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“Hairy” roots obtained through genetic transformation of plants by Agrobacterium rhizogenes, a soil phytopathogen, are valuable producers of important secondary metabolites possessing medicinal properties as well as a useful model system for studying plant responses to impacts of unfriendly environmental conditions. This study compares a postponed response of Althaea officinalis L. “hairy” roots to the impacts of short-term cold- and high-temperature stress factors. The results obtained by the study have shown that “hairy” roots from different A. officinalis lines (individual transformational events) are characterized by different sensitivity to short-term temperature stress impacts, regardless of the transformation vectors or the presence of the human interferon(ifn)-α2b gene. High temperature caused a significant level of growth inhibition in roots of all lines, except those with the highest flavonoid content under the control conditions. On the other hand, a short-term cultivation of “hairy” roots at a low temperature did not cause growth suppression. In parallel with growth inhibition caused by a temperature increase, the activation of flavonoid synthesis, which was probably a response of plants to high temperature as a stress factor, was observed. The study has shown a strong (R2 = 0.78) linear dependence between the antioxidant activity of extracts from “hairy” roots and their flavonoid content. Thus, it is obvious that flavonoids participate in the process of response and adaptation of roots to impacts of high-temperature stress.

Sunflower (Helianthus annuus L.) is one of the mainoilseeds in the world and by far one of the hardestcrops to cultivate in vitro. Currently, there is no registrated transgenic sunflower (Helianthus annuus L.) variety (clone, line, F1 hybrid, population). One ofthe main problems for obtaining transgenic plants isan efficient in vitro regeneration system, which willmake it possible to obtain morphologically typical fertile regenerants. It is known that the regenerative ability of sunflower can be studied in two ways: somatic embryogenesis and direct organogenesis. Due to the different ratio of growth regulators such as auxin and cytokinins, it is possible to influence the frequency of regeneration from different types of tissues. Thus, at high concentrations of auxins and low concentrations of cytokinins, it is possible to induce root regeneration. And for the induction of shoot regeneration, it is better to use high concentrations of cytokinin and lowconcentrations of auxins. However, knowing differentapproaches and methods of sunflower cultivation invitro regeneration culture, today there is no universalprotocol that is suitable for all sunflower genotypes.That is why the aim of our work was to investigatethe regenerative capacity of sunflower lines and develop an effective system that will later be used forgenetic research in order to improve economicallyvaluable traits of sunflower. This article presents data on sunflower regeneration (Helianthus annuus L.) by direct organogenesis.Cotyledons of immature seeds (21 days after pollination) were used as explants. The induction andproliferation of adventive buds was tested in a basicmedium with different concentrations of growth regulators. It was found that the optimal conditions for the induction and proliferation of adventive buds is a modified MS medium supplemented with vitamins for Gamborg, 5 mg/l AgNO3, 2 mg/l 2–isopentenyladenine (2–iP), 0.5 mg/l indole–3–acetic acid (IAA), 0.1 mg/L thidiazuron (TDZ).The elongation of adventitious shoots was carried out on a modified MS medium with Gamborg vitamins, 5 mg/L AgNO3, 1 mg/L 2-IP, 0.5 mg/L N6-benzylaminopurine (BAP). We were the development of an effective in vitro rooting system allowed to obtain seeds.

This study evaluated the potential of dwarf morning-glory Convolvulus tricolor (Convolvulaceae) plants and their hairy roots induced by Agrobacterium rhizogenes for rhizoextraction of heavy metals ions from the liquid medium under aseptic growth conditions. Both the young C. tricolor plants and the generated hairy root culture efficiently removed Cr6+, Ni2+, and Pb2+ ions from the liquid cultivation medium. As determined by atomic absorption spectroscopy, the hairy roots demonstrated a high level of heavy metal ions accumulation (μg/g dry weight): 3942 ± 210 of chromium, 1529 ± 312 of nickel, and 2613 ± 373 of lead. These data show that the hairy roots of morning glory might be of interest for some phytoremediation strategies due to their high bioaccumulation abilities. The comparison of bioaccumulation potential of established hairy roots and young C. tricolor plants give grounds to suppose that roots of C. tricolor play an active role in the absorption of Cr6+, Ni2+, and Pb2+ from liquid media, whereas the aboveground part rather serves as a storage for the accumulated metal ions.

Promoters are key elements regulating gene expression levels, therefore their selection is an important step in genetic engineering research. The reporter gene gfp, which encodes green fluorescent protein (GFP), was transiently expressed in leaf tissues of Aztec tobacco Nicotiana rustica L. Compared to other species of the Nicotiana genus, Aztec tobacco has a large potential for expression of heterologous proteins, a large vegetative biomass, can be easily infiltrated, and is unpretentious in cultivation. Six genetic constructs were used with different promoter sequences: the 35S promoter of Cauliflower Mosaic Virus (35S CaMV), the double-enhanced 35S promoter (D35S CaMV), promoters of the RbcS1B and RbcS2B genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isolated from Arabidopsis thaliana (L.) Heynh., and promoters of the LHB1B1 and LHB1B2 genes from A. thaliana encoding chlorophyll a/b binding proteins. The gfp gene expression was detected visually, spectrofluorimetrically, and by protein content (Bradford assay) on the seventh day after infiltration. The highest level of expression was observed using the double-enhanced 35S promoter (D35S CaMV) and the lowest using the LHB1B1 gene promoter.

