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Development of multiplex PCR system for identification of glyphosate-tolerant sugar beet | Создание мультиплексной системы ПЦР для идентификации сахарной свеклы, толерантной к воздействию глифосата | Розроблення мультиплексної системи ПЛР для ідентифікації цукрових буряків, толерантних до дії гліфосату Texte intégral
2016
Присяжнюк, Л. М. | Шитікова, Ю. В. | Волчков, О. О.
Purpose. To create a multiplex system for identification glyphosate-tolerant sugar beet by using PCR. Methods. Molecular genetic analysis. Results. The article presents the results of studies to determine the parameters of the polymerase chain reaction (PCR) in order to develop a multiplex system for identification of the structural elements of the design of transgenic gene cp 4 epsps, which provides tolerance to glyphosate. For amplicon target DNA sequences, the following values of temperature conditions of PCR were determined: step 1 (initial denaturation) 95 °C – 3 min; step 2 (specific reaction products accumulation): denaturation 95 °C – 45 s; hybridization of primers 55 °C – 50 s; elongation 72 °C – 1 min; number of cycles – 40; step 3 (final elongation) 72 °C – 6 min. A series of PCR were carried out for the purpose of selecting the optimal amount of DNA matrix for efficient estimate of transgenic sugar beet plants for the presence of specific sequences. Conclusions. To identify transgenic glyphosate-tolerant sugar beet, it is advisable to determine 35S promoter and gene cp 4 epsps in individual genotypes. It was found that during the selection of temperature parameters of multiplex reaction a 5 °C rise in primer hybridization temperature did not affect the identification of gene als that allowed to include specific primers for determination of this sequence as an internal control. Based on the results of test multiplex reactions, concentrations of dNTPs and Mg2+ ions were determined that allowed to exclude the possibility of non-specific fragments and false-negative results. The optimum amount of matrix DNA (100–150 ng) for an efficient estimate of transgenic sugar beet plants for the presence of specific sequences was determined. Obtained results allowed to develop a multiplex test system for identification of transgenic glyphosate-tolerant sugar beet which can be used for simultaneous determination of the 35S promoter, cp 4 epsps gene and als gene as an internal reaction control. | Цель. Создать мультиплексную систему для идентификации толерантной к глифосату сахарной свеклы с помощью ПЦР. Методы. Молекулярно-генетические методы анализа нуклеиновых кислот. Результаты. Приведены результаты исследований по определению параметров полимеразной цепной реакции (ПЦР) для разработки мультиплексной системы по идентификации структурных элементов трансгенной конструкции с геном сp 4 epsps, что обеспечивает толерантность к глифосату. Для получения ампликонов целевых последовательностей ДНК определены следующие значения температурных режимов ПЦР: шаг 1 (начальная денатурация) 95 °С – 3 мин; шаг 2 (наработка специфических продуктов реакции): денатурация 95 °С – 45 с; гибридизация праймеров 55 °С – 50 с; элонгация 72 °С – 1 мин; количество циклов – 40; шаг 3 (конечная элонгация) 72 °С – 6 мин. Проведена серия ПЦР с целью подбора оптимального количества матрицы ДНК для эффективной оценки трансгенных растений сахарной свеклы по наличию специфических последовательностей. Выводы. Для идентификации трансгенной сахарной свеклы, толерантной к воздействию глифосата, целесообразно определять в отдельных генотипах 35S промотор и ген cp 4 epsps. Установлено, что при подборе температурных параметров мультиплексной реакции на идентификацию гена als не влияло увеличение температуры гибридизации праймеров на 5 °С, что позволило включить специфические праймеры для определения этой последовательности в качестве внутреннего контроля. По результатам пробных мультиплексных реакций определены концентрации дНТФ (дезоксинуклеотидтрифосфатов) и ионов Mg2+, которые позволили исключить возможность получения неспецифических фрагментов и ложноотрицательных