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Brassica oleracea var. gongylodes (kohlrabi) is an economically important vegetable with edible stems. Due to the complicated in vitro regeneration, biotechnological cultivars with useful traits do not yet exist. This plant have never been subjected to genetic transformation of any kind. Therefore, the development of appropriate methods is an important task of modern plant science. A genetic construct targeting BoCPC gene have been introduced to Agrobacterium tumefaciens strain Agl0 in order to produce knockout mutants of kohlrabi. Addition of AgNO3 to the culture medium allowed to reduce browning after inoculation. Three glufosinate-resistant, PCR-positive transformants were obtained from cultivar Vienna White. In two of them expression level of target gene was significantly reduced. The described protocol allows for genetic transformation and genome editing of kohlrabi. This research opens up prospects for generation of new cultivars with improved agricultural traits such as stress tolerance, increased yields and altered metabolite content. According to the results of various studies, Vienna White can be used as a model cultivar of kohlrabi.

Endophytic

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.

BACKGROUND: Expansins are non-enzymatic proteins involved in the softening of cell walls, the mechanism of action of which is associated with the weakening and breaking of hydrogen bonds between xyloglucans and cellulose microfibrils and is aimed at ensuring cell expansion. THE AIM of our work was to obtain hairy roots of tobacco with constitutive expression of the NtEXPA5 expansin gene, their morphometric analysis and assessment of the state of their antioxidant system in response to stress factors. MATERIALS AND METHODS: The hairy roots were obtained from transgenic tobacco plants expressing the NtEXPA5 gene under the control of the 35S promoter. RESULTS: Constitutive expression of the NtEXPA5 gene promoted an increase in the length and dry weight of hairy roots both under normal conditions and under the action of salinity, copper sulfate, cadmium acetate, and mannitol. Both under normal conditions and under the action of stress factors in transgenic hairy roots, an increase in the activity of superoxide dismutase and the total antioxidate activity was recorded. CONCLUSION: Expansins exert their positive effect on the productivity and stress tolerance of plants not only through their influence on cell expansion, but also through the effect on the antioxidant system.

The emergence of the new SARS-CoV-2 coronavirus has given rise to many enigmas, to which there are no answers yet. However, the degree of threat to humanity, due to the fact that by the beginning of February 2021, more than 100 million people were ill in the world, of which 2 million died, led to the fact that the efforts of many researchers were aimed at combating this disease, including massive sequencing of the complete genomes of SARS-CoV-2, as this is necessary for diagnostics and prediction of the epidemiological situation, including in the long term. Currently, a fairly high level of conservativeness of the SARS-CoV-2 genome is shown, but there is also a significant variability of intra-host viral RNA, confirming the concept of the existence of quasispecies for RNA-containing viruses. As of February 2021, the complete genomes of almost half a million coronavirus isolates have been sequenced worldwide, and a number of nomenclatures have been proposed to streamline their analysis, including the convenient dynamic nomenclature Pango lineage. Variations of SARS-CoV-2 genomes in the form of consensus SNPs (Single Nucleotide Polymorphism) and intra-host iSNVs (intra-host Single Nucleotide Variant) were demonstrated. Taking into account iSNV and minor mutations, about 85% of the 29.9 thousand nucleotides viral genome were changed at least once, but only a very few of them turned into major mutations due to certain features that ensure the predominant distribution of such strains. The example of the S-protein gene, taking into account iSNV, minor and major mutations, shows its significant variability, which is detected when sequencing hundreds of thousands of SARS-CoV-2 genomes. On the basis of the analysis of 400 complete SARS-CoV-2 genomes isolated on the territory of the Russian Federation during 2020, the dynamics of the circulation of individual strains with acquired major mutations, the representation of which is slightly different from the changes in the SARS-CoV-2 genome in the rest world, is estimated. The possibility of long-term persistence of the new coronavirus in the human body is note, while the reservoirs for the latent existence of SARS-CoV-2, in contrast, for example, to the herpes simple virus, remain unknown. There is no consensus on the possibility of reactivation of SARS-CoV-2 or reinfection. The latter is theoretically possible in cases where SARS-CoV-2 strains belonging to other genetic lineages and clades are found in the body of the "re-infected". This, however, does not exclude the possibility of mutating the virus within a single host. Despite significant progress in monitoring the spread of SARS-CoV-2, many questions remain, but as knowledge of the biology of the new coronavirus accumulates, they will also be answered.