Aim. Acceleration of the sunflower lines homozygosity for isolation of the fertility restorer resistant to tribenuron methyl herbicide using in vitro culture of sunflower immature embryos. Methods. Methods of immature sunflower embryos cultivation in in vitro culture; methods of classical crossing. Results. As a result, homozygous genotype was created and isolated from three combinations (BH0118xSURES-2, BH0218xSURES-2 and BH0318xSURES-2) for 10 parent sunflower lines. Conclusions. The use of the technology of sunflower immature embryos cultivation in in vitro culture effectively accelerates the sunflower lines homozygosity for the fertility restorer isolation resistant to tribenuron methyl herbicide. The involvement of immature sunflower embryos cultivation in in vitro culture methods can reduce time for creation of initial parental sunflower lines twice. Keywords: sunflower, immature embryos, in vitro culture, herbicide resistance, tribenuron methyl.

An optimized regeneration method for commercial winter rape of the Bn1 line was proposed. Hypocotyl fragments of 6-day-old seedlings were used as explants. Regeneration occurred via organogenesis on an MS medium supplemented with 3 mg/L of 6-benzylaminopurine and 2 mg/L of 2-isopentyladenine. All obtained regenerated plants successfully formed roots on hormone-free media and were adapted to the soil conditions. The vernalization conditions were specified to promote budding and flowering at the level of 83.93 ± 5.33%. The PCR analysis using the ISSR 2 and ISSR 15 markers has shown that no somaclonal variations in Bn1 winter rape occur under the protocols based on the proposed methodology. Thus, the developed methodology is efficient and economically profitable for obtaining biotechnology-based winter rape plants.

The aim of the research was to establish efficient microclonal propagation conditions of endangered Crambe mitridatis plants in vitro and to study the possible effect of aseptic cultivation on biochemical composition (hydroxycinnamic acids, flavonoids, phenolic compounds) of plants. Methods. In vitro plant culture methods were applied. Seeds were used for aseptic culture initiation. Morphogenic potential of root, leaf and petiole explants was studied on Murashige-Skoog medium with addition of plant growth regulators. The content of biologically active compounds was measured using spectrometry in plants grown in aseptic conditions and in the greenhouse. Results. Morphogenic potential of root, leaf and petiole explants was studied and the highest regeneration frequency of plantlets was established for root explants (80 %), for petiole explants (50 %) and the lowest for leaf explants (20 %). It was found that plants cultivated in aseptic conditions have higher hydroxycinnamic acids, flavonoids and phenolic compounds compared to plants grown in vivo. Conclusions. It is advisable to multiply C. mitridatis plants in vitro via root and petiole explants. Aseptic cultivation contributes to synthesis of biologically active compounds (auxin synergists) in C. mitridatis plants.Keywords: in vitro culture, hydroxycinnamic acids, flavonoids, phenolic compounds, Crambe mіtridatis.

We investigated the influence of transgene interferon α2β synthesized in plants on human repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) in both cancer and non-cancer originated cells. Using Western-blot analysis we determined MGMT quantity in cancer-originated Hеp-2 cells (epidermoid carcinoma of the larynx) and non-cancer E8 ones (derived in our laboratory from germline cells) were treated with purified transgene interferon α2β in serum-free medium. Interferon α2β caused decrease of the MGMT protein amount in the cancer-originated Hеp-2 cells at all treated concentrations (2, 20, 200, 2000 IU/mL) relative to the control. In human non-cancer originated E8 cells we revealed a decrease in MGMT levels at the both highest concentrations: 200 and 2000 IU/mL, although only at 200 IU/mL the downregulation effect was statistically significant. Thus, the transgenic interferon inhibitory effect was more noticeable in the cancer-originated cells compared to the non-cancer ones.

Aim. To develop an effective protocol for callus induction from shoot apical meristem in Triticum spelta L. and T. aestivum L. Methods. Plant material: spelt “Europe” and common wheat “Bunchuk”. For this research we used shoot apical meristems from 3-days plants. For callus induction we proposed 4 media with different concentration of 2,4-D, picloram, NAA and AgNO3. Explants were growing in dark during 21 day at + 25 C. Results. Calli were transparent and mild, less than 8 mm. For callus induction positive effect were shown on media with 2,4-D and picloram. At the same time, NAA was not such effective. Conclusions. In our research was shown, that the best media for spelt callus induction should have 2 mg/l 2,4-D and 10 mg/l AgNO3. Keywords: callusogenesis, spelt (Triticum spelta L.), common wheat, callus, shoot apical meristem.

