Получение удвоенных гаплоидов огурца (Cucumis sativus L.)

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.Разработка и внедрение клеточных технологий существенно изменили селекционный процесс у сельскохозяйственных растений во всем мире. Производство чистых линий у сельскохозяйственных культур, особенно у перекрестноопыляемых растений, таких как огурец (Cucumis sativus L.), требует больших временных и трудовых затрат, а также и достаточных финансовых вложений. В связи с этим использование удвоенных гаплоидов (DH-растений) для получения полностью гомозиготных линий в течение одного года представляет большой интерес для современной селекции, в том числе у этой культуры. Важнейшим фактором, препятствующим использованию DH-растений в селекции огурца, является отсутствие эффективного способа их производства в больших масштабах. В обзоре представлены исторические факты и рассмотрены три основных способа получения удвоенных гаплоидов огурца: партеногенеза in situ (опыление неполноценной (облученная или обработанная химическими веществами) пыльцой); андрогенеза (культуры пыльников/микроспор in vitro); гиногенеза (культура неопыленных семяпочек in vitro). Произведен сравнительный анализ публикаций с учетом эффективности использующихся технологий, выявлены критические факторы, влияющие на выход гаплоидных и удвоенных гаплоидных растений, указаны преимущества и ограничения каждой из технологий

Усовершенствование методов создания гибридов капусты белокочанной

Relevance One of the basic directions of the cabbage crop breeding is the creation of F1 hybrids with a complex of economically valuable traits. This process is difficult and time-consuming as to get pure lines must be within 6-12 years hold inbreeding. Herewith not every line gives the desired heterotic effect that also requires additional verification. Methods Biotechnological method culture of isolated microspores in vitro, which allows in the first generation to receive a line with 100% homozygosity, was used to speed up the breeding process. Combination ability were performed in complete diallel cross on the basic morphological signs. Results Culture medium for cultivation of isolated microspores in vitro was optimized for each genotype of cabbage for the best embryoids regeneration. Maximum amount of embryoids was received on medium with pH 6.2 using ampicillin 100 mg/l and zeatin 1 mg/l: 466.7 ± 153.2 PCs/100 buds. A new source material for breeding – doubled haploid lines of cabbage was received. Lines – the best parents for F1 hybrids with high yield, compact rosette of leaves, with optimum inside and short outside cabbage stump was created. Studies have shown that optimization of breeding process in case of creation of pure lines of cabbage in 3 years with microspore culture requires to reduce the breeding process in 2 times.Актуальность Одним из основных направлений селекции капустных культур является создание гетерозисных гибридов F1 с комплексом хозяйственно ценных признаков. Этот процесс является длительным и трудоемким, поскольку для получения выровненных линий необходимо в течение 6-12 лет проводить инбридинг. При этом не каждая линия дает желаемый гетерозисный эффект, что, в свою очередь, требует дополнительной проверки. Методика Для ускорения некоторых этапов селекционного процесса в работе использовали биотехнологический метод культуры изолированных микроспор in vitro, который позволяет уже в первом поколении получать линии со 100% гомозиготностью. Комбинационную способность определяли в системе полных диаллельных скрещиваний по основным морфологическим признакам. Результаты В результате работы оптимизирован состав питательной среды культуры изолированных микроспор in vitro и индивидуально подобран для каждого генотипа капусты белокочанной для наибольшего выхода эмбриоидов. Максимальный выход эмбриоидов был получен на среде с рН 6,2 с использованием ампициллина 100 мг/л и зеатина 1 мг/л и составил 466,7±153,2 шт./100 бутонов. Получен принципиально новый исходный материал для селекции – удвоенные гаплоидные линии капусты белокочанной. Выделены линии, являющиеся наилучшими исходными формами при создании высокогетерозисных гибридов F1 по урожайности, линии с компактной розеткой листьев, с оптимальной наружной и короткой внутренней кочерыгой. Проведенные исследования показали, что с учетом оптимизации некоторых этапов селекционного процесса для создания чистых линий капусты белокочанной требуется 3 года, что сокращает селекционный процесс в 2 раза

