 3 and week 4 in CK, ROSox-1, and ROSox-2 (n = 6 biological replicates; 20 explants per replicate). \u003cstrong\u003ef\u003c/strong\u003e, Representative anthocyanin extracts from CK, ROSox-1, and ROSox-2 tissues collected at week 4. \u003cstrong\u003eg\u003c/strong\u003e, Anthocyanin content (mg g⁻¹ FW) at week 4 in CK, ROSox-1, and ROSox-2 tissues (n = 4 biological replicates; three technical replicates per biological replicate). Statistical significance was assessed using ANOVA at each time point (among three lines). Scale bars, 1 cm (a) and 5 mm (b).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/506aab947b672a3ddc752d54.png"},{"id":106560491,"identity":"001a181e-a57f-40c2-ac72-81a367372479","added_by":"auto","created_at":"2026-04-09 22:26:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":493845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROSEA1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ereduces plant regeneration during transformation in begonia and marigold. a\u003c/strong\u003e, Representative close-up bright-field and fluorescence images of transforming begonia explants under control (CK, top) and \u003cem\u003eROSEA1\u003c/em\u003e (ROSox, bottom) treatments at weeks 3 and 6. \u003cstrong\u003eb\u003c/strong\u003e, Representative Petri-dish overviews at week 9 for begonia CK (top) and ROSox (bottom), showing reduced regeneration in ROSox; red arrows indicate regenerating explants. \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, Regeneration frequency (%) (c) and shoots per explant (d) in begonia CK (green) and ROSox (purple) treatments. Data are presented as mean ± s.d. (n = 4 biological replicates; 15 explants per replicate). \u003cstrong\u003ee\u003c/strong\u003e, Representative Petri dishes of marigold ‘Marvel II Yellow’ explants during \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation under CK and ROSox treatments on two callus-induction media: CIM1 (0.5 mg L⁻¹ NAA + 0.75 mg L⁻¹ BAP) and CIM2 (0.5 mg L⁻¹ NAA + 2.0 mg L⁻¹ BAP), showing reduced regeneration in ROSox. \u003cstrong\u003ef\u003c/strong\u003e, Representative bright-field and fluorescence images of regenerating marigold tissues from CK (top) and ROSox (bottom) explants, showing reporter fluorescence in the corresponding panels. \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, Regeneration frequency (%) (g) and shoots per explant (h) for marigold CK (green) and ROSox (purple) across CIM1 and CIM2 conditions. Data are presented as mean ± s.d. (n = 5 biological replicates; 15 explants per replicate). Statistical significance was assessed using two-tailed Student’s \u003cem\u003et\u003c/em\u003e-tests; \u003cem\u003eP\u003c/em\u003e-values are indicated. Scale bars, 5 mm (a, f) and 1 cm (b, e).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/6b0b5d6dccbfc400c700977f.png"},{"id":106959486,"identity":"edeb1217-866f-400f-aff7-15e7506a4fb2","added_by":"auto","created_at":"2026-04-15 09:10:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7108860,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/1a3ad82e-721d-4402-946b-0ba989ec37dc.pdf"},{"id":106725099,"identity":"2fa2ba20-08cc-4d94-a090-806c3c6744f3","added_by":"auto","created_at":"2026-04-12 18:31:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14099,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/89f51da9d6ccdb07aa03408e.docx"},{"id":106560489,"identity":"b59aa524-89a8-478d-9e43-7c58773035d6","added_by":"auto","created_at":"2026-04-09 22:26:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1834454,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9298244/v1/2a2775a881a5cfad8345b6e0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ROSEA1-based visual selection reduces plant regeneration and alters developmental regulator expression","fulltext":[{"header":"Key Message","content":"\u003cp\u003e\u003cem\u003eROSEA1\u003c/em\u003e-based visual selection is not developmentally neutral, reduces regeneration, and can be partly mitigated by hormone optimization or genotype selection.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eVisual reporters that produce pigmentation in plant cells provide a convenient means to identify transgenic events without destructive assays or specialized equipment. Among these, the R2R3-MYB transcription factor \u003cem\u003eROSEA1\u003c/em\u003e from \u003cem\u003eAntirrhinum majus\u003c/em\u003e has become a widely adopted marker owing to its ability to induce robust anthocyanin accumulation across diverse plant species (Butelli et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bedoya et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Unlike conventional reporters such as β-glucuronidase (GUS), which require destructive histochemical assays and overnight incubation (Jefferson et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), or green fluorescent protein (GFP), which necessitates fluorescence microscopy and UV illumination (Baulcombe et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Stewart, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), \u003cem\u003eROSEA1\u003c/em\u003e, together with related anthocyanin-inducing transcription factors, enables real-time, non-destructive tracking of transgenic tissues through distinctive purple coloration visible to the unaided eye. This class of visual marker has been incorporated into transformation vectors for applications ranging from tracking viral infections to visual selection in rubber tree and hairy root systems (Bedoya et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Majer et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite this widespread adoption, the effects of \u003cem\u003eROSEA1\u003c/em\u003e expression on plant regeneration capacity, a critical determinant of transformation success, remain poorly characterized.\u003c/p\u003e \u003cp\u003e \u003cem\u003eROSEA1\u003c/em\u003e, a member of the R2R3-MYB transcription factor family, activates anthocyanin biosynthesis by forming a regulatory complex with endogenous basic helix-loop-helix (bHLH) and WD40 proteins, collectively known as the MYB-bHLH-WD40 (MBW) complex (Schwinn et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Koes et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This highly conserved regulatory module coordinates the expression of structural genes throughout the flavonoid pathway, including chalcone synthase (\u003cem\u003eCHS\u003c/em\u003e), chalcone isomerase (\u003cem\u003eCHI\u003c/em\u003e), flavanone 3-hydroxylase (\u003cem\u003eF3H\u003c/em\u003e), dihydroflavonol 4-reductase (\u003cem\u003eDFR\u003c/em\u003e), and anthocyanidin synthase (\u003cem\u003eANS\u003c/em\u003e) (Holton and Cornish, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Tanaka et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The broad conservation of endogenous bHLH partners across angiosperms enables \u003cem\u003eROSEA1\u003c/em\u003e expression alone to induce visible pigmentation in most plant species (Naing et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Albert et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Landmark work demonstrated that co-expression of \u003cem\u003eROSEA1\u003c/em\u003e and its bHLH partner, \u003cem\u003eDELILA\u003c/em\u003e, produced intensely purple tomato fruits with anthocyanin levels comparable to those of blueberries, thereby establishing the metabolic engineering potential of these transcription factors (Butelli et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, constitutive activation of flavonoid biosynthesis may also impose developmental and metabolic consequences beyond pigmentation itself.\u003c/p\u003e \u003cp\u003eAnthocyanin production requires substantial investment of carbon skeletons derived from phenylalanine and malonyl-CoA, potentially diverting metabolic flux from primary pathways and other phenylpropanoid branches, including lignin biosynthesis and protein synthesis (Winkel-Shirley, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Gould, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pourcel et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The synthesis and vacuolar sequestration of these pigments involve multiple biosynthetic enzymes and downstream tailoring reactions, including multiple glycosylation steps, representing considerable cellular resource commitment (Shi and Xie, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Winkel-Shirley, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Trade-offs among anthocyanin accumulation, vegetative growth, reproductive output, and stress tolerance have been documented across diverse species (Chalker-Scott, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Gould, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In maize, ectopic/inducible activation of the anthocyanin regulators C1 and R is sufficient to activate flavonoid pathway genes and drive strong anthocyanin accumulation, highlighting the scale of transcriptional and metabolic reprogramming required for high pigment output. In contrast, anthocyanin-rich tomato fruits exhibit altered ripening dynamics (Zhang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These observations suggest that strong anthocyanin-promoting programs could interfere with the regenerative capacity of cultured explants, in which cellular resources must support both pigment biosynthesis and the energetically demanding process of shoot organogenesis.\u003c/p\u003e \u003cp\u003eThe potential impact of anthocyanin accumulation on regeneration is particularly significant, as regeneration capacity is the primary bottleneck limiting the efficiency of plant transformation in economically important species. Genetic transformation underpins modern plant biotechnology, enabling functional genomics, precision breeding, and CRISPR/Cas-based genome editing (Altpeter et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The success of any transformation pipeline depends on two sequential processes: delivery of foreign DNA into plant cells and subsequent regeneration of whole plants from transformed tissues (Altpeter et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee and Wang, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While DNA delivery methods have been substantially optimized, regeneration recalcitrance, the inability of plant tissues to form organized structures such as shoots or somatic embryos under in vitro conditions, whether during tissue culture or after genetic transformation, continues to restrict the scope of genetic engineering across legumes, cereals, woody species, and many elite cultivars (Somers et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Gordon-Kamm et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Birch, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Moreover, the genotype-specificity of regeneration responses means that protocols optimized for one cultivar frequently fail when applied to related genotypes, and genome-editing tools can be applied only to the narrow subset of genotypes amenable to regeneration (Bregitzer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Debernardi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDe novo\u003c/em\u003e shoot organogenesis proceeds through a highly coordinated developmental sequence governed by precisely coordinated hormonal and transcriptional programs. The foundational work of Skoog and Miller established that the balance between auxin and cytokinin determines cell fate: high cytokinin-to-auxin ratios