In vitro culture can be used for endangered species preservation but its effect on biochemical properties and genetic stability of plants requires further research. Aim. The study on fatty acids content, phenolic compounds and phylogenetic relationships of Crambe aspera plants upon aseptic cultivation in vitro. Methods. Morphogenic potential of Crambe aspera leaf, root and petiole explants was studied in the Murashige and Skoog (MS) medium with different concentrations of growth factors. Fatty acids (FA) content was determined by gas chromatography-mass spectrophotometry of FA ethers, phenolic compounds were determined by spectrophotometry. Polymerase chain reactions were used to study phylogenetic relationships. Results. The efficient protocols of seed surface sterilization as well as methods of fast microclonal multiplication and obtention of C. aspera callus tissue for were developed. Conclusions. Leaves of both in vitro and in vivo cultured plants had a high content of α-linolenic acid whereas erucic acid was absent. At the same time, the difference in biochemical composition between the plants grown in aseptic and non-aseptic conditions was shown. In vivo populations of C. aspera showed a high level of polymorphism but its genome did not undergo changes after the in vitro establishment. Застосування культури іn vitro для збереження рідкісних видів має ряд переваг. Мета. Дослідження вмісту жирних кислот, фенольних сполук та філогенетичних зв’язків рослин Crambe aspera може допомогти оцінити вплив культивування in vitro. Методи. Було досліджено морфогенний потенціал листкових, кореневих та черешкових експлантів Crambe aspera на середовищі MS з різним вмістом регуляторів росту. Вміст жирних кислот визначали за допомогою газової хромато-мас спектрометрії ефірів жирних кислот. Вміст фенольних сполук визначали за допомогою спектрофотометра. Полімеразна ланцюгова реакція була застосована для дослідження філогенетичних зв’язків. Результати. Було встановлено ефективні протоколи поверхневої стерилізації насіння разом з методами швидкого мікроклонального розмноження та отримання калюсної культури для виду C. aspera. Висновки. Отримані результати свідчать про високий вміст α-ліноленової та відсутність еруковї кислоти у листках рослин що культивували як in vitro, так і in vivo. В той же час, було встановлено різницю біохімічного складу між рослинами з асептичних та грунтових умов. Популяції C. aspera що зростали в умовах in vivo показали високий рівень поліморфізму але їх геном після введення культуру in vitro був стабільним Использование культуры іn vitro с целью сохранения редких видов имеет ряд преимуществ. Цель. Исследование состава жирных кислот, фенольных соединений и филогенетических связей растений Crambe aspera может помочь в оценке влияния культивирования in vitro. Методы. Было установлено морфогенный потенциал листовых, корешковых и черешковых эксплантов Crambe aspera на среде MS с различным составом регуляторов роста. Содержание жирных кислот определяли с помощью газовой хромато-масс спектрометрии эфиров жирных кислот. Содержание фенольных соединений определяли на спектрофотомере. Полимеразная цепная реакция была использована для исследования филогенетических связей. Результаты. Было установлено эффективные протоколы поверхностной стерилизации семян вместе с методами быстрого микроклонального размножения и получения калюсной культуры для вида C.°aspera. Выводы. Полученные результаты свидетельствуют о высоком содержании α-линоленовой и отсутствии эруковой кислоты в листьях растений культивируемых как in vitro, так и in vivo. В то же время была установлена разница биохимического состава между растениями из асептических и грунтовых условий. Популяции C. aspera произрастающие в условиях in vivo показали высокий уровень полиморфизма, но их геном после введения в культуру in vitro был стабильным.

The present study reports an efficient in vitro plant regeneration system for amphidiploid (2n = 42) spelt wheat (Triticum spelta L.). Plant regeneration from mature embryos of winter spelt variety “Europe” was carried out as a two-step process. The first step was callus induction on a medium supplemented with 2 mg L−1 dichlorophenoxyacetic acid and 10 mg L−1 silver nitrate resulting in 96% callus formation. The second step resulted in 30% plant regeneration frequency from calluses transferred to Murashige and Skoog medium supplemented with 2 mg L−1 6-benzylaminopurine, 0.5 mg L−1 1-naphthaleneacetic acid, and 10 mg L−1 silver nitrate. Regeneration medium supplemented with 1 mg L−1 6-benzylaminopurine, 0.2 mg L−1 picloram, and the hormone-free medium turned out to be less effective in our experiments. Regenerated plants formed roots after transfer to half-strength Murashige and Skoog medium supplemented with 0.7 mg L−1 indole-3-butyric acid. About one-third of the resulting regenerated plants that formed roots were transferred to soil. The inter-simple sequence repeat markers were used to confirm the genetic homogeneity of regenerated plants. Thus, our regeneration protocol can be used for in vitro regeneration of spelt plants from mature embryos with minimal risk of genetic variability and provides an essential step towards developing an efficient strategy for spelt genetic transformation.

The aim of the study was to find an efficient culture medium for regeneration of Physalis species in vitro to provide their further propagation ex vitro and obtain fructiferous plants from the regenerants. Physalis peruviana L., P. ixocarpa Broth. (cv. Likhtaryk), and P. pubescens L. (cv. Zarynka) were taken as plant material for the research. Plant introduction into culture and regenerant production were carried out in vitro; the rooting of mature plants and obtaining plants with ripe fruits took place in a greenhouse and in open ground (ex vitro). To obtain regenerants, we used Murashige and Skoog (MC30) medium supplemented with the growth stimulant zeatin (Zea) at a concentration of 0.5–3 mg/l. The growth stimulant 6-benzylaminopurine (BAP) was used to elongate the regenerant stalks, and the growth stimulator α-naphthylacetic acid (NAA) was used to initiate root formation. Plant regeneration frequency and the number of regenerants per explant served as indicators of the efficiency of various zeatin concentrations on the physalis regenerative capacity. The most effective media for the shoot regeneration from cotyledonous leaf explants were MC30 + 1 mg/l Zea and MC30 + 2 mg/l Zea. Regeneration frequency on these media was 46.15 % and 53.84 % for P. ixocarpa (cv. Likhtaryk), 38.46 % and 45 % for P. peruviana, and 27 % and 34 % for P. pubescens (cv. Zarynka) respectively. The emerged regenerants were separated from explants and transferred to MC30 medium supplemented with 1 mg/l of BAP + 0.1 mg/l of NAA for stalk growth and rooting. After a month of cultivation, juvenile plants were obtained. They were transferred to a greenhouse for adaptation, and later to open ground at the experimental plot. Three months after the regenerant emergence, we obtained fertile plants, which bloomed and bore fruit. The regenerants for domestic varieties of P. ixocarpa (cv. Likhtaryk) and P. pubescens (cv. Zarynka) were obtained for the first time. We established a direct relationship between the concentration of zeatin and both the frequency of plant regeneration and the number of regenerants per explant.

The regenerative capability of three types of explants was studied on media with different compositions of growth regulators with the purpose of selecting optimal conditions of fast reproduction of endangered Crambe species that could be used as a relevant source of genetic material for the improvement of industrially valuable plants. PCR-analysis of genotypes of C. koktebelica and C. tataria plants was conducted to identify the influence of in vitro cultivation on the genetic stability of plants. The highest regeneration rates were observed with the use of petiole explants on MS medium with BA and NAA. The absence of somaclonal variability in C. koktebelica and C. tataria in vitro regenerated plants was demonstrated.

