First insights into colchicine-mediated polyploidy induction in Vaccinium floribundum Kunth

First insights into colchicine-mediated polyploidy induction in Vaccinium floribundum Kunth | 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 First insights into colchicine-mediated polyploidy induction in Vaccinium floribundum Kunth Cristian Mendoza, Alexander Uquiche This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8300888/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Polyploidy induction represents a promising yet challenging strategy to enhance the genetic potential of Vaccinium floribundum Kunth, an Andean crop wild relative of blueberry, with high nutritional and antioxidant value. This study explored for the first time the response of nine accessions to colchicine exposure as a preliminary step toward chromosome doubling. Axillary buds were treated with aqueous colchicine at 125 and 250 mg·L⁻¹ for 24, 48, or 96 h and subsequently cultivated in vitro. A generalized linear mixed model (GLMM) revealed that colchicine concentration was the principal determinant of explant mortality (β₁₂₅ = 4.658, β₂₅₀ = 5.037, p 0.05). Random effects captured pronounced inter-accession heterogeneity (σ² = 5.351), reflecting intrinsic variability in tolerance to colchicine toxicity. Modeled mortality increased consistently with concentration, yet certain accessions such as Enca 20 retained comparatively low mortality (< 45% across treatments) and partial regenerative competence, indicating genotype-dependent resilience. Flow cytometry confirmed that all regenerated plants remained diploid, indicating that chromosome doubling was not achieved under the tested conditions. These findings establish an empirical baseline for refining chromosome-doubling protocols through optimized concentrations and the selection of tolerant genotypes. Vaccinium floribundum Kunth polyploidy induction colchicine in vitro culture axillary buds Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Crop wild relatives (CWR) are wild progenitors or closely related species of cultivated plants,, featuring primitive and heterogeneous genotypes that can easily hybridize with cultivated forms, as well as more distantly related species in the same or related genera (Tyack et al., 2020 ). Having evolved under natural selective pressures, CWR retain extensive allelic diversity, constituting invaluable reservoirs of genetic variation that provides breeders with traits of agronomic relevance, including tolerance to drought, cold, and salinity, as well as the capacity to develop dynamic resistance to the shifting distribution and intensity of plant pathogens driven by climate change, thereby supporting long-term crop resilience (Dempewolf et al., 2014 ). Their integration into breeding programs over the past six decades has been pivotal for agricultural innovation, with numerous documented cases of successful gene introgressions that have strengthened yield stability and food security (Hajjar & Hodgkin, 2007 ). Globally, between 50,000 and 60,000 CWR species have been identified, of which approximately 10,000 are considered highly relevant to food production, and around 1,000 are closely related to the world’s most important crops (Maxted & Kell, 2009 ), collectively contributing an estimated ~ $ 186.3 billion to the global economy in 2020 (Tyack et al., 2020 ). Despite the potential of CWR, their direct use in breeding programs is often constrained by crossability barriers, which limit the incorporation of genes into cultivated varieties (Bohra et al., 2021 ). To overcome this limitation, breeders employ antimitotic agents to induce polyploidization, enabling the formation of stable hybrids between distantly related species and facilitating the introgression of beneficial alleles from wild relatives into crops (Tang et al., 2010 ; Zhang et al., 2019 ). This approach is widely adopted due to its capacity to generate substantial random and undirected morphological and physiological variation, resulting in individuals with novel or enhanced traits that surpass the limitations of their diploid progenitors (Jain, 2010 ). Such enhancements encompass increased resistance and tolerance to biotic and abiotic stresses, greater biomass production, and improvements in the quality and quantity of bioactive metabolites (Lin et al., 2011 ; van de Peer et al., 2021 ; Madani et al., 2021 ). Morphologically, polyploids commonly exhibit enlarged organs, extended flowering periods, more abundant inflorescences, and thicker leaves and petals (Alix et al., 2017 ; Touchell et al., 2020 ). At the physiological and biochemical levels, genome duplication can enhance enzymatic activity and metabolic efficiency, leading to gene overexpression or silencing and the synthesis of novel compounds (Niazian & Nalousi, 2020 ). Since the 1940s, polyploidization has been employed in the genus Vaccinium , one of the largest and most variable in the Ericaceae family (APG IV, 2016 ), starting with Vaccinium macrocarpon in which colchicine was utilized as the primary antimitotic agent (Dermen & Bain, 1944 ). From a cytogenetic perspective, Vaccinium exhibits substantial variation in ploidy levels, ranging from diploid (2n = 2x = 24) to hexaploid (2n = 6x = 72), with a basic chromosome number of x = 12 (Hummer et al., 2015 ), which has historically limited interspecific crosses due to endosperm-related barriers, commonly referred to as the “triploid block” (Håkansson & Ellerstromsee, 1950; Esen & Soost, 1973 ; Lafon-Placette et al., 2017 ). Within the genera, polyploidization efforts have evolved from early in vivo experiments to increasingly controlled in vitro protocols targeting explants such as axillary buds and adventitious shoots (Jarpa-Tauler, 2024 ). More recently, leaf explants have enabled regeneration from single cells, reducing mixoploidy and producing auto-octoploids and chimeras in V. corymbosum with induction rates up to 11% (Marangelli et al., 2022 ; Li et al., 2025 ). Peru harbors 13 identified wild relatives of Vaccinium crops, of which six are endemic (Mejía et al., 2016 ; Mostacero et al., 2017 ). Among these, Vaccinium floribundum Kunth (2n = 2x = 24) (Hummer et al., 2015 ), a perennial shrub primarily distributed in the high-altitude páramo and montane regions of Peru and Ecuador (Llivisaca-Contreras et al., 2022 ), rich nutritional and phytochemical profile. Its fruits provide nutrients, dietary fiber, and minerals such as iron, copper, zinc, and calcium, together with high concentrations of phenolic compounds and other secondary metabolites (Coba et al., 2012). Compared with cultivated blueberries such as V. corymbosum and V. virgatum , V. floribundum exhibits equal or even higher total anthocyanin content, reaching 335–477 mg C3G (cyanidin-3-glucoside equivalents) per 100 g FW (fresh weight) (Pérez et al., 2021 ; Andrade-Cuvi et al., 2017 ). In contrast, cultivated species generally range between 167 and 580 mg C3G/100 g FW depending on genotype and growing conditions (Shibata et al., 2021 ; Smrke et al., 2021 ). Its antioxidant activity is likewise remarkable, with FRAP (ferric reducing antioxidant power) values of about 19,653 µmol TE (Trolox equivalents)/100 g FW and ORAC (oxygen radical absorbance capacity) values near 40,220 µmol TE/100 g FW (Baenas et al., 2020 ), both well above those reported for commercial cultivars, with FRAP values of 2,480–6,160 µmol TE/100 g FW (Shibata et al., 2021 ) and ORAC values of 3,509–4,841 µmol TE/100 g FW (Milivojevic et al., 2012 ). These characteristics, together with favorable physicochemical traits such as high moisture content and optimal soluble solids (°Brix ≈ 11–16.9) (Alarcón-Barrera et al., 2018 ; Baenas et al., 2020 ), highlight V. floribundum as a strong candidate for breeding and the development of functional foods. As cultivated blueberries are predominantly tetraploid, direct hybridization with V. floribundum is limited by ploidy mismatches, which restrict the transfer of its desirable traits into breeding lines. To date, no studies have explored chromosome doubling in this species, and its response to colchicine or other antimitotic agents remains unknown. This study therefore represents a first exploratory attempt to induce polyploidy in vitro in V. floribundum by treating axillary buds with colchicine, while evaluating induction efficiency, explant survival, accession-specific responses, and shoot regeneration. The results provide a basis for optimizing chromosome-doubling protocols and support future hybridization with commercial cultivars. 2. METHODS 2.1. Plant material Plant material was originally collected in 2017–2019 from wild populations of V. floribundum located in the Cajamarca region of northern Peru,, under the General Access Resolution N.° 138-2019-MINAGRI-SERFOR-DGGSPFFS issued by the National Forest and Wildlife Service. A total of 67 accessions were obtained during this expedition and subsequently incorporated into the germplasm bank of the Centro de Investigación en Recursos Genéticos, Biotecnología y Bioseguridad (CIRGEBB) at Universidad Nacional Agraria La Molina, Peru, where they have since been maintained under in vitro conditions (Fig. 1 ). From this collection, nine accessions from five distinct collection sites within Cajamarca were selected in early 2024 for chromosome doubling experiments. Accession selection was based on previous evaluations identifying them as promising candidates due to their superior fruit size, with mean diameters greater than 0.7 cm. 2.1. Polyploidy induction with colchicine Shoot explants were treated with aqueous colchicine solutions (125 and 250 mg·L⁻¹) to induce polyploidy. Solutions were freshly prepared in distilled water containing 0.1% (v/v) ethanol and sterilized through 0.22 µm PES filters. Six explants per accession and treatment, each approximately 2 cm long with at least five nodes, were immersed in colchicine for 24, 48, or 96 h in darkness. Following exposure, explants were rinsed three times with sterile distilled water under aseptic conditions and transferred to WPM medium supplemented with 1 mg·L⁻¹ kinetin. Cultures were maintained for one week in darkness, followed by a 16 h photoperiod for 12 weeks. To account for immersion time effects, colchicine-free controls were included for each exposure duration (24, 48, and 96 h), resulting in six colchicine treatments (T1–T6) plus three time-matched controls (C1–C3) (Table 1 ). Table 1 Experimental treatments applied for the induction