Autopolyploidization and  in vitro regeneration of three blueberry cultivars from leaves and microstems.

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Gabriela Jarpa Tauler, Vera Martínez-Barradas, Jesús Lucina Romero-Romero, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4214823/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 Blueberries are a fruit with an increasing global demand due to their phytochemical and bioactive compounds content. They are promoted worldwide because of their health benefits. For optimal growth and productivity, blueberry crops need acidic soil pH, specific chilling hours, and an adequate atmospheric temperature. This delicate production equilibrium is under severe threat from climate change, potentially leading to reduced yields and increased cultivation costs unless new cultivars are developed for each edafoclimatic zone. Therefore, considering varietal replacements with more productive cultivars offering higher quality and better adaptability to local conditions is imperative. In this study, we employ polyploidization and in vitro tissue culture to promote variability and lay the foundation for new cultivar development. We report the successful induction of octoploids in three blueberry cultivars, namely 'Biloxi,' 'Legacy,' and 'Duke', through whole-genome duplication. Leaves and microstem explants were exposed to 0.1% colchicine for 24 and 48 hours in in vitro culture. After analyzing the polyploid level of 160 regenerated shoots using DNA flow cytometry, we obtained a total of 18 mutants, consisting of 8 mixoploids and 10 octoploids. The number of chloroplasts in the stomata was analysed by fluorescence microscopy, revealing the duplication of these organelles in the induced octoploid plants. To our knowledge, this represents the first successful induction of octoploids in three blueberry cultivars -'Biloxi,' 'Legacy,' and 'Duke'- achieved by exposing leaves and microstem explants to colchicine in in vitro culture. This technique holds promise as a valuable tool for the development of improved blueberry cultivars. Polyploidy breeding Vaccinium corymbosum L. Colchicine Whole genome duplication DNA flow cytometry Adventitious shoot regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Message This study presents the first successful induction of octoploids of three blueberry cultivars 'Biloxi', 'Legacy', and 'Duke' by inducing polyploidization exposing leaves and microstems explants to colchicine in in vitro culture. Introduction Blueberries belong to the genus Vaccinium , which comprises approximately 450–500 species (tropical and temperate) distributed in mountainous regions across most continents, excluding Oceania and Antarctica (Sleumer and Leyden 1966 ; Song and Hancock 2011 ). Blueberries belong to section Cyanococcus or true blueberries, and their fruit possesses health benefits due to its high concentration of phenolic compounds, potassium content, and dietary fiber (Prior et al. 1998 ). The health benefits of blueberries have led to a rising global demand, leading to sustained growth in blueberry production reaching 1,860,080 tons in 2022, and it is projected to surpass 2,800,000 tons by 2026 (Brazelton et al. 2023 ). Most genetic improvement initiatives began in the early 20th century in North America, involving active selection and conventional intraspecific-interspecific hybridization between more than half of the species within the section Cyanococcus, contributing to a greater or lesser way to the conformation of the genetic pool of the five types of blueberries that are currently commercialized: northern and southern highbush, half-high, rabbiteye and lowbush (Huber 2016 ). The primary cultivated blueberry worldwide is the highbush type, which is tetraploid (2n = 4x = 48), with Vaccinium corymbosum L. serving as the main species forming its genetic foundation. As consumption continues to rise, there is a pressing need to increase the yield per hectare cultivated due to limited land availability and the adverse effects of climate change. Therefore, it is crucial to increase genetic variability to develop cultivars that are more productive and better adapted to specific requirements, encompassing factors such as climate change, edaphoclimatic conditions, and extended postharvest durations, among others. Polyploidization has played a significant role in the evolution and generation of new species (Comai 2005 ). Polyploidization or whole genome duplication (WGD) represents a broad concept encompassing any mechanism resulting in the doubling of DNA content within cells or organisms, and it can occur naturally or artificially. At the cellular level, somatic duplication can occur due to failures during mitosis or through endopolyploidy (Bharadwaj 2015 ). At the organismal level, the formation of unreduced gametes (gametes with the somatic chromosome number) and polyspermy (Nakel et al. 2017 ) can lead to the development of complete polyploid organisms. A polyploid organism can be achieved artificially through the induction of unreduced gametes or genome duplication in somatic cells using chemical, physical, or biological methods. WGD results in substantial genetic and epigenetic alterations within the nuclear genome, leading to larger cell size, reduced surface area-to-volume ratio, and expanded intracellular storage capacity (Te Beest et al. 2012 ). More precisely, an increase in plant ploidy can offer several compelling agricultural advantages, including enhanced organ biomass (Guerra et al. 2014 ), such as bigger fruits (Sedov 2014 ); dwarfism in trees (Ma et al. 2016 ); altered flowering time (Dibyendu 2010 ); intensified flower color (Ghanbari et al. 2019 ); higher content of both primary (Tan et al. 2019 ) and secondary metabolites (De Jesus-Gonzalez and Weathers 2003 ); increased tolerance to abiotic (Zhang et al. 2010 ) and biotic stresses (Wang et al. 2018 ); and the production of seedless fruits. Several chemical methods consist on the application of antimitotic agents, such as colchicine, and herbicides like trifluralin or oryzalin, among others. Colchicine has been known for its usage in the treatment of gout since 1810, and in the 1930s, it began to be employed in plant breeding (Blakeslee and Avery 1937 ). This alkaloid is highly mutagenic as it acts by inhibiting the formation of microtubules, thereby preventing anaphase and the corresponding separation of sister chromatids during cell mitosis (Lu et al. 2012 ). Colchicine has been reported to induce autopolyploidy in various species, including those belonging to the genus Vaccinium (Neill and Contreras 2022 ; Valenzuela et al. 2022 ). The success of in vitro inducing autopolyploidization protocol hinges on several controllable factors, such as pre-treatment conditions (temperature, light exposure, hormone application, etc.), colchicine concentration and exposure time (penetration, agitation or shaker utilization), application method (dropping or soaked in liquid, or solid), induction medium (hormone composition, sugar concentration, etc.), incubation environment (temperature, light exposure, etc.), type of explants (meristematic or undifferentiated cells), all of which must be adjusted according to the genotypes under consideration (Dhooghe et al. 2011 ). The effectiveness of polyploidization can be evaluated by morphological evaluation, stomatal and/or pollen observations, or more precisely by chromosome counting or DNA flow cytometry. In this study, various concentrations of zeatin (ZT) were examined as growth promoters, followed by testing different durations of colchicine as inducers of autopolyploidy across three distinct types of explants. We successfully induced polyploidy among all three cultivars, evaluated by morphological and stomatal observations, and by flow cytometry, offering a valuable tool for enhancing blueberry improvement programs and establishing a fundamental basis for utilizing V. corymbosum resources in the development of innovative blueberry cultivars. Materials and methods Plant material V. corymbosum in vitro culture of 'Biloxi', 'Legacy', and 'Duke' cultivars were obtained from a national blueberry nursery company. Henceforth, when referencing the original genotype (2n = 4x = 48), these three cultivars will be labeled as 'Biloxi' control, 'Legacy' control, and 'Duke' control. For the in vitro propagation and maintenance of these cultivars, microshoot segments measuring 1–2 cm in length were cut and subcultured every 6–8 weeks in a vertical position in an in vitro propagation medium (PM) (Table 1 ). The medium was poured into glass vials (30–40 ml) and sealed with two layers of plastic wrap. Explants in media were incubated in a growth chamber at a temperature of 22°C ± 2°C and a photoperiod of 16 hours, provided by cool white, fluorescent lights emitting a photosynthetic photon flux density (PPFD) of 50–70 µmolm -2 s -1 . Table 1 Media components and zeatin concentration. Medium Components Zeatin (mg/L) Basal (BM) 2,45 mg/L of WPM with vitamins (Lloyd and McCown 1980 ), 30 g/L sucrose, 0.1 g/L myoinositol, 1 ml/L of Plant Preservation Mixture (Plant Cell Technology, Washington DC, USA), 2.45 g/L Phytagel ™ (Sigma-Aldrich Co. LLC) pH 5.1 adapted from (Qiu et al. 2018 ). 0.0 Propagation (PM) BM 0.5 Regeneration (RM) BM 1.0 Shoot regeneration PM and regeneration medium (RM), 0.5 and 1 mg/L of ZT respectively (Table 1 ), were assessed for their efficacy in inducing multiple shoot regeneration in vitro from blueberry leaves and 3-node stem segments (from now on called microstems) (Clapa et al. 2022 ). Explants in basal medium (BM) were used as control treatment (Table 1 ). Fully expanded leaves from in vitro cultures (8–12 weeks old in PM) were selected as leaf explants. The two youngest leaves from the apex were excluded. For the microstem explants, the source was part of the microshoot in vitro culture (8–12 weeks old in PM) located below the second or third youngest leaves, including three nodes without leaves. Each treatment consisted of four replicates of five explants each. Leaves were placed on solid medium, with the abaxial side of the leaf facing the media, from now on called “abaxial” explant, and adaxial side facing the media, from now on called “adaxial” explant. Microstems were placed horizontally in contact with the medium. Cultures were maintained under the previously described environmental conditions. Explants were changed to fresh media every 6 weeks. The percentage of sprouted explants per flask and the number of total sprouts per explant were evaluated at 9 weeks. Sprouted explants were considered as explants with at least one shoot equal to or greater than 2mm in length. Induction of polyploidy For polyploidy induction treatments, we employed RM (Table 1 ) containing 0.1% colchicine, with treatment durations of one and two days, modifying the approach presented by Perry ( 1984 ). A control group without the alkaloid was also included. Each treatment consisted of three replicates of ten explants per type as previously described in shoot regeneration section. After the colchicine treatment, explants were transferred to flasks containing RM (Table 1 ). Cultures were maintained under the previously described environmental conditions. Media was renewed every 6 weeks, and the percentage of sprouted explants per flask and the number of total sprouts per explant were measured at 9 weeks post-treatment. Ploidy analysis by DNA flow cytometry To carry out flow cytometry, fresh leaf samples were selected from regenerated shoots resulting from polyploidy colchicine induction treatments and their respective control. Cell nuclei from the samples were released using the razor blade chopping method as described by Galbraith et al. ( 1983 ) and with Arumuganathan and Earle ( 1991 ) protocol with one alteration. Propidium iodide was removed from the chopping buffer known as "Solution A" (Arumuganathan and Earle 1991 ). 