Purpose. To determine the dependence of the intensity of callus formation and organogenesis of Linum usitatissimum L. convar. elongatum in vitro on explant type and variety in order to optimize the cultivation protocol. Methods. For induction of callus formation and organogenesis, hypocotyls, cotyledons, leaves, immature embryos and anthers of flax varieties ‘Hlinum’, ‘Esman’, ‘Hladiator’, ‘Hlobus’ and ‘Charivnyi’ grown on Murashige and Skoog medium were treated with 0.05 mg/l 1­naphthylacetic acid and 1.0 mg/l 6­benzylaminopurine at a photoperiod of 16 h, light intensity 2500 lux, relative humidity 60–80% and air temperature 22–24 °C. Empirical data were interpreted using arithmetic mean, error of the sample mean, coefficient of variation, least significant difference and rank order. Results. The intensity of callus formation and organogenesis in the analysed varieties depended on the object of study, i.e. the genotype of the variety and the type of explant. The frequency of callus formation ranged from 9.4 (anthers of variety ‘Esman’) to 99.4% (leaf explants of variety ‘Hlinum’), the weight of callus – from 0.18 (anthers of variety ‘Esman’) to 3.18 g (anthers of variety ‘Hlobus’), the frequency of organogenesis – from 7.4 (anthers of variety ‘Esman’) to 97.3% (hypocotyls of variety ‘Hlinum’), number of shoots – from 0.6 (anthers of variety ‘Hladiator’ and immature embryos of variety ‘Hlobus’) to 4.0 (hypocotyls of variety ‘Hlinum’), height of shoots – from 0.34 (anthers of variety ‘Esman’) to 1.63 cm (anthers of variety ‘Hlobus’). Conclusions. Plants of all the varieties studied are capable of effective callus formation and organogenesis in vitro in the presence of phytohormones of exogenous origin. Certain types of explants (hypocotyls, cotyledons, leaves) respond stably to exogenous growth regulators that induce callus formation, whereas others, such as anthers, have a specific response that is largely determined by cultivar characteristics. To obtain diploid somaclones, it is optimal to use hypocotyls of varieties ‘Hlinum’ and ‘Charivnyi’, to obtain haploid regenerants – immature embryos and anthers of varieties ‘Hlobus’ and ‘Hladiator’, which ensures the highest reproduction rate of cultural plant objects. | Мета. Установити залежність інтенсивності калюсо­ й органогенезу Linum usitatissimum L. convar. elongatum в умовах in vitro від типу експланта та сорту для оптимізації протоколу культивування. Методи. Для індукції калюсо­ й органогенезу використовували гіпокотилі, сім’ядолі, листки, незрілі зародки та пиляки сортів льону­довгунця ‘Глінум’, ‘Есмань’, ‘Гладіатор’, ‘Глобус’ і ‘Чарівний’, які культивували на середовищі Мурасіге і Скуга, додаючи 0,05 мг/л 1­нафтилоцтової кислоти та 1,0 мг/л 6­бензиламінопурину, за фотоперіоду 16 год, інтенсивності освітлення 2500 лк, відносної вологості 60–80% і температури повітря 22–24 °С. Емпіричні дані інтерпретували за середнім арифметичним, похибкою вибіркової середньої, коефіцієнтом варіації, найменшою істотною різницею та ранжували. Результати. Інтенсивність калюсогенезу та органогенезу у проаналізованих сортів залежала від об’єкта дослідження. Водночас частота калюсогенезу варіювалася від 9,4 (пиляки сорту ‘Есмань’) до 99,4% (листкові експланти сорту ‘Глінум’), маса калюсу – від 0,18 (пиляки сорту ‘Есмань’) до 3,18 г (пиляки сорту ‘Глобус’), частота органогенезу – від 7,4 (пиляки сорту ‘Есмань’) до 97,3% (гіпокотилі сорту ‘Глінум’), кількість пагонів – від 0,6 (пиляки сорту ‘Гладіатор’ і незрілі зародки сорту ‘Глобус’) до 4,0 шт. (гіпокотилі сорту ‘Глінум’), висота пагонів – від 0,34 (пиляки сорту ‘Есмань’) до 1,63 см (пиляки сорту ‘Глобус’). Висновки. Рослини всіх досліджених сортів здатні до ефективного калюсо­ й органогенезу в культурі in vitro за наявності в середовищі фітогормонів екзогенного походження. Певні типи експлантів (гіпокотилі, сім’ядолі, листки) стабільно реагують на екзогенні регулятори росту, що індукують калюсогенез, а інші, як­от пиляки, мають специфічну реакцію, що значною мірою детермінується особливостями сорту. Для отримання диплоїдних сомаклонів оптимальним є використання гіпокотилів сортів ‘Глінум’ і ‘Чарівний’, для отримання гаплоїдних регенерантів – незрілих зародків та пиляків сортів ‘Глобус’ і ‘Гладіатор’, що забезпечує найвищий коефіцієнт розмноження культуральних рослинних об’єктів.

