Efficiency Improvement of In Vitro Chromosome Doubling in Melon haploid

Efficiency Improvement of In Vitro Chromosome Doubling in Melon haploid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficiency Improvement of In Vitro Chromosome Doubling in Melon haploid yan lelong, zhu songyu, wu kaimin, huang yao, zhang yaomei, yang jiaxi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5332893/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract In vitro chromosome doubling (ivCD) using colchicine is a main method for melon haploid chromosome doubling, but its doubling efficiency remains low. In this study, we aimed to increase the efficiency of melon haploid doubling. In Experiment 1, the impacts of genotype and explant age on the survival rate (SR) and sample doubling rate (SDR) of melon haploid through ivCD were studied. Nine melon haploid genotypes were treated with colchicine for 24 hours followed by inoculation into solid MS. The best doubling effect was achieved with explant age of 21 days. During this process, the explants exhibited a low SR (38.76 ± 9.72%) and SDR (19.04 ± 7.10%), but a high vitrification rate (VR) (37.36 ± 9.93%) and an extended period for explant regeneration for over 30 days. In Experiment 2, four representative genotypes were selected to screen suitable combinations of antimicrotubular agents and hormone-treatments to enhance the SR and SDR, and solve the other problems above. The results indicated that treatment with trifluralin for 24 hours, followed by inoculation into solid MS medium containing 6-BA for 15 days, yielded the highest SR (72.27 ± 9.44%) and SDR (42.12 ± 9.72%), but the lowest VR (2.35 ± 4.25%) and a shortened regeneration period of 15 days. Interestingly, in Experiment 2, doubled haploid (DH) obtained from treatment with trifluralin performed better in pollen viability rate (PVR), pollen deformity rate (PDR), and seed germination rate (SGR) compared to those treated with colchicine. In summary, we significantly increased the haploid chromosome doubling rate, reduced the regeneration time, and obtained DHs with relatively high fertility. Cucumi melo L. doubled haploid explant age trifluralin 6-BA Figures Figure 1 Figure 2 Figure 3 Figure 4 Key message This study improved the melon haploid doubling efficiency and shortened explant regeneration period of the ivCD, and finally obtained DH with relatively high fertility. Introduction Melon ( Cucumis melo L.), with its sweet taste and aromatic fragrance, is widely cultivated around the world, and its annual worldwide production reaches 28.5 million tons (FAOSTAT, 2022). Haploid breeding technology, with its characteristic of shortening the breeding cycle and enhance breeding efficiency(Zere Taskin and Bilgili 2023 , Qu et al. 2024 ), has been widely applied in the breeding of cucurbit crops such as melon(Sauton and Dumas de Vaulx 1987 ), cucumber(Przyborowski and Nlemirowicz-Szgzytt 1994 ), watermelon(Sari et al. 1994 ), pumpkin(Ertan et al. 2009 ), and summer squash(Kurtar et al. 2002 ). There are several methods that can accomplish haploid induction in cucurbit plants, such as anther culture(Asadi et al. 2018 ), interspecific pollination induced parthenogenesis(Sauton and Dumas de Vaulx 1987 ), ovule/ovary culture(Deng et al. 2020 ) and pollination with radiated pollen(Salehian et al. 2023 ). In melon, haploid induction is primarily achieved through pollination with irradiated pollen(Cuny et al. 1993 , Hooghvorst et al. 2020 ). However, its haploid induction rate is quite low, ranging only from 0–3.4%(Lotfi et al. 2003 , Ari et al. 2010 ), which has hampered the subsequent obtain of mass doubled haploid for further melon breeding. Thus, it is imperative to ensure that it can be successfully doubled to form a DH for each melon haploid. For this, two primary methods of chromosome doubling are employed: ivCD(Lim and Earle 2008 ) and in situ chromosome doubling(Hooghvorst et al. 2020 ). The former is favored for its high efficiency and low chimeras compared to the latter(Fu et al. 2019 ). Meanwhile, researchers have founded that several factors can influence the efficiency of the ivCD of melon haploid, including the plant's genotype(Mentewab and Sarrafi 1997 ), the type(Lim and Earle 2009 ) and age(Gałazka et al. 2015 ) of explant, the selections of antimicrotubular agents, their concentration and treatment duration(Dhooghe et al. 2011 ), as well as the hormone and its concentrations(Xu et al. 2018 ). Thus, to achieve a good effect of chromosome doubling of melon haploid, the ivCD technology system needs to be carefully designed or optimized. Affected by factors above, issues such as low SR and SDR(Lim and Earle 2008 , Lim and Earle 2009 ), high VR(Grzebelus and Adamus 2004 , Khan et al. 2020 ) and prolonged regeneration period, are likely to arise during the process of ivCD. Based on different haploid genotypes and doubling measures, the SDR ranges from 0 to 23.24%(Lotfi et al. 2003 , Lim and Earle 2008 , Solmaz et al. 2011 , Hooghvorst et al. 2020 ). Explant age affects the regenerative capacity(Mohebodini et al. 2011 , Liu et al. 2013 ), and selecting explants with appropriate age can significantly increase the frequency of regeneration(Thomas 2003 , Sun et al. 2009 ). Plant hormones, such as 6-BA, can regulate cell division and endoreduplication(Demeulenaere and Beeckman 2014 ), and promote explant regeneration(Yadav et al. 1996 , Lim and Earle 2009 ), thereby can improve the doubling efficiency. Colchicine is the most widely used reagent in this process, yet its low efficiency and high toxicity have been widely criticized(Bouvier et al. 1994 , Hooghvorst et al. 2020 ). Some low-toxicity antimicrotubular agents, such as trifluralin and oryzalin, are increasingly used in the doubling process and are gradually replacing colchicine in cucumber(Ebrahimzadeh et al. 2018 ). Here, in order to enhance the SR and SDR, and reduce VR and shorten axillary bud regeneration duration, we have carried out two experiments. In Experiment 1, we have screened out fit explant age with relative higher SR and SDR, but also found several issues such as high VR and long regeneration time during the doubling process. In Experiment 2, we have further reduced the VR and shortened the duration of regeneration time through the selection of suitable combinations of antimicrotubular agents and hormone treatments using four genotypes (BAI_1, G6B_1, G6_1, and JG_1) selected in Experiment 1. Our progress will promote and expand the application of haploid breeding technology in melon. Materials and methods Materials In our preliminary experiments, we used 15 different genotypes as parental materials to induce hundreds of haploid melons with various genotypes (Huang et al. 2024, online). The parental material was grown in polytunnel greenhouses at the Changshu Agricultural Science Institute in Changshu City, Jiangsu Province, China, which has a subtropical monsoon oceanic climate, and this work was carried out between 2022 and 2023. Among them, we selected nine relatively robust haploids which exhibited normal leaf, stem, and root growth as materials (Table 1) for ivCD in Experiment 1. Among nine genotypes, BAI_1，FW 5_1 and FW 8_1 are thick-skinned, G6B_1 and G6_B_2 are middle-skinned, G6_1, G6_2, JG_1, G61_1 and G6_2 are thin-skinned. Table 1 Haploid materials-genotype, type and origin in Experiment 1. Haploid code Donor parental materials Subspecies Origin BAI_1 BAIPICUI C. m. subsp. agrestis XAAS(Bi et al. 2024) FW 5_1 Flavor NO. 5 C. m. subsp. melo XAAS(Tang et al. 2010) FW 8_1 Flavor NO. 8 C. m. subsp. melo XAAS(Tang et al. 2010) G6B_1 PI420145 × BAIPICUI C. m. subsp. melo - G6B_2 PI420145 × BAIPICUI C. m. subsp. melo - G6_1 PI 420145 C. m. subsp. agrestis NAU(Wolukau et al. 2009) G6_2 PI 420145 C. m. subsp. agrestis NAU(Wolukau et al. 2009) JG_1 JINGGUA C. m. subsp. agrestis CV G61_1 PI420145 × PI140471 C. m. subsp. agrestis - Note: ‘XAAS’ means Xinjiang academic of agricultural sciences, ‘NAU’ means Nanjing agricultural university, ‘CV’ means commercial variety obtained in local market. In vitro chromosome doubling experiment design The basic experimental procedures (Fig. 1) are performed as follows: 1) Preparation of explants and antimicrotubular agent solution: haploid nodal explant with different age (Fig. 1a-c) were cut to a size of 2-3 cm(Fig. 1d), and antimicrotubular agents were dissolved in dimethyl sulfoxide (DMSO) (HUSHI, Shanghai), filtered through aseptic filtration, and then added to sterile liquid MS medium, with the volume fraction of DMSO adjusted to 2%. 2) Antimicrotubular agents’ treatment (Fig. 1e-g): the nodal explant was soaked in sterilized antimicrotubular agents’ solution, shaken on a shaker (CRY-1102C, Shanghai Rongyan) at a speed of 100 rpm, and maintained at a temperature of 25±1 °C under dark conditions. Subsequently, nodal explant segments were washed three times with sterilized distilled water. 3) Hormone treatment (Fig. 1h-m): This step was specific to Experiment 2, while for Experiment 1, the explants were treated with colchicine and then inoculated into hormone-free solid MS medium for a 15 days culture period. In Experiment 2, after treatment with antimicrotubular agents, the explants were inoculated into solid MS medium with hormone for further hormone-treatment — a process that lasts for 15 days. 4) Explant regeneration (Fig. 1n-p): For Experiment 1 and Experiment 2, the explants exhibit three different fates: vitrification (Fig. 1k), formation of callus at the base(Fig. 1l), and formation of both callus and roots at the base(Fig. 1m). Explants that became vitrified would eventually die, while green axillary buds from the latter two types would ultimately form regenerated plants when they were excised and inoculated into solid MS medium. The growth conditions for the explants were maintained at 2000 lx, 25±1°C, under a 12 hours light/12 hours dark cycle. Effect of genotype and explant age (Experiment 1) Referring to the method of Lim and Earle (2008), with appropriate adjustments, the nodal explants were treated in a solution containing 500 mg/L colchicine + 2% DMSO for 24 hours. Nine genotypes were selected, and explant of different ages (21 days, 28 days, and 35 days). After treated by colchicine, the explants were inserted into solid MS medium for 15 days, and during these days most of the explants exhibited vitrification (Fig. 1k). The axillary bud parts were then excised and inoculated into new solid MS culture medium, and after approximately 30 days complete regenerated plants were formed. Then, using the optimal explant age, further investigation of the effects of antimicrotubular agents and hormone treatments on ivCD was conducted. Effect of antimicrotubular agents and hormone-treatment (Experiment 2) Considering the issues encountered in Experiment 1, we performed experiments of replacing the antimicrotubular agents and using hormone treatments. In Experiment 2, we selected four genotypes (BAI_1, G6B_1, G6_1, and JG_1), based on various SDRs, to verify the impact of antimicrotubular agents (500 mg/L colchicine + 2% DMSO, 50 mg/L oryzalin + 2% DMSO, 50 mg/L trifluralin + 2% DMSO)(Ebrahimzadeh et al. 2018) and hormone treatments (free hormone, 0.2 mg/L 6-BA, 0.2 mg/L 6-BA + 0.15 mg/L NAA)(Sun et al. 2009) on the SR, SDR and VR. Unlike Experiment 1, after the explants were treated with antimicrotubular agents for 24 hours, they were inoculated into a hormone-containing culture medium for 15 days. After 15 days, most explants formed callus tissue at the base and some even developed roots. The axillary bud parts were then excised and inoculated into solid MS culture medium, where complete plants were formed after approximately 15-30 days. Regenerated plant ploidy identification Flow cytometry analysis Flow cytometry was used to identify the ploidy level of these regenerated plants (Lotfi et al. 2003, Lim and Earle 2008). Tender leaves were fragmented in 800 μL of dissociation solution for 10 minutes and filtered through a 40-micron cell strainer. Then 35 μL of 1 M propidium iodide (PI) (Solarbio, China) and 10 μL of 1 M RNase (Takara, Japan) were added to the filtrate and incubated in darkness under