The aim of the study was to study the state and reaction of cytoskeletal elements, microtubules and actin filaments, in root cells of Samsun tobacco plants and its transgenic line expressing the FeSOD1 gene from Arabidopsis thaliana with the pea rbcS leader sequence for the localization of the gene product in chloroplasts encoding Fe-dependent superoxide dismutase, constitutively inducing intracellular oxidative stress, by increasing the H2O2 pool for a long-term effect of moderate concentrations of NaCl and Na2SO4. The main hypothesis was to identify the positive protective effect of controlled constant oxidative stress on the stability of the most sensitive system that provides growth by division and growth by extension (the tubulin cytoskeleton) and effective intracellular transport and structural stability (the actin filament system). Localization of the microtubule cytoskeleton and actin filaments using antibodies to tubulin clone DM1α and actin clone 10-B3 by transmission electron microscopy and immunocytologically, detected by treatment with the second antibodies conjugated with Alexa‑488, made it possible to establish signs of reorganization and disassembly of the actin filament network under the action of NaCl and Na2SO4 as in control and in transgenic plants. At the same time, in transgenic plants, differences can be noted even without exposure, which indicates the effectiveness of this method for stimulating a protective response. These data suggest that the state of the system of the tubulin cytoskeleton and actin filaments may be an indicator of the resistance of FeSOD1 transgenic plants to salinity. A relationship has also been established between the reorganization of the cytoskeleton and vacuolization, especially with Na2SO4.

A detailed study into the structural features of the multilevel antipodal complex of wheat Triticum aestivum L. embryo sac was performed at different stages of the complex’s differentiation after double fertilization. The heterogeneity of nuclei ploidy in individual antipodal complexes caused by the asynchrony of the endoreduplication rounds of the nuclear DNA was revealed. The nuclei ploidy of basal, middle, and apical layers of the complexes was measured at the early, middle, and late stages of differentiation. At the early stage of differentiation, the nuclei ploidy of the antipodal complex’s basal layer adjacent to the chalasal region of the nucellus of the embryo sac reaches 13 C, the nuclei of the apical layer cells that contact the endosperm syncytium reaches 63 C, and the nuclei of the middle layer located between the basal and apical layers reach 30 C. At the middle stage of differentiation, the nuclei ploidy in the basal layer increases to 17 C. The nuclei ploidy of the apical layer cells increases to 95 C, and nuclei ploidy of the middle layer increases to 45 C. At the stage of late differentiation, the nuclei ploidy in the basal layer increases to 24 C; the apical layer ploidy increases to 215 C; the middle layer ploidy increases to 63 C. Changes in the shape and structure of the nuclei during differentiation were revealed. They manifest themselves in heterogeneity in shape, size and structure of chromatin; the formation of individual polytene chromosomes; nuclear membrane invaginations; and the variation in the number of nucleoli. Data on the distribution and structure of cytoplasmic organelles of the antipodal cells, endoplasmic reticulum, dictyosomes, mitochondria, and microtubules at different stages of differentiation of the antipodal complexes are fundamentally new. The increased number of cytoplasmic organelles was revealed. During the differentiation, prolong cisterns of the granular reticulum are replaced by concentric rings, mitochondria and plastids of extended and cupped shape appear, and the microtubule network is rebuilt. The features of the antipodal complex’s cell structure may reflect changes in the functions of the antipodal complex during the differentiation. At the early stage, all cells of the complex perform an osmoregulatory function, and cells of different layers of the complex specialize at the middle stage of differentiation. The ploidy level of cell nuclei with polytene chromosomes reflects their functional significance in the formation of endosperm at the nuclear stage of development, and, subsequently, of normal full-fledged grain.

