Salt-tolerance improvement of tomatoes is largely a task of modern selection and plant molecular genetics because of cultivation on dry and irrigated lands under salt stress. To reveal the salt resistance gene, we need quantitative real-time polymerase chain reaction (qRT-PCR) normalization through reference genes analysis. Sometimes, housekeeping gene expression changes in response to various stress factors, especially salinity. In this manuscript, we evaluated expression changes of elongation factor 1α X53043.1 (EF1α), actin BT013707.1 (ACT), ubiquitin NM_001346406.1 (UBI), nuclear transcript factor XM_026030313.2 (NFT-Y), β-tubulin NM_001247878.2 (TUB), glyceraldehyde-3 phosphate dehydrogenase NM_001247874.2 (GAPDH), phosphatase 2A catalytic subunit NM_001247587.2 (PP2a), and phosphoglycerate kinase XM_004243920.4 (PGK) in salt-sensitive Solanum lycopersicum L. YaLF line and salt tolerance Rekordsmen cv. under 100 mM NaCl. We also suggested potential correlations between relative water content (RWC), ion accumulation, and reference gene expression in tomato genotypes with contrasting salinity tolerance. We used geNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder algorithms to establish a set of the most reliable tomato candidate genes. The most stable genes for YaLF tomatoes were ACT, UBI, TUB, and PP2a. Despite differences in ranks, the NFT-Y was present in Rekordsmen’s stable set.

The influence of crown illumination on leaf damage of horse chestnut species (Aesculus hippocastanum L., Aesculus glabra Willd, Aesculus flava Aiton, Aesculus pavia L., Aesculus × carnea Hayne, Aesculus parviflora Walter, Aesculus chinensis Bunge) affected by ohrid leaf miner (Cameraria ohridella Deschka & Dymić) was studied using some accessions from the arboretum botanical tree collection. A. hippocastanum, A. glabra, A. flava had the lowest chl a content in the foliage on the sunlit side of the crown, while in A. pavia, A. parviflora and A. chinensis this indicator was the highest. The chl a content in the leaves of A. hippocastanum and A. flava under shaded conditions was 1.3 and 2.4 times higher than in the sunlit part, while in A. pavia, A. parviflora and A. chinensis the chl a content on the shaded side was 1.2, 1.6 and 1.3 times lower. The quantitative content of chl b in the sunlit part of the crown in A. hippocastanum and A. flava was significantly higher than in the other species. Moreover, while A. flava and A. parviflora had the highest chl b content in the foliage of the shaded part of the crown, A. glabra and A. × carnea had the lowest. Similarly, differences in proline levels were found in the leaves of different horse chestnut species on the sunny side of the crown. Higher proline levels in less infested species were identified. Water content imbalances due to feeding by leaf miners were most characteristic of the severely affected species. Chlorophyll fluorescence determination revealed high photochemical activity with an effective defense system in resistant species, while non-resistant species exhibited weak defense mechanisms in both sunlight and shade. To assess horse chestnut species the hyperspectral analysis indices (DSWI and SIPI) were also successfully applied. Changes in chl a and chl b content, proline levels, and leaf water-holding properties can be used to assess the resistance of horse chestnut species using classical physiological and biochemical methods. Hyperspectral analysis indices (DSWI and SIPI) can also be successfully applied.

The susceptibility of seven species of chestnuts to the horse chestnut leaf miner was studied in the arboretum of the Main Botanical Garden of the Russian Academy of Sciences (MBG RAS), taking into account their interspecific characteristics. Using pheromone delta traps, the highest number of Cameraria ohridella moths was shown for Aesculus hippocastanum and the lowest for Aesculus chinensis. A number of anatomical parameters of leaves were investigated, such as the thickness of the epidermal cell wall and the thickness of the palisade and spongy parenchyma layers. As a result, it was shown that the most infected chestnut species had a greater thickness of the nutritious parenchyma tissue. No dependence was found between the degree of susceptibility to the horse chestnut leaf miner and such indicators as the content of chlorophyll a + b and carotenoids in the leaves of seven species of chestnuts. Nevertheless, resistance of different species of the genus Aesculus to Cameraria ohridella under increased tannin content in leaves has been shown. Evaluation of phenolic compounds and flavonoids has not established their reliable role as repellents. The high levels of carbohydrates found during the study contributed to increased susceptibility to the horse chestnut leaf miner.

