Dihaploidization in Promising Summer Squash Genotypes ( Cucurbita pepo L.) via Irradiated Pollen Technique

: Summer squash ( Cucurbita pepo L.) is one of the important vegetable species preferred by the people of Turkey in terms of their nutritional value. F1 hybrid summer squash varieties are widely used both in the open field and protected cultivation. The majority of these varieties come from abroad, and our dependence on abroad continues. In this study, the effectiveness of the dihaploidization method, which has an important place in variety breeding studies, was investigated to reduce our import dependency and to produce new F1 hybrid varieties that are high yielded, quality, resistant to diseases and pests. In this context, 14 summer squash genotypes


Introduction
Turkey allows for the cultivation of many vegetable species related to the Cucurbitaceae family with its favorable climatic conditions and our total summer squash production is 474.527 tons (TÜİK, 2018). Cucurbita pepo L., defined as summer squash, has a very wide range in our country's food culture. Although squash is generally produced for fruit, its seeds are also used as appetizers, high-quality edible oil, and food production materials, which are demanded in the market, thus it is cultivated in both open field and greenhouse conditions. Some squash populations with mostly domestic names exist in many regions in Turkey. However, these local varieties are preferred their taste and flavor, they are not at the desired agricultural traits as high-yield, quality, and especially resistant to disease and pests. Thus, superior F1 hybrid summer squash varieties are widely used in vegetable production. As in many vegetable varieties, F1 hybrid summer squash varieties are mostly imported and our foreign dependency continues. Reducing foreign dependency on seed production is among the priority subject in Turkey. In this context, the use of the dihaploidization technique will provide a great convenience in breeding efforts to reduce our foreign dependency in summer squash, to reveal our domestic varieties that are high yielded, quality, resistant to diseases and pests, and to obtain new F1 hybrid varieties.
The dihaploidization technique is one of the plant tissue culture techniques and they are widely used in F1 hybrid seed production. The most important advantage of this technique shortens the cultivar breeding process. It also increases breeding efficiency by reducing the size of the population that needs to be addressed to achieve the targeted genotype. In classical breeding, the fact that selection cannot be done effectively in early generations is an important problem. Achieving double-haploid plants in a short time with the dihaploidization technique enables an effective selection in early generations and accelerates responding to changing consumer demands (Forster and Thomas, 2005).
Classical breeding processes take a long time because the flower types of the species that belong to the Cucurbitaceae family have very different characteristics (monoic, andromonoic, gynoic, gynomonoic, hermaphrodite) and naturally high rate of open-pollinated. However, the haploid plant obtaining technique, which has been used successfully in some species instead of classical breeding, has important advantages. The purpose of dihaploidization (DH) technique is to obtain male (antherpollen culture), female (ovule -ovary culture), and/or parthenogenesis (irradiated pollen technique) originating haploid (n) plants and to generate 100% pure (homozygous) lines (2n) by doubling the chromosome numbers of haploid plants with various antimitotic agents (colchicine, oryzalin, trifluralin, amiprofos methyl). Thus, the purification process, which takes many years (8-10 years) especially in open-pollinated species, can be carried out in a shorter time (1-2 years). The breeding of hybrid cultivar can be completed in a short time with the desired agronomic traits and high combination ability from these pure lines obtained.
Considering the species of the Cucurbitaceae family, irradiated pollen is the most used technique in dihaploidization process. In this technique, the pollen is irradiated by different irradiation source (usually with Co 60 -induced gamma-ray), but since the germination capabilities continue, it stimulates the female organ and causes the parthenogenetic haploid embryos. With the use of this technique, successful results have been obtained in watermelon (Gürsöz et al., 1991;Sarı et al., 1994), melon (Sauton and Dumas de Vaulx, 1987;Sarı et al., 1992;Abak et al., 1996;Lotfi et al., 2003), cucumber (Truong-Andre, 1988;Sauton, 1989;Çağlar and Abak, 1999;Dolcet-Sanjuan et al., 2006), summer squash (Kurtar, 1999;Kurtar et al., 2002;Berber, 2009;Bektemur et al., 2014;Kurtar et al., 2017), pumpkin (Kurtar et al., 2009) and winter squash (Kurtar and Balkaya, 2010) species. The most important factor that restricts the effectiveness of the irradiated pollen technique is genotype dependence. Besides, some factors such as irradiation and pollination studies, the composition of the media, culture conditions, embryo development stages, and growing season and conditions of donors can also affect the haploidy frequency.
In this study, it is aimed to determine the effectiveness of the dihaploidization process on producing pure lines in summer squash selected from our gene pool via irradiated pollen technique.