Селекция Cucumis sativus L. на устойчивость к фузариозу с применением фильтрата культуральной жидкости гриба Fusarium oxysporum Schlectend

Relevance Traditional breeding methods are based on crossing and selection of genotypes among hybrid offspring. In recent decades, along with traditional methods, more and more attention is paid to alternative methods of selection, based on biotechnological manipulations with plants. One of the most important methods of biotechnology is the method of cell selection, which is based on the replacement of the whole plant, as a unit of selection, on its cell. Applying biotechnology techniques from a single plant can produce millions of cells, which increases the chances of finding, eliminating the need for areas for the cultivation of tested plants. As well as accelerating the selection process due to the possibility to carry out the study in the offseason. Methods The studies used the linear material of C. sativus hybrids of All-Russian Scientific Research Institute of Vegetable Growing – Branch of the FSBSI Federal Scientific Vegetable Center and Agroholding "Poisk". Plants were cultivated in laboratory room conditions. As explants used hypocotyl 0.5-1 cm segments isolated from young plants. Results To obtain Cucumis sativus plants with increased resistance to Fusarium by cell selection method, it is recommended to alternate culturing of callus on a non – selective medium containing sucrose in a concentration of 30 g/l, agar – 7 g/l, 0.1 mg/l, NUC – 0.5 mg/l and the filter of the cultural fluid of the fungus in a concentration of 10% within 3 passages.Актуальность В последние десятилетия наряду с традиционными методами все больше внимания уделяется альтернативным методам селекции, в основе которых лежат биотехнологические манипуляции с растениями. Применяя методы биотехнологии из одного растения можно получить миллионы клеток, что увеличивает шансы поиска, исключая потребность в площадях для выращивания испытуемых растений, а также ускоряется селекционный процесс за счет возможности проводить исследования в межсезонье. Методика В исследованиях использовали линейный материал гибридов C. sativus селекции ВНИИО – филиала ФГБНУ ФНЦО и совместной селекции ВНИИО – филиала ФГБНУ ФНЦО с Агрохолдингом «Поиск». Материалом для исследования служили растения C. sativus, которые культивировали в вегетационных сосудах в условиях лабораторного помещения. В качестве эксплантов для получения пролиферирующей каллусной ткани, способной к морфогенезу, использовали гипокотильные сегменты размером 0,5-1 см, изолированные от молодых растений. Результаты Для получения растений Cucumis sativus L. с повышенной устойчивостью к фузариозу методом клеточной селекции рекомендуется чередование культивирования каллуса на неселективной и селективной средах, содержащих сахарозу в концентрации 30 г/л, агар – 7 г/л, БАП – 0,1мг/л, НУК – 0,5 мг/л и фильтрат культуральной жидкости гриба F. oxysporum в концентрации 10% в течение 3-х пассажей

Ускоренное создание гомозиготных линий листовых культур семейства Brassicaceae Burnett в культуре микроспор in vitro