The identification of reliable physiological and biochemical indicators for assessing tomato salt tolerance can increase the efficiency of plant breeding to create new varieties and lines. The purpose of our study was to identify available physiological and biochemical relevant characteristics for assessing the sensitivity to sodium chloride salinity of different tomato genotypes at the seedling stage. A complex analysis was carried out based on indicators such as biomass growth, water content of plant tissues, content of readily soluble salts, sodium and chlorine ions, photosynthetic pigments, carotenoids, phenolic compounds and flavonoids. Additionally, the stomata area of the upper and lower epidermis was taken into account. The comprehensive assessment carried out made it possible to reliably divide the eight studied tomato varieties into two groups: sensitive (Belyij Naliv, Geya, YaLF, Paradigma) and tolerant (Recordsmen, Yuryevskij, Bych’e Serdce, Astrakhanskij). Tomato genotypes that can be classified as sensitive (in order of increasing sensitivity to high concentrations of NaCl (150 mM)) were Belyij Naliv > Geya > YaLF ≥ Paradigma. Tomato genotypes that can be classified as resistant to salt stress (in order of increasing tolerance to high concentrations of NaCl (150 mM)) were Recordsmen < Yuryevskij < Bych’e Serdce < Astrakhanskij. The advisability of only using complex physiological and biochemical indicators to obtain relevant assessments for salinity tolerance at the early stages of tomato plant development has been demonstrated.

Understanding the mechanisms of plant salt tolerance as a complex trait is an integral part of many studies, the results of which have been used in the breeding process. The aim of this study was to compare the root response of two tomato (Solanum lycopersicum L.) genotypes (breeding line YaLF and cultivar Recordsmen) differing in salt tolerance. Rhizogenesis was induced in tomato shoots in vitro with different concentrations of NaCl in the culture medium. A number of morphobiological and cytological parameters were evaluated at the organ, tissue, and cellular levels for possible use in a comprehensive assessment of genotypes for salt tolerance. The influence of NaCl caused disruption of the cell cycle and redistribution of cells in the phases of the cell cycle. An increase in the degree of vacuolization was shown in cv Recordsmen at 75 and 150 mM NaCl and in the YaLF line at 150 mM NaCl. Under salt action, an increase/decrease in the length of cells such as columella cells (both genotypes) and epidermal cells (in cv Recordsmen at 75 and 150 mM NaCl) was shown. Differences between genotypes were demonstrated by changes in the area of the central cylinder and primary root cortex cells, as well as by changes of the Snucleolus/Snucleus ratio in these cells. Transmission electron microscopy (TEM) showed the modification of the chromatin structure in the root cells of these genotypes. Various cytoskeletal disorders were revealed in interphase cells of the tomato root of cv Recordsmen and the YaLF line by immunofluorescent staining under saline conditions. These morphometric and cytological parameters can be used for a comparative evaluation of genotypes differing in salt tolerance in a comprehensive assessment of varieties.

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.

The study of the leafmining moth of the chestnut miner (Cameraria ohridella Deschka & Dymić, 1986) was carried out through the planting of the common horse chestnut (Aesculus hippocastanum L.) in the Main Botanical Garden of the Russian Academy of Sciences. The effect of various degrees of insolation of horse chestnut plants on leaf morphology and the composition of secondary metabolites, as well as the relationship of these parameters with the number and density of C. ohridella populations during the growing season, was studied. The solar influence, it was noted, had a significant impact. Thus, the largest number of the pests was recorded on the leaves of the sunlit side of the tree crown, and the smallest on the leaves of the shady part of the crown. The low content of polyphenols in the pool of secondary metabolites in the tissues of the A. hippocastanum leaves did not deter C. ohridella and poorly protected the plants from this miner, while the significant content of carbohydrates in the leaves reduced the resistance of chestnut plants to damage by the Ohrid leaf miner.

В некоторых регионах засолённость почв является одной из самых больших проблем при возделывании культурных растений, поэтому использование районированных солеустойчивых сортов в промышленных масштабах может значительно повысить урожайность. Большинство всех растений, включая важные хозяйственные и продовольственные культуры, в том числе и томат, являются гликофитами. Они чрезвычайно чувствительны к концентрациям солей в почве, например, при 100-200 мМ может наблюдаться ингибирование роста или гибель растений (1). Поэтому одним из важнейших направлений селекции является идентификация солеустойчивых генотипов растений и их последующий отбор. In some regions, soil salinity is one of the biggest problems in the cultivation of cultivated plants, so the use of released salt-tolerant varieties on an industrial scale can significantly increase yields. Most of all plants, including important economic and food crops, including the tomato, are glycophytes. They are extremely sensitive to salt concentrations in the soil, for example, at 100-200 mM growth inhibition or plant death can be observed (1). Therefore, one of the most important areas of breeding is the identification of salt-tolerant plant genotypes and their subsequent selection.

