Yuri Sidorchuk

Russian Academy of Sciences | RAS · Institute of Cytology and Genetics, Siberian Branch

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carry...

Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast,...

The major problem associated with production of transgenic proteins in plant expression systems is the low level of their accumulation. Transplastomic plants with a transgene incorporated not in the nuclear but in the plastid genome (plastome) in some cases ensure elevation of the yield of the desired protein dozens of times and more. However, two...

The features of the expression of the uidA reporter gene, encoding the β-glucuronidase enzyme, under the control of AP3 and RPT2a tissue-specific gene promoters of Arabidopsis thaliana L. was studied in homozygous monoinserted transgenic plants of Nicotiana tabacum L. Both promoters ensured the expression of the reporter gene in the meristematic ti...

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding mod...

The peculiarities in the creation of genetic engineering tools for the knock-in variant of genome editing are considered in detail on the example of gfp gene delivery to two target regions (nucleolus organizer region and the region of one of histone H3 genes) of the Arabidopsis thaliana (L.) Heynh. genome using different methods of delivering exoge...

In this chapter we describe cytological techniques to study cytomixis, a process of nuclear migration between plant cells, in squashed plant male meiocytes of Nicotiana tabacum and Secale cereale. To perform immunostaining or fluorescence in situ hybridization (FISH) on meiotic cells involved in cytomixis common protocols are modified. During prepa...

Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell suspension cultures. Here, we describe a successful knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture. We transformed seven transgenic suspension cel...

The structure of cytoskeleton in the tobacco male meiocytes involved in the migration of nuclei between cells (cytomixis) is studied. The tubulin and actin components of cytoskeleton are examined using specific antibodies and phalloidin. The presence of microtubules and actin filaments inside cytomictic channels directly when the nuclei migrate thr...

A set of transgenic Arabidopsis thaliana monoclonal cell lines has been created by selecting individual cells (cell aggregates) with random GFP gene integration events after the Agrobacterium-mediated transformation. The yield of the recombinant GFP ranged from 0.07 to 2.37% of the total soluble protein. Three lines with the highest, about 2% of th...

Ultrastructural analysis of intercellular migration of DNA-containing organelles (nuclei, mitochondria, and plastids) in tobacco microsporogenesis during cytomixis was conducted. It was demonstrated for the first time that the migrating part of the nucleus is covered with ribosomes and can contain the accumulation of nuclear pores. The possibility...

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN...

We have proposed and tested a method for characterization of the signal sequences and determinations of target protein localization in a plant cell. This method, called the AgI-PrI, implies extraction of protoplasts from plant tissues after agroinfiltration. The suggested approach combines the advantages of two widely used methods for transient gen...

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6...

The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid,...

Microsporogenesis patterns of the polyploid (2n = 4x = 96) and diploid (2n = 2x = 48) Nicotiana tabacum L. (cv. Havana Petit line SR1) plants have been analyzed and compared. Four types of abnormal positions of the second division spindles-tripolar, parallel, proximal, and fused-have been observed. Of these abnormalities, only tripolar (2.4%) and p...

Confocal laser scanning microscopy for the first time is used to examine the structure of the tobacco microsporocytes involved in the intercellular migration of nuclei (cytomixis). As is observed, the cytomictic channels are distributed over the surface of tobacco microsporocytes in a non-random manner and their number depends on the meiotic stage....

Behavior of nucleolus during the nuclear migration between plant cells (cytomixis) is studied for the first time in the tobacco male meiosis. As is shown, the nucleolus is located in a nonrandom manner in the migrating nuclei. In the majority of cases, the nucleolus resides on the nuclear pole strictly opposite to the cytomictic channel. Owing to t...

The efficiencies of the induction of cytomixis in microsporogenesis by thermal stress are compared in tobacco (N. tabacum L.) and barley (H. distichum L.) It has been shown that different thermal treatment schedules (budding tobacco plants at 50°C and air-dried barley grains at 48°C) produce similar results in the species: the frequency of cytomixi...

Comparative cytological analysis of intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants has been performed for two cellular systems: the microsporocytes and the tapetum. Cytomixis was shown to be more common for intratissular interactions, and cytomixis in the tapetum exhibited taxon-specific...

The effect of plant ploidy level on the rate of cytomixis in microsporogenesis has been analyzed with the help of a unique model, the collection of tobacco plants of different ploidies (2n = 2x = 24, 4x = 48, 6x = 72, and 8x = 96). As has been shown, the rate of cytomixis proportionally increases in 6x and 8x cytotypes, being rather similar in 2x a...

