Irradiated Pollen Research Articles

Gamma irradiation of pollen and eradication of Israeli acute paralysis virus

Honeybees and bumblebees are the most important pollinators of agricultural crops. For this purpose honeybees and bumblebees are reared and transported. A pathogen-free status of bees in general, is crucial. Indeed anthropogenic transports of hosts carrying parasites could alter the natural host/pathogen association, inducing an extra pathogenic stress. Therefore the creation of a pathogen-free rearing environment is needed. For bumblebees this is possible, as these species are reared in a closed environment. Although, a link remains between reared bumblebees and the outside bee community, as honeybee-collected pollen is essential food for bumblebee mass rearing. Here we evaluated if gamma irradiation can minimize the risk of this potential route of exposure and can inactivate viral particles present in honeybee-collected pollen. We show that 16.9kGy gamma irradiation induced a 100–1000 fold reduction on the ability of IAPV to cause mortality after injections. This result opens avenues toward rearing pathogen-free bumblebees and towards eliminating the risks of pathogen spillover to native wild bee species.

Induction of parthenocarpic fruit set in watermelon by pollination with bottle gourd (Lagenaria siceraria (Molina) Standl.) pollen

Production of seedless watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) using soft X-ray irradiated pollen has the advantage that this method can be used with diploid cultivars; however, pistillate flowers must be covered with a bag before flowering to prevent normal pollination by insects. Therefore, we attempted to prevent normal pollen penetration by pollinating pistillate watermelon flowers with intergeneric pollen before anthesis. In the course of conducting these studies, we found a new method to produce seedless watermelon by pollinating with bottle gourd (Lagenaria siceraria (Molina) Standl.) pollen. When the pollen of bottle gourd was used to pollinate pistillate watermelon flowers, the fruit set rate was 57.1%. On the other hand, the fruit set rate by pollinating with watermelon pollen was 65.0%. Although the fruit set rate using bottle gourd pollen was slightly lower than the fruit set rate obtained when watermelon pollen was used, this percentage was higher than expected. Preservation of pollen collected from staminate flowers of bottle gourd was difficult, and fruit did not set. In contrast, fruit developed when bottle gourd pollen from staminate flowers stored at 4°C for 72h was used to pollinate watermelon. All parthenocarpic fruits produced by pollination with bottle gourd pollen were deformed (triangular or oblong shaped). There was a significant difference in the ratio of fruit length to fruit width; however, fruit weight, rind thickness, flesh color and Brix in the parthenocarpic fruit were almost the same as control fruit. There were no normal seeds except for small, white empty seeds in the fruit obtained from pollinating with bottle gourd pollen. Bottle gourd pollen tubes did not reach the ovules of watermelon ovaries. Therefore, we concluded that parthenocarpy resulting from pollination with bottle gourd pollen was stimulative parthenocarpy, not pseudo parthenocarpy (pseudogamy).

Effects of different genotypes and gamma ray doses on haploidization using irradiated pollen technique in squash

Fourteen genotypes and 3 different gamma ray doses were tested to develop an efficient haploidization protocol in squash. For this purpose, male flowers collected 1 day before anthesis were irradiated with 150, 200, and 300 Gy gamma ray doses, and female flowers were pollinated with the irradiated pollens the next day. In the first year of the study, 1858 embryos were obtained from 219 fruits. While 1358 of these were found in fruits irradiated with a 150 Gy gamma dose, the remaining 500 embryos were found in fruits irradiated with 200 Gy. From fruits irradiated with 150 and 200 Gy gamma doses, 9.12 and 3.53 haploid embryos per 100 seeds were obtained, respectively. While Genotype 3 was the most successful genotype with 12.42 embryos per 100 seeds, the minimum embryo numbers were obtained from Genotype 4 with 1.46 embryos per 100 seeds. In the second experimental year, 8 genotypes and the same gamma doses were used, and 2625 haploid and 1378 diploid embryos were obtained from 217 fruits. At irradiation doses of 150, 200, and 300 Gy, 2010, 539, and 76 haploid embryos were found, respectively. Genotype 6 was the most successful genotype with 13.35 embryos per 100 seeds.