Water caltrop Trapa natans L. is a relic aquatic plant protected in 36 regions of Russia. The most northern and isolated populations of this plant in Southern Ural and Altai are poorly studied. The results of a chemical analysis of water and aquarium culture experiments revealed that this plant could tolerate a wide range of environmental factors. The species appear to be sensitive to chlorides and low water temperatures, but resistant to sulfate salinization. Human factors are the main threats to the northern populations of water caltrop, which in Altai has already resulted in the extinction of this plant in Lake Aya and population declines in lakes Manzherok and Kanonerskoye.

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation. Protocols for the Agrobacterium-mediated and biolistic transformation of Trapa natans to induce hairy roots have been developed for the first time. All induced roots contained chloroplasts.

Kok-saghyz (Taraxacum kok-saghyz Rodin) is an alternative to Hevea brasiliensis source of high quality natural rubber that is able to grow in temperate climates. However, the field cultivation of kok-saghyz is associated with a number of problems, such as the need for seed stratification, seedlings that are very sensitive to stress factors, laboriousness in the fight against weeds and pests, uneven maturation of seeds, the lack of special equipment for sowing, collecting roots and seeds. In this regard, the use of hairy roots of kok-saghyz as a raw material for obtaining natural rubber seems to be promising. The aim of our work was to obtain cultures of hairy roots of kok-saghyz and to determine the content of natural rubber in them by the hexane method. In the course of the work, 10 lines of hairy roots of kok-saghyz were obtained, capable of growing in isolated cultures. It has been shown that these hairy roots accumulate an average of 7.5% hexane extract (rubber-like substances) per dry weight, which is about 1.5 times more than the roots of field kok-saghyz. Our data indicate that the use of the hairy roots of kok-saghyz is promising for the industrial production of natural rubber.

CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

В 2020 году исполнилось 100 лет термину «геном», предложенному немецким ботаником Г.Винклером при описании партеногенеза в растительном и животном царствах. Этимология данного слова неясна, но нам представляется, что Винклер использовал игру слов, соединив морфемы «ген», «сома» и получив “genosom” (нем.), после чего решил удалить один слог, что и привело к краткому и емкому слову «геном», объединяющему гены и составляющем для них нечто целое, что также совпало с окончаниями «–ом» отдельных биологических терминов, обозначающих некое множество (в данном случае для генома – генов). Первоначально под геномом подразумевался гаплоидный набор хромосом, и таким образом хромосомы служили элементарной единицей генома. Сейчас же с появлением возможности секвенировать полный геном его элементарной единицей стала пара нуклеотидов, но и хромосомы оказались задействованными, поскольку нуклеотидные последовательности распределяются в базах данных по хромосомам, когда такое по результатам полногеномного секвенирования становится возможным. На протяжении столетия менялись взгляды ученых, что считать геномом, подразумевая под этим его гаплоидную или диплоидную природу. Сейчас принято считать, что геном - это нуклеотидные последовательности всей совокупности генов и прочих участков ДНК гаплоидного набора хромосом конкретного вида организмов, однако и габитус и функциональное состояние каждого организма определяется его полным диплоидным геномом, тогда как при сборке гаплоидного генома a priori происходит игнорирование тех или иных азотистых оснований в отличающихся аллелях, поскольку выбирается лишь один какой-то нуклеотид, что сразу снижает ценность такого генома, которая все равно очень высока, поскольку секвенирование полных гаплоидных геномов уже очень большого числа видов организмов различного уровня генетической сложности дало весьма важную информацию о Живом. Тем не менее, для персонифицированной медицины будущего нужны знания об исключительно диплоидных геномах людей, получение которых пока представляет серьезную проблему, в отличие от нынешних квази-гаплоидных геномов, которые секвенируются почти массово. Диплоидные геномы растений также представляют интерес, в том числе для CRISPR/Cas геномного редактирования, когда необходимо произвести изменения в обоих аллелях парных хромосом, и для этого знать их возможно отличающиеся нуклеотидные последовательности крайне необходимо. Равно как и по завершении такого редактирования следует с помощью полногеномного диплоидного секвенирования выявить произведенные целевые и нецелевые мутации. Разрабатываемые подходы полногеномного секвенирования, в том числе протяженных участков ДНК вкупе с компьютерными программами, рассчитанными на фазировку данных, недавно вызвали к жизни новый термин «гаплотиг», представляющий собой гаплотип-специфичный контиг. Вне всякого сомнения, все это позволит со временем перейти к уверенному установлению полных диплоидных геномов, что придаст термину «геном» новый смысл, считая его для эукариотических организмов диплоидным, что будет к тому же отражать и саму сущность организации генома, имеющего в норме двуродительскую природу.