10 mg of leaves were placed in the center of a small petri dish containing 1 mL of "Solution A," and the tissue was chopped with a scalpel for 1 minute to release cell nuclei. Next, the nuclei suspension was filtered through a 42 µm disposable nylon mesh, and the liquid was transferred to a new 2 mL Eppendorf tube and kept on ice. Subsequently, DNA was stained with 50 µg/mL propidium iodide, with simultaneous addition of 50 µg/mL RNAse. Samples were incubated on ice, in the dark for a maximum of 1h before analysis. Nuclear DNA was measured by FACSCanto II flow cytometry (BD Biosciences) with an excitation laser of 488 nm wavelength and detection fluorescence emission between 543–627 nm. Data analysis was performed using the FlowJo ™ v10.8 Software (BD Life Sciences). Evaluation of morphological and anatomical characteristics Internodal length was measured in stems developed in the last acclimatization period, from mutated and control plants. Twelve stems were selected from three induced octoploid 'Biloxi' plants, ten stems from three induced mixoploid 'Biloxi' plants, and ten stems from four 'Biloxi' control plants. Stem length was measured from the first fully expanded leaf at the apex to the last leaf at the shoot's base. Then, to obtain the mean internodal length per stem, the length of the stem was divided by the number of nodes counted within that length. Stomatal density and guard cell length were evaluated in the 3rd, 5th, and 7th fully expanded leaves (counting downwards from the apex) from acclimatized mutated and control plants, and an impression of abaxial side was taken by applying transparent nail polish as described by Beaulieu et al. ( 2008 ). Photographs of the impressions were taken with a digital camera (Moticam 5 plus) coupled to a light microscope. Stomatal density was determined in four different fields in the middle part of the leaf, and stomatal guard cell length was measured on five stomata per field as described Beaulieu et al. ( 2008 ). Images were analyzed with Motic Images Plus 3.0 software (Motic Inc., LTD. Hong Kong, China). Chloroplast number was quantified in stomata guard cells from young leaves of mutated and control individuals. A 5 mm 2 sample, without veins from a thin new leaf, was cut with a scalpel blade and mounted with Fluoromount-G on a circular slide of 25 mm diameter. At least ten stomata per sample were photographed in 3–8 different fields under confocal laser scanning microscopy with the Airyscan system (LSM 880 Airyscan; Carl Zeiss AG) with a 63x oil-immersion objective (Plan-Apochromat; Carl Zeiss AG). Total chloroplasts per stoma were counted using Fiji software (Schindelin et al. 2012 ). Statistical analysis Regeneration and induction experiments were performed in completely randomized designs. A one-way ANOVA and a Tukey post hoc test ( P ≤ 0.05) was used to analyze differences between explant type and cultivar separately. Kruskal-Wallis test and Dunn´s multiple comparison test ( P ≤ 0.05) was performed for non-parametric data. Statistical analyses of internodal length, stomata length, stomatal density, and chloroplasts per stomata were performed with one-way ANOVA and Tukey’s post hoc test ( P ≤ 0.05). Analyses were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA. Values shown in text and figures stand for mean ± standard deviation (SD). Results Shoot regeneration The overall shoot regeneration capacity of the explants was enhanced by ZT, resulting in significant increases (P ≤ 0.05) in regeneration values when treated with 1 mg/L ZT (Fig. 1 ). Therefore, this concentration was selected for use in the regeneration medium (RM) (Table 1 ). However, 'Legacy' microstems deviated from this trend, exhibiting an equivalent regeneration rate even without ZT (Fig. 1 c). Despite this equivalence, ZT positively influenced the number of shoots per 'Legacy' microstem explant, increasing it from a mean of 1.1 in control explants to a mean of 4.1 shoots per explant with 1 mg/L ZT (Fig. 1 f). In contrast, ZT was needed to induce shoot regeneration in leaf explants (Fig. 1 a, b, d, and e). Among the three cultivars, 'Legacy' exhibited the highest regeneration in terms of both the percentage of sprouted explants (66%) and the number of shoots per explant (2.7), while 'Biloxi' showed the lowest regeneration rates (39%) and number of shoots per explant (1.1) (Supplementary Table 2). In terms of explant type, adaxial regeneration exhibited the lowest values, with a sprouted explant percentage of 34% and an average of 0.7 shoots per explant (Supplementary Tables 3 and 4). Microstem presented the highest percentage of sprouted explants (84%) and matched abaxial leaf in terms of the number of shoots per explant (2.5) (Supplementary Tables 3 and 4). Induction of polyploidy Microstem regeneration in all cultivars was significantly affected ( P ≤ 0.05) by colchicine treatments, with no significant effect observed from the treatment duration (Fig. 2 c and g). Among all cultivars and explant types, 'Duke' microstems exhibited the lowest regeneration rate after two days of colchicine treatment, with 3.3% (± 5.8) of sprouted microstems and 0.03 (± 0.06) shoots per microstem (Fig. 2 c and f). On the other hand, leaf regeneration was not affected by colchicine treatments, regardless of the cultivar, for either of the evaluated regeneration criteria: percentage of sprouted explant or number of shoots per explant (Fig. 2 a, b, d, and e). Furthermore, 'Legacy' leaves, both abaxial and adaxial, exhibited superior shoot regeneration rates and a greater number of regenerated shoots per explant compared to 'Duke' and 'Biloxi' (Fig. 2 a, b, d, and e). Ploidy analysis by DNA flow cytometry Ploidy detection of putative polyploid and control individuals was conducted via flow cytometry. Fluorescence emission data were gathered and visualized using FlowJo™ v10.8 Software (BD Life Sciences) to create histograms representing the number of nuclei based on their fluorescence intensity, which are indicative of their DNA content (Fig. 3 b, d, and f). Peaks in the histograms of the putative polyploid samples were juxtaposed with those originating from the control samples (4x) to determine the relative ploidy levels, categorizing them as either tetraploid, octoploid, or mixoploid (Fig. 3 b, d, and f). Analysis using flow cytometry identified 14 induced mutant individuals, representing 10.5% of the 133 shoots examined or 2.9% of the 483 regenerated shoots resulting from the two colchicine treatments (Table 2 ). Of these, six were identified as octoploids, and eight as mixoploids (Table 2 ). Induced mutant individuals were obtained from the three cultivars, all types of explants, and from the two exposure times to colchicine (Table 3 ). The highest induction rate was 6.7%, achieved by 'Legacy' and 'Biloxi' cultivars, but in different explants and treatments; abaxial, after 2 days of treatment, and microstem, after 1 day of treatment, respectively (Table 3 ). Furthermore, in a parallel assessment involving 27 regenerated shoots, derived from preliminary experiments employing 'Biloxi' with identical explants and treatment conditions, flow cytometry revealed the induction of four octoploid variants. Table 2 Results of the induction of polyploidization by cultivar: treated explants, regenerated shoots, analyzed shoots, and mutated shoots (octoploid and mixoploid). Cultivar Treated explants Regenerated shoots Analyzed shoots Mutated shoots y Number % x Octoploid Mixoploid Number % z Number % z 'Legacy' 180 338 51 15.1 3 1.7 2 1.1 'Duke' 180 88 48 54.6 1 0.6 0 0.0 'Biloxi' 180 57 34 59.7 2 1.1 6 3.3 TOTAL 540 483 133 27.5 6 1.1 8 1.5 x The rate is calculated on the number of regenerated shoots. y The mutated shoot count represents the sum of octoploids (8x) and mixoploids (4x + 8x) detected by DNA flow cytometry analysis of samples collected from some regenerated shoots resulting from the two treatments, 133 of a total of 483 regenerated shoots. z The rate is calculated on the number of treated explants. Table 3 Results of the induction of polyploidization by cultivar and explant: colchicine exposure, treated explants, survived explants and mutated shoots (octoploid and mixoploid). Cultivar Colchicine exposure (d) Number of explants Mutated shoots x Explant Treated Survived Octoploid Mixoploid Number % y Number % y 'Legacy' Microstem 1 30 27 0 0.0 2 6.7 2 30 30 0 0.0 0 0.0 Abaxial leaf 1 30 22 0 0.0 0 0.0 2 30 22 2 6.7 0 0.0 Adaxial leaf 1 30 30 0 0.0 0 0.0 2 30 30 1 3.3 0 0.0 'Duke' Microstem 1 30 21 1 3.3 0 0.0 2 30 23 0 0.0 0 0.0 Abaxial leaf 1 30 13 0 0.0 0 0.0 2 30 16 0 0.0 0 0.0 Adaxial leaf 1 30 30 0 0.0 0 0.0 2 30 30 0 0.0 0 0.0 'Biloxi' Microstem 1 30 30 2 6.7 4 13.3 2 30 30 0 0.0 2 6.7 Abaxial leaf 1 30 24 0 0.0 0 0.0 2 30 27 0 0.0 0 0.0 Adaxial leaf 1 30 30 0 0.0 0 0.0 2 30 30 0 0.0 0 0.0 x The mutated shoots count represents the sum of octoploids (8x) and mixoploids (4x + 8x) detected by DNA flow cytometry analysis of samples collected from some regenerated shoots resulting from the two treatments, 133 of a total of 483 regenerated shoots. y The rate is calculated on the number of treated explants. Evaluation of morphological and anatomical characteristics. Some induced octoploid plants differed phenotypicaly from the original cultivar, as shown in Fig. 3 a, c, and e, exhibiting thicker stems (Zeldin and McCown 2002 ; He et al. 2019 ) and shorter internodes (Dermen and Bain 1944 ; He et al. 2019 ). Supplementary Fig. 1 presents the mean of the internodal length of stem samples from control, octoploid, and mixoploid 'Biloxi' acclimatized plants. The mean internodal length of induced octoploids 'Biloxi' is significantly shorter than the tetraploids 'Biloxi' control (6.5 and 10.8 mm, respectively) (Supplementary Fig. 1). As shown in Fig. 4 c, induced octoploids 'Legacy-L116' and 'Legacy-L317' exhibit an increase of more than double (267% and 208%, respectively) in the number of chloroplasts per stoma compared to the tetraploid. The induced octoploid 'Duke-U7' showed a doubling of chloroplasts, a significant 38% increase in stomatal length, and a non-significant 23% decrease in stomatal density compared to the tetraploid (Fig. 4 ). In the case of 'Biloxi' mutants, the mixoploids, which exhibit a decrease in stomatal length, showed an increase in stomatal density, in contrast, the octoploids presented an equal stomatal size and density as the tetraploid (Fig. 4 