Styrian oil pumpkin pollen germinability at higher irradiation doses: optimization of the in vitro germination protocol and irradiation procedure

Protocols for haploid induction in cucurbits are based on pollination with irradiated pollen, but the induction frequency is low and the majority of obtained embryos are zygotic. The longevity of Cucurbita pepo L. pollen is short even under natural conditions; following irradiation, germinability is decreased even further. This study was initiated to develop an optimal in vitro germination medium for styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) pollen, which would enable accurate germination testing. Different pH values and the addition of sucrose, mannitol, and polyethylene glycol to the Brewbaker and Kwack germination medium were tested. The optimum medium condition was pH 9 and 12.5% (w/v) sucrose, while other tested components were not efficient. Using the optimized medium, X-ray-irradiated (100-700 Gy) pollen germinability was assessed under 2 air humidity conditions. Germinability of pollen irradiated at room humidity (RH) was generally lower than that of pollen irradiated at high humidity (HH). A major variability in pollen size (diameters ranged from 79.2 to 196.5 µm) and 2 subgroups were found in the pollen population. Following irradiation, HH conditions allowed germination of larger pollen grains than those of the nonirradiated control and RH.

유채(Brassica napus)화분에 대한 감마선 조사가 미생물 제어 및 화분의 품질특성에 미치는 영향

유채화분에 대한 일반성분 및 이화학적 변화를 최소화 시키면서 효과적으로 미생물을 제어할 수 있는 감마선 조사의 적정 선량을 확인하고자 유채화분에 0 kGy, 5 kGy, 10 kGy, 15 kGy로 감마선 조사를 실시하였다. 품질 특성 및 미생물 실험결과 조사선량에 따른 차이는 나타내었지만 전체적인 유채화분의 일반성분에 큰 차이는 없었으며 미생물의 경우 곰팡이 및 대장균군은 5 kGy 조사로 총균 및 효모는 10 kGy 조사로 검출되지 않았다(<10<SUP>2</SUP> CFU/g). 환원당 함량은 조사선량 증가에 따른 유의차를 보이지 않았다. 명도, 적색도, 황색도는 조사선량이 증가할수록 감소하는 경향을 나타내었으며 지질산화는 조사선량이 증가할수록 증가하는 경향을 나타낸 반면 휘발성 염기태 질소의 함량은 감마선 조사에 의해 영향을 받지 않았다. 따라서 감마선 조사에 대한 일반성분 및 이화학적 변화를 최소화하면서 화분에서 독소를 생성하는 곰팡이를 효과적으로 제어할 수 있는 감마선 조사선량은 5 kGy인 것으로 사료된다.

Effects of different genotypes and gamma ray doses on haploidization with irradiated pollen technique in watermelon (Citrullus lanatus L.)

Taşkın, H., Yücel, N. K., Baktemur, G., Çömlekçioğlu, S. and Büyükalaca, S. 2013. Effects of different genotypes and gamma ray doses on haploidization with irradiated pollen technique in watermelon ( Citrullus lanatus L.). Can. J. Plant Sci. 93: 1165–1168. Two watermelon genotypes, one commercial watermelon variety (Ustun F1) and five different doses of gamma rays coming from Co60 were tested to develop useful haploidization procedures in watermelon. For this purpose, male flowers collected a day before anthesis were irradiated with 50, 150, 200, 275 and 300 Gy doses of gamma rays, and female flowers were pollinated with irradiated pollen the next day. Seeds extracted from fruits harvested 25 d later were opened individually in a laminar flow hood. Embryos obtained via embryo rescue technique were placed in glass tubes containing CP medium with 30 g L−1 sucrose, 8 g L−1 agar, 0.08 mg L−1 B12, and 0.02 mg L−1 IAA. Sixty haploid embryos were obtained from 43 watermelon fruits in this study. Genotype 1 was found to be the most successful genotype with 3.57 haploid embryos per 100 seeds. Among tested irradiation doses, 275 Gy was better than other doses, with 5.26 haploid embryos per 100 seeds. Considered together with irradiation dose and genotypes, the maximum number of haploid embryos was obtained from Genotype 1 pollinated with 275 Gy irradiation dose, with 6.25 haploid embryos per 100 seeds.