Background. The perspective of research of small interfering RNA (siRNA) application in medical practice was proved by its efficiency of chemically synthesized siRNA in the experiment in vitro . The high cost of RNA production through chemical synthesis determines the importance of searching the biotechnological methods of obtaining these compounds. A promising area of biopharmaceuticals creation is a combination of active pharmaceutical substances with innovative tools of delivery, such as bio-encapsulation in plant cells. Objective. The aim of the paper is testing of capabilities and efficiency of transient expression of the gene encoding the small interfering RNA to mRNA \[\delta-isoform\] of human protein kinase C \[(anti-PKC\delta)\] in edible raw plant biomass of lettuce Lactuca sativa . Methods. Obtaining the biomass which accumulates anti-PKCd small interfering RNA molecules was carried out by the method that was developed by our research group for the expression of genes encoding recombinant proteins. Evaluation of small interfering RNA accumulation was performed by a reverse transcription method with the subsequent Real-Time Polymerase Chain Reaction (PCR). Results. The level of accumulation of target product is 22 fmol/g of lyophilized plant biomass. Conclusions. The method of transient expression allows obtaining the lettuce Lactuca sativa biomass, which contains small interfering RNA recombinant anti-PKCd (detected by a reverse transcription method with the subsequent Real-Time PCR).

Aim. For modifying of the plant organisms with genetic engineering techniques to produce genus stress resistant low temperatures or frosts it is necessary to check their physiological characteristics at high temperatures stress. Methods. In this study we used Nicotiana tabacum plants, expressing of cyanobacterial acyl-lipid desaturases genes (desA or desC), plants were tested for the level of malondialdehyde accumulation and gene expression by the reporter protein thermostable lichenase after exposure to thermal stress. Results. We discovered the reduced malondialdehyde accumulation in plants and increased expression desaturases genes after cold stress and high temperature stress. Conclusions. Cyanobacterial desaturases gene expression in Nicotiana tabacum plants did not increase their sensitivity to the high temperatures stress.Keywords: acyl-lipid desaturases, malondialdehyde, thermostable lichenase

The anatomical research of the vegetative organs of the cytoplasmatic hybrid grown in vitro and containing the Brassica napus nucleus and the Lesquerella fendleri chloroplasts was carried out and compared to the parental forms. It was found that the anatomical structure of the cybrid is similar to rapeseed. Anomalous changes in the epithelial, parenchymal, and connective tissue of the leaf, stalk, stem, and root of the cybrid were detected. The appearance of the anatomical defects can be explained by nuclear-cytoplasmatic incompatibility, which is the cause of low adaptability of the cybrid to in vivo conditions and takes place due to alien chloroplast gene expression in the remote species.

The aim of the study was to establish efficient protocols of seed surface sterilization with further multiplication in vitro for threatened species Crambe koktebelica (Junge) N. Busch and to show the effect of biotechnological approach (in vitro cultivation) of biodiversity conservation on plants biochemical properties. Seed surface sterilization was carried out according to the original method with further microclonal multiplication of aseptic sprouts from lateral buds on the Murashige and Skoog (MS) medium supplemented with different concentrations of growth regulators. Fatty acid content was determined using Gas chromatography-mass spectrophotometry of fatty acid ethers. Antioxidant activity was determined using 2.2-diphenyl-1-picrylhydrazyl radical scavenging method. Total soluble protein content was measured using Bradford method and polyfructan content determination was based upon ketosugars ability to color in the acidic environment with resorcinol. Plants that were grown under in vitro and in vivo conditions and seeds were used in this research. Efficient protocol of surface sterilization that resulted in 45% of aseptic seed material 50% of which has sprouted was elaborated for C. koktebelica as well as fast microclonal multiplication methods that provided with up to 5.25 ± 0.50 new formed plantlets from 1 lateral bud (on the MS medium that contained 1 mg/L of 6-benzylaminopurine). It was also shown that aseptic cultivation benefits to saturated fatty acid accumulation and increases protein content but on the other hand it reduces unsaturated fatty acid amount and polyfructan content as well as antioxidant activity of plant material. Obtained data confirms the prospect of biotechnology approach to biodiversity conservation and suggest the necessity of father in vitro cultivation effect on biochemical composition of plant study.

Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the salinity and osmotic stress tolerance by plant survival test in presence of NaCl and PEG in different concentrations. Results. Transgenic plants able to express recombinant thaumatin II gene (transcription proved for 60–100 %) were obtained by agrobacterial transformation. The transgenic carrot plant extracts inhibited the growth of the studied phytopathogenic bacteria strains but exhibited no antifungal activity. Survival level of transgenic plants under the salinity and osmotic stress effect was definitely higher comparing to the untransgenic ones. The analysis of the photosynthetic pigment content in the transgenic carrot plants showed no significant difference of this parameter under salinity stress that may indicate a possible protective activity of the recombinant protein. Conclusions. The obtained in our study transgenic carrot and celery plants able to express the recombinant thaumatin II gene were characterized by antibacterial activity and increased tolerance to salinity and osmotic stress factors. Мета Отримати трансгенні рослини моркви та селери, що експресують рекомбінантний тауматин ІІ, з метою підвищення стресостійкості цих культур. Методи. Для отримання трансгенних рослин проводили Agrobacterium-опосередковану трансформацію. Присутність та транскрипцію трансгенів підтверджували за допомогою ПЛР та ЗТ-ПЛР аналізів. Визначали стійкість отриманих рослин до біотичних стрес-факторів (аналіз антибактеріальної/антифунгальної активності in vitro) та до дії сольового/осмотичного стресу (тест на виживання рослин в присутності NaCl/ПЕГ у різних концентраціях). Результати. Трансгенні рослини моркви та селери, що експресують ген тауматину ІІ (транскрипцію підтверджено для 60–100 %) було отримано шляхом агро бактеріальної трансформації. Екстракти трансгенних рослин моркви інгібували ріст досліджуваних штамів фітопатогенних бактерій, але не проявляли антифунгальної активності. Рівень виживання трансгенних рослин при абіотичному стресі був значно вищим у порівнянні з нетрансгенними рослинами. Аналіз вмісту фотосинтетичних пігментів не показав достовірної різниці показників для трансгенних рослин при сольовому стресі, що може вказувати на можливу захисну активність рекомбінантного білка. Вис­новки. Отримані у наших дослідженнях трансгенні рослини селери та моркви, що експресують рекомбінантний тауматин II, характеризувались антибактеріальною активністю та підвищеною стійкістю до сольового та осмотичного стресу. Цель. Получить трансгенные растения моркови и сельдерея экспрессирующие рекомбинантный тауматин II с целью повышения стресс-устойчивости этих культур. Ме­то­ды. Для получения трансгенных растений проводили Agrobacterium-опосредованную трансформацию. Присутс­твие и транскрипцию трансгенов подтверждали с помощью ПЦР и ОТ-ПЦР анализов. Определяли устойчивость полученных растений к биотическим стресс-факторам (анализ антибактериальной/антифунгальной активности in vitro) и к действию солевого/ осмотического стресса (тест на выживывание растений в присутствии NaCl/ПЭГ в различных концентрациях). Результаты. Трансгенные растения моркови и сельдерея, экспрессирующие ген тауматина II (транскрипция подтверждена для 60–100 %) были получены путем агробактериальной трансформации. Экстракты трансгенных растений моркови ингибировали рост исследуемых штаммов фитопатогенных бактерий, но не проявляли антифунгальной активности. Уровень выживания трансгенных растений при абиотическом стрессе был значительно выше по сравнению с нетрансгенными растениями. Анализ содержания фотосинтетических пигментов не показал достоверной разницы показателей для трансгенных растений при солевом стрессе, что может указывать на возможную защитную активность рекомбинантного белка. Выводы. По­лученные в наших исследованиях трансгенные растения сельдерея и моркови, экспрессирующие рекомбинантный тауматин II, характеризовались антибактериальной активностью и повышенной устойчивостью к солевому и осмотическому стрессу.

Метою роботи було визначення наявності рекомбінантного протеїну ESAT6Ag85B(TMD)-6His із Mycobacterium tuberculosis та вивчення рівнів його накопичення у рослинах ряски ( Lemna minor L.). ESAT6 та Ag85B є секреторними протеїнами M. tuber culosis і запропоновані для створення нових вакцин проти туберкульозу. Отримані раніше шляхом опосередкованої Agrobacterium rhizogenes трансформації рослини ряски містили злиту послідовність генів: esxA-fbpB TMD. Для визначення рівнів накопичення туберкульозних антигенів у рослинах було одержано поліклональні мишачі антитіла шляхом імунізації лабораторних тварин рекомбінантним протеїном ESAT6-Ag85B(TMD)-6His, експресованим в E. coli. Методом вестерн-блот виявлено рекомбінантний туберкульозний антиген ESAT6-Ag85B(TMD)-6His в екстрактах із трансгенних рослин L. minor. Визначено рівень накопичення антигену, який становив 0,4-0,5 мкг протеїну на 1 г сирої маси рослин. Також вивчено можливість зберігання та ступінь деградації туберкульозного антигену в ліофілізованому рослинному матеріалі, що зберігався впродовж 1,5 року без охолодження чи заморожування. Отримані рослини ряски L. minor, які синтезують рекомбінантний антиген, аналог секреторних протеїнів ESAT6 та Ag85B M. tuberculosis, можуть бути об’єктом досліджень під час створення їстівних рослинних вакцин.Целью работы было определение наличия рекомбинантного протеина ESAT6-Ag85B (TMD)-6His из Mycobacterium tuberculosis и изучение уровней его накопления в растениях ряски ( Lemna minor L.). ESAT6 и Ag85B являются секреторными протеинами M. tuberculosis и предлагаются для создания новых вакцин против туберкулеза. Полученные ранее путем опосредованной Agrobacterium rhizogenes трансформации растения ряски содержали слитую последовательность генов: esxA fbpB TMD. Для определения уровней накопления туберкулезных антигенов в растениях были получены поликлональные мышиные антитела путем иммунизации лабораторных животных рекомбинантным протеином ESAT6Ag85B(TMD)-6His, экспрессированном в E. coli. Методом вестерн-блот выявлен рекомбинантный туберкулезный антиген ESAT6Ag85B(TMD)-6His в экстрактах из трансгенных растений L. minor. Определен уровень накопления антигена, соответствующий 0,4-0,5 мкг протеина на 1 г сырой массы растений. Также изучена возможность сохранения и степень деградации туберкулезного антигена в лиофилизированном растительном материале, который хранился в течение 1,5 года без охлаждения или замораживания. Полученные растения ряски, синтезирующие рекомбинантный антиген, аналог секреторных протеинов, ESAT6 и Ag85B M. tuberculosis, могут быть объектом исследований при создании съедобных растительных вакцин.Determination of the presence of the recombinant fusion protein (ESAT6-Ag85B(TMD)-6His) and its accumulation level in duckweed plants ( Lemna minor L.) was the aim of the research. ESAT6 and Ag85B are secretory proteins of Mycobacterium tuberculosis and are considered as potential candidates for development of new vaccine against tuberculosis. Transgenic duckweed plants were obtained previously by Agrobacterium rhizogenes -mediated transformation and possessed fusion gene sequence esxA-fbpB TMD. Specific polyclonal antibodies were produced in immunized mice to identify levels of accumulation of tuberculosis antigens in plants. Recombinant antigen used for mice immunization was obtained in our laboratory by expression in E. coli. Western blot analysis revealed the recombinant tuberculosis antigen ESAT6-Ag85B(TMD)-6His in extracts from transgenic L. minor plants. The level of accumulation of the protein corresponds to 0.4-0.5 μg protein per 1 g of fresh weight of plant. Additionally, the ability to save of recombinant protein was investigated in lyophilized transgenic plants after 1.5 year storage. Constructed duckweed plants accumulating a recombinant analogue of M. tuberculosis secretory proteins can be used for development of plant-based edible vaccines.