результатов. Определено оптимальное количество матрицы ДНК (100–150 нг) для эффективной оценки трансгенных растений сахарной свеклы по наличию специфических последовательностей. Полученные результаты позволили разработать мультиплексную тест-систему для идентификации трансгенной сахарной свеклы, толерантной к воздействию глифосата, с помощью которой можно одновременно определить 35S промотор, ген cp 4 epsps и ген als в качестве внутреннего контроля реакции. | Мета. Створити мультиплексну систему для ідентифікації толерантних до гліфосату буряків за допомогою ПЛР. Методи. Молекулярно-генетичні методи аналізу нуклеїнових кислот. Результати. Наведено результати досліджень з визначення параметрів полімеразної ланцюгової реакції (ПЛР) для розроблення мультиплексної системи для ідентифікації структурних елементів трансгенної конструкції з геном cp 4 epsps, що забезпечує толерантність до гліфосату. Для отримання ампліконів цільових послідовностей ДНК визначено такі значення температурних режимів ПЛР: крок 1 (початкова денатурація) 95 °С – 3 хв; крок 2 (напрацювання специфічних продуктів реакції): денатурація 95 °С – 45 с; гібридизація праймерів 55 °С – 50 с; елонгація 72 °С – 1 хв; кількість циклів – 40; крок 3 (кінцева елонгація) 72 °С – 6 хв. Проведено серію ПЛР з метою добору оптимальної кількості матриці ДНК для ефективної оцінки трансгенних рослин цукрових буряків за наявністю специфічних послідовностей. Висновки. Для ідентифікації трансгенних цукрових буряків, толерантних до дії гліфосату, доцільно визначати в окремих генотипах 35S промотор та ген cp 4 epsps. Встановлено, що в процесі добору температурних параметрів мультиплексної реакції на ідентифікацію гена als не впливало збільшення температури гібридизації праймерів на 5 °С, що дало змогу включити специфічні праймери для визначення цієї послідовності як внутрішнього контролю. За результатами пробних мультиплексних реакцій визначено концентрації дНТФ (дезоксинуклеотидтрифосфатів) та іонів Mg2+, які дали змогу виключити можливість отримання неспецифічних фрагментів та хибно негативних результатів. Визначено оптимальну кількість матриці ДНК (100–150 нг) для ефективної оцінки трансгенних рослин цукрових буряків за наявністю специфічних послідовностей. Отримані результати дали змогу розробити мультиплексну тест-систему для ідентифікації трансгенних цукрових буряків, толерантних до дії гліфосату, за допомогою якої можна одночасно визначити 35S промотор, ген cp 4 epsps та ген als як внутрішній контроль реакції.
Afficher plus [+] Moins [-]Morphological features of leaves in climbing roses varieties of the genus Rosa L. Texte intégral
2016
Т. О. Буйдіна | О. Ф. Рожок
Purpose. To study biometric characteristics of climbing roses leaves (length of compound leaf, the number of leaf blades, total leaf area and leaf blade area) and select the best varieties on this base. Methods. General biological (biometric) study and statistical evaluation. Results. As follows from the morphological analysis, a significant leaf size variation in the studied varieties of climbing roses was revealed. The leaf size in such varieties as ‘Kahovka’, ‘Flammentanz’, ‘Krasnyi Maiak’ was the largest, in ‘New Dawn’ and ‘Krymskoe Solnyshko’ – the smallest one. Based on the leaf biometric study of 9 varieties of climbing roses, the authors defined that ‘Flammentanz’, ‘Krasnyi Maiak’, ‘Kahovka’ varieties had the highest values of leaf parameters. The ‘Veilchenblau’ variety differed significantly from the other studied samples by the form and color of leaves that had a positive impact on its decorative value. Data obtained allowed to select varieties that can be used in landscaping not only because of flower decorative characteristics, but also due to attractive leaf appearance, as well as varieties for application in breeding programs related to this trait. Conclusions. Biometric characteristics of climbing roses leaves (length of compound leaf, the number of leaf blades, total leaf area and leaf blade area) were studied, and in compliance with the results the best varieties were selected that can be recommended for use in gardening and breeding.