Haploid induction in Mimulus aurantiacus Curtis obtained by pollination with gamma irradiated pollen

In this study we present a successful approach to haploid induction in Mimulus aurantiacus Curtis. In Europe, it is a promising ornamental plant and is intensively studied for its adaptive differentiation and speciation processes due to high ecological and phenotypic diversity of natural populations. The small nuclear DNA content, determined in our study as 0.607pg DNA (2C), indicated the suitability of this species for DNA sequencing approaches. Among several methods of haploid induction tested, pollination with gamma-irradiated pollen proved to be the only effective method and produced 4 haploid, 319 diploid, 5 triploid and 3 aneuploid plants. A new microsatellite marker was developed for determination of plantlets origin, from EST sequences deposited in NCBI GenBank. Together with the Mim-top6B microsatellite marker developed earlier, it revealed that 43 of the tested plants were homozygous and 171 heterozygous. The majority of heterozygotes were hybrids between female gamete and irradiated pollen, which retained its fertilization ability after irradiation. Genetic analysis of triploid and aneuploid regenerants provided novel insight into the mechanism of haploid embryo formation following pollination with irradiated pollen. On the basis of genetic analysis of a heterozygous aneuploid (with ploidy between haploid and diploid levels), we propose that paternal chromosome elimination might be the causative mechanism of haploid plant formation while the genetic structure of triploid regenerants provided evidence of regeneration from endospermal cells.

Haploid Induction in Hull-less Seed Pumpkin through Parthenogenesis Induced by X-ray-irradiated Pollen

Production of hull-less seeds of styrian oil pumpkin (Cucurbita pepo ssp. pepo var. styriaca) is increasing as a result of demand for aromatic seed oil and for other uses. Hybrid cultivars have recently been released but a method for haploid induction has not been established. We focused on the development of a parthenogenetic haploid induction protocol based on pollination with pollen irradiated with X-ray radiation at 0, 50, 100, 150, 200, 300, and 350 Gy. Fruit set largely decreased at 200 Gy, whereas a decline in embryo formation was observed at 100 Gy. Various accessions were tested as the female parent or as the pollen donor and large differences were found. The best parthenogenetic response was found in ‘Turkey #2’ (10.0%), ‘Gleisdorfer Ölkürbis’ (4.4%), and ‘Naked Seed’ (3.9%), whereas ‘GL Opal’ and ‘White Acorn’ were efficient as pollen donors. The ploidy level of 3830 putative parthenogenetic embryos was determined using flow cytometry. Four ploidy levels (n, 2n, 3n, and 4n) were found with the majority being diploid. Interestingly, a significant proportion was determined to be tetraploid and this was clearly correlated with increased radiation delivered to pollen grains. Using selected simple sequence repeat markers on diploid embryos, no spontaneous chromosome doubling could be confirmed. In this study, haploid induction in styrian oil pumpkin was elaborated for the first time. We also showed that X-ray pollen irradiation provides an alternative to gamma radiation treatment, yielding a sufficient percentage of haploid plantlets.

Review of research on haploid production in cucumber and other cucurbits

ABSTRACT This review provides a summary of haploid induction methods and factors affecting the efficacy of specific methodologies as applied to cucumber (Cucumis sativus L.), melon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum. &amp; Nakai), winter squash (Cucurbita maxima Duch. ex Lam.), summer squash (Cucurbita pepo L.) and other cucurbits. This report is focused on studies that were carried out during the last 20 years. The main objective of the research on the production of haploid cucurbit plants is to accelerate breeding programs through the use of homozygous double haploid lines (DHL) and to facilitate the selection of desired (e.g. disease-resistant) genotypes for breeding. Unfortunately, currently used protocols result in a low number of double haploids (DH). The most common and best-known method of obtaining haploid cucurbit plants is via pollination with irradiated pollen, which induces parthenogenetic development of haploid embryos in planta. The embryos are extracted from immature seeds and cultured in vitro to facilitate the maturation and development of plants. The studies described below were primarily aimed at the determination of an appropriate dose of radiation and the evaluation of the impact of the genotype and the time of year on the number of haploid embryos and plants obtained. A less popular method of haploid production - ovule and ovary culture - is based on in vitro gynogenesis. The studies related to this method concentrated on optimising the composition of the medium and pre-treatment conditions (primarily temperature) to which the flower buds were subjected. Recently, increasing attention has been paid to anther and microspore culture. As in the case of in vitro ovule and ovary culture, the medium composition and flower bud pre-treatment conditions were optimised. The most recent studies suggest that anther culture is comparable in effectiveness to the irradiated pollen technique.