of polyploidization in V. floribundum using colchicine. Treatment type Colchicine concentration (mg·L⁻¹) Exposure time (h) Code Control 0 24 C1 Control 0 48 C2 Control 0 96 C3 Treatment 125 24 T1 Treatment 250 24 T2 Treatment 125 48 T3 Treatment 250 48 T4 Treatment 125 96 T5 Treatment 250 96 T6 2.2. Statistical modeling of mortality response Mortality was assessed eight weeks after colchicine treatment to capture cumulative effects on explant survival. Explants that failed to produce at least one shoot were classified as dead, and each observation was recorded as a binary response (0 = alive, 1 = dead) for every combination of accession, colchicine concentration and exposure time. Mortality was analyzed using a generalized linear mixed model (GLMM) fitted with a binomial distribution and logit link function in the glmmTMB package (R). Fixed effects included exposure time, colchicine concentration, and their interaction, whereas accession was incorporated as a random effect to account for among-accession variability. The model was specified as follows: $$\:logit\left({p}_{ijk}\right)={\beta\:}_{0}+{\beta\:}_{1}\times\:{Time}_{i}+{\beta\:}_{2}\times\:{Concentration}_{j}+{\beta\:}_{3}\times\:({Time}_{i}\times\:{Concentration}_{j})+{u}_{k}$$ where p ijk denotes the probability of mortality for accession k under time i and concentration j ; β 0 -β 3 represent fixed-effect coefficients; and u k ​ is the random effect associated with accession k . Random-effect estimates were extracted using the ranef() function in glmmTMB to evaluate accession-specific patterns of colchicine tolerance and to refine fixed-effect estimates for exposure time and concentration. Pairwise contrasts between each treatment and the control within exposure times were performed with the contrast() function in emmeans, applying Tukey’s correction for multiple comparisons. Model fit and residual behavior were evaluated using simulated residuals with the DHARMa package (simulateResiduals()), ensuring appropriate model specification and the validity of the inferential framework. 2.3. Assessment of regenerative capacity Regenerative capacity was evaluated eight weeks after colchicine exposure by recording the number of shoots produced per explant for each accession–treatment combination. Mean regeneration values were calculated and analyzed separately for each accession using one-way analysis of variance (ANOVA), with treatment type as the fixed factor. When significant differences were detected (p < 0.05), Duncan’s multiple range test was applied to identify homogeneous groups. 2.4. Determination of ploidy level by flow cytometry Ploidy level was determined by flow cytometry at the Biotechnology Laboratory of the Research and Social Projection Program on Cereals and Native Grains at Universidad Nacional Agraria La Molina, using an Attune NxT cytometer (Thermo Fisher Scientific). The instrument was calibrated with diploid controls of V. corymbosum and untreated V. floribundum . Sample preparation followed Doležel et al. ( 2007 ) with minor modifications. Approximately 20 mg of young leaf tissue were finely chopped in 1 mL of Otto I buffer, incubated on ice for 15 min, filtered through a 42 µm nylon mesh, and centrifuged at 150 × g for 5 min. The pellet was resuspended in 100 µL of Otto I and mixed with 1 mL of Otto II containing propidium iodide (50 µg·mL⁻¹) and RNase A (50 µg·mL⁻¹), then incubated in darkness on ice for 15–60 min before analysis. At least 4 000 nuclei per sample were analyzed (λ_exc = 535 nm, λ_em = 617 nm). Histograms were gated to exclude debris and aggregates, and the main peak (2C) was identified for each sample. Ploidy was estimated as the ratio between the mean G₁ fluorescence intensity of the sample and that of the diploid standard. Samples with peak positions within ± 5% of the diploid reference and a coefficient of variation (CV) below 5% were classified as diploid, whereas peaks corresponding to approximately twice the fluorescence were classified as tetraploid. Intermediate or irregular profiles were interpreted as potential mixoploids. 3. RESULTS 3.1. Model validation and mortality response to colchicine exposure The GLMM fitted with a binomial distribution and logit link adequately described the mortality data, as verified by DHARMa residual diagnostics (Figure S1). The Kolmogorov–Smirnov test (p = 0.8758) and Levene’s test for homogeneity of variance (p > 0.05) indicated no deviation from uniformity or heteroscedasticity, supporting the adequacy of model specification (Figure S2). Observed and model-predicted mortalities exhibited consistent dose-dependent patterns across accessions (Fig. 2 ). Under control conditions (0 mg L⁻¹), observed mortality (Table S1) was generally low (< 20%) across accessions, with the exception of Namo 09, which exhibited comparatively higher baseline mortality values. In contrast, exposure to 125 mg L⁻¹ colchicine sharply increased mortality, reaching 100% in Namo 09, Enca 01, and Enca 11. At 250 mg L⁻¹, mortality exceeded 80% in G. Pita 08, Enca 01, and Enca 11, while Conch 03 and Taca 01 showed intermediate responses ranging from 30 to 70%, whereas Enca 20 exhibited the lowest observed mortality, remaining below 45% even at the highest concentration. Model-predicted probabilities closely paralleled these empirical proportions, accurately capturing the dose-response trends across accessions (Table 2 ). Minor deviations occurred in Conch 03 and Taca 01, where stochastic variation at low mortality levels led to slight overestimation of fitted values. Table 2 Modeled explant mortality (%) across nine V. floribundum accessions after colchicine treatments. Accession C1 T1 T2 C2 T3 T4 C3 T5 T6 Conch 03 0.72% 43.18% 52.60% 1.01% 40.15% 71.63% 0.31% 46.28% 65.38% Enca 01 4.24% 82.38% 87.22% 5.92% 80.49% 93.95% 1.88% 84.12% 92.07% Enca 08 0.72% 43.18% 52.60% 1.01% 40.15% 71.63% 0.31% 46.28% 65.38% Enca 10 1.65% 63.89% 72.09% 2.33% 60.96% 85.46% 0.72% 66.73% 81.47% Enca 11 13.34% 94.20% 95.95% 17.93% 93.48% 98.18% 6.23% 94.85% 97.58% Enca 20 0.21% 18.26% 24.59% 0.30% 16.47% 42.59% 0.09% 20.20% 35.70% G. Pita 08 10.44% 92.48% 94.72% 14.19% 91.56% 97.61% 4.79% 93.30% 96.83% Namo 09 82.15% 99.79% 99.86% 86.72% 99.77% 99.94% 66.52% 99.82% 99.92% Taca 01 0.40% 29.94% 38.43% 0.57% 27.39% 58.68% 0.17% 32.63% 51.51% Note. Values represent the percentage of explants showing mortality per treatment as estimated by a binomial GLMM. Fixed-effect estimates (Fig. 3a) confirmed that concentration was the primary driver of explant mortality, with β = 4.6584 for 125 mg L⁻¹ (p = 1.98 × 10⁻⁸) and β = 5.0370 for 250 mg L⁻¹ (p = 1.92 × 10⁻⁹), evidencing a strong concentration-dependent effect. In contrast, exposure duration and its interaction with concentration were non-significant, indicating that mortality remained largely independent of immersion time. Post hoc pairwise contrasts (Table S2) confirmed that exposure time did not significantly affect mortality, whereas the effect of concentration remained consistent across durations. Additionally, random effects attributable to accession (Fig. 3b) captured considerable variability in baseline mortality probability (variance = 5.351, SD = 2.313 log-odds units), with individual intercepts deviating markedly from the overall mean. Namo 09 (4.983) and Enca 11 (1.585) exhibited the largest positive deviations, indicating higher baseline mortality relative to the population average, whereas Enca 20 (–2.701) and Taca 01 (–2.052) showed the lowest baseline mortality. Accessions with values near zero, such as Enca 01 (0.340), had baseline probabilities similar to the overall mean. Figure 3a–b. Model-derived estimates describing colchicine-induced mortality responses in V. floribundum explants. Figure 3a. Forest plot of fixed-effect estimates from the generalized linear mixed model, where the intercept corresponds to 0 mg L⁻¹ colchicine at 24 h exposure. Figure 3b. Caterpillar plot of accession-level random intercepts (± 95% confidence intervals), illustrating among-genotype variability in baseline mortality probability. Significance codes: * (< 0.05), ** (< 0.01), *** (< 0.001), ns = not significant. 3.2. Regeneration capacity after colchicine treatment Shoot regeneration varied markedly among accessions and colchicine treatments (Table 3 ). Under control conditions, explants produced between 0.67 and 7.00 shoots per explant, evidencing wide intrinsic differences in morphogenic competence. Enca 08, Enca 10, and Enca 20 exhibited the highest baseline regeneration potential, with 7.00, 6.33, and 5.67 shoots per explant, respectively, whereas Namo 09 and G. Pita 08 remained largely unresponsive. Colchicine exposure led to a generalized decline in shoot regeneration, although the magnitude of inhibition was accession- and dose-dependent. Highly colchicine-sensitive accessions, such as Enca 01, Enca 11, G. Pita 08, and Namo 09, exhibited almost complete suppression across all treatments, with ≤ 0.67 shoots per explant even at the lowest colchicine levels. Enca 08 and Enca 10 also experienced substantial reductions with shoot production declining to 0.17–1.67 per explant. In contrast, Conch 03 and Taca 01 retained moderate regenerative capacity at intermediate colchicine concentrations, producing 1.17–3.33 shoots per explant. Notably, Enca 20 displayed relative tolerance, maintaining 1.67–5.33 shoots per explant across treatments, indicating partial resistance to colchicine-induced inhibition. Table 3 Shoot regenerative capacity of V. floribundum shoot explants after colchicine treatments Accession C1 T1 T2 C2 T3 T4 C3 T5 T6 Conch 03 5.00 a 1.83 bcd 2.33 bc 5.00 a 3.33 ab 0.17 d 3.50 ab 0.67 cd 0.67 cd Enca 01 2.67 b 0.00 c 0.00 c 3.33 ab 0.00 c 0000 c 4.00 a 0.00 c 0.67 c Enca 08 7.00 a 2.67 bcd 1.83 cde 4.83 b 3.00 bcd 0.17 e 4.00 bc 1.67 de 0.17 e Enca 10 6.33 a 1.83 c 1.00 cd 6.33 a 0.00 d 0.83 cd 4.67 b 0.83 cd 0.00 d Enca 11 4.50 a 0.00 d 0.00 d 4.00 b 0.00 d 0.00 d 3000 c 0.00 d 0.00 d Enca 20 5.67 ab 5.33 abc 2.33 d 5.67 ab 3.00 bcd 1.67 d 6.17 a 4.00 abcd 2.83 cd G. Pita 08 3.50 a 0.00 b 0.00 b 3.67 a 0.00 b 0.00 b 3.00 a 0.00 b 0.00 b Namo 09 0.00 b 0.00 b 0.00 b 0.00 b 0.00 b 0.00 b 0.67 a 0.00 b 0.00 b Taca 01 3.50 a 1.17 cd 1.33 cd 3.33 a 2.00 bc 1.17 cd 3.00 ab 1.50 cd 0.17 d Note : Duncan’s multiple range test (p < 0.05) was used to compare treatments within each accession; homogeneous groups are indicated by shared superscript letters. 