Afficher plus [+] Moins [-]Clonal micropropagation of peppermint (Mentha piperita L.) varieties of Ukrainian breeding Texte intégral
2016
Таланкова-Середа, Т. Є | Коломієць, Ю. В | Григорюк, І. П
Purpose. Developing technology for clonal micropropagation of peppermint (Mentha piperita L.) plants of Ukrainian breeding based on the complex of methods of isolated tissue and organ culture in vitro. Methods. During the experiment, such methods as isolated tissue and organ culture in vitro, clonal micropropagation, detached scion grafting, chemotherapy with adding of virucide Ribavirin to the nutrient medium, biometric and statistical ones were used. Results. The stepped procedure of sterilization that we have developed allows to receive 88–100% of sterile explants. For M. piperita L. introduction into culture and clonal micropropagation, Murashige and Skoog (MS) nutrient medium appeared to be optimal supplemented with 6-benzylaminopurine (0.75 mg/l), adenine (0.05 mg/l), indole-3-acetic acid (IAA) (0.05 mg/l) and gibberellic acid (0.5 mg/l) on which the reproduction ratio on the 28th day ranged between 1:7 and 1:15. For recovery of plants from viral infection, virucide Ribavirin at concentration of 10 mg/l was added to the nutrient medium. The proposed nutrient medium for rhizogenesis, that contained IAA (0.5 mg/l) and indole butyric acid (IBA) (0.5 mg/l), allows to obtain the frequency of rhizogenesis up to 84–100%. Regenerated plants were adapted to the conditions in vivo on substrate peat : universal soil : perlite : sand in the ratio 2:1:1:1. The survival rate for peppermint varieties amounted to 96–100%. Conclusions. Biotechnological scheme was developed that permits to get healthy, purebred planting material and intensively propagate plants for supplying breeding programs of the Experimental Station for Medicinal Plants of the Institute of Agroecology and Environmental Management of the National Academy of Agrarian Sciences of Ukraine, among which such varieties as ‘Lebedyna pisnia’ and ‘Ukrainska pertseva’ were selected as the most promising for clonal micropropagation.
Afficher plus [+] Moins [-]Features of accumulation of inorganic elements in seeds of white mustard (Sinapis alba L.) and black mustard (Brassica nigra L.) Texte intégral
2016
Рудник-Іващенко, О. І | Михальська, Л. М | Швартау, В. В
Purpose. To investigate special aspects of accumulation of inorganic elements including heavy metals in seeds of white and black mustard to be grown for obtaining drugs. Methods. Field experiments, microwave digestion, ICP-MS and statistical analysis. Results. The content of inorganic elements including heavy metals was determined in the seeds of white and black mustard grown in Kiev Oblast. It was revealed that during the growing season plants of white mustard were able to accumulate such elements as aluminum, barium, strontium, zinc in seeds in concentrations that exceed their content in black mustard seeds, while compounds of calcium, cesium, iron, magnesium, manganese, potassium in a greater degree were accumulated in black mustard seeds. Conclusions. As legal and regulatory documents for important chemical elements don’t contain the maximum permissible limits of their content in medicinal plants, it would make sense to launch a comprehensive research with the involvement of specialists of relevant profiles in order to establish such a gradation. Plants of white and black mustard in Kiev Oblast have accumulated high levels of such metals as Ba, Cu, Fe, Mn, Mo, Sr, Zn that exceed the known limits of accumulation, indicating a partial contamination of soils in the region. Consequently, these plants can be used for phytoremediation of soils. Considering the fact that in the pharmaceutical practice refined mustard seed oil is used, revealed alterations of metal accumulation in seeds will not affect the quality of the final drugs. According to the research results, white and black mustard is promising for cultivation in Kiev Oblast with a view to obtain raw material that can be processed into drugs.