DNA damage response in male gametes of Cyrtanthus mackenii during pollen tube growth

Male gametophytes of plants are exposed to environmental stress and mutagenic agents during the double fertilization process and therefore need to repair the DNA damage in order to transmit the genomic information to the next generation. However, the DNA damage response in male gametes is still unclear. In the present study, we analysed the response to DNA damage in the generative cells of Cyrtanthus mackenii during pollen tube growth. A carbon ion beam, which can induce DNA double-strand breaks (DSBs), was used to irradiate the bicellular pollen, and then the irradiated pollen grains were cultured in a liquid culture medium. The male gametes were isolated from the cultured pollen tubes and used for immunofluorescence analysis. Although inhibitory effects on pollen tube growth were not observed after irradiation, sperm cell formation decreased significantly after high-dose irradiation. After high-dose irradiation, the cell cycle progression of generative cells was arrested at metaphase in pollen mitosis II, and phosphorylated H2AX (γH2AX) foci, an indicator of DSBs, were detected in the majority of the arrested cells. However, these foci were not detected in cells that were past metaphase. Cell cycle progression in irradiated generative cells is regulated by the spindle assembly checkpoint, and modification of the histones surrounding the DSBs was confirmed. These results indicate that during pollen tube growth generative cells can recognize and manage genomic lesions using DNA damage response pathways. In addition, the number of generative cells with γH2AX foci decreased with culture prolongation, suggesting that the DSBs in the generative cells are repaired.

Comparison of Different Methods for Separation of Haploid Embryo Induced through Irradiated Pollen and Their Economic Analysis in Melon (Cucumis melovar.inodorus)

Irradiated pollen technique is the most successful haploidization technique within Cucurbitaceae. After harvesting of fruits pollinated with irradiated pollen, classical method called as “inspecting the seeds one by one” is used to find haploid embryos in the seeds. In this study, different methods were used to extract the embryos more easily, quickly, economically, and effectively. “Inspecting the seeds one by one” was used as control treatment. Other four methods tested were “sowing seeds direct nutrient media,” “inspecting seeds in the light source,” “floating seeds on liquid media,” and “floating seeds on liquid media after surface sterilization.” Y2 and Y3 melon genotypes selected from the third backcross population of Yuva were used as plant material. Results of this study show that there is no statistically significant difference among methods “inspecting the seeds one by one,” “sowing seeds direct CP nutrient media,” and “inspecting seeds in the light source,” although the average number of embryos per fruit is slightly different. No embryo production was obtained from liquid culture because of infection. When considered together with labor costs and time required for embryo rescue, the best methods were “sowing seeds directly in the CP nutrient media“ and ”inspecting seeds in the light source.”

Endosperm Tolerance of Paternal Aneuploidy Allows Radiation Hybrid Mapping of the Wheat D-Genome and a Measure of γ Ray-Induced Chromosome Breaks

Physical mapping and genome sequencing are underway for the ≈17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. Here, we report a novel approach to construct RH based physical maps of all seven D-genome chromosomes of the hexaploid wheat ‘Chinese Spring’, simultaneously. An 81-member subset of endosperm samples derived from 20-Gy irradiated pollen was genotyped for deletions, and 737 markers were mapped on seven D-genome chromosomes. Analysis of well-defined regions of six chromosomes suggested a map resolution of ∼830 kb could be achieved; this estimate was validated with assays of markers from a sequenced contig. We estimate that the panel contains ∼6,000 deletion bins for D-genome chromosomes and will require ∼18,000 markers for high resolution mapping. Map-based deletion estimates revealed a majority of 1–20 Mb interstitial deletions suggesting mutagenic repair of double-strand breaks in pollen provides a useful resource for RH mapping and map based cloning studies.

Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa

Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.