Combined effect of aluminum toxicity and acidic soil conditions is the major problem for barley productivity in certain areas of crop cultivation. Therefore the development of effective test systems for the identification and selection of aluminum-resistant barley genotypes is an important goal, especially for regions all over the world with high soil acidity. We used barley genotypes with different resistance to ionic toxicity (Belgorodsky 100, Belogorsky, Kupets, Elo, 999-93 and regenerant line 917-01) for characterization of the plant's reaction to the toxic effect of aluminum. Using methods of cytophotometry, light and electron microscopy we studied structural and functional changes of the root cap cells and obtained cytological markers suitable for early detection of the sensitivity of barley genotypes to aluminum. We showed that the response of plants to aluminum exposure was manifested in changes of mitotic index (MI) and phases of the cell cycle of meristematic cells, and differences in accumulation of starch grains in the root cap cells. It appeared that in aluminum-sensitive genotypes (variety Elo), aluminum induced destruction of starch grains in the root cap cells, whereas in aluminum-resistant genotypes (variety Belogorsky), the structure of starch grains was well preserved. We propose to use the live staining of starch grains in the root cap cells of the whole root as a test for the sensitivity of barley plants to the toxic effect of aluminum.

The transformation of the structural organization of interphase nuclei of a plant cell, depending on the type of tissue, ploidy, and the action of abiotic factors of the medium, has been studied by light and transmission electron microscopy. It is shown that the location and the quantitative relationship between condensed and decondensed chromatin, the presence and localization of nuclear bodies in the plant nucleus, and the presence of invaginations of the nuclear membrane and inclusions depend on the tissue type and cell age as well as the intensity, time, and type of exposure to abiotic factors. Examples of different degrees of chromatin condensation in one plant organism are given. It has been established that the degree of chromatin condensation and decondensation, the state of other domains and nonspecific inclusions of the cell nucleus can be artificially modeled for research purposes or subsequent modification. The issue of application of such technologies for the creation and selection of stable forms of agricultural plants taking into account the controlled modification of their genome is discussed. A possible mechanism for incorporation of nonspecific inclusions in a nuclear compartment is proposed.

Using the methods of light microscopy and DNA cytophotometry, structural organization of the calli of the barley hybrid line Duet x Bios grown in the absence and in the presence of aluminum ions (20 and 40 mg/L) was studied. In the morphogenic calli grown in the standard conditions without aluminum, three main cell types were identified: meristematic, differentiated, and dying. Meristematic cells form a morphogenic zone. A characteristic feature of differentiated cells is a progressive accumulation of vacuoles with storage substances. Structurally separated zones of meristematic cells were not found the calli surviving in the presence of aluminum. Besides, the number of differentiated cells with storage substances is dramatically reduced and the number of dying cells is increased in such calli. According to the cytophotometry data, morphological changes in the aluminum-tolerant calli are accompanied by partial suppression of proliferation, accumulation of cells in postsynthetic phase of the cell cycle, and polyploidization, presumably caused by the DNA endoreduplication. It is noteworthy that the aluminum-tolerant calli exhibit a high regenerative capacity: the proportion of morphogenic calli surviving in the presence of 40 mg/L aluminum is about 5 times greater than that in the calli grown in the control medium. To identify cellular targets of the aluminum effect in differentiated tissues, the roots of rhizogenic calli were studied. For this purpose, the calli derived from aluminum-tolerant plants (cultivar Kupetz), from aluminum-sensitive line 999-93, and from aluminum-tolerant line 917-1 obtained by the method of cell selection were used. It was found that aluminum induces the death of differentiated cells of epidermis and vascular system ingrowing roots of calli derived from non-tolerant lines, whereas undifferentiated cells of meristem remain undamaged. In contrast, rhizogenic calli derived from tolerant forms produce normal roots. This observation has an important prognostic value because it makes it possible to select aluminum-tolerant regenerants at the early stages of cell selection. The data suggest that most likely cause of the emergence of resistant lines is somaclonal variation induced by high concentrations of the selecting agent. Under stressful conditions such variability may result in the in vitro system from the activation of epigenetic factors, such as, for example, methylation of DNA or modification of the chromatin proteins.