The specific features in behavior of the nuclei and chromatin migrating through cytomictic channels as well as in formation of micronuclei in the cereal microsporogenesis have been studied. Immunofluorescence microscopy has allowed for demonstration that the tubulin cytoskeleton does not play a significant role in the intercellular migration of nuc...

Cytomixis is the migration of nuclei from one cell to another in higher plants, most frequently observable during microsporogenesis, which has a potential evolutionary significance. Currently, a major challenge is to label the chromatin migrating between cells to clarify its further fate. We have for the first time succeeded in visualizing the telo...

The bipolar spindle is a major cytoskeletal structure, which ensures an equal chromosome distribution between the daughter nuclei. The spindle formation in animal cells depends on centrosomes activity. In flowering plant cells the centrosomes have not been identified as definite structures. The absence of these structures suggests that plants assem...

The phenomenon of intercellular migration of nuclei in plant tissues (cytomixis) was discovered over a century ago, which has been followed by numerous attempts to clarify the essence of this process as well as to determine its causes and consequences. Most attention of researchers has been paid to cytomixis in microsporogenesis, since the transfer...

The formation of cytomictic channels (CCs) during the tobacco microsporogenesis has been analyzed by microscopy and cytochemical methods. Starting from the pachytene stage, CCs were formed between microsporocytes with involvement of specific organelles, the so-called spherosome-like vesicles. The presence of the enzyme callase, able to degrade call...

Spectinomycin resistant mutant carrot (Daucus carota L.) callus lines detected in the experiments on biolistic transformation of plastome were analyzed. It has been found that this antibiotic resistance is determined by point nucleotide substitutions at two distinct sites of the chloroplast gene rrn16, coding for 16S rRNA, namely, G1012T, G1012C, a...

Electron-microscopic analysis of cytomictic channels formation in the pollen mother cells in tobacco at the stage of meiosis prophase I of anthers has been conducted. The cytomictic channels in the pollen mother cells in tobacco have been established to be formed under the basis of both single plasmodesmata and de novo with the involvement of speci...

The cytomictic channels in pollen mother cells of tobacco can be formed by two distinct ways: on the basis of plasmodesmata and de novo without any relation to ones. Cytomictic channels formation it was shown to be possible on the basis of single plasmodesma. It is not unlikely that special electron-dense bodies involve de novo formation of the cha...

A comparative analysis of IAA and total cytokinin contents at various developmental stages of floral morphogenesis (I–XII) in transgenic tobacco (Nicotiana tabacum L.) plants with mutant phenotypes was performed. Disturbances in flower structure were correlated with changes in the content and dynamics of these phytohormones. In plants with the phen...

For the majority of dicotyledonous plants, cytokinesis in PMC is staged only once, i.e., after the completion of two cycles of caryokinesis. In the article, a cytological picture and the frequency characteristics of anomalies are shown, in which the cytokinesis in the PMCs of transgenic tobacco plants was already initiated after the first meiotic d...

The structure and dynamics of microtubular cytoskeleton and of callose walls in normal pollen mother cells (PMC) of tobacco N. tabacum L. and in cells with intercellular translocation of nuclear material (cytomictic) was studied in the course of the cell cycle. The microtubular cytoskeleton was established as playing no obvious role in the process...

The frequency characteristics and cytological picture of cytomixis in the course of male meiosis are described in transgenic tobacco plants (Nicotiana tabacum L.) with altered flower morphology and male sterility. Effects of cytomixis on qualitative composition of meiotic products are studied (formation of cytoplasts and polyads). Doubling of the c...

When variation of morphological traits was followed in 580 transgenic plants of tobacco (Nicotiana tabacum L.), 5.5% of genotypes in the population under study exhibited considerable changes (long styles, dwarfness, and male sterility). Longostyly was inherited as a trait linked to kanamycin resistance, and this evidence supports the insertional na...

14 samples of soybean G. max L. Merr. and G. ussuriensis L. were screened for their morphogenetic capacity in tissue culture. Two protocols of media were used for plant regeneration from immature embryos via somatic embryogenesis or organogenesis. The influence of different concentration of BAP and NAA on the morphogenetic responses frequency have...

Variation in morphological and biochemical traits was studied in regenerated alfalfa plants (Medicago varia Mart.), cv. Sibirskaya 8. Variation of morphological characteristics was shown to be very wide. Modifications of karyotype (aneuploidy, changes in ploidy) were not found. Qualitative variation in the composition of seed storage proteins was l...