In order to investigate the high temperature tolerance of spring canola plants (Brassica napus L.) constitutively expressing cyp11A1 gene which encodes bovine cytochrome P450SCC the growth features were analyzed under short time heat stress (42°C) in growth chamber. Earlier it was documented that results of the heat tolerance test positively correlated with improvement of high temperature resistance in field trial. Higher relative water content (by 13%) and superoxide dismutase (SOD) activity, lower electrolyte leakage (up 1.4-fold) and smaller increase in chlorophyll a and carotenoid contents in cyp11A1 canola leaves in comparison with wild-type plants under stress allowed to conclude cyp11A1 plants are more tolerant to high temperature than the control ones. We suppose that SOD activity increase which revealed in our transgenic canola in normal condition plays the defining role in the biochemical alterations in plant metabolism for the thermotolerance improvement. SOD activity increment could be caused by heterologous cytochrome P450SCC activity which resulted in the superoxide radical formation. Cyp11A1 canola plants might be resistant to the other stress conditions of different origin. Для исследования устойчивости трансгенных растений ярового рапса (Brassica napus L.), конститутивно экспрессирующих ген cyp11A1, который кодирует цитохром P450SCC быка, к высоким температурам были проанализированы особенности роста в условиях кратковременного высокотемпературного стресса (42 ºС) в климакамере. Результаты этого теста положительно коррелируют с повышением толерантности к действию высоких температур в полевых испытаниях. Увеличение относительного содержания воды (на 13 %) и активности супероксиддисмутазы (СОД) пониженный выход электролитов (в 1,4 раза) и меньшее повышение содержания хлорофилла a и каротиноидов в листьях трансгенных растений по сравнению с растениями дикого типа в условиях высокотемпературного стресса позволяют сделать вывод о том, что термоустойчивость cyp11A1 растений рапса выше, чем контрольных. Увеличение активности СОД, обнаруженное в наших трансгенных растениях рапса в нормальных условиях, играет предположительно определяющую роль в биохимических изменениях растительного метаболизма, что приводит к возрастанию устойчивости к повышенным температурам. Возрастание активности СОД возможно за счет экспрессии гетероло-гического цитохрома P450SCC, которая приводит к образованию супероксид радикалов. Полученные растения могут быть устойчивы к другим стрессовым воздействиям различного происхождения. Для вивчення стійкості трансгенних рослин ярого ріпаку (Brassica napus L.), які конститутивно експресують ген cyp11A1, що кодує цитохром P450SCC великої рогатої худоби, до дії високих температур проаналізовано особливості росту за умов коротко-тривалого високотемпературного стресу (42 ºС) вклімакамері. Результати цього тесту позитивно корелюють з підвищенням толерантності до дії високих температур в польових дослідах. На підставі збільшеного відносного вмісту води (на 13%) і підвищеної активності СОД, а також зменшеного виходу електролітів (в 1,4 раза) і меншого підвищення вмісту хлорофілу a і каротиноїдів в листках трансгенних рослин у порівнянні з рослинами дикого типу за умов стресу можна зробити висновок про підвищення термостійкості у рослин ріпаку з трансгеном cyp11A1 порівняно з контролем. Збільшення активності СОД в наших трансгенних рослинах ріпаку, виявлене за нормальних умов, відіграє визначальну роль у біохімічних змінах рослинного метаболізму, що спричиняє зростання стійкості до підвищених температур. Зростання активності СОД можливо за рахунок активності цитохрому P450SCC, в результаті якої відбувається утворення супероксид радикалів. Отримані рослини можуть бути стійкими до інших стресових впливів різного походження.

Spring rapeseed transgenic lines expressing human interferon alpha 2b were created by Agrobacteriummediated transformation of aseptic plant leaf explants. The maximum antiviral activity of the leaf extracts reached 4500 IU/g fresh weight. It was determined that the antioxidant activity and the activity of an enzyme of plant antioxidant system – superoxide dismutase (SOD) – in the leaf tissues of transgenic plants increased compared to controls. There were no correlations between the interferon and antioxidant activities, as well as between SOD and interferon activities. Using the obtained transgenic rapeseed plants with high interferon and antioxidant activities as a feed additive for animals might have preventive effect on their body, increasing resistance to infections of various origins. Методом агробактериальной трансформации листовых эксплантов асептических растений ярового рапса получены линии, экспрессирующие альфа 2b интерферон человека. Максимальная противо-вирусная активность экстрактов листьев достигала 4500 МЕ/сырого веса. Установлено, что антиоксидантная активность и активность одного из ферментов антиоксидантной системы растений – супероксиддисмутазы (СОД) – в тканях листьев трансгенных растений повышена по сравнению с контрольными. Не выявлено корреляции как между активностью интерферона и антиоксидантной активностью, так и между активностью интерферона и активностью СОД. Использование полученных трансгенных растений рапса с высокой активностью интерферона и повышенной антиоксидантной активностью как добавки к кормам животных могло бы оказывать профилактическое влияние на их организм, повышая устойчивость к различным инфекциям. Методом агробактеріальної трансформації листових експлантів асептичних рослин ярого ріпака отримано лінії, що експресують альфа 2b інтерферон людини. Максимальна противірусна активність екстрактів листків сягала 4500 МО/сирої ваги. Встановлено, що антиоксидантна активність і активність одного з ферментів антиоксидантної системи рослин – супероксиддисмутази (СОД) – в тканинах листків трансгенних рослин підвищена в порівнянні з контрольними. Не виявлено кореляції ні між активністю інтерферона і антиоксидантною активністю, ні між активністю інтерферона і активністю СОД. Використання отриманих трансгенних рослин ріпака з високою активністю інтерферона та підвищеною антиоксидантною активністю як добавки до кормів тварин мало б профілактично впливати на їхній організм, підвищуючи стійкість до інфекцій різного походження.