C использованием методов световой микроскопии и цитофотометрии ДНК проведен сравнительный анализ структурной организации каллусов гибридной линии ячменя Дуэт ? Биос, выращенных в стандартных условиях культивирования и на средах, содержащих ионы алюминия (20 и 40 мг/л). В морфогенных каллусах, растущих на среде без алюминия, идентифицировано три основных типа клеток: меристематические, дифференцированные и гибнущие. Меристематические клетки формируют морфогенные зоны, характерным признаком дифференцированных клеток является прогрессирующее накопление вакуолей с запасными веществами. В каллусах, выживающих в присутствии алюминия, отсутствуют структурно обособленные зоны меристематических клеток, резко снижается количество дифференцированных клеток с запасными веществами и возрастает количество гибнущих клеток. По данным цитофотометрии изменение морфологического статуса устойчивых каллусов сопровождается частичным подавлением пролиферации, накоплением клеток в постсинтетической фазе клеточного цикла и полиплоидизацией, по-видимому, вызванной эндоредупликацией ДНК. Принципиально важно, что устойчивые к токсическому действию алюминия каллусы обладают высокой способностью к регенерации: доля морфогенетически активных каллусов, выживших в среде с алюминием (40 мг/л) примерно в 5 раз превосходит долю морфогенных каллусов в контроле. Для выявления клеточных мишеней токсического действия алюминия в сформированных тканях изучены корешки ризогенных каллусов. С этой целью использовали каллусы, полученные от растений, толерантных к токсическому действию алюминия (сорт Купец), чувствительной линия 999-93 и полученной методом клеточной селекции линии, устойчивой к действию алюминия линия 917-1. Оказалось, что в растущих корнях каллусов, полученных от неустойчивых линий, алюминий индуцирует гибель дифференцированных клеток эпидермиса и проводящей системы, тогда как недифференцированные клетки меристемы остаются интактными. В противоположность этому ризогенные каллусы, полученные от устойчивых форм, дают нормальные корешки. Это наблюдение имеет важное прогностическое значение, так как позволяет отбирать устойчивые к алюминию регенеранты на ранних этапах клеточной селекции. Полученные данные позволяют высказать предположение, что появление устойчивых линий обусловлено, по-видимому, сомаклональной изменчивостью, индуцированной высокими концентрациями селективного агента. Такого рода изменчивость возникает в системе in vitro в условиях стресса, в результате активации таких эпигенетических факторов, как, например, метилирование ДНК или ацетилирование белков хроматина.

In this study, the morphological and cytoembryological analyses of the tomato plants transformed with the genes encoding chitin-binding proteins (ac and RS-intron-Shir) from Amaranthus caudatus L. and A. retroflexus L., respectively, as well as the gene amp2 encoding hevein-like antimicrobial peptides from Stellaria media L., have been performed. The transgenic lines were adapted to soil and grown in the greenhouse. The analysis of putative transgenic tomato plants revealed several lines that did not differ phenotypically from the wild type plants and three lines with disruption in differentiation of the inflorescence shoot and the flower, as well as the fruit formation (modified plants of each line were transformed with a single gene as noted before). Abnormalities in the development of the generative organs were maintained for at least six vegetative generations. These transgenic plants were shown to be defective in the mail gametophyte formation, fertilization, and, consequently, led to parthenocarpic fruits. The detailed analysis of growing ovules in the abnormal transgenic plants showed that the replacement tissue was formed and proliferated instead of unfertilized embryo sac. The structure of the replacement tissue differed from both embryonic and endosperm tissue of the normal ovule. The formation of the replacement tissue occurred due to continuing proliferation of the endothelial cells that lost their ability for differentiation. The final step in the development of the replacement tissue was its death, which resulted in the cell lysis. The expression of the genes used was confirmed by RTPCR in all three lines with abnormal phenotype, as well as in several lines that did not phenotypically differ from the untransformed control. This suggests that abnormalities in the organs of the generative sphere in the transgenic plants do not depend on the expression of the foreign genes that were introduced in the tomato genome. Here, we argue that agrobacterial transformation affects, directly or indirectly, expression of genes encoding for transcription factors that can activate a gene cascade responsible for the normal plant development.