promote shoot formation, while high auxin-to-cytokinin ratios favor root development (Skoog and Miller, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). This paradigm has guided the development of callus-induction media (CIM) and shoot-induction media (SIM), which are central to most transformation protocols (Valvekens et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). At the molecular level, key transcription factors orchestrate regeneration, including \u003cem\u003eWUSCHEL\u003c/em\u003e (\u003cem\u003eWUS\u003c/em\u003e), which serves as the master regulator of shoot apical meristem maintenance (Gallois et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gordon et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), the \u003cem\u003ePLETHORA\u003c/em\u003e genes (\u003cem\u003ePLT3\u003c/em\u003e, \u003cem\u003ePLT5\u003c/em\u003e, \u003cem\u003ePLT7\u003c/em\u003e) that promote pluripotency establishment (Kareem et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and \u003cem\u003eLATERAL ORGAN BOUNDARIES DOMAIN\u003c/em\u003e factors (\u003cem\u003eLBD16\u003c/em\u003e, \u003cem\u003eLBD29\u003c/em\u003e), which contribute to competence acquisition (Sugimoto et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Perturbations to the expression of these regulators are associated with impaired regeneration outcomes. Notably, several lines of evidence suggest potential mechanistic links between anthocyanin biosynthesis and regeneration pathways: cytokinin has been shown to stimulate both shoot organogenesis and anthocyanin accumulation (Deikman and Hammer, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), while the phenylpropanoid pathway shares metabolic precursors with auxin biosynthesis (Brown et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Consequently, constitutive \u003cem\u003eROSEA1\u003c/em\u003e expression may perturb regeneration through metabolic competition, hormonal crosstalk, or transcriptional interference with key developmental networks.\u003c/p\u003e \u003cp\u003eDespite the growing deployment of \u003cem\u003eROSEA1\u003c/em\u003e and related anthocyanin-inducing MYB factors as visible reporters in transformation systems, their effects on regeneration have not been systematically evaluated (Kortstee et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bedoya et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Fatihah et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Existing evidence suggests context-dependent outcomes. In cassava, anthocyanin induction via the R2R3-MYB \u003cem\u003eHbAN1\u003c/em\u003e did not significantly affect regeneration (Zhen et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In several dicots, anthocyanin-based markers such as MYB10 have been reported to be compatible with organogenesis during transformation, including in apple, strawberry, and potato (Kortstee et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). By comparison, maize anthocyanin regulators were first established as visible transformation markers in maize with the \u003cem\u003eLc\u003c/em\u003e gene (Ludwig et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), and maize \u003cem\u003eC1/B-peru\u003c/em\u003e regulators were later shown to induce pigmentation in transformed wheat tissues (Chawla et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), while related anthocyanin-based visible-marker applications have also been reported in sugarcane (Bower et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), although the developmental consequences for regeneration were not systematically compared across systems. However, no study has quantified the magnitude of any regeneration penalty across multiple species, profiled underlying transcriptional changes, or tested whether such effects are conserved across phylogenetically diverse lineages. This gap creates uncertainty when selecting visual markers and may contribute to suboptimal transformation outcomes, particularly in regeneration-sensitive genotypes. If such a penalty exists, defining its magnitude and mechanistic basis would enable mitigation strategies, such as hormone optimization, genotype selection, or developmental support, to retain the advantages of visual selection while minimizing regeneration deficits.\u003c/p\u003e \u003cp\u003eHere, we show that \u003cem\u003eROSEA1\u003c/em\u003e expression substantially impairs regeneration capacity across four plant species representing three eudicot families, both in stable transgenic lines and during plant transformation. These findings indicate that anthocyanin-based visual selection can impose a regeneration cost associated with disrupted developmental competence. We further identify hormone optimization, genotype selection, and developmental support as practical strategies to mitigate this trade-off.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003ePlant material and growth conditions\u003c/p\u003e \u003cp\u003eSeeds of tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e cv. 'Micro-Tom'), petunia (\u003cem\u003ePetunia hybrida\u003c/em\u003e cv. 'Mitchell'), and marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e cv. 'Marvel II Yellow' and \u003cem\u003eTagetes patula\u003c/em\u003e cv. 'Bonanza Yellow') were purchased from Ball Horticultural Company (West Chicago, IL, USA). \u003cem\u003eBegonia\u003c/em\u003e 'UF183-11', an advanced breeding line developed at the University of Florida, served as plant material. Seeds were surface-sterilized by immersion in 75% (v/v) ethanol for 1 min, followed by 15% (v/v) commercial bleach (6% sodium hypochlorite) for 10 min with gentle agitation, and then rinsed six times with sterile distilled water. Sterilized seeds were germinated on Murashige and Skoog (MS) basal medium supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar, pH adjusted to 5.8. Cultures were maintained at 25\u0026deg;C under a 16-h light/8-h dark photoperiod (100 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) in a controlled environment chamber. For begonia, mature leaves from greenhouse-grown stock plants were washed under running tap water for 30 min, then surface-sterilized in a laminar flow hood with 70% (v/v) ethanol for 1 min, followed by 1.5% (v/v) sodium hypochlorite for 10 min, and rinsed three to five times with sterile distilled water. For marigold, seeds were pretreated for 2 h in a solution containing Contrex AP detergent (4.0 mg L⁻\u0026sup1;) and Dithane M-45 fungicide (4 mg L⁻\u0026sup1;), rinsed with sterile distilled water, and then surface sterilized in a laminar flow hood with 20% (v/v) Clorox (7.5% sodium hypochlorite) plus two drops of Tween 20 for 20 min, followed by 10% (v/v) Clorox for 10 min. After five rinses with sterile distilled water, seeds were germinated on hormone-free MS medium, and cotyledons from 4- to 5-day-old seedlings were used as explants for regeneration experiments.\u003c/p\u003e \u003cp\u003eVector construction\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eROSEA1\u003c/em\u003e coding sequence from \u003cem\u003eAntirrhinum majus\u003c/em\u003e was cloned into the pOX135 binary vector backbone under the control of the CaMV 35S promoter for constitutive expression (Jiang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003ea). For transformation experiments comparing \u003cem\u003eROSEA1\u003c/em\u003e-expressing (ROSox) and control (CK) treatments, the empty vector lacking the \u003cem\u003eROSEA1\u003c/em\u003e insert served as the control. To generate stable overexpression lines (ROSox), the same 35S::\u003cem\u003eROSEA1\u003c/em\u003e construct was used. All constructs carried \u003cem\u003enptII\u003c/em\u003e and a \u003cem\u003eGFP\u003c/em\u003e fusion cassette driven by the CsVMV promoter, with \u003cem\u003enptII\u003c/em\u003e conferring kanamycin resistance. Construct integrity was verified by Sanger sequencing before transformation into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 harboring the binary vectors was cultured in 25 mL LB broth supplemented with spectinomycin (100 mg L⁻\u0026sup1;) and rifampicin (50 mg L⁻\u0026sup1;) at 28\u0026deg;C with shaking at 180 rpm overnight. A secondary culture was initiated by transferring 1 mL of the overnight culture into 10 mL of fresh LB broth containing the same antibiotics and growing it until the optical density at 600 nm (OD₆₀₀) reached 0.3\u0026ndash;0.5, except for marigold, for which cultures were grown to OD₆₀₀ = 0.6\u0026ndash;1.0. Bacterial cells were pelleted by centrifugation and resuspended in liquid MS medium supplemented with 100 \u0026micro;M acetosyringone for plant infection (Jiang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2026a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor tomato transformation, cotyledon explants excised from 7- to 10-day-old seedlings were immersed in the \u003cem\u003eAgrobacterium\u003c/em\u003e suspension for 10 min with gentle agitation. Inoculated explants were blotted on sterile filter paper and co-cultivated on MS medium (MS salts and vitamins, 30 g L⁻\u0026sup1; sucrose, 2.5 g L⁻\u0026sup1; Phytagel, pH 5.8) in the dark at 25\u0026deg;C for 2 days. Explants were then transferred to callus induction medium (CIM) consisting of MS medium supplemented with 2 mg L⁻\u0026sup1; zeatin, 0.2 mg L⁻\u0026sup1; NAA, 100 mg L⁻\u0026sup1; kanamycin, and 100 mg L⁻\u0026sup1; timentin. Cultures were maintained for 2\u0026ndash;5 weeks with subculturing onto fresh CIM every 2 weeks. Regenerating shoots were subsequently transferred to shoot induction medium (SIM) composed of MS medium with 1 mg L⁻\u0026sup1; zeatin, 100 mg L⁻\u0026sup1; kanamycin, and 100 mg L⁻\u0026sup1; timentin, followed by rooting on hormone-free MS medium containing 100 mg L⁻\u0026sup1; kanamycin and 100 mg L⁻\u0026sup1; timentin. Regenerated shoots exhibiting purple pigmentation, indicative of anthocyanin accumulation, were selected and transferred to a rooting medium. Rooted plantlets were acclimatized in a growth chamber and transferred to soil. T₀ transgenic plants were confirmed by PCR amplification of the \u003cem\u003eGFP\u003c/em\u003e transgene. Seeds from T₀ plants were germinated on kanamycin-containing medium to select T₁ progeny for subsequent regeneration assays.