a and b). Discussion Shoot regeneration In our study, ZT concentration, genotype, type of explant, and their interaction significantly influenced the percentage of sprouted explants and the number of shoots per explant (Supplementary Figs. 2 and 3). Notably, 'Legacy' exhibited a more robust regenerative response in the presence of ZT than the other cultivars (Fig. 1 ), supporting the regeneration disparities between genotypes documented in the literature (Clapa et al. 2022 ). This indicates that the addition of growth regulators notably interacts with explant´s endogenous hormones (Gahan and George 2008 ), generating different responses per cultivar depending on their proper inner hormone levels or sensitivity. This suggests that the medium with the proposed concentration of the growth regulator is more favorable for 'Legacy', either due to a better combination of 'Legacy's endogenous hormones with the applied growth regulator or because of a greater sensitivity of this genotype to ZT (Gahan and George 2008 ). Among the three types of explants, microstem regeneration was achieved even without ZT (Fig. 1 c and f). This phenomenon can be attributed to the presence of axilary buds in the microstem, capable of sprouting even in the absence of ZT (Debnath 2007 ). However, 1 mg/L ZT significantly increased the number of shoots produced per microstem (Fig. 1 f), as expected due to its known function in promoting axillary bud growth (Tamas 1995 ). With an average of three or more shoots per microstem explant, it confirmed the positive response of microstem regeneration to ZT (Clapa et al. 2022 ). In the case of leaf explants, no matter their position towards the medium, regeneration was not achieved without the application of ZT, extending the need for growth regulator application for this type of explants to our studied cultivars (Fig. 1 a, b, d and e). Our data suggest that the abaxial position of the leaves in contact with the medium containing ZT is more favorable than the adaxial position (see Supplementary Tables 3 and 4), contrary to what was reported by Debnath ( 2005 ). Generally, there is no consensus on the optimal orientation. However, in this instance, greater regeneration could have been achieved with the abaxial orientation, as it would provide a larger surface area of the explant in contact with the medium. This is because when the adaxial side of the leaf is positioned in contact with the medium, it tends to roll up at both ends (Ricci et al. 2020 ). 'Legacy' leaves in the abaxial position had the best responses among explants, achieving the highest number of shoots per explant (8.4) with 1mg/L ZT (Fig. 1 d). Induction of polyploidy The microstem explants of all cultivars were significantly affected by colchicine treatments (Fig. 2 c and g). In leaves, colchicine application caused a decrease of the percentage of sprouted explants and the number of shoots per explant (Fig. 2 a, b, d and f). This results reveal the impact of colchicine on physiological processes resulting in a cessation of growth in the treated tissues (Dermen and Bain 1944 ), especially in treated microstems. A hypothesis that could explain the significant susceptibility of microstems to colchicine treatments is that in axilary buds, there is growth of meristematic cells before the application of colchicine, which get affected by the chemical. In leaf explants, however, the increase in cells is induced simultaneously with the application of colchicine. 'Legacy' abaxial explant was the only exception, where 2 days of colchicine treatment did not affect shoot regeneration (Fig. 2 a and d). This phenomenon could be attributed to colchicine’s potential to harm the leaves due to its toxic nature (Dermen 1940 ; Espino and Vazquez 1981 ). Paradoxically, as documented in previous research (Callow et al. 1989 ), leaf injury may enhance sprouting capacity, possibly either due to the accumulation of endogenous hormones in that area or because more growth regulator enters via the wound (Gahan and George 2008 ), thereby mitigating the expected opposing effect of colchicine in reducing regeneration. Ploidy analysis by DNA flow cytometry DNA flow cytometry has the capability to analyze the ploidy of all layers within a tissue sample and can effectively detect mixoploids. In this study, mixoploids were exclusively obtained in microstem explants treated (Table 3 ), confirming Broertjes and Keen ( 1980 ) observation of a reduced occurrence of chimeric shoots or plantlets when induction was initiated before the formation of adventitious buds, as polyploid induction is considered a one-cell-event. We validated the findings of earlier studies (Cui et al. 2017 ; Ren et al. 2024 ) indicating that mixoploids had lower incidence or not produced when inducing polyploidy from leaf explants. Evaluation of morphological and anatomical characteristics. In this study, the number of chloroplasts per stomata (two guard cells) aligns with flow cytometry results, serving as an indicator of ploidy levels (Dhooghe et al. 2011 ). However, chloroplasts number does not discriminate mixoploids with polyploid epidermis (L1). Stomatal length and density have been recognized as valuable criteria for discriminating between polyploid variations, as there exists a positive and negative correlation, respectively across a broad spectrum of angiosperms (Beaulieu et al. 2008 ). Several authors have used increased stomatal (Norden 2017 ) and leaf (Yahata et al. 2022 ) size, and decreased stomatal density (Podwyszyńska and Pluta 2019 ) to distinguish ploidy levels after inducing polyploidization in the Vaccinium genus. The induced mixoploid 'Legacy-L9', following what the literature reports, shows an increase in stomatal length (Fig. 4 a), and a decreased stomatal density (Fig. 4 b), these values could suggest that the epidermis is polyploid, consequently the plant would be a periclinal chimera. In the induced octoploid 'Duke-U7' plant, although the stomatal length increases significantly (Fig. 4 a), and the stomatal density decreases (Fig. 4 b), but there is a high variability between the data, which did not allow to obtain a statistical difference. Previous reports had only screened shoots with increased diameter during the in vitro phase (Chandler and Lyrene 1982 ; Marangelli et al. 2022 ). Our research has uncovered that octoploids and mixoploids can emerge without exhibiting discernible morphological and anatomical disparities. If the induced octoploid clones, 'Biloxi-V160,' 'Biloxi-V203,' and 'Biloxi-W7', were classified solely based on stomatal length and density, they would have been excluded like octoploids (Fig. 4 a and b). We observed a sole mixoploid plant with phenological variations during the greenhouse acclimatization process, and no mixoploids during the in vitro culture phase. Only three octoploids were detected through phenotypical differences during in vitro culture such as thicker shoots, shorter internodes, and slow growth (Fig. 3 a). The induced octoploid 'Legacy-L116' clones exhibited thicker stems, shorter internodes, and slow growth in in vitro culture (Fig. 3 a). These clones also demonstrated low vigor during the acclimatization stage, a characteristic reported in several studies (Goldy 1983 ; Miyashita et al. 2009 ; Xue et al. 2017 ). The observed low vigor could be attributed to variations in gene expression, arising from differences in chromosome dosage or epigenetic modifications within WGD (Adams and Wendel 2005 ). Alternatively, it may be attributed to the fact that induced polyploids need to replicate larger amounts of nuclear DNA in larger cells, leading to extended cell cycle durations and contributing to slower development (Van Drunen and Husband 2018 ; Kashtwari et al. 2022 ). This lower growth rate of polyploidized plants could give them an advantage since it allows them to conserve resources and mitigate cellular damage in conditions of stress due to drought, cold or nutrients (Deng et al. 2012 ). We also observed that five induced octoploids exhibited dwarfism (Fig. 3 c and e). This reduced plant height could be caused by changes in gene expression after WGD involved in reducing hormone levels like indoleacetic acid (IAA) and ethylene reported in autotetraploid birch plants (Mu et al. 2012 ), or IAA and brassinosteroid reported in autotetraploid apple plants (Ma et al. 2016 ). Despite mixoploids not being the focus of polyploidization breeding programs, they could also possess valuable traits such as larger fruit size, fewer seeds, and thicker pericarp than diploid lines, as observed in Fortunella crassifolia periclinal chimeras (Nukaya et al. 2019 ). Conclusion Our results confirmed ZT as an adequate growth regulator to promote in vitro shoot regeneration in V. corymbosum. An ideal concentration for shoot promotion was established for three different explants: abaxial and adaxial leaves and microstems for three commercially valuable cultivars 'Biloxi', 'Legacy', and 'Duke'. In vitro shoot regeneration was standardized for these cultivars and explants, and it was a necessary step for in vitro polyploidization in blueberry breeding. Octoploids and mixoploids were successfully generated for the three cultivars. Polyploidization induction pursues to increase genetic variation within an individual, which in our case the observed phenotypes suggest was achieved due the phenotypical differences. Additionally, our research points to anatomical characteristics not being an adequate method for preliminary screening of polyploids in early developmental stages, pointing out flow cytometry as the most reliable method for polyploidy identification. The obtained octoploid plants of V. corymbosum hold significant potential for the development of superior cultivars and represent an asset for future breeding programs. Furthermore, it is essential to investigate the flowering patterns, fruit yield, and fruit quality of all the induced mutant blueberries in future studies. As well, the newly developed polyploid blueberry variants will need further investigation concerning their resistance levels against biotic and abiotic stressors. Declarations Competing Interests The authors declare that they have no relevant financial or non-financial interests to disclose. Funding GJT was supported by Agencia Nacional de Investigación y Desarrollo (Chile) 21191617 PhD scholarship and the 2018 Doctoral Scholarship from Vicerrectoría de Investigación of Pontificia Universidad Católica de Chile. VMB was supported by Consejo Nacional de Ciencia y Tecnología (México) 739582, and Agencia Nacional de Investigación y Desarrollo (Chile) 21200394 PhD scholarships. Author Contributions Patricio Arce-Johnson guided the research. Patricio Arce-Johnson and Gabriela Jarpa-Tauler conceived and designed the research. Gabriela Jarpa-Tauler conducted the material preparation, experiments, including induction treatment, seedling management, and plant acclimatization. Gabriela Jarpa-Tauler, Jesús Lucina Romero-Romero and Vera Martínez-Barradas analyzed the data. Gabriela Jarpa-Tauler wrote and revised the manuscript. Vera Martínez-Barradas revised the manuscript. All authors have read and approved the final manuscript. Acknowledgments The authors thank Alex Cabrera and Sergio Bustos for their technical assistance in the flow cytometry service, Pamela Naulin for her supervision in the plant experimentation unit center of the UC Faculty of Biological Science, and Nicole Salgado for her technical support with the confocal microscope in the advanced microscopy facility UMA UC, of the Pontificia Universidad Católica de Chile. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Adams KL, Wendel JF (2005) Polyploidy and genome evolution in plants. Curr Opin Plant Biol 8:135–141. https://doi.org/10.1016/j.pbi.2005.01.001 Arumuganathan K, Earle ED (1991) Estimation of nuclear DNA content of plants by flow cytometry. 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Acta Hortic 175–180. https://doi.org/10.17660/actahortic.2002.574.26 Zhang XY, Hu CG, Yao JL (2010) Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance. J Plant Physiol 167:88–94. https://doi.org/10.1016/j.jplph.2009.07.006 Supplementary Files Supplementary Figure 1 to 2 and Tables 1 to 4 are not available with this version. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 11 Apr, 2024 Reviewers invited by journal 10 Apr, 2024 Editor assigned by journal 10 Apr, 2024 First submitted to journal 03 Apr, 2024 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4214823","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289784863,"identity":"18540a4d-ce4b-4845-a201-0dece6cf217d","order_by":0,"name":"Gabriela Jarpa Tauler","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAnUlEQVRIiWNgGAWjYFCCAyDCBkQwHiBFSxqCSSw4TIIWecfDzz4X1JxP7G9gfkCcFsMDx4xnzzh2O3HGATYDIrU0HDBm5mG7nbiBgYdIhxk2HP/MzPPvHAla5BnOGDPzth0gQYsBw5li5pl9ycYzDhPrF/kZxzczF3yzk+1vb374gDhbbhxgYAazmIlSD7Klv4F4xaNgFIyCUTBCAQBkqjMRIHaPzQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-4082-2742","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":true,"prefix":"","firstName":"Gabriela","middleName":"Jarpa","lastName":"Tauler","suffix":""},{"id":289784864,"identity":"96fe6a53-29b1-40fe-8e08-700ccf78551c","order_by":1,"name":"Vera Martínez-Barradas","email":"","orcid":"","institution":"Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Vera","middleName":"","lastName":"Martínez-Barradas","suffix":""},{"id":289784865,"identity":"083d7646-13c2-4d82-8da1-13196888e6c9","order_by":2,"name":"Jesús Lucina Romero-Romero","email":"","orcid":"","institution":"Centro de Ciencias de Sinaloa","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"Lucina","lastName":"Romero-Romero","suffix":""},{"id":289784866,"identity":"4e7a4721-1b39-46f7-975b-7d60859709a5","order_by":3,"name":"Patricio Arce-Johnson","email":"","orcid":"","institution":"Universidad Autonoma de Chile - Sede Temuco: Universidad Autonoma de Chile","correspondingAuthor":false,"prefix":"","firstName":"Patricio","middleName":"","lastName":"Arce-Johnson","suffix":""}],"badges":[],"createdAt":"2024-04-03 21:25:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4214823/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4214823/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54714153,"identity":"292b5a24-5a75-4e36-a943-1c92b37bcaec","added_by":"auto","created_at":"2024-04-15 15:23:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of ZT concentration on shoot regeneration.\u003c/strong\u003e Effect of ZT concentration on percentages of sprouted explants (a, b, and c), and number of regenerated shoots (d, e, and f) per abaxial and adaxial side down on the media, and microstem horizontally on the media. Each cultivar, 'Legacy' (black color), 'Duke' (dark grey color), and 'Biloxi' (light grey color), was exposed to 0 (diagonally hatched bars), 0.5 ( horizontally hatched bars), and 1 mg/L ZT (filled bars) for nine weeks. The percentage of sprouted explants is expressed as the mean ± standard deviation (SD) of four replicates (five explants per vial). The number of shoots/explant is registered as the mean ± SD of four replicates (total number of sprouts per vial / total number of explants in the vial). One-way ANOVA was performed between explant type and cultivar separately. Tukey’s test (\u003cem\u003eP\u003c/em\u003e≤0.05) performed multiple comparisons of the averages. Kruskal-Wallis´s test was performed for samples with non-parametric distribution and multiple comparison of the averages was performed by Dunn´s multiple comparison test (\u003cem\u003eP\u003c/em\u003e≤0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4214823/v1/fc5526953d7ecd3647794210.png"},{"id":54713556,"identity":"bbddc4a7-3cc8-4033-b85f-2f1183ba3b64","added_by":"auto","created_at":"2024-04-15 15:15:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of exposure time to 0.1% colchicine treatment\u003c/strong\u003e \u003cstrong\u003eon shoot regeneration.\u003c/strong\u003e Effect of exposure time to 0.1% colchicine treatment on percentages of sprouted explants (a, b, and c), and number of regenerated shoots (d, e, and f) per abaxial and adaxial side down on the media, and microstem horizontally on the media. Results are shown separately by cultivar: 'Legacy' (black color), 'Duke' (dark grey color), and 'Biloxi' (light grey color). Explant groups were evaluated at 1 (horizontally hatched bars) and 2 days (diagonally hatched bars) under 0.1% colchicine treatment and compared with a no-colchicine control (filled bars) after nine weeks in regeneration media. The percentage of sprouted explants expressed as the mean ± SD of three replicates.The number of shoots/explant is presented as the mean ± SD of three replicates, after counting the total number of sprouts per vial and dividing it by the total number of explants in the vial (ten explants per vial). One-way ANOVA was performed between explant type and cultivar separately. Tukey’s test (\u003cem\u003eP\u003c/em\u003e≤0.05) performed multiple comparisons of the averages. Kruskal-Wallis´s test was performed for samples with non-parametric distribution and multiple comparison of the averages was performed by Dunn´s multiple comparison test (\u003cem\u003eP\u003c/em\u003e≤0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4214823/v1/bf97c2fef7e67c96620c847f.png"},{"id":54713552,"identity":"7ba5be1c-f493-4094-bc7a-b669922c6d2e","added_by":"auto","created_at":"2024-04-15 15:15:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePolyploidy detection through phenotypic differentiation and flow cytometry analysis.\u003c/strong\u003e (a) 'Legacy' \u003cem\u003ein vitro \u003c/em\u003eshoots. Induced octoploid 'Legacy-L116' \u003cem\u003ein vitro \u003c/em\u003eshoot (on the left) and tetraploid control 'Legacy' \u003cem\u003ein vitro \u003c/em\u003eshoot (on the right). Scale bar corresponds to 10 mm. (b) Histogram of DNA content of tetraploid control 'Legacy' \u003cem\u003ein vitro \u003c/em\u003eshoot (red) and induced octoploid 'Legacy-L116' \u003cem\u003ein vitro \u003c/em\u003eshoot(blue). (c) Acclimatized 'Duke' plants. Induced octoploid 'Duke-U7' plant (on the left) and tetraploid control 'Duke' plant (on the right). (d) Histogram of DNA content of tetraploid control 'Duke' (red) and induced octoploid of 'Duke-U7' (blue). (e) Acclimatized 'Biloxi' plants. Tetraploid 'Biloxi' control plant (on the left) and induced octoploid 'Biloxi-W7' plant (on the right). (f) Histogram of DNA content of tetraploid control 'Biloxi' plant (red) and induced octoploid 'Biloxi-W7' plant (blue).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4214823/v1/8e7a23701858415c4a6b7197.png"},{"id":54713558,"identity":"5b720f26-b4f5-47fa-9b49-c43747ff9dd2","added_by":"auto","created_at":"2024-04-15 15:15:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of stomatal length, stomatal density, and number of chloroplasts between mutated and control individuals. a)\u003c/strong\u003e Stomatal length (μm) \u003cstrong\u003e(b) \u003c/strong\u003eNumber of stomata per mm2 \u003cstrong\u003e(c) \u003c/strong\u003eNumber of chloroplasts per stomata. 'Legacy' (black color), 'Duke' (dark grey color), and 'Biloxi' (light grey color). Tetraploid control individuals (unfilled bars), induced octoploid individuals (filled bars), and induced mixoploid individuals (diagonally hatched bars). Data are presented as mean ± SD, different letters denote significant differences among ploidy (\u003cem\u003eP\u003c/em\u003e≤0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4214823/v1/dea2f140c7ebd1e055af6aa4.png"},{"id":54713567,"identity":"9b09b07e-9e55-460b-bef1-7d1c68404f69","added_by":"auto","created_at":"2024-04-15 15:15:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImages of stomata and chloroplasts of mutated and control individuals. (a) \u003c/strong\u003eAbaxial side´s leaf of an acclimatized tetraploid control 'Legacy' plant. Scale bar corresponds to 50 μm. \u003cstrong\u003e(b) \u003c/strong\u003eAbaxial side´s leaf of an acclimatized induced mixoploid 'Legacy-L9' plant. Scale bar corresponds to 50 μm.\u003cstrong\u003e (c)\u003c/strong\u003e Confocal fluorescence image of chloroplasts (pointed with white arrows) from a stoma of a tetraploid control 'Legacy' \u003cem\u003ein vitro \u003c/em\u003eshoot. \u003cstrong\u003e(d)\u003c/strong\u003e Confocal fluorescence image of chloroplasts (pointed with white arrows) from a stomata of an induced octoploid 'Legacy-L116'\u003cem\u003e in vitro \u003c/em\u003eshoot.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4214823/v1/c1c108cbb955f6d8f6e4fb8d.png"},{"id":54714810,"identity":"3c389599-9ad7-4090-b301-69dc4926549e","added_by":"auto","created_at":"2024-04-15 15:31:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":886223,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4214823/v1/ac6fc7c1-7d31-4d8e-a9fe-a04af729ca0a.pdf"}],"financialInterests":"","formattedTitle":"Autopolyploidization and in vitro regeneration of three blueberry cultivars from leaves and microstems.","fulltext":[{"header":"Key Message","content":"\u003cp\u003eThis study presents the first successful induction of octoploids of three blueberry cultivars \u0026apos;Biloxi\u0026apos;, \u0026apos;Legacy\u0026apos;, and \u0026apos;Duke\u0026apos; by inducing polyploidization exposing leaves and microstems explants to colchicine in \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eculture.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eBlueberries belong to the genus \u003cem\u003eVaccinium\u003c/em\u003e, which comprises approximately 450\u0026ndash;500 species (tropical and temperate) distributed in mountainous regions across most continents, excluding Oceania and Antarctica (Sleumer and Leyden \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Song and Hancock \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Blueberries belong to section Cyanococcus or true blueberries, and their fruit possesses health benefits due to its high concentration of phenolic compounds, potassium content, and dietary fiber (Prior et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The health benefits of blueberries have led to a rising global demand, leading to sustained growth in blueberry production reaching 1,860,080 tons in 2022, and it is projected to surpass 2,800,000 tons by 2026 (Brazelton et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Most genetic improvement initiatives began in the early 20th century in North America, involving active selection and conventional intraspecific-interspecific hybridization between more than half of the species within the section Cyanococcus, contributing to a greater or lesser way to the conformation of the genetic pool of the five types of blueberries that are currently commercialized: northern and southern highbush, half-high, rabbiteye and lowbush (Huber \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The primary cultivated blueberry worldwide is the highbush type, which is tetraploid (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48), with \u003cem\u003eVaccinium corymbosum\u003c/em\u003e L. serving as the main species forming its genetic foundation.