Pavel Khvatkov

Drought is one of the main stress factors significantly affecting the growth, development and yield of agricultural crops. This study investigated the impact of drought stress on the grapevine. The 30 cultivars were classified as drought-tolerant, intermediately tolerant or sensitive. The phenotypic characteristics the number of new leaves, the number of second-order roots and the length of second-order roots (NL, NR2 and LR2 respectively) were identified as the most sensitive biometric characteristics. These parameters can be used to determine the optimal level of stress exposure for plants. Using transcriptomic data from five cultivars with different levels of tolerance, differentially expressed genes (DEGs) were identified in control plants and in plants under stress, as well as DEGs between different varieties when exposed to 2% mannitol. General patterns of gene expression under drought stress were subsequently identified, including the activation of antioxidant defense systems and changes in the metabolism and biosynthesis of glucan, cellulose, polysaccharides, monocarboxylic acids, fatty acids and metal transport and splicing processes. It is hypothesized that drought tolerance is determined by the increased expression of genes associated with glutathione metabolism and methylation processes.

Drought is one of the main stress factors significantly affecting the growth, development and yield of agricultural crops. This study investigated the impact of drought stress on 30 grapevine genotypes. The cultivars were then ranked according to their degree of drought tolerance and classified as drought-tolerant, intermediately tolerant or sensitive. Phenotypic characteristics (NL, NR2 and LR2) were identified that can be used to determine the stress threshold. Using tran-scriptomic data from five cultivars with different levels of tolerance, differentially expressed genes (DEGs) were identified in control plants and in plants under stress, as well as DEGs between dif-ferent varieties when exposed to 2% mannitol. General patterns under drought stress were subsequently identified, including the activation of antioxidant defense systems and changes in the metabolism and biosynthesis of glucan, cellulose, polysaccharides, monocarboxylic acids, fatty acids and metal transport and splicing processes. It is hypothesised that drought tolerance is determined by increased expression of genes associated with glutathione metabolism and methylation processes.

Somatic embryogenesis is the preferred method for cell-to-plant regeneration of grapevine. In this study, we tested the embryogenic capacity of inflorescence-derived calli from 10 grape genotypes, representing a diverse group of Vitis vinifera and accessions of non-vinifera Vitis species. Explants produce pro-embryogenic calli when cultured on agarized Nitsch and Nitsch medium supplemented with 1.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 1.0 mg l^-1 6-benzyladenine. Embryogenic calli was developed upon transfer to liquid Murashige and Skoog medium supplemented with 2.0 mg l^-1 zeatin and 0.1 mg l^-1 2,4-dichlorophenoxyacetic acid. Somatic embryos were formed upon transfer to a agarized Murashige and Skoog medium supplemented with 0.1 mg l^-1 indolylacetic acid. Plantlets was developed when a obtained somatic embryos was transferred to liquid Murashige and Skoog medium supplemented with 0.2 mg l^-1 6-benzyladenine and 0.2 mg l^-1 gibberellic acid. Conversion into morphological normal plants was achieved by rooting the plantlets on agarized half strength Murashige and Skoog medium supplemented with 0.05 mg l^-1 α-naphthaleneacetic acid. This protocol has been successful for the regeneration of ‘Malbec’, ‘Saperavi’ ‘Aligote’, ‘Carménère’ and ‘Podarok Magaracha’ cultivars and ‘SO4’ rootstock. To future biotechnological use of studied cultivars, we established a stable transformation system for it based on Agrobacterium-mediated transformation. Following inoculation with the bacteria, somatic embryos were cultured for 4 weeks on liquid Murashige and Skoog medium containing 0.2 mg l^-1 6-benzyladenine, 0.2 mg l^-1 gibberellic acid, 100 mg l^-1 timentin and 50 mg l^-1 kanamycin. Explants were then transferred to agarized half strength Murashige and Skoog growth regulator-free media in the presence of 100 mg l^-1 timentin and 50.0 mg l^-1 kanamycin to select antibiotic-resistant somatic embryos. The resulting somatic embryos were re-cultured in liquid Murashige and Skoog medium supplemented with 0.2 mg l^-1 6-benzyladenine and 0.2 mg l^-1 gibberellic acid, 100 mg l^-1 timentin and 50 mg l^-1 kanamycin to develop transgenic sprouts. Then, the resulting sprouts were transferred to agarized half strength Murashige and Skoog medium to form transgenic shoots. The developed agro transformation conditions yielded stably transformed lines of the Aligote and the Podarok Magaracha cultivars, confirmed by Southern blotting, with an efficiency of 0.36–0.43% transgenes per 100 explants.

We present data on the ability for organogenesis in 22 genotypes of grapevine and developed a direct organogenesis protocol for the cultivar Podarok Magaracha and the rootstock Kober 5BB. The protocol does not require replacement of culture media and growth regulators, and the duration is 11 weeks. The cultivation of explants occurs on modified MS medium with the addition of 2.0 mg L⁻¹ benzyladenine and indole-3-butyric acid (0.15 mg L⁻¹ for the rootstock Kober 5BB or 0.05 mg L⁻¹ for the cultivar Podarok Magaracha). The direct organogenesis protocol consists of three time periods: (1) culturing explants for 2 weeks in dark conditions for meristematic bulk tissue, (2) followed by 4 weeks of cultivation in light conditions for regeneration, and (3) 5 weeks of cultivation in dark conditions for shoot elongation. Based on this protocol, conditions for the Agrobacterium-mediated transformation of the Podarok Magaracha cultivar were developed with an efficiency of 2.0% transgenic plants per 100 explants. Two stably transformed lines with integration into the genome of the pBin35SGFP plasmid construction, confirmed by Southern blotting, were obtained.

