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The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant alpha-tubulin gene (TUAm 1), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 microM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgene nature of plants. The analysis of seed of T1 oftransgene lines confirmed heterozygous character of inheritance of the resistance.

Impact of insect pests makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into plant genome is one of the promising strategies to ensure plant resistance. The aim of this work was to obtain sugar beet lines (based on the MM 1/2 line) transformed with cry2A and cry1Cgenes. We have optimized transformation protocol and direct plant let regeneration protocol from leaf explants using 1 mg/l benzylaminopurine as well as 0,25 mg/l benzylaminopurine and 0,1 mg/l indole-butyric acid. Consequently, transgenic sugar beet lines transformed with vector constructs pRD400-cry1C and pRD400-cry2A have been obtained. PCR analysis revealed integration of cry2A and cry1C into genome of transgenic lines and expression of these genes in leaf tissues was shown by reverse transcription PCR.

The results on in vitro culture establishment, plantlet regeneration and rooting of Camelina sativa cultivar sample Peremozhets and cultivar Mirazh are presented. Effective concentrations of sterilizing agents and duration of plant material treatment were estimated. Phytohormone ratio, sucrose concentration in nutrient medium that induce effective formation of C. sativa shoots and NAA concentration for plantlet rooting have been established. The method of Agrobacterium-mediated transformation of Camelina by using binary vector pGH217 carrying reporter beta-glucoronidase (gus) gene driven under 35S CaMV promoter and nos-terminator, and selective marker hpt gene conferring hygromycin-resistance in transgenic plant was elaborated.

The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind.

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

The performed morphologic study of transformation mechanisms of the intestinal reservoir mucous membrane using modern immunochemical and histochemical methods revealed three morphologic phases of enteric epithelium adaptation; reactive-inflammatory, compensatory-protective and atrophic. Enteric urinary reservoir preserves the cambial apparatus typical for the enteric phenotype with an inherent apoptosis activity and proliferation which confirms a low level of tumor transformation risk.

Induced apoptosis in urinary bladder cancer tumor cells of patients was studied using TUNEL reaction. It was shown that increase in induced apoptosis value had a definite correlation between corresponding features of tumor reaction as a response on Gemcitabine-Cisplatin neoadjuvant chemotherapy application. It was found that evaluation of induced apoptosis in urinary bladder cancer tumor cells using TUNEL method allows forecasting the effectiveness of chemotherapy on the cellular level in patients with this type of cancer.

Method of biolistic transformation was used for genetic improvement of commercial barley cultivars (Oksamitoviy, Vodogray and Getman). The plasmid pHLFTuBA was used for particle bombardment that consists of the hLF gene under the control of the barley glutelin B-1 promoter and a selectable marker gene, alpha-tubulin conferring resistance to trifluralin (dinitroanilinr herbicide). Preliminary screening of different trifluralin concentration range from 0,1 to 30 microM was tested for determination of effective selective agent concentration. Two transgenic barley line of genotype Oksamitiviy and transgenic callus line of cultivar Getman were obtained after selection on 10 microM of trifluralin. To confirm the transgenic nature of regenerated plants, the PCR analysis was carried out. The 734bp length fragment of hLFgene was amplified from both regenerated plants.

Shown antimicrotrubules activity of new 2,6-dinitroaniline compounds. Investigated their ability on apoptotic processes in a plant cell when used as a composition with inhibitors of acetyl-CoA carboxylase.

The aim of our work is the identification of protein kinases phosphorylating microtubule proteins in plant cells. Using bioinformatic approach, we found genes of putative homologues of microtubule-associated mammalian protein kinase MAST2 in higher plant genomes. The gene of closest MAST2 homologue, putative protein, named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI), was found in grape Vitis vinifera. We report here the cloning of cDNA of GMLK (A7NTE9) from Pinot Noir grape vine leaves.