Effects of pollen and artificial diets on the hypopharyngeal glands of newly hatched bees (Apis mellifera L.)

SummaryThe objectives of this experiment were to: 1. compare the hypopharyngeal gland development (by measuring the head weight) of caged newly hatched nurse bees fed on eight different pollen and artificial diets; and 2. examine the correlation between protein intake and head weight. The artificial diets: substitute 2; FeedBee®; substitute 1; and protein substitute stimulated (in order) significantly higher head weight than the pollen diets: pure irradiated mixed pollen; redgum (Corymbia calophylla); redgum + soyflour protein isolate; and pollen supplement. Only artificial diets stimulated overall head weight of more than 10 mg and maintained head weight above the emergent head weight for the duration of the experiment. Substitute 2 and FeedBee® stimulated the highest head weight, followed by substitute 1 and pollen substitute, pollen supplement, mixed pollen and redgum + soyflour protein isolate, and redgum. The only diets to have positive correlation (p < 0.05) between the crude protein consumption and head weight were substitute 1 and pollen substitute.

Alpha-Particles and 60Co y-Rays Have Different Biological Effects on Upland Cotton (Gossypium hirsutum L.) Pollen Grains

To compared the irradiation effects by y-rays and alpha-particles on the structure and function of upland cotton pollen grains during pollination, pollen grains from Gossypium hirsutum L. cultivar “Lumian 21” were irradiated by alpha-particles (1, 10, 30, 40 and 100 Gy) and y-rays (20, 30 and 40 Gy). We looked for changes in the ultrastructure of the exine and interior walls of the pollen grains. After germinating the irradiated pollen grains in liquid medium, we looked for changes in germination rate, the number of pollen tubes in styles after pollination and F-actin distribution in the pollen tubes. Alpha-particles penetrated and etched the exine and interior walls of the pollen grains and also damaged the organelles in the pollen grains. More pollen grains were destroyed as the alpha-particles dose increased. y-rays had no effects on the exine walls of the pollen grains but had stronger effects on the interior structure of the pollen grains than those induced by alpha-particle irradiation. The pollen grain germination rate, the number of pollen tubes in the styles and the average length of the pollen tubes in pollen grains treated with y-ray irradiation were decreased and shortened with increased y-rays irradiation dose, compared with that of control. However, under alpha-particle irradiation, these features initially increased at a low level of irradiation (1 Gy) and then decreased as the dose was increased. There was evident damage to F-actin structures in pollen tubes induced by the two kinds of mutagens. The mechanisms underlying the biological effects induced by alpha-particles and y-rays were different. The function and structure of pollen tubes in pollination and fertilization may be affected by the ultrastructure of pollen grains.

Comparison of in vivo and in vitro Colchicine Application for Production of Dihaploid Kirkagac and Yuva Hasanbey Melons

This study was performed to compare the efficiency of different colchicine application methods for doubling of haploid melon plants in melon breeding programs. Doubled haploids assist breeding by reducing time for the recovery of homozygous breeding lines. Irradiated pollen technique provides successful results in melons and pure breeding lines can be produced in relatively shorter time. Despite the advantages, low frequency of haploid embryos, difficulties for detecting and excising the embryos and low percentage of duplication rate are the limiting factors of this technique. The objective of this study was to determine the effect of different colchicine treatments to double haploid melon plants. The same dose of colchicine (0.5 %) was applied either in vitro or in vivo and the resulting doubled haploids were compared. The results indicated that the most convenient method was found in vivo application of colhicine (0.5 %) by shoot tip immersing method for 2 hours. The mean duplication rate was 10.13 % after in vitro colchicine application and 46.03 % after in vivo colchicine application among the genotypes of ‘Kirkagac’ and ‘Yuva Hasanbey melon groups. Key words: Irradiation, Co60, haploid, duplication, embryo rescue.