WRKYs are a multigenic family of transcription factors that are plant-specific and involved in the regulation of plant development and various stress response processes. However, the evolution of WRKY genes is not fully understood. This family has also been incompletely studied in grapevine, and WRKY genes have been named with different numbers in different studies, leading to great confusion. In this work, 62 Vitis vinifera WRKY genes were identified based on six genomes of different cultivars. All WRKY genes were numbered according to their chromosomal location, and a complete revision of the numbering was performed. Amino acid variability between different cultivars was assessed for the first time and was greater than 5% for some WRKYs. According to the gene structure, all WRKYs could be divided into two groups: more exons/long length and fewer exons/short length. For the first time, some chimeric WRKY genes were found in grapevine, which may play a specific role in the regulation of different processes: VvWRKY17 (an N-terminal signal peptide region followed by a non-cytoplasmic domain) and VvWRKY61 (Frigida-like domain). Five phylogenetic clades A–E were revealed and correlated with the WRKY groups (I, II, III). The evolution of WRKY was studied, and we proposed a WRKY evolution model where there were two dynamic phases of complexity and simplification in the evolution of WRKY.

We present data on the ability to organogenesis in 22 genotypes of grapevine and developed a direct organogenesis protocol for the cultivar Podarok Magaracha and the rootstock Kober 5BB. Protocol does not require replacement of culture media and growth regulators, the duration is 11 weeks. Cultivation of explants occurs on modified MS medium with the addition of 2.0 mg l^-1 benzyladenine and indole-3-butyric acid (0.15 mg l^-1 for the rootstock Kober 5BB or 0.05 mg l^-1 for the cultivar Podarok Magaracha). The direct organogenesis protocol consists of three time periods: (1) culturing explants for 2 weeks in dark conditions for callogenesis, (2) followed by 4 weeks of cultivation in light conditions for regeneration, (3) 5 weeks of cultivation in dark conditions for shoot elongation. Based on this protocol, conditions for the Agrobacterium-mediated transformation of the Podarok Magaracha cultivar were developed with an efficiency of 2.0% transgenic plants per 100 explants. Two stably transformed lines with integration into the genome of the pBin35SGFP plasmid construction, confirmed by Southern blotting, were obtained.

The development of new grape varieties with improved quality and resistance against environmental factors using biotechnological methods is a pressing task in the context of import substitution policy implementation. Due to the high heterogeneity of grape varieties, techniques for regenerating plants from embryogenic callus by somatic embryogenesis have been developed only for a small number of genotypes. This work presents a versatile approach to the development of globular, heart, and torpedo embryos in various grape genotypes. Experiments were carried out using cultures of colchicine embryogenic cell suspensions and somatic globular embryos of the E-342 hybrid form in liquid media. The developed methodology allows the protocols of embryogenesis and polyploidy induction to be effectively improved. The versatility of the presented methodology for polyploidy induction and somatic embryogenesis in grape plants is ensured by a fourfold reduction in the concentration of inorganic nitrogen and vitamins in the NN and PG media and the addition of organic nitrogen and other biological active substances at the stages of embryogenic callus development, colchicination, and development of somatic embryoids in liquid media. The methods of indirect ploidy determination by the properties of the grape leaf epidermis were studied and implemented. Nine promising somaclones were preliminary selected based on a set of four features. It was established that full-fledged transgenic grape plants can only be obtained by the method of transformation, which involves the production of embryogenic callus from a leaf explant, its subsequent passage into a liquid culture to obtain a liquid embryo culture, and the transformation of this culture. In total, 300 leaf explants of the Libya variety and 1 100 globular embryos of the Podarok Magaracha variety were transformed. As a result, six callus kanamycin-resistant lines of the Libya variety and six lines of transgenic plants of the Podarok Magaracha variety were selected. Following genotyping based on nine nuclear microsatellite loci, seven unknown forms with a seedless phenotype were identified. Among the population of hybrid seedlings, hybrids having alleles associated with seedlessness were revealed using the VMC7f2 and p3_VvAGL11 MAS markers.

The increased demand in vegetable oil for food purposes and high-protein feed for livestock and poultry encourages producers to expand the production of various oil crops, while occupying rather cold agroclimatic zones. Improved cold and frost resistance of cultivated crops would significantly increase the yield and expand the range of rape cultivation in a number of cold climate regions. Nine transgenic lines of winter rape containing genes encoding proteins with a cold shock domain (CspA и EsCSDP3) were obtained as a result of Agrobacterium transformation. In total, 260 explants were involved in transformation of rape using pBI121-CSPA-plant, with a transformation efficiency of 2.3%; among 750 explants using the pBI-EsCSDP3 construction, the efficiency was 0.4%. As a result of the studies, it was shown that the expression of the new gene Escsdp3 from the plant of Eutrema salsugineum was able to increase the cold and frost resistance of plants as effectively as the cspa gene from E. coli, which is classically used for this purpose. The cold resistance analysis of T₁ transgenic plants generation revealed four cold resistant winter rape lines (three lines with the cspA-plant gene and one line with the Escsdp3 gene). The transfer of Escsdp3 and cspA-plant genes into winter rape plants led to a significant increase in frost resistance of plants. Two winter rapeseed lines were resistant to freezing (with the cspA-plant gene and with the Escsdp3 gene). Non-hardened transgenic plants remained viable after 24 h of exposure to negative temperatures up to −5 °C, and plants that passed through the hardening stage survived after freezing at −16 °C.