Relevance Biotechnological methods are generally used to speed up breeding programs and to enhance genetic diversity, so the culture of isolated microspore in vitro can be regarded as one of very suitable methods. Nontraditional and uncommon vegetable crops belonging to Brassicaceae Burnett. are becoming more popular. Methods Accessions of sarepta mustard (Brassica juncea L. Czern.) and rocket salad (Eruca sativa Mill.) were taken for the study with the aim to optimize the basic protocol for these species. Results As a result of the study the optimum cultivation conditions have been determined for the species. Sizes of buds 2.5-3.5 mm long for sarepta mustard and 7.0-7.5 long for rocket salad which were used for cultivation had been experimentally defined. It was also shown that the cold pretreatment had improved the embryo yield. The nutritional NLN-13 medium with pH 6.1 and pretreatment at 32°C during a cultivation day had been shown to be more favourable for all accessions. All conditions that had been used were suitable for embryo formation. First divisions had been seen after 4 days of cultivation, while the embryos at primary cotyledonary stage only appeared after 2 weeks of cultivation. The embryo yield per 5 buds reached 25-30 and 5-7 in the sarepta mustard and the rocket salad, respectively. It is worth noticing that the root formation and plant adaptation had passed better and faster in sarepta mustard than in rocket salad. Thus, whole process of homozygous line developing can be completed for 4-5 months, making the breeding program 3 times shorter.Актуальность Для ускорения селекционного процесса и расширения генетического разнообразия необходимо вовлекать в селекционный процесс биотехнологические методы, одним из которых является метод культуры изолированных микроспор in vitro. Все большую популярность в России приобретают нетрадиционные, малораспространенные зеленные культуры семейства Brassicaceae Burnett. Материал и методика Объектами исследования были такие представители данного семейства, как горчица сарептская (Brassica juncea (L) Czern.) и индау посевной (Eruca sativa Mill.). Цель исследований заключалась в оптимизации базового протокола под изучаемые культуры. Результаты Были подобраны оптимальные условия культивирования для горчицы сарептской и индау посевного. Опытным путем был определен оптимальный для введения в культуру in vitro размер бутона, который составил у горчицы сарептской 2,5-3,5 мм, а у индау посевного – 7,0-7,5 мм. Показано, что холодовая предобработка бутонов в течение 1 суток значительно повышает выход эмбриоидов. Для большинства образцов наиболее благоприятным оказалось сочетание pH среды NLN-13, равное 6,1, и температурная обработка 32°С в течение 1 суток культивирования. Все вышеперечисленные условия культивирования способствовали успешному развитию эмбриоидов. Первые деления были отмечены на 4 сутки, а уже через 2 недели эмбриоиды находились на начальной семядольной стадии развития. Выход эмбриоидов достигал у горчицы сарептской – до 25-30 шт./5 бутонов, а у индау посевного – до 5-7 шт./5 бутонов. Отмечено, что у горчицы сарептской происходит быстрое укоренение и адаптация к условиям in vivo. То есть весь процесс получения чистой гомозиготной линии составляет 4-5 месяцев, что сокращает селекционный процесс в более чем в 3 раза

Production of Doubled Haploids in cucumber

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

Improvement of methods of creating hybrids of cabbage

Relevance One of the basic directions of the cabbage crop breeding is the creation of F1 hybrids with a complex of economically valuable traits. This process is difficult and time-consuming as to get pure lines must be within 6-12 years hold inbreeding. Herewith not every line gives the desired heterotic effect that also requires additional verification. Methods Biotechnological method culture of isolated microspores in vitro, which allows in the first generation to receive a line with 100% homozygosity, was used to speed up the breeding process. Combination ability were performed in complete diallel cross on the basic morphological signs. Results Culture medium for cultivation of isolated microspores in vitro was optimized for each genotype of cabbage for the best embryoids regeneration. Maximum amount of embryoids was received on medium with pH 6.2 using ampicillin 100 mg/l and zeatin 1 mg/l: 466.7 ± 153.2 PCs/100 buds. A new source material for breeding – doubled haploid lines of cabbage was received. Lines – the best parents for F1 hybrids with high yield, compact rosette of leaves, with optimum inside and short outside cabbage stump was created. Studies have shown that optimization of breeding process in case of creation of pure lines of cabbage in 3 years with microspore culture requires to reduce the breeding process in 2 times

Selection of <i>Cucumis sativus</i> L. for resistance to fusarium wilt using filtrate of the culture fluid of the fungus <i>Fusarium oxysporum</i> Schlectend