Specific amplification of nucleic acids is a convenient and quick alternative to the culture-based method of detecting bacterial cells. However, conventional PCR and other amplification reactions can not differentiate between live bacteria and dead or dormant ones, and are also capable of amplifying DNA that persists for a long time and in a cell-free state. Several methods have been developed in order to establish the viability of microorganisms by amplification of specific sequences of nucleic acids, both those controlled by changing temperatures and isothermal ones. For some of them, DNA modified by phenanthridine dyes serves as a target, and simultaneous use of monoazides of ethidium and propidium was shown to be preferable for the purpose. For other methods, the targets are directly RNA molecules or their cDNA copies. Pre-rRNA detection seems to be the most preferable approach, due to the presence of these types of RNA exclusively in living cells.

The paper considers the problematic aspects of detecting a new coronavirus infection using RT-PCR, which often lead to false negative diagnostic results, which occur both at the preanalytic stage and during nucleic acid amplification, including the interpretation of the obtained data. The viral load in humans was evaluated and assumptions were made about the expected number of viral particles in the studied oropharyngeal and nasopharyngeal samples. The list of diagnostic test systems approved for use in the Russian Federation for detecting SARS-CoV-2 with their brief characteristics is given. The necessity of simultaneous use of several targets in the coronavirus genome in one test system detected by probes with the same fluorochrome is noted, which on the one hand increases the probability of detection by increasing the signal, and on the other hand eliminates the false negative results that could occur in the case of mutations in the virus genome at the sites of annealing primers and hybridization probes.

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

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.

Hypothermia, drought, salinity and heavy metals are the most widespread stress factors negatively affecting plant growth and development. Plants respond to these stress factors on molecular, cellular, and physiological levels through the complicated mechanisms of signal perception and transduction, subsequently inducing various defense mechanisms. Transcription factors controlling the expression of numerous defense proteins are the most significant abiotic stress reaction regulators. Mainly, the negative environmental influence activates the AP2/ERF, WRKY, MYB, NAC, bZIP transcription factors. The numerous transcription factors genes can be used in genetic engineering of agricultural crops resistant to abiotic stress. These genes are also of great interest in marker assisted selection of cultivated plants. This review is dedicated to description of transcription factors and their genes, involved in plant response to hypothermia, drought, salinity and heavy metals.

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.

Red amaranth Amaranthus cruentus L. is a valuable fodder and grain crop. To generate new varieties of this plant, genetic transformation methods can be used, but for A. cruentus such methods remain undeveloped. The present study describes the results of our research in Agrobacterium-mediated transformation of epicotyl segments of A. cruentus variety “Bagryanyi” by the ARGOS-LIKE transgene of Arabidopsis thaliana controlled by the 35S promoter in the binary vector pCambia 1301 with a selective hygromycin B resistance gene. For shoot regeneration from epicotyl segments after Agrobacterium-mediated transformation, Murashige-Skoog (MS) medium containing 13 μM 6-benzylaminopurine and 1 μM 1-naphthylacetic acid was used. For the selection of transgenic shoots, 10 mg/L of hygromycin B was added to the MS medium. Rooting of shoots was performed on selective MS medium supplemented with 2 μM 3-indoleacetic acid. Three transgenic amaranth plants with the genetic engineering structure 35S::ARGOS-LIKE were generated. The efficiency of Agrobacterium-mediated transformation of A. cruentus was 4%. The amaranth plants transgenicity was confirmed by the PCR analysis for the presence of marker and target genes. Two transgenic plants were acclimatized to soil and open air conditions

Transgenic plants Nicotiana tabacum, Brassica napus, B. rapa, Eruca sativa with target gene AtGSTF11 were created. Their stress resistance is being studied.

Mutagenesis of Triticum sinskajae was performed using sodium azide. The optimal concentration was 0.1 mM. Changes were observed in the length of the spike and stem in the experimental plants as compared with the control ones.

Tobacco hairy roots with constitutive expression of the NtEXPA5 expansin gene were obtained and analyzed, which were characterized by higher growth rates and increased productivity under normal conditions, as well as the action of stress factors.

Experiments on CRISPR/Cas9 mediated knock-in approaches in the PDS gene of various genes of interest were planned. Biolistic bombardment mediated delivery of target vectors to plant explants was suggested.

During in vitro cultivation of various cultivars and lines of cotton, lines less prone to browning and forming healthy calli were revealed. The experiments on induction of somatic embryogenesis in these lines are conducted.

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).

Glutathione is the most important part of a plant’s antioxidant defense system. Two enzymes are involved in cellular glutathione biosynthesis: glutamylcysteine ligase and glutathione synthetase, of which the latter catalyzes the transfer of glycine to the glutamylcysteine dipeptide. Transgenic plants with an elevated expression level of the glutathione synthetase genes are known to be tolerant to heavy metals. However, our knowledge on their tolerance to other types of the abiotic stress is insufficient. The goal of this study is to produce transgenic tobacco plants with a constitutive expression of the glutathione synthetase gene BnGSH from rapeseed and to estimate their growth parameters under the normal conditions, as well as under the effects of salt, drought, and cold stresses. We generate 17 lines of transgenic plants with the rapeseed BnGSH gene under the control of the 35S promoter by agrobacterium-mediated transformation. The transgene presence is confirmed by the PCR and histochemical analysis of the activity of the GUS reporter gene. Twelve lines with the highest expression level of the BnGSH gene are chosen based on the results of RT-PCR. The following morphological parameters are measured: stem height, leaf area, flower length, fresh and dry weights of shoots, and root length. Some transgenic plants demonstrated increased productivity both under normal conditions and under the effect of a high salinity stress. However, no change in the tolerance to drought and cold was observed in the transgenic plants.

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)

A historical review of the advent and improvement of the methods for detecting multilocus DNA polymorphism that do not require preliminary knowledge of the individual gene and complete genome sequences of eukaryotes is presented. The first group of these methods includes approaches based on the use of primers with arbitrary sequence (random priming). Another group of methods to detect DNA polymorphism is based on the use of primers that consist of short repetitive sequences having anchor nucleotides at the 5'- or 3'-ends that position the annealing sites of these primers (microsatellite priming). Another approach for revealing polymorphism that does not require knowledge of the DNA sequence is based on cleavage of total DNA by a combination of restriction endonucleases (random cleavage) accompanied by PCR amplification. Considerable attention is paid to the opportunities of using these approaches to detect DNA polymorphism in the form of converting the obtained data to digital format and creation of integrative databases for all organisms, regardless of the methods used.

The role of xyloglucan endotransglycosylases in the regulation and promotion of plant growth in response to such widespread stress factors as drought, salinization, and hypothermia remains poorly understood. The tXET-B2 (SlXTH10) gene encodes one of the xyloglucan endotransglycosylases of tomato, which is most closely related in the nucleotide sequence to the AtXTH15 and AtXTH16 genes of Arabidopsis thaliana. At present, the specific functions of the tXET-B2 gene, as well as of its homologs, AtXTH15 and AtXTH16, remain obscure. To study the role of tXET-B2 in the regulation of growth and adaptation to abiotic stress factors, transgenic tobacco plants with estradiol-inducible expression of the tomato tXET-B2 gene were generated. Overexpression of this gene promoted tobacco root growth in a medium containing 50 mM NaCl. Under drought conditions, exogenous treatment with estradiol resulted in a considerable increase in fresh and dry weight in many of the studied transgenic lines. Under normal conditions, as well as under salinization and hypothermia stress, such positive effect was detected only for some transgenic lines. The obtained data point to the possibility of using genetically engineered constructs of the tXET-B2 gene to correct growth parameters of transgenic plants under the influence of stress factors.