low-temperature conditions. A Flow Cytometer (Beckman CytoFLEX) was used to analyze filtrate. Dissociation solution recipe is as follows: 10 mM MgSO 4 ·7H 2 O (Xilong Scientific, China), 50 mM KCl (Xilong Scientific, China), 5 mM HEPES (Shyuanye, China), 0.25% (v/v) Triton X-100 (Solarbio, China), and 1% (m/m) PVP (Vokai, China). Chromosome counting The chromosome counting method was referred to the work of Zhang et al. (2023), with appropriate modifications made on this basis. The root system of tissue-cultured seedlings was usually physiologically older, with fewer mitotic divisions in the root tip meristematic zone. We inoculated the explants into MS medium with 30 g/L sucrose (Solarbio, China) and 5 g/L agar (Solarbio, China), and once the explants developed roots with a length of 1-2 cm, they were ready for chromosome count observation. The basic procedure was as follows: the newly growth roots were treated with 0.002M Octahydroxyquinoline (Hushi, China) at 4 °C for 3 hours, rinsed with ddH2O, then fixed with Carnoy's solution (Solarbio, China) at 4 °C for 9 hours. After rinsing, the roots were treated with 1M HCl at 60°C for 4 minutes, and then residual hydrochloric acid was removed with distilled water. Subsequently, a 1 mm root tip was taken and stained with Carbol Fuchsin (Solarbio, China) at room temperature for 7 minutes, followed by slide preparation. Haploid and DH plant acclimatization and morphological observation The tissue culture plantlets for acclimatization were inoculated into solid MS medium with 30 g/L sucrose and 5 g/L agar, and grown in the acclimatization chamber for 15 days under 2000 lx, 25±1 °C, with a 12 hours light/dark cycle. The non-uncovered culture bottles were moved to outdoor growth for 5 days. Then we took out the seedling, removed the agar attached to the roots, transplanted them into sterilized substrate, thoroughly watered them with sterile water, and covered the nutrition bowl with a self-sealing bag to maintain local humidity. 5 days later, a corner of the bag was cut to reduce humidity, and after 10 days, the bag was removed to proceed for transplantation. To investigate the effects of colchicine and trifluralin on the DH regenerated plantlets, we statistically analyzed key fertility-related traits, including PVR, PDR, and SGR. Among them, PVR and PDR were assessed through microscopic observation (Olympus CX43) using 0.5% Triphenylchlorotetrazolium Chloride (TTC) staining solution(Xiaohua et al. 2020). Pollen grains were considered viable if they turned red after being immersed into the TTC solution at 25 °C for 15 minutes. SGR(Sohrabi et al. 2016) was assessed by soaking seeds in warm water (50 ℃, 1 hour) and then incubating them at 25 ± 1 °C for 1 day. To observe the morphological differences, including leaf length and width, fruit size, and male flowers, between haploid and DH, we conducted statistical analyses based on the method of Sarı (2017) . Leaf length and width were measured at the 13th, 14th, and 15th nodes of the main vine, and the number of petals on male flowers located on the lateral branches at these nodes was recorded. Once the fruits reached full maturity, their length and diameter were meticulously measured. Statistical analysis To characterize the effects of different treatments on the doubling of melon haploid, we used the SR, SDR and VR (Table 2) to quantify and present the results of the doubling process. The sample sizes of SR, SDR, and VR were not completely consistent, ranging from 9 to 12, and were analyzed using analysis of variance (ANOVA) and the Tukey-Kramer test. The statistical analysis of fertility-related indicators in DH regenerated plants, including PVR, PDR, and SGR (Table 2), was conducted with consistent sample sizes, using ANOVA and Tukey's post-hoc test. Each experiment was conducted with three replicates. The data were analyzed using Excel and R Studio. Table 2 Equations of ivCD-related indicators and fertility-related indicators Results In Experiment 1, nodal explants were treated with 500 mg/L colchicine and 2% DMSO for 24 hours followed by being inoculate into solid MS medium for 15 days. Then, the explants with green axillary buds were segmented and inserted to new solid MS medium. At 21 days of explant age, the highest SR (an average of 38.76±9.72%) and SDR (19.04±7.10%), as well as the lowest VR (37.36±9.93%), were obtained. Among the nine genotypes, G6_1 showed the highest SR (55.19±5.01%) and SDR (27.41±4.49%), with the lowest VR (20.74±1.28%). Conversely, BAI_1 had the lowest SR (24.85±4.20%) and SDR (9.39±0.52%), but the highest VR (50.00±4.55%) (Supplement 1). These results suggest the genetic variation in the ivCD process of melon haploids. Furthermore, explants subjected to vitrification rarely regenerated into complete plantlets and often died. Although a few explants could develop into complete plantlets，they failed to achieve successful chromosome doubling. And other few explants that do not form vitrification, the development of complete plants from axillary buds was notably slow, taking over 30 days In Experiment 2, to enhance SR and SDR while reducing VR and regeneration time, we modified the antimicrotubular agent and added hormones to the solid MS medium. We identified an optimal combination: 50 mg/L trifluralin + 2% DMSO and 0.2 mg/L 6-BA. By applying this combination, compared to the results of Experiment 1, the four genotypes (BAI_1, G6B_1, G6_1, and JG_1) of SR (72.27±9.44%) and SDR (42.12±9.72%) showed significant increases, while the VR (2.35±4.25%) (Supplement 5) significantly decreased by 30.82%, 22.62%, and -30.41%, respectively. This treatment combination not only increased the SR and SDR and reduced the VR, but also halved the time required for axillary buds to regenerate into complete plants, reducing it to just 15 days. Effect of genotype on SR, SDR and VR The variance analysis and post-hoc tests demonstrated significant differences (Table 3) on SR, SDR and VR in Experiment 1. The regenerative capacity was positively correlated with SR and SDR to a certain extent, and negatively correlated with VR. The haploids G6_1 and G6B_1 exhibited strong regenerative ability during the proliferation process, with robust root growth, and showed high SR and SDR, and relatively low VR during the doubling process. In contrast, BAI_1, FW 5_1, and FW 8_1 exhibited poor regenerative ability during the proliferation process, with underdeveloped root systems, and showed low SR and SDR, and high VR. This may indicate that the genotype affects the regenerative capacity, which in turn affects the efficiency of doubling. Table 3 Effect of genotype and explant age on the variance of SR, SDR and VR in Experiment 1 Experiment 1 SR (%) SDR (%) VR (%) Genotype (explant age = 21 days) BAI_1 24.85±4.20 c 9.39±0.52 e 50.00±4.55 a FW 5_1 31.21±4.67 bc 12.73±6.30 cde 43.64±3.15 ab FW 8_1 28.18±1.57 abc 15.76±5.84 de 46.97±2.62 a G6B_1 45.15±5.01 ab 25.76±5.17 ab 29.09±1.57 cd G6B_2 42.42±5.25 ab 24.24±5.25 bc 33.33±10.50 bcd G6_1 55.19±5.01 a 27.41±4.49 a 20.74±1.28 d G6_2 43.64±3.15 abc 21.82±4.81 ab 37.58±2.10 abc JG_1 40.61±4.58 abc 15.45±4.72 bc 31.21±4.67 bcd G61_1 37.58±2.10 abc 18.79±1.05 bcd 43.64±3.15 ab Explant age (day) 21 38.76±9.72 a 19.04±7.10 a 37.36±9.93 c 28 24.42±10.91 b 9.88±4.85 b 59.44±8.01 b 35 11.86±12.06 c 3.91±4.83 c 71.09±5.08 a Note: The Experiment utilized a full-factorial design, with each trial being conducted three times, and the sample size for each Experimental group varying between 9 and 12. The Tukey-Kramer test was applied, with the level of significance set at 5% (P > 0.05). Effect of explant age on SR, SDR and VR Variance analysis and post-hoc tests (Table 3) indicated that explant age significantly affects the SR, SDR, and VR. As the age of the explant increased, SR and SDR gradually decreased, while VR increased, with the best results of 38.76±9.72% (SR), 19.04±7.10% (SDR) and 37.36±9.93% (VR) at 21 days of explant age, respectively (Supplement 1). Furthermore, as the age of the explant advanced, the plant morphology underwent corresponding changes: at 21 days (Fig. 1a), the leaves being deep green without any signs of yellowing; by 28 days (Fig. 1b), the basal leaves beginning to yellow; and by 35 days (Fig. 1c), the yellowing spreading to the middle leaves. Concurrently, the plant's regenerative capacity progressively diminishes. Effect of antimicrotubular agents on SR, SDR and VR In Experiment 2, the results of the analysis of variance and post-hoc tests (Table 4) indicated that for SR, SDR, and VR, the treatment with 50 mg/L trifluralin + 2% DMSO yielded good results, with respective values of 65.23±9.17%, 30.48±11.21%, and 5.50±5.55% (Supplement 3). Meanwhile, we had observed that explants treated with colchicine required a regeneration period that was twice as long as that of trifluralin and oryzalin for the regeneration of complete plants from axillary buds during the second inoculation, taking over 30 days compared to the 15 days buffer period of trifluralin and oryzalin. During this process, the concentration of colchicine needed was 10 times that of trifluralin and oryzalin, exerting stronger phytotoxic effects on the plants, which were reflected in the prolonged duration, low SR and high VR compared to trifluralin and oryzalin. Table 4 Effect of antimicrotubular agent and hormone-treatment on the variance of SR, SDR and VR in Experiment 2 Experiment 2 SR (%) SDR (%) VR (%) Antimicrotubular agent(mg/L) 500 colchicine + 2% DMSO 55.95±9.90 b 30.95±10.96 a 22.33±10.31 a 50 oryzalin + 2% DMSO 62.81±9.12 a 22.62±8.72 b 7.04±5.42 b 50 trifluralin + 2% DMSO 65.24±9.17 a 30.48±11.21 a 5.50±5.55 b Hormone-treatment (mg/L) Free hormone 58.95±7.42 b 23.21± 7.12 b 15.79±11.30 a 0.2 6-BA 68.22±10.63 a 37.16±11.44 a 6.48±8.20 b 0.2 6-BA +0.15 NAA 56.83±8.32 b 23.68±7.49 b 12.60±10.17 a Note: The Experiment utilized a full-factorial design, with each trial being conducted three times, and the sample size for each Experimental group varying between 9 and 12. The Tukey-Kramer test was applied, with the level of significance set at 5% (P > 0.05). Effect of hormone-treatment on SR, SDR and VR Analysis of variance and post-hoc tests (Table 4) showed that the addition of 0.2 mg/L 6-BA to the MS medium significantly improved the SR and SDR, and minimized the VR compared to the free hormones and the addition of 0.2 mg/L 6-BA + 0.15 mg/L NAA, which were 68.22±10.63%, 37.16±11.44% and 6.48±8.20%, respectively (Supplement 4). In Experiments 1 and 2, a high prevalence of vitrification was observed, coupled with diminished SR and SDR, when nodal explants treated with antimicrotubular agents were subsequently cultured in a hormone-free medium. However, when the explants were inoculated into a medium containing 0.2 mg/L 6-BA, it significantly reduced the VR and improved the SR and SDR, indicated that 6-BA played a role in mitigating the damage caused by colchicine. In summary, 6-BA not only enhanced the SR and SDR but also reduced the VR. Polyploidy identification and tissue culture seedlings acclimation In Experiments 1 and 2, we obtained a total of 881 regenerated plants (Supplement 1 and Supplement 2). After flow cytometric ploidy analysis, there were 481 haploid plants (n=12; Fig. 2a,b) and 400 diploid plants (2n=24; Fig. 2c,d). Subsequently, we randomly selected 20 plants from both the haploid and diploid groups. Chromosome counting confirmed the results consistent with the flow cytometry analysis. By employing our enhanced acclimation technique, we have successfully achieved a survival rate of over 80% (Table 5) for both haploid and diploid plants. The use of 5 g/L agar facilitated the removal of agar attached to the root system, minimizing damage to the roots. Acclimating for 5 days outdoors with covering helped to enhance the plants' adaptability to the environment. At this stage, the explants have been grown for 20 days, and their root systems have become robust. Transplanting the plants into sterilized substrate and watering them with sterile water throughout the process created a low-bacterial environment. Using self-sealing bags to create a localized high-humidity environment for the plants, followed