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Promoters are key elements regulating gene expression levels, therefore their selection is an important step in genetic engineering research. The reporter gene gfp, which encodes green fluorescent protein (GFP), was transiently expressed in leaf tissues of Aztec tobacco Nicotiana rustica L. Compared to other species of the Nicotiana genus, Aztec tobacco has a large potential for expression of heterologous proteins, a large vegetative biomass, can be easily infiltrated, and is unpretentious in cultivation. Six genetic constructs were used with different promoter sequences: the 35S promoter of Cauliflower Mosaic Virus (35S CaMV), the double-enhanced 35S promoter (D35S CaMV), promoters of the RbcS1B and RbcS2B genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isolated from Arabidopsis thaliana (L.) Heynh., and promoters of the LHB1B1 and LHB1B2 genes from A. thaliana encoding chlorophyll a/b binding proteins. The gfp gene expression was detected visually, spectrofluorimetrically, and by protein content (Bradford assay) on the seventh day after infiltration. The highest level of expression was observed using the double-enhanced 35S promoter (D35S CaMV) and the lowest using the LHB1B1 gene promoter.

Aim. Acceleration of the sunflower lines homozygosity for isolation of the fertility restorer resistant to tribenuron methyl herbicide using in vitro culture of sunflower immature embryos. Methods. Methods of immature sunflower embryos cultivation in in vitro culture; methods of classical crossing. Results. As a result, homozygous genotype was created and isolated from three combinations (BH0118xSURES-2, BH0218xSURES-2 and BH0318xSURES-2) for 10 parent sunflower lines. Conclusions. The use of the technology of sunflower immature embryos cultivation in in vitro culture effectively accelerates the sunflower lines homozygosity for the fertility restorer isolation resistant to tribenuron methyl herbicide. The involvement of immature sunflower embryos cultivation in in vitro culture methods can reduce time for creation of initial parental sunflower lines twice. Keywords: sunflower, immature embryos, in vitro culture, herbicide resistance, tribenuron methyl.

Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods. Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression.

Aim. Genetic constructs creation for studying the influence effect of the viral posttranscriptional silencing protein suppressor p19 on transient reporter green fluorescent protein (GFP) expression and accumulation. Methods. The Golden Gate molecular cloning method was used to create the genetic constructs; the leafy tissues of the Aztec tobacco plants (Nicotiana rustica L.) were infiltrated with a suspension of Agrobacterium tumefaciens L.; the gfp gene expression level was determined by spectrofluorometric and quantitative protein (Bradford method) assays. Results. As a result of the work, the pSPV2324 genetic construct was created, which contained the reporter gene for the green fluorescent protein gfp and the gene for the synthesis of the viral posttranscriptional silencing protein suppressor p19 and its effect on the accumulation of the recombinant GFP protein was determined. A comparative analysis of the gfp gene expression level without and with the suppressor protein synthesis gene in the genetic vector showed that the fluorescence level of GFP protein in Aztec tobacco tissues was 1.3 times higher during spectrofluorimetric analysis using the p19 suppressor gene construct. Conclusions. The positive effect of the viral suppressor silencing P19 gene on the accumulation of recombinant GFP protein in tissues plants of N. rustica L. was shown for the first time. The increase in GFP protein fluorescence when using the p19 suppressor protein construct in spectrofluorimetric analysis coincides with an increase in the total concentration of total water-soluble proteins and the level fluorescence of GFP protein in their native electrophoretic separation. Keywords: cloning, genetic constructs, transient expression, silencing protein suppressor p19, green fluorescent protein (GFP).

An optimized regeneration method for commercial winter rape of the Bn1 line was proposed. Hypocotyl fragments of 6-day-old seedlings were used as explants. Regeneration occurred via organogenesis on an MS medium supplemented with 3 mg/L of 6-benzylaminopurine and 2 mg/L of 2-isopentyladenine. All obtained regenerated plants successfully formed roots on hormone-free media and were adapted to the soil conditions. The vernalization conditions were specified to promote budding and flowering at the level of 83.93 ± 5.33%. The PCR analysis using the ISSR 2 and ISSR 15 markers has shown that no somaclonal variations in Bn1 winter rape occur under the protocols based on the proposed methodology. Thus, the developed methodology is efficient and economically profitable for obtaining biotechnology-based winter rape plants.

