Efficient induction of parthenogenetic haploids in African marigold (Tagetes erecta) L.) using Co60 gamma-irradiated pollen of French Marigold (Tagetes patula L.)

Efficient induction of parthenogenetic haploids in African marigold (Tagetes erecta) L.) using Co60 gamma-irradiated pollen of French Marigold (Tagetes patula L.) | 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 Efficient induction of parthenogenetic haploids in African marigold (Tagetes erecta) L.) using Co60 gamma-irradiated pollen of French Marigold (Tagetes patula L.) Eram Arzoo, Kavita Dubey, Rashmi Arora, Kanwar Pal Singh, Sapna Panwar, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6683915/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 5 You are reading this latest preprint version Abstract Haploids and doubled haploids play a crucial role in crop improvement programs, including hybridisation, mutation breeding, and genetic and genomic studies. Induction of parthenogenesis using gamma-irradiated pollens is an efficient approach for inducing haploids and developing homozygous lines in horticultural crops. However, no research has been conducted using the induced parthenogenesis approach for haploid induction in the genus Tagetes. This study aims to assess the effectiveness of gamma irradiation at doses of 100, 200, 300, 400, and 500 Gy, using a Co60 (Cobalt-60) source, for haploid induction. The irradiated pollen of a distant tetraploid species, Tagetes patula genotype ‘Pusa Deep’, was used to induce haploids in the diploid marigold species, T. erecta genotypes ‘DAMH-24’ and ‘DAMH-55’. The pseudo-fertilized ovaries were cultured on modified MS medium supplemented with 0.75 mgl-1 TDZ, 0.2 mgl-1 NAA, and 0.5 mgl-1 GA3. To better understand the effect of γ irradiation doses on haploid induction rate, a correlation analysis was performed. Out of 50 regenerants, 20 were selected to determine ploidy levels using morphological traits, chloroplast count, chromosomal count, and flow cytometry analysis. A plant with 3-7 chloroplasts in stomatal guard cells, 2n=x=12 chromosomes, and 1C DNA (~54K mean Pi) was identified as haploid; the plant with 10-14 chloroplasts, 2n=2x=24 chromosomes, and 2C DNA (~108K mean Pi) was diploid, while the plant with 16-20 chloroplasts, 2n=2x=24 chromosomes, and 3C DNA (~154K mean Pi) was triploid. For efficient pollen inactivation and high-throughput haploid induction in Tagetes erecta, the 400 Gy treatment was found to be the most effective. Gamma (γ) irradiation Haploids Induced parthenogenesis Pseudo-fertilization Gynogenesis Flow cytometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Key message This study presents the first successful induction of haploids in Tagetes erecta via induced parthenogenesis using γ-irradiated pollen from the tetraploid species Tagetes patula . Introduction A haploid is defined as a plant that has a gametic number of chromosomes (n). The haploids are highly valuable for genetic and evolutionary studies and play a crucial role in crop improvement programmes like mutation, hybridisation, and transgenic development. The diploidisation of haploids leads to the complete fixation homozygosity in a single step, unlike traditional breeding approaches, and results in the production of doubled haploids (DHs). The DH, being considered an immortal population, is crucial for hybrid breeding (Pradeepkumara et al. 2023 ) and can be utilised for various genetic and genomic research (Schaart et al. 2021 ), QTLs analysis (Dash and Mishra 2024 ), and linkage mapping studies and genome sequencing (Scott et al. 2020 ). Marigold ( Tagetes sp .), a member of the Asteraceae family, is the leading flower crop grown in India and holds great significance in the livelihood security of small and marginal farmers. The genus Tagetes , a native to Mexico, comprises around 33 species, out of which only two species, namely, T. erecta L. (African marigold; 2n = 2x = 24) and T. patula L. (French marigold; 2n = 4x = 48) are commercially cultivated all over the world. In India, marigold rank first both in area (66,130 ha) and production (603180 T), occupying 21.7% of the total area and 19.7% of the total production under flower crops (Anonymous 2020 ). Besides its aesthetic importance, Tagetes erecta L. is a high-value industrial crop for the extraction of carotene, especially lutein, which is used as an eye tonic for healthy vision. The marigold petals are also valued as poultry feed for intensifying the colour of egg yolk and broiler skin (Valdez-Baro et al. 2024 ). The marigold crop is considered a viable option in sustainable agriculture owing to its role in controlling the nematode population. The allelopathic effect of chemical alpha-terthienyl, present in marigold, keeps the population of root-knot nematode under control when it is planted as an intercrop (Liu et al. 2020 ). Owing to the industrial importance of marigold, the demand for F 1 hybrids is immensely rising throughout the world. The F 1 hybrids in marigold possess novel traits, show tolerance to biotic and abiotic stresses and give higher returns to the farmers. In India, the indigenous F 1 hybrids are of meagre occurrence, and a significant amount of revenue is spent every year on the import of hybrid seeds. Recently, in India, institutes have taken up the systematic hybrid breeding program of marigold to meet the ever-increasing demands of farmers for hybrid seeds. Homozygous parental lines are crucial for hybrid seed production in any crop. Conventional breeding approaches require 6–8 generations of selfing to develop a completely homozygous line in this highly cross-pollinated crop. The several agricultural homozygosity can be fixed in a single generation within a tenure of ~ 1 year using the modern biotechnological approach based on doubled haploid. The induction of haploids via in vitro androgenesis ( via . microspore or anther culture) or gynogenesis ( via . unpollinated ovule/ovary culture) or induced parthenogenesis ( via. c ulture of ovaries after pollination) and their subsequent diploidisation to create doubled haploids (DHs) offer a viable option for the rapid development of homozygous lines in marigold. In vitro androgenesis is a well-established technique in marigold for the development of DHs in marigold (Kumar et al. 2019 ). However, in the petaloid male sterile marigold hybrids that completely lack male reproductive organs, i.e. anthers (Sukwiwat et al. 2023 ), the maternal haploid induction through culture of female gametophyte is the only viable alternative. Using female gametophytes, the haploids can be induced through unpollinated ovary culture ( in vitro gynogenesis; Kumar et al. 2020 ) and pollinated ovary culture approach (induced parthenogenesis). In pollinated ovary culture technique, the maternal haploid can be induced by either pollination with irradiated/inactive pollen or pollination with the pollen of distant-related species. The irradiated pollen mostly contains inactivated sperm cells; however, they retain the ability to produce standard pollen tubes that can penetrate ovules and eventually result in parthenocarpic seed development and haploid embryo induction (Tran et al. 2023 ). For successful haploid induction through induced parthenogenesis, ionising irradiations (X-rays and gamma rays) can be used. However, the application of gamma rays is more common because of their greater tissue penetration, less lethality, high mutation rate, and convenience (Kurtar and Balkaya 2010 ). The generative nuclei inside pollen degenerate due to high energy of irradiations and are unable to undergo mitosis-II to produce two sperm nuclei and thus inhibit double fertilisation. This leads to pseudo-fertilization as male gametes are eliminated from participating in the fertilisation process and stimulate haploid embryo induction through induced parthenogenesis. The irradiated pollen can provide a stimulus to the egg cell to induce parthenogenetic haploids. The dose of irradiation is crucial in induced parthenogenesis; the inactivation rate of the generative nuclei increases with the increase in doses of radiation and thus promotes more parthenogenic embryo development and haploid induction (Aktas et al., 2023). The successful induction of maternal haploids in Tagetes erecta L. using an unfertilised/unpollinated ovary culture ( in vitro gynogenesis ) approach has been described by Kumar et al. ( 2020 ). In the Asteraceae family, in vitro gynogenesis approach has been successfully utilised for haploid induction in sunflower (Aktas et al. 2023), gerbera (Li et al. 2020 ), niger (Bhat and Murthy 2007 ), and chrysanthemum (Miler et al. 2023 ). In most of these reports, very low haploid induction frequencies were observed. The induced parthenogenesis technique exploiting irradiated pollen has been efficiently used to improve the haploid induction frequency in several horticultural crops; however, to date, the potential of this approach has remained unexplored in Tagetes erecta L. In the Asteraceae family, γ-irradiated pollen of distantly related species/genera have been utilised to induce parthenogenetic haploids in Chichorium x Lactuca, Chichorium x Cicerbita (Dore et al. 1996), and Lactuca x Helianthus (Piosik 2016). In Iris pseudacorus , parthenogenetic haploids were successfully induced by utilising the X ray-irradiated pollen of I. spuria . Induced parthenogenesis through exploiting irradiated pollen of the same species has been successfully used in Helianthus annuus L. (Todorova et al. in 1997; Todorova and Ivanov 2000 ; Aktas et al. 2018; Wang et al. 2023 ), cyclamen (Tutuncu and Mendi 2022), Rosa damascena Mill. (Palvaneh et al. 2013 ), Rosa x hybrida (Meynet et al. 1994 ), Dianthus caryophyllus L. (Sato et al. 2000 ), and Mimulus aurantiacus Curtis (Murovec and Bohanec 2013 ). So far, no research has been undertaken on the use of the induced parthenogenesis approach for haploid induction in the genus Tagetes. Hence, we have conducted an in-depth study on the role of different gamma irradiation doses on pollen viability, germination, pseudo-fertilization and haploid induction in T. erecta L. This is the first report in marigold where the gamma-irradiated pollen of a tetraploid French marigold was used for the induction of parthenogenetic haploids in diploid African marigold. Materials and Methods The primary goal of this work was to assess the effectiveness of gamma irradiation on haploid induction frequency in Tagetes erecta L. It was hypothesised that irradiated pollen loses its capability of fertilisation, which may be because mutation leads to disorganisation of the nuclei as well as breakage of the chromosome. Still, the pollen retains the ability to germinate until the lethal dose. The irradiated pollens trigger unfertilised egg cells to divide, and this leads to the development of haploid embryos by ‘direct regeneration’. Further, we have chosen the distant species as a pollen source to reduce the risk of fertilisation. We have irradiated the pollen of tetraploid species (2n = 4x = 48) T. patula L. with different doses of γ ray (100, 200, 300, 400 and 500 Gy) in order to optimise the irradiation dose for parthenogenic haploid development in diploid (2n = 4x = 48) Tagetes erecta L. Plant material and its raising Two petaloid-type male sterile African marigold ( Tagetes erecta ) genotypes, DAMH-24 (OPMS-1 × AMS-3; dark orange) and DAMH-55 (YPMS-2 × AMS-18; lemon yellow) (Fig. 1 a-b) has been chosen to undertake haploid induction studies, while a French marigold ( Tagetes patula L.) genotype, ‘Pusa Deep’ (Fig. 2 c) was used as pollen parent for γ irradiation. The seeds of all three genotypes were sown at the research farm of the Division of Floriculture and Landscaping, ICAR - Indian Agricultural Research Institute (IARI), New Delhi, India. The seeds were initially sown in plug trays containing a mixture of cocopeat, perlite, and vermiculite (1:1:1) under net house conditions during the rainy seasons of 2021–2022. After one month of sowing, the seedlings were transplanted to the open field conditions at the research farm of the Division of Floriculture and Landscaping, ICAR-IARI, New Delhi (28.08 0 N and 77.12 0 E at 228.61 m above mean sea level). All the good agricultural practices as prescribed for marigold crop by IARI were followed to raise the healthy crop. The plants were weekly sprayed with liquid fertilisers @ 3 g l − 1 (N: P: K = 19:19:19) during the vegetative and @ 3 g l − 1 (N: P: K = 13:0:45) during the flowering phase. No chemicals were used for plant protection in any form to avoid viability reduction of female gametophytes (Kumar et al. 2019 ). Irradiation of Flowers The plants of the pollen parent, ‘Pusa Deep,’ were bagged at the initiation of flowering to avoid pollen contamination. In the morning hour (9.00 am to 10.00 am), the bagged flowers of ‘Pusa Deep’ were collected one day before anther dehiscence and placed in small transparent polypropylene boxes. Each box containing 6–8 flowers was irradiated with different doses of γ rays, 100, 200, 300, 400, and 500Gy, along with control (0 Gy) at the Nuclear Research Laboratory (NRL), New Delhi, in γ chamber 5000 (GC-5000) using Co 60 isotope. After irradiation, the anthers from the irradiated flowers were allowed to dehisce in the laboratory. The irradiated flowers were placed in water-filled test tubes, and a translucent butter paper bag was placed on each test tube to maintain proper hydration. The dehisced pollen from four irradiated flowers, along with the control, were used for pollination of two petalous male sterile genotypes of African marigold, DAMH-24 and DAMH-55. Two irradiated flowers from each treatment, along with the control, were utilised for pollen study. This whole process was repeated three times in a season. Pollen viability The freshly dehisced anthers were utilised for pollen viability studies, which were done using two methods, i.e. , pollen staining and germination. Pollen staining was estimated using two colourimetric tests, that is, 2, 3, 5, Triphenyl Tetrazolium Chloride (TTC) and Potassium Iodide (KI). The TTC stain was prepared by minor modification into black colour-stained pollen (Fig. 2 d ii). It was considered viable—the pollens, which were unable to take stain and remained yellow, were considered non-viable (Fig. (d iOberle and Watson's (1953) media, which included 1g TTC and 6g sucrose in 20 ml of distilled water, and the pH of the solution was adjusted to 5.7. At the same time, the Potassium iodide stain was prepared by dissolving 1g Potassium Iodide (KI) and 0.5g Iodine (I) in 100 ml of distilled water (Baker and Baker, 1979 ). The freshly dehisced pollen from the irradiated flowers (0, 100, 200, 300, 400, 500 Gy) were stained with TTC and KI solutions separately, and a cover slip was placed over them. For better absorption of the stain, the slides were kept in the dark for 30–60 minutes and then observed under an inverted light microscope. Three replications of ~ 40 pollen grains in each slide were observed (10X magnification). In the case of TTC, staining red-coloured pollen (Fig. 2 d i) was considered viable, while in the case of KI reddish- blue, ii)). For in-vitro pollen germination studies, Brewbaker and Kwack ( 1963 ) media consisting of 20% sucrose, 100 ppm Boric acid, 300 ppm Calcium nitrate, 200 ppm Magnesium sulphate, 100 ppm Potassium nitrate, and 0.2% Agar (pH: 5.2–5.8) was used. Pollen germination was estimated using the hanging drop method. In this method, the germinating media was placed over a coverslip, and pollens were dusted over it using a needle. The cover slip was then placed on a cavity slide sealed with petroleum jelly, and the slide was turned to hang the media over the coverslip. The slides were kept for incubation at 25 ºC under moist conditions for 12 hours for pollen germination. Three replications of ~ 100 pollen grains in each slide were observed (4X magnification). Pollen was considered germinated when its tube length was doubled the size of its diameter (Fig. 2 (d iii)). Pollination and Pseudo-fertilization The pollen of French marigold ( T. patula L.) genotype ‘Pusa Deep’ irradiated with different doses of gamma irradiations was used to pollinate the petaloid type male sterile genotypes of African marigold ‘DAMH-24’ and ‘DAMH-55’. The flower buds of African marigold genotypes DAMH-24 and DAMH-55 were covered with butter paper bags to prevent the chance of unwanted cross-pollination. The pollen from freshly dehiscence anthers of irradiated (0, 100, 200, 300, 400, 500 Gy) and non-irradiated (0 Gy) flowers of French marigold ( T. patula L.) genotype ‘Pusa Deep’ were utilised to pollinate the male sterile flowers of ‘DAMH-24’ and ‘DAMH-55’. A total of 60 flowers were pollinated in each genotype, i.e. , each treatment had 10 flowers (total of 6 treatments), and the experiment was repeated three times a season. The pollination was performed during the daytime (11:00 am to 1:00 pm) when the stigmas of female flowers were fully elongated, bifurcated and receptive. As marigold inflorescence (capitulum) consists of many florets and stigmas of different rows, they become receptive on other days; therefore, the pollination was repeated 2 to 3 times during 2 to 3 days’ time tenure. The irradiated pollen retains the ability to germinate on the stigmas. However, the capabilities of the male gametes to undergo fertilisation is significantly reduced, and hence, it promotes parthenogenic embryo development in pseudo-fertilized ovaries. The confirmation of