Our aim was to investigate the influence of some exogenous agents on the recombinant protein accumulation in plants via Agrobacterium-mediated transient expression. Methods. Agrobacterium-mediated transient expression method, spectrophotometric methods for protein analysis, statistical calculations. Results. It was shown that the tested compounds in different concentrations (namely, auxins, cytokinin, methyl jasmonate and suppressors of jasmonate biosynthesis (phenidon and diethyldithiocarbamic acid)) added to the infiltration buffer did not influence either GFP transient expression nor total protein accumulation in plant tissue. Conclusions. The results suggest that the tested factors are not substantial for Agrobacterium-mediated transient expression. Keywords: Nicotiana excelsior, Agrobacterium, transient expression, GFP. Мета. Дослідження впливу деяких екзогенних агентів на накопичення рекомбінантного білка в рослинах за допомогою Agrobacterium-опосередкованої транзієнтної експресії. Методи. Agrobacterium-опосередкована транзієнтна експресія, спектрофотометричні методи для аналізу білків, статистичні розрахунки. Результати. Показано, що протестовані речовини, а саме – деякі концентрації ауксинів, цитокініну, метилжасмонату і супресорів його біосинтезу (фенідону і диетилдитіокарбамату), додані до інфільтраційного буфера, не впливають на транзієнтну експресію GFP і загальний рівень вмісту білка в рослинній тканині. Висновки. Результати свідчать, що протестовані фактори не є суттєвими для проведення Agrobacterium-опосередкованої транзієнтної експресії. Ключові слова: Nicotiana excelsior, Agrobacterium, транзієнтна експресія, GFP. Цель. Исследование влияния некоторых экзогенных агентов на накопление рекомбинантного белка в растениях с помощью Agrobacterium-опосредованной транзиентной экспрессии. Методы. Agrobacterium-опосредованная транзиентная экспрессия, спектрофотометрические методы для анализа белков, статистические расчеты. Результаты. Показано, что тестируемые вещества, а именно – некоторые концентрации ауксинов, цитокинина, метилжасмоната и супрессоров его биосинтеза (фенидона и диэтилдитиокарбамата), добавленные к инфильтрационному буферу, не влияют на транзиентную экспрессию GFP и общий уровень содержания белка в растительной ткани. Выводы. Результаты свидетельствуют, что тестируемые факторы не являются существенными для проведения Agrobacterium-опосредованной транзиентной экспрессии. Ключевые слова: Nicotiana excelsior, Agrobacterium, транзиентная экспрессия, GFP.

"Hairy" roots of lettuce Lactuca sativa and regenerated plants with interferon-alpha2b gene had been obtained via Agrobacterium rhizogenes-mediated transformation. According to the results of PCR and rt-PCR analyses the studied plants had ifn-alpha2b gene. The regenerated plants differed from the plants of wild type by elongated internodes, early flower-bearing stem formation and purple coloration of leaves in artificial illumination conditions.

Transgenic plants containing either ifn-α2b gene encoding human leukocytic interferon or esxA::fbpBΔTMD genes encoding Mycobacterium tuberculosis antigens ESAT6 and Ag85B were regenerated from hairy root cultures after transformation of chicory cotyledons (Cichorium intybus L. var. Foliosum Hegi) with a wild-type A. rhizogenes A4 strain. The direct shoot regeneration from transgenic roots without callus formation phase on growth regulator-free nutrient medium was demonstrated. The transgenes transfer and transcription in the plants were confirmed by the results of RT-PCR and PCR analyses.

In vitro production of the meroterpene bakuchiol by Psoralea drupacea Bge (Fabaceae) has been studied using aseptically-grown plants, callus cultures of different origin, cell suspensions and transgenic hairy root cultures. The effect of phytohormones and methyl jasmonate on bakuchiol production was also investigated. Bakuchiol was not detected in cell suspensions or hairy root preparations of P. drupacea. In contrast, aerial parts of P. drupacea grown in vitro were found to accumulate up to 11% dry weight of bakuchiol and can therefore be regarded as a potentially useful source of this antimicrobial compound.

Hybrid plants resistant to phosphinothricin (PPT) are obtained as a result of experiments with somatic hybridization between Brassica napus L. cv. Kalinins’kyy and Orychophragmus violaceus L. O.E. Shulz. The hybrids inherited PPT resistance from O. violaceus plants that had been previously transformed by a vector containing the maize transposon system Spm/dSPm with bar gene located within the nonautonomous transposon. The morphologically obtained plants occupy an intermediate position between the initial forms, which is in agreement with the results of isoenzyme analyses (analysis of multiple forms of amylase and esterase) and PCR analysis (presence of the genes bar, gus, and SpmTPase). Inheritance of the plastome occurs from oilseed rape, while that of the mitochondrion, from O. violaceus, which is proved by means of PCR-RFLP analysis. The plant hybrids may be utilized for further selection research with oilseed rape following determination of the edible quality of its oil as well as in experiments with chloroplast transformation, a topic which is of critical importance for oilseed rape.