Purpose. To determine the dependence of the intensity of callus formation and organogenesis of Linum usitatissimum L. convar. elongatum in vitro on explant type and variety in order to optimize the cultivation protocol. Methods. For induction of callus formation and organogenesis, hypocotyls, cotyledons, leaves, immature embryos and anthers of flax varieties ‘Hlinum’, ‘Esman’, ‘Hladiator’, ‘Hlobus’ and ‘Charivnyi’ grown on Murashige and Skoog medium were treated with 0.05 mg/l 1­naphthylacetic acid and 1.0 mg/l 6­benzylaminopurine at a photoperiod of 16 h, light intensity 2500 lux, relative humidity 60–80% and air temperature 22–24 °C. Empirical data were interpreted using arithmetic mean, error of the sample mean, coefficient of variation, least significant difference and rank order. Results. The intensity of callus formation and organogenesis in the analysed varieties depended on the object of study, i.e. the genotype of the variety and the type of explant. The frequency of callus formation ranged from 9.4 (anthers of variety ‘Esman’) to 99.4% (leaf explants of variety ‘Hlinum’), the weight of callus – from 0.18 (anthers of variety ‘Esman’) to 3.18 g (anthers of variety ‘Hlobus’), the frequency of organogenesis – from 7.4 (anthers of variety ‘Esman’) to 97.3% (hypocotyls of variety ‘Hlinum’), number of shoots – from 0.6 (anthers of variety ‘Hladiator’ and immature embryos of variety ‘Hlobus’) to 4.0 (hypocotyls of variety ‘Hlinum’), height of shoots – from 0.34 (anthers of variety ‘Esman’) to 1.63 cm (anthers of variety ‘Hlobus’). Conclusions. Plants of all the varieties studied are capable of effective callus formation and organogenesis in vitro in the presence of phytohormones of exogenous origin. Certain types of explants (hypocotyls, cotyledons, leaves) respond stably to exogenous growth regulators that induce callus formation, whereas others, such as anthers, have a specific response that is largely determined by cultivar characteristics. To obtain diploid somaclones, it is optimal to use hypocotyls of varieties ‘Hlinum’ and ‘Charivnyi’, to obtain haploid regenerants – immature embryos and anthers of varieties ‘Hlobus’ and ‘Hladiator’, which ensures the highest reproduction rate of cultural plant objects.

Мета. Введення в культуру in vitro та одержання калюсів із зрілих зародків 3 зразків пшениці спельти та порівняння ефективності їхнього калюсогенезу із 2 зразками пшениці м’якої. Методи. Для проведення цього дослідження було обрано 5 зразків гексаплоїдної пшениці: 3 – спельти та 2 – пшениці м’якої. Стерилізацію зерна проводили 96% етиловим спиртом та 5% розчином гіпохлориту натрію. Для уведення в культуру in vitro експлантами брали зрілі зародки. Для калюсогенезу використали три типи живильних середовищ Мурасіге і Скуга (МS) з різним компонентним складом. Експланти культивували в темряві 21 добу. Результати. Підібрано оптимальні умови для індукції культури тканин Triticum spelta L. та T. aestivum L. із зрілих зародків. Отримані з різних зразків калюси, які вирощували на трьох типах модифікованого живильного середовища МS, не відрізнялись між собою морфологічно. У сортів спельти ‘Європа’ і пшениці м’якої ‘Бунчук’ та ‘Елегія Миронівська’ незалежно від складу середовища спостерігали високу ефективність калюсоутворення, у той час як на експлантах спельти сорту ‘Зоря України’ відбувалось повільне формування калюсу. Висновки. З експлантів зрілих зародків отримано культуру тканин 3 зразків спельти та 2 зразків пшениці м’якої. Встановлено, що найефективнішими для калюсоутворення з експлантів зрілих зародків пшениці м’якої та спельти було живильне середовище MS з 3% сахарози, доповнене 2 мг/л 2,4-Д, 10 мл/л арґентумом нітрату. Ефективність калюсогенезу на 21 добу культивування, залежно від зразка, варіювала в межах 80,2–100,0%. Досліджувані зразки відрізнялись між собою за здатністю формувати калюси на живильних середовищах із різним компонентним складом.