by a gradual reduction in humidity, ensured a high acclimation survival rate through these integrated measures. Table 5 The results of tissue culture seedlings acclimation genotype ploidy Number of acclimations Number of survival (Survival rate /%) BAI_1 Haploid 30 25(83.33) DH 30 26(86.67) G6B_1 Haploid 30 26(86.67) DH 30 28(93.33) G61_1 Haploid 30 25(83.33) DH 30 27(90.00) JG_1 Haploid 30 24(80.00) DH 30 26(86.67) Total 240 207(86.25) Observation of PVR, PDR, and SGR in DH regeneration plants In Experiment 2, trifluralin and colchicine exhibited similar efficiency on SDR. Thus, for further investigation into the impact of these two antimicrotubular agents on the fertility-related traits of DH, we had compiled the PVR, PDR, and SGR indices of the DH treated with colchicine and trifluralin. The results of variance analysis and post-hoc tests (Table 6) indicated that trifluralin caused less damage to fertility than colchicine, as evidenced by the higher PVR and SGR observed in DH regenerated plants treated with trifluralin, along with a markedly lower PDR compared to those treated with colchicine. Of course, these indicators are based on data obtained from the first generation of DH plants, which only reflect the short-term effects of the antimicrotubular agents on the explants. These results can only reflect the toxicity of different treatments to a certain extent. Table 6 Effects of colchicine and trifluralin on PVR, PDR and SGR of first generation of DH genotype Antimicrotubular agents PVR /% PDR /% SGR /% BAI_1 Colchicine 28.00±2.00 b 25.33±3.06 a 62.00±5.29 b Trifluralin 64.33±4.04 a 13.67±2.08 b 92.00±4.00 a G6B_1 Colchicine 44.00±4.00 b 21.67±2.31 a 83.33±3.06 b Trifluralin 71.00±3.61 a 13.00±4.58 b 93.33±4.16 a G61_1 Colchicine 41.00±1.00 b 24.33±3.21 a 78.00±2.00 b Trifluralin 59.67±2.08 a 12.33±2.52 b 92.67±3.06 a JG_1 Colchicine 30.00±2.00 b 21.67±1.53 a 63.33±3.06 b Trifluralin 71.67±3.51 a 10.33±2.31 b 93.33±4.16 a Note: Each trial was set up with three repetitions, with a sample size of 50 for each Experimental group. The Tukey test was used, with the significance level set at 5% (P > 0.05). Observation of haploid and DH Then, we further observed the characteristics of haploids and DH. There has been very little description of melon haploids, especially regarding fruit traits, because haploids contain only one set of chromosomes, and flowers often aborted during development(Gałazka et al. 2015). However, we found that G6B_1 and G6_1 could form mature male and hermaphroditic flowers, and after treatment with Thidiazuron, the hermaphroditic flowers developed into fruits. Their male flowers produced a small number of pollens, which were stained by TTC (Fig. 3). The color and stripes of the fruits were indistinguishable from those of DH, but the fruit length and diameter were visually smaller than those of DH. Furthermore, haploids exhibited a reduction in the size of various organs (Fig. 4). Table 7 The fruit, male flower, and leaf part trait of haploid and DH of melon Genotype Ploidy Fruit Male flower Leaf Length(cm) Width(cm) Petals Length(cm) Width(cm) BAI_1 Haploid - - - 9.99±0.70 b 8.75±0.31 b DH 12.88±0.86 12.59±0.79 5.00±0.00 14.86±1.63 a 14.51±0.73 a G6b_1 Haploid 6.05±0.66 b 3.54±0.58 b 6.00±0.00 b 10.17±0.88 b 7.62±0.17 b DH 12.57±0.65 a 6.62±0.46 a 6.33±0.58 a 14.56±0.67 a 13.87±0.74 a G6_1 Haploid 4.37±0.37 b 2.42±0.42 b 5.00±0.00 b 6.14±0.62 b 5.33±0.16 b DH 9.57±0.73 a 5.76±1.21 a 5.67±0.58 a 10.14±0.57 a 8.31±0.33 a JG_1 Haploid - - - 7.02±1.40 b 6.91±0.27 b DH 6.07±0.68 8.86±0.62 7.67±0.58 11.97±1.47 a 12.88±0.51 a Note: Each experiment is repeated three times, and three samples are collected for each treatment group. A t-test is conducted at a significance level of 0.05 (P > 0.05). Discussion Current research on ivCD of melon haploids primarily focuses on improving the efficiency of haploid doubling(Lotfi et al. 2003 , Lim and Earle 2008 , Lim and Earle 2009 , Solmaz et al. 2011 ). However, there has been insufficient attention paid to the use of antimicrotubular agents and issues that arise during the doubling process, such as high VR, prolonged explant regeneration duration, and the fertility of regenerated plants. This study has identified that using explants aged 21 days, replacing colchicine with trifluralin, and inoculating into the solid MS medium containing 6-BA after trifluralin treatment can not only significantly enhance the SDR compared to the former research(Lotfi et al. 2003 , Lim and Earle 2008 , Solmaz et al. 2011 , Hooghvorst et al. 2020 ), but shorten explant regeneration period and obtain DH with high fertility. This efficient processing can promote the application of haploid breeding in melon breeding. The role of explant age in the ivCD of melon haploids has been somewhat overlooked, despite its pivotal influence on the explant's regenerative capacity and doubling efficiency(Gałazka et al. 2015 ). This factor determines whether the explant can survive and regenerate after treatment with antimicrotubular agents, thereby directedly affecting the SR and SDR. The intricacies of this relationship are particularly pronounced in the development of genetic transformation systems, where younger tissues have a stronger regenerative ability, but excessively young tissues have weaker tolerance and eventually die(Cao et al. 2002 , Cui et al. 2020 ). Selecting the appropriate explant age can balance this relationship. In terms of SR and SDR, when explants are 21 days old, both are significantly higher than those when they are 28 or 35 days old. Currently, ivCD studies in melon haploid predominantly utilize colchicine as antimicrotubular agent. Yet it has a low binding affinity for plant microtubules(Dhooghe et al. 2011 ), requiring high concentrations of 500-5000mg/L for treatment(Ebrahimzadeh et al. 2018 , Hooghvorst et al. 2020 ), which not only increases experimental costs but also poses risks of damaging plant fertility and DNA, potentially leading to loss of fertility and DNA mutations(Luckett 1989 ). Additionally, colchicine's ability to bind to animal microtubules presents potential health hazards to researchers(Morejohn et al. 1984 ). In contrast, dinitroaniline compounds, such as oryzalin and trifluralin, offer safer alternatives due to their high binding efficiency with plant microtubules and lack of interaction with animal microtubules(Grosso et al. 2018 ). In our experiment, trifluralin was used at a dose one-tenth that of colchicine and was on par with colchicine in terms of SDR. The observed results (Table 7 ) indicated that DH regenerated plants treated with trifluralin have superior PVR, PDR, and SGR compared to those treated with colchicine, thus confirming this conclusion. After treatment with antimicrotubular agents, inoculating explants into solid MS medium with hormone for a period of time can enhance both the SR and SDR(Lim and Earle 2009 ). Not only that, but hormone treatment can also break the vitrification and growth stagnation of explants. 6-BA, as a common plant growth regulator, promotes cell division and the growth and differentiation of buds(Werner et al. 2001 ), and induces low endoreduplication activity, achieving polyploidization(Mahdad et al. 2023 ). In our Experiment 2, the addition of 0.2 mg/L 6-BA during the first inoculation, as opposed to free-hormone and 0.2 mg/L 6-BA + 0.15 mg/L NAA treatments, greatly reduces the occurrence of vitrification, thereby breaking the growth stagnation after colchicine treatment(Tabei et al. 1994 ). In addition, haploid contains a set of chromosomes(Blakeslee et al. 1922 ), and it is often manifested as plant leaf length, leaf width, and flower size being smaller than diploid(Chase 1964 , Xu et al. 2013 ). Our results (Table 7 ) also verify this phenomenon. Moreover, there are reports indicating that haploid plants are fully infertile(Nikolova and NiemirowiczSzczytt 1996 , Takahira et al. 2011 , Molenaar et al. 2019 ). However, we have observed that the haploids G6B_1 and G6_1 can form male and hermaphroditic flowers, which can produce a small amount of viable pollen and fruit respectively. The same phenomenon has also been observed in maize haploids(Chalyk 1994 ), and the underlying reason may be cell fusion leading to spontaneous doubling in some cells(Chase 1969 , Gayen and Sarkar 1996 , Testillano et al. 2004 ). The mechanism by which melon haploids produce unreduced gametes needs further investigation. Conclusion In this study, we successfully increased SR and SDR, shortened the regeneration period, and ultimately yielded doubled haploids with relative high fertility through an effective method of ivCD in melon, in which 21-day-old melon haploids were treated with 50 mg/L trifluralin + 2% DMSO for 24 hours, then cultured on medium with 0.2 mg/L 6-BA for 15 days. Declarations Data availability The datasets generated during the current study are available from the corresponding author upon reasonable request. Acknowledgments This work was supported by the Chinese program fund for Construction of National Agricultural Sustainable Development Experimental Demonstration Zone in Taizhou (tzyjy202302), Science and technology research program of Suzhou (SNG2022017, SNG2022023), Science and Technology Research Program of Changshu (CN202305), Jiangsu Province Seed Industry Revitalization Project (JBGS [2021]075) and Natural Science Foundation of Nantong City (JC2023049). Author Contributions Yan lelong and Qian chuntao contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Yan lelong. Ploidy identification and trait statistics were completed by Zhu songyu, Wu kaimin, and Huang yao. The draft of the manuscript was written by Yan lelong and revised by Qian chuntao, Zhu songyu, Wu kaimin, Huang yao, Wang kang, and Yang jiaxi. All authors read and approved the final manuscript. Conflict of interest There are no competing interests. References Ari, E., H. Ikten, M. Gocmen, R. Coskun and A. Eren (2010). Comparative evaluation of different embryo rescue techniques on parthenogenetic melon ( Cucumis Melo L.) fruits induced with irradiated pollen. African Journal of Biotechnology 9: 5347-5356. https://doi.org/10.5897/AJB10.514 Asadi, A., A. Zebarjadi, M. R. Abdollahi and J. M. Seguí-Simarro (2018). 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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-5332893","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373510980,"identity":"b109f019-a778-4f44-880f-35d5183dd1bd","order_by":0,"name":"yan lelong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACNvnDx39+qLDh4WdvbHyQUFFDWAufBFuCtMSZNDnJnsOHDR6cOUZYi5wEj4IEb9thY4MbaWmSD1uYiXCYdA+DgcSZw4kbbuSYVSQ2sDHwt3cn4Ncic/ZAQkFFeuLMM2/MbiTukGGQOHN2A34tDHkJByTOWCf2Hc8BajnDBrQzl5CWHMMG3jbmxIYDOWYFiW3MRGiRyDFm4G1zNhY4kZbGQJwWnmNpzLBAlkg4c4yHoF/k25uPMcKi8uOPiho5/vZe/FowAA9pykfBKBgFo2AUYAUAASxOSBqPQikAAAAASUVORK5CYII=","orcid":"","institution":"National Key Laboratory of Crop Genetics nad Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China","correspondingAuthor":true,"prefix":"","firstName":"yan","middleName":"","lastName":"lelong","suffix":""},{"id":373510981,"identity":"6958a834-ff0b-437e-ab70-eed311c3ce6c","order_by":1,"name":"zhu songyu","email":"","orcid":"","institution":"National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China","correspondingAuthor":false,"prefix":"","firstName":"zhu","middleName":"","lastName":"songyu","suffix":""},{"id":373510982,"identity":"47b26028-c52f-40fc-ac69-1f3bcb9bb78d","order_by":2,"name":"wu kaimin","email":"","orcid":"","institution":"National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agritultural University, Nanjing, China","correspondingAuthor":false,"prefix":"","firstName":"wu","middleName":"","lastName":"kaimin","suffix":""},{"id":373510983,"identity":"3934bc45-0511-4f73-a286-2cedf7e69583","order_by":3,"name":"huang yao","email":"","orcid":"","institution":"Nanjing Agricultural University, Nanjing; New Rural Development Research Institute of Nanjing Agricultural University, Suzhou, China","correspondingAuthor":false,"prefix":"","firstName":"huang","middleName":"","lastName":"yao","suffix":""},{"id":373510984,"identity":"25c16a44-783d-4307-a9cf-adf5f1d382e2","order_by":4,"name":"zhang