Afficher plus [+] Moins [-]Assessment of adaptive capacity of spring wheat lines under conditions of Forest-Steppe zone of Ukraine Texte intégral
2016
О. А. Демидов | С. О. Хоменко | І. В. Федоренко | Р. М. Близнюк | Є. А. Кузьменко
Purpose. Selecting spring wheat lines with increased adaptive capacity. Methods. Field experiments, statistical evaluation. Results. Lines of spring soft wheat with better adaptive capacity were identified (‘Erythrospermum 13-39’, ‘Lutescens 11-16’, ‘Lutescens 05-24’, ‘Lutescens 10-36’) and spring durum wheat (‘Melanopus 10-02’ (biotype II), ‘Melanopus 10-02’, ‘Melanopus 10-03’, ‘Hordeiforme 12-12’). Spring soft wheat varieties ‘Zlata’, ‘Dubravka’, ‘Oksamyt Myronivskyi’, ‘Bozhena’ and spring durum wheat varieties ‘MIP Raiduzhna’, ‘Magdalena’ with increased adaptive capacity were passed for State variety testing. Conclusions. With regard to the analysis of parameters of yield capacity and its variability under the influence of changing environmental factors, soft and durum spring wheat lines with increased adaptive capacity were identified. The best spring wheat lines for general adaptive capacity were passed for State variety testing. Soft spring wheat variety ‘Zlata’ and durum spring wheat variety ‘MIP Raiduzhna’, in 2014, and such soft spring wheat varieties as ‘Dubravka’, ‘Oksamyt Myronivskyi’, ‘Bozhena’ and spring durum wheat variety ‘Magdalena’, in 2015, were passed for State variety testing.
Afficher plus [+] Moins [-]Forming and maintaining a collection of plant genetic resources of the Buckwheat (Fagopyrum Mill.) genus Texte intégral
2016
Тригуб, О. В
Purpose. Providing information on the formation, composition, maintenance of the collection of Buckwheat (Fagopyrum Mill.) genus at Ustymivka Experimental Station of Plant Production (Poltava region), and areas of its use: introduction, preservation and study of the material, for the set of index, formation of special collections and providing research and education institutions of Ukraine with a valuable original material. Methods. Methodical developments dealing with formation and maintenance of crop collections, methodical literature on the study of buckwheat collection for breeding and agronomic performance. Results. The author represented results of the collection material study for the main breeding and economically valuable traits including yield, productivity, resistance to abiotic and biotic factors of the environment. The results of work are considered aimed on creating special collections of the gene pool – base, trait (for yield and large seeds; for productivity, drought and heat resistance; for suitability for mechanical cultivation), educational and core ones. Conclusions. All formed and registered collections are a valuable base material for solving a number of breeding goals to increase the quantity and improve the quality of plant production.
Afficher plus [+] Moins [-]Functional features of photosynthetic apparatus of modern high-yielding winter wheat varieties Texte intégral
2016
Моргун, В. В | Стасик, О. О | Кірізій, Д. А | Прядкіна, Г. О
Purpose. Comparative studies of the photosynthetic apparatus of winter wheat varieties of modern and earlier breeding in the relation to their productivity. Methods. Field, pot experiment, biometrical, gas analysis, analytical and statistical ones. Results. It is found that the modern high-yielding winter wheat varieties have a higher rate of flag leaf photosynthesis during generative period of development, particularly at grain filling period, improved ability to store assimilates in stem and effectively use them later for grain growth. The modern varieties form crop canopy with greater leaf area and chlorophyll indexes and larger photosynthetic capacity and maintain functional activity of photosynthetic apparatus longer at the end of the growing season. Conclusions. The superiority for grain productivity of modern high-yielding wheat varieties, originated from the Institute of Plant Physiology and Genetics, NAS of Ukraine, over varieties released before the “green revolution”, results from increased capacity and durability of functioning the crop photosynthetic apparatus, higher CO2 assimilation rate and optimization of source-sink relations in whole plant.