Purpose. To conduct in vitro screening of different genotypes of winter triticale for resistance to salinity in the shoot apical meristem culture. Methods. Plant tissue culture in vitro, in vitro breeding, statistical analysis. Results. It was found that the increase of sodium chloride concentration from 0.6 to 1.5% resulted in inhibition of the callus culture growth in all genotypes that was indicative of the toxic effect of the stress factor. It turns out that 1.2% sodium chloride concentration allowed to differentiate triticale genotypes for salt tolerance. The line ‘38/1296’ appeared to be the most resistant to salinity stress because under breeding conditions calli of this genotype were characterized by higher morphogenetic potential, had the highest crude mass increase, and plants-regenerants were obtained only from explants of this line after cultivation on the medium containing 1.5% sodium chloride. The ‘ADM 11’ variety was the most sensitive to saline stress as mass necrosis and lack of regenerative ability in its calli were observed under breeding conditions. In the studied forms, genotypic dependence of morphogenesis processes in vitro culture was registered. From the induced calli, plants-regenerants were obtained, and their completion of growing, root development and transfer to in vivo conditions were optimized. Conclusions. Genotypic response to salinity stress in the culture of shoot apical meristems of winter triticale was expressed by various crude mass increase and different morphogenetic potential on exposure to a stress factor. The line ‘38/1296’ can be used as a valuable material for further breeding of winter triticale. The culture of shoot apical meristems is recommended to apply as a test system for screening of triticale genotypes for resistance to salinity stress

Purpose. To study the expression of gus and gfp genes in callus explants of amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated genetic transformation. Methods. Winter spelt wheat of ‘Europa’ variety was chosen for transformation. Calli obtained from mature embryos were used as explants. Callus pre-cultivation was carried out on MS nutrient medium (Murashige–Skoog) supplemented with 2 mg/L 2.4-D (2.4-Dichlorophenoxyacetic acid) and 10 mg/L silver nitrate. For genetic transformation, Agrobacterium tumefaciens Conn., strain GV3101 and a genetic construct with reporter genes beta-glucuronidase (GUS) and green fluorescent protein (GFP) were used. Calli were transformed by inoculation with agrobacteria and vacuum infiltration. Then they were co-cultured on MS medium with 2 mg/L 2.4-D and 10 mg/L AgNO3, but without antibiotics. The expression of the gus gene was checked by histochemical and the gfp gene by visual analysis (fluorescence of the GFP protein in UV light). Gfp and gus gene expression levels were evaluated using ImajeJ software. The integration of the gfp and gus genes into the spelt genome was verified by PCR. Results. Genetic transformation of spelt callus explants by inoculation in a nutrient medium with agrobacteria and vacuum infiltration occurred at different frequencies. The level of expression of the gus gene during vacuum infiltration was 4.66 ± 0.74%, with inoculation – 4.00 ± 0.91%; and the gfp gene with vacuum infiltration – 3.66 ± 0.74%, with inoculation – 4.66 ± 1.39%. The level of expression of the gfp gene was higher when using inoculation with agrobacteria, and the gus gene was higher during vacuum infiltration. Using PCR analysis, the integration of the gfp and gus genes into the callus of spelt genome was confirmed. The length of the PCR product with primers for the gus gene was 240 bp, and 717 bp for the gfp gene. Conclusions. The use of vacuum infiltration and inoculation methods for spelt genetic transformation gave different results. The frequency of genetic transformation ranged from 3.66 to 4.66%. Agrobacterium-mediated genetic transformation of amphidiploid spelt wheat allows us to study the expression of gus and gfp reporter genes using callus explants derived from mature embryos

Purpose. To analyze the level of cross-resistance of obtained salt- and osmotolerant cell lines and plants regenerants of winter triticale to osmotic and salt stresses. Methods. Cultures of tissue and organs in vitro, in vitro breeding, biochemical, statistical analysis. Results. It was established that the stability of cross-resistance trait display to saline and osmotic stresses in obtained cell lines of winter triticale was rather high – from 50 to 76% of calli have survived to the end of the sixth passage. It has been shown that despite the presence of sublethal concentrations of the stress-factor (mannitol/sodium chloride) in selective medium, stable cell lines of the triticale actively continued to grow and accumulate biomass. It was found that in the line ‘38/1296’ cell lines 5L/sl and 5L/os respectively were the most resistant to osmotic and salt stresses, and lines 1C/s1 and 1C/os respectively in the ‘Obrii’ variety, since they had the highest percent of living calli and biomass increment