\u003c/p\u003e \u003cp\u003eFor petunia transformation, cotyledon explants from 7- to 10-day-old seedlings were inoculated as described above and co-cultivated on MS medium in the dark at 25\u0026deg;C for 2 days. Explants were then transferred to CIM comprising MS medium supplemented with 1 mg L⁻\u0026sup1; BAP, 0.1 mg L⁻\u0026sup1; NAA, 100 mg L⁻\u0026sup1; kanamycin, and 100 mg L⁻\u0026sup1; timentin. Because shoot regeneration occurred directly on CIM without a distinct induction phase, explants were maintained on the same medium for 2\u0026ndash;4 weeks with subculturing every 2 weeks until regenerated shoots were sufficiently developed for transfer to hormone-free MS medium containing 100 mg L⁻\u0026sup1; kanamycin and 100 mg L⁻\u0026sup1; timentin for elongation and rooting (Jiang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eBegonia\u003c/em\u003e transformation, leaf segments (approximately 0.5 \u0026times; 0.5 cm) from sterile \u003cem\u003ein vitro\u003c/em\u003e culture were inoculated with the \u003cem\u003eAgrobacterium\u003c/em\u003e suspension and co-cultivated on regeneration medium (REM), consisting of MS medium supplemented with 1.5 mg L⁻\u0026sup1; TDZ and 0.375 mg L⁻\u0026sup1; NAA, in the dark at 25\u0026deg;C for 2 days. The explants were then transferred to REM supplemented with 75 mg L⁻\u0026sup1; kanamycin and 100 mg L⁻\u0026sup1; timentin. Cultures were subcultured every 2 weeks. After 4\u0026ndash;9 weeks, regenerating explants were transferred to hormone-free MS medium with 75 mg L⁻\u0026sup1; kanamycin and 100 mg L⁻\u0026sup1; timentin to promote shoot elongation and rooting (Jiang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2026b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor marigold transformation, cotyledon explants from 4- to 5-day-old seedlings were vacuum-infiltrated with the \u003cem\u003eAgrobacterium\u003c/em\u003e suspension for 5\u0026ndash;10 min to enhance T-DNA delivery, then co-cultivated on MS medium in the dark at 25\u0026deg;C for 2 days. To evaluate the effect of cytokinin concentration on regeneration under \u003cem\u003eROSEA1\u003c/em\u003e expression, three CIM formulations differing only in BAP level were tested: CIM1 (MS medium\u0026thinsp;+\u0026thinsp;0.5 mg L⁻\u0026sup1; NAA\u0026thinsp;+\u0026thinsp;0.75 mg L⁻\u0026sup1; BAP), CIM2 (MS medium\u0026thinsp;+\u0026thinsp;0.5 mg L⁻\u0026sup1; NAA\u0026thinsp;+\u0026thinsp;2.0 mg L⁻\u0026sup1; BAP), and CIM3 (MS medium\u0026thinsp;+\u0026thinsp;0.5 mg L⁻\u0026sup1; NAA\u0026thinsp;+\u0026thinsp;2.5 mg L⁻\u0026sup1; BAP), all supplemented with 100 mg L⁻\u0026sup1; kanamycin and 100 mg L⁻\u0026sup1; timentin. After callus formation, explants from each CIM treatment were transferred to a corresponding shoot induction medium (SIM: MS\u0026thinsp;+\u0026thinsp;NAA 0.1 mg L⁻\u0026sup1; + BAP 1 mg L⁻\u0026sup1;) at the respective BAP concentration to maintain hormonal continuity during regeneration. Regenerated shoots were subsequently transferred to hormone-free MS medium containing 200 mg L⁻\u0026sup1; kanamycin and 100 mg L⁻\u0026sup1; timentin for elongation and rooting. Unless stated otherwise, all basal media consisted of MS salts and vitamins, 30 g L⁻\u0026sup1; sucrose, and 7 g L⁻\u0026sup1; agar (pH 5.8).\u003c/p\u003e \u003cp\u003eRegeneration assays\u003c/p\u003e \u003cp\u003eTwo categories of regeneration assay were conducted throughout this study: transformation-stage assays, in which freshly inoculated explants were tracked during \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation, and stable-line assays, in which explants derived from confirmed ROSox transgenic plants were compared with non-transgenic wild-type controls (CK) under identical culture conditions. Because all quantitative comparisons of regeneration capacity reported in this work originate from these assays, the scoring criteria and experimental design are described in detail below.\u003c/p\u003e \u003cp\u003eIn transformation-stage assays, explants were cultured on the species-specific CIM formulations described above. Regeneration was scored at discrete time points rather than across continuous intervals: at weeks 2, 3, and 4 for tomato; at weeks 1, 2, and 3 for petunia; at weeks 3, 5, 7, and 9 for begonia; and at weeks 2, 4, 6, and 8 for marigold. In stable-line assays, leaf or cotyledon explants excised from greenhouse-grown ROSox and CK plants were placed on the corresponding regeneration medium and cultured under the same light, temperature, and subculture regime used for transformation-stage experiments (Jiang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2026b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegeneration capacity was evaluated using two complementary metrics. Regeneration frequency was defined as the percentage of explants that produced at least one visible shoot\u0026thinsp;\u0026ge;\u0026thinsp;5 mm in length by a given scoring date. Shoots per explant were calculated as the total number of shoots (\u0026ge;\u0026thinsp;5 mm) divided by the total number of explants in that treatment. In addition, explant area was measured from calibrated overhead photographs at each time point using ImageJ (NIH, Bethesda, MD) to provide a quantitative record of tissue expansion independent of shoot formation.\u003c/p\u003e \u003cp\u003eEach treatment included a minimum of 15 explants per biological replicate, and at least three independent biological replicates were performed for every experiment unless otherwise noted. The exact number of explants (n) and replicates for each dataset is reported in the corresponding figure legends.\u003c/p\u003e \u003cp\u003eAnthocyanin extraction and quantification\u003c/p\u003e \u003cp\u003eAnthocyanins were extracted following the acidified methanol protocol with modifications. Fresh tissue (50\u0026ndash;100 mg) from regenerating explants or callus was weighed, flash-frozen in liquid nitrogen, and homogenized to a fine powder. Samples were extracted in 300 \u0026micro;L of acidified methanol [1% (v/v) HCl in methanol] by vortexing and incubating overnight at room temperature in the dark. Following extraction, 200 \u0026micro;L of Milli-Q water and 500 \u0026micro;L of chloroform were added to facilitate phase separation. Samples were vortexed vigorously and centrifuged (12,000 \u0026times; g, 5 min, 4\u0026deg;C). A 350-\u0026micro;L aliquot of the upper aqueous-methanolic phase was transferred to a fresh tube and diluted to a final volume of 1.05 mL with 415.8 \u0026micro;L methanol, 4.2 \u0026micro;L concentrated HCl, and 280 \u0026micro;L Milli-Q water. Absorbance was measured at 530 nm (anthocyanin peak) and 657 nm (chlorophyll interference) using a UV-visible spectrophotometer (NanoDrop 2000; Bio Tek). Corrected anthocyanin absorbance was calculated as Acorr\u0026thinsp;=\u0026thinsp;A₅₃₀ \u0026minus; 0.25 \u0026times; A₆₅₇. Anthocyanin content was expressed as milligrams of cyanidin-3-glucoside equivalents per gram fresh weight (mg g⁻\u0026sup1; FW) using the molar extinction coefficient ε\u0026thinsp;=\u0026thinsp;33,000 l mol⁻\u0026sup1; cm⁻\u0026sup1; and molecular weight 449.2 g mol⁻\u0026sup1;. Blank extractions processed without tissue were included to correct for background absorbance.\u003c/p\u003e \u003cp\u003eRNA isolation and quantitative real-time PCR\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from 80\u0026ndash;100 mg of flash-frozen regenerating tissue using RNAzol RT reagent (Molecular Research Center) following the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer; samples with A₂₆₀/A₂₈₀ ratios between 1.9 and 2.1 were used for cDNA synthesis. Genomic DNA contamination was eliminated by on-column DNase I digestion using the RNase-Free DNase Set (QIAGEN). First-strand cDNA was synthesized from 1 \u0026micro;g of DNase-treated RNA using the QuantiTect Reverse Transcription Kit (QIAGEN). The resulting cDNA was diluted 20- to 25-fold for use as qPCR template.\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR (qRT-PCR) was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). Each 10-\u0026micro;L reaction contained 5 \u0026micro;L of 2\u0026times; SYBR Green master mix, 4 \u0026micro;L of diluted cDNA template, and 0.5 \u0026micro;L of a 10 \u0026micro;M forward and reverse primer mixture. Gene-specific primers were designed using Primer-BLAST (NCBI) to span exon-exon junctions where possible and to generate amplicons of 100\u0026ndash;150 bp with melting temperatures of 58\u0026ndash;62\u0026deg;C. Target genes included \u003cem\u003eROSEA1\u003c/em\u003e (transgene expression), anthocyanin biosynthetic genes (\u003cem\u003eCHI\u003c/em\u003e, chalcone isomerase; \u003cem\u003eF3H\u003c/em\u003e, flavanone 3-hydroxylase; \u003cem\u003eF3\u003c/em\u003e'\u003cem\u003e5\u003c/em\u003e'\u003cem\u003eH\u003c/em\u003e, flavonoid 3\u0026prime;,5\u0026prime;-hydroxylase; \u003cem\u003eDFR\u003c/em\u003e, dihydroflavonol 4-reductase), and regeneration-associated transcription factors (\u003cem\u003ePLT5\u003c/em\u003e, PLETHORA5; \u003cem\u003eWUS\u003c/em\u003e, WUSCHEL; \u003cem\u003eLBD16\u003c/em\u003e, LATERAL ORGAN BOUNDARIES DOMAIN 16). These regeneration-associated regulators were selected based on their established roles in pluripotency acquisition and shoot meristem identity during de novo organogenesis. Primer sequences are provided in Supplementary Table\u0026nbsp;1. Thermal cycling conditions consisted of initial denaturation at 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s. Melt curve analysis was performed to verify primer specificity. Relative gene expression was calculated using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method with \u003cem\u003eACTIN2\u003c/em\u003e as the internal reference gene. Three biological replicates, each comprising three technical replicates, were analyzed per genotype and time point.\u003c/p\u003e \u003cp\u003eImaging and microscopy\u003c/p\u003e \u003cp\u003eMacroscopic images of regenerating explants and culture plates were captured using a digital camera under standardized lighting conditions. To confirm transgene expression and T-DNA integration, explants were visualized using a fluorescence stereomicroscope equipped with appropriate filter sets. Purple pigmentation indicative of anthocyanin accumulation was documented at each time point. Anthocyanin extracts were also photographed to illustrate differences in color intensity between genotypes.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data visualization and statistical analyses were performed using GraphPad Prism 10 (version 10.4.2). Biochemical and gene expression analyses were conducted using at least three independent biological replicates, with three technical replicates per biological sample for qRT-PCR. Morphological traits were quantified from a minimum of ten biological replicates per genotype. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.d. Statistical significance was assessed using two-tailed Student's t-tests for pairwise comparisons; \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Exact P-values and sample sizes are indicated in the corresponding figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eROSEA1\u003c/b\u003e \u003cb\u003eexpression compromises regeneration during tomato transformation and in stable transgenic lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe R2R3-MYB transcription factor \u003cem\u003eROSEA1\u003c/em\u003e from \u003cem\u003eAntirrhinum majus\u003c/em\u003e has been widely adopted as a visual reporter for plant transformation due to its ability to induce anthocyanin accumulation, resulting in a distinctive purple pigmentation that facilitates the identification of transgenic events (Butelli et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Schwinn et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). To determine whether \u003cem\u003eROSEA1\u003c/em\u003e-based visual selection influences regeneration during the transformation process itself, tomato explants were subjected to \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation with a \u003cem\u003eROSEA1\u003c/em\u003e-containing construct and compared with empty vector controls transformed in parallel under identical culture conditions. ROSox tissues developed purple pigmentation in regenerating regions, visible as discrete purple foci emerging from the wound sites, whereas control (CK) explants remained green throughout the culture period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This contrast confirmed strong \u003cem\u003eROSEA1\u003c/em\u003e activity and validated its utility as a visual selection marker. However, ROSox explants produced fewer visible regenerative structures than CK