Relevance. In onion breeding, quickly obtain aligned material is an urgent target for create parent forms of heterosis hybrids. Using classical methods with helping of backcrosses, this is achieved in 10-12 years. Using the technology of doubled haploids, it is possible to reduce these terms several times, and also to avoid the manifestation of inbred depression when obtaining lines by self-pollination. At the same time, the most effective in the production of haploids is the use of whole flower buds as an explant, unlike ovules and ovaries, the production of which is more time-consuming and labor-intensive.Methods. The doubled onion haploids were obtained by the method of ovule culture on the basis of the biotechnology laboratory of the Gavrish Breeding Center using the technology that based on the methodological recommendations of Monakhos S.G. et al., 2014. DH–onion plants with a developed root system and leaf apparatus were planted in the open ground and grown according to the technology generally accepted for the zone at the sites of the Gavrish breeding center, Krymsk, Krasnodar Territory. The obtained commercial bulbs were evaluated according to the RTG/46/2 method.Results. It was obtain doubled haploids, which were tested for ploidness by flow cytometry. 40 digaploid onion plants have been created. As a result of further development after transplantation into the open ground, storage, springization and selection, 3 uterine bulbs were obtained for further reproduction and inclusion in the breeding process.

Relevance. To create an effective technology for obtaining doubled haploids (DH-technology) of zucchini in unpollinatedseedpod culture in vitro it is necessary to select the optimal values of many factors, the degree of influence of each of which on gynogenesis can vary significantly. The aim of the study was to optimize the individual stages of the technology.Methods. Liquid and agarized (7 g/L) IMC medium with different sucrose concentrations (20 to 80 g/L) and different plant growth regulators (2 mg/L 2,4 D; 0.2 mg/L TDZ ; 0.8 mg/L 2,4 D and 1.2 mg/L NUC) were used for induction of embryogenesis.Results. Optimal for the studied zucchini genotypes was pre-isolated from the evening, plucked in the morning opened bud. Sterilization of zucchini ovaries by short-term burning after treatment with 96% alcohol, allows significant reduction of the time required for this step without loss of embryogenic potential. IMC nutrient medium with sucrose (20 to 40 g/l) can be used for induction of gynogenesis in the unpollinatedzucchini ovary culture. The use of nutrient media with 2 mg/l 2,4 D for most genotypes was more effective and resulted in higher number of embryoids compared to nutrient media containing 0.2 mg/l TDC and media with 0.8 mg/l 2,4 D and 1.2 mg/l NAA. Embryoid formation was observed after 5 weeks of cultivation.Conclusion. We were able to complete the full cycle of technology for obtaining doubled haploids in unpollinatedseedpod culture in vitro for 30 zucchini genotypes and obtain DHplants, which are valuable source material for both breeders and genetic research. Optimization of the individual steps of the technology made it possible to achieve the maximum result for individual genotypes – 55 embryoids per 100 cultivated ovules.

Doubled haploids have been widely used worldwide in breeding programs and fundamental research as valuable homozygous material for about 100 years. The species Cucurbita pepo L. are represented by a huge variety of forms, include highly productive vegetable crops and have a wide distribution in the world. Despite the great economic importance, the creation of effective protocols to ensure stable production of doubled haploids in this species remains an urgent task. DH plants are of interest not only because of the acceleration of the breeding process, but also because of the realization of the huge potential of gametoclonal variability inherent in this highly polymorphic species. In this review, we analyzed the main technologies used for obtaining doubled haploids in vegetable crops of C. pepo: parthenogenesis in situ stimulated by treated/irradiated pollen, gynogenesis in vitro (unpollinated ovule culture in vitro) and androgenesis in vitro (anther/microspore culture in vitro). An analysis is presented of the research carried out from the beginning of the discovery of haploid plants to the current advances and evaluation of the prospects in the field of DH plant production. The main critical factors influencing the efficiency of each technology and its individual steps are considered. The developed technology of doubled haploids obtaining using non-pollinated ovary culture in vitro is presented. This technology allows to obtain up to 55 embryoids per one cultivated ovary (28 embryoids/ 100 cultivated ovules) To introduce haploid technologies into the breeding process it is necessary to evaluate the obtained plants for ploidy level. The use of direct counting of chromosomes in apical cells may present a certain difficulty in this species due to their large number (2n=40) and their small size. Depending on the level of laboratory equipment, ploidy determination using flow cytometry of cell nuclei and counting the number of chloroplasts in stomatal guard cells in the epidermis of the abaxial side of the leaf may be more convenient methods. The prospects for the use of molecular markers for assessment for homozygosity in DH technologies used, including C. pepo, are discussed in the review.