under the selective conditions and their plant regenerant – the highest level of survival after the impact of the abiotic stressors complex. The salt-resistant cell lines of both genotypes of winter triticale as compared to the control were also characterized by significantly higher free proline content under the selective factors impact. The results obtained may indicate that the cell lines and triticale plant regenerants have a genetically determined trait of resistance to stress factors. Conclusions. Verification of traits of resistance to abiotic stressors has shown a significantly high level of cross-tolerance of the obtained cell lines of both triticale genotypes for saline and osmotic stresses. Resistance to saline and osmotic stresses of cells separated in vitro was preserved in induced plants and at the organism level has increased tolerance to abiotic environmental factors. It is shown that due to the general non-specific mechanisms of resistance, the capacity of the callus cultures of triticale to resist to one abiotic stressor can lead to increased tolerance for another one.

Callusogensis and morphogenesis of anthers of apple scab resistant varieties have been studied, with picloram applied. Auxin activity of this preparation has been revealed as in the darkness so in the light. The callusogenesis efficiency of studied varieties varied from 26,0 to 83,3% subject to the concentration of picloram in the darkness and from 11,3 to 74,9% in the conditions of low light. We consider 4 mg/l in the medium to be an optimal level of piloram concentration so far as this concentration provides the best callusogenesis indices of the tested apple varieties. The callus formed in the darkness in the medium with picloram is knobby and dense of white or milky color. The callus formed in the light is intensively green or greenishwhite, denser, with distinctly outlined meristem hearths. Morphological characters of calluses obtained in the medium of picloram are stable over years. The roots of Yubiley Moskvy variety have been induced under auxin:cytokinin ratio 1 : 2 (picloram : BAP, cytokinin). The roots of Svezhest cultivar were obtained under auxin : cytokinin ratio 3:9 (picloram : BAP, kinetin). Formation of four buds was recored in Orlovskoye polesie variety under auxin : cytokinin ratio 1:20.

Purpose. To reveal the frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction of various species of the genus Linum L. (Linaceae) in vitro. Methods. For in vitro induction of callus formation and organogenesis, hypocotyl segments of species Linum usitatissimum L. convar. elongatum and convar. usitatissimum, L. tenue Desf., L. bienne Mill., L. corymbulosum Pchb., L. nervosum Waldst. & Kit., L. flavum L., L. campanulatum L., L. perenne L., L. austriacum L., L. grandiflorum Desf., L. strictum L. were cultivated on Murashige and Skoog medium supplementedwith 0.05 mg/l 1-naphthylacetic acid and 1.0 mg/l 6-benzyl aminopurine at 22–24 °C, relative humidity of 60–80%,with 16 hours photoperiod (2500 flux). For microclonal reproduction Murashige and Skoog, White, Gamborg and Eveleigh media and their modifications were used. The measurement results were interpreted by the arithmetic mean, standard error for the sample mean, the leastsignificantdifference and ranked. Results. Different species of the genus Linum to a large extend are capable of forming callus and regenerating shoots under the specified cultivation conditions. The frequency of callus formation for the studied samples on the 35th day of cultivation varied within 81.25–100%, the mass of callus from one explant – 0.21–1.64 g, the frequency of organogenesis – 12.50–100%, the number of shoots – 1.8–7.6 pcs. and the height of the shoots was 0.82–2.12 cm. The following species: L. usitatissimum convar. elongatum, L. tenue, L. bienne and L. strictum were distinguished by a high intensity of callus formation. Intensive organogenesis was pecular to L. tenue, L. bienne, L. flavum, L. austriacum and L. grandiflorum. The efficiency of somaclone obtaining was quite low in L. nervosum and L. campanulatum. In total, for the microclonal reproduction of species of the genus Linum Murashige and Skoog, Gamborg and Eveleighmedia supplementedwith 12.5 g/l glucose were optimal. At the final stages of microclonal propagation, before transferring microclones in vivo, it is advisable to use White medium, which contributes to a high frequency of rhizogenesis. Varieties of L. usitatissimum convar. elongatum and convar. usitatissimum had diffe­rent responses to in vitro culture. Conclusions. The frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction depended on the genotype of a particular species; therefore it is advisable to select the composition of the nutrient medium and growth regulators for each of them. Some species of the genus Linum have not yet been studied in vitro, so the obtained results allow expanding the scope of their use in practice, in particular in breeding as a new source material with somaclonal variation, interspecific crosses, and ornamental floriculture.