explants, as evidenced by both magnified views and plate-level observations. The proportion of regenerating explants was reduced by 44.4% in ROSox compared with CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), indicating a substantial decline in regeneration initiation. Shoot yield was similarly affected, with shoots per explant reduced by 55.5% in ROSox (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Together, these results demonstrate that the visual marker function of \u003cem\u003eROSEA1\u003c/em\u003e is accompanied by a measurable reduction in regeneration efficiency during transformation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the regeneration deficit persisted beyond the transformation stage and reflected a stable phenotype associated with constitutive \u003cem\u003eROSEA1\u003c/em\u003e expression, stable transgenic tomato lines overexpressing \u003cem\u003eROSEA1\u003c/em\u003e (ROSox) were generated and subjected to regeneration assays. ROSox lines exhibited sustained purple pigmentation throughout three weeks of culture on regeneration medium, with coloration intensifying over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Shoot emergence in ROSox lagged behind CK from the earliest time point examined, and this difference widened from week 1 to week 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Although the pigment signal remained robust in stable lines, it was associated with reduced developmental output rather than serving solely as a neutral marker of transgenic tissue. Quantification analysis of regeneration parameters revealed substantial differences between genotypes. Regeneration frequency at week 2 was reduced by 60.0% in ROSox relative to CK, representing the maximum divergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). By week 3, regeneration frequency in ROSox had partially recovered but remained 43.5% lower than CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), indicating a sustained penalty despite overall progression of regeneration. Shoot production per explant was significantly reduced, declining by 52.4% at week 2 and by 42.6% at week 3 in ROSox relative to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To confirm that the pigmentation phenotype reflected elevated anthocyanin biosynthesis, pigments were extracted from regenerated tissues and quantified. Anthocyanin extracts from ROSox were intensely colored compared with the pale solutions obtained from CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Spectrophotometric quantification confirmed that anthocyanin levels were 20.7-fold and 7.6-fold higher in ROSox than CK at weeks 2 and 3, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test whether the regeneration penalty was specific to \u003cem\u003eROSEA1\u003c/em\u003e or a general consequence of anthocyanin pathway activation, stable transgenic tomato lines overexpressing the bHLH transcription factor DELILA (DELox) were evaluated in parallel (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, no regeneration penalty was observed in DELox lines (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). At week 3, DELox tissues accumulated 73.7% more anthocyanin than CK (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), confirming that \u003cem\u003eDELILA\u003c/em\u003e also enhanced anthocyanin biosynthesis, albeit to a lower level than \u003cem\u003eROSEA1\u003c/em\u003e. In contrast to ROSox, however, DELox did not exhibit reduced regenerative capacity; shoot production was 13.3% higher than in CK at the same stage, although this difference was not statistically significant (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). These results indicate that anthocyanin accumulation per se is insufficient to impair regeneration, and that the inhibitory effect observed in ROSox likely depends on the specific regulatory context and/or the magnitude of pathway activation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eROSEA1\u003c/b\u003e \u003cb\u003erewires gene expression during regeneration, coupling activation of anthocyanin synthesis with suppression of regeneration-associated regulators\u003c/b\u003e\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR (qRT-PCR) was performed on ROSox and CK tissues harvested at weeks 2 and 3 to characterize the transcriptional changes underlying the regeneration deficit. These time points were selected because week 2 corresponded to the period of maximum divergence in regeneration frequency between genotypes, whereas week 3 captured the partial recovery phase. \u003cem\u003eROSEA1\u003c/em\u003e transcript abundance in ROSox was readily detected at both time points and remained substantially higher than in CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), indicating sustained transgene expression throughout the culture period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnthocyanin pathway genes were coordinately activated in ROSox tissues. \u003cem\u003eCHI\u003c/em\u003e (chalcone isomerase), which catalyzes an early step in flavonoid biosynthesis, increased 117.5-fold at week 2 and 108.9-fold at week 3 relative to CK. \u003cem\u003eDFR\u003c/em\u003e (dihydroflavonol 4-reductase), which directs the metabolic flux toward anthocyanin production rather than alternative flavonoid branches, increased 9.1-fold at week 2 and 23.8-fold at week 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). \u003cem\u003eF3H\u003c/em\u003e (flavanone 3-hydroxylase) exhibited a more gradual response, increasing 1.8-fold at week 2 and 5.6-fold at week 3. \u003cem\u003eF3\u0026prime;5\u0026prime;H\u003c/em\u003e (HF1), encoding a cytochrome P450 flavonoid 3\u0026prime;,5\u0026prime;-hydroxylase typically regulated downstream of MYB-bHLH-WD40 complexes controlling late anthocyanin biosynthesis, increased 75.3-fold at week 2 and 39.2-fold at week 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The induction of multiple genes spanning both early and late steps of the pathway, rather than a single gene, is consistent with the role of \u003cem\u003eROSEA1\u003c/em\u003e as a transcriptional activator of the anthocyanin biosynthetic module (Schwinn et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the reduced regeneration capacity observed in ROSox lines, the expression of genes encoding key regulators of shoot regeneration was examined. \u003cem\u003ePLT5\u003c/em\u003e (PLETHORA5), \u003cem\u003eWUS\u003c/em\u003e (WUSCHEL), and \u003cem\u003eLBD16\u003c/em\u003e (LATERAL ORGAN BOUNDARIES DOMAIN 16) were selected for analysis because these transcription factors play central roles in establishing pluripotency and shoot meristem identity during de novo organogenesis (Kareem et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sugimoto et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003ePLT5\u003c/em\u003e promotes the acquisition of regenerative competence by facilitating the establishment of pluripotency during the early phase of callus formation (Kareem et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eWUS\u003c/em\u003e functions as master regulator of shoot apical meristem identity and is required for de novo shoot formation (Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eLBD16\u003c/em\u003e operates within the auxin-mediated lateral organ development pathway and contributes to the acquisition of cellular competence preceding regeneration (Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast to anthocyanin genes, regeneration-associated regulators were markedly repressed at the early time point. At week 2, \u003cem\u003ePLT5\u003c/em\u003e expression was reduced by 89.3%, \u003cem\u003eWUS\u003c/em\u003e by 71.1%, and \u003cem\u003eLBD16\u003c/em\u003e by 82.8% relative to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This repression coincided with the stage at which developmental competence is normally established during the regeneration process (Kareem et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By week 3, \u003cem\u003ePLT5\u003c/em\u003e rebounded to 2.4-fold above CK levels, \u003cem\u003eWUS\u003c/em\u003e to 1.4-fold above, whereas \u003cem\u003eLBD16\u003c/em\u003e remained 16.0% lower than CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This temporal pattern, characterized by early suppression followed by partial recovery, mirrored the regeneration phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), with maximal transcriptional repression at week 2 corresponding to the greatest reduction in regeneration capacity. Sustained \u003cem\u003eROSEA1\u003c/em\u003e activity was associated with persistent anthocyanin pathway induction and a dynamic perturbation of developmental regulators, characterized by early-stage suppression during the critical window of competence establishment.\u003c/p\u003e \u003cp\u003eTo test whether reinforcement of the regeneration program could counteract the inhibitory effect of \u003cem\u003eROSEA1\u003c/em\u003e, stable tomato lines co-expressing \u003cem\u003ePLT5\u003c/em\u003e and \u003cem\u003eROSEA1\u003c/em\u003e (P+ROSox) were evaluated alongside ROSox lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Relative to ROSox, P+ROSox lines showed a 73.7% increase in shoots per explant and a 41.9% increase in regeneration frequency, indicating that \u003cem\u003ePLT5\u003c/em\u003e co-expression substantially alleviated the regeneration defect associated with \u003cem\u003eROSEA1\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). In contrast, anthocyanin accumulation in P+ROSox tissues was reduced by 18.8% relative to ROSox (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c), demonstrating that restoration of regeneration was achieved with only a modest reduction in pigment output. These results indicate that regeneration inhibition in ROSox is not solely due to anthocyanin accumulation but is more likely driven by the suppression of regeneration-promoting developmental pathways. Importantly, this effect can be at least partially mitigated through \u003cem\u003ePLT5\u003c/em\u003e-mediated reinforcement of the regeneration program.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe regeneration penalty associated with\u003c/b\u003e \u003cb\u003eROSEA1\u003c/b\u003e \u003cb\u003eis conserved across diverse plant species\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test whether the regeneration deficit observed in tomato reflects a broader consequence of \u003cem\u003eROSEA1\u003c/em\u003e expression, stable petunia lines overexpressing \u003cem\u003eROSEA1\u003c/em\u003e (ROSox) were examined. In an initial screening experiment, a ROSox line (later designated ROSox-2) accumulated visible purple pigmentation throughout culture, whereas control (CK) explants remained green (Fig. S2a, b). Plate-level observations revealed that ROSox-2 cultures consistently produced fewer regenerants and exhibited delayed shoot progression relative to CK over a 1- to 3-week culture window (Fig. S2a, b). Quantification of explant expansion supported this macroscopic phenotype: explant area in ROSox-2 lagged behind CK at all time points, with the deficit increasing over time, being reduced by 18.3% at week 1, 32.2% at week 2, and 41.6% at week 3 relative to CK (Fig. S2c). Regeneration frequency was reduced by 47.2% at week 2 and remained 40.0% lower at week 3 (Fig. S2d). Anthocyanin extracts from week-3 tissues were visibly pink in ROSox-2 but colorless in CK, with pigment levels 21.0-fold higher than CK (Fig. S2e, f).