\u003c/p\u003e \u003cp\u003eAs consumption continues to rise, there is a pressing need to increase the yield per hectare cultivated due to limited land availability and the adverse effects of climate change. Therefore, it is crucial to increase genetic variability to develop cultivars that are more productive and better adapted to specific requirements, encompassing factors such as climate change, edaphoclimatic conditions, and extended postharvest durations, among others.\u003c/p\u003e \u003cp\u003ePolyploidization has played a significant role in the evolution and generation of new species (Comai \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Polyploidization or whole genome duplication (WGD) represents a broad concept encompassing any mechanism resulting in the doubling of DNA content within cells or organisms, and it can occur naturally or artificially. At the cellular level, somatic duplication can occur due to failures during mitosis or through endopolyploidy (Bharadwaj \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). At the organismal level, the formation of unreduced gametes (gametes with the somatic chromosome number) and polyspermy (Nakel et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) can lead to the development of complete polyploid organisms. A polyploid organism can be achieved artificially through the induction of unreduced gametes or genome duplication in somatic cells using chemical, physical, or biological methods. WGD results in substantial genetic and epigenetic alterations within the nuclear genome, leading to larger cell size, reduced surface area-to-volume ratio, and expanded intracellular storage capacity (Te Beest et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). More precisely, an increase in plant ploidy can offer several compelling agricultural advantages, including enhanced organ biomass (Guerra et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), such as bigger fruits (Sedov \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); dwarfism in trees (Ma et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); altered flowering time (Dibyendu \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e); intensified flower color (Ghanbari et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); higher content of both primary (Tan et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and secondary metabolites (De Jesus-Gonzalez and Weathers \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e); increased tolerance to abiotic (Zhang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and biotic stresses (Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); and the production of seedless fruits.\u003c/p\u003e \u003cp\u003eSeveral chemical methods consist on the application of antimitotic agents, such as colchicine, and herbicides like trifluralin or oryzalin, among others. Colchicine has been known for its usage in the treatment of gout since 1810, and in the 1930s, it began to be employed in plant breeding (Blakeslee and Avery \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1937\u003c/span\u003e). This alkaloid is highly mutagenic as it acts by inhibiting the formation of microtubules, thereby preventing anaphase and the corresponding separation of sister chromatids during cell mitosis (Lu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Colchicine has been reported to induce autopolyploidy in various species, including those belonging to the genus \u003cem\u003eVaccinium\u003c/em\u003e (Neill and Contreras \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Valenzuela et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe success of in vitro inducing autopolyploidization protocol hinges on several controllable factors, such as pre-treatment conditions (temperature, light exposure, hormone application, etc.), colchicine concentration and exposure time (penetration, agitation or shaker utilization), application method (dropping or soaked in liquid, or solid), induction medium (hormone composition, sugar concentration, etc.), incubation environment (temperature, light exposure, etc.), type of explants (meristematic or undifferentiated cells), all of which must be adjusted according to the genotypes under consideration (Dhooghe et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The effectiveness of polyploidization can be evaluated by morphological evaluation, stomatal and/or pollen observations, or more precisely by chromosome counting or DNA flow cytometry.\u003c/p\u003e \u003cp\u003eIn this study, various concentrations of zeatin (ZT) were examined as growth promoters, followed by testing different durations of colchicine as inducers of autopolyploidy across three distinct types of explants. We successfully induced polyploidy among all three cultivars, evaluated by morphological and stomatal observations, and by flow cytometry, offering a valuable tool for enhancing blueberry improvement programs and establishing a fundamental basis for utilizing \u003cem\u003eV. corymbosum\u003c/em\u003e resources in the development of innovative blueberry cultivars.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003e \u003cem\u003eV. corymbosum in vitro\u003c/em\u003e culture of 'Biloxi', 'Legacy', and 'Duke' cultivars were obtained from a national blueberry nursery company. Henceforth, when referencing the original genotype (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48), these three cultivars will be labeled as 'Biloxi' control, 'Legacy' control, and 'Duke' control. For the \u003cem\u003ein vitro\u003c/em\u003e propagation and maintenance of these cultivars, microshoot segments measuring 1\u0026ndash;2 cm in length were cut and subcultured every 6\u0026ndash;8 weeks in a vertical position in an \u003cem\u003ein vitro\u003c/em\u003e propagation medium (PM) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The medium was poured into glass vials (30\u0026ndash;40 ml) and sealed with two layers of plastic wrap. Explants in media were incubated in a growth chamber at a temperature of 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and a photoperiod of 16 hours, provided by cool white, fluorescent lights emitting a photosynthetic photon flux density (PPFD) of 50\u0026ndash;70 \u0026micro;molm\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\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\u003eMedia components and zeatin concentration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComponents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeatin\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal (BM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,45 mg/L of WPM with vitamins (Lloyd and McCown \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1980\u003c/span\u003e),\u003c/p\u003e \u003cp\u003e30 g/L sucrose,\u003c/p\u003e \u003cp\u003e0.1 g/L myoinositol,\u003c/p\u003e \u003cp\u003e1 ml/L of Plant Preservation Mixture (Plant Cell Technology, Washington DC, USA),\u003c/p\u003e \u003cp\u003e2.45 g/L Phytagel\u003csup\u003e\u0026trade;\u003c/sup\u003e (Sigma-Aldrich Co. LLC)\u003c/p\u003e \u003cp\u003epH 5.1\u003c/p\u003e \u003cp\u003eadapted from (Qiu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropagation (PM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegeneration (RM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \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=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eShoot regeneration\u003c/h2\u003e \u003cp\u003ePM and regeneration medium (RM), 0.5 and 1 mg/L of ZT respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), were assessed for their efficacy in inducing multiple shoot regeneration \u003cem\u003ein vitro\u003c/em\u003e from blueberry leaves and 3-node stem segments (from now on called microstems) (Clapa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Explants in basal medium (BM) were used as control treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fully expanded leaves from \u003cem\u003ein vitro\u003c/em\u003e cultures (8\u0026ndash;12 weeks old in PM) were selected as leaf explants. The two youngest leaves from the apex were excluded. For the microstem explants, the source was part of the microshoot \u003cem\u003ein vitro\u003c/em\u003e culture (8\u0026ndash;12 weeks old in PM) located below the second or third youngest leaves, including three nodes without leaves. Each treatment consisted of four replicates of five explants each. Leaves were placed on solid medium, with the abaxial side of the leaf facing the media, from now on called \u0026ldquo;abaxial\u0026rdquo; explant, and adaxial side facing the media, from now on called \u0026ldquo;adaxial\u0026rdquo; explant. Microstems were placed horizontally in contact with the medium. Cultures were maintained under the previously described environmental conditions. Explants were changed to fresh media every 6 weeks. The percentage of sprouted explants per flask and the number of total sprouts per explant were evaluated at 9 weeks. Sprouted explants were considered as explants with at least one shoot equal to or greater than 2mm in length.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInduction of polyploidy\u003c/h2\u003e \u003cp\u003eFor polyploidy induction treatments, we employed RM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) containing 0.1% colchicine, with treatment durations of one and two days, modifying the approach presented by Perry (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). A control group without the alkaloid was also included. Each treatment consisted of three replicates of ten explants per type as previously described in \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003eshoot regeneration\u003c/span\u003e section. After the colchicine treatment, explants were transferred to flasks containing RM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cultures were maintained under the previously described environmental conditions. Media was renewed every 6 weeks, and the percentage of sprouted explants per flask and the number of total sprouts per explant were measured at 9 weeks post-treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePloidy analysis by DNA flow cytometry\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo carry out flow cytometry, fresh leaf samples were selected from regenerated shoots resulting from polyploidy colchicine induction treatments and their respective control. Cell nuclei from the samples were released using the razor blade chopping method as described by Galbraith et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and with Arumuganathan and Earle (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) protocol with one alteration. Propidium iodide was removed from the chopping buffer known as \"Solution A\" (Arumuganathan and Earle \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). 