fertilisation and pseudo-fertilization was done after 30 days of pollination (DAP). These pseudo-fertilized ovaries failed to develop under field conditions. Ovaries showing dark black/brown colour are considered fertilised (triploid), while those showing cream to light purplish colour are deemed to be pseudo-fertilized (haploid). The percent of ovaries showing different colours was assessed, which showed the effectiveness of γ irradiation towards pseudo-fertilization . In vitro embryo culture To assess the role of γ irradiations on gynogenesis efficiency, the pseudo-fertilized ovaries were cultured on standardised media. As the pseudo-fertilised ovaries lacked endosperm and failed to support the development of parthenogenetic-induced embryos, hence ovary culture was attempted to rescue the haploid embryos. The pollinated and partially matured flowers (25–30 DAP) were plucked and sterilised as per the protocol described by Kumar et al. ( 2020 ). For surface sterilisation, the flower was treated with 0.2% HgCl 2 for 5 min, followed by 4–5 times with autoclaved double-distilled water to remove the traces of toxic remnants. Subsequently, the sterilised buds were dried on the autoclaved tissue papers. The pseudo-fertilized ovaries were carefully excised from the sterilised flowers with the help of forceps and inoculated on a standardised culture media containing MS, 0.75 mgl − 1 TDZ, 0.2 mgl − 1 NAA, 0.5 mgl − 1 GA 3 , 125 mgl − 1 PVP, 250 mgl − 1 Casein hydrolysate, 50mll − 1 Coconut water mix, 40 gl − 1 Sucrose, 2.6 gl − 1 Gelrite at 5.7 pH (Arzoo et al. 2022; Murashige and Skoog, 1962 ). A total of three Petri dishes, each containing ~ 30 ovaries, were cultured in each treatment, and each treatment consisted of three replications. The cultured plates were sealed with parafilm followed by phyta-wrap to avoid contamination and maintained at 25 ± 1°C temperature in the dark. After the induction of optimum responses (swelling/bulging of ovaries or embryo emergence), plates were moved to light conditions in a photoperiod of 16:8 hours of light and dark cycles under fluorescent white light (47µmol/m²/s). Plant regeneration, proliferation, and hardening The direct regenerated embryos, as well as the embryogenic calli induced from pseudo fertilised ovaries, were transferred to MS medium supplemented with 0.25 mgl − 1 GA 3, 0.1mgl − 1 NAA, 0.2mgl − 1 BAP, 125mgl − 1 Casein hydrolysate, 125 mg − 1 PVP, 40 gl − 1 Sucrose and 8 gl − 1 Agar for shoot formation and elongation. The regenerated micro-shoots were proliferated on MS medium supplemented with 0.25 mgl − 1 KIN, 0.25 mgl − 1 GA 3, 0.1mgl − 1 NAA, 125 mgl − 1 PVP, 40 gl − 1 Sucrose with 8 gl − 1 Agar and the well-developed shoots were transferred to ½ MS medium supplemented with 0.5 mgl − 1 NAA, 125 mgl − 1 PVP, 45 gl − 1 Sucrose with 8 gl − 1 Agar for rooting. All the cultures were maintained at the same temperature and light condition as the embryo. The well-rooted plantlets were gradually acclimatized in a plastic pot covered with polythene containing sterile coco peat, perlite and vermiculite mixture (3:1:1) under controlled conditions (Arzoo et al. 2022). Ploidy determination Ploidy levels of plants induced from ovaries were determined using direct (chromosome counting and flow cytometry) and indirect (chloroplast number of the guard cells and morphological observations) methods of ploidy evaluation. The flow diagram in Fig. 1 below depicts all the four direct and indirect approaches used for ploidy determination. Statistical Analysis The experiments were laid out in a completely randomised design (CRD) to test the significance of the mean difference among treatments. The different treatments comprising irradiation doses (0, 100, 200, 300, 400, 500 Gy) in combination with genotypes were replicated three times. The percentage data were subjected to Arc Sin transformation. We had some data with zero (0) values, but for statistical analysis, we could not use zero values. Therefore, the values of the entire set of data were transformed through square root transformation. After the transformation of original values, statistical analysis was performed by using ANOVA. Further, the Karl Pearson correlation coefficient was computed by using the cor() and corrplot mixed() function in “ R software ” to study the relationship of irradiation doses with other variables under study. Results and Discussion Irradiated pollen induces parthenogenesis or apogamy and can also lead to efficient haploid induction in several economically important crops (Wang et al. 2023 ). At a specific dose, the capabilities of the irradiated pollen (male gametophytes) to undergo fertilisation are significantly reduced. However, it retains its ability to provide stimulus on the stigma for fertilisation that leads to cell division as well as embryo development from egg cells. Hence, an effective dose of gamma irradiation can enhance the production of parthenogenic embryos rather than zygotic embryos and lead to the development of haploid plants (Tran et al. 2023 ). Effects of gamma irradiation on pollen viability and pollen germination The pollen viability of irradiation and non-irradiation (control) flowers of Tagetes patula L. genotype ‘Pusa Deep’ was assessed through staining and in vitro germination tests. In both stains, the number of pollens obtaining stains reduced drastically when the exposure time to the irradiations (dose) was enhanced. In the Tetrazolium chloride (TTC) staining, the control (non-irradiated) pollen exhibited maximum staining, revealing maximum pollen viability. As the dose was increased from 100 to 200 Gy, there was a sharp decline (~ 68 percent) in the percentage of pollen taking the stain, and only 18.34% of the pollen remained viable on exposure to 200 Gy. The percentage of pollen taking up stain reduced to below 5.0% when the flowers were exposed to 400 and 500 Gy doses of gamma irradiations, indicating the drastic reduction in pollen viability on exposure to higher doses of gamma irradiation (Fig. 3 ). Similarly, with Potassium Iodide (KI) staining the maximum pollen staining ~ 95.0% was observed in non-irradiated pollen, which reduces to almost 60.0% on exposure to 100 Gy. The exposure of flowers to higher doses of gamma irradiations, i.e. 300 and 400 Gy, led to the reduction in pollen viability to as low as 5.0% (Fig. 3 ). A negative correlation between radioresistance and pollen viability was observed earlier by Guler et al . (2017). In both TTC and KI stains, exposure of flowers to 400 Gy of gamma irradiation led to the maximum reduction in staining and hence resulted in the least viability of pollen (0.84% in TTC & 3.33% in KI); therefore, this dose can be considered as the optimum dose for parthenogenic haploid induction in marigold genotypes. Among different parts of a flower, pollen is regarded as the most sensitive to irradiation and reduction in pollen viability on exposure to irradiation can be attributed to pollen dehydration (Kundu and Dubey, 2022 ). One of the reasons behind this is that the treatment of pollen grains by ionising radiation (X-ray, γ-ray) may also cause mutations, breakage of the chromosomes and disorganisation of the nucleus (Froelicher et al. 2007 ). We have also assessed the effects of gamma irradiations on in vitro pollen germination of irradiated and non-irradiated (control) flowers of Pusa Deep. The maximum in vitro pollen germination (71%) was found in the non-irradiated flowers (Fig. 4 ). A sharp decline in pollen germination percentage was observed when gamma irradiation was applied to the flowers. A reduction of 85.0% germination was observed in pollen when flowers were subjected to 100Gy of gamma radiation (only 10.0% pollen germinated), while further increasing the irradiation doses to 300Gy reduced the germination to as low as 5.0%. The effects of irradiation on viability and in vitro germination of pollen were earlier studied by Thaneshwari et al. (2019) in Marigold, who noticed that pollen treated with 300Gy gamma irradiations exhibited minimum pollen viability and pollen germination. Salehian et al. ( 2023 ) also proved that with increasing doses of gamma rays, pollen viability, germination, and tube elongation of cucumber continuously decreased compared to non-irradiated pollen. Similar revelations were earlier proposed by Blasco et al. ( 2016 ) in Loquat and Kurtar et al. ( 2009 ) in Pumpkin, where the pollen grains completely failed to germinate under in vitro conditions when flowers were irradiated with 400Gy. Hence, this dose was considered as the desirable dose for gynogenic haploid induction in marigold. Similar to γ rays, X-rays also reduced the pollen viability and germination in Iris spuria at a comparable dose (Grouh et al. 2015 ). The loss of viability and inability of the pollen to germinate on irradiation exposure is attributed to the loss of cytoplasmic fluid inside the pollen that keeps the generative cells wet and alive inside the pollen (Pacini and Franchi, 2020 ). In our experiments, we have also observed the difficulties in anther dehiscence on exposing the flowers to 500 Gy, signifying the sensitivity of marigold genotypes to irradiations. Meynet et al. ( 1994 ) have also revealed a similar problem with the higher dose of irradiations ~ 500Gy dose in Rose. The exposure of flowers to higher doses of γ rays leads to pollen dehydration. Which in turn gives rise to poor germination (Fig. 4 ). The irradiated pollen technique can be effectively used to improve the haploid induction frequency. Still, before applying this technique on a commercial scale, the dose of γ irradiation that can inactivate the generative nuclei in a maximum number of pollen grains and thus reduce the pollen viability to as low as zero must be optimised. Effects of gamma irradiation on fertilisation The fertilisation, as indicated by a change of the ovary colour to black, was severely hampered by irradiation. Pollination of African marigold genotypes DAMH-24 & DAMH-55 with the irradiated pollen of French marigold genotype ‘PD’ exhibited only a slight change in the colour of the ovaries to purplish red, indicating resulted in pseudo-fertilization . The earlier studies have established the fact that the irradiated pollen retains its capability to germinate on the stigma and can grow within the style to reach the embryo sac. Still, it cannot fertilise the egg cell and the polar nuclei (Froelicher et al. 2007 ). The irradiated pollen thus stimulates the division of the egg cell and apogamic/parthenogenic embryo development, which was evident from the change in the colour of the ovaries in the marigold. The full penetration of pollen tubes into the ovaries (even in the absence of fertilization) can sufficiently trigger seed sets (Tran et al. 2023 ). Aslam et al. (2023) delineated that seeds produced in irradiated crosses were 45% lighter compared with seeds of control (non-irradiated pollen). Both colour change and weight loss of seed were because the irradiated pollen stimulated cell division from haploid cells of the embryo sac other than the zygote formation (diploid cell) and induced haploid embryos (Falque, 1994 ). The maximum frequency of fertilisation (indicated by blackening of ovaries; 93.67%) was observed when the African marigold flowers were pollinated with the non-irradiated pollen of PD (Fig. 5 ). Pollination with 400 Gy irradiated pollen (Fig. 5 ) resulted in the lowest percentage of fertilised ovaries (1.83%). This treatment was found to be statistically similar to 500 Gy (Fig. 5 ). Similar observations have been noticed by Kundu and Dubey ( 2022 ) in citrus on pseudo-fertilized seed development pattern when C. grandis was pollinated with γ irradiated pollen (100–500 Gy) of C. limetta . They observed maximum pseudo fertilisation at 300 Gy dose. The degree of pseudo-fertilization was dependent on both the genotype and irradiation doses. The genotypes × treatments interaction revealed that the minimum percentage (1.67%) of pseudo-fertilization was recorded in DAMH-24×PD when pollinated with the 400 Gy. Thus, 400 Gy has been considered the optimum dose for induced parthenogenesis-mediated haploid production in marigold. (Fig. 5 ). Godbole et al. (2012) also successfully induced parthenogenesis in Snapmelon with 250 Gy of γ-irradiation, whereas low (150 and 200 Gy) or high (300 and 350 Gy) irradiation doses were not found desirable for parthenogenesis. Contrary to our results, Wang et al. ( 2023 ) achieved the highest frequency of the parthenogenetic haploid induction in sunflower with a lower dose of irradiation, i.e. with 100 Gy. The irradiation doses for induced parthenogenesis were found to be highly crop and genotype-specific. The lower doses of γ irradiation were found effective for parthenogenic embryo development in citron watermelon (200 to 250 Gy; Kurtar et al. 2025 ), bottle gourd (100 to 125 Gy; Zhao et al. 2023 ) and in Winter Squash (50 and 100 Gy; Kurtar and Balakaya, 2010). In Muskmelon, Cuny et al. ( 1993 ) noticed that a dose less than 1600 Gy was not adequate for pseudo-fertilisation , while pollen irradiated at 2500 Gy showed a maximum number of aborted/ pseudo-fertilised fruits. Effects of gamma irradiation on haploid induction through induced parthenogenesis The irradiation doses significantly affected the response and embryo regeneration (direct or indirect) in both the genotypes of African marigold (Fig. 6 – 9 & Table 1 ). The minimum days to visible response (swelling/bulging of ovaries and embryo emergence) was shown by ovaries pollinated with non-irradiated pollen, which may be due to ease in fertilisation and embryo development. In contrast, with the increase in irradiation doses, the days to visible response from the pseudo-fertilized ovaries increased significantly up to 300Gy. However, we observed a slight reduction in days to response at 400Gy, indicating higher efficiency of pseudo-fertilization . The maximum response from cultured ovaries (24%) was observed when African marigold genotypes were pollinated with non-irradiated pollen, while the minimum response was observed with 200 Gy dose (2.76%). We have observed 400Gy as the optimum dose for induced parthenogenesis in marigold. It led to the optimum pseudo-fertilization as reflected by the highest percentage of direct regeneration in the shortest possible time. Besides direct regeneration, we have also observed indirect regenerations (callusing). The maximum percent callusing was recorded with 0 Gy (control) and 100 Gy doses. Kundu et al. (2017) have revealed that pollination of Citrus grandis with gamma-irradiated (300 and 400 Gy doses) pollen of C. limetta and C. sinensis (respective doses) stimulated the process of parthenogenesis and haploid induction. At the same time, doses below 300 Gy were found to be ineffective in inducing haploids in either of the cross combinations. A high irradiation dose of 500Gy was not found suitable for regeneration (either direct or indirect) because higher irradiation prevents parthenogenic or zygotic embryo formation. These results are supported by the findings of Salehian et al. ( 2023 ) in Sunflower, where they could not induce any haploids at 500 Gy because of higher penetration energy. They have standardised 300 Gy as an optimum dose for parthenogenic embryo development and haploid induction. The optimum dose of γ irradiation for parthenogenic haploid induction was found to vary among the flower crops: 250 Gy in Rosa damascena (Palvaneh et al. 2013 ), 300 Gy in Sunflower (Todorova et al. 1997 ), and 500 Gy in Rosa x hybrida (Meynet et al. 1994 ). The number of induced embryos was significantly reduced with the increase in the dose of irradiations. The embryo response and regeneration were not only related to the irradiation doses but also to the genotypes. DAMH-24 x PD showed the maximum direct regeneration (Fig. 6 ; Table 1 ), while no direct regeneration and only indirect regeneration (callogenic embryo) was observed in DAMH-55 x PD (Fig. 7 ; Table 1 ). The maximum regeneration for efficient haploid induction was found when 400 Gy irradiated pollen of PD were used to pollinate DAMH-24. Keles et al. (2016) also observed varietal variations for haploid induction in Spinach; out of three varieties, haploid embryo formation was observed in Favorit F1 when pollinated with 400 Gy irradiation pollen of the same variety. Contrary to these, Aktas et al. (2023) obtained efficient haploid induction in Sunflower when a higher dose of irradiation, i.e. 750 Gy, was used. They obtained haploid induction only in three genotypes (K3AD SN:8, IMI 069 and IMI 044) out of the 16 tested genotypes. Similarly, Zhao et al. ( 2023 ) observed the highest embryo induction bottle gourd genotype, BG-4, when 50 Gy irradiated pollen of the same variety was used for pollination. Correlation among different variables of in vitro cultured pseudo fertilised ovaries of and irradiation doses For a better understanding of the relationship between haploid induction through induced parthenogenesis and γ irradiation doses, a correlation matrix was established between different variables of haploid induction and irradiation doses. In Fig. 10 , a correlation matrix scale showing the values ranging from r = 1 to -1 represents the strength and direction of the correlation between variables; the brown colour denotes a negative/indirect correlation, while the blue colour depicts a positive/direct correlation between both the parameters (Bakhshandeh et al. 2023 ). All the parameters are correlated after taking