Изучен процесс эмбриогенеза и отработаны элементы технологии получения удвоенных гаплоидов брокколи B. oleracea L. convar. botrytis (L.) Alef. var. italica Plenck в культуре микроспор in vitro. Было выявлено, что успешное развитие эмбриоидов происходит из микроспор, изолированных из бутонов длиной 4 и 5 мм, где преимущественно содержатся микроспоры-на поздней вакуолизированной, пыльца-на ранней двухклеточной стадии развития. Оптимальным режимом температурной обработки является обработка 32°С в течение первых 2-х суток после введения в культуру. Эмбриоиды были получены из пяти образцов брокколи: Arcadia F 1 , Everest, Green Valiant, Marathon F 1 , Furio. Наибольший выход эмбриоидов был получен у образца Green Valiant, где он составил до 140 эмбриоидов на чашку Петри, а наименьший-у Furio (до 3 эмбриоидов/чашку Петри). Первые деления в культуре микроспор у всех образцов наблюдали уже на 2-3 сутки культивирования. Дальнейшее развитие эмбриоидов шло по двум направлениямпутем прямого развития или с образованием суспензороподобных структур. Эмбриоиды, содержащие суспензор, развивались медленнее, чем бессуспензорные. Мы наблюдали образование эмбриоида не только на дистальном (по отношению к микроспоре) конце суспензороподобной структуры, но и образование цепочки из эмбриоидов, а также все возможные близнецовые комбинации эмбриоидов. Было показано, что метод получения эмбриоидов в культуре микроспор in vitro может быть использован не только для получения удвоенных гаплоидных растений, но и служить моделью для фундаментальных исследований по изучению этапов развития зиготических эмбриоидов и суспензоров.

Over the recent years the market demand for scaling up the production of European radish (Raphanus sativus L.) varieties and hybrids for open and protected production, varying in ripeness group, root shape and color, has drastically increased. Therefore, the expansion of genetic diversity and acceleration of the selection process are important. Doubled haploid technology considerably curtails the time required for creation of homozygous constant parental cell lines when in vitro microspore culture is used as the most promising method. For the first time, we were able to realize the full production cycle of DH plants of European radish by in vitro microspore culture up to inclusion of the produced material into the selection process. We have selected: preferable flower bud size, heat shock parameters, induction and regeneration media. It was revealed that linear length on the flower buds with the best possible stage of microspore development is genotype-specific: the flower bud length 2.8–3.3 mm is optimal for accessions of Rhodes and 3.7–4.2 mm is optimal for accessions of Teplichny Gribovsky. Heat shock at 32 °C for 48 hours is the most suitable for most genotypes. For the first time Murashige and Skoog based culture medium has been used for embryogenesis induction, and a major dependence of embryogenesis induction on the genotype × medium interaction was found. At regeneration and tiller stage it is advisable to add 1 mg/mL of benzylaminopurine and 0.1 mg/L of gibberellic acid to the medium, and rotting of micro-sprouts is performed with the use of hormone-free medium. Analysis of the produced regenerant plants by chromosome count and cell nucleus flow cytometry showed that 69 % of plants have a diploid chromosome set, 9 % have a haploid chromosome set, and 22 % have mixoploids and aneuploids chromosome sets. The seed progeny from doubled haploids and mixoploids were obtained by self-pollination, where all R1 plants had a doubled set of chromosomes. This study launches the development of an efficient method of radish doubled haploid production to be used in the selection process.

Turnip is a biennial crop and, consequently, the creation of pure lines for breeding is a time-consuming process. The production of pure turnip lines using doubled haploids produced in isolated microspore culture has not been sufficiently developed. The aim of the present work was to determine some key factors inducing embryogenesis in the isolated microspore culture of turnip, as well as investigating the manners of embryo development. It was shown that the acidity of the medium is an important factor in embryo production; different optimal pH levels ranging from 6.2 to 6.6 corresponded to individual genotypes. Such factors as the cold treatment of buds and the addition of activated charcoal to the nutrient medium increased the responsiveness of all genotypes studied. The turnip variety ‘Ronde witte roodkop herfst’ demonstrated a genetic disorder in the development of microspores; namely, non-separation of some microspores from tetrads. In the in vitro culture, each of the daughter microspores developed on its own. This indicates the dependence of the possibility of embryogenesis in the turnip microspore culture on the genotype. Results suggest that the initiation of secondary embryogenesis in primary embryos leads to an increase in the proportion of doubled haploid plants.

The process of embryogenesis in isolated microspore culture was studied in eight carrot accessions of different origin. The ½NLN-13 medium supplemented with 0.2 mg/L 2,4D and 0.2mg/L kinetin was used to induce embryogenesis. The temperature treatment was performed at 5–6 °C for three days, followed by cultivation at 25 °C in darkness. As was shown, the first embryogenesis was only observed in microspores at the late vacuolated stage when the nucleus moved from the center to one pole following the long cell axis. Depending on the nucleus position, the microspore can divide into two equal or two different sized cells. Following divisions occurred either in one of these cells or in two. However, microspores that divided into two unequal cells were morphologically different form bi-cellular pollen grain. Embryogenic divisions in bi-cellular pollen grains were not observed. First divisions began by the third day of cultivation, and continued until the globular embryoid stage that was well-seen after the fourth week of cultivation. The already-formed embryoids can develop the secondary embryoids on their surface. Depending on the genotype, up to 1000 secondary embryoids can be produced from one embryoid in the liquid MSm medium supplemented with 0.1 mg/L of kinetin for regeneration. All carrot accessions studied were split into three groups: responsive genotypes, weakly responsive genotypes, and reluctant genotypes. The highest yield was 53 initial embryoids per a 6 cm diameter petri dish. Thus, the Nantskaya 4 cultivar totally produced 256 initial embryoids, out of which 94 developed into green plantlets and 162 into albino plantlets, whereas 97 initial embryoids with 45 albino plantlets formed from them were obtained from Chantenay cultivar.

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