10 mg of leaves were placed in the center of a small petri dish containing 1 mL of \"Solution A,\" and the tissue was chopped with a scalpel for 1 minute to release cell nuclei. Next, the nuclei suspension was filtered through a 42 \u0026micro;m disposable nylon mesh, and the liquid was transferred to a new 2 mL Eppendorf tube and kept on ice. Subsequently, DNA was stained with 50 \u0026micro;g/mL propidium iodide, with simultaneous addition of 50 \u0026micro;g/mL RNAse. Samples were incubated on ice, in the dark for a maximum of 1h before analysis. Nuclear DNA was measured by FACSCanto II flow cytometry (BD Biosciences) with an excitation laser of 488 nm wavelength and detection fluorescence emission between 543\u0026ndash;627 nm. Data analysis was performed using the FlowJo\u003csup\u003e\u0026trade;\u003c/sup\u003e v10.8 Software (BD Life Sciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of morphological and anatomical characteristics\u003c/h2\u003e \u003cp\u003eInternodal length was measured in stems developed in the last acclimatization period, from mutated and control plants. Twelve stems were selected from three induced octoploid 'Biloxi' plants, ten stems from three induced mixoploid 'Biloxi' plants, and ten stems from four 'Biloxi' control plants. Stem length was measured from the first fully expanded leaf at the apex to the last leaf at the shoot's base. Then, to obtain the mean internodal length per stem, the length of the stem was divided by the number of nodes counted within that length.\u003c/p\u003e \u003cp\u003eStomatal density and guard cell length were evaluated in the 3rd, 5th, and 7th fully expanded leaves (counting downwards from the apex) from acclimatized mutated and control plants, and an impression of abaxial side was taken by applying transparent nail polish as described by Beaulieu et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Photographs of the impressions were taken with a digital camera (Moticam 5 plus) coupled to a light microscope. Stomatal density was determined in four different fields in the middle part of the leaf, and stomatal guard cell length was measured on five stomata per field as described Beaulieu et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Images were analyzed with Motic Images Plus 3.0 software (Motic Inc., LTD. Hong Kong, China).\u003c/p\u003e \u003cp\u003eChloroplast number was quantified in stomata guard cells from young leaves of mutated and control individuals. A 5 mm\u003csup\u003e2\u003c/sup\u003e sample, without veins from a thin new leaf, was cut with a scalpel blade and mounted with Fluoromount-G on a circular slide of 25 mm diameter. At least ten stomata per sample were photographed in 3\u0026ndash;8 different fields under confocal laser scanning microscopy with the Airyscan system (LSM 880 Airyscan; Carl Zeiss AG) with a 63x oil-immersion objective (Plan-Apochromat; Carl Zeiss AG). Total chloroplasts per stoma were counted using Fiji software (Schindelin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eRegeneration and induction experiments were performed in completely randomized designs. A one-way ANOVA and a Tukey post hoc test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) was used to analyze differences between explant type and cultivar separately. Kruskal-Wallis test and Dunn\u0026acute;s multiple comparison test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) was performed for non-parametric data.\u003c/p\u003e \u003cp\u003eStatistical analyses of internodal length, stomata length, stomatal density, and chloroplasts per stomata were performed with one-way ANOVA and Tukey\u0026rsquo;s post hoc test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05). Analyses were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA. Values shown in text and figures stand for mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eShoot regeneration\u003c/h2\u003e \u003cp\u003eThe overall shoot regeneration capacity of the explants was enhanced by ZT, resulting in significant increases (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) in regeneration values when treated with 1 mg/L ZT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, this concentration was selected for use in the regeneration medium (RM) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, 'Legacy' microstems deviated from this trend, exhibiting an equivalent regeneration rate even without ZT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Despite this equivalence, ZT positively influenced the number of shoots per 'Legacy' microstem explant, increasing it from a mean of 1.1 in control explants to a mean of 4.1 shoots per explant with 1 mg/L ZT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). In contrast, ZT was needed to induce shoot regeneration in leaf explants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b, d, and e). Among the three cultivars, 'Legacy' exhibited the highest regeneration in terms of both the percentage of sprouted explants (66%) and the number of shoots per explant (2.7), while 'Biloxi' showed the lowest regeneration rates (39%) and number of shoots per explant (1.1) (Supplementary Table\u0026nbsp;2). In terms of explant type, adaxial regeneration exhibited the lowest values, with a sprouted explant percentage of 34% and an average of 0.7 shoots per explant (Supplementary Tables\u0026nbsp;3 and 4). Microstem presented the highest percentage of sprouted explants (84%) and matched abaxial leaf in terms of the number of shoots per explant (2.5) (Supplementary Tables\u0026nbsp;3 and 4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInduction of polyploidy\u003c/h2\u003e \u003cp\u003eMicrostem regeneration in all cultivars was significantly affected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) by colchicine treatments, with no significant effect observed from the treatment duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and g). Among all cultivars and explant types, 'Duke' microstems exhibited the lowest regeneration rate after two days of colchicine treatment, with 3.3% (\u0026plusmn;\u0026thinsp;5.8) of sprouted microstems and 0.03 (\u0026plusmn;\u0026thinsp;0.06) shoots per microstem (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and f). On the other hand, leaf regeneration was not affected by colchicine treatments, regardless of the cultivar, for either of the evaluated regeneration criteria: percentage of sprouted explant or number of shoots per explant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, d, and e). Furthermore, 'Legacy' leaves, both abaxial and adaxial, exhibited superior shoot regeneration rates and a greater number of regenerated shoots per explant compared to 'Duke' and 'Biloxi' (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, d, and e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePloidy analysis by DNA flow cytometry\u003c/h2\u003e \u003cp\u003ePloidy detection of putative polyploid and control individuals was conducted via flow cytometry. Fluorescence emission data were gathered and visualized using FlowJo\u0026trade; v10.8 Software (BD Life Sciences) to create histograms representing the number of nuclei based on their fluorescence intensity, which are indicative of their DNA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, d, and f). Peaks in the histograms of the putative polyploid samples were juxtaposed with those originating from the control samples (4x) to determine the relative ploidy levels, categorizing them as either tetraploid, octoploid, or mixoploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, d, and f). Analysis using flow cytometry identified 14 induced mutant individuals, representing 10.5% of the 133 shoots examined or 2.9% of the 483 regenerated shoots resulting from the two colchicine treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of these, six were identified as octoploids, and eight as mixoploids (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Induced mutant individuals were obtained from the three cultivars, all types of explants, and from the two exposure times to colchicine (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest induction rate was 6.7%, achieved by 'Legacy' and 'Biloxi' cultivars, but in different explants and treatments; abaxial, after 2 days of treatment, and microstem, after 1 day of treatment, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, in a parallel assessment involving 27 regenerated shoots, derived from preliminary experiments employing 'Biloxi' with identical explants and treatment conditions, flow cytometry revealed the induction of four octoploid variants.\u003c/p\u003e \u003cp\u003e \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\u003eResults of the induction of polyploidization by cultivar: treated explants, regenerated shoots, analyzed shoots, and mutated shoots (octoploid and mixoploid).\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=\"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=\"left\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCultivar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTreated explants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRegenerated shoots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eAnalyzed shoots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e \u003cp\u003eMutated shoots\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e%\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eOctoploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eMixoploid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e%\u003csup\u003ez\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e%\u003csup\u003ez\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e'Legacy'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e338\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e'Duke'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e'Biloxi'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTOTAL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e540\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e483\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e133\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e27.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e1.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003ex\u003c/sup\u003e The rate is calculated on the number of regenerated shoots.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003ey\u003c/sup\u003e The mutated shoot count represents the sum of octoploids (8x) and mixoploids (4x\u0026thinsp;+\u0026thinsp;8x) detected by DNA flow cytometry analysis of samples collected from some regenerated shoots resulting from the two treatments, 133 of a total of 483 regenerated shoots.