Afficher plus [+] Moins [-]Bioinformatic analysis of maize gene encoding starch branching enzyme SBEIIb Texte intégral
2016
Сліщук, Г. І | Жернаков, Т. Ю | Волкова, Н. Е
Purpose. Investigation of maize ae1 gene polymorphism by bioinformatic methods. Methods. Global and local alignment of the nucleotide and amino acid sequences, in silico translation and transcription, translates modeling, primers design, phylogenetic analysis. Results. 255 nucleotide sequences of maize аe1 gene, 500 amino acid sequences of homology translates of maize ae1 gene (SBEIIb enzyme homologs) and 100 mRNA expressed from the maize ae1 gene were analyzed to establish phylogenetic relationships. Polymorphism of maize ae1 gene different regions was investigated by bioinformatic methods. Modeling of the maize enzyme SBEIIb was performed. Conclusions. According to the results of amino acid sequences of SBEIIb enzyme homologs alignment, it was found that ae1 gene orthologs are present only in monocots, paralogs – in monocots, dicots, and other taxa, including algae and animals. Based on the results of alignment of plants mRNA from which enzyme SBEIIb is translated, maize ae1 gene orthologs and the nearest paralogs encoding starch branching enzymes with chloroplast localization were defined; this suggests a possible origin of ae1 gene due to duplication of the gene encoding the 1,4-alpha-glucan-branching enzyme 2 with chloroplast or amyloplast localization. In the maize ae1 gene structure, regions were found that include polymorphic sites not defined previously. For the polymorphic sites design primers were developed that allowed to differentiate the maize lines. It was determined that the detection of polymorphism in theory can influence the enzyme function and, as a result, change the concentration of amylopectin in maize grain.
Afficher plus [+] Moins [-]Bioecological peculiarities of mid-ripening varieties of strawberry (Fragaria ananassa Duch.) in the northern part of the Forest-Steppe zone of Ukraine Texte intégral
2016
Павлюк, В. В | Ковальчук, Н. С | Павлюк, Н. В
Purpose. Improvement of a range of strawberry varieties based on comprehensive study and introduction of new Ukrainian and West European cultivars. Methods. Cultivars and elite forms were studied using the methods of primary variety studying and state variety testing at three-year strawberry plantations of the Institute of Horticulture of the National Academy of Sciences of Ukraine (the city of Kyiv) and its variety testing stations. Results. It was found that more frequent spring and summer droughts as well as black frosts in winter adversely affected the strawberry productivity. The best varieties were selected among new ones for the complex of traits, they included ‘Elegance’ from the Great Britain, ‘Gercules’ from Ukraine (both are dessert grapes), ‘Elsanta’ from Holland (for universal use), ‘Alba’ from Italia (as raw material). The highly adaptive and productive ‘Istochnik’ and ‘Festyvalna romashka’ varieties (as raw material) are recommended for the north regions. Conclusions. In the context of profound climatic change, high-profitable cultivation of mid-ripening varieties and a strawberry as such in the Northern Forest-Steppe zone is expedient under controlled conditions – in high film tunnels, in hot-houses and under perennial irrigation that already has become the European tendency.
Afficher plus [+] Moins [-]Biological peculiarities of stem softwood cuttings of ornamental garden plants and their use in landscaping Texte intégral
2016
Мамчур, Т. В
Purpose. To study biological peculiarities of softwood cuttings of three species of ornamental garden plants of the Hydrangeaceae family introduced under the conditions of a botanical nursery of Uman National University of Horticulture, and the prospects of planting material use in landscaping. Methods. Field study, laboratory test, statistical and mathematical analysis. Results. Bioecological and ornamental peculiarities of grafters of investigated species and their preservation for supplying the store of initial plant graft material were studied. The dependence of regeneration ability of softwood cuttings of investigated varieties on preplant treatment by a biologically active substance (KANO), type of a shoot, time of grafting, etc was analyzed. Substantially intensive rooting of basal drafts with three nods and less intensive rooting of apical drafts was observed during the whole period of rooting. Conclusions. The treatment of softwood cuttings with a biologically active substance at the concentration of 10–15 mg/l in the aqueous solution stimulates root formation and activates growth of own root plants. Investigated plant species of Hydrangeaсeae family are promising for landscaping on urbanized areas of various functional use.
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