3.3. Ploidy analysis A total of 48 shoots were analyzed by flow cytometry. The external standard of V. floribundum showed a fluorescence value of 12,218, used as the diploid reference. Experimental samples exhibited a mean fluorescence intensity of 11,682.42 with a coefficient of variation of 3.55%, values consistent with diploidy and within the accepted range relative to the standard (± 610.9) (Table 4 ). The histograms showed a single fluorescence peak around 11 × 10⁴ in all experimental samples (Fig. 4 a), while the calibration control displayed two distinct peaks corresponding to the diploid standard (2x) and the tetraploid positive control of V. corymbosum (4x) (Fig. 4 b). The absence of any shift toward the tetraploid region confirmed the nuclear homogeneity of the samples and the lack of subpopulations with differing ploidy levels. Table 4 Flow cytometry fluorescence measurements and ploidy levels of V. floribundum explants Experimental group n Mean fluorescence % CV Ploidy level V. floribundum 48 11682.42 3.55 2x V. floribundum (External standard) 1 12218 2.77 2x V. corymbosum (Positive control) 1 26494 3.12 4x 4. DISCUSSIONS 4.1. Dose-dependent colchicine-induced mortality in V. floribundum explants Polyploidy induction is a complex, multivariate, and difficult-to-predict process, influenced by both biological and technical factors that determine treatment efficacy (Sattler et al., 2016 ; Niazian & Nalousi, 2020 ). In this study, the response of V. floribundum to colchicine occurred on two levels: a dose-dependent effect defining the critical toxicity limit (Fig. 3a) and an accession-associated modulation that determined survival within that range (Fig. 3b). The interaction between these components accounted for the pronounced contrasts in modeled explant mortality, even under controlled conditions (Fig. 2 ). V. floribundum reached its toxicity threshold at 125 mg·L⁻¹, a concentration at which mortality became critical and further increases produced only marginal changes. Additionally, the mortality observed in control treatments (Table 2 ) suggests a slight basal susceptibility to the immersion process itself. Although this colchicine sensitivity was more pronounced than that reported for other species of the genus, it similarly lacked a linear relationship between concentration or exposure time and mortality. In V. corymbosum , Tsuda et al. ( 2012 ) observed mortality rates of up to 53.3% at 10 mg·L⁻¹ after 96 h of exposure, which then decreased to 3.3% at 50 mg·L⁻¹ under the same conditions. Likewise, Yahata et al. ( 2022 ) reported a survival rate of 76.7% at 500 mg·L⁻¹ for 72 h, which increased to 100% at 2000 mg·L⁻¹ for 48 h in V. sieboldii . However, the GLMM provided a clearer picture of the underlying trend: increasing colchicine concentration consistently raised the probability of explant mortality, whereas neither exposure time nor their interaction had detectable effects. This relationship became evident once accession was included as a random factor, allowing the model to account for the intrinsic variability among genotypes. Accessions such as Namo 09, Enca 11, and G. Pita 08 exhibited near-total mortality even at 125 mg·L⁻¹, whereas Enca 20 and Taca 01 maintained moderate survival rates even at the highest doses. This pattern aligned with observations in V. corymbosum , where mortality at 2000 mg·L⁻¹ for 24 h ranged from 53 to 100% depending on the genotype (Goldy & Lyrene, 1984 ). Similarly, in V. fuscatum (Walter et al., 2025 ), V. ashei , and V. elliotii (Goldy & Lyrene, 1984 ), genotypic sensitivity determined protocol efficiency. The magnitude of these differences suggests the presence of accession-specific mechanisms that define tolerance or susceptibility, possibly linked to the efficiency of mitotic damage repair and the differential regulation of ABC-type transporters or detoxifying enzymes, as previously proposed for V. corymbosum (Tsuda et al., 2012 ; Jarpa-Tauler et al., 2024 ). 4.2. Regenerative capacity of V. floribundum explants in response to colchicine exposure The Duncan test (p < 0.05) revealed that, in some accessions, regenerative capacity among controls varied significantly, again suggesting a basal susceptibility to the immersion process (Table 3 ). Although the ethanol present in the treatment could potentially be toxic to axillary buds, concentrations below 1.75% generally do not cause physiological damage (Zenda, 2025 ), but rather induce adaptive responses to abiotic stresses such as heat, drought, or salinity (Diot et al., 2024 ). Consequently, this variation could be attributed to the prolonged immersion of explants, implying that an optimized protocol should prioritize the use of solid media for the generation of polyploid mutants. As expected, an inverse relationship was observed between mortality and shoot regeneration. Accessions Conch 03, Taca 01, Enca 10, and Enca 08 experienced a pronounced inhibition of organogenesis with increasing colchicine concentration and exposure time, showing decreases between 60 and 90% compared to the control. In contrast, Enca 01 consistently produced few shoots (≤ 3) across treatments, indicating an intrinsically limited organogenic capacity independent of colchicine effects. Finally, Enca 20 exhibited a more stable response, without abrupt transitions between significance levels, indicating superior regenerative plasticity. Its response not only matched the control in some cases but also maintained the highest shoot regeneration rates under the most intense treatments. 4.3. V. floribundum polyploidy clones regeneration The recovery of tetraploid clones in this study was null, as no evidence of polyploidization was detected in the regenerated material. These findings reflect the low success rates and high variability reported within the genus Vaccinium , where protocol efficiency is highly dependent on the genotype used. For instance, in V. fuscatum , duplication rates of nearly 100% were achieved under 2000 mg·L⁻¹ colchicine in liquid medium for 48 h, including an isolated case of octoploidy (Walter et al., 2025 ). In contrast, V. corymbosum exposed to similar conditions reached only 4.78% (Goldy & Lyrene, 1984 ), while V. ashei and V. elliotii showed intermediate values (Lyrene & Perry, 1982 ). Even within V. corymbosum , responses ranged from 13.3% under 2000 mg·L⁻¹ for 96 h (Tsuda et al., 2012 ) to maximum rates of 9.1% or 6.7% in more recent immersion-based approaches (Jarpa-Tauler et al., 2024 ; Yahata et al., 2020). In V. floribundum , our results revealed a particularly restricted response, as no chromosomal duplications were detected in any of the treatments. Increasing colchicine concentration raised mortality without producing a corresponding rise in duplication frequency, which was counterintuitive given that the highest mutation rates are typically achieved at doses near the lethal threshold (Leitão, 2012 ). The combination of high lethality and absence of polyploids indicates that further dose escalation was not a viable strategy to enhance induction in this species. Consequently, alternative approaches based on solid media (Podwyszynska et al., 2021 ), prolonged exposure times (He et al., 2019 ), or repeated treatments (Heraldez et al., 2025 ) may represent more promising strategies, particularly when combined with resistant accessions such as Enca 20, which could promote a more stable chromosomal reorganization. Likewise, pretreatments involving cold or darkness could be explored, as these have been shown in V. corymbosum to improve cell division synchronization (Goldy & Lyrene, 1984 ), facilitating coincidence with the mitotic stage at which microtubules are most sensitive to antimitotic agents. Another alternative is the incorporation of permeabilizing agents such as dimethyl sulfoxide (1–2%), which enhances cellular uptake of the inhibitor (Shi et al., 2015 ; Tavan et al., 2015 ), or surfactants such as Tween 20, whose addition improves absorption efficiency but requires species- and explant-specific optimization (Talebi et al., 2017 ; Gantait & Mukherjee, 2021 ). 5. CONCLUSIONS This study is the first documented attempt to induce in vitro polyploidy in Vaccinium floribundum and provides new insight into its physiological response to colchicine. The results revealed a clear dose-dependent toxicity threshold and a marked genotypic influence on explant survival and regeneration. Although no polyploid plants were recovered, the species showed a narrow tolerance range strongly shaped by accession-specific variability. These findings offer valuable guidance for the refinement of polyploidization protocols and open future perspectives for developing stable polyploid lines to enhance breeding and conservation strategies within the genus Vaccinium . Abbreviations ANOVA: one-way analysis of variance; C3G: cyanidin-3-glucoside equivalents; CIRGEBB: Centro de Investigación en Recursos Genéticos, Biotecnología y Bioseguridad; CWR: crop wild relatives; FW: fresh weight; FRAP: ferric reducing antioxidant power; GLMM: generalized linear mixed model; ORAC: oxygen radical absorbance capacity; SERFOR: National Forest and Wildlife Service; TE: Trolox equivalents. Declarations Acknowledgments: Technical assistance, laboratory infrastructure, and plant material were provided by the Centro de Investigación en Recursos Genéticos, Biotecnología y Bioseguridad. This research was funded by PROCIENCIA (Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica – CONCYTEC/PROCIENCIA), contract No. PE501090007-2024-PROCIENCIA. Compliance with Ethical Standards Conflict of Interest: Not applicable. Data Availability: The datasets generated and analyzed during the current study are included in the supplementary material of this manuscript. Ethics declaration: Not applicable. References Andrade-Cuvi MJ, Moreno C, Zaro MJ, Vicente AR, Concellón A (2017) Improvement of the Antioxidant Properties and Postharvest Life of Three Exotic Andean Fruits by UV-C Treatment. 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Discovery Plants 2:268. https://doi.org/10.1007/s44372-025-00346-6 Zhang L, Shivute FN, Shahid MQ, Kamara N, Wu J, Liu X (2019) In vitro induction of auto-allotetraploid in a newly developed wild rice line from Oryza alta Swallen. Plant Cell, Tissue and Organ Culture, 139 (3), 577–587. https://doi.org/10.1007/s11240-019-01701-8 Supplementary Files TableS1.docx FigureS1.tiff FigureS2.tiff Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 10 Dec, 2025 Reviewers invited by journal 08 Dec, 2025 Editor assigned by journal 08 Dec, 2025 First submitted to journal 07 Dec, 2025 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. 