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003ez\u003c/sup\u003e The rate is calculated on the number of treated explants.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \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\u003eResults of the induction of polyploidization by cultivar and explant: colchicine exposure, treated explants, survived explants and mutated shoots (octoploid and mixoploid).\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=\"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=\"left\" 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=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCultivar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eColchicine exposure (d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNumber of explants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e \u003cp\u003eMutated shoots\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExplant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSurvived\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eOctoploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eMixoploid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e%\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e%\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e'Legacy'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMicrostem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAbaxial leaf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAdaxial leaf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e'Duke'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMicrostem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAbaxial leaf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdaxial leaf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e'Biloxi'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMicrostem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAbaxial leaf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAdaxial leaf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003ex\u003c/sup\u003e The mutated shoots count represents the sum of octoploids (8x) and mixoploids (4x\u0026thinsp;+\u0026thinsp;8x) detected by DNA flow cytometry analysis of samples collected from some regenerated shoots resulting from the two treatments, 133 of a total of 483 regenerated shoots.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003ey\u003c/sup\u003e The rate is calculated on the number of treated explants.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of morphological and anatomical characteristics.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSome induced octoploid plants differed phenotypicaly from the original cultivar, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, c, and e, exhibiting thicker stems (Zeldin and McCown \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; He et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and shorter internodes (Dermen and Bain \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1944\u003c/span\u003e; He et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Supplementary Fig.\u0026nbsp;1 presents the mean of the internodal length of stem samples from control, octoploid, and mixoploid 'Biloxi' acclimatized plants. The mean internodal length of induced octoploids 'Biloxi' is significantly shorter than the tetraploids 'Biloxi' control (6.5 and 10.8 mm, respectively) (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, induced octoploids 'Legacy-L116' and 'Legacy-L317' exhibit an increase of more than double (267% and 208%, respectively) in the number of chloroplasts per stoma compared to the tetraploid. The induced octoploid 'Duke-U7' showed a doubling of chloroplasts, a significant 38% increase in stomatal length, and a non-significant 23% decrease in stomatal density compared to the tetraploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the case of 'Biloxi' mutants, the mixoploids, which exhibit a decrease in stomatal length, showed an increase in stomatal density, in contrast, the octoploids presented an equal stomatal size and density as the tetraploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eShoot regeneration\u003c/h2\u003e \u003cp\u003eIn our study, ZT concentration, genotype, type of explant, and their interaction significantly influenced the percentage of sprouted explants and the number of shoots per explant (Supplementary Figs.\u0026nbsp;2 and 3). Notably, 'Legacy' exhibited a more robust regenerative response in the presence of ZT than the other cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), supporting the regeneration disparities between genotypes documented in the literature (Clapa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This indicates that the addition of growth regulators notably interacts with explant\u0026acute;s endogenous hormones (Gahan and George \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), generating different responses per cultivar depending on their proper inner hormone levels or sensitivity. This suggests that the medium with the proposed concentration of the growth regulator is more favorable for 'Legacy', either due to a better combination of 'Legacy's endogenous hormones with the applied growth regulator or because of a greater sensitivity of this genotype to ZT (Gahan and George \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the three types of explants, microstem regeneration was achieved even without ZT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and f). This phenomenon can be attributed to the presence of axilary buds in the microstem, capable of sprouting even in the absence of ZT (Debnath \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, 1 mg/L ZT significantly increased the number of shoots produced per microstem (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), as expected due to its known function in promoting axillary bud growth (Tamas \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). With an average of three or more shoots per microstem explant, it confirmed the positive response of microstem regeneration to ZT (Clapa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the case of leaf explants, no matter their position towards the medium, regeneration was not achieved without the application of ZT, extending the need for growth regulator application for this type of explants to our studied cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b, d and e). Our data suggest that the abaxial position of the leaves in contact with the medium containing ZT is more favorable than the adaxial position (see Supplementary Tables\u0026nbsp;3 and 4), contrary to what was reported by Debnath (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Generally, there is no consensus on the optimal orientation. However, in this instance, greater regeneration could have been achieved with the abaxial orientation, as it would provide a larger surface area of the explant in contact with the medium. This is because when the adaxial side of the leaf is positioned in contact with the medium, it tends to roll up at both ends (Ricci et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 'Legacy' leaves in the abaxial position had the best responses among explants, achieving the highest number of shoots per explant (8.4) with 1mg/L ZT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInduction of polyploidy\u003c/h2\u003e \u003cp\u003eThe microstem explants of all cultivars were significantly affected by colchicine treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and g). In leaves, colchicine application caused a decrease of the percentage of sprouted explants and the number of shoots per explant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, d and f). This results reveal the impact of colchicine on physiological processes resulting in a cessation of growth in the treated tissues (Dermen and Bain \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1944\u003c/span\u003e), especially in treated microstems. A hypothesis that could explain the significant susceptibility of microstems to colchicine treatments is that in axilary buds, there is growth of meristematic cells before the application of colchicine, which get affected by the chemical. In leaf explants, however, the increase in cells is induced simultaneously with the application of colchicine.\u003c/p\u003e \u003cp\u003e'Legacy' abaxial explant was the only exception, where 2 days of colchicine treatment did not affect shoot regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and d). This phenomenon could be attributed to colchicine\u0026rsquo;s potential to harm the leaves due to its toxic nature (Dermen \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1940\u003c/span\u003e; Espino and Vazquez \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Paradoxically, as documented in previous research (Callow et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), leaf injury may enhance sprouting capacity, possibly either due to the accumulation of endogenous hormones in that area or because more growth regulator enters via the wound (Gahan and George \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), thereby mitigating the expected opposing effect of colchicine in reducing regeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePloidy analysis by DNA flow cytometry\u003c/h2\u003e \u003cp\u003eDNA flow cytometry has the capability to analyze the ploidy of all layers within a tissue sample and can effectively detect mixoploids. In this study, mixoploids were exclusively obtained in microstem explants treated (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming Broertjes and Keen (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) observation of a reduced occurrence of chimeric shoots or plantlets when induction was initiated before the formation of adventitious buds, as polyploid induction is considered a one-cell-event. We validated the findings of earlier studies (Cui et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ren et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) indicating that mixoploids had lower incidence or not produced when inducing polyploidy from leaf explants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of morphological and anatomical characteristics.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, the number of chloroplasts per stomata (two guard cells) aligns with flow cytometry results, serving as an indicator of ploidy levels (Dhooghe et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, chloroplasts number does not discriminate mixoploids with polyploid epidermis (L1).\u003c/p\u003e \u003cp\u003eStomatal length and density have been recognized as valuable criteria for discriminating between polyploid variations, as there exists a positive and negative correlation, respectively across a broad spectrum of angiosperms (Beaulieu et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Several authors have used increased stomatal (Norden \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and leaf (Yahata et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) size, and decreased stomatal density (Podwyszyńska and Pluta \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to distinguish ploidy levels after inducing polyploidization in the \u003cem\u003eVaccinium\u003c/em\u003e genus.