Relevance Traditional breeding methods are based on crossing and selection of genotypes among hybrid offspring. In recent decades, along with traditional methods, more and more attention is paid to alternative methods of selection, based on biotechnological manipulations with plants. One of the most important methods of biotechnology is the method of cell selection, which is based on the replacement of the whole plant, as a unit of selection, on its cell. Applying biotechnology techniques from a single plant can produce millions of cells, which increases the chances of finding, eliminating the need for areas for the cultivation of tested plants. As well as accelerating the selection process due to the possibility to carry out the study in the offseason. Methods The studies used the linear material of C. sativus hybrids of All-Russian Scientific Research Institute of Vegetable Growing – Branch of the FSBSI Federal Scientific Vegetable Center and Agroholding "Poisk". Plants were cultivated in laboratory room conditions. As explants used hypocotyl 0.5-1 cm segments isolated from young plants. Results To obtain Cucumis sativus plants with increased resistance to Fusarium by cell selection method, it is recommended to alternate culturing of callus on a non – selective medium containing sucrose in a concentration of 30 g/l, agar – 7 g/l, 0.1 mg/l, NUC – 0.5 mg/l and the filter of the cultural fluid of the fungus in a concentration of 10% within 3 passages

Embryogenesis induction of carrot (Daucus carota L.) in isolated microspore culture

Haploid technologies are used to create homozygous lines for accelerated breeding. We aimed to optimize the technology for using the isolated microspore culture in vitro to obtain doubled haploids of the carrot (Daucus carota L.). We studied two carrot varieties with different responsiveness to embryogenesis, Altajskaya lakomka and Breeding line 17. Carrot microspores were isolated from buds and cultivated in liquid nutrient media supplemented with an antibiotic and activated carbon in vitro. They were exposed to different thermal treatments. The experiment showed the benefits of combining cold pre-treatment of buds (5°C for 1 day) with heat shock of isolated microspores in vitro (32°C for 2 days). The induction of embryogenesis on the NLN-13 medium was twice as high as on the MSm-13 medium. The use of 1% activated carbon in 0.5% agarose increased the yield of embryoids by more than 1.5 times. 100 mg/L of ampicillin was found to be the most efficient concentration. After 30 days of cultivation under optimized conditions, the yield was 161.3 and 44.0 embryoids per Petri dish for the cultivar Altajskaya lakomka and Breeding line 17, respectively. The induction of carrot embryogenesis is determined by the type and duration of thermal stress, the composition of the nutrient medium, the use of activated carbon as a sorbent, the addition of β-lactam antibiotics, and the type of explant exposed to thermal treatment. Our technology enabled us to obtain homozygous doubled haploid lines of carrots during a year, and these lines were included in the breeding process to create F1 hybrids

Rapid development of homozygous lines through culture of isolated microspores in leafy crops of <i>Brassicaceae</i> Burnett

Relevance Biotechnological methods are generally used to speed up breeding programs and to enhance genetic diversity, so the culture of isolated microspore in vitro can be regarded as one of very suitable methods. Nontraditional and uncommon vegetable crops belonging to Brassicaceae Burnett. are becoming more popular. Methods Accessions of sarepta mustard (Brassica juncea L. Czern.) and rocket salad (Eruca sativa Mill.) were taken for the study with the aim to optimize the basic protocol for these species. Results As a result of the study the optimum cultivation conditions have been determined for the species. Sizes of buds 2.5-3.5 mm long for sarepta mustard and 7.0-7.5 long for rocket salad which were used for cultivation had been experimentally defined. It was also shown that the cold pretreatment had improved the embryo yield. The nutritional NLN-13 medium with pH 6.1 and pretreatment at 32°C during a cultivation day had been shown to be more favourable for all accessions. All conditions that had been used were suitable for embryo formation. First divisions had been seen after 4 days of cultivation, while the embryos at primary cotyledonary stage only appeared after 2 weeks of cultivation. The embryo yield per 5 buds reached 25-30 and 5-7 in the sarepta mustard and the rocket salad, respectively. It is worth noticing that the root formation and plant adaptation had passed better and faster in sarepta mustard than in rocket salad. Thus, whole process of homozygous line developing can be completed for 4-5 months, making the breeding program 3 times shorter