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01:23:04","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23549,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/4a6d2e6c213020558692b819.png"},{"id":97934559,"identity":"2e245cce-6fa7-4d3b-8b3d-5f92d3cae8a1","added_by":"auto","created_at":"2025-12-11 01:23:04","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23102,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/fba885343e73622de64a134f.png"},{"id":98421707,"identity":"576cfd85-b191-4a34-b410-c6f3e11b1547","added_by":"auto","created_at":"2025-12-17 16:29:02","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30651,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/8517fea581bb66a238a2b7a6.png"},{"id":97934569,"identity":"8794efb9-00d6-421b-ba72-6eca3168da32","added_by":"auto","created_at":"2025-12-11 01:23:04","extension":"xml","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186395,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25009190structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/858fce3a536a9ce313ea0cec.xml"},{"id":98421642,"identity":"b76236b5-3d55-4ab1-9069-afe92c73fe7a","added_by":"auto","created_at":"2025-12-17 16:28:46","extension":"html","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":191156,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/6a10161a0d80d2c419db8a66.html"},{"id":97934539,"identity":"70221ccf-c4b3-4d37-bdd8-b5260b9f45e3","added_by":"auto","created_at":"2025-12-11 01:23:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":366527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e culture of \u003cem\u003eV. floribundum\u003c/em\u003e established from nodal explants under controlled conditions at CIRGEBB. Explants are placed horizontally on the culture medium to enhance axillary shoot proliferation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/ffdc0fa0346c4ec26757cdde.png"},{"id":97934540,"identity":"db40081b-bb27-447b-a8a2-65671d08fb94","added_by":"auto","created_at":"2025-12-11 01:23:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399123,"visible":true,"origin":"","legend":"\u003cp\u003eComparative mortality profiles for nine \u003cem\u003eV. floribundum\u003c/em\u003e accessions under nine colchicine treatments. Dark gray bars denote observed mortality and light gray bars represent model-estimated values.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/b41d6245dc3dbe1211a9ee90.png"},{"id":98422028,"identity":"141877be-f1eb-431f-b9d9-006348f0cf12","added_by":"auto","created_at":"2025-12-17 16:30:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":359455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea–b.\u003c/strong\u003eModel-derived estimates describing colchicine-induced mortality responses in \u003cem\u003eV. floribundum\u003c/em\u003e explants. \u003cu\u003eFigure 3 a.\u003c/u\u003e Forest plot of fixed-effect estimates from the generalized linear mixed model, where the intercept corresponds to 0 mg L⁻¹ colchicine at 24 h exposure. \u003cu\u003eFigure 3 b\u003c/u\u003e. Caterpillar plot of accession-level random intercepts (±95% confidence intervals), illustrating among-genotype variability in baseline mortality probability. Significance codes: * (\u0026lt;0.05), ** (\u0026lt;0.01), *** (\u0026lt;0.001), ns = not significant.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/8c2acdb3930f91661d1fc151.png"},{"id":97934542,"identity":"6192b5fe-d149-4e01-b5cf-29d8fca2f99e","added_by":"auto","created_at":"2025-12-11 01:23:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1032445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea–b. \u003c/strong\u003eFlow cytometric histograms showing nuclear DNA content in \u003cem\u003eVaccinium\u003c/em\u003e samples. \u003cu\u003eFigure 4 a.\u003c/u\u003e Experimental sample Enca 20 (\u003cem\u003eV. floribundum\u003c/em\u003e) displaying a single fluorescence peak corresponding to the diploid level (2x). \u003cu\u003eFigure 4 b.\u003c/u\u003e Calibration control exhibiting two distinct peaks: the first corresponding to the diploid external standard (2x) and the second to the tetraploid positive control (\u003cem\u003eV. corymbosum\u003c/em\u003e, 4x).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/e314001a47f3f9087f1442df.png"},{"id":98622744,"identity":"eff86e2d-1949-4e9e-a97c-ca20af42993a","added_by":"auto","created_at":"2025-12-19 17:01:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3486025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/f0cf78a6-3d6f-46f3-8b53-89ef9c3ca0c7.pdf"},{"id":97934541,"identity":"fe83ad50-9a9a-47d2-8789-8a2c7e9dcd93","added_by":"auto","created_at":"2025-12-11 01:23:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8931,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/5432ab60f347d8dc95fdc502.docx"},{"id":98421798,"identity":"56c7b637-e497-4070-90f1-bf47492383b5","added_by":"auto","created_at":"2025-12-17 16:29:29","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2167692,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/b3171b76460c6b51f2efd530.tiff"},{"id":98421687,"identity":"bbe51cbd-977a-4fdc-9615-04d9f9eb0f05","added_by":"auto","created_at":"2025-12-17 16:28:57","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2167692,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8300888/v1/3cfa5356849165a8d8872712.tiff"}],"financialInterests":"","formattedTitle":"First insights into colchicine-mediated polyploidy induction in Vaccinium floribundum Kunth","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eCrop wild relatives (CWR) are wild progenitors or closely related species of cultivated plants,, featuring primitive and heterogeneous genotypes that can easily hybridize with cultivated forms, as well as more distantly related species in the same or related genera (Tyack et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Having evolved under natural selective pressures, CWR retain extensive allelic diversity, constituting invaluable reservoirs of genetic variation that provides breeders with traits of agronomic relevance, including tolerance to drought, cold, and salinity, as well as the capacity to develop dynamic resistance to the shifting distribution and intensity of plant pathogens driven by climate change, thereby supporting long-term crop resilience (Dempewolf et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Their integration into breeding programs over the past six decades has been pivotal for agricultural innovation, with numerous documented cases of successful gene introgressions that have strengthened yield stability and food security (Hajjar \u0026amp; Hodgkin, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Globally, between 50,000 and 60,000 CWR species have been identified, of which approximately 10,000 are considered highly relevant to food production, and around 1,000 are closely related to the world\u0026rsquo;s most important crops (Maxted \u0026amp; Kell, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), collectively contributing an estimated ~\u003cspan\u003e$\u003c/span\u003e186.3\u0026nbsp;billion to the global economy in 2020 (Tyack et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the potential of CWR, their direct use in breeding programs is often constrained by crossability barriers, which limit the incorporation of genes into cultivated varieties (Bohra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To overcome this limitation, breeders employ antimitotic agents to induce polyploidization, enabling the formation of stable hybrids between distantly related species and facilitating the introgression of beneficial alleles from wild relatives into crops (Tang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This approach is widely adopted due to its capacity to generate substantial random and undirected morphological and physiological variation, resulting in individuals with novel or enhanced traits that surpass the limitations of their diploid progenitors (Jain, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Such enhancements encompass increased resistance and tolerance to biotic and abiotic stresses, greater biomass production, and improvements in the quality and quantity of bioactive metabolites (Lin et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; van de Peer et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Madani et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Morphologically, polyploids commonly exhibit enlarged organs, extended flowering periods, more abundant inflorescences, and thicker leaves and petals (Alix et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Touchell et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At the physiological and biochemical levels, genome duplication can enhance enzymatic activity and metabolic efficiency, leading to gene overexpression or silencing and the synthesis of novel compounds (Niazian \u0026amp; Nalousi, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSince the 1940s, polyploidization has been employed in the genus \u003cem\u003eVaccinium\u003c/em\u003e, one of the largest and most variable in the Ericaceae family (APG IV, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), starting with \u003cem\u003eVaccinium macrocarpon\u003c/em\u003e in which colchicine was utilized as the primary antimitotic agent (Dermen \u0026amp; Bain, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1944\u003c/span\u003e). From a cytogenetic perspective, \u003cem\u003eVaccinium\u003c/em\u003e exhibits substantial variation in ploidy levels, ranging from diploid (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24) to hexaploid (2n\u0026thinsp;=\u0026thinsp;6x\u0026thinsp;=\u0026thinsp;72), with a basic chromosome number of x\u0026thinsp;=\u0026thinsp;12 (Hummer et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which has historically limited interspecific crosses due to endosperm-related barriers, commonly referred to as the \u0026ldquo;triploid block\u0026rdquo; (H\u0026aring;kansson \u0026amp; Ellerstromsee, 1950; Esen \u0026amp; Soost, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Lafon-Placette et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Within the genera, polyploidization efforts have evolved from early \u003cem\u003ein vivo\u003c/em\u003e experiments to increasingly controlled \u003cem\u003ein vitro\u003c/em\u003e protocols targeting explants such