The effect of different types of serine/thereonine protein kinases inhibitors (cyclin-dependent, Ca2+-calmodulin-dependent and protein kinase C) on microtubules organization in cells of Arabidopsis thaliana main primary root zones were investigated in vivo. The microtubules in epidermal and cortex cells in the transition and elongation zones as well as microtubules in trichoblasts and atrichoblasts in the differentiation zone showed the greatest sensitivity to protein kinases inhibitors studied. It was established that microtubules in these cell types modified their initial transverse/oblique orientation to a chaotic or longitudinal relative to the major axis of primary root as a result of serinethereonine protein kinases inhibition. The microtubules in cells in root meristematic zone as well as in root hairs were less sensitive to influence of protein kinases inhibitors tested. Alterations of microtubules orientation in the cells in primary root zones under the influence of serinethereonine protein kinases inhibitors led to further disturbances in growth and differentiation processes. It was assumed that phosphorylation of microtubules proteins, especially tubulin, might be involved in the regulation of these processes.

Eleven plant homologs of animal and yeast Ste20-like protein kinases were identified. It was shown that the nearest plant homologs of the Ste20-like protein kinases are the unknown proteins A9RVK0 from Physcomitrella patens ssp. patens and A7P2E2 from Vitis vinifera. Cladistic analysis showed a protein kinase dstl from Dictyostelium discoideum as the closest protein to the newly found plant homologs. A predicted spatial structure of the A9RVK0 from P. patens ssp. patens catalytic domain is presented.

Agrobacterium tumefaciens was used to transform fiber flax with the pBITUBA8 plasmid carrying the mutant alpha-tubulin gene imparting resistance to dinitroaniline herbicides and the nptII selective marker gene imparting resistance to canamycin. The transformants were selected in parallel on media containing canamycin and trifluralin (a dinitroaniline herbicide). The transgenic nature of the resultant regenerants resistant to dinitroaniline herbicides was confirmed by means of Southern blotting and polymerase chain reaction (PCR) analysis using specific probes for the ntpll gene and the gene of alpha-tubulin.

The identification of point mutation locations on beta-tubulin molecules of amiprophosmethyl- and trifluralin-resistant Nicotiana plumbaginifolia lines have described in the work. It was shown that in the first case this mutation is connected with the substitution ofserine residue on proline in position 248; in the second case--with the substitution of phenilalanine on serine in position 317 of beta-tubulin amino acid sequence. Three-dimensional models of beta-tubulin molecule from Chlamydomonas with well-known location of mutations conferring dinitroaniline- and phosphorotioamidate resistance (substitution of lysine residue to methionine on position 350), and beta-tubulin from Nicotiana plumbaginifolia have been reconstructed. On the basis of analysis of site of interaction with dinitroanilines and phosphorotioamides on Chlamydomonas beta-tubulin molecule it was concluded that the revealed mutations on Nicotiana plumbaginifolia beta-tubulin affect amino acid residues participating in formation of this site.

The results of Allium-test screening of new 2,4- and 2,6-dinitroaniline derivates on antimitotic activity and phytotoxicity are presented in the work. It is revealed that all studied compounds which are derivates of 2,4-dinitroaniline, 2,6-dinitro-(4-fluoromethyl)-aniline as well as (methylsulfonyl) nitrobenzol, can evoke a change in mitotic index value, an appearance of cytogenetic damages and also have phytotoxic effect on Allium cepa seedling roots. On data obtained the continuation of investigation the action of N-(2,4-dinitrophenyl)-orto-aminobenzoic acid, N,N-diethyl-2,6-dinitro-4-(trifluoromethyl)-aniline and 1-methylsulfonyl-3-nitrobenzol as potential herbicides is proposed.

The data on in vitro culture establishment and on estimation of callus formation and plant regeneration for eight barley Hordeum vulgare varieties (Getman, Tabora, Adagio, Galactic, Europrestige, Korona, Nevada and Stalker) zoned in Ukraine is represented. Mature embryos of these genotypes were used to study the callus induction and plant regeneration. Using of this approach can rather simplify and intensify the work. Medium, that consists of MS salts supplemented with caseine hydrolysate (1 g/L), L-proline (690 mg/L), thiamine-HCl (1 mg/L), maltose (30 g/L), 2,4-D (2 mg/L), CuSO4 (12.5 mg/L), myoinositol (250 mg/l), gerlite (3.5 g/L) pH 5.6-5.8 was used for both callus formation and plant regeneration. Formation of callus from mature embryos was observed in all studied varieties and showed high frequency (from 65 +/- 3.4% to 100%). It was found that cultivars Korona (88 +/- 2.8%), Europrestige (89 +/- 6.5%), Tabora (93 +/- 3.4%), Getman (99 +/- 0.8%) and Nevada (100%) had the highest regeneration potential. Organogenesis and somatic embryogenesis were the two ways of plant regeneration from callus tissues in barley. The highest general regeneration potential of cultivar Getman (50 +/- 5%) was observed. This cultivar was selected for the further work for development of effective genetic transformation protocol.

