Improvement of embryogenesis in cucumber (Cucumis Sativus L.) parthenogenesis by polyamine and cycocel treatments

Improvement of embryogenesis in cucumber (Cucumis Sativus L.) parthenogenesis by polyamine and cycocel treatments | 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 Improvement of embryogenesis in cucumber (Cucumis Sativus L.) parthenogenesis by polyamine and cycocel treatments Sakineh Farhadi-Tooli, Mehran E. Shariatpanahi, Mohammadvali Habibi Silabi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8326911/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract This study evaluated the combined application of polyamines (putrescine, spermidine) and cycocel (CCC) to maternal plants in relation to the effect of pollen irradiation with gamma rays on seed formation, embryo formation and haploid production in three cucumber genotypes (Storm, Sahm, 547) using pollen irradiation at doses of 400 to 600 Gy. Maternal plants were treated with combination of putrescine (500 mg/L), spermidine (50 mg/L) and CCC (50 mg/L) from 12 days before to 12 days after pollination. Male flowers were irradiated with gamma rays (400, 500, 600 Gy) and pollination was performed. Embryos were rescued in vitro and regenerated plantlets were analyzed for ploidy level using flow cytometry and SSR marker to confirm haploid status. Haploid plants produced were treated with oryzalin for chromosome doubling. The combined treatment of polyamine and CCC significantly affected embryo induction and seed formation, with the Storm genotype producing the highest number of embryos and regenerated plants at a dose of 600 Gy. Embryogenesis and regeneration varied by genotype, with Storm and 547 showing the strongest responses; Sahm was the least responsive. Flow cytometry confirmed haploid/diploid status, and SSR validated homozygosity in doubled haploid (DH) lines. Higher gamma doses not only increased empty seeds but also embryo numbers, underscoring genotype-dependent radiation sensitivity. Controlled pollen irradiation combined with foliar polyamine/CCC pretreatment effectively induce haploid embryos and DH lines in cucumber, with strong genotype dependency and optimal effects at 600 Gy for certain genotypes. These findings support genotype-informed optimization of DH production and offer practical guidelines for accelerating cucumber breeding. Cucumber haploid embryogenesis gamma radiation polyamine cycocel Figures Figure 1 Key Message The combined application of polyamines and “CCC“ on the maternal plant, plus gamma-irradiated pollen, yields genotype-dependent embryogenesis improvements and haploid production in cucumber, showing Storm and 547 as the responsive varieties. Introduction Cucurbitaceae is among the most widely cultivated and economically important vegetable families. Cucumis sativus L. (2n = 2x = 14) is a key cucurbitaceous crop valued commercially and widely used as a model in vegetable breeding and functional genomics (Dey et al. 2022 ). It is grown globally under diverse agroclimatic conditions, in both open-field and protected environments. The species originates from India, with a secondary center of diversity in China and the Near East. As an out breeding crop without inbreeding depression, cucumber provides an exemplary system for investigating a range of developmental and molecular pathways, aided by its relatively small genome (~ 372 Mbp) (Huang et al. 2009 ). The classification of Cucumis species into primary, secondary, and tertiary gene pools has been established through comprehensive assessments of cross-compatibility, genetic analyses, phylogenetic studies, and molecular evidence (Dey et al. 2022 ). Application of doubled haploid (DH) technology offers a powerful route to accelerate conventional breeding by enabling the rapid production of completely homozygous lines, thus bypassing multiple generations of selfing (Germana 2006; Ahmadiet al., 2018 ). Traditional hybrid breeding relies on inbred parental lines, typically requiring 6–8 years of selfing in cucumber to achieve homozygosity. By contrast, DH induction via gynogenesis and androgenesis provides a single‑step route to homozygous parental lines, with demonstrated applicability across several vegetable crops (Behera et al. 2022 ; Dey et al. 2022 ; Bhatia et al. 2018 ; Mineykina et al. 2021 ; Romanova et al. 2023 ; Mulyana et al. 2023 ; Zhang et al. 2023 ). Successfully produced in vitro DH plants, whether via androgenesis or gynogenesis, provide valuable resources for research and breeding, including the development of mapping among biotechnological strategies, androgenesis ( in vitro anther/microspore culture), gynogenesis ( in vitro ovule/ovary culture), and parthenogenesis (egg cell induction by irradiated pollen followed by in vitro haploid embryo rescue) have proven more efficient and sustainable than conventional methods (Shariatpanahi and Ahmadi 2016 ). Notably, gynogenesis often yieldshaploids at higher frequencies than androgenesis in cucurbitaceous crops, as female haploid cells within the embryo sac/ovules tend to respond more readily than male haploid cells produced in the same flower. Structural differences haploid cells residing inside ovules and encased by nucellar and integument tissues versus male haploids confined to the anther wall contribute to the greater complexity and longer timelines of ovary/culture approaches, necessitating advanced technical expertise(populations, inbred lines, and expanded genetic diversity for breeding programs(Segui-Simarro 2021 ; Ferrie 2011 ). Polyamines are natural polycationic amines produced in living cells, including putrescine, spermidine, and spermine. Polyamines influence many biological processes, including cell division, differentiation, organogenesis, pollen formation, fertilization, gene expression, DNA and protein synthesis, apoptosis, and stress responses (reviewed by Tiburcio et al. 2014 ). There is compelling evidence for their crucial roles in somatic/gametic embryogenesis. Higher polyamine levels in unfertilized ovaries correlate with stronger gynogenesis ( Wei et al. 2010 ; Ahmadi et al. 2014;), and adding polyamines to induction media markedly enhances somatic/gametic embryogenesis and subsequent embryo-to-plant conversion across multiple species (Kumar et al.2004;Ponce et al. 2006 ;Thiru-vengadam et al.2013; Ahmadi et al. 2014;Ebrahimzadeh et al. 2018 ). However, the underlying mechanism through which polyamines exert an effect is largely unknown. The plant growth regulator (CCC) is widely used to reduce stem elongation, promote flowering, and improve bud formation. CCC inhibits gibberellin production early in development (Zhang et al. 2009 ; Ebrahimzadehetet al. 2018). The regulatory role of CCC in in vitro somatic/gametic embryogenesis is less clear: CCC in induction media can retard embryo formation in some cases, while applying CCC to donor plants can markedly improve in vitro embryo formation, germination, and plant regeneration in certain species such as Vitis vinifera (Alifar et al. 2015 ; Ebrahimzadeh et al. 2018 ). Application of irradiation to pollen as a means to induce haploidy remains a widely used and efficient approach in cucurbits, with ultraviolet (UV), gamma rays, and X-rays serving as recalcitrant agents to stimulate haploid formation. Among these, gamma irradiation is particularly favored in practical programs due to its simplicity, good penetrance, reproducibility, relatively high mutation frequency, and manageable abortion rates, making it the most commonly adopted method for haploid induction in many cucurbit breeding initiatives(Gałązka and Niemirowicz-Szczytt 2013; Chahal and Gosal 2002 ). Parthenogenesis has emerged as the most effective strategy to achieve complete homozygosity in Cucumis sativus L. in a single generation, although its routine deployment in breeding is limited by low haploid production rates(reviewed by Gałązka and Niemirowicz-Szczytt 2013; Dong et al. 2016 ). In irradiated pollen, observations include chromosome misbehavior and physio-biochemical alterations, with reports noting instances where a single male gamete is available for fertilization of either the egg cell or the fused polar nuclei; the former can lead to maternal homozygous diploids, while the latter may result in haploid parthenogenesis (pseudoembryony) triggered by irradiated pollen. Irradiated pollen can be still germinated on the stigma, extended through the style, and reached the embryo sac without fertilizing the egg cell or polar nuclei, allowing for parthenogenetic induction or gynogenic haploid production (Zhang 1991 ; De Witte 2000 ;Cuny 1993 ). Induction of haploid parthenogenetic embryos by in situ pollination with irradiated pollen has been reported in multiple Cucurbitaceae species, including Cucumis melo , C. sativus , Citrullus lanatus , Cucurbita pepo, C. moschata , and C. maxima , with varying efficiencies dependent on genotype, growth conditions, irradiation source and dose, and the developmental stage of embryos at excision (Sari et al. 1992 ; Taner et al.2000; Lotfi et al. 2003 ; Ebrahimzadeh et al. 2018 ; Ebrahimzadeh et al. 2013 ; Kurtar et al. 2009 ;Kurtar and Balkaya 2010 ). In this study, the concurrent effects of spraying maternal plants with putrescine, spermidine, and CCC from 12 days before to 12 days after pollination at 1-day intervals were examined, using three doses of gamma irradiation, on the frequency of haploid plant production. Materials and Methods Plant material, Pollen Irradiation and In Vivo Fertilization This study was conducted in the greenhouse of the Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj. The”Sahm”, “547” and “Storm” genotypes were used as maternal plants, with the “Beta Alpha” cultivar serving as the pollen donor. Plants were grown and maintained in a glasshouse under a 16-hour light/8-hour dark photoperiod, at a day temperature of 25 ± 2°C and a night temperature of 20 ± 2°C. To support plant growth, daily fertilization was performed with Hoagland’s nutrient solution. Mother plants were sprayed with concentrations of putrescine, spermidine, and CCC each at 500, 50 and 50 mg/l, ranging from12 days before until 12 days after anthesis with 1-day intervals. One day before the anthers opened and dehisced, male flowers (greenish-yellow) were collected and exposed to gamma radiation from a cobalt-60 source at a dose rate of 0.2 Gy/s with various gamma doses (400, 500 and 600 Gy). The female flowers of the F1 hybrids, serving as pollen recipients, were bagged to prevent contamination by unwanted pollen, up to dusk on the same day. After irradiation, the male flowers were kept at room temperature until pollination in the following day. Early the next morning, when the stigma was receptive to pollen, pollination was performed using pollen grains from the irradiated anthers. For each flower, 2–3 anthers were sufficient. The female flowers were re-bagged for 3–4 days with their petals closed to prevent cross-pollination. Embryo Rescue Growth sprouts were harvested after 35 days and surface-sterilized with 70% ethanol to reduce greenhouse contaminants. After washing, the fruits were opened and the seeds were extracted from running tap water and cleaned. Seed surface sterilization was performed under a laminar flow hood with 1% sodium hypochlorite for 10 minutes, followed by three washes with sterile distilled water for 5 minutes each.To facilitate identification and embryo extraction, the sterilized seeds were transferred to liquid medium E20 (Sauton and Dumas de Vaulx, 1987 ) supplemented with 3% sucrose and 0.01 mg/L indole-3-acetic acid (IAA) according to Lotfi et al. ( 2003 ). Approximately 25 seeds per dish containing 25 mL liquid E20 medium were used, ensuring seeds were submerged. Dishes containing immature embryos were sealed with Parafilm and placed in a growth chamber under standard conditions for tissue culture: a 16-hour photoperiod (light) and an 8-hour dark period, at a temperature of 22 ± 2°C. The dishes were gently agitated several times per week to maintain aeration. After 10–20 days, embryos were examined and isolated using a suitable light source. Isolated embryos were transferred to solid E20 medium (Fig. 1 a,b).One to three weeks after culture, the developed plantlets (epigeal plantlets) were transferred to glass jars containing MS medium supplemented with BAP at 2.0 mg/L and NAA at 0.05 mg/L for further growth (Fig. 1 c). After three weeks, for better growth of small plantlets, the culture was transferred to MS medium without supplements (Fig. 1 d). Ploidy Level Determination Flow cytometry Small section of young leaf tissue (0.5 cm) from diploid control plants (control) and plants with unknown ploidy, produced by induced apomixis, was separately excised with a fine scalpel in 500 µL of hypotonic propidium iodide (PI) lysis buffer, cooled on ice. The tissue was finely chopped.Subsequently, 2000 µL of nuclear staining solution and 6 µL of RNase were added, followed by 12 µL of propidium iodide (PI) to the crude mixture.To reduce the effects of cytosolic and phenolic compounds on PI fluorescence, 1% PVP-40 was also added to the buffer.The suspension was filtered through a 50 µm filter to remove cell debris and isolate nuclei. Ploidy level of the samples was analyzed with a flow cytometer (Partec, Münster, Germany) using a 488 nm Argon laser to excite the fluorochrome PI and a FL-2 detector with a 585/42 nm emission filter. Samples were run at low pressure and a minimum of 10,000 nuclei per sample were analyzed. Subsequently, DNA fluorescence per nucleus was measured and results were displayed as histograms. Since the injected sample contained a mixture of haploid and diploid plant extracts, the ploidy level of the sample was determined based on the fixed peak positions for haploid and diploid references. Induction of doubled haploids in regenerated haploid