\u003c/p\u003e \u003cp\u003eMotivated by this reproducible phenotype, we next tested whether the regeneration penalty scales with \u003cem\u003eROSEA1\u003c/em\u003e-driven pigment output by comparing two independent ROSox lines with distinct anthocyanin intensities (ROSox-1, moderate; ROSox-2, strong) in a time-course assay extending to week 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Both lines maintained purple pigmentation throughout culture and produced fewer regenerants with delayed shoot progression at the plate level relative to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Quantification of explant expansion confirmed that reduced tissue growth emerged early and persisted: compared with CK, mean explant area was reduced by 27.6% in ROSox-1 and 29.2% in ROSox-2 at week 1, by 46.2% in ROSox-1 and 42.1% in ROSox-2 at week 2, and by 42.8% in ROSox-1 and 47.1% in ROSox-2 at week 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This growth deficit was accompanied by impaired regeneration performance: at week 3, mean regeneration frequency was reduced by 51.0% in ROSox-1 and 59.8% in ROSox-2 relative to CK; by week 4, ROSox-1 remained 29.3% lower than CK, whereas ROSox-2 remained 64.1% lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Likewise, regenerated shoots per explant at week 4 were reduced by 63.2% in ROSox-2, with a milder effect in ROSox-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Biochemical analysis confirmed the intended pigment gradient: anthocyanin content at week 4 was 4.3-fold higher in ROSox-1 and 21.7-fold higher in ROSox-2 relative to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, g). These results demonstrate that, as in tomato, strong \u003cem\u003eROSEA1\u003c/em\u003e-driven anthocyanin accumulation in petunia is associated with reduced regeneration capacity and tissue expansion, and the severity of the penalty scales with pigment output across independent transgenic lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBegonia, phylogenetically distant from \u003cem\u003eSolanaceae\u003c/em\u003e and representing the order Cucurbitales, was included to broaden the taxonomic scope of the analysis and test whether the \u003cem\u003eROSEA1\u003c/em\u003e-associated penalty extends beyond the Solanaceae family. \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation introduced \u003cem\u003eROSEA1\u003c/em\u003e into begonia leaf explants, and the success of transformation was confirmed by co-expression of a fluorescent reporter. Fluorescence signals and developing structures were observed in ROSox explants at weeks 3 and 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), confirming successful T-DNA integration and ruling out failed transformation as an explanation for any subsequent differences in regeneration. At week 9, when regeneration scoring was performed, CK cultures had produced visibly more regenerants than ROSox cultures at the plate level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Quantification supported this observation: regeneration frequency was reduced by 57.4% in ROSox relative to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), while shoots per explant were reduced by 70.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The \u003cem\u003eROSEA1\u003c/em\u003e-associated penalty thus extends beyond Solanaceae into a phylogenetically distinct eudicot clade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate context dependence, marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e), a member of the \u003cem\u003eAsteraceae\u003c/em\u003e family, was examined under varying cytokinin regimes. Cytokinins are key determinants of regeneration efficiency in many species, and manipulating hormone levels represents a potential strategy to mitigate regeneration deficits (Lowe et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of marigold 'Marvel II Yellow' was carried out under two callus-induction media differing in cytokinin content: CIM1 (0.5 mg L⁻\u0026sup1; NAA\u0026thinsp;+\u0026thinsp;0.75 mg L⁻\u0026sup1; BAP), representing a lower cytokinin condition, and CIM2 (0.5 mg L⁻\u0026sup1; NAA\u0026thinsp;+\u0026thinsp;2.0 mg L⁻\u0026sup1; BAP), representing an intermediate cytokinin condition. ROSox explants exhibited reduced regenerative progression relative to CK on both formulations at the plate level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Reporter fluorescence in regenerating tissues verified that these outcomes arose from successfully transformed cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). On CIM1, regeneration frequency was reduced by 33.1% in ROSox relative to CK, whereas on CIM2, regeneration frequency was reduced by 30.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Shoot output followed a similar trend, decreasing by 35.2% on CIM1 and by 16.2% on CIM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). Across both hormone regimes, \u003cem\u003eROSEA1\u003c/em\u003e expression was associated with reduced regeneration, although the apparent penalty was less pronounced under higher cytokinin conditions.\u003c/p\u003e \u003cp\u003eThe attenuation of the regeneration penalty at elevated cytokinin levels prompted us to ask whether further increases could fully offset the \u003cem\u003eROSEA1\u003c/em\u003e effect. To test this, \u0026lsquo;Marvel II Yellow\u0026rsquo; explants were cultured on CIM3 (0.5 mg L⁻\u0026sup1; NAA\u0026thinsp;+\u0026thinsp;2.5 mg L⁻\u0026sup1; BAP), which supported substantially higher baseline regeneration than CIM1 or CIM2. Under CIM3 conditions, CK and ROSox cultures appeared macroscopically similar throughout the culture period (Fig. S3a). No significant differences were detected between treatments for either regeneration frequency (Fig. S3b) or shoots per explant (Fig. S3c). Collectively, these results suggest that the \u003cem\u003eROSEA1\u003c/em\u003e-associated regeneration penalty becomes progressively less apparent with increasing cytokinin and is not detectable under strongly cytokinin-dominant conditions.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eROSEA1\u003c/em\u003e-associated penalty also appeared to vary with genotype. In \u0026lsquo;Bonanza Yellow\u0026rsquo; cultured on CIM1, baseline regeneration was low for both CK and ROSox treatments, and plate phenotypes appeared similar (Fig. S3d). Neither regeneration frequency nor shoot output differed significantly between treatments (Fig. S3e,f); ROSox values for regeneration showed a slight, non-significant increase relative to CK (Fig. S3e,f). The inherently low regeneration capacity of this genotype may have masked additional \u003cem\u003eROSEA1\u003c/em\u003e-associated effects, as floor effects can obscure modest reductions in already low-performing systems. Together, these results suggest that the impact of \u003cem\u003eROSEA1\u003c/em\u003e on regeneration in marigold depends on both hormone regime and genotype-specific regeneration capacity and further indicate that mitigation strategies based on cytokinin optimization and genotype selection could influence transformation outcomes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVisual reporters that enable non-destructive identification of transgenic events are valuable tools in plant transformation; however, their developmental neutrality has rarely been systematically evaluated. In this study, constitutive \u003cem\u003eROSEA1\u003c/em\u003e expression was consistently associated with reduced regeneration across multiple eudicot species, although the magnitude of this effect varied with species, genotype, and hormone regime (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In tomato, this phenotype coincided with activation of anthocyanin biosynthetic genes and reduced expression of key regeneration-associated regulators during an early regeneration window (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), providing a mechanistic basis for the observed trade-off. Together, these findings demonstrate that \u003cem\u003eROSEA1\u003c/em\u003e-based visual selection is not inherently developmentally neutral and that marker systems capable of reprogramming secondary metabolism may significantly influence developmental plasticity during tissue culture.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMechanistic basis of the\u003c/b\u003e \u003cb\u003eROSEA1\u003c/b\u003e\u003cb\u003e\u0026ndash;regeneration trade-off\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe data presented here argue against a simple model in which anthocyanin accumulation alone directly suppresses regeneration. This distinction is most clearly illustrated by the \u003cem\u003eDELILA\u003c/em\u003e comparison: although DELox tomato lines accumulated 73.7% more anthocyanin than the control at week 3, they did not exhibit reduced regenerative capacity (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). By contrast, ROSox lines showed substantially greater anthocyanin induction, reaching 20.7-fold above the control at week 2 and 7.6-fold at week 3, accompanied by a marked reduction in regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A similar dosage-dependent pattern was observed in petunia, where the more intensely pigmented ROSox-2 line exhibited a stronger regeneration defect than the moderately pigmented ROSox-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Together, these comparisons indicate that the severity of the phenotype correlates more closely with the extent of \u003cem\u003eROSEA1\u003c/em\u003e-driven pathway activation than with pigment accumulation per se. This interpretation is consistent with the biology of \u003cem\u003eROSEA1\u003c/em\u003e in \u003cem\u003eAntirrhinum\u003c/em\u003e, where it functions as an R2R3-MYB regulator of floral pigmentation intensity and patterning rather than as a canonical developmental regulator (Schwinn et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and with previous reports that ectopic \u003cem\u003eROSEA1\u003c/em\u003e expression can alter cellular programs beyond pigmentation, including abiotic stress responses (Naing et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA key line of support comes from the transcriptional data. During the competence acquisition phase, \u003cem\u003eROSEA1\u003c/em\u003e overexpression was associated with reduced expression of \u003cem\u003ePLT5, WUS\u003c/em\u003e, and \u003cem\u003eLBD16\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), coinciding with the developmental stage at which the divergence in regeneration between ROSox and the control was maximal (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This temporal overlap is notable because these genes occupy central positions in regeneration: PLT factors promote the acquisition of pluripotency (Kareem et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), WUS is required for shoot formation (Gallois et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and LBD16 contributes to the establishment of organogenic competence upstream of WUS (Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Consistent with this framework, lines co-expressing \u003cem\u003ePLT5\u003c/em\u003e and \u003cem\u003eROSEA1\u003c/em\u003e (P+ROSox) exhibited a 73.7% increase in shoots per explant and a 41.9% increase in regeneration frequency relative to ROSox, whereas anthocyanin levels declined by only 18.