Materials and Methods
The research was carried out in the laboratory and greenhouse of Selcuk University Faculty of Agriculture, Department of Horticulture, Vegetable Growing and Breeding in 2018 and 2019. Seeds of 14 genotypes obtained from the gene pool of this department were sown in trays filled with peat-moss on May 4, 2018, and seedlings at the 2-3-leaf stage were planted in the greenhouse at a distance of 1.2 x 1 m on 21 May 2018. Twenty plants were used for each genotype (Figure 1). In the growing period, considering the soil analysis results, 8 kg N (Nitrogen), 6 kg P2O5 (Phosphorus), and 7 kg K2O (Potassium) were supplied per decare with drip irrigation, and pest and disease management were applied regularly. Anthers were collected one day before the anthesis from genotypes when the plants were in the full-flowering period and the female flowers to be pollinated the next day were isolated with cloth bags. The anthers were irradiated on the same day with a dose of 150 Gy gamma rays in TAEK (Turkish Atomic Energy Authority) ( Figure 2). The isolated female flowers were pollinated with irradiated pollen the next morning and re-isolated to prevent undesired pollen contamination. After 2-3 days of pollination, cloth bags were removed and the fruits were labeled. About 3-4 weeks after pollination, the fruits were harvested before they were not fully ripened and the embryos were not aged and lost their regeneration ability. Harvested fruits were washed under tap water before extraction, cleaned from external contamination, weighed on a digital scale with a sensitivity of 2 g after drying. Then the fruit stems were removed and fruits were soaked in a 20% commercial bleach solution for 30 min. Finally, after thoroughly pulverizing with 96% ethyl alcohol under a sterile bench, surface sterilization was performed by burning. The surface-sterilized fruits were carefully cut with the help of a sterile knife without damaging the seeds, and all the seeds (except very small, dice or scar) were opened one by one and the embryos were cultured.
All the embryos obtained from the opened seeds, less than half the seed size were taken into culture tubes containing approximately 10 ml of modified E20A medium. Because, according to our previous experiences, embryos larger than half the seed size have formed completely diploid plants. During this period, embryos were kept in the climate room, whose temperature was 28 ± 1 o C, the photoperiod was 16/8 day-night, and the illumination was set to 5000 lux with daylight type fluorescent lamps (Kurtar and Balkaya, 2010) (Figure 3). The composition of the modified E20A medium is presented in Table 1.  To prevent the loss and increase the number of plants, micro-propagation was carried out in developed plants (had a favorable root and shoot ratio) under laboratory conditions for 2-3 times. Micro-propagation was realized in MS medium (Murashige and Skoog, 1962) with the addition of 0.1 mg l -1 IAA and 1.0 mg l -1 BAP (Figure 4).
The plantlets were acclimatized to the open field conditions as reported by Kurtar and Balkaya (2010). The ploidy levels of the plants were determined by both stomatal (stoma number and stoma size) and morphological observations. As a result of ploidy analyses, haploid plants were doubled by applying 1% colchicine solution to the shoot-tips of the plants (

Data analyses
Since the number of fruits of the genotypes is not equal and a different number of materials are used, only the averages of the data are provided.

Effects of pollination with irradiated pollen on fruit weight, seed formation, and embryo stimulation
In pollination studies, the number of pollinated flowers (PF), the number of fruit set (FS), the fruit set rate (FR), and the average fruit weight (AW) were evaluated and presented in Table 2. As a result of the pollination studies, 165 flowers were pollinated, 64 fruits were obtained from the pollinated flowers and the fruit set rate was 38.8%. Fruit set ratio varied between 20% (G6) and 61.5% (G7) and the average fruit weight (AW) ranged from 1650 g (G6) to 4465 g (G4). As a result of the extraction of 64 fruits, 7034 seeds and 521 embryos were obtained. The average number of seeds per fruit was 109.9 and the number of embryos per fruit was 8.14. Of the 521 embryos obtained, 144 transformed into a complete plant and the conversion rate to the plant was 27.6%. Among the genotypes, the average number of seeds ranged from 69 (G8) to 183 (G9), the number of embryos per fruit was between 2.2 (G14) and 14.7 (G3), and the conversion rate to the plant was between 19.0% (G2) and 38.2% (G12) ( Table 3). These results are following our previous studies in the summer squash (Kurtar, 1999;Kurtar et al., 2002;Kurtar et al., 2017).

Ploidy analyses and dihaploidization process
The ploidy levels of the plants were determined by both stomatal (stoma number, stoma size) and morphological observations. While the average stoma length and stoma widths were 32.24 and 21.67 μm in diploids, these values were determined as 20.41 and 16.84 μm in haploids. While the number of stomata per unit area (mm 2 ) was 306.2 in diploids, it was 435.1 in haploids.
While diploid plants formed larger leaves and habitus, haploid plants had smaller leaves and plants. Besides, pollen formation was not observed in the anthers of haploid plants (Figure 7). Similar results have been found in the previous studies in squash (Kurtar, 1999;Kurtar et al., 2002;Kurtar et al., 2017), pumpkin (Kurtar et al., 2009), and winter squash (Kurtar and Balkaya, 2010).  The double-haploid plants have been shown to very different growth characteristics and it has been demonstrated that the dihaploidization technique is also important for creating variation beyond obtaining pure lines (Figure 8).

Conclusion and perspective
The dihaploidization technique, which enables obtention of pure homozygous lines in many vegetable types and greatly shortens the breeding time, improves the breeding efficiency, and is successfully applied in the summer squash. However, the most important factor preventing the widespread use of the technique is that it is genotype-dependent and the haploid frequency shows a large variation among the genotypes.
It has been observed that the dihaploid lines we have obtained have very different growth characteristics and that the dihaploidization technique is an important technique for creating variation besides pure line production. In the light of our results, we believe that it will increase the success of working with genotypes with high haploid frequency, hybridization between high frequency and lowfrequency genotypes, the spread of irradiation and pollination studies to a wider vegetation period in future studies. Also, as an alternative to the irradiated pollen technique, anther-microspore or ovuleovary cultures should be engaged (Kurtar et al., 2020).
In this context, our goals are to continue irradiation and pollination activities with high haploid frequency genotypes, on the other hand, to produce suitable candidates for the breeding of F1 hybrid summer squash with promising dihaploid lines that have desired agronomic traits we obtained as a result of the study.