plants Haploid plantlets regenerated were micro-propagated in vitro before chromosome doubling. Young shoots, about 1–3 cm in length and leafless, were excised and transferred to liquid medium E20 containing 0.2 mg/L BAP, 80 mg/L Fe-EDDHA, and 50 mg/L oryzalin. The antimutagenic agent (oryzalin) was dissolved in dimethyl sulfoxide (DMSO, 2% v/v) and then added to the culture medium. The samples were incubated for 18 hours in a incubator at a constant temperature of 27°C with a speed of 150 rpm. Subsequently, the samples were washed three times with sterile distilled water, and the microexplants were transferred to solid E20 medium containing 0.01 mg/L IAA, 0.2 mg/L BAP, and 80 mg/L Fe-EDDHA. All cultures were maintained in a growth chamber under standard light conditions (photoperiod:16 h light/8 h dark) and at a temperature of 22 ± 2°C. After 3 weeks, each regenerated plantlet was individually transferred to flasks containing MS medium. Distinguishing doubled haploids from diploid plants In this study, the SSR marker CMCCA145 was used to identify the embryo type (diploid or doubled haploid). More than 20 SSR markers taken from the cucumber genome (from the Soul genomic network) were screened and the CMCCA145 marker was selected due to its apparent polymorphism. DNA extraction from 200 mg of young leaf tissue was performed using CTAB reagent according to the protocol with minor modifications. The quality and quantity of DNA were assessed using gel electrophoresis and fluorometric assay. PCR amplification was performed in a 96-well Variti thermal cycler (Applied Biosystem, USA) using the following cycles: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and a final extension step at 72°C for 5 min. The amplified products were examined using a 8% acrylamide gel in TBE buffer stained with Gelred using vertical electrophoresis. They were then visualized under UV light (Gelduc., UK). The size of the amplified fragments was determined using a 50bp Gen Ruler DNA ladder (Thermo Scientific, USA). Experimental design and data analysis method The experiment was a factorial experiment in a completely randomized design (the first factor was genotype and the second factor was gamma ray dose) with three replications. Two fruits were examined from each replication. SPSS software was used for data normalization and SAS software version 9.4 was used for data analysis. Results Simultaneous spraying of putercine, spermidine and CCC on mother plants with concentrations of (500, 50 and 50 mg/L) respectively along with irradiation with different doses of gamma rays to sterilize pollen grains showed that the interaction effect of genotype with gamma ray dose had a significant effect at the 5% probability level on the number of seeds per fruit. The results showed that the highest number of seeds per fruit was in the Storm genotype with a gamma ray dose of 600 Gy (205 pcs.) and the lowest number of seeds per fruit was in the 547 genotype (66.88 pcs.) and the gamma ray dose treatment was 400 Gy.The results showed that with increasing dose from 500 to 600Gy, the number of seeds per fruit increased in 3 tested cultivars (Table 1 ).Also, the genotype and gamma ray dose had a significant effect on the number and percentage of full and unfilled seeds in cucumber fruit at the 5% probability level. The results showed that in all 3 varieties, the control treatment had the highest number of seeds. The highest number of filled seeds (124.66) was related to the 547 genotype, and the lowest number was observed in the same genotype and gamma ray dose treatment at 600 Gy. Considering that the number and percentage of full seeds decreased with increasing gamma ray dose, the role of gamma ray, especially at higher doses, in the sterilization of pollen grains can be justified (Table 1 ). The response of each genotype under the same conditions was different, indicating that the effect of genotype is also effective in the success rate of parthenocarpy. Regarding the number and percentage of whole and empty seeds, the results showed that the interaction between genotype and gamma radiation dose had a significant effect at the 5% probability level. The Storm genotype pollinated with pollen irradiated with a dose of 600 Gy had the highest number of whole and empty seeds (153), and the lowest number (21.66) was observed in the Saham genotype and the control treatment. Our results showed that by increasing gamma radiation dose, the number of whole and empty seeds in the fruit increased, and also the response of different genotypes was different. Considering that the embryo is obtained from empty seeds, the increase in the number of empty seeds due to irradiation indicates the efficiency of gamma radiation in sterilizing anthers. Data on the number of embryogenesis and regenerated plants in cucumber fruit through pollen irradiation showed that the interaction effect of genotype with gamma radiation dose had a significant effect at the 5% probability level on the number of embryos and regenerated plants (Table 2 ), such that the Storm genotype with a radiation dose of 600 Gy showed the greatest effect on the number of embryos and regenerated plants (6 and 5, respectively), while in the control treatment no embryo formation was observed in any of the three cultivars. Examination of the main effect of gamma radiation dose showed that the dose of 600 Gy had the greatest effect on the number of embryos and regenerated plants in all three cultivars studied. Table 1 Analysis of the comparison of means of the interaction effect of genotype and gamma radiation dose on the total number of seeds in the fruit, whole and full seeds, percentage of whole and full seeds, whole and empty seeds and percentage of whole and empty seeds in the three genotypes of cucumber ( Cucumis sativus L.). Genotype Gamma irradiationdose (Gy) Total seeds/fruit (TSF) Filledseeds/fruit (FSF) Filledseeds (%) Emptyseeds/fruit (ESF) Emptyseeds (%) ” sahm F1” Control 400 500 600 138.33 de 90.66 g 126 ef 133.33 de 88.33 b 28.66 cd 29.66 cd 20.66 d − f 63.85 c 31.64 d 23.53 e 15.5 g 21.66 h 43.33 gh 69.33 ef 83.66 cd 15.68 g 47.8 f 55.01 e 62.77 d ” stoorm F1” Control 400 500 600 180.33 bc 156 dc 189 ab 205 a 117.66 a 37.66 c 22.33 de 16.66 ef 65.13 b 24.16 e 11.83 h 8.12 j 31.33 gh 88 c 128.66 b 153 a 17.77 g 56.46 e 68.12 c 74.53 a ” 547 F1” Control 400 500 600 187 ab 95.33 g 88.66 g 103 gf 124.66 a 19.66 d − f 10.66 f 10 f 66.66 a 20.62 f 12.03 h 9.69 i 29.33 h 60.33 f 62 ef 74.66 de 15.68 g 63.21 d 70 bc 72.58 ab Shared superscrip tletters indicate that means do not differ significantly at the 5% probability level (p ≤ 0.05). Table 2 Analysis of the comparison of means of the interaction effect of genotype and gamma radiation dose on the number of embryos, percentage of embryogenesis, number of regenerated plants and resistance to regeneration in the three genotypes of cucumber ( Cucumis sativus L.). Genotype Gamma irradiationdose (Gy) Embryos (No.) Embryogenesis (%) Regeneratedplants (No.) Regeneration (%) ” sahm F1” Control 400 500 600 0 g 3.66 d − f 3.33 ef 2.66 f 0 d 2.75 cb 3.57 b 2.13 c 0 f 2.33 de 3 c − e 2 e 0 d 63.88 bc 93.88 a 72.22 bc ” stoorm F1” Control 400 500 600 0 g 4 c − e 4.66 b − d 6 a 0 d 2.61 cb 2.46 2.92 c 0 f 3.33 b − d 3 c − e 5 a 0 d 83.33 ab 65 bc 83.8 ab ” 547 F1” Control 400 500 600 0 g 3.33 ef 5 a − c 5.66 ab 0 d 3.45 b 5.68 a 5.52 a 0 f 2 e 3.66 bc 4.33 ab 0 d 58.33 c 73.88 a − c 76.66 a − c Shared superscrip tletters indicate that means do not differ significantly at the 5% probability level (p ≤ 0.05). Ploidy analysis using a flow cytometry to determine the ploidy level of regenerated plants resulting from in vitro embryo rescue showed that all regenerated plantlets contained the gametic chromosome number (n = x = 7) of the parental plants. As shown in (Fig. 1 ), diploid and haploid samples showed a single, distinct peak, while in haploid plants, the number of peaks was approximately half that of diploid plants (Fig. 1 j ,i). In contrast, a mixed sample containing diploid and haploid nuclei showed three distinct peaks. The first peak (which may be due to instrument noise), a second peak with a higher height indicating the G1 phase of the cell cycle, and a third peak indicating the G2/M phase, located approximately twice as far away as the previous peak ( Fig. 1 k).Oryzalin was applied to the nodal sections at a rate of 50 mg for 18 hours at a speed of 150 rpm and showed a favorable effect on the rate of chromosome doubling and the generation of doubled haploid plants(Fig. 1 e).The SSR marker was also used to assess the ploidy status of plants for which chromosome doubling was performed. Banding profiles on 8% acrylamide gels showed that haploid samples produced a single band of low intensity and samples in which doubling was performed had a similar band but with stronger intensity, probably due to the homozygous nature of their genome, in contrast to diploid plants which had two distinct bands due to heterozygosity (Fig. 1 i). Discussion We evaluated the immediate effectiveness of simultaneously applying polyamines and CCC. This approach resulted in high embryogenesis rates, though embryogenesis efficiency varied among genotypes. We found that embryo induction in in vitro cultures of the Storm, 547 and Sahm genotypes was significantly increased by the combined application of three compounds putrescine at a concentration of 500 mg/L, Spermidine and CCC (each 50 mg/L) on the mother plant from 12 days before to 12 days after flowering. Previous cucumber observations reported by Ebrahimizadeh et al. (2018) showed that separate applications of these substances at similar concentrations increased embryogenesis and plant regeneration in our assay. Furthermore, prior observations indicated that higher levels of these compounds were detrimental, with embryogenesis and regenerated plantlets from embryos grown at higher concentrations markedly reduced (Ebrahizadeh et al., 2018). Additionally, a high concentration of Putrescine at 5000 mg/L and CCC 500–5000 mg/L relative to fruit weight significantly reduced fruit set and seed formation (Ebrahimzade et al. 2018).Exogenous putrescine at low concentrations markedly enhances somatic embryogenesis across several species, including cotton ( Gossypium hirsutum L.), spine gourd ( Momordica dioica Roxb.), Brassica napus L., and wheat (Triticum aestivum L.). Polyamines are involved in a range of processes, including cell division, embryogenesis induction, flowering initiation, fruit development, and maturation. External application of polyamines has been reported to enhance fruit set and performance in several species, including Cucumis sativus L. (Ebrahimzade et al. 2018), Pistachia vera L. (Kamiab et al. 2015 ), Phoenix dactylifera L. (Abd El-Migeed et al. 2013 ), and Gossypium hirsutum L. (Bibi et al. 2012 ). Compared with polyamines, there is limited information on the role CCC in the induction of somatic/gametic embryogenesis. The enhancing effect of CCC and other inhibitors of gibberellin biosynthesis on the induction of somatic/gametic embryogenesis has been reported in species such as Cucumis sativus L. (Ebrahimzade et al. 2018), Pinus taeda L. (Pullman et al. 2005 ), A. cepa L. ( Ponce et al. 2006 )d aestivum L. (Miroshnichenko et al. 2009 ). Fruit weight and seed set were significantly improved by applying CCC low levels (50 mg L − 1 ) to mother plants in cucumber (Ebrahimzade et al. 2018). Similarly, foliar spraying (500 mg L − 1 CCC) on autumn lettuce ( Lactuca sativa L.) increased the number of inflorescence branches per plant and also seed yield (Pasam et al. 2008). In tomato ( Solanumlycopersicum ), the greatest fruit weight was obtained with 100–300 mg L − 1 CCC (Altinas 2011 ).The results show that the simultaneous use of these mixtures varies in different cultivars. This phenomenon seems to be somewhat genotype-dependent, as the Sahm genotype showed lower embryogenesis compared to Storm and 547. Although spraying these mixtures was useful for inducing embryogenesis in vitro . Careful cultivation of Storm and 547 mother plants in greenhouse conditions followed by the use of 600 Gy gamma-irradiated pollen grains resulted in higher frequencies of embryogenesis. Gamma irradiation, which has been used in recent years, is one of the effective strategies for inducing embryogenesis through parthenogenesisin Cucurbitaceae. The results showed that the interaction effects of genotype and gamma radiation dose were significant on the total number of seeds, the number of whole and filled seeds, the number of whole and empty seeds in the fruit, and the number of embryos and regenerated plants. The highest number of seeds per fruit was obtained from the genotype "Storm" with an average of 205 at a dose of 600 Gy. These findings are consistent with previous studies showing that controlled pollen irradiation can enhance haploid induction and parthenocarpic embryogenesis (Pradeepkumara et al. 2023 ; Dey et al. 2022 ). These results highlight the importance of selecting the appropriate genotype and adjusting the irradiation dose for each genotype for seed production. Regarding the number of whole and filled seeds, the highest number was observed in the genotypes "547" and "Storm" with averaging 124.66 and 117.66 seeds respectively under the control treatment. In contrast, the genotype "547" exhibited the lowest number of whole and filled seeds, with an average of 10. The effect of radiation dose showed that the control treatment produced the highest number of whole and filled seeds, and increasing radiation doses led to a significant decrease in this trait. In contrast, the highest number of whole and empty seeds in all 3 cultivars was in the treatment with the highest radiation dose of 600 Gy. The results of this study show that radiation dose and genotype had significant effects on the percentage of full and empty seeds. Increasing the radiation dose to 600 Gy increased the number of whole and empty seeds in the Storm genotype, but lower doses reduced this trend, indicating a stimulating effect of higher doses and an effect on pollen and mother cells. Gamma radiation has a distinct negative effect on full seed production, and the response of genotypes to radiation dose is different. Lower doses of irradiation were able to maintain the number of full seeds, while higher doses caused a severe reduction in the number of seeds. The findings of this study are consistent with previous results in cucumber and other Cucurbitaceae, which showed that the use of controlled pollen irradiation can improve haploid induction and parthenocarpic embryo formation in cucumber, while simultaneously increasing the quality and number of seeds under the optimal dose. However, high doses reduce the quality and number of full seeds (Pradeepkumara et al. 2023 ; Dey et al. 2022 ).The findings related to embryogenesis showed that the genotypes "Storm" and 547 produced the highest number of embryos (6 and 66.5), respectively, while the control treatments did not produce any embryos. Also, the radiation dose of 600 Gy had the greatest effect on the number of embryos and regenerated plants. These results indicate that gamma radiation has an effective role in embryo induction from empty seeds and the selection of the optimal dose can facilitate the production of haploid lines. This finding is consistent with the results reported in other species such as cucumber and cucurbits, which have shown that genotypic sensitivity and radiation dose play an important role in the success of parthenocarpic embryogenesis (Khoshkam et al. 2024 ). These results are in contrast to the findings of Ebrahimzadeh et al. ( 2018 ) and Gonzalo et al. ( 2011 ), who reported that the percentage of embryo induction decreases with increasing radiation dose. The possible reason for this discrepancy could be the dose range used and the difference in genotypic sensitivity to radiation, which could be an important factor in this difference in results. Flow cytometry analysis confirmed the accuracy of the regeneration data. DNA content index values in haploid samples were approximately half those of diploid samples. Despite the utility of flow cytometry in estimating DNA content, this method is inherently unable to distinguish between homozygous diploids and heterozygous diploids, especially when both show the the same ploidy level. To overcome this limitation, co-dominant SSR markers were included as a complementary tool to confirm genetic uniformity. In particular, the 3B27marker consistently showed single-band patterns in gel electrophoresis assays and provided molecular confirmation of homozygosity in selected DH lines. Therefore, the integration of nuclear DNA quantification and molecular marker-based genotyping is a comprehensive and reliable strategy for DH identification, providing critical support for accelerating breeding programs and ensuring genetic stability in restored populations (Keleş et al. 2015 ; Pareeth 2015 ). Among antimitotic agents, oryzaline has shown better efficacy in producing doubled haploid plants, which highlights that choosing the right agent and carefully adjusting the dose and contact time are essential for successful chromosome doubling (Ebrahimzadeh et al., 2018 ). Furthermore, animal cells are immune to this agent and it has less negative effect on humans. Conclusion The present study aimed to produce pure lines of cucumber ( Cucumis sativus L.) through pollen irradiation. Results showed that increasing the gamma radiation dose, particularly at level 600 Gy, had a significant inhibitory effect on the formation of whole and fill seeds and was associated with an increase in the number of whole and empty seeds as well as embryos, identifying 600 Gy as the optimal dose for this study. To mitigate the adverse effects of radiation and improve reproductive conditions, foliar spraying of polyamines including putrescine (500 mg/L), spermidine (50 mg/L)and CCC (50 mg/L) was applied 12 days before and 12 days after pollination on an alternate-day basis. Foliar application of polyamines and CCC increased the proportion of fruits formed and preserved viable embryos. Overall, combined treatment with a high radiation dose (600 Gy) and polyamines plus CCC showed the greatest efficiency in inducing haploid embryos. Thus, these results indicate that controlled levels of gamma radiation can be an effective tool for pollen-induced haploidy in cucumber, though dose and genotype play decisive roles in the success of the process. Additionally, polyamines and CCC can serve as an ancillary strategy to reduce radiation-induced oxidative stress and to improve the growth and viability of gametic cells. Statements & Declarations Abbreviations BAP: 6-Benzylaminopurine CCC: Cycocel CTAB: CetylTerimetil Ammonium Bromide DH: Doubled haploid DNA: Deoxyribonucleic acid EDTA: Ethylene diamine tetra acetic acid Gy Gamma irradiation IAA: Indole-3-acetic acid MS: Murashige and Skoog medium NAA: 1-Naphthaleneacetic acid PI: Propidium iodide PCR: Polymerase chain reaction Rpm: Revolutions per minute SSR: Simple sequence repeat SAS: Statistical Analysis System SPSS: Statistical Package for the Social Sciences TBE : Tris- borate -EDTA buffer Declarations Funding: This work was funded by the Agricultural Biotechnology Research Institute of Iran (Project no. 34-05-0533-014-020240 Competing Interests The authors declare that they have no conflict of interest. Authors' contributions: Authors' contributions: MES conceived of the presented idea and planned the experiments. SFT developed the theory conducted the research experiments and wrote the manuscript with the support of all co-authors. MHS analysis of related data preparation. MT assisted in project implementation. Zahra Hosseini provided laboratory materials. All authors discussed the results and contributed to the final manuscript Acknowledgments: The authors sincerely thank the Agricultural Biotechnology Research Institute of Iran (Project no. 34-05-0533-014-020240) for financial support and the Iran National Science Foundation for postdoctoral support, and Dr. Mehrshad Zeinalabedini for molecular analyses Data availability: The data that support the findings of this study is available Ethices decleration: not applicable References Abd El-Migeed MMM, Mostafa EAM, Ashour NE, Hassan HSA, Mohamed DM, Saleh MMS (2013) Effect of potassium and polyamine sprays on fruit set, fruit retention, yield and fruit quality of Amhat date palm. Int J Agric Res 8(2):77–86. 10.3923/ijar.2013.77.86 Ahamdi B, Shariatpanahi ME, Ojaghkandi MA, Heydari AA (2014) Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaximeandvancomycininBrassicanapus L. Plant Cell Tissue Organ Cult 118:497–505. 10.1007/s11240-014-0501-9 Ahmadi B, Ahmadi M, Teixeira da Silva JA (2018) Microspore embryogenesis in Brassica:calcium signaling, epigenetic modification, and programmed cell death. Planta 248(6):1339–1350. 10.1111/pce.70177 Alifar M, Ebadi A, FatahiMoghadamMR (2015) Effects of putrescineandchlormequat pre-flowering treatment on embryo rescue techniquesuccess in diallelecrossesofgrape Cultivars Flame Seedless, perlette and Yaghooti. Iran J HorticSci 46(2):179–192. 10.22059/IJHS.2015.54614 Altinas S (2011) Effects of chlormequat chloride and different rates of prohexadione-calcium on seedling growth, flowering, fruit development and yield of tomato. Afr J Biotechnol 10(75):17160–17169. 10.5897/AJB11.2706 Behera TK, Boopalakrishnan G, Jat GS, Das MA, Choudhary H, Ravindran A, Kumari R, Horticulture (2022) Environ Biotechnol 63(2):263–274. 10.1186/s40529-024-00436-w Bhatia R, Dey SS, Parkash C, Sharma K, Sood S, Kumar R (2018) Modification of important factors for efficient microspore embryogenesis and doubled haploid production in field grown white cabbage ( Brassica oleracea var. capitata L.) genotypes in India. SciHort233:178–187. https://doi.org/10.1016/j. scienta.2018.01.017 Bibi A, Oosterhuis D, Gonias E (2012) Changes in the ovarian polyamine content and seed set efficiency of cotton by the plant growthregulatorBM86″. Am J Plant Sci 3(9):1328–1335. 10.4236/ajps.2012.39160 Chahal G, Gosal S (2002) Principles and procedures of plant breeding: biotechnological and conventional approaches. Alpha Science Int'l Ltd. United Kingdom.ISBN 1-84265-036-X Cuny F (1993) Processusd'inductiond'embryonshaploïdes par du pollen irradié chez le melon ( Cucumis melo L.) Réponses du pollen à l'irradiation gamma. These de Docteur, Universite´ d’Avignonet des Pays de Vaucluse, Avignon De Witte K, Keulemans J (2000) Homozygous plant production in apple by parthenogenesis in situ: improvement of the pollination stimulus for parthenogenic egg cell development. Acta Hortic 538:315–320. DIO: 10.17660/ActaHortic.2000.538.54 Dey SS, Singh S, Munshi AD, Behera TK (2022) Classical genetics and traditional breeding. In: Pandey S, Weng Y, Behera TK, Bo K (eds) The Cucumber Genome. Compendium of Plant Genomes. Springer, Cham. DOI: 10.5772/intechopen.97593 Dong YQ, Zhao WX, Li XH, Liu XC, Gao NN, Huang JH, Wang WY, Xu XL, Tang ZH (2016) Androgenesis, gynogenesis, and parthenogenesis haploids in cucurbit species. Plant Cell Rep 35(10):1991–2019. 10.1007/s00299-016-2018-7 Ebrahimzadeh H, Lotfi M, Azizinia S, Ghanavati F (2013) Production of haploids in Cucurbita pepo L. through parthenogenesis induced by gamma-irradiated pollen. Crop Biotechnol 3:99–108. DOI: 20.1001.1.22520783.1392.3.4.11.4 Ebrahimzadeh H, Shariatpanahi ME, Ahmadi B, Soltanloo H, Lotfi M, Zarif E (2018) Efficient parthenogenesis induction and in vitro haploid plant regeneration in cucumber ( Cucumis sativus L.) using putrescine, spermidine, and cycocel. J Plant Growth Regul 37:1127–1134. 10.1007/s00344-018-9803-1 Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104(3):301–309. 10.1007/s11240-010-9800-y Galazka J, Niemirowicz-Szczytt K (2013) Review of research on haploid production in cucumber and other cucurbits. Folia Hortic 25(1):67–78. 10.2478/fhort-2013-000 Germanà MA (2006) Doubled haploid production in fruit crops. Plant Cell Tissue Organ Cult 86:131–146. 10.2478/fhort-2013-0008 Gonzalo MJ, Claveria E, Monforte AJ, Dolcet-Sanjuan R (2011) Parthenogenic haploids in melon: Generation and molecular characterization of a doubled haploid line population. J Am Soc forHorticultural Sci 136(2):145–154. 10.21273/JASHS.136.2.145 Huang S, Li R, Zhang Z, Li LI, Gu X, Fan W, Li S (2009) The genome of the cucumber. Cucumis sativus L Nat Genet 41(12):1275–1128. 10.1038/ng.475 Kamiab F, Salehabad MH, Zamanibahramabadi E (2015) Evaluation of the effects of foliar treatments of polyamines and some organic acids on quantitative and qualitative traits in some pistachio cultivars. J Nuts 6(2):131–142. 10.22034/jon.2015.516322 Khoshkam S, Samsampour D, Shariat Panahi ME, Khiabani BN (2024) Investigating the possibility of producing double haploid lines in some selected cultivars of greenhouse cucumber through induction of parthenogenesis. Int J Multiphys, 18(4) Keleş D, Pınar H, Ata A, Taşkın H, Yıldız S, Büyükalaca S (2015) Effect of pepper types on obtaining spontaneous doubled haploid plants via anther culture. HortScience 50:1671–1676. 10.21273/HORTSCI.50.11.1671 Kumar HGA, Ravishankar BV, Murthy HN (2004) The influence of polyamines on androgenesis of Cucumis sativus L. Eur J Hortic Sci 69(5):201–205. 10.17660/eJHS.2004/23936 Kurtar ES, Balkaya A (2010) Production of in vitro haploid plants from in situ induced haploid embryos in winter squash ( Cucurbita maxima Duchesne ex Lam.) via irradiated pollen. Plant Cell Tissue Organ Cult 102:267–277. 10.1007/s11240-010-9729-1 Kurtar ES, Balkaya A, Ozbakir M, Ofluoglu T (2009) Induction of haploid embryo and plant regeneration via irradiated pollen technique in pumpkin ( Cucurbita moschata Duchesne ex Poir). Afr J Biotechnol 8:5944–5951DOI. 10.5897/AJB09.730 Lotfi M, Alan A, Henning M, Jahn M, Earle E (2003) Production of haploid and doubled haploid plants of melon ( Cucumis melo L.) for use in breeding for multiple virus resistance. Plant Cell Rep 21:1121–1128. 10.1007/s00299-003-0636-3 Mineykina A, Bondareva L, Soldatenko A, Domblides E (2021) Androgenesis of red cabbage in isolated microspore culture in vitro. Plants 10(9):1950. 10.3390/plants10091950 Miroshnichenko D, Filippov M, Doglov S (2009) Effects of daminozide on somatic embryogenesis from immature and mature embryos of wheat. Aust J Crop Sci 3(2):83–94 Mulyana A, Purwoko BS, Dewi IS, Maharijaya A (2023) Comparison of six anther culture methods for the production of doubled haploids in eggplant ( Solanum melongena L). Euphytica 219(4):44. 10.1007/s10681-023-03171-8 Pareeth CM, Purushothama MG (2015) Development of double haploids in bell pepper Passam HC, Koutri AC, Karapanos IC (2008) The effect of chlormequat chloride (CCC) Application at the bolting stage on the flowering and seed production of lettuce plants previously treated with water or gibberellic acid (GA3). Sci Hortic 116(2):117–121. 10.1016/j.scienta.2007.11.004 Ponce M, Martinez L, Galmarini C (2006) Influence of CCC, putrescine and gellan gum concentration on gynogenic embryo induction in Allium cepa. Biol Plant 50(3):425–428. 10.1007/s10535-006-0061-x Pradeepkumara N, Dey SS, Munshi AD, Behera TK, Bhatia R, Kumari K, Talukdar A (2023) Cucumber F hybrid derived from two contrasting inbreds ensures high frequency gynogenesis for induction of haploids through a modified in vitro based protocol. South Afr J Bot 157:314–324. 10.1016/j. sajb.2023.03.066 Pullman GS, Mein J, Johnson S, Zhang Y (2005) Gibberellin inhibitors improve embryogenic tissue initiation in conifers. Plant Cell Rep 23:596–605. 10.1007/s00299-004-0880-1 Romanova OV, Vjurtts TS, Mineykina AI, Tukuser YP, Kulakov YV, AkhramenkoDomblides EA (2023) Embryogenesis induction of carrot ( Daucus carota L.) in isolated microspore culture. Food Raw Mater 11:25–34. 