yaomei","email":"","orcid":"","institution":"National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China","correspondingAuthor":false,"prefix":"","firstName":"zhang","middleName":"","lastName":"yaomei","suffix":""},{"id":373510985,"identity":"1bd3f8fd-ee7d-4f4c-97a1-8a5d0be5adb7","order_by":5,"name":"yang jiaxi","email":"","orcid":"","institution":"National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China","correspondingAuthor":false,"prefix":"","firstName":"yang","middleName":"","lastName":"jiaxi","suffix":""},{"id":373510986,"identity":"cadfcec6-854f-443c-847c-2ae7dbbe73f5","order_by":6,"name":"wang kang","email":"","orcid":"","institution":"Jiangsu Yanjing Institute of Agricultural Sciences, Jiangsu Academy of Agricultural Sciences, Nantong, China","correspondingAuthor":false,"prefix":"","firstName":"wang","middleName":"","lastName":"kang","suffix":""},{"id":373510987,"identity":"0a0c585d-dc1b-48b0-8427-f157ceed2ed9","order_by":7,"name":"qian chuntao","email":"","orcid":"https://orcid.org/0009-0008-9387-2196","institution":"National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China","correspondingAuthor":false,"prefix":"","firstName":"qian","middleName":"","lastName":"chuntao","suffix":""}],"badges":[],"createdAt":"2024-10-25 13:56:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5332893/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5332893/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-02995-7","type":"published","date":"2025-02-07T15:58:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68974332,"identity":"19537554-cf56-4dfe-90ba-b9085ebef55d","added_by":"auto","created_at":"2024-11-14 06:27:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7258047,"visible":true,"origin":"","legend":"\u003cp\u003eThe whole procedure of\u003cem\u003e \u003c/em\u003eivCD. 1) Preparation of explants and antimicrotubular agent solution: a: Haploid plantlet age: 21 d; b: Haploid plantlet age: 28 d; c: Haploid plantlet age is 35 d; d: Melon haploids are sectioned into 2-3 cm segments; 2) Antimicrotubular agents treatment: e-g: Explants are treated with 500 mg/L colchicine + 2% DMSO, 50 mg/L oryzalin + 2% DMSO and 50 mg/L trifluralin + 2% DMSO for 24 h, respectively; 3) Hormone-treatment:h-j: Explants are inoculated on MS medium with free-hormone, 0.2 mg/L 6-BA and 0.2 mg/L 6-BA + 0.15 mg/L NAA for 15 d, respectively; k: Explant vitrification; i: Callus forms at the base of the explant; m: Callus forms at the base of the explant and develops a root system; 4) Explant regeneration: n) Excise the green axillary bud section from the explant; o: Green axillary bud inoculate into MS medium and p: Axillary buds regenerate into complete plants.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5332893/v1/247b6a9a17bcfbad7080bce3.jpeg"},{"id":68974330,"identity":"5fad6aaf-b557-4d08-86f4-7e0a51dc50a8","added_by":"auto","created_at":"2024-11-14 06:27:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2146816,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis and chromosome counting of melon haploid and DH. a and c: The first normal peak appears at PE-A:2.2 E+5 for haploid and PE-A: 4.4 E+5 forDH. b and d: Chromosome number for haploid (n=12) and DH(2n=24).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5332893/v1/bbea73775de9d7a9e8b13edb.png"},{"id":68974334,"identity":"06253e50-b4a5-4a4f-b6e4-6c972dbb31c7","added_by":"auto","created_at":"2024-11-14 06:27:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17086287,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of stained pollen. a: Microscopic images of pollen; b: Abnormally shaped pollen, not stainable, lacking vitality; c: Normally shaped pollen, stainable and viable, and d: Normally shaped pollen, but not stainable, lacking vitality.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5332893/v1/3fd7dc0a00b87bf804c931c5.png"},{"id":68974331,"identity":"4fa4e2c0-ede7-4c30-a882-a48cef8afd63","added_by":"auto","created_at":"2024-11-14 06:27:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2774473,"visible":true,"origin":"","legend":"\u003cp\u003eComparisons of fruit, male flowers, and leaf of haploid and DH; a: Fruits: Haploid fruits are smaller than DHs, with no shapedifferences; b: Male flowers: Haploid male flowers are smaller and have fewer petals than those of DHs, and c: Leaves: Haploid leaves are smaller than those of DHs, with no significant differences in leaf shape from those of DHs .\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5332893/v1/0cee37efea414b2e342cebf0.png"},{"id":75931332,"identity":"1341c964-e52a-42a8-afe7-aaf911890144","added_by":"auto","created_at":"2025-02-10 16:14:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":46468852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5332893/v1/d278c54d-3665-4530-a6b7-400a5fec959a.pdf"},{"id":68974329,"identity":"4480e927-266e-4d92-9caf-de273d4c6580","added_by":"auto","created_at":"2024-11-14 06:27:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32268,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-5332893/v1/6292c77ed1828304bbdb4fd6.docx"}],"financialInterests":"","formattedTitle":"Efficiency Improvement of In Vitro Chromosome Doubling in Melon haploid","fulltext":[{"header":"Key message","content":"\u003cp\u003eThis study improved the melon haploid doubling efficiency and shortened explant regeneration period of the ivCD, and finally obtained DH with relatively high fertility.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eMelon (\u003cem\u003eCucumis melo\u003c/em\u003e L.), with its sweet taste and aromatic fragrance, is widely cultivated around the world, and its annual worldwide production reaches 28.5\u0026nbsp;million tons (FAOSTAT, 2022). Haploid breeding technology, with its characteristic of shortening the breeding cycle and enhance breeding efficiency(Zere Taskin and Bilgili \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Qu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), has been widely applied in the breeding of cucurbit crops such as melon(Sauton and Dumas de Vaulx \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), cucumber(Przyborowski and Nlemirowicz-Szgzytt \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), watermelon(Sari et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), pumpkin(Ertan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and summer squash(Kurtar et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are several methods that can accomplish haploid induction in cucurbit plants, such as anther culture(Asadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), interspecific pollination induced parthenogenesis(Sauton and Dumas de Vaulx \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), ovule/ovary culture(Deng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and pollination with radiated pollen(Salehian et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In melon, haploid induction is primarily achieved through pollination with irradiated pollen(Cuny et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Hooghvorst et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, its haploid induction rate is quite low, ranging only from 0\u0026ndash;3.4%(Lotfi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Ari et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which has hampered the subsequent obtain of mass doubled haploid for further melon breeding. Thus, it is imperative to ensure that it can be successfully doubled to form a DH for each melon haploid.\u003c/p\u003e \u003cp\u003eFor this, two primary methods of chromosome doubling are employed: ivCD(Lim and Earle \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and \u003cem\u003ein situ\u003c/em\u003e chromosome doubling(Hooghvorst et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The former is favored for its high efficiency and low chimeras compared to the latter(Fu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Meanwhile, researchers have founded that several factors can influence the efficiency of the ivCD of melon haploid, including the plant's genotype(Mentewab and Sarrafi \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), the type(Lim and Earle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and age(Gałazka et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) of explant, the selections of antimicrotubular agents, their concentration and treatment duration(Dhooghe et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), as well as the hormone and its concentrations(Xu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, to achieve a good effect of chromosome doubling of melon haploid, the ivCD technology system needs to be carefully designed or optimized.\u003c/p\u003e \u003cp\u003eAffected by factors above, issues such as low SR and SDR(Lim and Earle \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Lim and Earle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), high VR(Grzebelus and Adamus \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Khan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and prolonged regeneration period, are likely to arise during the process of ivCD. Based on different haploid genotypes and doubling measures, the SDR ranges from 0 to 23.24%(Lotfi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Lim and Earle \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Solmaz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Hooghvorst et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Explant age affects the regenerative capacity(Mohebodini et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and selecting explants with appropriate age can significantly increase the frequency of regeneration(Thomas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Sun et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Plant hormones, such as 6-BA, can regulate cell division and endoreduplication(Demeulenaere and Beeckman \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and promote explant regeneration(Yadav et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Lim and Earle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), thereby can improve the doubling efficiency. Colchicine is the most widely used reagent in this process, yet its low efficiency and high toxicity have been widely criticized(Bouvier et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Hooghvorst et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Some low-toxicity antimicrotubular agents, such as trifluralin and oryzalin, are increasingly used in the doubling process and are gradually replacing colchicine in cucumber(Ebrahimzadeh et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, in order to enhance the SR and SDR, and reduce VR and shorten axillary bud regeneration duration, we have carried out two experiments. In Experiment 1, we have screened out fit explant age with relative higher SR and SDR, but also found several issues such as high VR and long regeneration time during the doubling process. In Experiment 2, we have further reduced the VR and shortened the duration of regeneration time through the selection of suitable combinations of antimicrotubular agents and hormone treatments using four genotypes (BAI_1, G6B_1, G6_1, and JG_1) selected in Experiment 1. Our progress will promote and expand the application of haploid breeding technology in melon.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eIn our preliminary experiments, we used 15 different genotypes as parental materials to induce hundreds of haploid melons with various genotypes (Huang et al. 2024, online). The parental material was grown in polytunnel greenhouses at the Changshu Agricultural Science Institute in Changshu City, Jiangsu Province, China, which has a subtropical monsoon oceanic climate, and this work was carried out between 2022 and 2023. Among them, we selected nine relatively robust haploids which exhibited normal leaf, stem, and root growth as materials (Table 1) for ivCD in\u0026nbsp;Experiment\u0026nbsp;1. Among nine genotypes, BAI_1，FW 5_1 and FW 8_1 are thick-skinned, G6B_1 and G6_B_2 are middle-skinned, G6_1, G6_2, JG_1, G61_1 and G6_2 are thin-skinned.