Relevance. The development of F1 hybrids distinguishing it from cultivars by high productivity, plant uniformity in ripening date, fruit sizes and quality is the promising trend in breeding program in cucumber (Cucumis sativus L.).The aim of the study was to optimize the gynogenesis induction condition in culture of unpollinated ovules in vitro in order to broad the generation of new breeding forms and to accelerate homozygous line production.Materials and methods. Eight promising cucumber accessions from Laboratory of Cucurbit Breeding and Seed Production (FSBSI FSVC) were taken for the study. The protocol developed in Laboratory of Biotechnology (FSBSI FSVC) for production of doubled haploid in Cucurbitaceae family was used in the experiment. The medium IMC with 30 g/L sucrose and 7g/L agar supplemented with 200 mg/L ampicillin and 0.2 mg/L thidiazuron (TDZ) was applied to induce gynogenic development.Results. The half-open bud or flower was shown to be the most suitable to be taken as an explant for cultivation. Highest number of embryo-like structures in all accessions developed from ovaries 2.1-2.6 cm long. Exposure to sterilization solution of sodium hypochlorite for 15 min made ovary wall softer and ovules can be then easily extracted without traumatizing. The traumatized ovule resulted in inhibited gynogenic development. Embryoids and calli had developed in all studied cucumber accessions, but well-formed plants were only obtained in six accessions. In total 26 plants were produced. The maximum gynogenesis induction equal to 63.1% was achieved in accession 1810. Maximum number of plant produced was twelve in accession 1763, but the greatest plant outcome 7.7% of the ovules with induced gynogenesis was observed in accession 1807.

Relevance. The induction of gynogenic development of the culture of unpollinated ovules of Cucumis sativus L., as a part of solid nutrient media agar-agar or Phytagel™ are used as a gelling agent. The gelling agent determines the mobilization of substances in the composition of the nutrient medium and has various effects on the explants, which affects the quality of the resulting regenerates. However, there are no scientific works that explaines the effect of these gelling agents on the development of ovules and compare them with each other for C. sativus L. The aim: investigate the effect of various gelling agent in the nutrient medium on the induction of gynogenesis and the development of cucumber unpollinated ovules.Materials and methods. There are two promising collection specimens of cucumber №58 and №831 of the laboratory of cucurbits crop breeding and seed production of FSBSI FSVC were included into research. IMC nutrient medium (Induction Medium for Cucurbitaceae) with 30 g/l sucrose, 200 mg/l ampicillin, 0.2 mg/l thidiazuron (TDZ) was used for the induction of gynogenesis; agar-agar at a concentration of 7 g/l or Phytagel™ at a concentration of 3.5 g/l was used as a gel-forming agents. Ovules were isolated from ovaries in the phase of half-opened flower (FL-1) and fully opened flower (FL). Cultivation was carried out in plastic Petri dishes with a diameter of 60 mm with an air gap of 28.8 cm3 – "KS No. 1", and glass culture jars with an air gap of 140 cm3 – "KS No. 2".Results. On containing Phytagel™ or agar-agar nutrient medium, the area of ovules during 30 days of cultivation increases irregularly. Coefficients of ovule enlargement between gelling agents differed from 1.7 to 2.6 times depending on the cultivation time. The average growth rate of ovules on media with Phytagel™ was 0.08 mm2/day, while on media with agar-agar it was 0.02 mm2/day. Gelling agents type and cucumber phenotype are significant factors affecting the increase in area of entered into unpollinated cucumber ovules culture. Herewith the share of the gelling agent effect was 55.01%, and the share of genotype effect was 14.53%. The effect of flower development stage or culture vessel type has not found for both of genotypes. In the study, it was possible to achieve the induction of gynogenesis in 67% of the unpollinated ovules genotype №831 on nutrient medium with agar-agar.Conclusion. Ovules development were faster on a nutrient medium using Phytagel™ as a gelling agent than on agaragar. At the same time, the percentage of induced ovules was significally higher on nutrient medium with agar-agar for both phenotypes. Gelling agents type and cucumber phenotype were found as significant factors of the induction and the development of unpollinated ovules.