In vitro evaluation of tomato seeds and seedlings for salt tolerance has undoubted advantages (high productivity, as well as stability and reproducibility of the obtained experimental data due to the maintenance of constant controlled conditions) in comparison with open-field system and pot experiments. However, even high-quality seeds greatly differ in the uniformity of germination capacity and germination energy. Heterogeneous germination in the habit and developmental stage of plant material significantly distorts the obtaining of relevant experimental data suitable for correct interpretation. In our study, we propose a simple and effective bioassay method suitable to comparative in vitro study of tomato salt tolerance using shoot apex of seedlings at the early first-true-leaf stage. Shoot apexes cultured the on the root induction medium (RIM) supplemented with 0.2 mg/L indole-3-butyric acid (IBA) and NaCl at different concentrations (0–250 mM NaCl) revealed significant differences between two tomato genotypes (line YaLF and cv. Rekordsmen) at the organismal (measurements of CO2 gas exchange), organ (rhizogenesis frequency; number and length of de novo regenerated roots; root fresh (RFW) and dry (RDW) weights; shoot fresh (SFW) and dry (SDW) weights), tissue (the average cross-sectional area of epidermal and mesophylls cotyledonary cells) and cellular (ultrastructure of chloroplast and nuclear compartments) development levels. In addition, a quantitative comparison of proline and photosynthetic pigments contents under 75 and 150 mm NaCl treatments showed a different response between two tomato genotypes. The proposed methodological approach can be used for other plants with a high response to auxin-induced rhizogenesis in vitro, as well as for the comparative in vitro assessment of other abiotic stresses.

The population of the Ohrid miner on infested horse chestnut trees growing in different light conditions was evaluated. The dynamics of development, expressed in the ratio of caterpillars and pupae of different ages was found to depend on the conditions of natural lighting. The need to take this factor into account when monitoring a pest and developing protective measures is discussed.

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

The study was devoted to morphological and cytoembryological analysis of disorders in the anther and pollen development of transgenic tomato plants with a normal and abnormal phenotype, which is characterized by the impaired development of generative organs. Various abnormalities in the structural organization of anthers and microspores were revealed. Such abnormalities in microspores lead to the blocking of asymmetric cell division and, accordingly, the male gametophyte formation. Some of the non-degenerated microspores accumulate a large number of storage inclusions, forming sterile mononuclear pseudo-pollen, which is similar in size and appearance to fertile pollen grain (looks like pollen grain). It was discussed that the growth of tapetal cells in abnormal anthers by increasing the size and ploidy level of nuclei contributes to this process. It has been shown that in transgenic plants with a normal phenotype, individual disturbances are also observed in the development of both male and female gametophytes. The reason for the developmental arrest of some ovules was the death of endosperm at different stages of the globular embryo. At the same time, noticeable hypertrophy of endothelial cells performing a secretory function was observed. In the ovules of transgenic plants with abnormalities, the endothelium forms a pseudo-embryo instead of the embryo sac, stimulating the development of parthenocarpic fruits. The data obtained in this study can be useful for a better understanding of the genetic and molecular mechanisms of cytoplasmic male sterility and parthenocarpic fruit development in tomatoes.

A comparative analysis of tomato roots regenerated in vitro on media supplemented with different NaCl concentrations (0–250 mM) has been carried out. A morphogenetic study performed at the organ level has determined rhizogenesis-inhibiting NaCl concentrations, while the morphometric analysis of regenerated roots in juvenile seedlings has registered such characteristics as their number, length, and fresh/dry weight. A cytological study has revealed some tissue disorders, such as changes in the vacuolization of root cap cells and root cortex cells (RCC) under salinity conditions. At some NaCl concentrations, significant changes in the cells of these tissues have been observed in relation to such parameters as the root cap length (50–100 and 250 mM NaCl), the number of root cap layers (50, 75, 150–250 mМ NaCl), the area of central cylinder cells (CCCs, 75–150 and 250 mM NaCl), the RCC area (100–200 mM NaCl), and the nucleolus to nucleus ratio (25, 50, and 200 mM NaCl). Using cytophotometry, we have shown there to be an increase in the number of interphase cells of the root meristem in the G2 phase with a simultaneous decrease of this parameter in the G1 phase. Immunofluorescent labeling has revealed various disorganizations in the alpha-tubulin cytoskeleton of interphase root meristem cells. Using transmission electron microscopy, we have revealed structural changes in plastids of root cap cells, RCC, and CCC, as well as changes in the organization of a nuclear compartment in RCC occurred in the presence of 150 mM NaCl. The studied characteristics can be used for a comparative evaluation of tomato genotypes under salinity at different levels of their organization. The proposed approach can be also used for crops that have no difficulties with induced rhizogenesis in vitro.

Various abiotic stresses cause the appearance of reactive oxygen species (ROS) in plant cells, which seriously damage the cellular structures. The engineering of transgenic plants with higher production of ROS-scavenging enzyme in plant cells could protect the integrity of such a fine intracellular structure as the cytoskeleton and each cellular compartment. We analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na2SO4 solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv (WT) and FeSOD-transgenic line, we examined the distribution of ROS and enzyme-linked immunosorbent detection of α-tubulin. In addition, longitudinal sections of the root apexes were compared. Transmission electronic microscopy of atypical cytoskeleton structures was also performed. The differences in the microtubules cortical network between WT and transgenic plants without salt stress were detected. The differences were found in the cortical network of microtubules between WT and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of WT tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of WT plants were manifested under the NaCl treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. Similar changes, including the cortical microtubules disorganization, possibly associated with the formation of atypical tubulin polymers as a response to salt stress caused by Na2SO4 treatment, were also observed. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.