as axillary buds and adventitious shoots (Jarpa-Tauler, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). More recently, leaf explants have enabled regeneration from single cells, reducing mixoploidy and producing auto-octoploids and chimeras in \u003cem\u003eV. corymbosum\u003c/em\u003e with induction rates up to 11% (Marangelli et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePeru harbors 13 identified wild relatives of \u003cem\u003eVaccinium\u003c/em\u003e crops, of which six are endemic (Mej\u0026iacute;a et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mostacero et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among these, \u003cem\u003eVaccinium floribundum\u003c/em\u003e Kunth (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24) (Hummer et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), a perennial shrub primarily distributed in the high-altitude p\u0026aacute;ramo and montane regions of Peru and Ecuador (Llivisaca-Contreras et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), rich nutritional and phytochemical profile. Its fruits provide nutrients, dietary fiber, and minerals such as iron, copper, zinc, and calcium, together with high concentrations of phenolic compounds and other secondary metabolites (Coba et al., 2012). Compared with cultivated blueberries such as \u003cem\u003eV. corymbosum\u003c/em\u003e and \u003cem\u003eV. virgatum\u003c/em\u003e, \u003cem\u003eV. floribundum\u003c/em\u003e exhibits equal or even higher total anthocyanin content, reaching 335\u0026ndash;477 mg C3G (cyanidin-3-glucoside equivalents) per 100 g FW (fresh weight) (P\u0026eacute;rez et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Andrade-Cuvi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, cultivated species generally range between 167 and 580 mg C3G/100 g FW depending on genotype and growing conditions (Shibata et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Smrke et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Its antioxidant activity is likewise remarkable, with FRAP (ferric reducing antioxidant power) values of about 19,653 \u0026micro;mol TE (Trolox equivalents)/100 g FW and ORAC (oxygen radical absorbance capacity) values near 40,220 \u0026micro;mol TE/100 g FW (Baenas et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), both well above those reported for commercial cultivars, with FRAP values of 2,480\u0026ndash;6,160 \u0026micro;mol TE/100 g FW (Shibata et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and ORAC values of 3,509\u0026ndash;4,841 \u0026micro;mol TE/100 g FW (Milivojevic et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These characteristics, together with favorable physicochemical traits such as high moisture content and optimal soluble solids (\u0026deg;Brix\u0026thinsp;\u0026asymp;\u0026thinsp;11\u0026ndash;16.9) (Alarc\u0026oacute;n-Barrera et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Baenas et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), highlight \u003cem\u003eV. floribundum\u003c/em\u003e as a strong candidate for breeding and the development of functional foods.\u003c/p\u003e\u003cp\u003eAs cultivated blueberries are predominantly tetraploid, direct hybridization with \u003cem\u003eV. floribundum\u003c/em\u003e is limited by ploidy mismatches, which restrict the transfer of its desirable traits into breeding lines. To date, no studies have explored chromosome doubling in this species, and its response to colchicine or other antimitotic agents remains unknown. This study therefore represents a first exploratory attempt to induce polyploidy \u003cem\u003ein vitro\u003c/em\u003e in \u003cem\u003eV. floribundum\u003c/em\u003e by treating axillary buds with colchicine, while evaluating induction efficiency, explant survival, accession-specific responses, and shoot regeneration. The results provide a basis for optimizing chromosome-doubling protocols and support future hybridization with commercial cultivars.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant material\u003c/h2\u003e\u003cp\u003ePlant material was originally collected in 2017\u0026ndash;2019 from wild populations of \u003cem\u003eV. floribundum\u003c/em\u003e located in the Cajamarca region of northern Peru,, under the General Access Resolution N.\u0026deg; 138-2019-MINAGRI-SERFOR-DGGSPFFS issued by the National Forest and Wildlife Service. A total of 67 accessions were obtained during this expedition and subsequently incorporated into the germplasm bank of the Centro de Investigaci\u0026oacute;n en Recursos Gen\u0026eacute;ticos, Biotecnolog\u0026iacute;a y Bioseguridad (CIRGEBB) at Universidad Nacional Agraria La Molina, Peru, where they have since been maintained under \u003cem\u003ein vitro\u003c/em\u003e conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). From this collection, nine accessions from five distinct collection sites within Cajamarca were selected in early 2024 for chromosome doubling experiments. Accession selection was based on previous evaluations identifying them as promising candidates due to their superior fruit size, with mean diameters greater than 0.7 cm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Polyploidy induction with colchicine\u003c/h2\u003e\u003cp\u003eShoot explants were treated with aqueous colchicine solutions (125 and 250 mg\u0026middot;L⁻\u0026sup1;) to induce polyploidy. Solutions were freshly prepared in distilled water containing 0.1% (v/v) ethanol and sterilized through 0.22 \u0026micro;m PES filters. Six explants per accession and treatment, each approximately 2 cm long with at least five nodes, were immersed in colchicine for 24, 48, or 96 h in darkness. Following exposure, explants were rinsed three times with sterile distilled water under aseptic conditions and transferred to WPM medium supplemented with 1 mg\u0026middot;L⁻\u0026sup1; kinetin. Cultures were maintained for one week in darkness, followed by a 16 h photoperiod for 12 weeks. To account for immersion time effects, colchicine-free controls were included for each exposure duration (24, 48, and 96 h), resulting in six colchicine treatments (T1\u0026ndash;T6) plus three time-matched controls (C1\u0026ndash;C3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental treatments applied for the induction of polyploidization in \u003cem\u003eV. floribundum\u003c/em\u003e using colchicine.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColchicine concentration (mg\u0026middot;L⁻\u0026sup1;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExposure time (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCode\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Statistical modeling of mortality response\u003c/h2\u003e\u003cp\u003eMortality was assessed eight weeks after colchicine treatment to capture cumulative effects on explant survival. Explants that failed to produce at least one shoot were classified as dead, and each observation was recorded as a binary response (0\u0026thinsp;=\u0026thinsp;alive, 1\u0026thinsp;=\u0026thinsp;dead) for every combination of accession, colchicine concentration and exposure time.\u003c/p\u003e\u003cp\u003eMortality was analyzed using a generalized linear mixed model (GLMM) fitted with a binomial distribution and logit link function in the glmmTMB package (R). Fixed effects included exposure time, colchicine concentration, and their interaction, whereas accession was incorporated as a random effect to account for among-accession variability. The model was specified as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:logit\\left({p}_{ijk}\\right)={\\beta\\:}_{0}+{\\beta\\:}_{1}\\times\\:{Time}_{i}+{\\beta\\:}_{2}\\times\\:{Concentration}_{j}+{\\beta\\:}_{3}\\times\\:({Time}_{i}\\times\\:{Concentration}_{j})+{u}_{k}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eijk\u003c/em\u003e\u003c/sub\u003e denotes the probability of mortality for accession \u003cem\u003ek\u003c/em\u003e under time \u003cem\u003ei\u003c/em\u003e and concentration \u003cem\u003ej\u003c/em\u003e; β\u003csub\u003e0\u003c/sub\u003e-β\u003csub\u003e3\u003c/sub\u003e represent fixed-effect coefficients; and \u003cem\u003eu\u003c/em\u003e\u003csub\u003e\u003cem\u003ek\u003c/em\u003e\u003c/sub\u003e​ is the random effect associated with accession \u003cem\u003ek\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eRandom-effect estimates were extracted using the ranef() function in glmmTMB to evaluate accession-specific patterns of colchicine tolerance and to refine fixed-effect estimates for exposure time and concentration. Pairwise contrasts between each treatment and the control within exposure times were performed with the contrast() function in emmeans, applying Tukey\u0026rsquo;s correction for multiple comparisons.\u003c/p\u003e\u003cp\u003eModel fit and residual behavior were evaluated using simulated residuals with the DHARMa package (simulateResiduals()), ensuring appropriate model specification and the validity of the inferential framework.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Assessment of regenerative capacity\u003c/h2\u003e\u003cp\u003eRegenerative capacity was evaluated eight weeks after colchicine exposure by recording the number of shoots produced per explant for each accession\u0026ndash;treatment combination. Mean regeneration values were calculated and analyzed separately for each accession using one-way analysis of variance (ANOVA), with treatment type as the fixed factor. When significant differences were detected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Duncan\u0026rsquo;s multiple range test was applied to identify homogeneous groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Determination of ploidy level by flow cytometry\u003c/h2\u003e\u003cp\u003ePloidy level was determined by flow cytometry at the Biotechnology Laboratory of the Research and Social Projection Program on Cereals and Native Grains at Universidad Nacional Agraria La Molina, using an Attune NxT cytometer (Thermo Fisher Scientific). The instrument was calibrated with diploid controls of \u003cem\u003eV. corymbosum\u003c/em\u003e and untreated \u003cem\u003eV. floribundum\u003c/em\u003e. Sample preparation followed Doležel et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) with minor modifications. Approximately 20 mg of young leaf tissue were finely chopped in 1 mL of Otto I buffer, incubated on ice for 15 min, filtered through a 42 \u0026micro;m nylon mesh, and centrifuged at 150 \u0026times; g for 5 min. The pellet was resuspended in 100 \u0026micro;L of Otto I and mixed with 1 mL of Otto II containing propidium iodide (50 \u0026micro;g\u0026middot;mL⁻\u0026sup1;) and RNase A (50 \u0026micro;g\u0026middot;mL⁻\u0026sup1;), then incubated in darkness on ice for 15\u0026ndash;60 min before analysis.