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The disproportionate recovery of regeneration relative to pigment reduction supports the conclusion that the \u003cem\u003eROSEA1\u003c/em\u003e-associated penalty is not driven solely by anthocyanin accumulation but instead reflects suppression of regeneration-promoting developmental pathways that can be partially restored by PLT5. Importantly, these data do not indicate that \u003cem\u003eROSEA1\u003c/em\u003e itself directly regulates regeneration; rather, ectopic \u003cem\u003eROSEA1\u003c/em\u003e activity likely perturbs the transcriptional environment required for shoot induction, thereby indirectly suppressing the regeneration program.\u003c/p\u003e \u003cp\u003eOne plausible mechanistic layer underlying this effect is metabolic burden. Anthocyanin biosynthesis requires a substantial investment of phenylalanine-derived carbon skeletons and malonyl-CoA, potentially diverting metabolic flux from pathways that support cell proliferation and differentiation (Gould, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Anthocyanin accumulation is closely linked to growth, energy allocation, and environmental responsiveness rather than representing a metabolically neutral output (Shi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and engineered high-anthocyanin tomato genotypes have been reported to show reduced vegetative growth and yield (Cerqueira et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A similar principle may extend beyond anthocyanin-based systems. In ornamental eggplant, constitutive overexpression of the betalain marker cassette \u003cem\u003eRUBY\u003c/em\u003e caused profound developmental alterations, including complete reproductive sterility due to defects in both male and female organs (Tanwir et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2026\u003c/span\u003e, in revision). Although that system involves betalain rather than anthocyanin biosynthesis, it similarly demonstrates that strong constitutive activation of visible pigment pathways may not be developmentally neutral and can impose substantial physiological trade-offs. These studies focused on whole-plant growth rather than regeneration-stage tissues, but they are broadly consistent with our observation that the regeneration penalty became most apparent when anthocyanin induction was especially pronounced (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, S1), compatible with a threshold-like effect. However, the partial rescue observed in P+ROSox lines, in which regeneration improved markedly despite only modest reductions in pigment output (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggests that metabolic cost alone may not fully account for the phenotype.\u003c/p\u003e \u003cp\u003eA complementary explanation is broader regulatory pleiotropy. As a transcription factor, \u003cem\u003eROSEA1\u003c/em\u003e may influence pathways beyond anthocyanin biosynthesis. Indeed, transcriptomic and metabolomic studies of tomato expressing snapdragon anthocyanin regulators have revealed widespread reprogramming across flavonoid and phenylpropanoid networks rather than a confined increase in pigment accumulation (Tohge et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The coordinate suppression of \u003cem\u003ePLT5, WUS\u003c/em\u003e, and \u003cem\u003eLBD16\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) may therefore reflect either a shared upstream perturbation or parallel interference across multiple developmental pathways. Supporting this view, callus cultures of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e expressing \u003cem\u003eROSEA1\u003c/em\u003e and DELILA under a strong constitutive promoter exhibited enhanced pigmentation but reduced shoot formation compared with those driven by a weaker, tissue-specific promoter (Fatihah et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zheng et al., 2007). Although regeneration was not quantitatively assessed in that study, the qualitative trend aligns with the present findings, in which stronger regulatory activation is associated with a more pronounced regeneration penalty (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, S1).\u003c/p\u003e \u003cp\u003eA further layer likely involves altered hormone signaling. While there is currently no evidence that \u003cem\u003eROSEA1\u003c/em\u003e directly represses \u003cem\u003ePLT5, WUS\u003c/em\u003e, or \u003cem\u003eLBD16\u003c/em\u003e, it is plausible that intermediate effects on auxin distribution, cytokinin responsiveness, or other signaling pathways contribute to the phenotype. Nevertheless, the biological plausibility of such a link is considerable because anthocyanin-associated regulation is closely integrated with sugar signaling and multiple phytohormone pathways (Das et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; LaFountain and Yuan, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, elevated cytokinin largely alleviated the regeneration penalty in marigold (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026ndash;h; Fig. S3), indicating that exogenous hormone supply can buffer at least part of the \u003cem\u003eROSEA1\u003c/em\u003e-associated inhibition, consistent with the broader principle that regeneration barriers can often be mitigated through hormonal optimization (Valvekens et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, the mechanistic evidence supports a hierarchical model in which ectopic \u003cem\u003eROSEA1\u003c/em\u003e expression drives extensive activation of the anthocyanin network, accompanied by broader metabolic and transcriptional reprogramming that compromises regeneration competence, at least in part through suppression of \u003cem\u003ePLT5, WUS\u003c/em\u003e, and \u003cem\u003eLBD16\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Within this framework, metabolic burden, transcription-factor pleiotropy, and altered hormone signaling should be viewed not as competing explanations but as interconnected components of a broader mechanistic framework.\u003c/p\u003e\n\u003ch3\u003eConservation of the regeneration penalty across species and genotypes\u003c/h3\u003e\n\u003cp\u003eThe regeneration penalty associated with \u003cem\u003eROSEA1\u003c/em\u003e extended across three eudicot families: Solanaceae (tomato and petunia; Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), Begoniaceae (begonia; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026ndash;d), and Asteraceae (marigold; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026ndash;h), indicating that the phenotype reflects broadly conserved developmental constraints rather than a species-specific anomaly. This phylogenetic breadth is consistent with the deep conservation of both the MYB-bHLH-WD40 module, which controls anthocyanin biosynthesis, and the WUS-, PLT-, and LBD-associated pathways that govern regeneration (Albert et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lloyd et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, because \u003cem\u003eROSEA1\u003c/em\u003e is a transcriptional regulator rather than a metabolic endpoint, these findings are best generalized to \u003cem\u003eROSEA1\u003c/em\u003e-based visual selection rather than to anthocyanin markers as a class. This distinction is reinforced by the DELox comparison, which demonstrates that moderate anthocyanin enhancement does not necessarily compromise organogenesis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results support a regulator- and context-dependent model in which the developmental cost is determined by regulator identity, expression level, and endogenous partner context.\u003c/p\u003e \u003cp\u003eThe magnitude of the regeneration penalty appeared to vary across species, ranging from approximately 30% in marigold under moderate cytokinin conditions to nearly 70% in begonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This variation may reflect differences in baseline regenerative capacity, the endogenous availability of bHLH and WD40 partners that may be needed to assemble functional MBW complexes with \u003cem\u003eROSEA1\u003c/em\u003e (Naing et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and species-specific differences in phenylpropanoid metabolism. Genotype-specific responses within marigold further suggest this complexity. In \u0026lsquo;Bonanza Yellow\u0026rsquo;, which showed relatively low baseline regeneration, no significant penalty was detected (Fig. S3), possibly because of a floor effect. In contrast, genotypes with higher regenerative capacity tended to show a clearer \u003cem\u003eROSEA1\u003c/em\u003e-associated reduction in regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026ndash;h). This pattern is consistent with the well-established genotype dependence of regeneration capacity (Bregitzer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Gordon-Kamm et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and may also suggest genotype-by-marker-system interactions, with the developmental burden associated with \u003cem\u003eROSEA1\u003c/em\u003e potentially becoming more evident in genotypes with moderate to high regenerative potential.\u003c/p\u003e\n\u003ch3\u003ePractical implications for transformation pipeline design\u003c/h3\u003e\n\u003cp\u003eThe distinctive purple pigmentation induced by \u003cem\u003eROSEA1\u003c/em\u003e enables rapid, non-destructive identification of transformed sectors under ambient lighting, offering clear advantages over β-glucuronidase (GUS), which requires destructive histochemical assays, and green fluorescent protein (GFP), which often necessitates specialized fluorescence imaging equipment (Jefferson et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Stewart, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Leclercq et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bedoya et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Our findings do not diminish these advantages but indicate that they can be offset by reduced regeneration efficiency, particularly in systems with moderate regenerative competence (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Importantly, this cost is not an inherent consequence of anthocyanin production per se. The DELox comparison demonstrates that increased pigment output does not necessarily impair regeneration (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating that regulator identity and expression context are key determinants of whether a developmental penalty arises.\u003c/p\u003e \u003cp\u003eSeveral mitigation strategies emerge from these findings. Hormone optimization, particularly increased cytokinin, can partially or fully compensate for the phenotype, as demonstrated in marigold (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026ndash;h; Fig. S3), consistent with the broader principle that regeneration barriers can often be alleviated by adjusting the hormonal environment (Valvekens et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Co-expression of \u003cem\u003ePLT5\u003c/em\u003e partially rescued the ROSox regeneration defect while only modestly reducing anthocyanin accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that reinforcement of developmental competence is an effective complementary strategy. Inducible or tissue-specific expression systems, such as the XVE promoter (Zuo et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), could delay \u003cem\u003eROSEA1\u003c/em\u003e activation until after regeneration is established. Alternative reporters, such as \u003cem\u003eRUBY\u003c/em\u003e, which drives betalain rather than anthocyanin pigmentation, may impose a distinct developmental burden (He et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Finally, genotype pre-screening is advisable because the \u003cem\u003eROSEA1\u003c/em\u003e-associated penalty is most pronounced in genotypes with intermediate regeneration capacity; selecting highly regenerable backgrounds can help maintain both visual selection efficiency and acceptable transformation frequencies.