Aim. To establish the optimal concentration of glyphosate for efficient selection of transgenic tissue culture in biotechnology of rapeseed by in vitro selection of glyphosate-resistant and non-resistant winter rape. Methods. As an explants 7–10 mm fragments of 6-day rapeseed hypocotyls were used to cultivate on the medium, supplemented with 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) for 12 days at 24°C under dark conditions for the initiation of callusogenesis. For regeneration of plants, the MS nutrient medium was supplemented with 3 mg/L of 6-benzylaminopurine (BAP), 2 mg/L of zeatin, 0.1, 0.5 and 1 mM of glyphosate, respectively. As a control, glyphosate-free regenerative nutrient medium was used. The results were statistically processed using Microsoft Excel. Results. It has been shown that glyphosate-susceptible rape explants form leaves and shoots with a frequency up to 8.3 % ± 0.8 % on the medium supplemented with 0.1 mM glyphosate compared to 57.2 % ± 0.6 % in the control. However, the morphogenic structures did not pass selection after transferring shoots to the elongation medium supplemented with 0.1 mM glyphosate. Conclusions. For the in vitro selection of rapeseed 0.1 mM glyphosate can be used in a nutrient medium at the stage of adventitious buds formation. But at the regeneration stage, the amount of herbicide should be increased to avoid false-positive results. Keywords: winter rapeseed, Brassica napus L., in vitro culture, selective marker, herbicide, glyphosate.

Aim. To develop an effective protocol for callus induction from shoot apical meristem in Triticum spelta L. and T. aestivum L. Methods. Plant material: spelt “Europe” and common wheat “Bunchuk”. For this research we used shoot apical meristems from 3-days plants. For callus induction we proposed 4 media with different concentration of 2,4-D, picloram, NAA and AgNO3. Explants were growing in dark during 21 day at + 25 C. Results. Calli were transparent and mild, less than 8 mm. For callus induction positive effect were shown on media with 2,4-D and picloram. At the same time, NAA was not such effective. Conclusions. In our research was shown, that the best media for spelt callus induction should have 2 mg/l 2,4-D and 10 mg/l AgNO3. Keywords: callusogenesis, spelt (Triticum spelta L.), common wheat, callus, shoot apical meristem.

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

The present study reports an efficient in vitro plant regeneration system for amphidiploid (2n = 42) spelt wheat (Triticum spelta L.). Plant regeneration from mature embryos of winter spelt variety “Europe” was carried out as a two-step process. The first step was callus induction on a medium supplemented with 2 mg L−1 dichlorophenoxyacetic acid and 10 mg L−1 silver nitrate resulting in 96% callus formation. The second step resulted in 30% plant regeneration frequency from calluses transferred to Murashige and Skoog medium supplemented with 2 mg L−1 6-benzylaminopurine, 0.5 mg L−1 1-naphthaleneacetic acid, and 10 mg L−1 silver nitrate. Regeneration medium supplemented with 1 mg L−1 6-benzylaminopurine, 0.2 mg L−1 picloram, and the hormone-free medium turned out to be less effective in our experiments. Regenerated plants formed roots after transfer to half-strength Murashige and Skoog medium supplemented with 0.7 mg L−1 indole-3-butyric acid. About one-third of the resulting regenerated plants that formed roots were transferred to soil. The inter-simple sequence repeat markers were used to confirm the genetic homogeneity of regenerated plants. Thus, our regeneration protocol can be used for in vitro regeneration of spelt plants from mature embryos with minimal risk of genetic variability and provides an essential step towards developing an efficient strategy for spelt genetic transformation.

The genetic improvement of soft wheat (Triticum aestivum L.) and other hexaploid wheat species (T. spelta, T. spherococcum, T. petropavlovskyi, and T. compactum) is important. This can be done by transferring genes of interest (resistance to biotic and abiotic factors). For this purpose, phylogenetic connections of the studied genotypes should be investigated. This problem can be solved by using a multilocus system based on ISSR markers. The marker system is highly polymorphic and convenient for analysis. The study presents the results of a comparison of 20 different hexaploid wheat genotypes based on ISSR markers. In addition, the level of polymorphism was determined and a dendrogram reflecting the phylogenetic connections between the studied genotypes was constructed. It was shown that the species located next to each other according to the systematics turned out to be more remote after the ISSR-marker analysis, and, vice versa, the species that were systematically more distant showed a higher level of kinship.