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;1\u0026nbsp;Haploid materials-genotype, type and origin\u0026nbsp;in Experiment 1.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eHaploid code\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003eDonor parental materials\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003eSubspecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eOrigin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eBAI_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003eBAIPICUI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003eagrestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eXAAS(Bi et al. 2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eFW 5_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003eFlavor NO. 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003emelo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eXAAS(Tang et al. 2010)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eFW 8_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003eFlavor NO. 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003emelo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eXAAS(Tang et al. 2010)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eG6B_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003ePI420145 \u0026times; BAIPICUI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003emelo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eG6B_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003ePI420145 \u0026times; BAIPICUI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003emelo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eG6_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003ePI 420145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003eagrestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eNAU(Wolukau et al. 2009)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eG6_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003ePI 420145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003eagrestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eNAU(Wolukau et al. 2009)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eJG_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003eJINGGUA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003eagrestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3061%;\"\u003e\n \u003cp\u003eG61_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.5306%;\"\u003e\n \u003cp\u003ePI420145 \u0026times; PI140471\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.449%;\"\u003e\n \u003cp\u003e\u003cem\u003eC. m.\u003c/em\u003e subsp. \u003cem\u003eagrestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.7143%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: \u0026lsquo;XAAS\u0026rsquo; means Xinjiang academic of agricultural sciences, \u0026lsquo;NAU\u0026rsquo; means Nanjing agricultural university, \u0026lsquo;CV\u0026rsquo; means commercial variety\u0026nbsp;obtained in local market.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vitro\u003c/em\u003e chromosome doubling\u0026nbsp;experiment design\u003c/h2\u003e\n\u003cp\u003eThe basic experimental procedures (Fig. 1) are performed as follows: 1) Preparation of explants and antimicrotubular agent solution: haploid nodal explant with different age (Fig. 1a-c) \u0026nbsp; were cut to a size of 2-3 cm(Fig. 1d), and antimicrotubular agents were dissolved in dimethyl sulfoxide (DMSO) (HUSHI, Shanghai), filtered through aseptic filtration, and then added to sterile liquid MS medium, with the volume fraction of DMSO adjusted to 2%. 2) Antimicrotubular agents\u0026rsquo; treatment (Fig. 1e-g): the nodal explant was soaked in sterilized antimicrotubular agents\u0026rsquo; solution, shaken on a shaker (CRY-1102C, Shanghai Rongyan) at a speed of 100 rpm, and maintained at a temperature of 25\u0026plusmn;1 \u0026deg;C under dark conditions. Subsequently, nodal explant segments were washed three times with sterilized distilled water. 3) Hormone treatment (Fig. 1h-m): This step was specific to Experiment 2, while for Experiment 1, the explants were treated with colchicine and then inoculated into hormone-free solid MS medium for a 15 days culture period. In Experiment 2, after treatment with antimicrotubular agents, the explants were inoculated into solid MS medium with hormone for further hormone-treatment \u0026mdash; a process that lasts for 15 days. 4) Explant regeneration (Fig. 1n-p): For Experiment 1 and Experiment 2, the explants \u0026nbsp;exhibit three different fates: vitrification (Fig. 1k), formation of callus at the base(Fig. 1l), and formation of both callus and roots at the base(Fig. 1m). Explants that became vitrified would eventually die, while green axillary buds from the latter two types would ultimately form regenerated plants when they were excised and inoculated into solid MS medium. The growth conditions for the explants were maintained at 2000 lx, 25\u0026plusmn;1\u0026deg;C, under a 12 hours light/12 hours dark cycle.\u003c/p\u003e\n\u003ch3\u003eEffect of genotype and explant age (Experiment 1)\u003c/h3\u003e\n\u003cp\u003eReferring to the method of Lim and Earle (2008), with appropriate adjustments, the nodal explants were treated in a solution containing 500 mg/L colchicine + 2% DMSO for 24 hours. Nine genotypes were selected, and explant of\u0026nbsp;different ages (21 days, 28 days, and 35 days). After treated by colchicine, the explants were inserted into solid MS medium for 15 days, and\u0026nbsp;during these days most of the explants exhibited vitrification (Fig. 1k). The axillary bud parts were then excised and inoculated into new solid MS culture medium, and after approximately 30 days complete\u0026nbsp;regenerated plants were formed. Then, using the optimal explant age, further investigation of the effects of\u0026nbsp;antimicrotubular agents and hormone treatments on ivCD was conducted.\u003c/p\u003e\n\u003ch3\u003eEffect of antimicrotubular agents and hormone-treatment (Experiment 2)\u003c/h3\u003e\n\u003cp\u003eConsidering\u0026nbsp;the issues encountered in Experiment 1, we performed experiments of\u0026nbsp;replacing the antimicrotubular agents and using hormone\u0026nbsp;treatments. In Experiment 2, we selected four genotypes (BAI_1, G6B_1, G6_1, and JG_1),\u0026nbsp;based on various SDRs, to verify the impact of antimicrotubular agents (500 mg/L colchicine + 2% DMSO, 50 mg/L oryzalin + 2% DMSO, 50 mg/L trifluralin + 2% DMSO)(Ebrahimzadeh et al. 2018)\u0026nbsp; and hormone\u0026nbsp;treatments (free hormone, 0.2 mg/L 6-BA, 0.2 mg/L 6-BA + 0.15 mg/L NAA)(Sun et al. 2009) on the SR, SDR and VR. Unlike Experiment 1, after the explants were treated with antimicrotubular agents for 24 hours, they were inoculated into a hormone-containing culture medium for 15 days. After 15 days, most explants formed callus tissue at the base\u0026nbsp;and some even developed roots. The axillary bud parts were then excised and inoculated into solid MS culture medium, where complete plants were formed after approximately 15-30 days.\u003c/p\u003e\n\u003ch2\u003eRegenerated plant ploidy identification\u003c/h2\u003e\n\u003ch3\u003eFlow cytometry\u0026nbsp;analysis\u003c/h3\u003e\n\u003cp\u003eFlow cytometry was used to identify the ploidy level of these regenerated plants\u0026nbsp;(Lotfi et al. 2003, Lim and Earle 2008). Tender leaves were fragmented in 800 \u0026mu;L of dissociation solution for 10 minutes and filtered through a 40-micron cell strainer. Then 35 \u0026mu;L of 1 M propidium iodide (PI) (Solarbio, China) and 10 \u0026mu;L of 1 M RNase (Takara, Japan) were added to the filtrate and incubated in darkness under low-temperature conditions. A Flow Cytometer (Beckman CytoFLEX) was used to analyze filtrate. Dissociation solution recipe is as follows: 10 mM MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (Xilong Scientific, China), 50 mM KCl (Xilong Scientific, China), 5 mM HEPES (Shyuanye, China), 0.25% (v/v) Triton X-100 (Solarbio, China), and 1% (m/m) PVP (Vokai, China).\u003c/p\u003e\n\u003ch3\u003eChromosome counting\u003c/h3\u003e\n\u003cp\u003eThe chromosome counting method was\u0026nbsp;referred to the work of\u0026nbsp;Zhang et al. (2023), with appropriate modifications made on this basis. The root system of tissue-cultured seedlings was usually physiologically older, with fewer mitotic divisions in the root tip meristematic zone. We inoculated the explants into MS medium with 30 g/L sucrose\u0026nbsp;(Solarbio, China) and 5 g/L agar\u0026nbsp;(Solarbio, China), and once the explants developed roots with a length\u0026nbsp;of 1-2 cm, they were ready for chromosome count observation. The basic procedure was as follows: the newly growth roots were treated with 0.002M Octahydroxyquinoline (Hushi, China) at 4\u0026nbsp;\u0026deg;C for 3 hours, rinsed with ddH2O, then fixed with Carnoy\u0026apos;s solution (Solarbio, China) at 4\u0026nbsp;\u0026deg;C for 9 hours. After rinsing, the roots were treated with 1M HCl at 60\u0026deg;C for 4 minutes, and then residual hydrochloric acid\u0026nbsp;was removed with distilled water. Subsequently, a 1\u0026nbsp;mm root tip was taken and stained with Carbol Fuchsin (Solarbio, China) at room temperature for 7 minutes, followed by slide preparation.\u003c/p\u003e\n\u003ch2\u003eHaploid and DH plant acclimatization and\u0026nbsp;morphological\u0026nbsp;observation\u003c/h2\u003e\n\u003cp\u003eThe tissue culture plantlets for acclimatization were inoculated into solid MS medium with 30 g/L sucrose and 5 g/L agar, and grown in the acclimatization chamber for 15 days under 2000 lx, 25\u0026plusmn;1 \u0026deg;C, with a 12 hours light/dark cycle. The\u0026nbsp;non-uncovered culture bottles were moved to outdoor growth for 5 days. Then we took out the seedling, removed the agar attached to the roots, transplanted them into sterilized substrate, thoroughly watered them with sterile water, and covered the nutrition bowl with a self-sealing bag to maintain local humidity. 5 days later, a corner of the bag was cut to reduce humidity, and after 10 days, the bag was removed to proceed for transplantation.\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of colchicine and trifluralin on the DH regenerated plantlets, we statistically analyzed key fertility-related traits, including PVR, PDR, and SGR. Among them, PVR and PDR were assessed through microscopic observation (Olympus CX43) using 0.5% Triphenylchlorotetrazolium Chloride (TTC) staining solution(Xiaohua et al. 2020). Pollen grains were considered viable if they turned red after being immersed into the TTC solution at 25 \u0026deg;C for 15 minutes. SGR(Sohrabi et al. 2016) was assessed by soaking seeds in warm water (50 ℃, 1 hour) and then incubating them at 25 \u0026plusmn; 1 \u0026deg;C for 1 day.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo observe the morphological differences, including leaf length and width, fruit size, and male flowers, between haploid and DH, we conducted statistical analyses based on the method of\u0026nbsp;Sarı (2017)\u0026nbsp;. Leaf length and width were measured at the 13th, 14th, and 15th nodes of the main vine, and the number of petals on male flowers located on the lateral branches at these nodes was recorded. Once the fruits reached full maturity, their length and diameter were meticulously measured.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eTo characterize the effects of different treatments on the doubling of melon haploid, we used the SR, SDR and VR (Table 2) to quantify and present the results of the doubling process. The sample sizes of SR, SDR, and VR were not completely consistent, ranging from 9 to 12, and were analyzed using analysis of variance (ANOVA) and the Tukey-Kramer test. The statistical analysis of fertility-related indicators in DH regenerated plants, including PVR, PDR, and SGR (Table 2), was conducted with consistent sample sizes, using ANOVA and Tukey\u0026apos;s post-hoc test. Each experiment was conducted with three replicates. The data were analyzed using Excel and R Studio.