\u003c/p\u003e \u003cp\u003eMore broadly, these findings highlight a critical interference between secondary metabolism and developmental plasticity. The coordinated reduction in \u003cem\u003ePLT5, WUS\u003c/em\u003e, and \u003cem\u003eLBD16\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) may arise from direct transcriptional effects, indirect metabolic perturbation, or a combination of both. Dissecting these mechanisms will be essential for designing next-generation visual reporters that retain strong pigmentation while minimizing developmental cost. Comparative analysis using different anthocyanin regulators under matched expression systems will be particularly informative for distinguishing pigment-associated effects from regulator-specific pleiotropy.\u003c/p\u003e \u003cp\u003eIn conclusion, \u003cem\u003eROSEA1\u003c/em\u003e-based visual selection can impose a regeneration cost associated with reduced expression of key developmental regulators during competence acquisition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This effect extends across phylogenetically diverse species (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) but can be mitigated through hormone optimization, co-expression of developmental regulators, and genotype selection. The findings provide a mechanistic framework for understanding how a secondary-metabolism regulator can influence regeneration and offer practical guidance for the informed deployment of \u003cem\u003eROSEA1\u003c/em\u003e in transformation pipelines.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jaideep Chandranshu Cherukula for plant and facility management, and Keila Emily Rodriguez for the previously generated transgenic lines used in this project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.J. conceived and designed the study. S.E.T. performed plant regeneration experiments in tomato. F.L. performed plant regeneration experiments in petunia. S.E.T. conducted the qRT-PCR analysis. Y.N. performed the plant regeneration experiments in marigold, and W.H.A. performed those in begonia. F.H. and S. Z. maintained plant growth in the greenhouse. T.J. conducted data analysis and visualization. T.J. wrote the manuscript with input from all authors. H.H. and T.J. revised the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by USDA-NIFA (grant number 2019-67013-29236) and the USDA HATCH program (grant number FLA-MFC-006387) to H.H.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCode availability\u003c/p\u003e\n\u003cp\u003eNo custom code was developed for this study. All analyses were performed using publicly available software as described in the Methods section.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbert, N.W., Davies, K.M., Lewis, D.H., Zhang, H., Montefiori, M., Brendolise, C., Boase, M.R., Ngo, H., Jameson, P.E. and Schwinn, K.E., 2014. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. \u003cem\u003eThe Plant Cell\u003c/em\u003e, 26(3), pp.962\u0026ndash;980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltpeter, F., Springer, N.M., Bartley, L.E., Blechl, A.E., Brutnell, T.P., Citovsky, V., Conrad, L.J., Gelvin, S.B., Jackson, D.P., Kausch, A.P., Lemaux, P.G., Medford, J.I., Orozco-C\u0026aacute;rdenas, M.L., Tricoli, D.M., Van Eck, J., Voytas, D.F., Walbot, V., Wang, K., Zhang, Z.J. and Stewart, C.N., 2016. Advancing crop transformation in the era of genome editing. \u003cem\u003eThe Plant Cell\u003c/em\u003e, 28(7), pp.1510\u0026ndash;1520.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaulcombe, D.C., Chapman, S. and Santa Cruz, S., 1995. Jellyfish green fluorescent protein as a reporter for virus infections. \u003cem\u003eThe Plant Journal\u003c/em\u003e, 7(6), pp.1045\u0026ndash;1053.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBedoya, L.C., Mart\u0026iacute;nez, F., Orz\u0026aacute;ez, D. and Dar\u0026ograve;s, J.-A., 2012. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. \u003cem\u003ePlant Physiology\u003c/em\u003e, 158(3), pp.1130\u0026ndash;1138.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirch, R.G., 1997. Plant transformation: problems and strategies for practical application. \u003cem\u003eAnnual Review of Plant Physiology and Plant Molecular Biology\u003c/em\u003e, 48, pp.297\u0026ndash;326.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBower, R., Elliott, A.R., Potier, B.A.M. and Birch, R.G., 1996. High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. \u003cem\u003eMolecular Breeding\u003c/em\u003e, 2(3), pp.239\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBregitzer, P., Dahleen, L.S. and Campbell, R.D., 1998. Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars. \u003cem\u003ePlant Cell Reports\u003c/em\u003e, 17(12), pp.941\u0026ndash;945.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown, D.E., Rashotte, A.M., Murphy, A.S., Normanly, J., Tague, B.W., Peer, W.A., Taiz, L. and Muday, G.K., 2001. Flavonoids act as negative regulators of auxin transport in vivo in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003ePlant Physiology\u003c/em\u003e, 126(2), pp.524\u0026ndash;535.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButelli, E., Titta, L., Giorgio, M., Mock, H.-P., Matros, A., Peterek, S., Schijlen, E.G.W.M., Hall, R.D., Bovy, A.G., Luo, J. and Martin, C., 2008. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. \u003cem\u003eNature Biotechnology\u003c/em\u003e, 26(11), pp.1301\u0026ndash;1308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerqueira, J.V.A., Zhu, F., Mendes, K., Nunes-Nesi, A., Martins, S.C.V., Benedito, V., Fernie, A.R. and Zs\u0026ouml;g\u0026ouml;n, A., 2023. Promoter replacement of ANT1 induces anthocyanin accumulation and triggers the shade avoidance response through developmental, physiological and metabolic reprogramming in tomato. \u003cem\u003eHorticulture Research\u003c/em\u003e, 10(2), uhac254.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChalker-Scott, L., 1999. Environmental significance of anthocyanins in plant stress responses. \u003cem\u003ePhotochemistry and Photobiology\u003c/em\u003e, 70(1), pp.1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChawla, H.S., Cass, L.A. and Simmonds, J.A., 1999. Developmental and environmental regulation of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes. \u003cem\u003eIn Vitro Cellular \u0026amp; Developmental Biology-Plant\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(5), pp.403\u0026ndash;408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas, P.K., Shin, D.-H., Choi, S.-B. and Park, Y.-I., 2012. Sugar-hormone cross-talk in anthocyanin biosynthesis. \u003cem\u003eMolecules and Cells\u003c/em\u003e, 34(6), pp.501\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebernardi, J.M., Tricoli, D.M., Ercoli, M.F., Hayta, S., Ronald, P., Palatnik, J.F. and Dubcovsky, J., 2020. A GRF\u0026ndash;GIF chimera boosts regeneration and transformation in wheat. \u003cem\u003eNature Biotechnology\u003c/em\u003e, 38(12), pp.1274\u0026ndash;1279.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeikman, J. and Hammer, P.E., 1995. Induction of anthocyanin accumulation by cytokinins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003ePlant Physiology\u003c/em\u003e, 108(1), pp.47\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFatihah, H.N.N., Mo\u0026ntilde;ino L\u0026oacute;pez, D., van Arkel, G.,\"; Schaart, J.G., Visser, R.G.F. and Krens, F.A., 2019. The ROSEA1 and DELILA transcription factors control anthocyanin biosynthesis in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and \u003cem\u003eLilium\u003c/em\u003e flowers. \u003cem\u003eScientia Horticulturae\u003c/em\u003e, 243, pp.327\u0026ndash;337.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallois, J.-L., Nora, F.R., Mizukami, Y. and Sablowski, R., 2004. WUSCHEL induces shoot stem cell activity and developmental plasticity in the root meristem. \u003cem\u003eGenes \u0026amp; Development\u003c/em\u003e, 18(4), pp.375\u0026ndash;380.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGordon, S.P., Heisler, M.G., Reddy, G.V., Ohno, C., Das, P. and Meyerowitz, E.M., 2007. Pattern formation during de novo assembly of the \u003cem\u003eArabidopsis\u003c/em\u003e shoot meristem. \u003cem\u003eDevelopment\u003c/em\u003e, 134(19), pp.3539\u0026ndash;3548.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGordon-Kamm, B., Sardesai, N., Arling, M., Lowe, K., Hoerster, G. and Betts, S., 2019. Using morphogenic genes to improve recovery and regeneration of transgenic plants. \u003cem\u003ePlants\u003c/em\u003e, 8(2), 38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGould, K.S., 2004. Nature's Swiss army knife: the diverse protective roles of anthocyanins in leaves. \u003cem\u003eJournal of Biomedicine and Biotechnology\u003c/em\u003e, 2004(5), pp.314\u0026ndash;320.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, Y., Zhang, T., Sun, H., Zhan, H. and Zhao, Y., 2020. A reporter for noninvasively monitoring gene expression and plant transformation. \u003cem\u003eHorticulture Research\u003c/em\u003e, 7, 152.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolton, T.A. and Cornish, E.C., 1995. Genetics and biochemistry of anthocyanin biosynthesis. \u003cem\u003eThe Plant Cell\u003c/em\u003e, 7(7), pp.1071\u0026ndash;1083.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, T., Xin, S., Fang, Y., Chen, T., Chang, J., Ko, N.C.K., Huang, H. and Hua, Y., 2021. Use of a novel R2R3-MYB transcriptional activator of anthocyanin biosynthesis as visual selection marker for rubber tree (Hevea brasiliensis) transformation. \u003cem\u003eIndustrial Crops and Products\u003c/em\u003e, \u003cem\u003e174\u003c/em\u003e, p.114225.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkeuchi, M., Favero, D.S., Sakamoto, Y., Iwase, A., Coleman, D., Rymen, B. and Sugimoto, K., 2019. Molecular mechanisms of plant regeneration. \u003cem\u003eAnnual Review of Plant Biology\u003c/em\u003e, 70, pp.377\u0026ndash;406.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJefferson, R.A., Kavanagh, T.A. and Bevan, M.W., 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. \u003cem\u003eThe EMBO Journal\u003c/em\u003e, 6(13), pp.3901\u0026ndash;3907.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, T., Lian, Z., Rodriguez, K., Liu, F., Karn, A., Handoyo, W., Kyum, M., Tanwir, S.E., Van Deynze, A. Bradford, K., and Huo, H., 2026b. Synergistic developmental regulators enable efficient plant regeneration and transformation across species.