В работе проведен морфологический и цитоэмбриологический анализ растений томата, трансформированных генами, кодирующими хитинсвязывающие белки из Amaranthus caudatus L. (ac) и A. retroflexus L. (RS-intron-Shir), а также гевеиноподобные антимикробные пептиды из Stellaria media L. (amp2). Полученные трансгенные линии были адаптированы к почвенным условиям и выращены в защищенном грунте. Анализ трансгенных растений поколения Т0 выявил линии, фенотипически не отличающиеся от растений дикого типа, и три линии (по одной линии с каждым из вышеперечисленных генов), которые имели существенные нарушения в дифференцировке цветоносных побегов, строении цветков и плодов. Продемонстрировано сохранение нарушений в развитии генеративных органов в 6 вегетативных поколениях. Показано, что у трансгенных растений с вегетативно наследуемыми аномалиями наблюдаются нарушения в формировании мужского гаметофита, отсутствие нормального оплодотворения и, как следствие, развитие партенокарпических плодов. Детальный анализ растущих семяпочек аномальных трансгенных линий показал, что на месте неоплодотворенного зародышевого мешка формируется и разрастается замещающая ткань, по структуре отличающаяся как от зародышевой, так и от эндоспермальной ткани нормальной семяпочки. Формирование замещающей ткани происходит в результате продолжающейся пролиферации клеток эндотелия, утративших способность к нормальной дифференцировке. Конечным этапом развития замещающей ткани является ее гибель, сопровождающаяся лизисом клеток. Методом ОТ-ПЦР экспрессия целевых генов была подтверждена у всех трех линий с аномальным фенотипом, а также у ряда линий, фенотипически не отличающихся от нетрансформированного контроля. Это означает, что нарушения органов генеративной сферы у трансформированных растений не зависят от экспрессии привнесенных в геном томата гетерологичных генов. Обсуждается, что причиной возникновения нарушений у трансгенных растений является прямое или опосредованное влияние агробактериальной трансформации на изменение экспрессии генов, кодирующих транскрипционные факторы и контролирующих включение каскада генов, необходимых для нормального развития растений.

Dynamics of an antipodal complex formation in wheat ( Tritiñum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of “truncated” ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.

The structural organization of roots, induced in the callus cultures of barley genotypes differing in aluminum tolerance: cv Kupetz, cv 999-93 and its somaclonal form R 999-93 was studied. The goal of this research was to determine how the toxic effects of aluminum at low pH affects the structure of the root and any cells damage of different tissues meristematic zone. At pH 5.8–6.0 root callus from different genotypes had specific features, manifested in the structure root cap, the epidermis, the form of roots. In the presence of 40 mg/1 Al3+ ions and pH 3.7–3.8 in the roots, callus was obtained from the cv Kupetz, there is strong damage to the epidermis, root cap and some other tissues, which, although and resulted in the death of cells, however, were not lethal character. In roots, callus derived from varieties 999-93, under the influence Al3+ ions was observed irreversible changes that lead either to compaction cytoplasm and nuclei, especially in the central part of the root or to the plasmolysis and the destruction of cells, especially in the meristematic zone. On transverse sections was found damaged cells of the central part of the root, formation of the densified outer cell wall of epidermal cells. At the investigation of R 999-93 callus roots it was be shown that most of the cells of meristematic zone of root retained their viability at influence of Al3+ ions. So, the somaclon from cv 999-93, obtained earlier in callus culture by selection on Al-tolerance, differs from the original form at the level of mesostructure.