\u003c/p\u003e\u003cp\u003eAt least 4 000 nuclei per sample were analyzed (λ_exc\u0026thinsp;=\u0026thinsp;535 nm, λ_em\u0026thinsp;=\u0026thinsp;617 nm). Histograms were gated to exclude debris and aggregates, and the main peak (2C) was identified for each sample. Ploidy was estimated as the ratio between the mean G₁ fluorescence intensity of the sample and that of the diploid standard. Samples with peak positions within \u0026plusmn;\u0026thinsp;5% of the diploid reference and a coefficient of variation (CV) below 5% were classified as diploid, whereas peaks corresponding to approximately twice the fluorescence were classified as tetraploid. Intermediate or irregular profiles were interpreted as potential mixoploids.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Model validation and mortality response to colchicine exposure\u003c/h2\u003e\u003cp\u003eThe GLMM fitted with a binomial distribution and logit link adequately described the mortality data, as verified by DHARMa residual diagnostics (Figure S1). The Kolmogorov\u0026ndash;Smirnov test (p\u0026thinsp;=\u0026thinsp;0.8758) and Levene\u0026rsquo;s test for homogeneity of variance (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) indicated no deviation from uniformity or heteroscedasticity, supporting the adequacy of model specification (Figure S2).\u003c/p\u003e\u003cp\u003eObserved and model-predicted mortalities exhibited consistent dose-dependent patterns across accessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under control conditions (0 mg L⁻\u0026sup1;), observed mortality (Table S1) was generally low (\u0026lt;\u0026thinsp;20%) across accessions, with the exception of Namo 09, which exhibited comparatively higher baseline mortality values. In contrast, exposure to 125 mg L⁻\u0026sup1; colchicine sharply increased mortality, reaching 100% in Namo 09, Enca 01, and Enca 11. At 250 mg L⁻\u0026sup1;, mortality exceeded 80% in G. Pita 08, Enca 01, and Enca 11, while Conch 03 and Taca 01 showed intermediate responses ranging from 30 to 70%, whereas Enca 20 exhibited the lowest observed mortality, remaining below 45% even at the highest concentration. Model-predicted probabilities closely paralleled these empirical proportions, accurately capturing the dose-response trends across accessions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Minor deviations occurred in Conch 03 and Taca 01, where stochastic variation at low mortality levels led to slight overestimation of fitted values.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eModeled explant mortality (%) across nine \u003cem\u003eV. floribundum\u003c/em\u003e accessions after colchicine treatments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAccession\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eT5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eT6\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConch 03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.72%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43.18%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52.60%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.01%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e40.15%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e71.63%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.31%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e46.28%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e65.38%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.24%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e82.38%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e87.22%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.92%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e80.49%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e93.95%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1.88%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e84.12%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e92.07%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.72%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43.18%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52.60%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.01%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e40.15%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e71.63%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.31%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e46.28%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e65.38%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.65%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e63.89%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e72.09%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e60.96%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e85.46%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.72%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e66.73%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e81.47%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.34%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e94.20%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e95.95%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17.93%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e93.48%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e98.18%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6.23%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e94.85%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e97.58%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.21%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18.26%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24.59%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.30%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16.47%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e42.59%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.09%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e20.20%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e35.70%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG. Pita 08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.44%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e92.48%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e94.72%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.19%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e91.56%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e97.61%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e4.79%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e93.30%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e96.83%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNamo 09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e82.15%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99.79%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e99.86%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e86.72%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e99.77%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e99.94%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e66.52%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e99.82%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e99.92%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTaca 01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.40%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29.94%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e38.43%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.57%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e27.39%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e58.68%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.17%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e32.63%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e51.51%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003cb\u003eNote.\u003c/b\u003e Values represent the percentage of explants showing mortality per treatment as estimated by a binomial GLMM.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFixed-effect estimates (Fig.\u0026nbsp;3a) confirmed that concentration was the primary driver of explant mortality, with β\u0026thinsp;=\u0026thinsp;4.6584 for 125 mg L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;1.98 \u0026times; 10⁻⁸) and β\u0026thinsp;=\u0026thinsp;5.0370 for 250 mg L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;1.92 \u0026times; 10⁻⁹), evidencing a strong concentration-dependent effect. In contrast, exposure duration and its interaction with concentration were non-significant, indicating that mortality remained largely independent of immersion time. Post hoc pairwise contrasts (Table S2) confirmed that exposure time did not significantly affect mortality, whereas the effect of concentration remained consistent across durations. Additionally, random effects attributable to accession (Fig.\u0026nbsp;3b) captured considerable variability in baseline mortality probability (variance\u0026thinsp;=\u0026thinsp;5.351, SD\u0026thinsp;=\u0026thinsp;2.313 log-odds units), with individual intercepts deviating markedly from the overall mean. Namo 09 (4.983) and Enca 11 (1.585) exhibited the largest positive deviations, indicating higher baseline mortality relative to the population average, whereas Enca 20 (\u0026ndash;2.701) and Taca 01 (\u0026ndash;2.052) showed the lowest baseline mortality. Accessions with values near zero, such as Enca 01 (0.340), had baseline probabilities similar to the overall mean.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;3a\u0026ndash;b.