Comparison of in vivo and in vitro Colchicine Application for Production of Dihaploid ‘Kirkagac’ and ‘Yuva Hasanbey’ Melons

This study was performed to compare the efficiency of different colchicine application methods for doubling of haploid melon plants in melon breeding programs. Doubled haploids assist breeding by reducing time for the recovery of homozygous breeding lines. Irradiated pollen technique provides successful results in melons and pure breeding lines can be produced in relatively shorter time. Despite the advantages, low frequency of haploid embryos, difficulties for detecting and excising the embryos and low percentage of duplication rate are the limiting factors of this technique. The objective of this study was to determine the effect of different colchicine treatments to double haploid melon plants. The same dose of colchicine (0.5 %) was applied either in vitro or in vivo and the resulting doubled haploids were compared. The results indicated that the most convenient method was found in vivo application of colhicine (0.5 %) by shoot tip immersing method for 2 hours. The mean duplication rate was 10.13 % after in vitro colchicine application and 46.03 % after in vivo colchicine application among the genotypes of ‘Kirkagac’ and ‘Yuva Hasanbey melon groups.

Production of Haploids in Persian Walnut through Parthenogenesis Induced by Gamma-irradiated Pollen

We report the first successful regeneration of haploid lines in persian walnut (Juglans regia) developed by in situ parthenogenesis followed by embryo rescue. Female flowers of cultivars Hartley and Pedro and two native Iranian selections (Z63 and Z67) were pollinated using pollen of selections Z53 and Z30 that had been irradiated with gamma rays at five doses (50, 150, 300, 600, and 900 Gy). Gamma-irradiated pollen induced fruit set and development of some parthenogenetic embryos. The immature embryos were excised 30 and 45 days after pollination, cultured in vitro, and then stratified for 30 days at 4 °C to overcome dormancy. Ploidy level of the resulting plantlets was determined by chromosome counting and flow cytometry. Haploid plants were obtained from ‘Hartley’, ‘Pedro’, Z63, and Z67 after pollination using pollen irradiated at 300 and 600 Gy. Plants obtained from pollen irradiated at 50 and 150 Gy were all diploid. Molecular marker analysis using four simple sequence repeat (SSR) markers also showed that all the diploid plants recovered were zygotic and no spontaneous double haploid plants were obtained in this work. Also, the haploid plantlets presented only one allele of their female parents. These profiles confirmed the parthenogenetic origin of the obtained haploid plants. The techniques used to induce haploid walnut plants by irradiated pollen were successful and could be used in breeding programs and accelerate genome analysis in this plant in which the genome size is approximately three times the size of the human genome.

Comparative evaluation of different embryo rescue techniques on parthenogenetic melon (Cucumis melo L.) fruits induced with irradiated pollen.

embryos were obtained. Germination rate of haploid was 96%. When the M1, M2 and M3 were compared, the required time for opening a fruit was found to be 162, 125 and 49 min, respectively. The M3 method was identified as the most successful, economic and effective. The results are expected to lower the cost and increase the effeciency of dihaploidization use in melon breeding.

MELON BREEDING FOR RESISTANCE TO FUSARIUM WILT USING MOLECULAR MARKERS AND DIHAPLOIDIZATION

In this breeding program aimed at developing resistant varieties to Fusarium wilt in Turkish melon type, molecular markers and dihaploidization techniques were used. Cum334 was used as the donor parent in a backcross program and the haploid plants were obtained via pollination with the irradiated pollen from the BC3 population to shorten the breeding period. Four different embryo rescue methods were tested to extract haploid embryos. According to the results, the best methods were found to be inspecting seeds on the light and sowing seeds directly to nutrient media, and a total of the 122 haploid plants were obtained. The haploid plants were screened by molecular markers for fom1 and fom2 genes. A Simple Sequence Repeat (SSR) and a Randomly Amplified Polymorphic DNA (RAPD) marker were used for fom2 gene and a Cleaved Amplified Polymorphic Sequences (CAPS) and three Sequence Characterized Amplified Region (SCAR) markers were used for fom1 gene. While SSR (SSR180) did not segregate the haploid plants the RAPD marker (OPG17) segregated those haploid plants for fom2 gene. Haploid plants were screened for fom1 by a SCAR marker (SB17). Among the tested haploids, 41% of plants were found to be resistant and the rest of 59% were susceptible to the Fusarium wilt races 0 and 2. On the other hand while 34% of plants were resistant 66% were susceptible to the races 0 and 1 of the Fusarium oxysporum f.sp. melonis.