the average of both crosses (DAMH-24 × PD and DAMH-24 × PD) with different gamma-ray doses. According to our study, as the irradiation dose increased, the time taken for the response (r = 0.85) and days to direct regeneration (r = 0.73) showed a strong direct correlation. In contrast, percent direct regeneration (r = 0.58) showed a moderate direct correlation and days to callus initiation (r = 0.42) exhibited a direct correlation. Similarly, with some other parameters as doses increase the percentage of ovaries showing colour change (r = -0.90) exhibited a robust indirect correlation while percent response (r = -0.58) and percent callusing (r = -0.52) exhibited a moderate indirect correlation. Similar to the present findings, Ulukapi and Ozmen ( 2018 ) also studied the correlation effects of various levels of γ-rays (100 to 500 Gy; 50 Gy interval) on the survival rates of the seedlings and vegetative traits in Common bean ( Phaseolus vulgaris L.) plants. They found a significant positive correlation between root length and shoot length (r = 0.90), between root fresh weight and shoot fresh weight (r = 0.667), and between leaf length and chlorophyll index in M1 plants of the F16 variety. Ploidy determination of gynogenically developed plants Determination of the ploidy level is a vital step during haploid and doubled haploid production and their utilisation in breeding programmes. The methods for ploidy determination in marigold have been described earlier by Kumar et al. ( 2019 ). A total of 52 plants, regenerated from the cultured ovaries of DAMH-24 × PD and DAMH-24 × PD) were assessed for their ploidy confirmation along with the mother plants. The ploidy was initially assessed by indirect and later confirmed by direct methods (Fig. 11 ; Table 2). The morphological evaluation of regenerated plants revealed the prevalence of albino plants at higher doses, ~ 400 & 500 Gy. At higher irradiation doses, haploid embryo induction might have resulted due to induced parthenogenesis. Hence, more plants with physical deformities were obtained. The haploid plants were mostly albino and exhibited high mortality rates, and if they survived, they also then failed to flower; this phenomenon may be due to the ‘Hertwig effect’ (Pandey and Phung, 1982 ). The number of chloroplasts in guard cells of stomata varied from 3–7 in haploids, 10–14 in diploids and 16–20 in triploid plants (Fig. 11 ). The plants of different ploidy levels, including haploids, diploids, and mixoploids have also been reported during the androgenesis and gynogenesis in marigold by Kumar et al. (2018) and Kumar et al. ( 2020 ) and Bhatia et al. ( 2017 ) in cauliflower. The cytological analysis (chromosome counting) further revealed that all the regenerants from 0 Gy and a few from 100, 200 and 300 Gy were triploid showing chromosome number 2n = 3x = 36 in their root cells (Fig. 11 k). A total of 8 haploids (2n = x = 12; 15.38% Fig. 11 c), 27 diploids (2n = 2x = 24; Fig. 11 g ) and 17 triploids (2n = 3x = 36; Fig. 11 k) were identified. The maximum haploid induction regenerants were identified in DAMH-24 ×PD. The maximum haploid induction was observed within DAMH-24 ×PD with 400 Gy (7 out of 12) followed by 200 Gy (1 out of 5), while no haploid was obtained in DAMH-55× PD even on irradiations. The higher dose of gamma irradiation might have led to the inactivation of male gamete in the pollen; however, it supported the direct embryogenesis from haploid cells of the embryo sac and subsequent haploid production. In this study, we have obtained a large number of diploid regenerants, which could be a result of regeneration from the diploid cells of the ovary wall/ embryo sac. The triploid plants could have regenerated as a result of successful fertilisation between diploid African marigold and tetraploid French marigold species. Both these species are cross-compatible, and successful interspecific hybrids among these two species have been reported by Zhang et al. ( 2022 ). Some of these diploids could also be doubled haploids that might have resulted from spontaneous chromosome doubling of haploid embryos. Spontaneous chromosome doubling is a common phenomenon during haploid production and has been in a large number of plant species, including Brassica vegetables (Bhatia et al. 2016 & 2018 ). After chromosome counting, the ploidy levels of the regenerants were further confirmed by flow cytometry analysis. Here, we have haploids have C DNA content with ~ 54K mean Pi A (Fig. 11 d), diploids with 2C DNA content with ~ 108K mean Pi (Fig. 11 h), and triploids with 3C DNA content with ~ 154K mean Pi (Fig. 11 i). The flow cytometry analysis also revealed that out of the 52 randomly selected plants 8 were haploids (15.38%), 27 were diploid (51.92%) and 17 were triploid (32.69%) (Fig. 12 ). The haploid plants were successfully recorded using induced parthenogenesis approach in cucumber (Pradeep et al. 2024). This study has proved the utility of the irradiated pollen technique (induced parthenogenesis) in haploid induction in African marigold. In the future, this approach can be utilised to achieve high throughput haploid induction in different marigold genotypes. Conclusion For the first time, we have conducted an in-depth study to determine the role of gamma irradiations on haploid induction in marigold genotypes. The irradiated pollen of tetraploid marigold species, Tagetes patula genotype Pusa Deep ‘PD’ (2n = 4x = 48) was used to induce the haploids in the diploid marigold species, T. erecta genotypes ‘DAMH-24’ and DAMH-55’ (African marigold; 2n = 2x = 24). The irradiated pollen technique was found effective in improving the haploid induction frequency in marigold. An irradiation dose of 400 Gy was found to be the optimum dose for efficient pollen inactivation and high-throughput induction of haploids. This research also provided insight into the effectiveness of gamma irradiation on successful haploid induction in marigold. This protocol will be instrumental in strengthening the F 1 hybrid breeding and genetic and genomic research in marigolds. Abbreviations Gy Gray (=100 rad) KI Potassium Iodide TTC 2, 3, 5, Triphenyl Tetrazolium Chloride DHs Doubled Haploids DAP Days after Pollination DAI Days after Inoculation PVP Polyvinylpyrrolidone TDZ Thidiazuron QTLs Quantitative Trait Locus Declarations Acknowledgements The first author acknowledges the Indian Council of Agricultural Research (ICAR), New Delhi, India for awarding a Junior Research Fellowship during the entire period of this experiment. The funding received from the Science and Engineering Research Board, Department of Science and Technology, Government of India (Sanction Order: EEQ/2019/000632) for facilitating this research from 2019 to 2023 is also highly acknowledged. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests Funding This work was funded by the Science and Engineering Research Board, Department of Science and Technology, Government of India (Sanction Order: EEQ/2019/000632). Author contributions RB comprehended and supervised the experiments. RB, KPS, SP, and AT designed the experiments. EA and KD executed field/lab experiments and collected experimental data. EA and BS conducted the gamma irradiation experiment. EA, MF and RA have done pollen and pollination studies. EA, KD, and MR have determined the ploidy. RB, EA, and B did data analysis and interpretation. EA wrote the first draft of the manuscript, and RB has made all the corrections. All other authors have proofread and approved the final manuscript. Availability of data and materials All the data and other details used for this study can be requested from the corresponding author. Conflict of interest All the authors declare no competing interests regarding the publication of this study. References Aktaş YE, Aydin Y, Uncuoglu AA (2023) Induction of haploid plants for speed-up breedıng in sunflower ( Helianthus annuus L.) by pollen irradiation. Genetics & Applications. 7(1):35-44. https://doi.org/10.31383/ga.vol7iss1ga05 Aktaş YE, Uncuoğlu AA, Aydin Y (2018) Induction of parthenogenetic haploid embryos and plants after pollination by irradiated pollen in sunflower. In: Proceedings of the International Agricultural, Biological and Life Science Conference, Edirne, Turkey, pp. 2-5. Anonymous (2020). Indian Horticulture Database. http://nhb.gov.in. Accessed 23 Jan 2024. Arzoo E (2022) In vitro gynogenesis studies in African marigold. M.Sc dissertation, ICAR-IARI, New Delhi, India-110012. Aswath C, Prakash T, Kumari P, Thakur N, Sharma R (2019) Effect of gamma radiation on pollen viability and pollen germination of marigold cultivar. Think India Journal. 22(30):621-628. Baker HG, Baker I (1979) Starch in angiosperm pollen grains and its evolutionary significance. American Journal of Botany. 66(5):591-600. https://doi.org/10.1002/j.1537-2197.1979.tb06262.x Bakhshandeh E, Hosseini Sanehkoori F, Ghorbani H, Nematzadeh GA, Sekrafi M, Abdellaoui R, Yaghoubi Khanghahi M, Crecchio C (2023) Quantifying plant biomass and seed production in camelina ( Camelina sativa (L.) Crantz) across a large range of plant densities: Modelling approaches. Annals of Applied Biology. 183(1):23-32. https://doi.org/10.1111/aab.12830 Bhat JG, Murthy HN (2007) Factors affecting in vitro gynogenic haploid production in niger ( Guizotia abyssinica (L. f.) Cass.). Plant growth regulation. 52:241-248. https://doi.org/10.1007/s10725-007-9196-9 Bhatia R, Dey S S, Sood S, Sharma K, Parkash C, Kumar R (2017) Efficient microspore embryogenesis in cauliflower ( Brassica oleracea var. botrytis L.) for development of plants with different ploidy level and their use in a breeding programme. Scientia Horticulture 216:83–92. https://doi.org/10.1016/j. scienta.2016.12.020 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. Sci Hort 233:178–187. https://doi.org/10.1016/j. scienta.2018.01.017 Bhatia R, Dey SS, Sood S, Sharma K, Sharma VK, Parkash C, Kumar R (2016) Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower ( B. oleracea var. botrytis L.) in India. Euphytica. 212:439-54. https://doi.org/10.1007/s10681-016-1775-2 Blasco M, Badenes ML, del Mar Naval M (2016) Induced parthenogenesis by gamma-irradiated pollen in loquat for haploid production. Breeding science. 66(4):606-12. https://doi.org/10.1270/jsbbs.16021 Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. American journal of botany. 50(9):859-65. https://doi.org/10.2307/2439772 Cuny F, Grotte M, De Vaulx RD, Rieu A (1993) Effects of gamma irradiation of pollen on parthenogenetic haploid production in muskmelon ( Cucumis melo L.). Environmental and Experimental Botany. 33(2):301-12. https://doi.org/10.1016/0098-8472(93)90076-r Dash M, Mishra A (2024) QTL Mapping: Principle, Approaches, and applications in crop improvement. In Smart Breeding. Apple Academic Press.31-60. https://doi.org/10.1201/9781003361862-2 Doré C, Prigent J, Desprez B (1996) In situ gynogenetic haploid plants of chicory ( Cichorium intybus L.) after intergeneric hybridization with Cicerbita alpina Walbr. Plant cell reports. 15(10):758-761. https://doi.org/10.1007/bf00232223 Falque M (1994) Pod and seed development and phenotype of the M1 plants after pollination and fertilization with irradiated pollen in cacao ( Theobroma cacao L.). Euphytica. 75(1):19-25. https://doi.org/10.1007/bf00024527 Froelicher Y, Bassene JB, Jedidi-Neji E, Dambier D, Morillon R, Bernardini G, Costantino G, Ollitrault P (2007) Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. Plant Cell Reports. 26:937-44. https://doi.org/10.1007/s00299-007-0314-y Godbole M, Murthy HN (2012) Parthenogenetic haploid plants using gamma irradiated pollen in snapmelon ( Cucumis melo var. momordica ). Plant Cell, Tissue and Organ Culture (PCTOC). 109:167-70. https://doi.org/10.1007/s11240-011-0066-9 Grouh MS, Sousaraei N, Akbari M, Rahimi V, Bayat H (2015) Induction of haploid plants in iris ( Iris pseudacorus ) by pollen irradiation. Turkish Journal of Agriculture and Forestry. 39(4):596-600. https://doi.org/10.3906/tar-1407-18 Keleş D, Özcan C, Pınar H, Ata A, Denli N, Yücel NK, Taşkın H, Büyükalaca S. (2016) First report of obtaining haploid plants using tissue culture techniques in spinach. HortScience. 51(6):742-9. https://doi.org/10.21273/hortsci.51.6.742 Kumar KR, Singh KP, Bhatia R, Raju DV, Panwar S (2019) Optimising protocol for successful development of haploids in marigold ( Tagetes spp.) through in vitro androgenesis. Plant Cell, Tissue and Organ Culture (PCTOC). 138:11-28. https://doi.org/10.1007/s11240-019-01598-3 Kumar KR, Singh KP, Raju DV, Bhatia R, Panwar S (2020) Maternal haploid induction in African marigold ( Tagetes erecta L.) through in vitro culture of unfertilized ovules. Plant Cell, Tissue and Organ Culture (PCTOC). 143:549-64. https://doi.org/10.1007/s11240-020-01940-0 Kundu M, Dubey AK (2022) Anatomical studies of In vitro raised haploid and diploid citrus plants generated through gamma irradiated pollen. Indian Journal of Horticulture. 79(2):136-44. https://doi.org/10.5958/0974-0112.2022.00019.6 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 and Organ Culture (PCTOC). 102:267-77. https://doi.org/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). African Journal of Biotechnology. 8(21):5944-51. https://doi.org/10.5897/ajb09.730 Kurtar ES, Metin D, Atakul Z, Kal U, Seymen M, Takcı E, Yılmaz HN, Alan AR, Toprak FC (2025) Effects of gamma ray sources and irradiation doses on the haploidization frequency of citron watermelon ( Citrullus lanatus var. citroides ). Plant Cell, Tissue and Organ Culture (PCTOC), 160(3), p.62. https://doi.org/10.1007/s11240-025-03004-7 Li F, Cheng Y, Zhao X, Yu R, Li H, Wang L, Li S, Shan Q (2020) Haploid induction via unpollinated ovule culture in Gerbera hybrida . Scientific Reports. 10(1):1702. https://doi.org/10.1038/s41598-020-58552-z Liu H, Wang Y, Liang C, Yang Q, Wang S, Wang B, Zhang F, Zhang L, Cheng H, Song S, Zhang L (2020) Utilization of marigold ( Tagetes erecta ) flower fermentation wastewater as a fertilizer and its effect on microbial community structure in maize rhizosphere and non-rhizosphere soil. Biotechnology & Biotechnological Equipment. 34(1):522-31. https://doi.org/10.1080/13102818.2020.1781548 Meynet J, Barrade R, Duclos A, Siadous R (1994) Dihaploid plants of roses ( Rosa x hybrida , cv'Sonia') obtained by parthenogenesis induced using irradiated pollen and in vitro culture of immature seeds. Agronomie. 14(3):169-75. https://doi.org/10.1051/agro:19940303 Miler N, Tymoszuk A, Rewers M, Kulus D (2023) In vitro regeneration of chrysanthemum from ovaries and ovules treated with thermal and chemical stimuli: morphogenic and cytogenetic effects. Agriculture. 13(11):2069. https://doi.org/10.3390/agriculture13112069 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia plantarum. 15(3). https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Murovec J, Bohanec B (2013) Haploid induction in Mimulus aurantiacus Curtis obtained by pollination with gamma irradiated pollen. Scientia horticulturae. 162:218-225. https://doi.org/10.1016/j.scienta.2013.08.012 Oberle GD, Watson RI (1953) The use of 2, 3, 5-triphenyl tetrazolium chloride in viability tests of fruit pollens. Journal of the American Society for Horticultural Science. 61: 299–303. https://doi.org/10.1093/ajcp/23.5_ts.512 Pacini E, Franchi GG (2020) Pollen biodiversity–why are pollen grains different despite having the same function? A review. Botanical Journal of the Linnean Society. 193(2):141-64. https://doi.org/10.1093/botlinnean/boaa014 Palvaneh K, Ghamari Zare A, Lotfi M, Naraghi TS, Asadi-Corom F, Tabaei-Aghdaei SR (2013) Investigation of gamma ray effects on pollen germination and parthenogenic haploid embryogenesis in Rosa damascena Mill. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research. 21(1):45-55. Pandey KK, Phung M (1982) ‘Hertwig effect’ in plants: induced parthenogenesis through the use of irradiated pollen. Theoretical and Applied Genetics. 62:295-300. https://doi.org/10.1007/bf00275088 Piosik Ł, Zenkteler E, Zenkteler M (2016) Development of haploid embryos and plants of Lactuca sativa induced by distant pollination with Helianthus annuus and H. tuberosus . Euphytica. 208(3):439-751. https://doi.org/10.1007/s10681-015-1578-x Pradeepkumara N, Dey SS, Munshi AD, Behera TK, Bhatia R, Kumari K, Prakash P, Bhattacharya RC, Talukdar A (2023) Cucumber F1 hybrid derived from two contrasting inbreds ensures high frequency gynogenesis for induction of haploids through a modified in vitro based protocol. South African Journal of Botany. 157(1):314-24. https://doi.org/10.1016/j.sajb.2023.03.066 Salehian H, Shahnazi S, Nazari M (2023) Production of doubled haploid plants in cucumber ( Cucumis sativus L.) via parthenogenesis. In Vitro Cellular & Developmental Biology-Plant. 59(4):467-74. https://doi.org/10.1007/s11627-023-10368-y Sato S, Katoh N, Yoshida H, Iwai S, Hagimori M (2000) Production of doubled haploid plants of carnation ( Dianthus caryophyllus L.) by pseudofertilized ovule culture. Scientia horticulturae. 83(3-4):301-310. https://doi.org/10.1016/s0304-4238(99)00090-4 Schaart JG, van de Wiel CC, Smulders MJ (2021) Genome editing of polyploid crops: prospects, achievements and bottlenecks. Transgenic Research. 30(4):337-51. https://doi.org/10.1007/s11248-021-00251-0 Scott MF, Ladejobi O, Amer S, Bentley AR, Biernaskie J, Boden SA, Clark M, Dell’Acqua M, Dixon LE, Filippi CV, Fradgley N (2020) Multi-parent populations in crops: a toolbox integrating genomics and genetic mapping with breeding. Heredity. 