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. Проанализирована способность lox-сайтов Cre/lox системы рекомбинации бактериофага Р1 влиять на экспрессию трансгенов при расположении этой последовательности непосредственно возле правого бордера (RB) перед кодирующей последовательностью гена. Нативная и мутированная последовательность lox-сайта были размещены в векторах для трансформации возле гена bar и проведена генетическая трансформация растений с помощью агробактерии и биолистическим методом. Lox-опосредованная экспрессия гена bar, обусловливающая устойчивость растений к фосфинотрицину, наблюдалась только у растений, которые получены с помощью агробактериальной трансформации. Методом РТ-ПЦР анализа подтверждено, что в трансгенных растениях, устойчивых к фосфинотрицину, происходит транскрипция гена bar. Сконструирован вектор, в котором ген gus и предшествующий ему lox-сайт размещены вблизи правого бордера, и проведена трансформация табака этим вектором. Экспрессия гена gus задетектирована в листьях трансгенных растений. Векторы, у которых последовательность lox-сайта предшествует гену bar возле правого бордера (RB-lox-bar), успешно использованы для получения устойчивых к фосфинотрицину трансгенных растений таких видов, как Beta vulgaris, Brassica napus, Lactuca sativa и Solanum tuberosum. Наши результаты подтверждают возможность использования последовательности lox-сайта возле правого бордера для контроля экспрессии гена bar в трансгенных растениях.

Phosphinothricin (PPT) resistant hybrid plants between Brassica napus L. cv. Kalinovsky and Orychophragmus violaceus (L.) O.E. Shulz. were obtained as a result of somatic hybridization experiments. The hybrids inherited PPT resistance from O. violaceus plants which were previously transformed by the vector containing Spm/dSpm Zea mays transposon system with bar gene located within the nonautonomous transposon. The obtained plants had intermediate morphology. Their hybrid nature has been confirmed by isozyme (esterase and amilase activity) and PCR (bar, gus, Spm/dSpm integration) analyses. The hybrids combined B. napus plastom and O. violaceus mithochondrion that was revealed by PCR-RFLP. The hybrid plants might be included to rapeseed breeding programme after examination of their oil quality as well as to chloroplast transformation experiments that is still urgent for B. napus.

Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.

Plastid genetic transformation has been performed using both the PEG-treatment of protoplasts of somatic hybrids of B. oleracea carrying A. thaliana chloroplasts and the particle bombardment of regenerable calluses of B. napus sv. Westar. The chloroplast transformation vector pCB040 carried resistance (aadA) gene flanked by rapeseed plastid DNA sequences to target its insertion between the trnV-rps7 fragments. Selection of transplastomic cell lines has been performed according to their ability to grow on the medium supplied with spectinomycin and streptomycin in high concentrations. Antibiotic resistant cell lines have been obtained using the both transformation methods. The presence of the aadA gene in the A. thaliana and B. napus plastomes was confirmed by PCR analysis for two cell lines of B. oleracea (+ A. thaliana) and three lines of B. napus.

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) by vacuum infiltration has been developed. Transgenic sugarbeet plants of Ukraine breeding were selected for their resistance either to the antibiotic kanamycin or to the herbicide glufosinate ammonium. Integration of transgenes was confirmed by PCR and GUS-assay.

Intertribal somatic hybridization between wild type Brassica juncea (L.) Czern.Coss. and transgenic A. thaliana L. has been carried out. Genome of A. thaliana plants contained heterologous transposable element Spm/dSpm, reporter GUS gene, selective genes for kanamycin- (npt II) and phosphinothricin (bar) resistance. Hybrid nature of obtained plants was confirmed with their morphology, GUS hystochemical assay, PCR-RFLP, RAPD and isozyme analyses. It was determined that heterologous transposable element Spm/dSpm is able to function in hybrid plants. There was no complete elimination of A. thaliana genetic material in the hybrids and the transgenes were stably maintained.

Aim. Agrobacterium-mediated transient expression using viral-based vectors is one of the most effective method for recombinant protein production in plant. Transient expression of GFP using viral-based module system was studied in plant tissue culture in vitro. Methods. Regenerated shoots and callus clones of Nicotiana benthamiana were agroinfiltrated with viral-based module system. Protein extracts from GFP-positive tissues were resolved by non-reducing polyacrylamide gel electrophoresis. GFP from gel was eluted and GFP fluorescence was measured by fluorescence spectrometry with excitation filter at 395 nm and emission filter at 510 nm. Results. Regenerated shoots and callus clones lines accumulated GFP at 242.0±0.7 and 221.6±4.1 μg per g fresh tissue, respectively. The obtained level of transient expression is comparable with other plant production systems in early stage development. Conclusions. The developed technique shows promise for production of therapeutic proteins and antigens in the short term (14–16 days) by transient expression system in plant tissue culture in vitro. Keywords: viral-based module system, transient expression, recombinant proteins, plant tissue culture, GFP.

Aim. The production of alfalfa and peanut cultivars with new properties is necessary. The purpose of this work was to develop Agrobacterium-mediated transformation protocol and to construct transgenic alfalfa and peanut plants resistant to herbicide Pursuit Methods. Genetic transformation was carried out using cocultivation of peanut and alfalfa explants with Agrobacterium tumefaciens strain GV3101 carrying genetic construct pCB004 containing mutant ahas/als gene and nptII gene. Selection was held on the solidified callus inducing medium with 50 mkg/l Pursuit. The selected callus clones were put on the regeneration medium with the same selective agents. Obtained regeneration lines were analysed using PCR-analysis. Results. 17 peanut and 14 alfalfa regeneration lines had positive signals after PCR analysis with DNA fragments of required molecular size for ahas/als and nptII genes. Conclusions. Transgenic alfalfa and peanut plants resistant to the herbicide Pursuit were obtained.Keywords: alfalfa, peanut, ahas/als gene, transformation.

This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein.