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One of the pressing tasks of current biotechnology is the production of stress-resistant cultures of hairy roots and elaboration of procedures of their long-term storage without frequent passages and the use of expensive and labor-consuming methods of cryopreservation. Plant resistance to stress factors very much depends on their antioxidant system and its component glutathione-S-transferases. In this work, we describe the production and examination of tobacco (Nicotiana tabacum L.) hairy roots with constitutive expression of glutathione-S-transferase gene AtGSTF11 of Arabidopsis thaliana. Tobacco hairy roots carrying this transgene were notable for an intense accumulation of biomass and greater resistance to salinization, heavy metals, and heat stress. Moreover, transgenic hairy roots better survived long-term storage without passages to fresh nutrient medium. Constitutive expression of gene AtGSTF11 in the cultures of hairy roots under stress conditions was accompanied by a decrease in the content of hydrogen peroxide, proline, and malonic dialdehyde and was associated with a rise in the rate of superoxide anion production and total antioxidant capacity.

Natural rubber (NR) remains an indispensable raw material with unique properties that is used in the manufacture of a large number of products and the global demand for it is growing every year. The only industrially important source of NR is the tropical tree Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg., thus alternative sources of rubber are required. For the temperate zone, the most suitable source of high quality rubber is the Russian (Kazakh) dandelion Taraxacum kok-saghyz L.E. Rodin (TKS). An obstacle to the widespread industrial cultivation of TKS is its high heterozygosity, poor growth energy, and low competitiveness in the field, as well as inbreeding depression. Rapid cultivation of TKS requires the use of modern technologies of marker-assisted and genomic selection, as well as approaches of genetic engineering and genome editing. This review is devoted to describing the progress in the field of molecular genetics, genomics, and genetic engineering of TKS. Sequencing and annotation of the entire TKS genome made it possible to identify a large number of SNPs, which were subsequently used in genotyping. To date, a total of 90 functional genes have been identified that control the rubber synthesis pathway in TKS. The most important of these proteins are part of the rubber transferase complex and are encoded by eight genes for cis-prenyltransferases (TkCPT), two genes for cis-prenyltransferase-like proteins (TkCPTL), one gene for rubber elongation factor (TkREF), and nine genes for small rubber particle proteins (TkSRPP). In TKS, genes for enzymes of inulin metabolism have also been identified and genome-wide studies of other gene families are also underway. Comparative transcriptomic and proteomic studies of TKS lines with different accumulations of NR are also being carried out, which help to identify genes and proteins involved in the synthesis, regulation, and accumulation of this natural polymer. A number of authors already use the knowledge gained in the genetic engineering of TKS and the main goal of these works is the rapid transformation of the TKS into an economically viable rubber crop. There are no great successes in this area so far, therefore work on genetic transformation and genome editing of TKS should be continued, considering the recent results of genome-wide studies.

Oilseed rape (Brassica napus) is an economically important crop. In a temperate climate, powdery mildew Erysiphe crucifertaum can drastically reduce its yield. Nevertheless, cultivars resistant to this fungal disease have not yet been selected. Glutathione S-transferase GSTF11 is involved in glucosinolate (GSL) biosynthesis and response to stress, including fungal deceases. However, the impact of exogenous GSTF11 gene expression on resistance to powdery mildew has not yet been confirmed and requires further investigation. Transgenic B. napus was generated for this purpose. It demonstrated increased GST activity and a higher GSH:GSSG ratio under normal conditions. Powdery mildew Erysiphe crucifertaum caused 50% mortality in wild type (WT) plants. In most of transgenic plants, mycelium growth was inhibited. The infection contributed to higher GSTF11 expression and increased levels of glutathione (GSH) and oxidized glutathione (GSSG) in both transgenic and WT plants. In contrast, GSTF11 mRNA content, GST activity and GSSG level were lower only in WT plants. In transgenic plants, increased resistance to powdery mildew correlated with a lower GSH:GSSG ratio, indicating a higher content of neutralized toxic molecules. GSTF11 expression was also affected by cold stress, but not drought. At −1 °C, the expression level increased only in transgenic plants. Therefore, GSTF11 appears to be nonspecific and is able to protect plants under several types of stress. This gene could be used as a target in the production of stress tolerant cultivars.

Endophytic Bacillus subtilis is a non-pathogenic beneficial bacterium which promotes plant growth and tolerance to abiotic stresses, including drought. However, the underlying physiological mechanisms are not well understood. In this study, the potential role that endogenous salicylic acid (SA) plays in regulating endophytic B. subtilis-mediated drought tolerance in wheat (Triticum aestivum L.) was examined. The study was conducted on genotypes with contrasting levels of intrinsic drought tolerance (drought-tolerant (DT) cv. Ekada70; drought-susceptible (DS) cv. Salavat Yulaev). It was revealed that B. subtilis 10-4 promoted endogenous SA accumulation and increased the relative level of transcripts of the PR-1 gene, a marker of the SA-dependent defense pathway, but two wheat cultivars responded differently, with the highest levels exhibited in DT wheat seedlings. These had a positive correlation with the ability of strain 10-4 to effectively protect DT wheat seedlings against drought injury by decreasing osmotic and oxidative damages (i.e., proline, water holding capacity (WHC), and malondialdehyde (MDA)). However, the use of the SA biosynthesis inhibitor 1-aminobenzotriazole prevented endogenous SA accumulation under normal conditions and the maintenance of its increased level under stress as well as abolished the effects of B. subtilis treatment. Particularly, the suppression of strain 10-4-induced effects on proline and WHC, which are both contributing factors to dehydration tolerance, was found. Moreover, the prevention of strain 10-4-induced wheat tolerance to the adverse impacts of drought, as judged by the degree of membrane lipid peroxidation (MDA) and plant growth (length, biomass), was revealed. Thus, these data provide an argument in favor of a key role of endogenous SA as a hormone intermediate in triggering the defense responses by B. subtilis 10-4, which also afford the foundation for the development of the bacterial-induced tolerance of these two different wheat genotypes under dehydration.

CRISPR/Cas genome editing of plants is realized in three basic variants, including knockout mutations as indels, insertion of alien DNA fragments, and base editing via deamination of nitrogenous bases. The most important stages of the CRISPR/Cas-based genome editing are the choice of a target site, design of guide RNAs, creation of genetically engineered constructions, and delivery of CRISPR/Cas components into a plant cell. Rapid developments in the field of plant genome editing with the use of CRISPR/Cas systems requires more detailed consideration of the last stage, so this review is dedicated to the description of the main ways to deliver CRISPR/Cas components into cells of higher plants. In the first studies on the genome editing of different plant species, these components were delivered to the target site mainly by Agrobacterium tumefaciens. This approach supposes integration of T-DNA into a genome and a stable expression of CRISPR/Cas components or their transient expression in the case of agroinfiltration. Another widespread approach included the use of plant viruses as delivery platforms; in this case, viruses were used mainly for production of an increased amount of guide RNAs that significantly improved the efficiency of genome editing. Another approach provides for the use of another bacterium, A. rhizogenes, as a platform for delivery of CRISPR/Cas components. This bacterium induces hairy root formation that may be an indirect confirmation of successful genome editing and assist in the selection of genetically modified forms. Other common ways to obtain genetically edited plants are the biolistic delivery of genetically engineered constructions into explants and various protoplast transformation technologies. The review also considers some issues transgenic and GM status of CRISPR/Cas-edited plants to transgenic and GM plants. There are a number of cases in which new organisms created by a CRISPR/Cas genome editing without any introduction of alien DNA were not considered as transgenic ones; it is quite possible that such plants will not fall under Russian legislation prohibiting GMO cultivation.

Although changes in root architecture in response to the environment can optimize mineral and water nutrient uptake, mechanisms regulating these changes are not well-understood. We investigated whether P deprivation effects on root development are mediated by abscisic acid (ABA) and its interactions with other hormones. The ABA-deficient barley mutant Az34 and its wild-type (WT) were grown in P-deprived and P-replete conditions, and hormones were measured in whole roots and root tips. Although P deprivation decreased growth in shoot mass similarly in both genotypes, only the WT increased primary root length and number of lateral roots. The effect was accompanied by ABA accumulation in root tips, a response not seen in Az34. Increased ABA in P-deprived WT was accompanied by decreased concentrations of cytokinin, an inhibitor of root extension. Furthermore, P-deficiency in the WT increased auxin concentration in whole root systems in association with increased root branching. In the ABA-deficient mutant, P-starvation failed to stimulate root elongation or promote branching, and there was no decline in cytokinin and no increase in auxin. The results demonstrate ABA&rsquo, s ability to mediate in root growth responses to P starvation in barley, an effect linked to its effects on cytokinin and auxin concentrations.

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).

The purpose of the present study was to develop effective methods for callus induction, shoot regeneration, and rooting for Parasponia andersonii. Leaf explants of P. andersonii were placed on Lloyd and McCown’s (WPM) medium supplemented with various concentrations of TDZ and NAA for callus induction. Callus induction was observed on media containing 0.1 - 0.2 mg/l TDZ with 0.05 mg/l NAA. Maximum shoot regeneration was observed when the calluses were cultured on MS supplemented with TDZ and IBA. Shoots cultured on WPM medium supplemented with 0.5 mg/l IBA had the maximum rooting percentage (100) in 3 weeks. Rooted plants were transplanted to a potting mixture containing vermiculite (50%) and peat (50%) (v/v). After 2 months, more than 20% of plants survived and were transferred to the greenhouse. Thus, a new effective method has been developed for P. andersonii micropropagation that can be used in studies of plant-Rhizobium symbiosis and for the generation of transgenic Parasponia plants.Plant Tissue Cult. & Biotech. 28(1): 45-55, 2018 (June)

The peculiarities of root growth and stress tolerance of transgenic tobacco plants with constitutive expression of NtEXPA1 and NtEXPA5 genes, as well as plants with reduced expression of NtEXPA4 gene encoding α-expansins of Nicotiana tabacum, were studied during prolonged cultivation under conditions of drought, salinity, and low positive temperatures. Increased expression of expansin genes led to an increase in the growth rate and root length both under normal plant growth conditions and at 12°C and 50 mM NaCl. Increased expression of expansin genes influenced the changes in the fresh and dry mass of a shoot, leading to an increase in their exposure to hypothermia. Transgenic plants with a reduced level of NtEXPA4 expansin gene expression were characterized by a reduction in the fresh and dry weight of a shoot due to drought and low positive temperatures. The totality of the data obtained may indicate the involvement of NtEXPA1, NtEXPA4, and NtEXPA5 tobacco expansin genes in the regulation of growth under hypothermia, drought, and salinity.

The expression level of the gene PtrXTH1 encoding xyloglucan endo-transglycosylase in the leaves of Populus tremula L. of wild-type and in response to exogenous phytohormones treatment was analyzed. The highest level of transcripts of PtrXTH1 was detected in young, intensively growing leaves of aspen. In young aspen leaves, the expression of PtrXTH1 was induced by cytokinins, auxins, and brassinosteroids. The content of PtrXTH1 transcripts increased under the constitutive expression of the PnARGOS-LIKE gene. Bioinformatic analysis of PtXTH1 putative promoter region in P. trichocarpa Torr. and A. Gray ex. Hook showed the presence of cis-regulatory elements associated with the regulation of growth and stress resistance. To determine the role of the gene under study, we also created transgenic tobacco plants with constitutive expression of the PnXTH1 gene (the ortholog of PtrXTH1 from the black poplar P. nigra). Transgenic tobacco plants were characterized by an increase in leaf size and fresh and dry weight of the aboveground part under normal growth conditions. When grown under conditions of salinization and drought, transgenic plants were distinguished by increased stress resistance due to the maintenance of cell expansion in roots and stems at a higher level and the ability to more effectively retain water in leaves compared with wild-type plants.

Taraxacum hybernum Steven, also known as krym‐saghyz, is one of the potential sources of natural rubber. The authors studied the aseptic germination of the T. hybernum seeds using various sterilizing agents and their effect on contamination. In vitro T. hybernum plants were used for Agrobacterium rhizogenesmediated transformation. A4 and 15834 strains of A. rhizogenes showed similar effectiveness after inoculation by injection into the hypocotyls. Authors generated the hairy roots of T. hybernum and the transgenic forms of this plant using A. rhizogenes‐mediated transformation.Plant Tissue Cult. & Biotech. 27(2): 141-151, 2017 (December)

Despite the intensive study of xyloglucan endotransglucosylases/hydrolases, their multifaceted role in plant growth regulation in changing environmental conditions is not yet clarified. The functional role of the large number of genes encoding this group of enzymes is also still unclear. NtEXGT gene encodes one of xyloglucan endotransglucosylases/hydrolases (XTHs) of Nicotiana tabacum L. The highest level of NtEXGT gene expression was detected in young flowers and leaves near the shoot apex. Expression of the NtEXGT gene in leaves was induced by cytokinins, auxins, brassinosteroids and gibberellins. NtEXGT gene was also up-regulated by salinity, drought, cold, cadmium and 10 μM abscisic acid treatments and down-regulated in response to 0 °C and 100 μM abscisic acid. Pretreatment of leaves with fluridone contributed to smaller increase in the level of NtEXGT transcripts in response to drought stress. These data suggest that NtEXGT gene is ABA-regulated and probably implicated in ABA-dependent signaling in response to stress factors. 35S::NtEXGT plants of tobacco showed higher rate of root growth under salt-stress conditions, greater frost and heat tolerance as compared with the wild type tobacco plants.

A small family of ARGOS genes encodes transmembrane proteins that act as negative regulators of ethylene signaling. Recent studies show that ARGOS genes are involved in the regulation of plant growth under the influence of stress factors. However, the role of ARGOS genes in this process is poorly known. Thereby, our goal was to determine the expression profile of these genes in Arabidopsis thaliana and Nicotiana tabacum in response to phytohormone treatment and stress factors. We discovered that expression of the AtARGOS and AtARGOS-LIKE genes of A. thaliana is regulated by ethylene and depends on environmental conditions. The highest expression level of the NtARGOS-LIKE1 gene of tobacco (NtARL1) was observed in blooming flowers and young organs. It was induced by auxins, ethylene, ABA, methyl jasmonate as well as hypothermia, drought, salinity and heat stresses. To evaluate the impact of ARGOS genes on plant growth under stress, we created transgenic tobacco plants with constitutive expression of the AtARGOS-LIKE gene of A. thaliana (AtARL), controlled by a strong Dahlia mosaic virus promoter. Overexpression of the AtARL gene contributed to an increase in the volume and quantity of mesophyll cells in the leaves of tobacco under normal conditions, and also to an improvement in root growth under salinity, cold and cadmium treatment. The AtARL transgene produced a positive effect on shoot growth when exposed to drought and high salinity, and a negative effect under cold stress. Accordingly, genes of the ARGOS family can be recommended as targets for genetic engineering and genome editing in order to enhance productivity and stress tolerance of economically important plants.

We identified a Populus nigra auxin-regulated gene involved in organ size (PnARGOS)-LIKE, encoding one organ size related protein in black poplar. It is homologous to AtARGOS and AtARGOS-LIKE genes of Arabidopsis thaliana. ABRE-like, G-box, GATA and I-box motifs were discovered in the promoter region of the poplar ARGOS-LIKE gene. In wild type aspen (Populus tremula) plants, an ortholog of the PnARGOS-LIKE gene (PtrARGOS-LIKE) was noticeably expressed in actively dividing and expanding young leaves and calli, whereas its mRNA content increased in response to exogenous 6-benzylaminopurine, 1-naphthaleneacetic acid, and 24-epibrassinolide. Expression of the PtrARGOS-LIKE gene was reduced under a salinity treatment. In addition, we generated transgenic tobacco and aspen plants with an up-regulated expression of the PnARGOS-LIKE gene. A constitutive expression of the gene contributed to an increase in size of stems and leaves of the transgenic tobacco plants. In the transgenic aspen, a constitutive expression of the PnARGOS-LIKE gene promoted an increase in the frequency of leaf initiations and in leaf length and area. The size of transgenic tobacco and aspen leaves increased due to the enlargement of individual cells. The results show the significance of the PnARGOS-LIKE gene for control of leaf initiation and organ growth by cell expansion in poplar.

Diverse functions of ethylene in plants may depend on its ability to interact with other hormones. We studied the participation of ethylene in the regulation of accumulation and metabolism of cytokinins comparing ethylene-insensitive mutant plants of arabidopsis (Arabidopsis thaliana [L.] Heynh., etr1-1) with the plants of original ecotype Columbia (Col-0). Because cytokinins can regulate growth of both leaves and roots, we determined the weights of these organs and the ratio between them. The content of zeatin and its riboside in the roots of etr1-1 plants was two times greater than in Col-0 plants, which could be accounted for by inhibition of conversion of these forms of cytokinins into 9-N-glucosides. In the leaves of mutant plants, expression of IPT3 gene responsible for the synthesis of cytokinins was more intense than in Col-0 plants, which could also contribute to a rise in the content of cytokinins. In this case, the weight of roots in etr1-1 mutants was lower than in the plants of original ecotype. Because high concentrations of cytokinins can inhibit root growth, suppression of accumulation of their biomass in mutant plants may be related to a greater content of cytokinins therein. The obtained results suggest that ethylene can suppress accumulation of cytokinins and, thereby, maintain redistribution of biomass in favor of the roots, which is important for plant adaptation to a shortage of water and ions.

We generated transgenic tobacco plants (Nicotiana tabacum L.) with overexpression of the Arabidopsis thaliana CLAVATA3 (CLV3) gene which is known to be a negative regulator of cell division. Surprisingly, most of the 35S::CLV3 transgenic plants showed no phenotypic differences with the wild type plants. However, there were considerable changes in the morphological parameters between 35S::CLV3 overexpressors and wild type plants. As expected, the number of meristematic cells in the shoot apical meristem was reduced in 35S::CLV3 plants as compared to the wild type plants. Moreover, overexpression of CLV3 exerted morphological changes not only to shoot apical meristem but also to leaves and flowers. Thus, transgenic plants were characterized by reduced number of epidermal and mesophyll cells as well as stomatal pores in mature leaves. However, there was a compensatory increase in leaf cell size of 35S::CLV3 plants that contributed to maintenance of organ size within the normal range. We observed that expression of cell expansion-promoted genes, expansin NtEXPA4 and endo-xyloglucan transferase NtEXGT, were elevated in mature leaves. In contrast, there was a decrease in the transcript level of the cell division-related AINTEGUMENTA-like (NtANTL) gene in 35S::CLV3 transgenic plants. In addition, we detected an increase in cytokinin level without any changes in the contents of IAA and ABA in 35S::CLV3 overexpressors. Interestingly, cytokinin treatment was shown to stimulate the expression of NtEXPA4 and NtEXGT genes in 35S::CLV3 transgenic plants. We propose that observed compensatory cell expansion in leaves of 35S::CLV3 transgenic plants may be due, at least in part, to a possible link between cytokinin signalling and cell expansion-related genes.

The Nicotiana tabacum AINTEGUMENTA-like gene (NtANTL), encoding one of AP2/ERF transcription factors, is a putative ortholog of the AtANT gene from Arabidopsis thaliana. In wild-type tobacco plants, the NtANTL gene was expressed in the actively dividing young flowers, shoot apices, and calluses, while the level of its mRNA increased considerably after treatment with exogenous 6-benzylaminopurine, indoleacetic acid and 24-epibrassinolide. We found a positive correlation among the expression levels of NtANTL, cyclin NtCYCD3;1 and cyclin-dependent kinase NtCDKB1-1 genes, suggesting possible molecular links between AINTEGUMENTA and cell cycle regulators in tobacco plants. However, no correlation was observed between NtANTL, NtCYCD3;1 and NtCDKB1-1 expression levels in response to NaCl and ABA. These observations indicate that the transcription factor NtANTL was not involved in the regulation of the cellular response to salinity nor did it affect the expression of NtCYCD3;1 and NtCDKB1-1 when tobacco plants were exposed to salt stress and ABA. In addition, we generated transgenic tobacco plants with both up-regulated and down-regulated expression of the NtANTL gene. Constitutive expression of the NtANTL gene contributed to an increase in the size of leaves and corolla of transgenic plants. Transgenic plants with reduced expression of the NtANTL gene had smaller leaves, flowers and stems, but showed a compensatory increase in the cell size of leaves and flowers. The results show the significance of the NtANTL gene for the control of organ growth by both cell division and expansion in tobacco plants.

The effect of cadmium acetate (200 and 400 μM Cd(CH3COO)2) on the transcriptional promoter activity of rice (Oryza sativa L.) phytochelatin synthase gene 900 bp in size was studied. The reporter gene of glucuronidase was placed under the control of this promoter in transgenic tobacco (Nicotiana tabacum L.) plants. For determining boundaries of this gene promoter responsive to cadmium, deletion constructs were created and their transcriptional activity was measured under the influence of different cadmium concentrations. Deletion of 189 bp from the 5′-terminus of this gene promoter resulted in some transcription activation, whereas deletion of successive 95 bp from the 5′-terminus resulted in cadmium-independent activation of the promoter.

Changes in the expression levels of tobacco expansin genes NtEXPA1, NtEXPA4, NtEXPA5, and NtEXPA6 were studied in different organs of tobacco (Nicotiana tabacum L.) as well as in response to phytohormone and stress treatments. It was shown that NtEXPA1, NtEXPA4 and NtEXPA5 transcripts were predominantly expressed in the shoot apices and young leaves, but almost absent in mature leaves and roots. The NtEXPA6 mRNA was found at high levels in calluses containing a large number of undifferentiated cells, but hardly detectable in the leaves of different ages and roots. In young leaves, expression levels of NtEXPA1, NtEXPA4 and NtEXPA5 genes were induced by cytokinins, auxins and gibberellins. Cytokinins and auxins were also found to increase NtEXPA6 transcripts in young leaves but to the much lower levels than the other expansin mRNAs. Expression analysis demonstrated that brassinosteroid phytohormones were able either to up-regulate or to down-regulate expression of different expansins in leaves of different ages. Furthermore, transcript levels of NtEXPA1, NtEXPA4, and NtEXPA5 genes were increased in response to NaCl, drought, cold, heat, and 10μM abscisic acid (ABA) treatments but reduced in response to more severe stresses, i.e. cadmium, freezing, and 100μM ABA. In contrast, no substantial changes were found in NtEXPA6 transcript level after all stress treatments. In addition, we examined the involvement of tobacco expansins in the regulation of abiotic stress tolerance by transgenic approaches. Transgenic tobacco plants with constitutive expression of NtEXPA1 and NtEXPA5 exhibited improved tolerance to salt stress: these plants showed higher growth indices after NaCl treatment and minimized water loss by reducing stomatal density. In contrast, NtEXPA4-silenced plants were characterized by a considerable growth reduction under salinity and enhanced water loss. Our findings indicate that expression levels of all studied tobacco expansins genes are modulated by plant hormones whereas NtEXPA1, NtEXPA4, and NtEXPA5 expansins may be involved in the regulation of stress tolerance in tobacco plants.

The AINTEGUMENTA gene encodes a transcription factor involved in the regulation of growth of both reproductive and vegetative plant organs. Two conserved regions of this gene were amplified and cloned in antisense orientation. The produced genetic constructs were used to obtain transgenic tobacco plants with the AINTEGUMENTA gene expression downregulated. Over half of the transgenic plants were characterized by smaller leaves, stems, and flowers with their shapes and symmetry not changed. The transgenic plants ceased to grow earlier, flowered later, and had fewer flowers than controls. The main reason for smaller organs was shown to be the lower cell number in an organ with cell sizes unchanged. The genetic constructs can be used to obtain transgenic plants of various species with smaller organs.

In the process of obtaining transgenic plants suitable for phytoremediation of heavy metal-contaminated soils, a synthetic pseudophytochelatin gene coding for a phytochelatin analog Met(GluCys)6Gly was de novo designed and cloned. Contrary to natural enzymatically synthesized phytochelatins, this peptide can be made by a template synthesis. A construct carrying the gene in question under the control of the viral constitutive 35S promoter was made on the basis of a binary vector pCAMBIA 1305.1. This construct was used to express the pseudophytochelatin gene in the model transgenic tobacco plants (Nicotiana tabacum L.), and the above plants acquired additional resistance to various cadmium concentrations.

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.

The auxin-inducible gene ARGOS from Arabidopsis thaliana is expressed in growing tissues and controls the plant organ size by regulating cell proliferation and meristematic competence. The promoter of the dahlia (Dahlia pinnata Cav.) mosaic virus (DMV) resembles the well-known cauliflower mosaic virus 35S promoter but shows a higher activity in transgenic tobacco plants (Nicotiana tabacum L.). We obtained transgenic tobacco plants expressing the Arabidopsis ARGOS gene under the control of the DMV promoter. Several of the T0 generation plants exhibited an accelerated transition to flowering, a slight increase in flower size, and a significant increase in the leaf size. The T1 transgenic plants were characterized by faster growth, the increased leaf size, and somewhat enlarged flowers as compared with control plants. These phenotypic traits, as well as stability and inheritance of the transgene were demonstrated also in T2 transgenic plants.

By the techniques of DNA shuffling, PCR, and restriction-ligation, chimeric forms of cauliflower (Brassica oleracea) mosaic virus (CaMV), dahlia (Dahlia pinnata) mosaic virus (DMV), and carnation (Dianthus caryophillus) etching ring virus (CERV) promoters were obtained at various combinations. Twelve chimeric promoters were cloned into pCambia binary vectors comprising the reporter GUS gene, and their activities in transgenic tobacco (Nicotiana tabacum) plants were determined fluorimetrically. 35S promoter and those of DMV (442 bp) and CERV (371 and 501 bp) were used as controls. Seven of analyzed promoters displayed higher and seven promoters lower activity in transgenic tobacco plants than 35S promoter. The highest activity was characteristic of natural DMV promoter, and the least one — natural CERV promoter 501 bp in size. The CERV promoter 371 bp in size was approximately similar in strength to 35S promoter.

Promoters of carnation etched ring virus (CERV) and dahlia mosaic virus (DMV) were cloned into binary vectors pCambia 1304, pCambia 1281Z, and pCambia 1291Z with reporter GFP and GUS genes. Activities of these promoters in tobacco protoplasts and transgenic plants were determined using these constructs. Histochemical GUS analysis demonstrated the absence of tissue-specificity in transgenic plants transformed with these promoters. The quantitative analysis of these promoter activities in transgenic tobacco plants, using 4-methylumbelliferone as a substrate, showed that 35S CaMV, CERV, and DMV promoters displayed approximately similar activities in transgenic tobacco plants.