125(6):396-416. https://doi.org/10.1038/s41437-020-0336-6 Sukwiwat K, Kumchai J, Bundithya W, Potapohn N (2023) Apetaloid and petaloid female performance on horticultural characteristics of F1 American marigold ( Tagetes erecta L.) hybrids. SABRAO J. Breed. Genet. 55(5):1754-67. https://doi.org/10.54910/sabrao2023.55.5.27 Todorova M, Ivanov P, Shindrova P, Christov M, Ivanova I (1997) Doubled haploid production of sunflower ( Helianthus annuus L.) through irradiated pollen-induced parthenogenesis. Euphytica. 97(3):249-54. https://doi.org/10.1023/a:1002966824988 Todorova MI, Ivanov PE (2000) Induced parthenogenesis in sunflower ( Helianthus annuus L.): Effect of gamma-irradiation doses. In: Proc of the 15th International Sunflower Conference, Toulouse, France, p. 46-51. https://doi.org/10.2298/hel0441067t Tran LT, Sugimoto K, Kasozi M, Mitalo OW, Ezura H (2023) Pollination, pollen tube growth, and fertilization independently contribute to fruit set and development in tomato. Frontiers in Plant Science. 14:1205816. https://doi.org/10.3389/fpls.2023.1205816 Tütüncü M, Mendi YY (2022) Effect of pollination with gamma irradiated pollen on in vitro regeneration of ovule culture in Cyclamen. Turkish Journal of Agriculture-Food Science and Technology. 10(12):2415-20. https://doi.org/10.24925/turjaf.v10i12.2415-2420.5409 Ulukapi K, Ozmen SF (2018) Study of the effect of irradiation ( 60 Co) on M1 plants of common bean ( Phaseolus vulgaris L.) cultivars and determined of proper doses for mutation breeding. Journal of Radiation Research and Applied Sciences. 11(2):157-61. https://doi.org/10.1016/j.jrras.2017.12.004 Valdez-Baro O, Jiménez-Ortega LA, Cabanillas-Bojórquez LÁ, Gutiérrez-Grijalva EP, Heredia JB (2024) Food byproducts as potential functional ingredients for animal feeding. In: Food byproducts management and their utilization. Apple Academic Press, p 417-452. https://doi.org/10.1201/9781003377801-20 Wang H, Hou H, Jan CC, Chao WS (2023) Irradiated pollen-induced parthenogenesis for doubled haploid production in sunflowers ( Helianthus spp.). Plants. 12(13):2430. https://doi.org/10.3390/plants12132430 Zhang H, Lina SO, Lifang LI, Haibo XI, Rongfeng CU, Zijing LI, Shiwei ZH, Zunzheng WE (2022) Interspecific hybridization with African marigold ( Tagetes erecta ) can improve flower-related performance in French marigold ( T. patula ). Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 50(4):12808-. https://doi.org/10.15835/nbha50312808 Zhao Q, Zhang M, Qi H, Yang X (2023) Haploid induction and plant production in bottle gourd by pollination with gamma irradiated pollen. Plant Cell, Tissue and Organ Culture (PCTOC). 152(2):331-8. https://doi.org/10.1007/s11240-022-02410-5 Tables Table 1 Effect of different doses of gamma irradiation on direct and indirect regeneration response from pseudo-fertilized ovaries. Table 2 Ploidy estimation of in vitro regenerated plants of DAMH-24 x PD and DAMH-55 x PD by different indirect and direct methods. Supplementary Files Graphicalabstract.png Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Editorial decision: Minor revisions 25 Jul, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviewers invited by journal 17 Jun, 2025 Editor assigned by journal 22 May, 2025 First submitted to journal 19 May, 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-6683915","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472403024,"identity":"27ba09f6-eb71-4cbe-8d75-0f76fa6a84d5","order_by":0,"name":"Eram Arzoo","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Eram","middleName":"","lastName":"Arzoo","suffix":""},{"id":472403025,"identity":"abfddf13-addf-4867-9531-d4a1eecbdeca","order_by":1,"name":"Kavita Dubey","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Kavita","middleName":"","lastName":"Dubey","suffix":""},{"id":472403026,"identity":"9ef9df90-651f-4adb-a719-407b376c7e82","order_by":2,"name":"Rashmi Arora","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Rashmi","middleName":"","lastName":"Arora","suffix":""},{"id":472403027,"identity":"31d84641-fbd9-4baf-b5b5-028f7c6a6f68","order_by":3,"name":"Kanwar Pal Singh","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Kanwar","middleName":"Pal","lastName":"Singh","suffix":""},{"id":472403028,"identity":"a0a631e3-4f07-4d68-8bbc-64074d11c77f","order_by":4,"name":"Sapna Panwar","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sapna","middleName":"","lastName":"Panwar","suffix":""},{"id":472403029,"identity":"d0ee2f98-95a4-429e-9203-48fbd8ece7fd","order_by":5,"name":"Akshay Talukdar","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Akshay","middleName":"","lastName":"Talukdar","suffix":""},{"id":472403030,"identity":"7e74cb0d-0b5b-489d-b41c-7c252e2beecb","order_by":6,"name":"Bhupinder Singh","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Bhupinder","middleName":"","lastName":"Singh","suffix":""},{"id":472403031,"identity":"fb7a5b96-f273-4d81-92ef-cc51e6989eff","order_by":7,"name":"Mahesh Rao","email":"","orcid":"","institution":"ICAR - National Institute for Plant Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Mahesh","middleName":"","lastName":"Rao","suffix":""},{"id":472403032,"identity":"cd285c80-0dd6-4a68-b8b5-72bbaa7b354e","order_by":8,"name":"Mariyam Firdous","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Mariyam","middleName":"","lastName":"Firdous","suffix":""},{"id":472403033,"identity":"ed6ff57f-5fd3-440b-a1bb-499ab19fe3e0","order_by":9,"name":"Bharti Bharti","email":"","orcid":"","institution":"Indian Agricultural Statistics Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Bharti","middleName":"","lastName":"Bharti","suffix":""},{"id":472403034,"identity":"5d0fb31e-2a8a-44da-9101-f29b43a0ee9a","order_by":10,"name":"Reeta Bhatia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYNACAwYGNvYGBmZi1TM2gLXwHCBJCwhIJBCpxby9+fiDHwV2+XySbww/F1TYMPC3dyfg1SJz5lhiY49BsmWbdI6x9IwzaQwSZ85uwKtFQiLHsIHHgNmATTrHQJq37TCDgUQuAS3y7z82/jGoN2CTPGP8mzgtEjyMzTwGhw3YJHjMiLSFJ81wtozBcQM2nrQya54zaTyE/cJ++MHHN3+qDeTbD2++zVNhI8ff3otfCxLgMACRPMQqBwH2B6SoHgWjYBSMghEEAPDuPcvJxFTZAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0668-6272","institution":"Indian Agricultural Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Reeta","middleName":"","lastName":"Bhatia","suffix":""}],"badges":[],"createdAt":"2025-05-17 01:31:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6683915/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6683915/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03201-4","type":"published","date":"2025-09-24T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84967725,"identity":"da420650-1fba-4c18-9a49-8de62d173e4a","added_by":"auto","created_at":"2025-06-19 10:09:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":361713,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent methods of ploidy determination.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/73332a03a851592f5353a05a.png"},{"id":84968072,"identity":"d7709ef1-5d81-43c8-a643-57e4662e6e0c","added_by":"auto","created_at":"2025-06-19 10:17:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":366623,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gamma irradiation on pollen viability and fertilisation. Female parents: (\u003cstrong\u003ea) \u003c/strong\u003eDAMH-24, \u003cstrong\u003eb \u003c/strong\u003eDAMH-55 and pollen parent (\u003cstrong\u003ec) \u003c/strong\u003ePusa Deep. (\u003cstrong\u003ed) \u003c/strong\u003ePollen study:\u003cstrong\u003e \u003c/strong\u003eviable pollen (LHS) - (\u003cstrong\u003ei) \u003c/strong\u003ered stained (Tetrazolium chloride), and (\u003cstrong\u003eii) \u003c/strong\u003ezed blue stain (Potassium Iodide), while non-viable pollen (RHS) (\u003cstrong\u003ei-ii)\u003c/strong\u003ewithout stain; (\u003cstrong\u003eiii) \u003c/strong\u003eviable germinated pollen. Fertilised and \u003cem\u003epseudo-fertilized \u003c/em\u003eovary 30 DAP:\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(e) \u003c/strong\u003eDAMH-24 × PD (0 Gy) and \u003cstrong\u003e(f)\u003c/strong\u003e DAMH-55 × PD (0 Gy), \u003cstrong\u003e(g) \u003c/strong\u003eDAMH-24 × PD (400 Gy) and \u003cstrong\u003e(h)\u003c/strong\u003e DAMH-55 × PD (400 Gy).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/28b6839d9215a279a41d9190.png"},{"id":84968612,"identity":"a1bfcb65-a210-4a3f-8cc3-21e8fc31fd22","added_by":"auto","created_at":"2025-06-19 10:25:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128313,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different doses of gamma irradiation on \u003cem\u003ein vitro \u003c/em\u003epollen staining and pollen viability in \u003cem\u003eTagetes patula \u003c/em\u003ecv. Pusa Deep.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/21f4c6a9a48cdb7ae831d0d5.png"},{"id":84967706,"identity":"4ffb29ee-032e-4394-a11c-2cc9014c6291","added_by":"auto","created_at":"2025-06-19 10:09:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60044,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different doses of gamma irradiation on \u003cem\u003ein vitro \u003c/em\u003epollen germination in \u003cem\u003eTagetes patula \u003c/em\u003ecv. Pusa Deep.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/cbf50566dadfd5b5c51dcb28.png"},{"id":84968871,"identity":"fa4f8ed0-8d3a-4f2a-8955-10b4d4e3db15","added_by":"auto","created_at":"2025-06-19 10:33:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100962,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different doses of gamma irradiation on fertilisation/ pseudo fertilisation in other crosses.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/4b67c22c5fbcc027de798b06.png"},{"id":84967723,"identity":"b9f91646-2868-42ec-8be5-ccb1790237a3","added_by":"auto","created_at":"2025-06-19 10:09:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":796105,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gamma irradiation on\u003cstrong\u003e \u003c/strong\u003eDAMH-24 x PD (direct regeneration from \u003cem\u003ein vitro\u003c/em\u003ecultured ovaries ) on pollination with different doses of γ irradiated pollen: \u003cstrong\u003e(a)\u003c/strong\u003e healthy ovary on the day of culture; \u003cstrong\u003e(b) \u003c/strong\u003eembryo emergence\u003cstrong\u003e \u003c/strong\u003e5 days after culture\u003cstrong\u003e (\u003c/strong\u003e100 Gy); (\u003cstrong\u003ec) \u003c/strong\u003eembryo emergence from\u003cstrong\u003e the \u003c/strong\u003e\u003cem\u003epseudo-fertilised\u003c/em\u003eovary (400 Gy),\u003cstrong\u003e (d) \u003c/strong\u003edried/degenerated ovary (500 Gy); e-j represents direct regeneration with true cotyledonary leaves: 200 Gy (\u003cstrong\u003ee \u0026amp; h)\u003c/strong\u003e; 400 Gy (\u003cstrong\u003ef \u0026amp;i)\u003c/strong\u003e; 500 Gy (\u003cstrong\u003eg \u0026amp; j)\u003c/strong\u003e; \u003cstrong\u003ek- n\u003c/strong\u003edepicted the regenerated plants induced via parthenogenesis: (\u003cstrong\u003ek) \u003c/strong\u003e20 days old shoots (200 Gy); (\u003cstrong\u003em) \u003c/strong\u003e40 days old shoors (200 Gy); (\u003cstrong\u003el) \u003c/strong\u003e20 days old shoots (400 Gy), (\u003cstrong\u003en) \u003c/strong\u003e40 days old shoots (400 Gy).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/fc2a9b961d3939f0cbf6fac2.png"},{"id":84967714,"identity":"d3377321-8dda-4913-8bc3-b50a10efcb8b","added_by":"auto","created_at":"2025-06-19 10:09:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":802955,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gamma irradiation on\u003cstrong\u003e \u003c/strong\u003eDAMH-55 x PD (indirect regeneration/Callogenic embryogenesis from \u003cem\u003ein vitro\u003c/em\u003e cultured ovaries) on pollination with different doses of γ irradiated pollen: (\u003cstrong\u003ea)\u003c/strong\u003e healthy ovary on the day of culture; (\u003cstrong\u003eb) \u0026amp; (e) \u003c/strong\u003eembryogenic callus induction from ovaries (100 Gy ); (\u003cstrong\u003ec) \u0026amp; (f) \u003c/strong\u003ehealthy vigorous embryos (400 Gy),\u003cstrong\u003e (d) \u0026amp; (g) \u003c/strong\u003edryed/degenerated embryo at higher gamma irradiation (500 Gy); (\u003cstrong\u003eh) \u003c/strong\u003eglobular shaped embryo (300 Gy), (\u003cstrong\u003ei) \u003c/strong\u003eheart shape embryo (400 Gy), (\u003cstrong\u003ej) \u003c/strong\u003eembryo with mutated orange coloured shoot (500 Gy); \u003cstrong\u003e(k-n) \u003c/strong\u003eCotyledonary embryo formation from ovary: (\u003cstrong\u003ek)\u003c/strong\u003e 200 Gy and\u003cstrong\u003e (l)\u003c/strong\u003e 400 Gy; (\u003cstrong\u003em-n)\u003c/strong\u003e Proliferated Shoot from parthenogenically induced ovaries (40 DAI): (\u003cstrong\u003em) \u003c/strong\u003e200 Gy, and (\u003cstrong\u003en) \u003c/strong\u003e400 Gy.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/65eb1097de28eee952e99a8a.png"},{"id":84967729,"identity":"7b190b41-d022-49e2-a5f0-6205e741c22b","added_by":"auto","created_at":"2025-06-19 10:09:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":107672,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different doses of gamma irradiation on the number of days for visible response in \u003cem\u003epseudo-fertilized \u003c/em\u003eovaries.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/357db03cd60eb176ac74cf0c.png"},{"id":84968080,"identity":"e747eb8b-f8e7-49c5-b46f-081d10995e8a","added_by":"auto","created_at":"2025-06-19 10:17:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":124575,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different doses of gamma irradiation on \u003cem\u003ein vitro\u003c/em\u003e response of \u003cem\u003epseudo-fertilized \u003c/em\u003eovaries.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/e71b625c45b41a282e8b8d50.png"},{"id":84967741,"identity":"83085885-716f-4abf-bd39-d94e247cd20b","added_by":"auto","created_at":"2025-06-19 10:09:05","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":246983,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation among different variables and doses of gamma irradiation from \u003cem\u003ein vitro \u003c/em\u003ecultured \u003cem\u003epseudo-fertilized \u003c/em\u003eovaries.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/c137ddbc59652fd6eabc8ea5.png"},{"id":84967718,"identity":"1719a191-6da5-4526-b1df-429b22e63822","added_by":"auto","created_at":"2025-06-19 10:09:04","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":498125,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the ploidy level of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003ein vitro\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003einduced regenerated haploid (a-d; \u003cem\u003epseudo-fertilized\u003c/em\u003eparthenogenically induced), doubled haploid (e-h) and triploid (i-l) plants of DAMH-24 x PD on pollination with different doses of γ irradiated pollen. Here, (a), (e) \u0026amp; (i) shows morphological; (b), (f) \u0026amp; (j) show chloroplast count; (c), (g) \u0026amp; (k) show chromosomal count; and (d), (h) \u0026amp; (l) depicted flow cytometry analysis of haploid, doubled haploid as well as triploid respectively.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/e85a6a65a6241dc75dfdf677.png"},{"id":84968613,"identity":"68f87d82-38ee-4df4-915b-ecb54ecab5a6","added_by":"auto","created_at":"2025-06-19 10:25:04","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":67050,"visible":true,"origin":"","legend":"\u003cp\u003ePie chart showing the overall percentage of regenerated plants. Out of 52 plants, 15.38% were haploids, 51.92% were diploids, and 32.69% were triploids.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/14984837474a679931321edd.png"},{"id":92431189,"identity":"d63ed83e-fbbb-4486-a6db-d68ef31c00c1","added_by":"auto","created_at":"2025-09-29 16:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5111890,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/0f0afb41-df4e-492a-8da3-8371ad2f48ae.pdf"},{"id":84968075,"identity":"6d7ef30a-c956-4cb7-a3ef-420f4dd75526","added_by":"auto","created_at":"2025-06-19 10:17:05","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":623226,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6683915/v1/d3a284e96d02b1775bf01d25.png"}],"financialInterests":"","formattedTitle":"Efficient induction of parthenogenetic haploids in African marigold (Tagetes erecta) L.) using Co60 gamma-irradiated pollen of French Marigold (Tagetes patula L.)","fulltext":[{"header":"Key message","content":"\u003cp\u003eThis study presents the first successful induction of haploids in \u003cem\u003eTagetes erecta\u003c/em\u003e via induced parthenogenesis using \u0026gamma;-irradiated pollen from the tetraploid species \u003cem\u003eTagetes patula\u003c/em\u003e.