Implementation of cell technologies has essentially improved the plant breeding process in agricultural crops in the world. The production of pure lines in cultivated crops, particularly among cross-pollinated species such as cucumber (Cucumis sativus L.) requires much time, labor and expense. Thus, the use of DH-plants for production of fully homozygous lines for one year becomes a very promising method for near cucumber breeding program. The major factor limiting the wide use of DH is a lack of effective protocol for large-scale plant production. In this review the historical facts with description of three main methods of DH-plant production were presented. By now these three methods have been such as parthenogenesis in situ induced by pollination with irradiated or chemically treated pollen; androgenesis in vitro including anther and isolated microspore cultivation in vitro; gynogenesis through ovule cultivation in vitro. Comparative analysis of published data with regard to the efficiency of the technology for DH-plant production was shown as well as advantages and limitations of each technology were described.

147 new forms were obtained only from nine responsive unpollinated ovules of summer squash in 2015-2017. The cytological analysis is used to estimate the level of ploidy in regenerated plants R0. The small size of mitotic chromosomes and large number in Cucurbita genus make it difficult to count them even though they are well separated. As a result of analysis the optimal method of chromosome staining has been chosen with the use of modified propiono- lacmoid cytological technique. The rootlet tips and apical meristems were used to make smear preparations. As it was shown the summer squash was a difficult species as a cytological object because of low mitosis frequency and low number of metaphase plates with well scattered chromosomes. The photos of chromosomes in squash C. pepo subsp. brevicaulis var. giraumons Duch; distant hybrid (breeding accession N37) between variety ‘Cornishon’ and winter squash C. pepo subsp. longicaulis Greb. var. pepo and doubled haploid plants produced from them were made. In spite of the small size 2 μm the chromosomes observed were clearly seen. Nearly all regenerated plants that had been analyzed passed well the adaptation in vivo and occurred to be a doubled haploids (2n = 2x=40). The seed progeny was then obtained through self-pollination. About 20% of plants R0 analyzed were mixploids and supposedly only 7% were haploids.

The culture of unpollinated ovules in vitro in summer squash was used to develop fully homozygous breeding lines with the aim of the speeding-up breeding program. As a result of assessment for economically valuable traits, the seven promising DH-lines obtained from summer squash accessions differed by fruit shapes and colours were selected out. All breeding lines produced showed high homogeneity that retained in following generations and also have an appropriate set of economically valuable traits. DH-lines belonging to female type have up to 96% female flowers and only 4% male flowers. It is very important for breeding when the male flowers appeared in two weeks just after the female flower began blooming. The development of morphologically abnormal female and male flowers, along with gynandromorphy flowers was noted on selected DH-lines. During vegetation period from 26 to 36 flowers appeared on the plant, where out of them 19-21 ones were normally developed female flowers, 3-5 ones were normally developed male flowers, and up to 10-11 ones showed an abnormal way of development. The percentage of abnormal flowers stayed invariable when growing in greenhouse condition with high humidity and temperature as well as in open field condition. As it was shown the development of deformed abnormal flowers inherited and manifested in the following generation after self-pollination. As a result of the study, the occurred anomalies in course of male and female flower development in summer squash (C. pepo L.,) DH-lines produced through a cultivation of unpollinated ovules in vitro were described in details for the first time.

The perspective of utilization of in vitro culture of unpollinated ovules for development of dihaploid lines of pumpkin is described in the article. The ovules of five hybrids were cultivated on CBM medium. The following combinations of growth regulators for gynogenesis induction were used: a) 0.2 mg/L of thidiazuron and 0,0001 мкМ of epibrassinolide; b) 2 mg/l of 2,4-D. The frequency of embryo development depended on cultivation medium and genotype. The maximum amount of embryos developed from unpollinated ovules of one ovary was 6-9 ones.

The role of plant extracts in the process of induction of embryogenic structures and its following development in plants is revealed. Thus, the addition in the culture medium of the extracts diluted in DMSO increases the rate of gynoand androgenesis on 2.4% as compared with control. Experimentally proved that the plant extract derived from the reproductive organs of certain genotype increases the morphogenetic potential of isolated organs only of the same genotype.