\u003c/b\u003e Model-derived estimates describing colchicine-induced mortality responses in \u003cem\u003eV. floribundum\u003c/em\u003e explants. Figure\u0026nbsp;3a. Forest plot of fixed-effect estimates from the generalized linear mixed model, where the intercept corresponds to 0 mg L⁻\u0026sup1; colchicine at 24 h exposure. Figure\u0026nbsp;3b. Caterpillar plot of accession-level random intercepts (\u0026plusmn;\u0026thinsp;95% confidence intervals), illustrating among-genotype variability in baseline mortality probability. Significance codes: * (\u0026lt;\u0026thinsp;0.05), ** (\u0026lt;\u0026thinsp;0.01), *** (\u0026lt;\u0026thinsp;0.001), ns\u0026thinsp;=\u0026thinsp;not significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Regeneration capacity after colchicine treatment\u003c/h2\u003e\u003cp\u003eShoot regeneration varied markedly among accessions and colchicine treatments (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Under control conditions, explants produced between 0.67 and 7.00 shoots per explant, evidencing wide intrinsic differences in morphogenic competence. Enca 08, Enca 10, and Enca 20 exhibited the highest baseline regeneration potential, with 7.00, 6.33, and 5.67 shoots per explant, respectively, whereas Namo 09 and G. Pita 08 remained largely unresponsive.\u003c/p\u003e\u003cp\u003eColchicine exposure led to a generalized decline in shoot regeneration, although the magnitude of inhibition was accession- and dose-dependent. Highly colchicine-sensitive accessions, such as Enca 01, Enca 11, G. Pita 08, and Namo 09, exhibited almost complete suppression across all treatments, with \u0026le;\u0026thinsp;0.67 shoots per explant even at the lowest colchicine levels. Enca 08 and Enca 10 also experienced substantial reductions with shoot production declining to 0.17\u0026ndash;1.67 per explant. In contrast, Conch 03 and Taca 01 retained moderate regenerative capacity at intermediate colchicine concentrations, producing 1.17\u0026ndash;3.33 shoots per explant. Notably, Enca 20 displayed relative tolerance, maintaining 1.67\u0026ndash;5.33 shoots per explant across treatments, indicating partial resistance to colchicine-induced inhibition.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eShoot regenerative capacity of \u003cem\u003eV. floribundum\u003c/em\u003e shoot explants after colchicine treatments\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAccession\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eC3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eT5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eT6\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConch 03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.83\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.17\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.67\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.67\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0000\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.67\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.67\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.83\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.00\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.17\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.67\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.17\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.83\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.00\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.83\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.83\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3000\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnca 20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.33\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.00\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.67\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.00\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.83\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG. Pita 08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNamo 09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTaca 01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.17\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.00\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.17\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.00\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.50\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.17\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003cb\u003eNote\u003c/b\u003e: Duncan\u0026rsquo;s multiple range test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was used to compare treatments within each accession; homogeneous groups are indicated by shared superscript letters.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Ploidy analysis\u003c/h2\u003e\u003cp\u003eA total of 48 shoots were analyzed by flow cytometry. The external standard of \u003cem\u003eV. floribundum\u003c/em\u003e showed a fluorescence value of 12,218, used as the diploid reference. Experimental samples exhibited a mean fluorescence intensity of 11,682.42 with a coefficient of variation of 3.55%, values consistent with diploidy and within the accepted range relative to the standard (\u0026plusmn;\u0026thinsp;610.9) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The histograms showed a single fluorescence peak around 11 \u0026times; 10⁴ in all experimental samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), while the calibration control displayed two distinct peaks corresponding to the diploid standard (2x) and the tetraploid positive control of \u003cem\u003eV. corymbosum\u003c/em\u003e (4x) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The absence of any shift toward the tetraploid region confirmed the nuclear homogeneity of the samples and the lack of subpopulations with differing ploidy levels.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFlow cytometry fluorescence measurements and ploidy levels of \u003cem\u003eV. floribundum\u003c/em\u003e explants\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean fluorescence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e% CV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePloidy level\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eV. floribundum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11682.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eV. floribundum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(External standard)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eV. corymbosum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(Positive control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26494\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSIONS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Dose-dependent colchicine-induced mortality in \u003cem\u003eV. floribundum\u003c/em\u003e explants\u003c/h2\u003e\u003cp\u003ePolyploidy induction is a complex, multivariate, and difficult-to-predict process, influenced by both biological and technical factors that determine treatment efficacy (Sattler et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Niazian \u0026amp; Nalousi, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the response of \u003cem\u003eV. floribundum\u003c/em\u003e to colchicine occurred on two levels: a dose-dependent effect defining the critical toxicity limit (Fig.\u0026nbsp;3a) and an accession-associated modulation that determined survival within that range (Fig.\u0026nbsp;3b). The interaction between these components accounted for the pronounced contrasts in modeled explant mortality, even under controlled conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eV. floribundum\u003c/em\u003e reached its toxicity threshold at 125 mg\u0026middot;L⁻\u0026sup1;, a concentration at which mortality became critical and further increases produced only marginal changes. Additionally, the mortality observed in control treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) suggests a slight basal susceptibility to the immersion process itself. Although this colchicine sensitivity was more pronounced than that reported for other species of the genus, it similarly lacked a linear relationship between concentration or exposure time and mortality. In \u003cem\u003eV. corymbosum\u003c/em\u003e, Tsuda et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) observed mortality rates of up to 53.3% at 10 mg\u0026middot;L⁻\u0026sup1; after 96 h of exposure, which then decreased to 3.3% at 50 mg\u0026middot;L⁻\u0026sup1; under the same conditions. Likewise, Yahata et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported a survival rate of 76.7% at 500 mg\u0026middot;L⁻\u0026sup1; for 72 h, which increased to 100% at 2000 mg\u0026middot;L⁻\u0026sup1; for 48 h in \u003cem\u003eV. sieboldii\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eHowever, the GLMM provided a clearer picture of the underlying trend: increasing colchicine concentration consistently raised the probability of explant mortality, whereas neither exposure time nor their interaction had detectable effects. This relationship became evident once accession was included as a random factor, allowing the model to account for the intrinsic variability among genotypes. Accessions such as Namo 09, Enca 11, and G. Pita 08 exhibited near-total mortality even at 125 mg\u0026middot;L⁻\u0026sup1;, whereas Enca 20 and Taca 01 maintained moderate survival rates even at the highest doses. This pattern aligned with observations in \u003cem\u003eV. corymbosum\u003c/em\u003e, where mortality at 2000 mg\u0026middot;L⁻\u0026sup1; for 24 h ranged from 53 to 100% depending on the genotype (Goldy \u0026amp; Lyrene, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Similarly, in \u003cem\u003eV. fuscatum\u003c/em\u003e (Walter et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), V. \u003cem\u003eashei\u003c/em\u003e, and \u003cem\u003eV. elliotii\u003c/em\u003e (Goldy \u0026amp; Lyrene, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), genotypic sensitivity determined protocol efficiency. The magnitude of these differences suggests the presence of accession-specific mechanisms that define tolerance or susceptibility, possibly linked to the efficiency of mitotic damage repair and the differential regulation of ABC-type transporters or detoxifying enzymes, as previously proposed for \u003cem\u003eV. corymbosum\u003c/em\u003e (Tsuda et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jarpa-Tauler et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Regenerative capacity of \u003cem\u003eV. floribundum\u003c/em\u003e explants in response to colchicine exposure\u003c/h2\u003e\u003cp\u003eThe Duncan test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed that, in some accessions, regenerative capacity among controls varied significantly, again suggesting a basal susceptibility to the immersion process (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although the ethanol present in the treatment could potentially be toxic to axillary buds, concentrations below 1.75% generally do not cause physiological damage (Zenda, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), but rather induce adaptive responses to abiotic stresses such as heat, drought, or salinity (Diot et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, this variation could be attributed to the prolonged immersion of explants, implying that an optimized protocol should prioritize the use of solid media for the generation of polyploid mutants.