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, T., Liu, F., Tanwir, S.E., Shaheen, N., Wang, G., Copsey, L., Lian, Z. and Huo, H., 2026a. Identification and functional characterization of AmbHLH002 as a conserved bHLH regulator of anthocyanin biosynthesis in Antirrhinum majus. \u003cem\u003eHorticultural Plant Journal\u003c/em\u003e, 12 (3), pp. 704\u0026ndash;719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, T., Rodriguez, K., Tanwir, S.E., Liu, F., Hussain, F., Cherukula, J.C. and Huo, H., 2025. Regulatory role of MicroRNA164 in heat and salinity stress responses via candidate target genes during seed germination in petunia. \u003cem\u003eHorticulture Advances\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(1), p.15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKareem, A., Durgaprasad, K., Sugimoto, K., Du, Y., Pulianmackal, A.J., Trivedi, Z.B., Abhayadev, P.V., Pinon, V., Meyerowitz, E.M., Scheres, B. and Prasad, K., 2015. PLETHORA genes control regeneration by a two-step mechanism. \u003cem\u003eCurrent Biology\u003c/em\u003e, 25(8), pp.1017\u0026ndash;1030.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoes, R., Verweij, W. and Quattrocchio, F., 2005. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. \u003cem\u003eTrends in Plant Science\u003c/em\u003e, 10(5), pp.236\u0026ndash;242.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKortstee, A.J., Khan, S.A., Helderman, C., Trindade, L.M., Wu, Y., Visser, R.G.F., Brendolise, C., Allan, A., Schouten, H.J. and Jacobsen, E., 2011. Anthocyanin production as a potential visual selection marker during plant transformation. \u003cem\u003eTransgenic Research\u003c/em\u003e, 20(6), pp.1253\u0026ndash;1264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaFountain, A.M. and Yuan, Y.-W., 2021. Repressors of anthocyanin biosynthesis. \u003cem\u003eNew Phytologist\u003c/em\u003e, 231(3), pp.933\u0026ndash;949.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeclercq, J., Lardet, L., Martin, F., Chapuset, T., Oliver, G. and Montoro, P., 2010. The green fluorescent protein as an efficient selection marker for \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation in \u003cem\u003eHevea brasiliensis\u003c/em\u003e (M\u0026uuml;ll. Arg.). \u003cem\u003ePlant Cell Reports\u003c/em\u003e, 29(5), pp.513\u0026ndash;522.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, K. and Wang, K., 2023. Strategies for genotype-flexible plant transformation. \u003cem\u003eCurrent Opinion in Biotechnology\u003c/em\u003e, 79, 102848.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, J., Hu, X., Qin, P., Prasad, K., Hu, Y. and Xu, L., 2018. The WOX11\u0026ndash;LBD16 pathway promotes pluripotency acquisition in callus cells during de novo shoot regeneration in tissue culture. \u003cem\u003ePlant and Cell Physiology\u003c/em\u003e, 59(4), pp.739\u0026ndash;748.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLloyd, A., Brockman, A., Aguirre, L., Campbell, A., Bean, A., Cantero, A. and Gonzalez, A., 2017. Advances in the MYB\u0026ndash;bHLH\u0026ndash;WD repeat (MBW) pigment regulatory model: addition of a WRKY factor and co-option of an anthocyanin MYB for betalain regulation. \u003cem\u003ePlant and Cell Physiology\u003c/em\u003e, 58(9), pp.1431\u0026ndash;1441.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLowe, K., Wu, E., Wang, N., Hoerster, G., Hastings, C., Cho, M.-J., Scelonge, C., Lenderts, B., Chamberlin, M., Cushatt, J., Wang, L., Ryan, L., Khan, T., Chow-Yiu, J., Hua, W., Yu, M., Banh, J., Bao, Z., Brink, K., Igo, E., Rudrappa, B., Shamseer, P.M., Bruce, W., Newman, L., Shen, B., Zheng, P., Bidney, D., Falco, C., Register, J., Zhao, Z.-Y., Xu, D., Jones, T. and Gordon-Kamm, W., 2016. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. \u003cem\u003eThe Plant Cell\u003c/em\u003e, 28(9), pp.1998\u0026ndash;2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLudwig, S.R., Bowen, B., Beach, L. and Wessler, S.R., 1990. A regulatory gene as a novel visible marker for maize transformation. \u003cem\u003eScience\u003c/em\u003e, 247(4941), pp.449\u0026ndash;450.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajer, E., Dar\u0026ograve;s, J.-A. and Zwart, M.P., 2013. Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome. \u003cem\u003eViruses\u003c/em\u003e, 5(9), pp.2153\u0026ndash;2168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaing, A.H., Ai, T.N., Lim, K.B., Lee, I.J. and Kim, C.K., 2018. Overexpression of Rosea1 from snapdragon enhances anthocyanin accumulation and abiotic stress tolerance in transgenic tobacco. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, 9, 1070.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePourcel, L., Irani, N.G., Lu, Y., Riedl, K., Schwartz, S. and Grotewold, E., 2010. The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. \u003cem\u003eMolecular plant\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(1), pp.78\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E., Oyama, R., Bailey, P., Davies, K. and Martin, C., 2006. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus \u003cem\u003eAntirrhinum\u003c/em\u003e. \u003cem\u003eThe Plant Cell\u003c/em\u003e, 18(4), pp.831\u0026ndash;851.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, L., Li, X., Fu, Y. and Li, C., 2023. Environmental stimuli and phytohormones in anthocyanin biosynthesis: a comprehensive review. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e, 24(22), 16415.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, M.-Z. and Xie, D.-Y., 2014. Biosynthesis and metabolic engineering of anthocyanins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eRecent Patents on Biotechnology\u003c/em\u003e, 8(1), pp.47\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkoog, F. and Miller, C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. \u003cem\u003eSymposia of the Society for Experimental Biology\u003c/em\u003e, 11, pp.118\u0026ndash;130.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSomers, D.A., Samac, D.A. and Olhoft, P.M., 2003. Recent advances in legume transformation. \u003cem\u003ePlant Physiology\u003c/em\u003e, 131(3), pp.892\u0026ndash;899.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart, C.N., Jr., 2001. The utility of green fluorescent protein in transgenic plants. \u003cem\u003ePlant Cell Reports\u003c/em\u003e, 20(5), pp.376\u0026ndash;382.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugimoto, K., Jiao, Y. and Meyerowitz, E.M., 2010. \u003cem\u003eArabidopsis\u003c/em\u003e regeneration from multiple tissues occurs via a root development pathway. \u003cem\u003eDevelopmental Cell\u003c/em\u003e, 18(3), pp.463\u0026ndash;471.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, Y., Sasaki, N. and Ohmiya, A., 2008. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. \u003cem\u003eThe Plant Journal\u003c/em\u003e, 54(4), pp.733\u0026ndash;749.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanwir, S.E., Jiang, T., Creech, M.R., Kim, J., Cherukula, J.C., Wang, S. and Huo, H. (2026) Engineering betalain biosynthesis via RUBY enhances ornamental aesthetics but alters fertility in \u003cem\u003eSolanum aethiopicum\u003c/em\u003e. \u003cem\u003eMolecular Horticulture\u003c/em\u003e, Manuscript in revision.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTohge, T., Zhang, Y., Peterek, S., Matros, A., Rallapalli, G., Tandr\u0026oacute;n, Y.A., Butelli, E., Kallam, K., Hertkorn, N., Mock, H.-P., Martin, C. and Fernie, A.R., 2015. Ectopic expression of snapdragon transcription factors facilitates the identification of genes encoding enzymes of anthocyanin decoration in tomato. \u003cem\u003eThe Plant Journal\u003c/em\u003e, 83(4), pp.686\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValvekens, D., Van Montagu, M. and Van Lijsebettens, M., 1988. \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e root explants by using kanamycin selection. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e, 85(15), pp.5536\u0026ndash;5540.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVandenbussche, M., Zethof, J., Souer, E., Koes, R., Tornielli, G.B., Pezzotti, M., Ferrario, S., Angenent, G.C. and Gerats, T., 2003. Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia. \u003cem\u003eThe Plant Cell\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(11), pp.2680\u0026ndash;2693.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinkel-Shirley, B., 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. \u003cem\u003ePlant Physiology\u003c/em\u003e, 126(2), pp.485\u0026ndash;493.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, T.-Q., Lian, H., Zhou, C.-M., Xu, L., Jiao, Y. and Wang, J.-W., 2017. A two-step model for de novo activation of WUSCHEL during plant shoot regeneration. \u003cem\u003eThe Plant Cell\u003c/em\u003e, 29(5), pp.1073\u0026ndash;1087.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y., Butelli, E. and Martin, C., 2013. Engineering anthocyanin biosynthesis in plants. \u003cem\u003eCurrent Opinion in Plant Biology\u003c/em\u003e, 16(2), pp.218\u0026ndash;223.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y., Malzahn, A.A., Sretenovic, S. and Qi, Y., 2019. The emerging and uncultivated potential of CRISPR technology in plant science. \u003cem\u003eNature Plants\u003c/em\u003e, 5(8), pp.778\u0026ndash;794.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhen, X.H., Pan, R.R., Lu, X.H., Ge, Y.J., Li, R.M., Liu, J., Wang, Y.J., Yi, K.X., Li, C.X., Guo, J.C. and Yao, Y., 2024. An anthocyanin-based visual reporter system for genetic transformation and genome editing in cassava. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(21), p.11808.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuo, J., Niu, Q.-W. and Chua, N.-H., 2000. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. \u003cem\u003eThe Plant Journal\u003c/em\u003e, 24(2), pp.265\u0026ndash;273.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ROSEA1, Anthocyanin biosynthesis, Regeneration capacity, Metabolic flux, Visual markers","lastPublishedDoi":"10.21203/rs.3.rs-9298244/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9298244/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnthocyanin-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 \u003cem\u003eROSEA1\u003c/em\u003e expression reduces shoot regeneration across four eudicot species from three families. In tomato and petunia, stable \u003cem\u003eROSEA1\u003c/em\u003e-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 \u003cem\u003eROSEA1\u003c/em\u003e coordinately activated anthocyanin biosynthetic genes, including \u003cem\u003eCHI, F3H, F3′5′H,\u003c/em\u003e and \u003cem\u003eDFR\u003c/em\u003e, while downregulating the regeneration regulators \u003cem\u003ePLT5, WUS,\u003c/em\u003e and \u003cem\u003eLBD16\u003c/em\u003eduring the early regeneration phase. Co-expression of \u003cem\u003ePLT5\u003c/em\u003e with \u003cem\u003eROSEA1\u003c/em\u003epartially 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 \u003cem\u003eROSEA1\u003c/em\u003e-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.\u003c/p\u003e","manuscriptTitle":"ROSEA1-based visual selection reduces plant regeneration and alters developmental regulator expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 22:26:09","doi":"10.21203/rs.3.rs-9298244/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7fbcc186-fa21-4f54-89ef-30305f67eba9","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-17T13:16:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-17T07:38:51+00:00","index":15,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T09:55:05+00:00","index":14,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T13:30:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 22:26:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9298244","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9298244","identity":"rs-9298244","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}