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eA haploid is defined as a plant that has a gametic number of chromosomes (n). The haploids are highly valuable for genetic and evolutionary studies and play a crucial role in crop improvement programmes like mutation, hybridisation, and transgenic development. The diploidisation of haploids leads to the complete fixation homozygosity in a single step, unlike traditional breeding approaches, and results in the production of doubled haploids (DHs). The DH, being considered an immortal population, is crucial for hybrid breeding (Pradeepkumara et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and can be utilised for various genetic and genomic research (Schaart et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), QTLs analysis (Dash and Mishra \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and linkage mapping studies and genome sequencing (Scott et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMarigold (\u003cem\u003eTagetes sp\u003c/em\u003e.), a member of the Asteraceae family, is the leading flower crop grown in India and holds great significance in the livelihood security of small and marginal farmers. The genus \u003cem\u003eTagetes\u003c/em\u003e, a native to Mexico, comprises around 33 species, out of which only two species, namely, \u003cem\u003eT. erecta\u003c/em\u003e L. (African marigold; 2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24) and \u003cem\u003eT. patula\u003c/em\u003e L. (French marigold; 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48) are commercially cultivated all over the world. In India, marigold rank first both in area (66,130 ha) and production (603180 T), occupying 21.7% of the total area and 19.7% of the total production under flower crops (Anonymous \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Besides its aesthetic importance, \u003cem\u003eTagetes erecta\u003c/em\u003e L. is a high-value industrial crop for the extraction of carotene, especially lutein, which is used as an eye tonic for healthy vision. The marigold petals are also valued as poultry feed for intensifying the colour of egg yolk and broiler skin (Valdez-Baro et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The marigold crop is considered a viable option in sustainable agriculture owing to its role in controlling the nematode population. The allelopathic effect of chemical alpha-terthienyl, present in marigold, keeps the population of root-knot nematode under control when it is planted as an intercrop (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOwing to the industrial importance of marigold, the demand for F\u003csub\u003e1\u003c/sub\u003e hybrids is immensely rising throughout the world. The F\u003csub\u003e1\u003c/sub\u003e hybrids in marigold possess novel traits, show tolerance to biotic and abiotic stresses and give higher returns to the farmers. In India, the indigenous F\u003csub\u003e1\u003c/sub\u003e hybrids are of meagre occurrence, and a significant amount of revenue is spent every year on the import of hybrid seeds. Recently, in India, institutes have taken up the systematic hybrid breeding program of marigold to meet the ever-increasing demands of farmers for hybrid seeds. Homozygous parental lines are crucial for hybrid seed production in any crop. Conventional breeding approaches require 6\u0026ndash;8 generations of selfing to develop a completely homozygous line in this highly cross-pollinated crop. The several agricultural homozygosity can be fixed in a single generation within a tenure of ~\u0026thinsp;1 year using the modern biotechnological approach based on doubled haploid. The induction of haploids via \u003cem\u003ein vitro\u003c/em\u003e androgenesis (\u003cem\u003evia\u003c/em\u003e. microspore or anther culture) or gynogenesis (\u003cem\u003evia\u003c/em\u003e. unpollinated ovule/ovary culture) or induced parthenogenesis (\u003cem\u003evia. c\u003c/em\u003eulture of ovaries after pollination) and their subsequent diploidisation to create doubled haploids (DHs) offer a viable option for the rapid development of homozygous lines in marigold. \u003cem\u003eIn vitro androgenesis is a\u003c/em\u003e well-established technique in marigold for the development of DHs in marigold (Kumar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, in the petaloid male sterile marigold hybrids that completely lack male reproductive organs, i.e. anthers (Sukwiwat et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the maternal haploid induction through culture of female gametophyte is the only viable alternative. Using female gametophytes, the haploids can be induced through unpollinated ovary culture (\u003cem\u003ein vitro\u003c/em\u003e gynogenesis; Kumar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and pollinated ovary culture approach (induced parthenogenesis).\u003c/p\u003e \u003cp\u003eIn pollinated ovary culture technique, the maternal haploid can be induced by either pollination with irradiated/inactive pollen or pollination with the pollen of distant-related species. The irradiated pollen mostly contains inactivated sperm cells; however, they retain the ability to produce standard pollen tubes that can penetrate ovules and eventually result in parthenocarpic seed development and haploid embryo induction (Tran et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For successful haploid induction through induced parthenogenesis, ionising irradiations (X-rays and gamma rays) can be used. However, the application of gamma rays is more common because of their greater tissue penetration, less lethality, high mutation rate, and convenience (Kurtar and Balkaya \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The generative nuclei inside pollen degenerate due to high energy of irradiations and are unable to undergo mitosis-II to produce two sperm nuclei and thus inhibit double fertilisation. This leads to \u003cem\u003epseudo-fertilization\u003c/em\u003e as male gametes are eliminated from participating in the fertilisation process and stimulate haploid embryo induction through induced parthenogenesis. The irradiated pollen can provide a stimulus to the egg cell to induce parthenogenetic haploids. The dose of irradiation is crucial in induced parthenogenesis; the inactivation rate of the generative nuclei increases with the increase in doses of radiation and thus promotes more parthenogenic embryo development and haploid induction (Aktas et al., 2023).\u003c/p\u003e \u003cp\u003eThe successful induction of maternal haploids in \u003cem\u003eTagetes erecta\u003c/em\u003e L. using an unfertilised/unpollinated ovary culture (\u003cem\u003ein vitro gynogenesis\u003c/em\u003e) approach has been described by Kumar et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the Asteraceae family, \u003cem\u003ein vitro\u003c/em\u003e gynogenesis approach has been successfully utilised for haploid induction in sunflower (Aktas et al. 2023), gerbera (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), niger (Bhat and Murthy \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and chrysanthemum (Miler et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In most of these reports, very low haploid induction frequencies were observed. The induced parthenogenesis technique exploiting irradiated pollen has been efficiently used to improve the haploid induction frequency in several horticultural crops; however, to date, the potential of this approach has remained unexplored in \u003cem\u003eTagetes erecta\u003c/em\u003e L. In the Asteraceae family, γ-irradiated pollen of distantly related species/genera have been utilised to induce parthenogenetic haploids in \u003cem\u003eChichorium\u003c/em\u003e x \u003cem\u003eLactuca, Chichorium\u003c/em\u003e x \u003cem\u003eCicerbita\u003c/em\u003e (Dore et al. 1996), and \u003cem\u003eLactuca\u003c/em\u003e x \u003cem\u003eHelianthus\u003c/em\u003e (Piosik 2016). In \u003cem\u003eIris pseudacorus\u003c/em\u003e, parthenogenetic haploids were successfully induced by utilising the X ray-irradiated pollen of \u003cem\u003eI. spuria\u003c/em\u003e. Induced parthenogenesis through exploiting irradiated pollen of the same species has been successfully used in \u003cem\u003eHelianthus annuus\u003c/em\u003e L. (Todorova et al. in 1997; Todorova and Ivanov \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Aktas et al. 2018; Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), cyclamen (Tutuncu and Mendi 2022), \u003cem\u003eRosa damascena\u003c/em\u003e Mill. (Palvaneh et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eRosa\u003c/em\u003e x \u003cem\u003ehybrida\u003c/em\u003e (Meynet et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), \u003cem\u003eDianthus caryophyllus\u003c/em\u003e L. (Sato et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and \u003cem\u003eMimulus aurantiacus\u003c/em\u003e Curtis (Murovec and Bohanec \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, no research has been undertaken on the use of the induced parthenogenesis approach for haploid induction in the genus \u003cem\u003eTagetes.\u003c/em\u003e Hence, we have conducted an in-depth study on the role of different gamma irradiation doses on pollen viability, germination, \u003cem\u003epseudo-fertilization and\u003c/em\u003e haploid induction in \u003cem\u003eT. erecta L.\u003c/em\u003e This is the first report in marigold where the gamma-irradiated pollen of a tetraploid French marigold was used for the induction of parthenogenetic haploids in diploid African marigold.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe primary goal of this work was to assess the effectiveness of gamma irradiation on haploid induction frequency in \u003cem\u003eTagetes erecta\u003c/em\u003e L. It was hypothesised that irradiated pollen loses its capability of fertilisation, which may be because mutation leads to disorganisation of the nuclei as well as breakage of the chromosome. Still, the pollen retains the ability to germinate until the lethal dose. The irradiated pollens trigger unfertilised egg cells to divide, and this leads to the development of haploid embryos by \u0026lsquo;direct regeneration\u0026rsquo;. Further, we have chosen the distant species as a pollen source to reduce the risk of fertilisation. We have irradiated the pollen of tetraploid species (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48) \u003cem\u003eT. patula\u003c/em\u003e L. with different doses of γ ray (100, 200, 300, 400 and 500 Gy) in order to optimise the irradiation dose for parthenogenic haploid development in diploid (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48) \u003cem\u003eTagetes erecta\u003c/em\u003e L.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and its raising\u003c/h2\u003e \u003cp\u003eTwo petaloid-type male sterile African marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e) genotypes, DAMH-24 (OPMS-1 \u0026times; AMS-3; dark orange) and DAMH-55 (YPMS-2 \u0026times; AMS-18; lemon yellow) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b) has been chosen to undertake haploid induction studies, while a French marigold (\u003cem\u003eTagetes patula\u003c/em\u003e L.) genotype, \u0026lsquo;Pusa Deep\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) was used as pollen parent for γ irradiation. The seeds of all three genotypes were sown at the research farm of the Division of Floriculture and Landscaping, ICAR - Indian Agricultural Research Institute (IARI), New Delhi, India. The seeds were initially sown in plug trays containing a mixture of cocopeat, perlite, and vermiculite (1:1:1) under net house conditions during the rainy seasons of 2021\u0026ndash;2022. After one month of sowing, the seedlings were transplanted to the open field conditions at the research farm of the Division of Floriculture and Landscaping, ICAR-IARI, New Delhi (28.08 \u003csup\u003e0\u003c/sup\u003eN and 77.12 \u003csup\u003e0\u003c/sup\u003eE at 228.61 m above mean sea level). All the good agricultural practices as prescribed for marigold crop by IARI were followed to raise the healthy crop. The plants were weekly sprayed with liquid fertilisers @ 3 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N: P: K\u0026thinsp;=\u0026thinsp;19:19:19) during the vegetative and @ 3 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N: P: K\u0026thinsp;=\u0026thinsp;13:0:45) during the flowering phase. No chemicals were used for plant protection in any form to avoid viability reduction of female gametophytes (Kumar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIrradiation of Flowers\u003c/h3\u003e\n\u003cp\u003eThe plants of the pollen parent, \u0026lsquo;Pusa Deep,\u0026rsquo; were bagged at the initiation of flowering to avoid pollen contamination. In the morning hour (9.00 am to 10.00 am), the bagged flowers of \u0026lsquo;Pusa Deep\u0026rsquo; were collected one day before anther dehiscence and placed in small transparent polypropylene boxes. Each box containing 6\u0026ndash;8 flowers was irradiated with different doses of γ rays, 100, 200, 300, 400, and 500Gy, along with control (0 Gy) at the Nuclear Research Laboratory (NRL), New Delhi, in γ chamber 5000 (GC-5000) using Co\u003csup\u003e60\u003c/sup\u003e isotope. After irradiation, the anthers from the irradiated flowers were allowed to dehisce in the laboratory. The irradiated flowers were placed in water-filled test tubes, and a translucent butter paper bag was placed on each test tube to maintain proper hydration. The dehisced pollen from four irradiated flowers, along with the control, were used for pollination of two petalous male sterile genotypes of African marigold, DAMH-24 and DAMH-55. Two irradiated flowers from each treatment, along with the control, were utilised for pollen study. This whole process was repeated three times in a season.\u003c/p\u003e\n\u003ch3\u003ePollen viability\u003c/h3\u003e\n\u003cp\u003eThe freshly dehisced anthers were utilised for pollen viability studies, which were done using two methods, \u003cem\u003ei.e.\u003c/em\u003e, pollen staining and germination. Pollen staining was estimated using two colourimetric tests, that is, 2, 3, 5, Triphenyl Tetrazolium Chloride (TTC) and Potassium Iodide (KI). The TTC stain was prepared by minor modification into black colour-stained pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed ii). It was considered viable\u0026mdash;the pollens, which were unable to take stain and remained yellow, were considered non-viable (Fig. (d iOberle and Watson's (1953) media, which included 1g TTC and 6g sucrose in 20 ml of distilled water, and the pH of the solution was adjusted to 5.7. At the same time, the Potassium iodide stain was prepared by dissolving 1g Potassium Iodide (KI) and 0.5g Iodine (I) in 100 ml of distilled water (Baker and Baker, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). The freshly dehisced pollen from the irradiated flowers (0, 100, 200, 300, 400, 500 Gy) were stained with TTC and KI solutions separately, and a cover slip was placed over them. For better absorption of the stain, the slides were kept in the dark for 30\u0026ndash;60 minutes and then observed under an inverted light microscope. Three replications of ~\u0026thinsp;40 pollen grains in each slide were observed (10X magnification). In the case of TTC, staining red-coloured pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed i) was considered viable, while in the case of KI reddish- blue, ii)). For \u003cem\u003ein-vitro\u003c/em\u003e pollen germination studies, Brewbaker and Kwack (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) media consisting of 20% sucrose, 100 ppm Boric acid, 300 ppm Calcium nitrate, 200 ppm Magnesium sulphate, 100 ppm Potassium nitrate, and 0.2% Agar (pH: 5.2\u0026ndash;5.8) was used. Pollen germination was estimated using the hanging drop method. In this method, the germinating media was placed over a coverslip, and pollens were dusted over it using a needle. The cover slip was then placed on a cavity slide sealed with petroleum jelly, and the slide was turned to hang the media over the coverslip. The slides were kept for incubation at 25 \u0026ordm;C under moist conditions for 12 hours for pollen germination. Three replications of ~\u0026thinsp;100 pollen grains in each slide were observed (4X magnification). Pollen was considered germinated when its tube length was doubled the size of its diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (d iii)).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePollination and\u003c/b\u003e \u003cb\u003ePseudo-fertilization\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe pollen of French marigold (\u003cem\u003eT. patula\u003c/em\u003e L.) genotype \u0026lsquo;Pusa Deep\u0026rsquo; irradiated with different doses of gamma irradiations was used to pollinate the petaloid type male sterile genotypes of African marigold \u0026lsquo;DAMH-24\u0026rsquo; and \u0026lsquo;DAMH-55\u0026rsquo;. The flower buds of African marigold genotypes DAMH-24 and DAMH-55 were covered with butter paper bags to prevent the chance of unwanted cross-pollination. The pollen from freshly dehiscence anthers of irradiated (0, 100, 200, 300, 400, 500 Gy) and non-irradiated (0 Gy) flowers of French marigold (\u003cem\u003eT. patula\u003c/em\u003e L.) genotype \u0026lsquo;Pusa Deep\u0026rsquo; were utilised to pollinate the male sterile flowers of \u0026lsquo;DAMH-24\u0026rsquo; and \u0026lsquo;DAMH-55\u0026rsquo;. A total of 60 flowers were pollinated in each genotype, \u003cem\u003ei.e.