The unique and balanced components of the biochemical composition, together with high antioxidant activity, make the red beet necessary a dietary vegetable crop, much contributing to healthy food ration. The application of the technology for producing gynogenic plants in vitro increases the genetic diversity and significantly reduces the period of time required to obtain the appropriate homozygous lines used to create the F1 hybrids that are demanded in the market. For induction of gynogenesis, we used IMB medium developed by us with the addition of 55 g/L sucrose, 3 g/L phytogel, 200 mg/L ampicillin, and 0.4 mg/L thidiazuron (TDZ) and cultured at 28 °C in the dark for 4–6 weeks. Shoot regeneration from embryoids and callus was performed on MS medium with 20 g/L sucrose, 3 g/L phytogel, 1 mg/L 6-benzylaminopurine (BAP), and 0.1 mg/L gibberellic acid (GA₃). Immersion of the obtained microshoots with 5–7 well-developed leaves for 10–15 s into concentrated sterile indole-3-butyric acid (IBA) solution (50 mg/L) followed by their cultivation on solid medium ½ _IMB with 2% sucrose and 3 g/L phytogel was the most efficient method for root formation. The addition of silver nitrate (22 mg/L) to the nutrient medium provoked an increase in the number of induced ovules up to nine per Petri dish (up to 25% of induced ovules). Gynogenic development was produced in six out of 11 genotypes studied, and the plants that were then acclimatized to ex vitro conditions were obtained in three genotypes (Nezhnost’, Dobrynya, b/a 128). The evaluation of ploidy of gynogenic plants that was carried out by flow cytometry and direct counting of chromosomes stained with propion-lacmoide revealed that all obtained gynogenic plants were haploids (2n = x = 9).

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).