Background. Triticum sinskajae A. Filat. et Kurk. was discovered in the early 70th in the last century at the regular reproduction in the Central Asian and Dagestan VIR-stations of T. monococcum samples. Materials and methods. The objects of the study were 4 species of diploid wheat Triticum urartu Thum. ex Gandil. (lines k-62477, k-62465), Triticum monococcum L. (lines k-20970, k-39471), Triticum boeoticum Boiss. (lines k-59161, k-28132, k-40118) and Triticum sinskajae A. Filat. et Kurk. (line k-48993). Results. We found differences between T. sinskajaeand T. monococcum in the variable region of the histone gene H3.2, and the RAPD analysis showed the presence of unique polymorphic loci in T. sinskajae. Conclusion. In gene ral, T. boeoticum, T. monococcum, and T. sinskajae are most likely to be closely related species of diploid wheat, whereas T. urartu is quite significantly different from them.

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)

The genes of α-expansins of woody plants are of great interest for genetic engineering, since they can potentially be used to improve the tree growth parameters. In the flora of Russia, model woody plants for plant biotechnology are aspen (Populus tremula L.) and black poplar (Populus nigra L.). The objective of this study was to determine the role of α-expansin-encoding genes, aspen PtrEXPA3 and black poplar PnEXPA3, in the regulation and maintenance of woody plant growth. To achieve this goal, the PtrEXPA3 expression level were determined upon exogenous phytohormone treatment, the action of stress factors, and constitutive expression of the PnARGOS-LIKE gene. In addition, transgenic aspen plants with constitutive expression of the black poplar PnEXPA3 gene were generated, and their morphological analysis was carried out. The highest PtrEXPA3 mRNA level was detected in young intensely growing aspen leaves, and furthermore, expression of the gene was induced by exogenous cytokinins and auxins. In response to NaCl and constitutive expression of the PnARGOS-LIKE gene, the PtrEXPA3 mRNA level decreased. Transgenic aspen plants with constitutive PnEXPA3 expression were characterized by the decreased size of leaves, petioles, and internodes, as well as the increased size of leaf epidermal cells, while the stem size remained unchanged. Taken together, the data obtained enable the suggestion that the PtrEXPA3 and PnEXPA3 genes encode cytokinin- and auxin-regulated, leaf-specific expansins that are involved in the cell expansion.

Summary: Glutathione is the most important part of plant antioxidant defense system. Biosynthesis of glutathione in the cells is performed by two enzymes: glutamylcysteine ligase and glutathione synthetase, the latter catalyzing the attachment of glycine to a dipeptide glutamylcysteine. In literature there is information on the improvement of heavy metal-tolerance of transgenic plants due to the increase in the expression level of glutathione synthetase genes. However there is not enough data on the tolerance of these plants to other types of abiotic stress. Therefore the aim of our research was to make transgenic tobacco plants with constitutive expression of rapeseed glutathione synthetase gene BnGSH and to estimate their growth parameters in normal conditions and under salt, drough and cold stress. Using agrobacterial transformation method, we generated 17 lines of transgenic plants containing rapeseed BnGSH gene under control of 35S promoter. The presence of transgenes was confirmed by PCR method and histochemical analysis of the activity of GUS reporter gene. 12 lines with the highest expression of BnGSH gene were chosen on the basis of the results of RT-PCR. We performed morphological analysis, including measurements of stem hight, leaf area, flower length, fresh and dry weight of shoots and root length. Some transgenic plants demonstrated increased productivity in normal conditions as well as under NaCl stress. However, no change in drought and cold tolerance was observed in transgenic plants.