\u003c/em\u003e, each treatment had 10 flowers (total of 6 treatments), and the experiment was repeated three times a season. The pollination was performed during the daytime (11:00 am to 1:00 pm) when the stigmas of female flowers were fully elongated, bifurcated and receptive. As marigold inflorescence (capitulum) consists of many florets and stigmas of different rows, they become receptive on other days; therefore, the pollination was repeated 2 to 3 times during 2 to 3 days\u0026rsquo; time tenure. The irradiated pollen retains the ability to germinate on the stigmas. However, the capabilities of the male gametes to undergo fertilisation is significantly reduced, and hence, it promotes parthenogenic embryo development in pseudo-fertilized ovaries. The confirmation of fertilisation and \u003cem\u003epseudo-fertilization\u003c/em\u003e was done after 30 days of pollination (DAP). These pseudo-fertilized ovaries failed to develop under field conditions. Ovaries showing dark black/brown colour are considered fertilised (triploid), while those showing cream to light purplish colour are deemed to be \u003cem\u003epseudo-fertilized\u003c/em\u003e (haploid). The percent of ovaries showing different colours was assessed, which showed the effectiveness of γ irradiation towards \u003cem\u003epseudo-fertilization\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eembryo culture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the role of γ irradiations on gynogenesis efficiency, the \u003cem\u003epseudo-fertilized\u003c/em\u003e ovaries were cultured on standardised media. As the pseudo-fertilised ovaries lacked endosperm and failed to support the development of parthenogenetic-induced embryos, hence ovary culture was attempted to rescue the haploid embryos. The pollinated and partially matured flowers (25\u0026ndash;30 DAP) were plucked and sterilised as per the protocol described by Kumar et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For surface sterilisation, the flower was treated with 0.2% HgCl\u003csub\u003e2\u003c/sub\u003e for 5 min, followed by 4\u0026ndash;5 times with autoclaved double-distilled water to remove the traces of toxic remnants. Subsequently, the sterilised buds were dried on the autoclaved tissue papers. The \u003cem\u003epseudo-fertilized\u003c/em\u003e ovaries were carefully excised from the sterilised flowers with the help of forceps and inoculated on a standardised culture media containing MS, 0.75 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TDZ, 0.2 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, 0.5 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3\u003c/sub\u003e, 125 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PVP, 250 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Casein hydrolysate, 50mll\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Coconut water mix, 40 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Sucrose, 2.6 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Gelrite at 5.7 pH (Arzoo et al. 2022; Murashige and Skoog, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). A total of three Petri dishes, each containing\u0026thinsp;~\u0026thinsp;30 ovaries, were cultured in each treatment, and each treatment consisted of three replications. The cultured plates were sealed with parafilm followed by phyta-wrap to avoid contamination and maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C temperature in the dark. After the induction of optimum responses (swelling/bulging of ovaries or embryo emergence), plates were moved to light conditions in a photoperiod of 16:8 hours of light and dark cycles under fluorescent white light (47\u0026micro;mol/m\u0026sup2;/s).\u003c/p\u003e\n\u003ch3\u003ePlant regeneration, proliferation, and hardening\u003c/h3\u003e\n\u003cp\u003eThe direct regenerated embryos, as well as the embryogenic calli induced from pseudo fertilised ovaries, were transferred to MS medium supplemented with 0.25 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3,\u003c/sub\u003e 0.1mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, 0.2mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP, 125mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Casein hydrolysate, 125 mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PVP, 40 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Sucrose and 8 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eAgar for shoot formation and elongation. The regenerated micro-shoots were proliferated on MS medium supplemented with 0.25 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KIN, 0.25 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GA\u003csub\u003e3,\u003c/sub\u003e 0.1mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, 125 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PVP, 40 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Sucrose with 8 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eAgar and the well-developed shoots were transferred to \u0026frac12; MS medium supplemented with 0.5 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, 125 mgl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PVP, 45 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Sucrose with 8 gl\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Agar for rooting. All the cultures were maintained at the same temperature and light condition as the embryo. The well-rooted plantlets were gradually acclimatized in a plastic pot covered with polythene containing sterile coco peat, perlite and vermiculite mixture (3:1:1) under controlled conditions (Arzoo et al. 2022).\u003c/p\u003e\n\u003ch3\u003ePloidy determination\u003c/h3\u003e\n\u003cp\u003ePloidy levels of plants induced from ovaries were determined using direct (chromosome counting and flow cytometry) and indirect (chloroplast number of the guard cells and morphological observations) methods of ploidy evaluation. The flow diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below depicts all the four direct and indirect approaches used for ploidy determination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe experiments were laid out in a completely randomised design (CRD) to test the significance of the mean difference among treatments. The different treatments comprising irradiation doses (0, 100, 200, 300, 400, 500 Gy) in combination with genotypes were replicated three times. The percentage data were subjected to \u003cem\u003eArc Sin\u003c/em\u003e transformation. We had some data with zero (0) values, but for statistical analysis, we could not use zero values. Therefore, the values of the entire set of data were transformed through square root transformation. After the transformation of original values, statistical analysis was performed by using ANOVA. Further, the Karl Pearson correlation coefficient was computed by using the \u003cem\u003ecor()\u003c/em\u003e and \u003cem\u003ecorrplot mixed()\u003c/em\u003e function in \u0026ldquo;\u003cem\u003eR software\u003c/em\u003e\u0026rdquo; to study the relationship of irradiation doses with other variables under study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eIrradiated pollen induces parthenogenesis or apogamy and can also lead to efficient haploid induction in several economically important crops (Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). At a specific dose, the capabilities of the irradiated pollen (male gametophytes) to undergo fertilisation are significantly reduced. However, it retains its ability to provide stimulus on the stigma for fertilisation that leads to cell division as well as embryo development from egg cells. Hence, an effective dose of gamma irradiation can enhance the production of parthenogenic embryos rather than zygotic embryos and lead to the development of haploid plants (Tran et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eEffects of gamma irradiation on pollen viability and pollen germination\u003c/h3\u003e\n\u003cp\u003eThe pollen viability of irradiation and non-irradiation (control) flowers of \u003cem\u003eTagetes patula\u003c/em\u003e L. genotype \u0026lsquo;Pusa Deep\u0026rsquo; was assessed through staining and \u003cem\u003ein vitro\u003c/em\u003e germination tests. In both stains, the number of pollens obtaining stains reduced drastically when the exposure time to the irradiations (dose) was enhanced. In the Tetrazolium chloride (TTC) staining, the control (non-irradiated) pollen exhibited maximum staining, revealing maximum pollen viability. As the dose was increased from 100 to 200 Gy, there was a sharp decline (~\u0026thinsp;68 percent) in the percentage of pollen taking the stain, and only 18.34% of the pollen remained viable on exposure to 200 Gy. The percentage of pollen taking up stain reduced to below 5.0% when the flowers were exposed to 400 and 500 Gy doses of gamma irradiations, indicating the drastic reduction in pollen viability on exposure to higher doses of gamma irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, with Potassium Iodide (KI) staining the maximum pollen staining\u0026thinsp;~\u0026thinsp;95.0% was observed in non-irradiated pollen, which reduces to almost 60.0% on exposure to 100 Gy. The exposure of flowers to higher doses of gamma irradiations, i.e. 300 and 400 Gy, led to the reduction in pollen viability to as low as 5.0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A negative correlation between radioresistance and pollen viability was observed earlier by Guler \u003cem\u003eet al\u003c/em\u003e. (2017). In both TTC and KI stains, exposure of flowers to 400 Gy of gamma irradiation led to the maximum reduction in staining and hence resulted in the least viability of pollen (0.84% in TTC \u0026amp; 3.33% in KI); therefore, this dose can be considered as the optimum dose for parthenogenic haploid induction in marigold genotypes. Among different parts of a flower, pollen is regarded as the most sensitive to irradiation and reduction in pollen viability on exposure to irradiation can be attributed to pollen dehydration (Kundu and Dubey, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). One of the reasons behind this is that the treatment of pollen grains by ionising radiation (X-ray, γ-ray) may also cause mutations, breakage of the chromosomes and disorganisation of the nucleus (Froelicher et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe have also assessed the effects of gamma irradiations on \u003cem\u003ein vitro\u003c/em\u003e pollen germination of irradiated and non-irradiated (control) flowers of Pusa Deep. The maximum \u003cem\u003ein vitro\u003c/em\u003e pollen germination (71%) was found in the non-irradiated flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A sharp decline in pollen germination percentage was observed when gamma irradiation was applied to the flowers. A reduction of 85.0% germination was observed in pollen when flowers were subjected to 100Gy of gamma radiation (only 10.0% pollen germinated), while further increasing the irradiation doses to 300Gy reduced the germination to as low as 5.0%. The effects of irradiation on viability and \u003cem\u003ein vitro\u003c/em\u003e germination of pollen were earlier studied by Thaneshwari et al. (2019) in Marigold, who noticed that pollen treated with 300Gy gamma irradiations exhibited minimum pollen viability and pollen germination. Salehian et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also proved that with increasing doses of gamma rays, pollen viability, germination, and tube elongation of cucumber continuously decreased compared to non-irradiated pollen. Similar revelations were earlier proposed by Blasco et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in Loquat and Kurtar et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) in Pumpkin, where the pollen grains completely failed to germinate under \u003cem\u003ein vitro\u003c/em\u003e conditions when flowers were irradiated with 400Gy. Hence, this dose was considered as the desirable dose for gynogenic haploid induction in marigold. Similar to γ rays, X-rays also reduced the pollen viability and germination in \u003cem\u003eIris spuria\u003c/em\u003e at a comparable dose (Grouh et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The loss of viability and inability of the pollen to germinate on irradiation exposure is attributed to the loss of cytoplasmic fluid inside the pollen that keeps the generative cells wet and alive inside the pollen (Pacini and Franchi, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our experiments, we have also observed the difficulties in anther dehiscence on exposing the flowers to 500 Gy, signifying the sensitivity of marigold genotypes to irradiations. Meynet et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) have also revealed a similar problem with the higher dose of irradiations\u0026thinsp;~\u0026thinsp;500Gy dose in Rose. The exposure of flowers to higher doses of γ rays leads to pollen dehydration. Which in turn gives rise to poor germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The irradiated pollen technique can be effectively used to improve the haploid induction frequency. Still, before applying this technique on a commercial scale, the dose of γ irradiation that can inactivate the generative nuclei in a maximum number of pollen grains and thus reduce the pollen viability to as low as zero must be optimised.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffects of gamma irradiation on fertilisation\u003c/h2\u003e \u003cp\u003eThe fertilisation, as indicated by a change of the ovary colour to black, was severely hampered by irradiation. Pollination of African marigold genotypes DAMH-24 \u0026amp; DAMH-55 with the irradiated pollen of French marigold genotype \u0026lsquo;PD\u0026rsquo; exhibited only a slight change in the colour of the ovaries to purplish red, indicating resulted in \u003cem\u003epseudo-fertilization\u003c/em\u003e. The earlier studies have established the fact that the irradiated pollen retains its capability to germinate on the stigma and can grow within the style to reach the embryo sac. Still, it cannot fertilise the egg cell and the polar nuclei (Froelicher et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The irradiated pollen thus stimulates the division of the egg cell and apogamic/parthenogenic embryo development, which was evident from the change in the colour of the ovaries in the marigold. The full penetration of pollen tubes into the ovaries (even in the absence of fertilization) can sufficiently trigger seed sets (Tran et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Aslam et al. (2023) delineated that seeds produced in irradiated crosses were 45% lighter compared with seeds of control (non-irradiated pollen). Both colour change and weight loss of seed were because the irradiated pollen stimulated cell division from haploid cells of the embryo sac other than the zygote formation (diploid cell) and induced haploid embryos (Falque, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The maximum frequency of fertilisation (indicated by blackening of ovaries; 93.67%) was observed when the African marigold flowers were pollinated with the non-irradiated pollen of PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Pollination with 400 Gy irradiated pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) resulted in the lowest percentage of fertilised ovaries (1.83%). This treatment was found to be statistically similar to 500 Gy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similar observations have been noticed by Kundu and Dubey (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) in citrus on \u003cem\u003epseudo-fertilized\u003c/em\u003e seed development pattern when \u003cem\u003eC. grandis\u003c/em\u003e was pollinated with γ irradiated pollen (100\u0026ndash;500 Gy) of \u003cem\u003eC. limetta\u003c/em\u003e. They observed maximum \u003cem\u003epseudo fertilisation\u003c/em\u003e at 300 Gy dose. The degree of \u003cem\u003epseudo-fertilization\u003c/em\u003e was dependent on both the genotype and irradiation doses. The genotypes \u0026times; treatments interaction revealed that the minimum percentage (1.67%) of \u003cem\u003epseudo-fertilization\u003c/em\u003e was recorded in DAMH-24\u0026times;PD when pollinated with the 400 Gy. Thus, 400 Gy has been considered the optimum dose for induced parthenogenesis-mediated haploid production in marigold. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Godbole et al. (2012) also successfully induced parthenogenesis in Snapmelon with 250 Gy of γ-irradiation, whereas low (150 and 200 Gy) or high (300 and 350 Gy) irradiation doses were not found desirable for parthenogenesis. Contrary to our results, Wang et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) achieved the highest frequency of the parthenogenetic haploid induction in sunflower with a lower dose of irradiation, i.e. with 100 Gy. The irradiation doses for induced parthenogenesis were found to be highly crop and genotype-specific. The lower doses of γ irradiation were found effective for parthenogenic embryo development in citron watermelon (200 to 250 Gy; Kurtar et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), bottle gourd (100 to 125 Gy; Zhao et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and in Winter Squash (50 and 100 Gy; Kurtar and Balakaya, 2010). In Muskmelon, Cuny et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) noticed that a dose less than 1600 Gy was not adequate for \u003cem\u003epseudo-fertilisation\u003c/em\u003e, while pollen irradiated at 2500 Gy showed a maximum number of aborted/\u003cem\u003epseudo-fertilised\u003c/em\u003e fruits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of gamma irradiation on haploid induction through induced parthenogenesis\u003c/h2\u003e \u003cp\u003eThe irradiation doses significantly affected the response and embryo regeneration (direct or indirect) in both the genotypes of African marigold (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e \u0026amp; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The minimum days to visible response (swelling/bulging of ovaries and embryo emergence) was shown by ovaries pollinated with non-irradiated pollen, which may be due to ease in fertilisation and embryo development. In contrast, with the increase in irradiation doses, the days to visible response from the \u003cem\u003epseudo-fertilized\u003c/em\u003e ovaries increased significantly up to 300Gy. However, we observed a slight reduction in days to response at 400Gy, indicating higher efficiency of \u003cem\u003epseudo-fertilization\u003c/em\u003e. The maximum response from cultured ovaries (24%) was observed when African marigold genotypes were pollinated with non-irradiated pollen, while the minimum response was observed with 200 Gy dose (2.76%). We have observed 400Gy as the optimum dose for induced parthenogenesis in marigold. It led to the optimum pseudo-fertilization as reflected by the highest percentage of direct regeneration in the shortest possible time. Besides direct regeneration, we have also observed indirect regenerations (callusing). The maximum percent callusing was recorded with 0 Gy (control) and 100 Gy doses. Kundu et al. (2017) have revealed that pollination of \u003cem\u003eCitrus grandis\u003c/em\u003e with gamma-irradiated (300 and 400 Gy doses) pollen of C. limetta and C. sinensis (respective doses) stimulated the process of parthenogenesis and haploid induction. At the same time, doses below 300 Gy were found to be ineffective in inducing haploids in either of the cross combinations. A high irradiation dose of 500Gy was not found suitable for regeneration (either direct or indirect) because higher irradiation prevents parthenogenic or zygotic embryo formation. These results are supported by the findings of Salehian et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) in Sunflower, where they could not induce any haploids at 500 Gy because of higher penetration energy. They have standardised 300 Gy as an optimum dose for parthenogenic embryo development and haploid induction. The optimum dose of γ irradiation for parthenogenic haploid induction was found to vary among the flower crops: 250 Gy in \u003cem\u003eRosa damascena\u003c/em\u003e (Palvaneh et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), 300 Gy in Sunflower (Todorova et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), and 500 Gy in \u003cem\u003eRosa x hybrida\u003c/em\u003e (Meynet et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe number of induced embryos was significantly reduced with the increase in the dose of irradiations. The embryo response and regeneration were not only related to the irradiation doses but also to the genotypes. DAMH-24 x PD showed the maximum direct regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), while no direct regeneration and only indirect regeneration (callogenic embryo) was observed in DAMH-55 x PD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The maximum regeneration for efficient haploid induction was found when 400 Gy irradiated pollen of PD were used to pollinate DAMH-24. Keles et al. (2016) also observed varietal variations for haploid induction in Spinach; out of three varieties, haploid embryo formation was observed in Favorit F1 when pollinated with 400 Gy irradiation pollen of the same variety. Contrary to these, Aktas et al. (2023) obtained efficient haploid induction in Sunflower when a higher dose of irradiation, i.e. 750 Gy, was used. They obtained haploid induction only in three genotypes (K3AD SN:8, IMI 069 and IMI 044) out of the 16 tested genotypes. Similarly, Zhao et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) observed the highest embryo induction bottle gourd genotype, BG-4, when 50 Gy irradiated pollen of the same variety was used for pollination.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation among different variables of\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003ecultured\u003c/b\u003e \u003cb\u003epseudo fertilised\u003c/b\u003e \u003cb\u003eovaries of and irradiation doses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor a better understanding of the relationship between haploid induction through induced parthenogenesis and γ irradiation doses, a correlation matrix was established between different variables of haploid induction and irradiation doses. In Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e, a correlation matrix scale showing the values ranging from r\u0026thinsp;=\u0026thinsp;1 to -1 represents the strength and direction of the correlation between variables; the brown colour denotes a negative/indirect correlation, while the blue colour depicts a positive/direct correlation between both the parameters (Bakhshandeh et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). All the parameters are correlated after taking the average of both crosses (DAMH-24 \u0026times; PD and DAMH-24 \u0026times; PD) with different gamma-ray doses. According to our study, as the irradiation dose increased, the time taken for the response (r\u0026thinsp;=\u0026thinsp;0.85) and days to direct regeneration (r\u0026thinsp;=\u0026thinsp;0.73) showed a strong direct correlation. In contrast, percent direct regeneration (r\u0026thinsp;=\u0026thinsp;0.58) showed a moderate direct correlation and days to callus initiation (r\u0026thinsp;=\u0026thinsp;0.42) exhibited a direct correlation. Similarly, with some other parameters as doses increase the percentage of ovaries showing colour change (r = -0.90) exhibited a robust indirect correlation while percent response (r = -0.58) and percent callusing (r = -0.52) exhibited a moderate indirect correlation. Similar to the present findings, Ulukapi and Ozmen (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) also studied the correlation effects of various levels of γ-rays (100 to 500 Gy; 50 Gy interval) on the survival rates of the seedlings and vegetative traits in Common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.) plants. They found a significant positive correlation between root length and shoot length (r\u0026thinsp;=\u0026thinsp;0.90), between root fresh weight and shoot fresh weight (r\u0026thinsp;=\u0026thinsp;0.667), and between leaf length and chlorophyll index in M1 plants of the F16 variety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePloidy determination of gynogenically developed plants\u003c/h2\u003e \u003cp\u003eDetermination of the ploidy level is a vital step during haploid and doubled haploid production and their utilisation in breeding programmes. The methods for ploidy determination in marigold have been described earlier by Kumar et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A total of 52 plants, regenerated from the cultured ovaries of DAMH-24 \u0026times; PD and DAMH-24 \u0026times; PD) were assessed for their ploidy confirmation along with the mother plants. The ploidy was initially assessed by indirect and later confirmed by direct methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e; Table\u0026nbsp;2). The morphological evaluation of regenerated plants revealed the prevalence of albino plants at higher doses, ~ 400 \u0026amp; 500 Gy. At higher irradiation doses, haploid embryo induction might have resulted due to induced parthenogenesis. Hence, more plants with physical deformities were obtained. The haploid plants were mostly albino and exhibited high mortality rates, and if they survived, they also then failed to flower; this phenomenon may be due to the \u0026lsquo;Hertwig effect\u0026rsquo; (Pandey and Phung, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). The number of chloroplasts in guard cells of stomata varied from 3\u0026ndash;7 in haploids, 10\u0026ndash;14 in diploids and 16\u0026ndash;20 in triploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The plants of different ploidy levels, including haploids, diploids, and mixoploids have also been reported during the androgenesis and gynogenesis in marigold by Kumar et al. (2018) and Kumar et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Bhatia et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) in cauliflower. The cytological analysis (chromosome counting) further revealed that all the regenerants from 0 Gy and a few from 100, 200 and 300 Gy were triploid showing chromosome number 2n\u0026thinsp;=\u0026thinsp;3x\u0026thinsp;=\u0026thinsp;36 in their root cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003ek). A total of 8 haploids (2n\u0026thinsp;=\u0026thinsp;x\u0026thinsp;=\u0026thinsp;12; 15.38% Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003ec), 27 diploids (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eg ) and 17 triploids (2n\u0026thinsp;=\u0026thinsp;3x\u0026thinsp;=\u0026thinsp;36; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003ek) were identified. The maximum haploid induction regenerants were identified in DAMH-24 \u0026times;PD. The maximum haploid induction was observed within DAMH-24 \u0026times;PD with 400 Gy (7 out of 12) followed by 200 Gy (1 out of 5), while no haploid was obtained in DAMH-55\u0026times; PD even on irradiations. The higher dose of gamma irradiation might have led to the inactivation of male gamete in the pollen; however, it supported the direct embryogenesis from haploid cells of the embryo sac and subsequent haploid production. In this study, we have obtained a large number of diploid regenerants, which could be a result of regeneration from the diploid cells of the ovary wall/ embryo sac. The triploid plants could have regenerated as a result of successful fertilisation between diploid African marigold and tetraploid French marigold species. Both these species are cross-compatible, and successful interspecific hybrids among these two species have been reported by Zhang et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Some of these diploids could also be doubled haploids that might have resulted from spontaneous chromosome doubling of haploid embryos. Spontaneous chromosome doubling is a common phenomenon during haploid production and has been in a large number of plant species, including Brassica vegetables (Bhatia et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e \u0026amp; \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). After chromosome counting, the ploidy levels of the regenerants were further confirmed by flow cytometry analysis. Here, we have haploids have C DNA content with ~\u0026thinsp;54K mean Pi A (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003ed), diploids with 2C DNA content with ~\u0026thinsp;108K mean Pi (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eh), and triploids with 3C DNA content with ~\u0026thinsp;154K mean Pi (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003ei). The flow cytometry analysis also revealed that out of the 52 randomly selected plants 8 were haploids (15.38%), 27 were diploid (51.92%) and 17 were triploid (32.69%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The haploid plants were successfully recorded using induced parthenogenesis approach in cucumber (Pradeep et al. 2024). This study has proved the utility of the irradiated pollen technique (induced parthenogenesis) in haploid induction in African marigold. In the future, this approach can be utilised to achieve high throughput haploid induction in different marigold genotypes.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFor the first time, we have conducted an in-depth study to determine the role of gamma irradiations on haploid induction in marigold genotypes. The irradiated pollen of tetraploid marigold species, \u003cem\u003eTagetes patula\u003c/em\u003e genotype Pusa Deep \u0026lsquo;PD\u0026rsquo; (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48) was used to induce the haploids in the diploid marigold species, \u003cem\u003eT. erecta\u003c/em\u003e genotypes \u0026lsquo;DAMH-24\u0026rsquo; and DAMH-55\u0026rsquo; (African marigold; 2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24). The irradiated pollen technique was found effective in improving the haploid induction frequency in marigold. An irradiation dose of 400 Gy was found to be the optimum dose for efficient pollen inactivation and high-throughput induction of haploids. This research also provided insight into the effectiveness of gamma irradiation on successful haploid induction in marigold. This protocol will be instrumental in strengthening the F\u003csub\u003e1\u003c/sub\u003e hybrid breeding and genetic and genomic research in marigolds.\u003c/p\u003e"},{"header":"Abbreviations ","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eGy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eGray (=100 rad)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eKI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003ePotassium Iodide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eTTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e2, 3, 5, Triphenyl Tetrazolium Chloride\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eDHs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eDoubled Haploids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eDAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eDays after Pollination\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eDAI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eDays after Inoculation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003ePVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003ePolyvinylpyrrolidone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eTDZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eThidiazuron\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eQTLs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003eQuantitative Trait Locus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first author acknowledges the Indian Council of Agricultural Research (ICAR), New Delhi, India for awarding a Junior Research Fellowship during the entire period of this experiment. The funding received from the Science and Engineering Research Board, Department of Science and Technology, Government of India (Sanction Order: EEQ/2019/000632) for facilitating this research from 2019 to 2023 is also highly acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Science and Engineering Research Board, Department of Science and Technology, Government of India (Sanction Order: EEQ/2019/000632).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRB comprehended and supervised the experiments. RB, KPS, SP, and AT designed the experiments. EA and KD executed field/lab experiments and collected experimental data. EA and BS conducted the gamma irradiation experiment. EA, MF and RA have done pollen and pollination studies. EA, KD, and MR have determined the ploidy. RB, EA, and B did data analysis and interpretation. EA wrote the first draft of the manuscript, and RB has made all the corrections. All other authors have proofread and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data and other details used for this study can be requested from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare no competing interests regarding the publication of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAktaş YE, Aydin Y, Uncuoglu AA (2023) Induction of haploid plants for speed-up breedıng in sunflower (\u003cem\u003eHelianthus annuus\u003c/em\u003e L.) by pollen irradiation. Genetics \u0026amp; Applications. 7(1):35-44. https://doi.org/10.31383/ga.vol7iss1ga05 \u003c/li\u003e\n\u003cli\u003eAktaş YE, Uncuoğlu AA, Aydin Y (2018) Induction of parthenogenetic haploid embryos and plants after pollination by irradiated pollen in sunflower. In: Proceedings of the International Agricultural, Biological and Life Science Conference, Edirne, Turkey, pp. 2-5.\u003c/li\u003e\n\u003cli\u003eAnonymous (2020). Indian Horticulture Database. http://nhb.gov.in. Accessed 23 Jan 2024.\u003c/li\u003e\n\u003cli\u003eArzoo E (2022)\u003cem\u003e In vitro\u003c/em\u003e gynogenesis studies in African marigold.\u003cem\u003e \u003c/em\u003eM.Sc dissertation, ICAR-IARI, New Delhi, India-110012.\u003c/li\u003e\n\u003cli\u003eAswath C, Prakash T, Kumari P, Thakur N, Sharma R (2019) Effect of gamma radiation on pollen viability and pollen germination of marigold cultivar. Think India Journal. 22(30):621-628.\u003c/li\u003e\n\u003cli\u003eBaker HG, Baker I (1979) Starch in angiosperm pollen grains and its evolutionary significance. American Journal of Botany. 66(5):591-600. https://doi.org/10.1002/j.1537-2197.1979.tb06262.x \u003c/li\u003e\n\u003cli\u003eBakhshandeh E, Hosseini Sanehkoori F, Ghorbani H, Nematzadeh GA, Sekrafi M, Abdellaoui R, Yaghoubi Khanghahi M, Crecchio C (2023) Quantifying plant biomass and seed production in camelina (\u003cem\u003eCamelina sativa\u003c/em\u003e (L.) Crantz) across a large range of plant densities: Modelling approaches. Annals of Applied Biology. 183(1):23-32. https://doi.org/10.1111/aab.12830 \u003c/li\u003e\n\u003cli\u003eBhat JG, Murthy HN (2007) Factors affecting \u003cem\u003ein vitro\u003c/em\u003e gynogenic haploid production in niger (\u003cem\u003eGuizotia abyssinica\u003c/em\u003e (L. f.) Cass.). Plant growth regulation. 52:241-248. https://doi.org/10.1007/s10725-007-9196-9 \u003c/li\u003e\n\u003cli\u003eBhatia R, Dey S S, Sood S, Sharma K, Parkash C, Kumar R (2017) Efficient microspore embryogenesis in cauliflower (\u003cem\u003eBrassica oleracea\u003c/em\u003e var. \u003cem\u003ebotrytis \u003c/em\u003eL.) for development of plants with different ploidy level and their use in a breeding programme. Scientia Horticulture 216:83\u0026ndash;92. https://doi.org/10.1016/j. scienta.2016.12.020 \u003c/li\u003e\n\u003cli\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. \u003cem\u003ecapitata \u003c/em\u003eL.) genotypes in India. Sci Hort 233:178\u0026ndash;187. https://doi.org/10.1016/j. scienta.2018.01.017 \u003c/li\u003e\n\u003cli\u003eBhatia R, Dey SS, Sood S, Sharma K, Sharma VK, Parkash C, Kumar R (2016) Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (\u003cem\u003eB. oleracea\u003c/em\u003e var. \u003cem\u003ebotrytis\u003c/em\u003e L.) in India. Euphytica. 