10.21603/2308-4057-2023-1-548 Sari N, Abak K, Pitrat M, Dumas de Vaulx R (1992) Induction of parthenogenetic haploid embryos and plant obtention in melon ( Cucumis melo L. var. inodorus Naud & C. melo L. var. reticulatus Naud) Turk J Agric For 16:302–314. 10.21273/HORTSCI.29.10.1189 Sauton A, Dumas de Vaulx R (1987) Production of haploid plants in melon ( Cucumis melo L.) as a result of gynogenesis induced by irradiated pollen. Agronomie 7(2):141–147. 10.1051/agro:19870209 Segui-Simarro JM (2021) Doubled haploid technology. Springer. org/10.1007/978-1-0716-1331-3 Shariatpanahi ME, Ahmadi B (2016) Isolated microspore culture and its applications in plant breeding and genetics. In: Anis M, Ahmad N (eds) Plant tissue culture: propagation, conservation and crop improvement. Springer Science + Business Media Singapore, pp 487–507. 10.1007/978-981-10-1917-3_21 Taner K, Yanmaz R, Kunter B (2000) The effects of irradiation dose and harvest period on haploid plant formation via irradiated pollen in snake cucumber ( Cucumis melo var. flexuosus Naud.). IIIrd National Vegetable Culture Symposium, 11–13 September 2000, Isparta, Turkey, pp 177–181. 10.17660/ActaHortic.1999.492.41 Thiruvengadam M, Rekha KT, Jayabalan N, Praveen N, Kin EH, Chung IM (2013) Effect of exogenous polyamines on somatic embryogenesis via suspension cultures of spine gourd (Momordica dioica Roxb. ex Willd). Aust J Crop Sci 7(3):446–453 Tiburcio A, Altabella T, Bitrián M, Alcázar R (2014) The roles of polyamines during the lifespan of plants: from development to stress. Planta 240(1):1–18. 10.1007/s00425-014-2055-9 Wei A, Du S, Han Y, Zhang G (2010) A study on the relationship between cucumber gynogenesis and content of ovary hormones and polyamines. Acta Hortic 871:625–630. 10.17660/ActaHortic.2010.871.86 Zhang L, Nie FJ, Gong L, Gan XY, Zhang GH, Liu X, Song Y (2023) Regenerative plantlets with improved agronomic characteristics caused by anther culture of tetraploid potato (Solanum tuberosum L). PeerJ 11:e14984. 10.7717/peerj.14984 Zhang T, Wang X, Wang Y, Han J, Mao P, Majerus M (2009) Plant growth regulator effects on balancing vegetative and reproductive phases in alfalfa seed yield. Agron J 101:1139–1145. 10.2134/agronj2009.0017 Zhang YX, Lespinasse Y (1991) Pollination with gamma-irradiated pollen and development of fruits. 10.1007/bf00145636 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 23 Jan, 2026 Reviewers invited by journal 22 Jan, 2026 Editor assigned by journal 15 Dec, 2025 First submitted to journal 13 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8326911","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578757684,"identity":"47562399-56e2-40c3-a489-b141394ce956","order_by":0,"name":"Sakineh Farhadi-Tooli","email":"","orcid":"","institution":"ABRII: Agricultural Biotechnology Research Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Sakineh","middleName":"","lastName":"Farhadi-Tooli","suffix":""},{"id":578757685,"identity":"8c8c654a-b74e-41b7-8021-0948ce2f13d1","order_by":1,"name":"Mehran E. Shariatpanahi","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9634-6716","institution":"Agricultural Biotechnology Research Institute of Iran","correspondingAuthor":true,"prefix":"","firstName":"Mehran","middleName":"E.","lastName":"Shariatpanahi","suffix":""},{"id":578757686,"identity":"2f69725c-7a56-4045-a6e2-bfa666c5486b","order_by":2,"name":"Mohammadvali Habibi Silabi","email":"","orcid":"","institution":"ABRII: Agricultural Biotechnology Research Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Mohammadvali","middleName":"Habibi","lastName":"Silabi","suffix":""},{"id":578757687,"identity":"1405a153-d557-4c85-970b-3209c546bbef","order_by":3,"name":"Maryam Tavakoli","email":"","orcid":"","institution":"ABRII: Agricultural Biotechnology Research Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Tavakoli","suffix":""},{"id":578757688,"identity":"e4369e01-1b6e-4bb6-8b94-849669f2da8a","order_by":4,"name":"Zahra Sadat Hosseini","email":"","orcid":"","institution":"ABRII: Agricultural Biotechnology Research Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"Sadat","lastName":"Hosseini","suffix":""}],"badges":[],"createdAt":"2025-12-10 11:34:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8326911/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8326911/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101338134,"identity":"1db2fcef-f347-47d2-937d-2cd8d0d56ffd","added_by":"auto","created_at":"2026-01-28 15:44:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":825957,"visible":true,"origin":"","legend":"\u003cp\u003eSelected steps in Parthenogenesis procedurein cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.); a) embryo rescue; b) embryo growth and its transformation into a seedling, c) transfer of the seedling into a glass for further growth, d) micropropagation of haploid plants, e) regeneration of plants after doubling treatment, e) acclimatization; f-g)seeding from the fruit;i)SSR marker profiles on a 8% acrylamide gel used for distinguishing haploid, spontaneously doubled haploid, and diploid,Lane M: molecular size marker (DNA ladder,Lane 1: diploid,Lane 2: haploid;Lane 3: spontaneously doubled haploid, Flowcytometry histogram of a haploid sample, showing a single peak representing C DNA content;j)Flow cytometry analysis of ploidy levels in regenerated plants:k) Flowcytometry histogram of a mixed sample containing both diploid and haploid nuclei, with the third peak corresponding to diploid, the second to haploid, and the first to background noise; l) Flowcytometric histogram of a diploid sample, showing a single peak representing 2C DNA content\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8326911/v1/7cd211a4d4ecd5cdce87abfb.png"},{"id":101338135,"identity":"d0346ce6-214c-4493-9497-8db06043cb54","added_by":"auto","created_at":"2026-01-28 15:44:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1908742,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8326911/v1/449c32df-012c-4182-acc1-34647976e822.pdf"}],"financialInterests":"","formattedTitle":"Improvement of embryogenesis in cucumber (Cucumis Sativus L.) parthenogenesis by polyamine and cycocel treatments","fulltext":[{"header":"Key Message","content":"\u003cp\u003eThe combined application of polyamines and \u0026ldquo;CCC\u0026ldquo; on the maternal plant, plus gamma-irradiated pollen, yields genotype-dependent embryogenesis improvements and haploid production in cucumber, showing Storm and 547 as the responsive varieties.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCucurbitaceae is among the most widely cultivated and economically important vegetable families. \u003cem\u003eCucumis sativus\u003c/em\u003e L. (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;14) is a key cucurbitaceous crop valued commercially and widely used as a model in vegetable breeding and functional genomics (Dey et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is grown globally under diverse agroclimatic conditions, in both open-field and protected environments. The species originates from India, with a secondary center of diversity in China and the Near East. As an out breeding crop without inbreeding depression, cucumber provides an exemplary system for investigating a range of developmental and molecular pathways, aided by its relatively small genome (~\u0026thinsp;372 Mbp) (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The classification of Cucumis species into primary, secondary, and tertiary gene pools has been established through comprehensive assessments of cross-compatibility, genetic analyses, phylogenetic studies, and molecular evidence (Dey et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApplication of doubled haploid (DH) technology offers a powerful route to accelerate conventional breeding by enabling the rapid production of completely homozygous lines, thus bypassing multiple generations of selfing (Germana 2006; Ahmadiet al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Traditional hybrid breeding relies on inbred parental lines, typically requiring 6\u0026ndash;8 years of selfing in cucumber to achieve homozygosity. By contrast, DH induction via gynogenesis and androgenesis provides a single‑step route to homozygous parental lines, with demonstrated applicability across several vegetable crops (Behera et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dey et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bhatia et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mineykina et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Romanova et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mulyana et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Successfully produced in vitro DH plants, whether via androgenesis or gynogenesis, provide valuable resources for research and breeding, including the development of mapping among biotechnological strategies, androgenesis (\u003cem\u003ein vitro\u003c/em\u003e anther/microspore culture), gynogenesis (\u003cem\u003ein vitro\u003c/em\u003e ovule/ovary culture), and parthenogenesis (egg cell induction by irradiated pollen followed by \u003cem\u003ein vitro\u003c/em\u003e haploid embryo rescue) have proven more efficient and sustainable than conventional methods (Shariatpanahi and Ahmadi \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Notably, gynogenesis often yieldshaploids at higher frequencies than androgenesis in cucurbitaceous crops, as female haploid cells within the embryo sac/ovules tend to respond more readily than male haploid cells produced in the same flower. Structural differences haploid cells residing inside ovules and encased by nucellar and integument tissues versus male haploids confined to the anther wall contribute to the greater complexity and longer timelines of ovary/culture approaches, necessitating advanced technical expertise(populations, inbred lines, and expanded genetic diversity for breeding programs(Segui-Simarro \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ferrie \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePolyamines are natural polycationic amines produced in living cells, including putrescine, spermidine, and spermine. Polyamines influence many biological processes, including cell division, differentiation, organogenesis, pollen formation, fertilization, gene expression, DNA and protein synthesis, apoptosis, and stress responses (reviewed by Tiburcio et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). There is compelling evidence for their crucial roles in somatic/gametic embryogenesis. Higher polyamine levels in unfertilized ovaries correlate with stronger gynogenesis ( Wei et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ahmadi et al. 2014;), and adding polyamines to induction media markedly enhances somatic/gametic embryogenesis and subsequent embryo-to-plant conversion across multiple species (Kumar et al.2004;Ponce et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e;Thiru-vengadam et al.2013; Ahmadi et al. 2014;Ebrahimzadeh et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the underlying mechanism through which polyamines exert an effect is largely unknown. The plant growth regulator (CCC) is widely used to reduce stem elongation, promote flowering, and improve bud formation. CCC inhibits gibberellin production early in development (Zhang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ebrahimzadehetet al. 2018). The regulatory role of CCC in \u003cem\u003ein vitro\u003c/em\u003e somatic/gametic embryogenesis is less clear: CCC in induction media can retard embryo formation in some cases, while applying CCC to donor plants can markedly improve \u003cem\u003ein vitro\u003c/em\u003e embryo formation, germination, and plant regeneration in certain species such as \u003cem\u003eVitis vinifera\u003c/em\u003e (Alifar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ebrahimzadeh et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApplication of irradiation to pollen as a means to induce haploidy remains a widely used and efficient approach in cucurbits, with ultraviolet (UV), gamma rays, and X-rays serving as recalcitrant agents to stimulate haploid formation. Among these, gamma irradiation is particularly favored in practical programs due to its simplicity, good penetrance, reproducibility, relatively high mutation frequency, and manageable abortion rates, making it the most commonly adopted method for haploid induction in many cucurbit breeding initiatives(Gałązka and Niemirowicz-Szczytt 2013; Chahal and Gosal \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Parthenogenesis has emerged as the most effective strategy to achieve complete homozygosity in \u003cem\u003eCucumis sativus\u003c/em\u003e L. in a single generation, although its routine deployment in breeding is limited by low haploid production rates(reviewed by Gałązka and Niemirowicz-Szczytt 2013; Dong et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In irradiated pollen, observations include chromosome misbehavior and physio-biochemical alterations, with reports noting instances where a single male gamete is available for fertilization of either the egg cell or the fused polar nuclei; the former can lead to maternal homozygous diploids, while the latter may result in haploid parthenogenesis (pseudoembryony) triggered by irradiated pollen. Irradiated pollen can be still germinated on the stigma, extended through the style, and reached the embryo sac without fertilizing the egg cell or polar nuclei, allowing for parthenogenetic induction or gynogenic haploid production (Zhang \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; De Witte \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e;Cuny \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Induction of haploid parthenogenetic embryos by \u003cem\u003ein situ\u003c/em\u003e pollination with irradiated pollen has been reported in multiple Cucurbitaceae species, including \u003cem\u003eCucumis melo\u003c/em\u003e, \u003cem\u003eC. sativus\u003c/em\u003e, \u003cem\u003eCitrullus lanatus\u003c/em\u003e, Cucurbita pepo, \u003cem\u003eC. moschata\u003c/em\u003e, and \u003cem\u003eC. maxima\u003c/em\u003e, with varying efficiencies dependent on genotype, growth conditions, irradiation source and dose, and the developmental stage of embryos at excision (Sari et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Taner et al.2000; Lotfi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Ebrahimzadeh et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ebrahimzadeh et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kurtar et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e;Kurtar and Balkaya \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, the concurrent effects of spraying maternal plants with putrescine, spermidine, and CCC from 12 days before to 12 days after pollination at 1-day intervals were examined, using three doses of gamma irradiation, on the frequency of haploid plant production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003ePlant material, Pollen Irradiation and\u003c/b\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eFertilization\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study was conducted in the greenhouse of the Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj. The\u0026rdquo;Sahm\u0026rdquo;, \u0026ldquo;547\u0026rdquo; and \u0026ldquo;Storm\u0026rdquo; genotypes were used as maternal plants, with the \u0026ldquo;Beta Alpha\u0026rdquo; cultivar serving as the pollen donor. Plants were grown and maintained in a glasshouse under a 16-hour light/8-hour dark photoperiod, at a day temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and a night temperature of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. To support plant growth, daily fertilization was performed with Hoagland\u0026rsquo;s nutrient solution. Mother plants were sprayed with concentrations of putrescine, spermidine, and CCC each at 500, 50 and 50 mg/l, ranging from12 days before until 12 days after anthesis with 1-day intervals.\u003c/p\u003e \u003cp\u003eOne day before the anthers opened and dehisced, male flowers (greenish-yellow) were collected and exposed to gamma radiation from a cobalt-60 source at a dose rate of 0.2 Gy/s with various gamma doses (400, 500 and 600 Gy). The female flowers of the F1 hybrids, serving as pollen recipients, were bagged to prevent contamination by unwanted pollen, up to dusk on the same day. After irradiation, the male flowers were kept at room temperature until pollination in the following day. Early the next morning, when the stigma was receptive to pollen, pollination was performed using pollen grains from the irradiated anthers. For each flower, 2\u0026ndash;3 anthers were sufficient. The female flowers were re-bagged for 3\u0026ndash;4 days with their petals closed to prevent cross-pollination.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo Rescue\u003c/h2\u003e \u003cp\u003eGrowth sprouts were harvested after 35 days and surface-sterilized with 70% ethanol to reduce greenhouse contaminants. After washing, the fruits were opened and the seeds were extracted from running tap water and cleaned. Seed surface sterilization was performed under a laminar flow hood with 1% sodium hypochlorite for 10 minutes, followed by three washes with sterile distilled water for 5 minutes each.To facilitate identification and embryo extraction, the sterilized seeds were transferred to liquid medium E20 (Sauton and Dumas de Vaulx, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) supplemented with 3% sucrose and 0.01 mg/L indole-3-acetic acid (IAA) according to Lotfi et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Approximately 25 seeds per dish containing 25 mL liquid E20 medium were used, ensuring seeds were submerged. Dishes containing immature embryos were sealed with Parafilm and placed in a growth chamber under standard conditions for tissue culture: a 16-hour photoperiod (light) and an 8-hour dark period, at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. The dishes were gently agitated several times per week to maintain aeration. After 10\u0026ndash;20 days, embryos were examined and isolated using a suitable light source. Isolated embryos were transferred to solid E20 medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b).One to three weeks after culture, the developed plantlets (epigeal plantlets) were transferred to glass jars containing MS medium supplemented with BAP at 2.0 mg/L and NAA at 0.05 mg/L for further growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). After three weeks, for better growth of small plantlets, the culture was transferred to MS medium without supplements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePloidy Level Determination\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eSmall section of young leaf tissue (0.5 cm) from diploid control plants (control) and plants with unknown ploidy, produced by induced apomixis, was separately excised with a fine scalpel in 500 \u0026micro;L of hypotonic propidium iodide (PI) lysis buffer, cooled on ice. The tissue was finely chopped.Subsequently, 2000 \u0026micro;L of nuclear staining solution and 6 \u0026micro;L of RNase were added, followed by 12 \u0026micro;L of propidium iodide (PI) to the crude mixture.To reduce the effects of cytosolic and phenolic compounds on PI fluorescence, 1% PVP-40 was also added to the buffer.The suspension was filtered through a 50 \u0026micro;m filter to remove cell debris and isolate nuclei. Ploidy level of the samples was analyzed with a flow cytometer (Partec, M\u0026uuml;nster, Germany) using a 488 nm Argon laser to excite the fluorochrome PI and a FL-2 detector with a 585/42 nm emission filter. Samples were run at low pressure and a minimum of 10,000 nuclei per sample were analyzed. Subsequently, DNA fluorescence per nucleus was measured and results were displayed as histograms. Since the injected sample contained a mixture of haploid and diploid plant extracts, the ploidy level of the sample was determined based on the fixed peak positions for haploid and diploid references.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInduction of doubled haploids in regenerated haploid plants\u003c/h3\u003e\n\u003cp\u003eHaploid plantlets regenerated were micro-propagated \u003cem\u003ein vitro\u003c/em\u003e before chromosome doubling. Young shoots, about 1\u0026ndash;3 cm in length and leafless, were excised and transferred to liquid medium E20 containing 0.2 mg/L BAP, 80 mg/L Fe-EDDHA, and 50 mg/L oryzalin. The antimutagenic agent (oryzalin) was dissolved in dimethyl sulfoxide (DMSO, 2% v/v) and then added to the culture medium. The samples were incubated for 18 hours in a incubator at a constant temperature of 27\u0026deg;C with a speed of 150 rpm. Subsequently, the samples were washed three times with sterile distilled water, and the microexplants were transferred to solid E20 medium containing 0.01 mg/L IAA, 0.2 mg/L BAP, and 80 mg/L Fe-EDDHA. All cultures were maintained in a growth chamber under standard light conditions (photoperiod:16 h light/8 h dark) and at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. After 3 weeks, each regenerated plantlet was individually transferred to flasks containing MS medium.\u003c/p\u003e\n\u003ch3\u003eDistinguishing doubled haploids from diploid plants\u003c/h3\u003e\n\u003cp\u003eIn this study, the SSR marker CMCCA145 was used to identify the embryo type (diploid or doubled haploid). More than 20 SSR markers taken from the cucumber genome (from the Soul genomic network) were screened and the CMCCA145 marker was selected due to its apparent polymorphism. DNA extraction from 200 mg of young leaf tissue was performed using CTAB reagent according to the protocol with minor modifications. The quality and quantity of DNA were assessed using gel electrophoresis and fluorometric assay. PCR amplification was performed in a 96-well Variti thermal cycler (Applied Biosystem, USA) using the following cycles: initial denaturation at 94\u0026deg;C for 3 min, followed by 35 cycles of denaturation at 94\u0026deg;C for 20 s, annealing at 58\u0026deg;C for 30 s, extension at 72\u0026deg;C for 30 s, and a final extension step at 72\u0026deg;C for 5 min. The amplified products were examined using a 8% acrylamide gel in TBE buffer stained with Gelred using vertical electrophoresis. They were then visualized under UV light (Gelduc., UK). The size of the amplified fragments was determined using a 50bp Gen Ruler DNA ladder (Thermo Scientific, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and data analysis method\u003c/h2\u003e \u003cp\u003eThe experiment was a factorial experiment in a completely randomized design (the first factor was genotype and the second factor was gamma ray dose) with three replications. Two fruits were examined from each replication. SPSS software was used for data normalization and SAS software version 9.4 was used for data analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSimultaneous spraying of putercine, spermidine and CCC on mother plants with concentrations of (500, 50 and 50 mg/L) respectively along with irradiation with different doses of gamma rays to sterilize pollen grains showed that the interaction effect of genotype with gamma ray dose had a significant effect at the 5% probability level on the number of seeds per fruit. The results showed that the highest number of seeds per fruit was in the Storm genotype with a gamma ray dose of 600 Gy (205 pcs.) and the lowest number of seeds per fruit was in the 547 genotype (66.88 pcs.) and the gamma ray dose treatment was 400 Gy.The results showed that with increasing dose from 500 to 600Gy, the number of seeds per fruit increased in 3 tested cultivars (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).Also, the genotype and gamma ray dose had a significant effect on the number and percentage of full and unfilled seeds in cucumber fruit at the 5% probability level. The results showed that in all 3 varieties, the control treatment had the highest number of seeds. The highest number of filled seeds (124.66) was related to the 547 genotype, and the lowest number was observed in the same genotype and gamma ray dose treatment at 600 Gy. Considering that the number and percentage of full seeds decreased with increasing gamma ray dose, the role of gamma ray, especially at higher doses, in the sterilization of pollen grains can be justified (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The response of each genotype under the same conditions was different, indicating that the effect of genotype is also effective in the success rate of parthenocarpy. Regarding the number and percentage of whole and empty seeds, the results showed that the interaction between genotype and gamma radiation dose had a significant effect at the 5% probability level. The Storm genotype pollinated with pollen irradiated with a dose of 600 Gy had the highest number of whole and empty seeds (153), and the lowest number (21.66) was observed in the Saham genotype and the control treatment. Our results showed that by increasing gamma radiation dose, the number of whole and empty seeds in the fruit increased, and also the response of different genotypes was different. Considering that the embryo is obtained from empty seeds, the increase in the number of empty seeds due to irradiation indicates the efficiency of gamma radiation in sterilizing anthers. Data on the number of embryogenesis and regenerated plants in cucumber fruit through pollen irradiation showed that the interaction effect of genotype with gamma radiation dose had a significant effect at the 5% probability level on the number of embryos and regenerated plants (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), such that the Storm genotype with a radiation dose of 600 Gy showed the greatest effect on the number of embryos and regenerated plants (6 and 5, respectively), while in the control treatment no embryo formation was observed in any of the three cultivars. Examination of the main effect of gamma radiation dose showed that the dose of 600 Gy had the greatest effect on the number of embryos and regenerated plants in all three cultivars studied.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of the comparison of means of the interaction effect of genotype and gamma radiation dose on the total number of seeds in the fruit, whole and full seeds, percentage of whole and full seeds, whole and empty seeds and percentage of whole and empty seeds in the three genotypes of cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGamma irradiationdose (Gy)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal seeds/fruit (TSF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFilledseeds/fruit (FSF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFilledseeds (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEmptyseeds/fruit (ESF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEmptyseeds (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026rdquo; sahm F1\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e138.33\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e90.66\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e126\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e133.33\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e28.66\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e29.66\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e20.66\u003csup\u003ed\u0026thinsp;\u0026minus;\u0026thinsp;f\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e31.64\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e23.53\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e15.5\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.66\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e43.33\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e69.33\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e83.66\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.68\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e47.8\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e55.01\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e62.77\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026rdquo; stoorm F1\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180.33\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e156\u003csup\u003edc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e189\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e205\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e117.