\u003c/p\u003e \u003cp\u003eThe induced mixoploid 'Legacy-L9', following what the literature reports, shows an increase in stomatal length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and a decreased stomatal density (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), these values could suggest that the epidermis is polyploid, consequently the plant would be a periclinal chimera. In the induced octoploid 'Duke-U7' plant, although the stomatal length increases significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and the stomatal density decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), but there is a high variability between the data, which did not allow to obtain a statistical difference.\u003c/p\u003e \u003cp\u003ePrevious reports had only screened shoots with increased diameter during the \u003cem\u003ein vitro\u003c/em\u003e phase (Chandler and Lyrene \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Marangelli et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our research has uncovered that octoploids and mixoploids can emerge without exhibiting discernible morphological and anatomical disparities. If the induced octoploid clones, 'Biloxi-V160,' 'Biloxi-V203,' and 'Biloxi-W7', were classified solely based on stomatal length and density, they would have been excluded like octoploids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). We observed a sole mixoploid plant with phenological variations during the greenhouse acclimatization process, and no mixoploids during the \u003cem\u003ein vitro\u003c/em\u003e culture phase. Only three octoploids were detected through phenotypical differences during \u003cem\u003ein vitro\u003c/em\u003e culture such as thicker shoots, shorter internodes, and slow growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The induced octoploid 'Legacy-L116' clones exhibited thicker stems, shorter internodes, and slow growth in \u003cem\u003ein vitro\u003c/em\u003e culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These clones also demonstrated low vigor during the acclimatization stage, a characteristic reported in several studies (Goldy \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Miyashita et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Xue et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The observed low vigor could be attributed to variations in gene expression, arising from differences in chromosome dosage or epigenetic modifications within WGD (Adams and Wendel \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Alternatively, it may be attributed to the fact that induced polyploids need to replicate larger amounts of nuclear DNA in larger cells, leading to extended cell cycle durations and contributing to slower development (Van Drunen and Husband \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kashtwari et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This lower growth rate of polyploidized plants could give them an advantage since it allows them to conserve resources and mitigate cellular damage in conditions of stress due to drought, cold or nutrients (Deng et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also observed that five induced octoploids exhibited dwarfism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and e). This reduced plant height could be caused by changes in gene expression after WGD involved in reducing hormone levels like indoleacetic acid (IAA) and ethylene reported in autotetraploid birch plants (Mu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), or IAA and brassinosteroid reported in autotetraploid apple plants (Ma et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite mixoploids not being the focus of polyploidization breeding programs, they could also possess valuable traits such as larger fruit size, fewer seeds, and thicker pericarp than diploid lines, as observed in \u003cem\u003eFortunella crassifolia\u003c/em\u003e periclinal chimeras (Nukaya et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur results confirmed ZT as an adequate growth regulator to promote \u003cem\u003ein vitro\u003c/em\u003e shoot regeneration in \u003cem\u003eV. corymbosum.\u003c/em\u003e An ideal concentration for shoot promotion was established for three different explants: abaxial and adaxial leaves and microstems for three commercially valuable cultivars 'Biloxi', 'Legacy', and 'Duke'. \u003cem\u003eIn vitro\u003c/em\u003e shoot regeneration was standardized for these cultivars and explants, and it was a necessary step for \u003cem\u003ein vitro\u003c/em\u003e polyploidization in blueberry breeding.\u003c/p\u003e \u003cp\u003eOctoploids and mixoploids were successfully generated for the three cultivars. Polyploidization induction pursues to increase genetic variation within an individual, which in our case the observed phenotypes suggest was achieved due the phenotypical differences.\u003c/p\u003e \u003cp\u003eAdditionally, our research points to anatomical characteristics not being an adequate method for preliminary screening of polyploids in early developmental stages, pointing out flow cytometry as the most reliable method for polyploidy identification.\u003c/p\u003e \u003cp\u003eThe obtained octoploid plants of \u003cem\u003eV. corymbosum\u003c/em\u003e hold significant potential for the development of superior cultivars and represent an asset for future breeding programs. Furthermore, it is essential to investigate the flowering patterns, fruit yield, and fruit quality of all the induced mutant blueberries in future studies. As well, the newly developed polyploid blueberry variants will need further investigation concerning their resistance levels against biotic and abiotic stressors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eGJT was supported by Agencia Nacional de Investigaci\u0026oacute;n y Desarrollo (Chile) 21191617 PhD scholarship and the 2018 Doctoral Scholarship from Vicerrector\u0026iacute;a de Investigaci\u0026oacute;n of Pontificia Universidad Cat\u0026oacute;lica de Chile. VMB was supported by Consejo Nacional de Ciencia y Tecnolog\u0026iacute;a (M\u0026eacute;xico) 739582, and Agencia Nacional de Investigaci\u0026oacute;n y Desarrollo (Chile) 21200394 PhD scholarships.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003ePatricio Arce-Johnson guided the research. Patricio Arce-Johnson and Gabriela Jarpa-Tauler conceived and designed the research. Gabriela Jarpa-Tauler conducted the material preparation, experiments, including induction treatment, seedling management, and plant acclimatization. Gabriela Jarpa-Tauler, Jes\u0026uacute;s Lucina Romero-Romero and Vera Mart\u0026iacute;nez-Barradas analyzed the data. Gabriela Jarpa-Tauler wrote and revised the manuscript. Vera Mart\u0026iacute;nez-Barradas revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors thank Alex Cabrera and Sergio Bustos for their technical assistance in the flow cytometry service, Pamela Naulin for her supervision in the plant experimentation unit center of the UC Faculty of Biological Science, and Nicole Salgado for her technical support with the confocal microscope in the advanced microscopy facility UMA UC, of the Pontificia Universidad Cat\u0026oacute;lica de Chile.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams KL, Wendel JF (2005) Polyploidy and genome evolution in plants. 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J Plant Physiol 167:88\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jplph.2009.07.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jplph.2009.07.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"},{"header":"Supplementary Files","content":"\u003cp\u003eSupplementary Figure 1 to 2 and Tables 1 to 4 are not available with this version.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Polyploidy breeding, Vaccinium corymbosum L., Colchicine, Whole genome duplication, DNA flow cytometry, Adventitious shoot regeneration","lastPublishedDoi":"10.21203/rs.3.rs-4214823/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4214823/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBlueberries are a fruit with an increasing global demand due to their phytochemical and bioactive compounds content. They are promoted worldwide because of their health benefits. For optimal growth and productivity, blueberry crops need acidic soil pH, specific chilling hours, and an adequate atmospheric temperature. This delicate production equilibrium is under severe threat from climate change, potentially leading to reduced yields and increased cultivation costs unless new cultivars are developed for each edafoclimatic zone. Therefore, considering varietal replacements with more productive cultivars offering higher quality and better adaptability to local conditions is imperative. In this study, we employ polyploidization and \u003cem\u003ein vitro\u003c/em\u003e tissue culture to promote variability and lay the foundation for new cultivar development. We report the successful induction of octoploids in three blueberry cultivars, namely 'Biloxi,' 'Legacy,' and 'Duke', through whole-genome duplication. Leaves and microstem explants were exposed to 0.1% colchicine for 24 and 48 hours in \u003cem\u003ein vitro\u003c/em\u003e culture. After analyzing the polyploid level of 160 regenerated shoots using DNA flow cytometry, we obtained a total of 18 mutants, consisting of 8 mixoploids and 10 octoploids. The number of chloroplasts in the stomata was analysed by fluorescence microscopy, revealing the duplication of these organelles in the induced octoploid plants. To our knowledge, this represents the first successful induction of octoploids in three blueberry cultivars -'Biloxi,' 'Legacy,' and 'Duke'- achieved by exposing leaves and microstem explants to colchicine in \u003cem\u003ein vitro\u003c/em\u003e culture. This technique holds promise as a valuable tool for the development of improved blueberry cultivars.\u003c/p\u003e","manuscriptTitle":"Autopolyploidization and in vitro regeneration of three blueberry cultivars from leaves and microstems.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-15 15:15:32","doi":"10.21203/rs.3.rs-4214823/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-11T07:54:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-10T15:41:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-10T06:34:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2024-04-03T17:25:03+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":"04d93aac-df64-474e-976b-24b43d118fde","owner":[],"postedDate":"April 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-14T08:06:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-15 15:15:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4214823","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4214823","identity":"rs-4214823","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}