Мета. Введення в культуру in vitro та одержання калюсів із зрілих зародків 3 зразків пшениці спельти та порівняння ефективності їхнього калюсогенезу із 2 зразками пшениці м’якої. Методи. Для проведення цього дослідження було обрано 5 зразків гексаплоїдної пшениці: 3 – спельти та 2 – пшениці м’якої. Стерилізацію зерна проводили 96% етиловим спиртом та 5% розчином гіпохлориту натрію. Для уведення в культуру in vitro експлантами брали зрілі зародки. Для калюсогенезу використали три типи живильних середовищ Мурасіге і Скуга (МS) з різним компонентним складом. Експланти культивували в темряві 21 добу. Результати. Підібрано оптимальні умови для індукції культури тканин Triticum spelta L. та T. aestivum L. із зрілих зародків. Отримані з різних зразків калюси, які вирощували на трьох типах модифікованого живильного середовища МS, не відрізнялись між собою морфологічно. У сортів спельти ‘Європа’ і пшениці м’якої ‘Бунчук’ та ‘Елегія Миронівська’ незалежно від складу середовища спостерігали високу ефективність калюсоутворення, у той час як на експлантах спельти сорту ‘Зоря України’ відбувалось повільне формування калюсу.Висновки. З експлантів зрілих зародків отримано культуру тканин 3 зразків спельти та 2 зразків пшениці м’якої. Встановлено, що найефективнішими для калюсоутворення з експлантів зрілих зародків пшениці м’якої та спельти було живильне середовище MS з 3% сахарози, доповнене 2 мг/л 2,4-Д, 10 мл/л арґентумом нітрату. Ефективність калюсогенезу на 21 добу культивування, залежно від зразка, варіювала в межах 80,2–100,0%. Досліджувані зразки відрізнялись між собою за здатністю формувати калюси на живильних середовищах із різним компонентним складом. | Purpose. Introduction to in vitro culture and obtaining of callus from mature embryos of 3 spelt samples and comparing the effectiveness of their callusogenesis with 2 soft wheat samples. Methods. Five samples of hexaploid wheat (three of spelt and two of soft wheat) were taken for experiments. Surface sterilization of grains was carried out in 96% ethanol and 5% sodium hypochlorite solution. Mature embryos were used as explants. Three types of MS culture media with different component compositions were used for callusogenesis. Explants were cultivated in the dark for 21 days. Results. The optimal conditions for the induction of tissue culture of Triticum spelta L. and T. aestivum L. from mature embryos were selected. Received calli from different samples, which were grown on three types of culture media MS, did not differ morphologically from each other. A genetic predisposition to callus formation was observed for specimens of ‘Evropa’ spelt variety and soft wheat ‘Bunchuk’ and ‘Elehiia Myronivska’ wheat samples regardless of the composition of the MS medium, while callus formation was slow on the explants of ‘Zoria Ukrainy’ cultivar. Conclusions. A tissue culture of 3 spelt samples and 2 soft wheat samples was obtained using mature embryos as explants. It was found that a nutrient medium containing 3% sucrose and supplemented with 2 mg/L 2,4-D, 10 ml/L silver nitrate was the most effective for callus formation from mature germ explants of soft wheat and spelt. The efficiency of the calluso­genesis on the 21st day of cultivation, depending on the sample, varied in the range of 80.2–100.0%. The studied samples differed among themselves in their ability to form calli on nutrient media with different component composition. The efficiency of spelt callusogenesis was first studied in Ukraine. | Цель. Введение