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;2\u0026nbsp;Equations of ivCD-related indicators and fertility-related indicators\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"914\" height=\"196\"\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn Experiment 1, nodal explants were treated with 500 mg/L colchicine and 2% DMSO for 24 hours\u0026nbsp;followed by being inoculate into solid MS medium for 15 days. Then, the explants with green axillary buds were segmented and inserted to new solid MS medium. At 21 days of explant age, the highest SR\u0026nbsp;(an average of 38.76\u0026plusmn;9.72%) and SDR\u0026nbsp;(19.04\u0026plusmn;7.10%), as well as the lowest VR\u0026nbsp;(37.36\u0026plusmn;9.93%), were obtained. Among the nine genotypes,\u0026nbsp;G6_1 showed the highest SR (55.19\u0026plusmn;5.01%) and SDR (27.41\u0026plusmn;4.49%), with the lowest VR (20.74\u0026plusmn;1.28%). Conversely, BAI_1 had the lowest SR (24.85\u0026plusmn;4.20%) and SDR (9.39\u0026plusmn;0.52%), but the highest VR (50.00\u0026plusmn;4.55%)\u0026nbsp;(Supplement 1). These results suggest\u0026nbsp;the genetic variation in the ivCD process of melon haploids. Furthermore, explants subjected to vitrification rarely regenerated into complete plantlets and often died. Although a few explants could develop into complete plantlets，they failed to achieve successful chromosome doubling. And other few explants that do not form vitrification, the development of complete plants from axillary buds was notably slow, taking over 30 days\u003c/p\u003e\n\u003cp\u003eIn Experiment 2, to enhance SR and SDR while reducing VR and regeneration time, we modified the antimicrotubular agent and added hormones to the solid MS medium. We identified an optimal combination: 50 mg/L trifluralin + 2% DMSO and 0.2 mg/L 6-BA. By applying this combination, compared to the results of Experiment 1, the four genotypes (BAI_1, G6B_1, G6_1, and JG_1) of SR\u0026nbsp;(72.27\u0026plusmn;9.44%)\u0026nbsp;and SDR\u0026nbsp;(42.12\u0026plusmn;9.72%) showed significant increases, while the VR\u0026nbsp;(2.35\u0026plusmn;4.25%) (Supplement 5)\u0026nbsp;significantly decreased\u0026nbsp;by 30.82%, 22.62%, and -30.41%, respectively. This treatment combination not only increased the SR and SDR and reduced the VR, but also\u0026nbsp;halved the time required for axillary buds to regenerate into complete plants, reducing it to just 15 days.\u003c/p\u003e\n\u003ch2\u003eEffect of genotype\u0026nbsp;on SR, SDR and VR\u003c/h2\u003e\n\u003cp\u003eThe variance analysis and post-hoc tests demonstrated significant differences (Table 3) on SR, SDR and VR in Experiment 1. The regenerative capacity was positively correlated with SR and SDR to a certain extent, and negatively correlated with VR. The haploids G6_1 and G6B_1 exhibited strong regenerative ability during the proliferation process, with robust root growth, and showed high SR and SDR, and relatively low VR during the doubling process. In contrast, BAI_1, FW 5_1, and FW 8_1 exhibited poor regenerative ability during the proliferation process, with underdeveloped root systems, and showed low SR and SDR, and high VR. This may indicate that the genotype affects the regenerative capacity, which in turn affects the efficiency of doubling.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;3\u0026nbsp;Effect of genotype and explant age on the variance of SR, SDR and VR in Experiment 1\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eExperiment 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003eSR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003eSDR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003eVR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003cp\u003e(explant age = 21\u0026nbsp;days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eBAI_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e24.85\u0026plusmn;4.20\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e9.39\u0026plusmn;0.52\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e50.00\u0026plusmn;4.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eFW 5_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e31.21\u0026plusmn;4.67\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e12.73\u0026plusmn;6.30\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e43.64\u0026plusmn;3.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eFW 8_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e28.18\u0026plusmn;1.57\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e15.76\u0026plusmn;5.84\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e46.97\u0026plusmn;2.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eG6B_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e45.15\u0026plusmn;5.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e25.76\u0026plusmn;5.17\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e29.09\u0026plusmn;1.57\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eG6B_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e42.42\u0026plusmn;5.25\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e24.24\u0026plusmn;5.25\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e33.33\u0026plusmn;10.50\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eG6_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e55.19\u0026plusmn;5.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e27.41\u0026plusmn;4.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e20.74\u0026plusmn;1.28\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eG6_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e43.64\u0026plusmn;3.15\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e21.82\u0026plusmn;4.81\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e37.58\u0026plusmn;2.10\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eJG_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e40.61\u0026plusmn;4.58\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e15.45\u0026plusmn;4.72\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e31.21\u0026plusmn;4.67\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eG61_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e37.58\u0026plusmn;2.10\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e18.79\u0026plusmn;1.05\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e43.64\u0026plusmn;3.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003eExplant age (day)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e38.76\u0026plusmn;9.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e19.04\u0026plusmn;7.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e37.36\u0026plusmn;9.93\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e24.42\u0026plusmn;10.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e9.88\u0026plusmn;4.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e59.44\u0026plusmn;8.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.9588%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e11.86\u0026plusmn;12.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e3.91\u0026plusmn;4.83\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.6804%;\"\u003e\n \u003cp\u003e71.09\u0026plusmn;5.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: The Experiment utilized a full-factorial design, with each trial being conducted three times, and the sample size for each Experimental group varying between 9 and 12. The Tukey-Kramer test was applied, with the level of significance set at 5% (P \u0026gt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEffect of explant age\u0026nbsp;on SR, SDR and VR\u003c/h2\u003e\n\u003cp\u003eVariance analysis and post-hoc tests (Table 3) indicated that explant age significantly affects the SR, SDR, and VR. As the age of the explant increased, SR and SDR gradually decreased, while VR increased, with the best results of\u0026nbsp;38.76\u0026plusmn;9.72% (SR), 19.04\u0026plusmn;7.10% (SDR) and 37.36\u0026plusmn;9.93% (VR)\u0026nbsp;at 21 days of explant age,\u0026nbsp;respectively (Supplement 1). Furthermore, as the age of the explant advanced, the plant morphology underwent corresponding changes: at 21 days (Fig. 1a), the leaves being deep green without any signs of yellowing; by 28 days (Fig. 1b), the basal leaves beginning to yellow; and by 35 days (Fig. 1c), the yellowing spreading to the middle leaves. Concurrently, the plant\u0026apos;s regenerative capacity progressively diminishes.\u003c/p\u003e\n\u003ch2\u003eEffect of antimicrotubular agents\u0026nbsp;on\u0026nbsp;SR, SDR and VR\u003c/h2\u003e\n\u003cp\u003eIn Experiment 2, the results of the analysis of variance and post-hoc tests (Table 4) indicated that for SR, SDR, and VR, the treatment with 50 mg/L trifluralin + 2% DMSO yielded good results, with respective values of 65.23\u0026plusmn;9.17%, 30.48\u0026plusmn;11.21%, and 5.50\u0026plusmn;5.55%\u0026nbsp;(Supplement 3). Meanwhile, we had observed that explants treated with colchicine required a regeneration period that was twice as long as that of trifluralin and oryzalin for the regeneration of complete plants from axillary buds during the second inoculation, taking over 30 days compared to the 15\u0026nbsp;days buffer period of trifluralin and oryzalin. During this process, the concentration of colchicine needed was 10 times that of trifluralin and oryzalin, exerting stronger phytotoxic effects on the plants, which were reflected in the prolonged duration, low SR and high VR compared to trifluralin and oryzalin.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;4\u0026nbsp;Effect of antimicrotubular agent and hormone-treatment on the variance of SR, SDR and VR in Experiment 2\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eExperiment 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003eSR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003eSDR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003eVR (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eAntimicrotubular agent(mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003e500 colchicine + 2% DMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e55.95\u0026plusmn;9.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e30.95\u0026plusmn;10.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e22.33\u0026plusmn;10.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003e50 oryzalin + 2% DMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e62.81\u0026plusmn;9.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e22.62\u0026plusmn;8.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e7.04\u0026plusmn;5.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003e50 trifluralin + 2% DMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e65.24\u0026plusmn;9.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e30.48\u0026plusmn;11.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e5.50\u0026plusmn;5.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eHormone-treatment (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003eFree hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e58.95\u0026plusmn;7.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e23.21\u0026plusmn; 7.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e15.79\u0026plusmn;11.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003e0.2 6-BA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e68.22\u0026plusmn;10.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e37.16\u0026plusmn;11.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e6.48\u0026plusmn;8.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.2371%;\"\u003e\n \u003cp\u003e0.2 6-BA +0.15 NAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e56.83\u0026plusmn;8.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e23.68\u0026plusmn;7.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.5876%;\"\u003e\n \u003cp\u003e12.60\u0026plusmn;10.