212:439-54. https://doi.org/10.1007/s10681-016-1775-2 \u003c/li\u003e\n\u003cli\u003eBlasco M, Badenes ML, del Mar Naval M (2016) Induced parthenogenesis by gamma-irradiated pollen in loquat for haploid production. Breeding science. 66(4):606-12. https://doi.org/10.1270/jsbbs.16021 \u003c/li\u003e\n\u003cli\u003eBrewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. American journal of botany. 50(9):859-65. https://doi.org/10.2307/2439772 \u003c/li\u003e\n\u003cli\u003eCuny F, Grotte M, De Vaulx RD, Rieu A (1993) Effects of gamma irradiation of pollen on parthenogenetic haploid production in muskmelon (\u003cem\u003eCucumis melo\u003c/em\u003e L.). Environmental and Experimental Botany. 33(2):301-12. https://doi.org/10.1016/0098-8472(93)90076-r \u003c/li\u003e\n\u003cli\u003eDash M, Mishra A (2024) QTL Mapping: Principle, Approaches, and applications in crop improvement. In Smart Breeding. Apple Academic Press.31-60. https://doi.org/10.1201/9781003361862-2 \u003c/li\u003e\n\u003cli\u003eDor\u0026eacute; C, Prigent J, Desprez B (1996) \u003cem\u003eIn situ\u003c/em\u003e gynogenetic haploid plants of chicory (\u003cem\u003eCichorium intybus\u003c/em\u003e L.) after intergeneric hybridization with \u003cem\u003eCicerbita alpina\u003c/em\u003e Walbr. Plant cell reports. 15(10):758-761. https://doi.org/10.1007/bf00232223 \u003c/li\u003e\n\u003cli\u003eFalque M (1994) Pod and seed development and phenotype of the M1 plants after pollination and fertilization with irradiated pollen in cacao (\u003cem\u003eTheobroma cacao\u003c/em\u003e L.). Euphytica. 75(1):19-25. https://doi.org/10.1007/bf00024527 \u003c/li\u003e\n\u003cli\u003eFroelicher Y, Bassene JB, Jedidi-Neji E, Dambier D, Morillon R, Bernardini G, Costantino G, Ollitrault P (2007) Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. Plant Cell Reports. 26:937-44. https://doi.org/10.1007/s00299-007-0314-y \u003c/li\u003e\n\u003cli\u003eGodbole M, Murthy HN (2012) Parthenogenetic haploid plants using gamma irradiated pollen in snapmelon (\u003cem\u003eCucumis melo\u003c/em\u003e var. \u003cem\u003emomordica\u003c/em\u003e). Plant Cell, Tissue and Organ Culture (PCTOC). 109:167-70. https://doi.org/10.1007/s11240-011-0066-9 \u003c/li\u003e\n\u003cli\u003eGrouh MS, Sousaraei N, Akbari M, Rahimi V, Bayat H (2015) Induction of haploid plants in iris (\u003cem\u003eIris pseudacorus\u003c/em\u003e) by pollen irradiation. Turkish Journal of Agriculture and Forestry. 39(4):596-600. https://doi.org/10.3906/tar-1407-18 \u003c/li\u003e\n\u003cli\u003eKeleş D, \u0026Ouml;zcan C, Pınar H, Ata A, Denli N, Y\u0026uuml;cel NK, Taşkın H, B\u0026uuml;y\u0026uuml;kalaca S. (2016) First report of obtaining haploid plants using tissue culture techniques in spinach. HortScience. 51(6):742-9. https://doi.org/10.21273/hortsci.51.6.742 \u003c/li\u003e\n\u003cli\u003eKumar KR, Singh KP, Bhatia R, Raju DV, Panwar S (2019) Optimising protocol for successful development of haploids in marigold (\u003cem\u003eTagetes\u003c/em\u003e spp.) through \u003cem\u003ein vitro\u003c/em\u003e androgenesis. Plant Cell, Tissue and Organ Culture (PCTOC). 138:11-28. https://doi.org/10.1007/s11240-019-01598-3 \u003c/li\u003e\n\u003cli\u003eKumar KR, Singh KP, Raju DV, Bhatia R, Panwar S (2020) Maternal haploid induction in African marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e L.) through \u003cem\u003ein vitro\u003c/em\u003e culture of unfertilized ovules. Plant Cell, Tissue and Organ Culture (PCTOC). 143:549-64. https://doi.org/10.1007/s11240-020-01940-0 \u003c/li\u003e\n\u003cli\u003eKundu M, Dubey AK (2022) Anatomical studies of \u003cem\u003eIn vitro \u003c/em\u003eraised haploid and diploid citrus plants generated through gamma irradiated pollen. Indian Journal of Horticulture. 79(2):136-44. https://doi.org/10.5958/0974-0112.2022.00019.6 \u003c/li\u003e\n\u003cli\u003eKurtar ES, Balkaya A (2010) Production of \u003cem\u003ein vitro\u003c/em\u003e haploid plants from \u003cem\u003ein situ\u003c/em\u003e induced haploid embryos in winter squash (\u003cem\u003eCucurbita maxima\u003c/em\u003e Duchesne ex Lam.) \u003cem\u003evia\u003c/em\u003e irradiated pollen. Plant Cell, Tissue and Organ Culture (PCTOC). 102:267-77. https://doi.org/10.1007/s11240-010-9729-1 \u003c/li\u003e\n\u003cli\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). African Journal of Biotechnology. 8(21):5944-51. https://doi.org/10.5897/ajb09.730 \u003c/li\u003e\n\u003cli\u003eKurtar ES, Metin D, Atakul Z, Kal U, Seymen M, Takcı E, Yılmaz HN, Alan AR, Toprak FC (2025) Effects of gamma ray sources and irradiation doses on the haploidization frequency of citron watermelon (\u003cem\u003eCitrullus lanatus\u003c/em\u003e var. \u003cem\u003ecitroides\u003c/em\u003e). Plant Cell, Tissue and Organ Culture (PCTOC), 160(3), p.62. https://doi.org/10.1007/s11240-025-03004-7 \u003c/li\u003e\n\u003cli\u003eLi F, Cheng Y, Zhao X, Yu R, Li H, Wang L, Li S, Shan Q (2020) Haploid induction via unpollinated ovule culture in \u003cem\u003eGerbera hybrida\u003c/em\u003e. Scientific Reports. 10(1):1702. https://doi.org/10.1038/s41598-020-58552-z \u003c/li\u003e\n\u003cli\u003eLiu H, Wang Y, Liang C, Yang Q, Wang S, Wang B, Zhang F, Zhang L, Cheng H, Song S, Zhang L (2020) Utilization of marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e) flower fermentation wastewater as a fertilizer and its effect on microbial community structure in maize rhizosphere and non-rhizosphere soil. Biotechnology \u0026amp; Biotechnological Equipment. 34(1):522-31. https://doi.org/10.1080/13102818.2020.1781548 \u003c/li\u003e\n\u003cli\u003eMeynet J, Barrade R, Duclos A, Siadous R (1994) Dihaploid plants of roses (\u003cem\u003eRosa\u003c/em\u003e x \u003cem\u003ehybrida\u003c/em\u003e, cv\u0026apos;Sonia\u0026apos;) obtained by parthenogenesis induced using irradiated pollen and \u003cem\u003ein vitro\u003c/em\u003e culture of immature seeds. Agronomie. 14(3):169-75. https://doi.org/10.1051/agro:19940303 \u003c/li\u003e\n\u003cli\u003eMiler N, Tymoszuk A, Rewers M, Kulus D (2023) \u003cem\u003eIn vitro\u003c/em\u003e regeneration of chrysanthemum from ovaries and ovules treated with thermal and chemical stimuli: morphogenic and cytogenetic effects. Agriculture. 13(11):2069. https://doi.org/10.3390/agriculture13112069 \u003c/li\u003e\n\u003cli\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia plantarum. 15(3). https://doi.org/10.1111/j.1399-3054.1962.tb08052.x \u003c/li\u003e\n\u003cli\u003eMurovec J, Bohanec B (2013) Haploid induction in \u003cem\u003eMimulus aurantiacus\u003c/em\u003e Curtis obtained by pollination with gamma irradiated pollen. Scientia horticulturae. 162:218-225. https://doi.org/10.1016/j.scienta.2013.08.012 \u003c/li\u003e\n\u003cli\u003eOberle GD, Watson RI (1953) The use of 2, 3, 5-triphenyl tetrazolium chloride in viability tests of fruit pollens. Journal of the American Society for Horticultural Science. 61: 299\u0026ndash;303. https://doi.org/10.1093/ajcp/23.5_ts.512 \u003c/li\u003e\n\u003cli\u003ePacini E, Franchi GG (2020) Pollen biodiversity\u0026ndash;why are pollen grains different despite having the same function? A review. Botanical Journal of the Linnean Society. 193(2):141-64. https://doi.org/10.1093/botlinnean/boaa014 \u003c/li\u003e\n\u003cli\u003ePalvaneh K, Ghamari Zare A, Lotfi M, Naraghi TS, Asadi-Corom F, Tabaei-Aghdaei SR (2013) Investigation of gamma ray effects on pollen germination and parthenogenic haploid embryogenesis in \u003cem\u003eRosa damascena\u003c/em\u003e Mill. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research. 21(1):45-55.\u003c/li\u003e\n\u003cli\u003ePandey KK, Phung M (1982) \u0026lsquo;Hertwig effect\u0026rsquo; in plants: induced parthenogenesis through the use of irradiated pollen. Theoretical and Applied Genetics. 62:295-300. https://doi.org/10.1007/bf00275088 \u003c/li\u003e\n\u003cli\u003ePiosik Ł, Zenkteler E, Zenkteler M (2016) Development of haploid embryos and plants of \u003cem\u003eLactuca sativa\u003c/em\u003e induced by distant pollination with \u003cem\u003eHelianthus annuus\u003c/em\u003e and \u003cem\u003eH. tuberosus\u003c/em\u003e. Euphytica. 208(3):439-751. https://doi.org/10.1007/s10681-015-1578-x \u003c/li\u003e\n\u003cli\u003ePradeepkumara N, Dey SS, Munshi AD, Behera TK, Bhatia R, Kumari K, Prakash P, Bhattacharya RC, Talukdar A (2023) Cucumber F1 hybrid derived from two contrasting inbreds ensures high frequency gynogenesis for induction of haploids through a modified in vitro based protocol. South African Journal of Botany. 157(1):314-24. https://doi.org/10.1016/j.sajb.2023.03.066 \u003c/li\u003e\n\u003cli\u003eSalehian H, Shahnazi S, Nazari M (2023) Production of doubled haploid plants in cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.) via parthenogenesis. \u003cem\u003eIn Vitro\u003c/em\u003e Cellular \u0026amp; Developmental Biology-Plant. 59(4):467-74. https://doi.org/10.1007/s11627-023-10368-y \u003c/li\u003e\n\u003cli\u003eSato S, Katoh N, Yoshida H, Iwai S, Hagimori M (2000) Production of doubled haploid plants of carnation (\u003cem\u003eDianthus caryophyllus\u003c/em\u003e L.) by \u003cem\u003epseudofertilized \u003c/em\u003eovule culture. Scientia horticulturae. 83(3-4):301-310. https://doi.org/10.1016/s0304-4238(99)00090-4 \u003c/li\u003e\n\u003cli\u003eSchaart JG, van de Wiel CC, Smulders MJ (2021) Genome editing of polyploid crops: prospects, achievements and bottlenecks. Transgenic Research. 30(4):337-51. https://doi.org/10.1007/s11248-021-00251-0 \u003c/li\u003e\n\u003cli\u003eScott MF, Ladejobi O, Amer S, Bentley AR, Biernaskie J, Boden SA, Clark M, Dell\u0026rsquo;Acqua M, Dixon LE, Filippi CV, Fradgley N (2020) Multi-parent populations in crops: a toolbox integrating genomics and genetic mapping with breeding. Heredity. 125(6):396-416. https://doi.org/10.1038/s41437-020-0336-6 \u003c/li\u003e\n\u003cli\u003eSukwiwat K, Kumchai J, Bundithya W, Potapohn N (2023) Apetaloid and petaloid female performance on horticultural characteristics of F1 American marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e L.) hybrids. SABRAO J. Breed. Genet. 55(5):1754-67. https://doi.org/10.54910/sabrao2023.55.5.27 \u003c/li\u003e\n\u003cli\u003eTodorova M, Ivanov P, Shindrova P, Christov M, Ivanova I (1997) Doubled haploid production of sunflower (\u003cem\u003eHelianthus annuus\u003c/em\u003e L.) through irradiated pollen-induced parthenogenesis. Euphytica. 97(3):249-54. https://doi.org/10.1023/a:1002966824988 \u003c/li\u003e\n\u003cli\u003eTodorova MI, Ivanov PE (2000) Induced parthenogenesis in sunflower (\u003cem\u003eHelianthus annuus\u003c/em\u003e L.): Effect of gamma-irradiation doses. In: Proc of the 15th International Sunflower Conference, Toulouse, France, p. 46-51. https://doi.org/10.2298/hel0441067t \u003c/li\u003e\n\u003cli\u003eTran LT, Sugimoto K, Kasozi M, Mitalo OW, Ezura H (2023) Pollination, pollen tube growth, and fertilization independently contribute to fruit set and development in tomato. Frontiers in Plant Science. 14:1205816. https://doi.org/10.3389/fpls.2023.1205816 \u003c/li\u003e\n\u003cli\u003eT\u0026uuml;t\u0026uuml;nc\u0026uuml; M, Mendi YY (2022) Effect of pollination with gamma irradiated pollen on \u003cem\u003ein vitro\u003c/em\u003e regeneration of ovule culture in Cyclamen. Turkish Journal of Agriculture-Food Science and Technology. 10(12):2415-20. https://doi.org/10.24925/turjaf.v10i12.2415-2420.5409 \u003c/li\u003e\n\u003cli\u003eUlukapi K, Ozmen SF (2018) Study of the effect of irradiation (\u003csup\u003e60\u003c/sup\u003eCo) on M1 plants of common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.) cultivars and determined of proper doses for mutation breeding. Journal of Radiation Research and Applied Sciences. 11(2):157-61. https://doi.org/10.1016/j.jrras.2017.12.004 \u003c/li\u003e\n\u003cli\u003eValdez-Baro O, Jim\u0026eacute;nez-Ortega LA, Cabanillas-Boj\u0026oacute;rquez L\u0026Aacute;, Guti\u0026eacute;rrez-Grijalva EP, Heredia JB (2024) Food byproducts as potential functional ingredients for animal feeding. In: Food byproducts management and their utilization. Apple Academic Press, p 417-452. https://doi.org/10.1201/9781003377801-20 \u003c/li\u003e\n\u003cli\u003eWang H, Hou H, Jan CC, Chao WS (2023) Irradiated pollen-induced parthenogenesis for doubled haploid production in sunflowers (\u003cem\u003eHelianthus\u003c/em\u003e spp.). Plants. 12(13):2430. https://doi.org/10.3390/plants12132430 \u003c/li\u003e\n\u003cli\u003eZhang H, Lina SO, Lifang LI, Haibo XI, Rongfeng CU, Zijing LI, Shiwei ZH, Zunzheng WE (2022) Interspecific hybridization with African marigold (\u003cem\u003eTagetes erecta\u003c/em\u003e) can improve flower-related performance in French marigold (\u003cem\u003eT. patula\u003c/em\u003e). Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 50(4):12808-. https://doi.org/10.15835/nbha50312808 \u003c/li\u003e\n\u003cli\u003eZhao Q, Zhang M, Qi H, Yang X (2023) Haploid induction and plant production in bottle gourd by pollination with gamma irradiated pollen. Plant Cell, Tissue and Organ Culture (PCTOC). 152(2):331-8. https://doi.org/10.1007/s11240-022-02410-5 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eEffect of different doses of gamma irradiation on direct and indirect regeneration response from \u003cem\u003epseudo-fertilized\u0026nbsp;\u003c/em\u003eovaries.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg src=\"data:image/png;base64,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\" width=\"606\" height=\"190\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Ploidy estimation of \u003cem\u003ein vitro\u003c/em\u003e regenerated plants of DAMH-24 x PD and DAMH-55 x PD by different indirect and direct methods.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"566\" height=\"315\"\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gamma (γ) irradiation, Haploids, Induced parthenogenesis, Pseudo-fertilization, Gynogenesis, Flow cytometry","lastPublishedDoi":"10.21203/rs.3.rs-6683915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6683915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Haploids and doubled haploids play a crucial role in crop improvement programs, including hybridisation, mutation breeding, and genetic and genomic studies. Induction of parthenogenesis using gamma-irradiated pollens is an efficient approach for inducing haploids and developing homozygous lines in horticultural crops. However, no research has been conducted using the induced parthenogenesis approach for haploid induction in the genus Tagetes. This study aims to assess the effectiveness of gamma irradiation at doses of 100, 200, 300, 400, and 500 Gy, using a Co60 (Cobalt-60) source, for haploid induction. The irradiated pollen of a distant tetraploid species, Tagetes patula genotype ‘Pusa Deep’, was used to induce haploids in the diploid marigold species, T. erecta genotypes ‘DAMH-24’ and ‘DAMH-55’. The pseudo-fertilized ovaries were cultured on modified MS medium supplemented with 0.75 mgl-1 TDZ, 0.2 mgl-1 NAA, and 0.5 mgl-1 GA3. To better understand the effect of γ irradiation doses on haploid induction rate, a correlation analysis was performed. Out of 50 regenerants, 20 were selected to determine ploidy levels using morphological traits, chloroplast count, chromosomal count, and flow cytometry analysis. A plant with 3-7 chloroplasts in stomatal guard cells, 2n=x=12 chromosomes, and 1C DNA (~54K mean Pi) was identified as haploid; the plant with 10-14 chloroplasts, 2n=2x=24 chromosomes, and 2C DNA (~108K mean Pi) was diploid, while the plant with 16-20 chloroplasts, 2n=2x=24 chromosomes, and 3C DNA (~154K mean Pi) was triploid. For efficient pollen inactivation and high-throughput haploid induction in Tagetes erecta, the 400 Gy treatment was found to be the most effective.","manuscriptTitle":"Efficient induction of parthenogenetic haploids in African marigold (Tagetes erecta) L.) using Co60 gamma-irradiated pollen of French Marigold (Tagetes patula L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 10:08:50","doi":"10.21203/rs.3.rs-6683915/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-07-25T13:12:28+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-17T17:35:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T07:59:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-22T13:11:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-05-19T11:49:06+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":"9f4aab0a-d5aa-4cf1-96d4-50f36b5fe04b","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:07:39+00:00","versionOfRecord":{"articleIdentity":"rs-6683915","link":"https://doi.org/10.1007/s11240-025-03201-4","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-09-24 15:57:22","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-06-19 10:08:50","video":"","vorDoi":"10.1007/s11240-025-03201-4","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03201-4","workflowStages":[]},"version":"v1","identity":"rs-6683915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6683915","identity":"rs-6683915","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}