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e37.66\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e22.33\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e16.66\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e24.16\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e11.83\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e8.12\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31.33\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e88\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e128.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e153\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.77\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e56.46\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e68.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e74.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026rdquo; 547 F1\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e187\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e95.33\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e88.66\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e103\u003csup\u003egf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e19.66\u003csup\u003ed\u0026thinsp;\u0026minus;\u0026thinsp;f\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e10.66\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e10\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e20.62\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e12.03\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e9.69\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29.33\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e60.33\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e62\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e74.66\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.68\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e63.21\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e70\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e72.58\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eShared superscrip tletters indicate that means do not differ significantly at the 5% probability level (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of the comparison of means of the interaction effect of genotype and gamma radiation dose on the number of embryos, percentage of embryogenesis, number of regenerated plants and resistance to regeneration in the three genotypes of cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGamma irradiationdose (Gy)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmbryos (No.)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEmbryogenesis (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRegeneratedplants (No.)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRegeneration (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026rdquo; sahm F1\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.66\u003csup\u003ed\u0026thinsp;\u0026minus;\u0026thinsp;f\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.33\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2.66\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2.75\u003csup\u003ecb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2.13\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2.33\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3\u003csup\u003ec\u0026thinsp;\u0026minus;\u0026thinsp;e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e63.88\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e93.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e72.22\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026rdquo; stoorm F1\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e4\u003csup\u003ec\u0026thinsp;\u0026minus;\u0026thinsp;e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e4.66\u003csup\u003eb\u0026thinsp;\u0026minus;\u0026thinsp;d\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2.61\u003csup\u003ecb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2.46\u003c/p\u003e \u003cp\u003e2.92\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.33\u003csup\u003eb\u0026thinsp;\u0026minus;\u0026thinsp;d\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3\u003csup\u003ec\u0026thinsp;\u0026minus;\u0026thinsp;e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e83.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e65\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e83.8\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026rdquo; 547 F1\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.33\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e5\u003csup\u003ea\u0026thinsp;\u0026minus;\u0026thinsp;c\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e5.66\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e5.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e5.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e2\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e3.66\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e4.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e58.33\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e73.88\u003csup\u003ea\u0026thinsp;\u0026minus;\u0026thinsp;c\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e76.66\u003csup\u003ea\u0026thinsp;\u0026minus;\u0026thinsp;c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eShared superscrip tletters indicate that means do not differ significantly at the 5% probability level (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePloidy analysis using a flow cytometry to determine the ploidy level of regenerated plants resulting from \u003cem\u003ein vitro\u003c/em\u003e embryo rescue showed that all regenerated plantlets contained the gametic chromosome number (n\u0026thinsp;=\u0026thinsp;x\u0026thinsp;=\u0026thinsp;7) of the parental plants. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), diploid and haploid samples showed a single, distinct peak, while in haploid plants, the number of peaks was approximately half that of diploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ej ,i). In contrast, a mixed sample containing diploid and haploid nuclei showed three distinct peaks. The first peak (which may be due to instrument noise), a second peak with a higher height indicating the G1 phase of the cell cycle, and a third peak indicating the G2/M phase, located approximately twice as far away as the previous peak ( Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ek).Oryzalin was applied to the nodal sections at a rate of 50 mg for 18 hours at a speed of 150 rpm and showed a favorable effect on the rate of chromosome doubling and the generation of doubled haploid plants(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).The SSR marker was also used to assess the ploidy status of plants for which chromosome doubling was performed. Banding profiles on 8% acrylamide gels showed that haploid samples produced a single band of low intensity and samples in which doubling was performed had a similar band but with stronger intensity, probably due to the homozygous nature of their genome, in contrast to diploid plants which had two distinct bands due to heterozygosity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe evaluated the immediate effectiveness of simultaneously applying polyamines and CCC. This approach resulted in high embryogenesis rates, though embryogenesis efficiency varied among genotypes. We found that embryo induction in \u003cem\u003ein vitro\u003c/em\u003e cultures of the Storm, 547 and Sahm genotypes was significantly increased by the combined application of three compounds putrescine at a concentration of 500 mg/L, Spermidine and CCC (each 50 mg/L) on the mother plant from 12 days before to 12 days after flowering. Previous cucumber observations reported by Ebrahimizadeh et al. (2018) showed that separate applications of these substances at similar concentrations increased embryogenesis and plant regeneration in our assay. Furthermore, prior observations indicated that higher levels of these compounds were detrimental, with embryogenesis and regenerated plantlets from embryos grown at higher concentrations markedly reduced (Ebrahizadeh et al., 2018). Additionally, a high concentration of Putrescine at 5000 mg/L and CCC 500\u0026ndash;5000 mg/L relative to fruit weight significantly reduced fruit set and seed formation (Ebrahimzade et al. 2018).Exogenous putrescine at low concentrations markedly enhances somatic embryogenesis across several species, including cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e L.), spine gourd (\u003cem\u003eMomordica dioica\u003c/em\u003e Roxb.), \u003cem\u003eBrassica napus\u003c/em\u003e L., and wheat \u003cem\u003e(Triticum aestivum\u003c/em\u003e L.). Polyamines are involved in a range of processes, including cell division, embryogenesis induction, flowering initiation, fruit development, and maturation. External application of polyamines has been reported to enhance fruit set and performance in several species, including \u003cem\u003eCucumis sativus\u003c/em\u003e L. (Ebrahimzade et al. 2018), \u003cem\u003ePistachia vera\u003c/em\u003e L. (Kamiab et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003ePhoenix dactylifera\u003c/em\u003e L. (Abd El-Migeed et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and \u003cem\u003eGossypium hirsutum\u003c/em\u003e L. (Bibi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Compared with polyamines, there is limited information on the role CCC in the induction of somatic/gametic embryogenesis. The enhancing effect of CCC and other inhibitors of gibberellin biosynthesis on the induction of somatic/gametic embryogenesis has been reported in species such as \u003cem\u003eCucumis sativus\u003c/em\u003e L. (Ebrahimzade et al. 2018), \u003cem\u003ePinus taeda\u003c/em\u003e L. (Pullman et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), A. \u003cem\u003ecepa\u003c/em\u003e L. ( Ponce et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e)d \u003cem\u003eaestivum\u003c/em\u003e L. (Miroshnichenko et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Fruit weight and seed set were significantly improved by applying CCC low levels (50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to mother plants in cucumber (Ebrahimzade et al. 2018). Similarly, foliar spraying (500 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CCC) on autumn lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e L.) increased the number of inflorescence branches per plant and also seed yield (Pasam et al. 2008). In tomato (\u003cem\u003eSolanumlycopersicum\u003c/em\u003e), the greatest fruit weight was obtained with 100\u0026ndash;300 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CCC (Altinas \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).The results show that the simultaneous use of these mixtures varies in different cultivars. This phenomenon seems to be somewhat genotype-dependent, as the Sahm genotype showed lower embryogenesis compared to Storm and 547. Although spraying these mixtures was useful for inducing embryogenesis \u003cem\u003ein vitro\u003c/em\u003e. Careful cultivation of Storm and 547 mother plants in greenhouse conditions followed by the use of 600 Gy gamma-irradiated pollen grains resulted in higher frequencies of embryogenesis. Gamma irradiation, which has been used in recent years, is one of the effective strategies for inducing embryogenesis through parthenogenesisin Cucurbitaceae. The results showed that the interaction effects of genotype and gamma radiation dose were significant on the total number of seeds, the number of whole and filled seeds, the number of whole and empty seeds in the fruit, and the number of embryos and regenerated plants. The highest number of seeds per fruit was obtained from the genotype \"Storm\" with an average of 205 at a dose of 600 Gy. These findings are consistent with previous studies showing that controlled pollen irradiation can enhance haploid induction and parthenocarpic embryogenesis (Pradeepkumara et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dey et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results highlight the importance of selecting the appropriate genotype and adjusting the irradiation dose for each genotype for seed production. Regarding the number of whole and filled seeds, the highest number was observed in the genotypes \"547\" and \"Storm\" with averaging 124.66 and 117.66 seeds respectively under the control treatment. In contrast, the genotype \"547\" exhibited the lowest number of whole and filled seeds, with an average of 10. The effect of radiation dose showed that the control treatment produced the highest number of whole and filled seeds, and increasing radiation doses led to a significant decrease in this trait. In contrast, the highest number of whole and empty seeds in all 3 