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: The Experiment utilized a full-factorial design, with each trial being conducted three times, and the sample size for each Experimental group varying between 9 and 12. The Tukey-Kramer test was applied, with the level of significance set at 5% (P \u0026gt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEffect of hormone-treatment\u0026nbsp;on\u0026nbsp;SR, SDR and VR\u003c/h2\u003e\n\u003cp\u003eAnalysis of variance and post-hoc tests (Table 4) showed that the addition of 0.2 mg/L 6-BA to the MS medium significantly improved the SR and SDR, and minimized the VR compared to the free hormones and the addition of 0.2 mg/L 6-BA + 0.15 mg/L NAA, which were\u0026nbsp;68.22\u0026plusmn;10.63%,\u0026nbsp;37.16\u0026plusmn;11.44% and\u0026nbsp;6.48\u0026plusmn;8.20%, respectively (Supplement 4). In Experiments 1 and 2, a high prevalence of vitrification was observed, coupled with diminished SR and SDR, when nodal explants treated with antimicrotubular agents were subsequently cultured in a hormone-free medium. However, when the explants were inoculated into a medium containing 0.2 mg/L 6-BA, it significantly reduced the VR and improved the SR and SDR, indicated that 6-BA played a role in mitigating the damage caused by colchicine. In summary, 6-BA not only enhanced the SR and SDR but also reduced the VR.\u003c/p\u003e\n\u003ch2\u003ePolyploidy identification and tissue culture seedlings acclimation\u003c/h2\u003e\n\u003cp\u003eIn Experiments 1 and 2, we obtained a total of 881 regenerated plants\u0026nbsp;(Supplement 1\u0026nbsp;and\u0026nbsp;Supplement 2). After flow cytometric ploidy analysis, there were 481 haploid plants (n=12; Fig. 2a,b) and 400 diploid plants (2n=24; Fig. 2c,d). Subsequently, we randomly selected 20 plants from both the haploid and diploid groups. Chromosome counting confirmed the results consistent with the flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003eBy employing our enhanced acclimation technique, we have successfully achieved a survival rate of over 80%\u0026nbsp;(Table 5) for both haploid and diploid plants. The use of 5\u0026nbsp;g/L agar facilitated the removal of agar attached to the root system, minimizing damage to the roots. Acclimating for 5 days outdoors with covering helped to enhance the plants\u0026apos; adaptability to the environment. At this stage, the explants have been grown for 20 days, and their root systems have become robust. Transplanting the plants into sterilized substrate and watering them with sterile water throughout the process created a low-bacterial environment. Using self-sealing bags to create a localized high-humidity environment for the plants, followed by a gradual reduction in humidity, ensured a high acclimation survival rate through these integrated measures.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;5\u0026nbsp;The results of\u0026nbsp;tissue culture seedlings acclimation\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003egenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eploidy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003eNumber of acclimations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003eNumber of survival (Survival rate /%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eBAI_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e25(83.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e26(86.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eG6B_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e26(86.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e28(93.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eG61_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e25(83.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e27(90.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eJG_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e24(80.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e26(86.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 22%;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37%;\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40%;\"\u003e\n \u003cp\u003e207(86.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003eObservation of PVR, PDR, and SGR in DH regeneration plants\u003c/h2\u003e\n\u003cp\u003eIn Experiment 2,\u0026nbsp;trifluralin\u0026nbsp;and\u0026nbsp;colchicine\u0026nbsp;exhibited similar efficiency on SDR. Thus, for further investigation into the impact of these two antimicrotubular agents on the fertility-related traits of DH, we had compiled the PVR, PDR, and SGR indices of the DH treated with colchicine and trifluralin.\u003c/p\u003e\n\u003cp\u003eThe results of variance analysis and post-hoc tests (Table 6) indicated that trifluralin caused less damage to fertility than colchicine, as evidenced by the higher PVR and SGR observed in DH regenerated plants treated with trifluralin, along with a markedly lower PDR compared to those treated with colchicine. Of course, these indicators are based on data obtained from the first generation of DH plants, which only reflect the short-term effects of the antimicrotubular agents on the explants. These results can only reflect the toxicity of different treatments to a certain extent.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;6\u0026nbsp;Effects of colchicine and trifluralin on PVR, PDR and SGR of first generation of DH\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003egenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eAntimicrotubular agents\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003ePVR /%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003ePDR /%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003eSGR /%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 14%;\"\u003e\n \u003cp\u003eBAI_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eColchicine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e28.00\u0026plusmn;2.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e25.33\u0026plusmn;3.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e62.00\u0026plusmn;5.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eTrifluralin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e64.33\u0026plusmn;4.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e13.67\u0026plusmn;2.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e92.00\u0026plusmn;4.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 14%;\"\u003e\n \u003cp\u003eG6B_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eColchicine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e44.00\u0026plusmn;4.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e21.67\u0026plusmn;2.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e83.33\u0026plusmn;3.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eTrifluralin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e71.00\u0026plusmn;3.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e13.00\u0026plusmn;4.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e93.33\u0026plusmn;4.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 14%;\"\u003e\n \u003cp\u003eG61_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eColchicine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e41.00\u0026plusmn;1.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e24.33\u0026plusmn;3.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e78.00\u0026plusmn;2.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eTrifluralin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e59.67\u0026plusmn;2.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e12.33\u0026plusmn;2.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e92.67\u0026plusmn;3.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 14%;\"\u003e\n \u003cp\u003eJG_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eColchicine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e30.00\u0026plusmn;2.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e21.67\u0026plusmn;1.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e63.33\u0026plusmn;3.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 32%;\"\u003e\n \u003cp\u003eTrifluralin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e71.67\u0026plusmn;3.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e10.33\u0026plusmn;2.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17%;\"\u003e\n \u003cp\u003e93.33\u0026plusmn;4.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Each trial was set up with three repetitions, with a sample size of 50 for each Experimental group. The Tukey test was used, with the significance level set at 5% (P \u0026gt; 0.05).\u003c/p\u003e\n\u003ch2\u003eObservation of haploid and DH\u003c/h2\u003e\n\u003cp\u003eThen, we further observed the characteristics of haploids and DH. There has been very little description of melon haploids, especially regarding fruit traits, because haploids contain only one set of chromosomes, and flowers often aborted during development(Gałazka et al. 2015). However, we found that G6B_1 and G6_1 could form mature male and hermaphroditic flowers, and after treatment with Thidiazuron, the hermaphroditic flowers developed into fruits. Their male flowers produced a small number of pollens, which were stained by TTC\u0026nbsp;(Fig. 3). The color and stripes of the fruits were indistinguishable from those of DH, but the fruit length and diameter were visually smaller than those of DH. Furthermore, haploids exhibited a reduction in the size of various organs\u0026nbsp;(Fig. 4).\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;7\u0026nbsp;The fruit, male flower, and leaf part trait of haploid and DH of melon\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003ePloidy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 28%;\"\u003e\n \u003cp\u003eFruit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003eMale flower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 32%;\"\u003e\n \u003cp\u003eLeaf\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003eLength(cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003eWidth(cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003ePetals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003eLength(cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003eWidth(cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eBAI_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e9.99\u0026plusmn;0.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e8.75\u0026plusmn;0.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e12.88\u0026plusmn;0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e12.59\u0026plusmn;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e5.00\u0026plusmn;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e14.86\u0026plusmn;1.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e14.51\u0026plusmn;0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eG6b_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e6.05\u0026plusmn;0.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e3.54\u0026plusmn;0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e6.00\u0026plusmn;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e10.17\u0026plusmn;0.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e7.62\u0026plusmn;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e12.57\u0026plusmn;0.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e6.62\u0026plusmn;0.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e6.33\u0026plusmn;0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e14.56\u0026plusmn;0.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e13.87\u0026plusmn;0.74\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eG6_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e4.37\u0026plusmn;0.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e2.42\u0026plusmn;0.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e5.00\u0026plusmn;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e6.14\u0026plusmn;0.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e5.33\u0026plusmn;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e9.57\u0026plusmn;0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e5.76\u0026plusmn;1.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e5.67\u0026plusmn;0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e10.14\u0026plusmn;0.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e8.31\u0026plusmn;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11%;\"\u003e\n \u003cp\u003eJG_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eHaploid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e7.02\u0026plusmn;1.