An important stage in doubled haploid (DH) production is to evaluate and to differentiate the ploidy level of regenerant plants at least two–three times during the technology. Therefore, rapid and reliable methods are necessary for particular species taken into the technology. In this study, Cucurbita pepo regenerants obtained through unpollinated ovule culture in vitro were evaluated including three different methods: direct chromosome counting in apical meristems, flow cytometry of the cell nucleus, and estimation of morphological parameters of the abaxial epidermis. Methods were optimized for each of three evaluations, and main criteria were determined for ploidy level differentiation. As a result, four ploidy levels, namely, 2n, 3n, 4n, and 8n, were defined among regenerant plants adapted to ex vitro conditions, while true haploids were only found among plants that remained in the in vitro culture. In total, 32.35%, 26.47%, 33.82%, 4.41%, and 2.94% of regenerant plants of courgette and patisson were diploid, triploid, tetraploid, octaploid, and aneuploid, respectively. According to results of flow cytometry of the cell nucleus, two cytotypes in diploid samples with DNA content of 2C = 1.07 ± 0.03 pg for courgette belonging to subsp. pepo and 2C = 0.95 ± 0.03 pg for patisson samples belonging to subsp. ovifera were revealed. The images of metaphase chromosomes of haploid, triploid, and tetraploid C. pepo specimens obtained using the propion–lacmoid chromosome staining method were presented for the first time. Parameters of abaxial epidermis in diploid samples of courgette and patisson grown in open-field and greenhouse conditions were described and compared. It was shown that the most robust parameter not depending on external factors was the number of chloroplasts in stomatal guard cells, which contained 9.41 to 11.31, 14.84 to 16.3, and up to 17.58 chloroplasts in diploid, triploid, and tetraploid samples, respectively. The application of several methods for estimation enables avoiding the misidentification of ploidy levels in adapted regenerant plants produced with the use of DH technology.

The great economic importance of sugar beet determines the ongoing biotechnological studies conducted worldwide to improve the technology of obtaining doubled haploids (DHs) using the method of unpollinated ovule culture in vitro. To improve the induction of gynogenesis, we tested the effect of thidiazuron (TDZ), temperature bud pretreatment, different concentrations of sucrose, and culturing on liquid or solid medium. Three genotypes were tested in this study. The use of TDZ at a concentration of 0.4 mg/L in solid IMB (induction medium for Beta vulgaris) induction nutrient medium with 3 g/L phytagel, 50 g/L sucrose, 200 mg/L ampicillin and cultivation at 28◦C in the dark produced up to 16.7% induced ovules. The liquid nutrient medium of the same composition induced up to 8% ovules. Increasing TDZ concentration to 0.8 mg/L resulted in reduction or total inhibition of gynogenesis, depending on the genotype. Reducing the sucrose concentration to 20 g/L or increasing it to 80 g/L was not effective. In all three genotypes, the absence of temperature pretreatment of buds (5–6 °C) showed the best results. The plant regeneration with MS nutrient medium of 20 g/L sucrose, 3 g/L phytagel, 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L gibberellic acid (GA₃) resulted in up to seven shoots from one induced ovule in the most responsive genotype. We showed by flow cytometry, chromosome counting and chloroplast number assessment that all regenerant plants were haploid (2n = x = 9).

This literature review systematizes current data on the development of triploid seedless hybrids in the Cucurbitaceae Juss family. The absence of seeds simplifies the consumption and industrial preparation of products from cucurbits. In addition, triploids showed larger plant habitus, field resistance to infections, extended shelf life, and higher fruit quality. Phenotypic differences in polyploids can stem from altered chromatin organization and gene regulation, as the nucleus must accommodate a doubled chromosome set. The triploid watermelon cultivation method developed in 1951 failed to gain traction among other crops in the gourd (Cucurbitaceae) family. The challenges of triploid seed production and use include the need for the development of tetraploid and diploid parental lines, as well as bypassing the problem of the low viability of tetraploid parent pollen and the issue of thick seed coats and underdeveloped embryos in triploids. The research findings presented in this review can be applied to the development of triploid seedless hybrids for other members of the Cucurbitaceae family.

Species Cucurbita pepo L., which includes marrow, squash, hard-barked pumpkin and a number of other crops, has a high polymorphism both in plant structure and in shape, size, color of the bark and fruit pulp. The color of the bark of the fruits of this species can be white, cream, yellow, green, orange with or without a pattern. Appearance can greatly influence the marketing success of new hybrids among the target audience. This is explained by the fact that there are strong regional consumer preferences regarding the color of fruits, although most often, on the shelves of chain stores, in fresh or canned form, you can find fruits of bright, saturated colors and shades. At the same time, brightly colored fruits are more often used for canning, while light-colored ones are used for processing into caviar [1]. Paris H.S. and Brown R.N. summarized the identification of more than 80 gene loci that affect fruit color in Cucurbita pepo L., some of which are multi-allelic [2]. The color of the fruit in hard-barked pumpkin depends on the presence and interaction between the loci responsible for this trait, and many other factors, including growing conditions [3,4]. In cucurbits, in different phenophases, different genes are responsible for color [5–7]. All this makes the selection of summer squash and scallop on the basis of the color of the fruit bark very difficult. Knowing the genetics of coloration can make this task easier. In this article, we tried to summarize the results of studies presented in foreign literature sources on the study of Cucurbita pepo L. genes responsible for the color of the surface of the fruit and pulp, its intensity, the presence or absence of a pattern of various colors and configurations. No domestic studies on this topic could be found.

The aim of this work was to study the influence of various factors (sucrose concentration, silver nitrate, activated charcoal, and plant growth regulators 2.4-D (2.4-Dichlorophenoxyacetic Acid) and TDZ (Thidiazuron)) on the induction of gynogenesis and their effect on eight genotypes of summer squash on the ploidy level of regenerated summer squash plants (Cucurbita pepo L.) in isolated ovule culture in vitro. IMC (Induction Medium for Cucurbitaceae) nutrient medium supplemented with 2 mg/L 2.4-D or 0.2 mg/L TDZ was used. It was shown that the optimal sucrose concentration for embryogenesis was 30 g/L; at 70 g/L, embryogenesis was absent. The addition of 10 mg/L AgNO₃ in combination with 2.4-D in the genotype Faraon increased the frequency of embryogenesis up to 80%. It was found that a low concentration of activated charcoal (5 mg/L) together with TDZ increased the yield of embryoids by 8.5 times (68% embryogenesis) in the genotype Sangrum. Both growth regulators effectively induce embryogenesis, but their action strongly depends on the genotype and the composition of the nutrient medium. Flow cytometry analysis of 247 regenerant plants adapted to ex vitro conditions of the studied genotypes obtained in this study showed that diploids (2n) accounted for 47.2%, tetraploids (4n) 45.7%, triploids 3.7%, and octoploids 3.4%. Genotype-specificity of growth regulator action was demonstrated: in the genotypes Sangrum and Faraon, TDZ promoted a higher yield of tetraploids, while in the genotype Yakor, 2.4-D was more effective. The obtained tetraploid lines can be used to create seedless triploid hybrids without colchicine treatment. The developed combinations of factors will allow more efficient induction of gynogenesis in summer squash in vitro.