в культуру in vitro и получение каллюса от зрелых зародышей 3 образцов спельты и сравнение эффективности их каллюсогенеза с 2 образцами пшеницы мягкой. Методы. Для работы взяты 5 образцов гексаплоидной пшеницы – 3 спельты и 2 пшеницы мягкой. Поверхностную стерилизацию зерна проводили в 96% этиловом спирте и 5% растворе гипохлорита натрия. В качестве эксплантов использовализрелые зародыши. Для калюсогенеза использовали три типа питательных сред МS с различным компонентным составом. Экспланты культивировали в темноте 21 день. Результаты. Подобраны оптимальные условия для индукции культуры тканей Triticum spelta L. и T. aestivum L. из зрелых зародышей. Полученные каллюсы из разных образцов, которые выращивали на трех типах питательных сред МS, не отличались между собой морфологически. Наблюдали генетическую предрасположенность к каллюсообразованию образцов спельты сорта ‘Європа’ и пшеницы мягкой сортов ‘Бунчук’ и ‘Елегія Миронівська’ независимо от состава среды MS в то время, как на эксплантах спельты сорта ‘Зоря України’ происходило медленное формирование каллюса. Выводы. Получено культуру тканей 3 образцов спельты и 2 образцов пшеницы мягкой с использованием в качестве эксплантов зрелых зародышей. Установлено, что наиболее эффективной для каллюсообразования из эксплантов зрелых зародышей пшеницы мягкой и спельты была питательная среда, дополненная 2 мг/л 2,4-Д, 10 мл/л нитрата серебра и содержащая 3% сахарозы. Среднее значение эффективности каллюсогенеза при этом составляло 80,2–100,0% на 21 сутки выращивания. Исследуемые образцы отличались между собой способностью формировать каллюс на питательных средах с различным компонентным составом. Впервые в Украине исследована эфективность каллюсогенеза спельты.

Purpose. To obtain Miscanthus sacchariflorus (Maxim.) Hack and Miscanthus sinensis Andersson in vitro culture by indirect morphogenesis. Methods. Biotechnological procedures, mathematical and statistical analyses. Results. Composition of nutrient medium was developed intended for induction of callusogenesis from Miscanthus seeds with a poor germination and viability of seedlings – Murashige and Skoog (MS) medium was modified for the amount of macroelements (half-dose) that was supplemented with amino acids (300 mg/l of glutamic acid, 50 mg/l of aspartic acid, 5 mg/l of tyrosine, 3 mg/l of arginine, 2 mg/l of hydroxyproline) and plant growth regulators [2,5 mg/l of 2.4D (2.4-Dichlorophenoxyacetic acid), 0,6 mg/l of BAP (6-Benzyl-aminopurine) and 0,3 mg/l of ABA (Abscisic acid)]. Composition of nutrient medium was developed for regeneration of microplants from callus – agar MS medium was modified for the amount of macroelements (half-dose) supplemented with vitamins: 10 mg/l of thiaminum, 1,0 mg/l of pyridoxine, 1,0 mg/l of nicotinic acid (by White), 1,0 mg/l of ascorbic acid, 250 mg/l of glutamic acid, 2,0 mg/l of BAP, 0,3 mg/l of NAA (Naphthaleneacetic acid). On this medium, 100% regeneration of M. sacchariflorus (Maxim.) Hack and 50% regeneration of M. sinensis Andersson was obtained. Due to media modification aimed at initiating callusogenesis and microplants regeneration, reproduction factor of M. sinensis was increased 20 times at the average, M. sacchariflorus – 35–40 times. Conclusions. Plants of M. sacchariflorus (Maxim.) Hack and M. sinensis Andersson were obtained in vitro culture by initiation of callusogenes and microplants regeneration from the Miscanthus seeds with poor germination and viability on nutrient media of certain composition.