\u003c/p\u003e\u003cp\u003eAs expected, an inverse relationship was observed between mortality and shoot regeneration. Accessions Conch 03, Taca 01, Enca 10, and Enca 08 experienced a pronounced inhibition of organogenesis with increasing colchicine concentration and exposure time, showing decreases between 60 and 90% compared to the control. In contrast, Enca 01 consistently produced few shoots (\u0026le;\u0026thinsp;3) across treatments, indicating an intrinsically limited organogenic capacity independent of colchicine effects. Finally, Enca 20 exhibited a more stable response, without abrupt transitions between significance levels, indicating superior regenerative plasticity. Its response not only matched the control in some cases but also maintained the highest shoot regeneration rates under the most intense treatments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.3. \u003cem\u003eV. floribundum\u003c/em\u003e polyploidy clones regeneration\u003c/h2\u003e\u003cp\u003eThe recovery of tetraploid clones in this study was null, as no evidence of polyploidization was detected in the regenerated material. These findings reflect the low success rates and high variability reported within the genus \u003cem\u003eVaccinium\u003c/em\u003e, where protocol efficiency is highly dependent on the genotype used. For instance, in \u003cem\u003eV. fuscatum\u003c/em\u003e, duplication rates of nearly 100% were achieved under 2000 mg\u0026middot;L⁻\u0026sup1; colchicine in liquid medium for 48 h, including an isolated case of octoploidy (Walter et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, \u003cem\u003eV. corymbosum\u003c/em\u003e exposed to similar conditions reached only 4.78% (Goldy \u0026amp; Lyrene, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), while \u003cem\u003eV. ashei\u003c/em\u003e and \u003cem\u003eV. elliotii\u003c/em\u003e showed intermediate values (Lyrene \u0026amp; Perry, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Even within \u003cem\u003eV. corymbosum\u003c/em\u003e, responses ranged from 13.3% under 2000 mg\u0026middot;L⁻\u0026sup1; for 96 h (Tsuda et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) to maximum rates of 9.1% or 6.7% in more recent immersion-based approaches (Jarpa-Tauler et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yahata et al., 2020).\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eV. floribundum\u003c/em\u003e, our results revealed a particularly restricted response, as no chromosomal duplications were detected in any of the treatments. Increasing colchicine concentration raised mortality without producing a corresponding rise in duplication frequency, which was counterintuitive given that the highest mutation rates are typically achieved at doses near the lethal threshold (Leit\u0026atilde;o, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The combination of high lethality and absence of polyploids indicates that further dose escalation was not a viable strategy to enhance induction in this species. Consequently, alternative approaches based on solid media (Podwyszynska et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), prolonged exposure times (He et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), or repeated treatments (Heraldez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) may represent more promising strategies, particularly when combined with resistant accessions such as Enca 20, which could promote a more stable chromosomal reorganization.\u003c/p\u003e\u003cp\u003eLikewise, pretreatments involving cold or darkness could be explored, as these have been shown in V. corymbosum to improve cell division synchronization (Goldy \u0026amp; Lyrene, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), facilitating coincidence with the mitotic stage at which microtubules are most sensitive to antimitotic agents. Another alternative is the incorporation of permeabilizing agents such as dimethyl sulfoxide (1\u0026ndash;2%), which enhances cellular uptake of the inhibitor (Shi et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tavan et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), or surfactants such as Tween 20, whose addition improves absorption efficiency but requires species- and explant-specific optimization (Talebi et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gantait \u0026amp; Mukherjee, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"5. CONCLUSIONS","content":"\u003cp\u003eThis study is the first documented attempt to induce in vitro polyploidy in \u003cem\u003eVaccinium floribundum\u003c/em\u003e and provides new insight into its physiological response to colchicine. The results revealed a clear dose-dependent toxicity threshold and a marked genotypic influence on explant survival and regeneration. Although no polyploid plants were recovered, the species showed a narrow tolerance range strongly shaped by accession-specific variability. These findings offer valuable guidance for the refinement of polyploidization protocols and open future perspectives for developing stable polyploid lines to enhance breeding and conservation strategies within the genus \u003cem\u003eVaccinium\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA: one-way analysis of variance; C3G: cyanidin-3-glucoside equivalents; CIRGEBB: Centro de Investigaci\u0026oacute;n en Recursos Gen\u0026eacute;ticos, Biotecnolog\u0026iacute;a y Bioseguridad; CWR: crop wild relatives; FW: fresh weight; FRAP: ferric reducing antioxidant power; GLMM: generalized linear mixed model; ORAC: oxygen radical absorbance capacity; SERFOR: National Forest and Wildlife Service; TE: Trolox equivalents.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTechnical assistance, laboratory infrastructure, and plant material were provided by the Centro de Investigaci\u0026oacute;n en Recursos Gen\u0026eacute;ticos, Biotecnolog\u0026iacute;a y Bioseguridad. This research was funded by PROCIENCIA (Consejo Nacional de Ciencia, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n Tecnol\u0026oacute;gica \u0026ndash; CONCYTEC/PROCIENCIA), contract No. PE501090007-2024-PROCIENCIA.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated and analyzed during the current study are included in the supplementary material of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndrade-Cuvi MJ, Moreno C, Zaro MJ, Vicente AR, Concell\u0026oacute;n A (2017) Improvement of the Antioxidant Properties and Postharvest Life of Three Exotic Andean Fruits by UV-C Treatment. 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Discovery Plants 2:268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s44372-025-00346-6\u003c/span\u003e\u003cspan address=\"10.1007/s44372-025-00346-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Shivute FN, Shahid MQ, Kamara N, Wu J, Liu X (2019) \u003cem\u003eIn vitro\u003c/em\u003e induction of auto-allotetraploid in a newly developed wild rice line from \u003cem\u003eOryza alta\u003c/em\u003e Swallen. \u003cem\u003ePlant Cell, Tissue and Organ Culture, 139\u003c/em\u003e(3), 577\u0026ndash;587. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-019-01701-8\u003c/span\u003e\u003cspan address=\"10.1007/s11240-019-01701-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Vaccinium floribundum Kunth, polyploidy induction, colchicine, in vitro culture, axillary buds","lastPublishedDoi":"10.21203/rs.3.rs-8300888/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8300888/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolyploidy induction represents a promising yet challenging strategy to enhance the genetic potential of \u003cem\u003eVaccinium floribundum\u003c/em\u003e Kunth, an Andean crop wild relative of blueberry, with high nutritional and antioxidant value. This study explored for the first time the response of nine accessions to colchicine exposure as a preliminary step toward chromosome doubling. Axillary buds were treated with aqueous colchicine at 125 and 250 mg\u0026middot;L⁻\u0026sup1; for 24, 48, or 96 h and subsequently cultivated in vitro. A generalized linear mixed model (GLMM) revealed that colchicine concentration was the principal determinant of explant mortality (β₁₂₅ = 4.658, β₂₅₀ = 5.037, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas exposure duration and interaction terms were non-significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Random effects captured pronounced inter-accession heterogeneity (σ\u0026sup2; = 5.351), reflecting intrinsic variability in tolerance to colchicine toxicity. Modeled mortality increased consistently with concentration, yet certain accessions such as Enca 20 retained comparatively low mortality (\u0026lt;\u0026thinsp;45% across treatments) and partial regenerative competence, indicating genotype-dependent resilience. Flow cytometry confirmed that all regenerated plants remained diploid, indicating that chromosome doubling was not achieved under the tested conditions. These findings establish an empirical baseline for refining chromosome-doubling protocols through optimized concentrations and the selection of tolerant genotypes.\u003c/p\u003e","manuscriptTitle":"First insights into colchicine-mediated polyploidy induction in Vaccinium floribundum Kunth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 01:22:58","doi":"10.21203/rs.3.rs-8300888/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-12-10T09:37:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T13:28:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T06:07:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-12-07T11:00:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"18111b5e-61d5-4367-b3b2-fb9793837b41","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-11T01:22:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 01:22:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8300888","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8300888","identity":"rs-8300888","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}