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e6.91\u0026plusmn;0.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11%;\"\u003e\n \u003cp\u003eDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14%;\"\u003e\n \u003cp\u003e6.07\u0026plusmn;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13%;\"\u003e\n \u003cp\u003e8.86\u0026plusmn;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15%;\"\u003e\n \u003cp\u003e7.67\u0026plusmn;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e11.97\u0026plusmn;1.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16%;\"\u003e\n \u003cp\u003e12.88\u0026plusmn;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: Each experiment is repeated three times, and three samples are collected for each treatment group. A t-test is conducted at a significance level of 0.05 (P \u0026gt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrent research on ivCD of melon haploids primarily focuses on improving the efficiency of haploid doubling(Lotfi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Lim and Earle \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Lim and Earle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Solmaz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, there has been insufficient attention paid to the use of antimicrotubular agents and issues that arise during the doubling process, such as high VR, prolonged explant regeneration duration, and the fertility of regenerated plants. This study has identified that using explants aged 21 days, replacing colchicine with trifluralin, and inoculating into the solid MS medium containing 6-BA after trifluralin treatment can not only significantly enhance the SDR compared to the former research(Lotfi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Lim and Earle \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Solmaz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Hooghvorst et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but shorten explant regeneration period and obtain DH with high fertility. This efficient processing can promote the application of haploid breeding in melon breeding.\u003c/p\u003e \u003cp\u003eThe role of explant age in the ivCD of melon haploids has been somewhat overlooked, despite its pivotal influence on the explant's regenerative capacity and doubling efficiency(Gałazka et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This factor determines whether the explant can survive and regenerate after treatment with antimicrotubular agents, thereby directedly affecting the SR and SDR. The intricacies of this relationship are particularly pronounced in the development of genetic transformation systems, where younger tissues have a stronger regenerative ability, but excessively young tissues have weaker tolerance and eventually die(Cao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Selecting the appropriate explant age can balance this relationship. In terms of SR and SDR, when explants are 21 days old, both are significantly higher than those when they are 28 or 35 days old.\u003c/p\u003e \u003cp\u003eCurrently, ivCD studies in melon haploid predominantly utilize colchicine as antimicrotubular agent. Yet it has a low binding affinity for plant microtubules(Dhooghe et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), requiring high concentrations of 500-5000mg/L for treatment(Ebrahimzadeh et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Hooghvorst et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which not only increases experimental costs but also poses risks of damaging plant fertility and DNA, potentially leading to loss of fertility and DNA mutations(Luckett \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Additionally, colchicine's ability to bind to animal microtubules presents potential health hazards to researchers(Morejohn et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). In contrast, dinitroaniline compounds, such as oryzalin and trifluralin, offer safer alternatives due to their high binding efficiency with plant microtubules and lack of interaction with animal microtubules(Grosso et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In our experiment, trifluralin was used at a dose one-tenth that of colchicine and was on par with colchicine in terms of SDR. The observed results (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) indicated that DH regenerated plants treated with trifluralin have superior PVR, PDR, and SGR compared to those treated with colchicine, thus confirming this conclusion.\u003c/p\u003e \u003cp\u003eAfter treatment with antimicrotubular agents, inoculating explants into solid MS medium with hormone for a period of time can enhance both the SR and SDR(Lim and Earle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Not only that, but hormone treatment can also break the vitrification and growth stagnation of explants. 6-BA, as a common plant growth regulator, promotes cell division and the growth and differentiation of buds(Werner et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and induces low endoreduplication activity, achieving polyploidization(Mahdad et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our Experiment 2, the addition of 0.2 mg/L 6-BA during the first inoculation, as opposed to free-hormone and 0.2 mg/L 6-BA\u0026thinsp;+\u0026thinsp;0.15 mg/L NAA treatments, greatly reduces the occurrence of vitrification, thereby breaking the growth stagnation after colchicine treatment(Tabei et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, haploid contains a set of chromosomes(Blakeslee et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1922\u003c/span\u003e), and it is often manifested as plant leaf length, leaf width, and flower size being smaller than diploid(Chase \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1964\u003c/span\u003e, Xu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our results (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) also verify this phenomenon. Moreover, there are reports indicating that haploid plants are fully infertile(Nikolova and NiemirowiczSzczytt \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Takahira et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Molenaar et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, we have observed that the haploids G6B_1 and G6_1 can form male and hermaphroditic flowers, which can produce a small amount of viable pollen and fruit respectively. The same phenomenon has also been observed in maize haploids(Chalyk \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), and the underlying reason may be cell fusion leading to spontaneous doubling in some cells(Chase \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1969\u003c/span\u003e, Gayen and Sarkar \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Testillano et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The mechanism by which melon haploids produce unreduced gametes needs further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we successfully increased SR and SDR, shortened the regeneration period, and ultimately yielded doubled haploids with relative high fertility through an effective method of ivCD in melon, in which 21-day-old melon haploids were treated with 50 mg/L trifluralin\u0026thinsp;+\u0026thinsp;2% DMSO for 24 hours, then cultured on medium with 0.2 mg/L 6-BA for 15 days.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Chinese program fund for Construction of National Agricultural Sustainable Development Experimental Demonstration Zone in Taizhou (tzyjy202302), Science and technology research program of Suzhou (SNG2022017, SNG2022023), Science and Technology Research Program of Changshu (CN202305), Jiangsu Province Seed Industry Revitalization Project (JBGS [2021]075) and Natural Science Foundation of Nantong City (JC2023049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYan lelong and Qian chuntao contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Yan lelong. Ploidy identification and trait statistics were completed by Zhu songyu, Wu kaimin, and Huang yao. The draft of the manuscript was written by Yan lelong and revised by Qian chuntao,\u0026nbsp;Zhu songyu, Wu kaimin, Huang yao, Wang kang, and Yang jiaxi. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no competing interests.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAri, E., H. Ikten, M. Gocmen, R. Coskun and A. Eren (2010). Comparative evaluation of different embryo rescue techniques on parthenogenetic melon (\u003cem\u003eCucumis Melo \u003c/em\u003eL.) fruits induced with irradiated pollen. 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New Zealand Journal of Crop and Horticultural Science 51: 341-354. https://doi.org/10.1080/01140671.2021.1971267\u003c/li\u003e\n\u003c/ol\u003e"}],"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":"Cucumi melo L., doubled haploid, explant age, trifluralin, 6-BA","lastPublishedDoi":"10.21203/rs.3.rs-5332893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5332893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e chromosome doubling (ivCD) using colchicine is a main method for melon haploid chromosome doubling, but its doubling efficiency remains low. In this study, we aimed to increase the efficiency of melon haploid doubling. In Experiment 1, the impacts of genotype and explant age on the survival rate (SR) and sample doubling rate (SDR) of melon haploid through ivCD were studied. Nine melon haploid genotypes were treated with colchicine for 24 hours followed by inoculation into solid MS. The best doubling effect was achieved with explant age of 21 days. During this process, the explants exhibited a low SR (38.76\u0026thinsp;\u0026plusmn;\u0026thinsp;9.72%) and SDR (19.04\u0026thinsp;\u0026plusmn;\u0026thinsp;7.10%), but a high vitrification rate (VR) (37.36\u0026thinsp;\u0026plusmn;\u0026thinsp;9.93%) and an extended period for explant regeneration for over 30 days. In Experiment 2, four representative genotypes were selected to screen suitable combinations of antimicrotubular agents and hormone-treatments to enhance the SR and SDR, and solve the other problems above. The results indicated that treatment with trifluralin for 24 hours, followed by inoculation into solid MS medium containing 6-BA for 15 days, yielded the highest SR (72.27\u0026thinsp;\u0026plusmn;\u0026thinsp;9.44%) and SDR (42.12\u0026thinsp;\u0026plusmn;\u0026thinsp;9.72%), but the lowest VR (2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25%) and a shortened regeneration period of 15 days. Interestingly, in Experiment 2, doubled haploid (DH) obtained from treatment with trifluralin performed better in pollen viability rate (PVR), pollen deformity rate (PDR), and seed germination rate (SGR) compared to those treated with colchicine. In summary, we significantly increased the haploid chromosome doubling rate, reduced the regeneration time, and obtained DHs with relatively high fertility.\u003c/p\u003e","manuscriptTitle":"Efficiency Improvement of In Vitro Chromosome Doubling in Melon haploid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 06:27:37","doi":"10.21203/rs.3.rs-5332893/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-11-04T12:59:54+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-03T20:16:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-29T05:53:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2024-10-26T07:06:19+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":"219f5a9e-74ab-497a-a3fd-fe110bfc5507","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:10:08+00:00","versionOfRecord":{"articleIdentity":"rs-5332893","link":"https://doi.org/10.1007/s11240-025-02995-7","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-02-07 15:58:21","publishedOnDateReadable":"February 7th, 2025"},"versionCreatedAt":"2024-11-14 06:27:37","video":"","vorDoi":"10.1007/s11240-025-02995-7","vorDoiUrl":"https://doi.org/10.1007/s11240-025-02995-7","workflowStages":[]},"version":"v1","identity":"rs-5332893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5332893","identity":"rs-5332893","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}