cultivars was in the treatment with the highest radiation dose of 600 Gy. The results of this study show that radiation dose and genotype had significant effects on the percentage of full and empty seeds. Increasing the radiation dose to 600 Gy increased the number of whole and empty seeds in the Storm genotype, but lower doses reduced this trend, indicating a stimulating effect of higher doses and an effect on pollen and mother cells. Gamma radiation has a distinct negative effect on full seed production, and the response of genotypes to radiation dose is different. Lower doses of irradiation were able to maintain the number of full seeds, while higher doses caused a severe reduction in the number of seeds. The findings of this study are consistent with previous results in cucumber and other Cucurbitaceae, which showed that the use of controlled pollen irradiation can improve haploid induction and parthenocarpic embryo formation in cucumber, while simultaneously increasing the quality and number of seeds under the optimal dose. However, high doses reduce the quality and number of full seeds (Pradeepkumara et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dey et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).The findings related to embryogenesis showed that the genotypes \"Storm\" and 547 produced the highest number of embryos (6 and 66.5), respectively, while the control treatments did not produce any embryos. Also, the radiation dose of 600 Gy had the greatest effect on the number of embryos and regenerated plants. These results indicate that gamma radiation has an effective role in embryo induction from empty seeds and the selection of the optimal dose can facilitate the production of haploid lines. This finding is consistent with the results reported in other species such as cucumber and cucurbits, which have shown that genotypic sensitivity and radiation dose play an important role in the success of parthenocarpic embryogenesis (Khoshkam et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These results are in contrast to the findings of Ebrahimzadeh et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Gonzalo et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), who reported that the percentage of embryo induction decreases with increasing radiation dose. The possible reason for this discrepancy could be the dose range used and the difference in genotypic sensitivity to radiation, which could be an important factor in this difference in results. Flow cytometry analysis confirmed the accuracy of the regeneration data. DNA content index values in haploid samples were approximately half those of diploid samples. Despite the utility of flow cytometry in estimating DNA content, this method is inherently unable to distinguish between homozygous diploids and heterozygous diploids, especially when both show the the same ploidy level. To overcome this limitation, co-dominant SSR markers were included as a complementary tool to confirm genetic uniformity. In particular, the 3B27marker consistently showed single-band patterns in gel electrophoresis assays and provided molecular confirmation of homozygosity in selected DH lines. Therefore, the integration of nuclear DNA quantification and molecular marker-based genotyping is a comprehensive and reliable strategy for DH identification, providing critical support for accelerating breeding programs and ensuring genetic stability in restored populations (Keleş et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pareeth \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong antimitotic agents, oryzaline has shown better efficacy in producing doubled haploid plants, which highlights that choosing the right agent and carefully adjusting the dose and contact time are essential for successful chromosome doubling (Ebrahimzadeh et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, animal cells are immune to this agent and it has less negative effect on humans.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study aimed to produce pure lines of cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.) through pollen irradiation. Results showed that increasing the gamma radiation dose, particularly at level 600 Gy, had a significant inhibitory effect on the formation of whole and fill seeds and was associated with an increase in the number of whole and empty seeds as well as embryos, identifying 600 Gy as the optimal dose for this study. To mitigate the adverse effects of radiation and improve reproductive conditions, foliar spraying of polyamines including putrescine (500 mg/L), spermidine (50 mg/L)and CCC (50 mg/L) was applied 12 days before and 12 days after pollination on an alternate-day basis. Foliar application of polyamines and CCC increased the proportion of fruits formed and preserved viable embryos. Overall, combined treatment with a high radiation dose (600 Gy) and polyamines plus CCC showed the greatest efficiency in inducing haploid embryos. Thus, these results indicate that controlled levels of gamma radiation can be an effective tool for pollen-induced haploidy in cucumber, though dose and genotype play decisive roles in the success of the process. Additionally, polyamines and CCC can serve as an ancillary strategy to reduce radiation-induced oxidative stress and to improve the growth and viability of gametic cells.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatements \u0026amp; Declarations\u003c/h2\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBAP: \u0026nbsp; 6-Benzylaminopurine\u003c/p\u003e\n\u003cp\u003eCCC: Cycocel\u003c/p\u003e\n\u003cp\u003eCTAB: CetylTerimetil Ammonium Bromide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDH: \u0026nbsp; Doubled haploid\u003c/p\u003e\n\u003cp\u003eDNA: Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eEDTA: Ethylene diamine tetra acetic acid\u003c/p\u003e\n\u003cp\u003eGy \u0026nbsp; \u0026nbsp; \u0026nbsp;Gamma irradiation\u003c/p\u003e\n\u003cp\u003eIAA: \u0026nbsp; Indole-3-acetic acid\u003c/p\u003e\n\u003cp\u003eMS: \u0026nbsp; Murashige and Skoog medium\u003c/p\u003e\n\u003cp\u003eNAA: 1-Naphthaleneacetic acid\u003c/p\u003e\n\u003cp\u003ePI: \u0026nbsp; Propidium iodide\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRpm: \u0026nbsp; Revolutions per minute\u003c/p\u003e\n\u003cp\u003eSSR: Simple sequence repeat\u003c/p\u003e\n\u003cp\u003eSAS: \u0026nbsp; Statistical Analysis System\u003c/p\u003e\n\u003cp\u003eSPSS: Statistical Package for the Social Sciences\u003c/p\u003e\n\u003cp\u003eTBE : Tris- borate -EDTA buffer \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Agricultural Biotechnology Research Institute of Iran (Project no.\u0026nbsp;34-05-0533-014-020240\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: MES conceived of the presented idea and planned the experiments. SFT developed the theory conducted the research experiments and wrote the manuscript with the support of all co-authors. MHS analysis of related data preparation. MT assisted in project implementation. Zahra Hosseini provided laboratory materials. All authors discussed the results and contributed to the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank the Agricultural Biotechnology Research Institute of Iran (Project no.\u0026nbsp;34-05-0533-014-020240) for financial support and the Iran National Science Foundation for postdoctoral support, and Dr. Mehrshad Zeinalabedini for molecular analyses\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study is available\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthices decleration:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd El-Migeed MMM, Mostafa EAM, Ashour NE, Hassan HSA, Mohamed DM, Saleh MMS (2013) Effect of potassium and polyamine sprays on fruit set, fruit retention, yield and fruit quality of Amhat date palm. Int J Agric Res 8(2):77\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3923/ijar.2013.77.86\u003c/span\u003e\u003cspan address=\"10.3923/ijar.2013.77.86\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhamdi B, Shariatpanahi ME, Ojaghkandi MA, Heydari AA (2014) Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaximeandvancomycininBrassicanapus L. Plant Cell Tissue Organ Cult 118:497\u0026ndash;505. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11240-014-0501-9\u003c/span\u003e\u003cspan address=\"10.1007/s11240-014-0501-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmadi B, Ahmadi M, Teixeira da Silva JA (2018) Microspore embryogenesis in \u003cem\u003eBrassica:calcium\u003c/em\u003e signaling, epigenetic modification, and programmed cell death. Planta 248(6):1339\u0026ndash;1350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/pce.70177\u003c/span\u003e\u003cspan address=\"10.1111/pce.70177\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlifar M, Ebadi A, FatahiMoghadamMR (2015) Effects of putrescineandchlormequat pre-flowering treatment on embryo rescue techniquesuccess in diallelecrossesofgrape Cultivars Flame Seedless, perlette and Yaghooti. Iran J HorticSci 46(2):179\u0026ndash;192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.22059/IJHS.2015.54614\u003c/span\u003e\u003cspan address=\"10.22059/IJHS.2015.54614\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltinas S (2011) Effects of chlormequat chloride and different rates of prohexadione-calcium on seedling growth, flowering, fruit development and yield of tomato. Afr J Biotechnol 10(75):17160\u0026ndash;17169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5897/AJB11.2706\u003c/span\u003e\u003cspan address=\"10.5897/AJB11.2706\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehera TK, Boopalakrishnan G, Jat GS, Das MA, Choudhary H, Ravindran A, Kumari R, Horticulture (2022) Environ Biotechnol 63(2):263\u0026ndash;274. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40529-024-00436-w\u003c/span\u003e\u003cspan address=\"10.1186/s40529-024-00436-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatia R, Dey SS, Parkash C, Sharma K, Sood S, Kumar R (2018) Modification of important factors for efficient microspore embryogenesis and doubled haploid production in field grown white cabbage (\u003cem\u003eBrassica oleracea\u003c/em\u003e var. capitata L.) genotypes in India. SciHort233:178\u0026ndash;187. https://doi.org/10.1016/j. scienta.2018.01.017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBibi A, Oosterhuis D, Gonias E (2012) Changes in the ovarian polyamine content and seed set efficiency of cotton by the plant growthregulatorBM86\u0026Prime;. Am J Plant Sci 3(9):1328\u0026ndash;1335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4236/ajps.2012.39160\u003c/span\u003e\u003cspan address=\"10.4236/ajps.2012.39160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChahal G, Gosal S (2002) Principles and procedures of plant breeding: biotechnological and conventional approaches. Alpha Science Int'l Ltd. United Kingdom.ISBN 1-84265-036-X\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuny F (1993) Processusd'inductiond'embryonshaplo\u0026iuml;des par du pollen irradi\u0026eacute; chez le melon (\u003cem\u003eCucumis melo\u003c/em\u003e L.) R\u0026eacute;ponses du pollen \u0026agrave; l'irradiation gamma. These de Docteur, Universite\u0026acute; d\u0026rsquo;Avignonet des Pays de Vaucluse, Avignon\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Witte K, Keulemans J (2000) Homozygous plant production in apple by parthenogenesis in situ: improvement of the pollination stimulus for parthenogenic egg cell development. Acta Hortic 538:315\u0026ndash;320. DIO:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17660/ActaHortic.2000.538.54\u003c/span\u003e\u003cspan address=\"10.17660/ActaHortic.2000.538.54\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey SS, Singh S, Munshi AD, Behera TK (2022) Classical genetics and traditional breeding. In: Pandey S, Weng Y, Behera TK, Bo K (eds) The Cucumber Genome. Compendium of Plant Genomes. Springer, Cham. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5772/intechopen.97593\u003c/span\u003e\u003cspan address=\"10.5772/intechopen.97593\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong YQ, Zhao WX, Li XH, Liu XC, Gao NN, Huang JH, Wang WY, Xu XL, Tang ZH (2016) Androgenesis, gynogenesis, and parthenogenesis haploids in cucurbit species. Plant Cell Rep 35(10):1991\u0026ndash;2019. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00299-016-2018-7\u003c/span\u003e\u003cspan address=\"10.1007/s00299-016-2018-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahimzadeh H, Lotfi M, Azizinia S, Ghanavati F (2013) Production of haploids in Cucurbita pepo L. through parthenogenesis induced by gamma-irradiated pollen. Crop Biotechnol 3:99\u0026ndash;108. DOI: 20.1001.1.22520783.1392.3.4.11.4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahimzadeh H, Shariatpanahi ME, Ahmadi B, Soltanloo H, Lotfi M, Zarif E (2018) Efficient parthenogenesis induction and in vitro haploid plant regeneration in cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.) using putrescine, spermidine, and cycocel. J Plant Growth Regul 37:1127\u0026ndash;1134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00344-018-9803-1\u003c/span\u003e\u003cspan address=\"10.1007/s00344-018-9803-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104(3):301\u0026ndash;309. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11240-010-9800-y\u003c/span\u003e\u003cspan address=\"10.1007/s11240-010-9800-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalazka J, Niemirowicz-Szczytt K (2013) Review of research on haploid production in cucumber and other cucurbits. Folia Hortic 25(1):67\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2478/fhort-2013-000\u003c/span\u003e\u003cspan address=\"10.2478/fhort-2013-000\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerman\u0026agrave; MA (2006) Doubled haploid production in fruit crops. Plant Cell Tissue Organ Cult 86:131\u0026ndash;146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2478/fhort-2013-0008\u003c/span\u003e\u003cspan address=\"10.2478/fhort-2013-0008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalo MJ, Claveria E, Monforte AJ, Dolcet-Sanjuan R (2011) Parthenogenic haploids in melon: Generation and molecular characterization of a doubled haploid line population. J Am Soc forHorticultural Sci 136(2):145\u0026ndash;154. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21273/JASHS.136.2.145\u003c/span\u003e\u003cspan address=\"10.21273/JASHS.136.2.145\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang S, Li R, Zhang Z, Li LI, Gu X, Fan W, Li S (2009) The genome of the cucumber. Cucumis sativus L Nat Genet 41(12):1275\u0026ndash;1128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ng.475\u003c/span\u003e\u003cspan address=\"10.1038/ng.475\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamiab F, Salehabad MH, Zamanibahramabadi E (2015) Evaluation of the effects of foliar treatments of polyamines and some organic acids on quantitative and qualitative traits in some pistachio cultivars. J Nuts 6(2):131\u0026ndash;142. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.22034/jon.2015.516322\u003c/span\u003e\u003cspan address=\"10.22034/jon.2015.516322\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhoshkam S, Samsampour D, Shariat Panahi ME, Khiabani BN (2024) Investigating the possibility of producing double haploid lines in some selected cultivars of greenhouse cucumber through induction of parthenogenesis. Int J Multiphys, 18(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeleş D, Pınar H, Ata A, Taşkın H, Yıldız S, B\u0026uuml;y\u0026uuml;kalaca S (2015) Effect of pepper types on obtaining spontaneous doubled haploid plants via anther culture. HortScience 50:1671\u0026ndash;1676. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21273/HORTSCI.50.11.1671\u003c/span\u003e\u003cspan address=\"10.21273/HORTSCI.50.11.1671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar HGA, Ravishankar BV, Murthy HN (2004) The influence of polyamines on androgenesis of Cucumis sativus L. Eur J Hortic Sci 69(5):201\u0026ndash;205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17660/eJHS.2004/23936\u003c/span\u003e\u003cspan address=\"10.17660/eJHS.2004/23936\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurtar ES, Balkaya A (2010) Production of in vitro haploid plants from in situ induced haploid embryos in winter squash (\u003cem\u003eCucurbita maxima\u003c/em\u003e Duchesne ex Lam.) via irradiated pollen. Plant Cell Tissue Organ Cult 102:267\u0026ndash;277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11240-010-9729-1\u003c/span\u003e\u003cspan address=\"10.1007/s11240-010-9729-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurtar ES, Balkaya A, Ozbakir M, Ofluoglu T (2009) Induction of haploid embryo and plant regeneration via irradiated pollen technique in pumpkin (\u003cem\u003eCucurbita moschata\u003c/em\u003e Duchesne ex Poir). Afr J Biotechnol 8:5944\u0026ndash;5951DOI. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5897/AJB09.730\u003c/span\u003e\u003cspan address=\"10.5897/AJB09.730\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLotfi M, Alan A, Henning M, Jahn M, Earle E (2003) Production of haploid and doubled haploid plants of melon (\u003cem\u003eCucumis melo\u003c/em\u003e L.) for use in breeding for multiple virus resistance. Plant Cell Rep 21:1121\u0026ndash;1128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00299-003-0636-3\u003c/span\u003e\u003cspan address=\"10.1007/s00299-003-0636-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMineykina A, Bondareva L, Soldatenko A, Domblides E (2021) Androgenesis of red cabbage in isolated microspore culture in vitro. Plants 10(9):1950. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/plants10091950\u003c/span\u003e\u003cspan address=\"10.3390/plants10091950\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiroshnichenko D, Filippov M, Doglov S (2009) Effects of daminozide on somatic embryogenesis from immature and mature embryos of wheat. Aust J Crop Sci 3(2):83\u0026ndash;94\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMulyana A, Purwoko BS, Dewi IS, Maharijaya A (2023) Comparison of six anther culture methods for the production of doubled haploids in eggplant (\u003cem\u003eSolanum melongena\u003c/em\u003e L). Euphytica 219(4):44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10681-023-03171-8\u003c/span\u003e\u003cspan address=\"10.1007/s10681-023-03171-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePareeth CM, Purushothama MG (2015) Development of double haploids in bell pepper\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePassam HC, Koutri AC, Karapanos IC (2008) The effect of chlormequat chloride (CCC) Application at the bolting stage on the flowering and seed production of lettuce plants previously treated with water or gibberellic acid (GA3). Sci Hortic 116(2):117\u0026ndash;121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.scienta.2007.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2007.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePonce M, Martinez L, Galmarini C (2006) Influence of CCC, putrescine and gellan gum concentration on gynogenic embryo induction in Allium cepa. Biol Plant 50(3):425\u0026ndash;428. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10535-006-0061-x\u003c/span\u003e\u003cspan address=\"10.1007/s10535-006-0061-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePradeepkumara N, Dey SS, Munshi AD, Behera TK, Bhatia R, Kumari K, Talukdar A (2023) Cucumber F hybrid derived from two contrasting inbreds ensures high frequency gynogenesis for induction of haploids through a modified in vitro based protocol. South Afr J Bot 157:314\u0026ndash;324. 10.1016/j. sajb.2023.03.066\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePullman GS, Mein J, Johnson S, Zhang Y (2005) Gibberellin inhibitors improve embryogenic tissue initiation in conifers. Plant Cell Rep 23:596\u0026ndash;605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00299-004-0880-1\u003c/span\u003e\u003cspan address=\"10.1007/s00299-004-0880-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomanova OV, Vjurtts TS, Mineykina AI, Tukuser YP, Kulakov YV, AkhramenkoDomblides EA (2023) Embryogenesis induction of carrot (\u003cem\u003eDaucus carota\u003c/em\u003e L.) in isolated microspore culture. Food Raw Mater 11:25\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21603/2308-4057-2023-1-548\u003c/span\u003e\u003cspan address=\"10.21603/2308-4057-2023-1-548\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSari N, Abak K, Pitrat M, Dumas de Vaulx R (1992) Induction of parthenogenetic haploid embryos and plant obtention in melon (\u003cem\u003eCucumis melo\u003c/em\u003e L. var. inodorus Naud \u0026amp;\u003cem\u003eC. melo\u003c/em\u003e L. var. reticulatus Naud) Turk J Agric For 16:302\u0026ndash;314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21273/HORTSCI.29.10.1189\u003c/span\u003e\u003cspan address=\"10.21273/HORTSCI.29.10.1189\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSauton A, Dumas de Vaulx R (1987) Production of haploid plants in melon (\u003cem\u003eCucumis melo\u003c/em\u003e L.) as a result of gynogenesis induced by irradiated pollen. Agronomie 7(2):141\u0026ndash;147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1051/agro:19870209\u003c/span\u003e\u003cspan address=\"10.1051/agro:19870209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSegui-Simarro JM (2021) Doubled haploid technology. Springer. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eorg/10.1007/978-1-0716-1331-3\u003c/span\u003e\u003cspan address=\"10.1007/978-1-0716-1331-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShariatpanahi ME, Ahmadi B (2016) Isolated microspore culture and its applications in plant breeding and genetics. In: Anis M, Ahmad N (eds) Plant tissue culture: propagation, conservation and crop improvement. Springer Science\u0026thinsp;+\u0026thinsp;Business Media Singapore, pp 487\u0026ndash;507. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-981-10-1917-3_21\u003c/span\u003e\u003cspan address=\"10.1007/978-981-10-1917-3_21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaner K, Yanmaz R, Kunter B (2000) The effects of irradiation dose and harvest period on haploid plant formation via irradiated pollen in snake cucumber (\u003cem\u003eCucumis melo\u003c/em\u003e var. flexuosus Naud.). IIIrd National Vegetable Culture Symposium, 11\u0026ndash;13 September 2000, Isparta, Turkey, pp 177\u0026ndash;181. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17660/ActaHortic.1999.492.41\u003c/span\u003e\u003cspan address=\"10.17660/ActaHortic.1999.492.41\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThiruvengadam M, Rekha KT, Jayabalan N, Praveen N, Kin EH, Chung IM (2013) Effect of exogenous polyamines on somatic embryogenesis via suspension cultures of spine gourd (Momordica dioica Roxb. ex Willd). Aust J Crop Sci 7(3):446\u0026ndash;453\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiburcio A, Altabella T, Bitri\u0026aacute;n M, Alc\u0026aacute;zar R (2014) The roles of polyamines during the lifespan of plants: from development to stress. Planta 240(1):1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00425-014-2055-9\u003c/span\u003e\u003cspan address=\"10.1007/s00425-014-2055-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei A, Du S, Han Y, Zhang G (2010) A study on the relationship between cucumber gynogenesis and content of ovary hormones and polyamines. Acta Hortic 871:625\u0026ndash;630. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17660/ActaHortic.2010.871.86\u003c/span\u003e\u003cspan address=\"10.17660/ActaHortic.2010.871.86\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Nie FJ, Gong L, Gan XY, Zhang GH, Liu X, Song Y (2023) Regenerative plantlets with improved agronomic characteristics caused by anther culture of tetraploid potato (Solanum tuberosum L). PeerJ 11:e14984. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7717/peerj.14984\u003c/span\u003e\u003cspan address=\"10.7717/peerj.14984\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Wang X, Wang Y, Han J, Mao P, Majerus M (2009) Plant growth regulator effects on balancing vegetative and reproductive phases in alfalfa seed yield. Agron J 101:1139\u0026ndash;1145. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2134/agronj2009.0017\u003c/span\u003e\u003cspan address=\"10.2134/agronj2009.0017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YX, Lespinasse Y (1991) Pollination with gamma-irradiated pollen and development of fruits. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/bf00145636\u003c/span\u003e\u003cspan address=\"10.1007/bf00145636\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cucumber, haploid, embryogenesis, gamma radiation, polyamine, cycocel","lastPublishedDoi":"10.21203/rs.3.rs-8326911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8326911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the combined application of polyamines (putrescine, spermidine) and cycocel (CCC) to maternal plants in relation to the effect of pollen irradiation with gamma rays on seed formation, embryo formation and haploid production in three cucumber genotypes (Storm, Sahm, 547) using pollen irradiation at doses of 400 to 600 Gy. Maternal plants were treated with combination of putrescine (500 mg/L), spermidine (50 mg/L) and CCC (50 mg/L) from 12 days before to 12 days after pollination. Male flowers were irradiated with gamma rays (400, 500, 600 Gy) and pollination was performed. Embryos were rescued \u003cem\u003ein vitro\u003c/em\u003e and regenerated plantlets were analyzed for ploidy level using flow cytometry and SSR marker to confirm haploid status. Haploid plants produced were treated with oryzalin for chromosome doubling. The combined treatment of polyamine and CCC significantly affected embryo induction and seed formation, with the Storm genotype producing the highest number of embryos and regenerated plants at a dose of 600 Gy. Embryogenesis and regeneration varied by genotype, with Storm and 547 showing the strongest responses; Sahm was the least responsive. Flow cytometry confirmed haploid/diploid status, and SSR validated homozygosity in doubled haploid (DH) lines. Higher gamma doses not only increased empty seeds but also embryo numbers, underscoring genotype-dependent radiation sensitivity. Controlled pollen irradiation combined with foliar polyamine/CCC pretreatment effectively induce haploid embryos and DH lines in cucumber, with strong genotype dependency and optimal effects at 600 Gy for certain genotypes. These findings support genotype-informed optimization of DH production and offer practical guidelines for accelerating cucumber breeding.\u003c/p\u003e","manuscriptTitle":"Improvement of embryogenesis in cucumber (Cucumis Sativus L.) parthenogenesis by polyamine and cycocel treatments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 15:44:09","doi":"10.21203/rs.3.rs-8326911/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-23T09:39:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T12:46:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-16T04:15:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-12-14T04:09:12+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":"3188acb0-aaf7-42a2-910c-928386f875e0","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T15:44:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 15:44:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8326911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8326911","identity":"rs-8326911","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}