The interaction of dinitroaniline compounds with tubulin molecules is characterized by extraordinary selectivity--these matters effectively associate with plant as well as protozoan tubulin and practically don't interact with fungal and animal tubulin in spite of extraordinarily high level of similarity of their sequences. Structural features and mechanisms of this interaction are generalized and in detail analysed in this research. In particular, the regularities of dinitroaniline binding sites' structure and localization on surfaces of tubulin different subunits and tubulins of different origin are characterized. Dinitroaniline binding sites are disposed on the surfaces of longitudinal contacts between tubulin subunits, contain residues of diamine amino acids (lysine or arginine) coupling al nitrile group (s) of dinitroanilines. Binding site location on the surfaces of the same subunit of different origin (for example, plant and protozoan alpha-tubulins) is coincided, however site localisation on surface of alpha- and beta-subunits is distinct. The described sites potentially can be the binding areas for another antimicrotubular compounds, in particular, cyanoacrilates.

Bioinformatic search of plant homologues of human protein kinases SLK, PAK6, PAK7, MARK1, MAST2, TTBK1, TTBK2, AURKA, PLK1, PLK2 and PASK participating in microtubular protein phosphorylation and cell division regulation is carried out. The homologues of protein kinases SLK, MAST2 and AURKA were identified. It is found that closest homologue of human AURKA protein kinase is a protein with unknown function A7PY12_VITVI (STALK--Serine-Threonine Aurora-Like Kinase) from grape (Vitis vinifera). Reconstruction and analysis of three-dimensional structure of STALK protein confirmed its relation to the group of AURKA-like protein kinases.

Effects of exogenic NO donor, sodium nitroprusside, on orientation and organization of cortical microtubules in Arabidopsis thaliana root cells that express GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10-500 microM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in differentiation zone. The influence of sodium nitroprusside revealed in alterations of cortical: microtubules orientation and organization in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of elongation zone where native transverse orientation of cortical microtubules turned into random, oblique or longitudinal relatively to primary root axis. We suppose that NO as one of the intracellular secondary messengers triggers cells differentiation by cortical microtubules reorientation possibly via tubulin nitrotyrosination.

To investigate the functional role of protein hyperphosphorylation in plant cells the general morphology of Arabidopsis thaliana primary roots and structural-functional property of cortical microtubules were studied after treatment with okadaic acid, specific inhibitor of protein phosphatases PPI and PP2A. It has been estimated that okadaic acid affects microtubule organization in a different manner depending from the type of the cells and functional zones of the primary root. It was found that the microtubules in epidermis and cortex cells of distal elongation zone which depolymerized after inhibitor treatment were the most sensitive to 0, 1, 1 and 10 nM okadaic acid concentrations. In trichoblasts, antichoblasts and cortex cells of differentiation zone treatment with okadaic acid caused the microtubules stabilization. Okadaic acid influences were particularly evident in root hair morphology, root hairs swelling and branching as a result of abnormal microtubules orientation observed. According to the data obtained, we can suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays crucial key in plant cell microtubule organization.

Biolistic transformation of soybean by construction carrying a mutant alpha-tubulin gene as selective marker gene conferring resistance to dinitroaniline herbicides, and by additional construction pAHTUBI carrying a full-length barley beta-tubulin gene for correct co-expression of exogeneous tubulin in cells of transgenic soybean lines has been realized. It was established that 10 microM trifluralin is the most optimal selective concentration to pick up transformed soybean lines. Transgenic nature of offained regenerants was confirmed by Southern blotting hybridization using specific probe to selective alpha-tubulin gene.

Data on selection and characterizing of finger millet Eleusine coracana (L.) Gaertn. somaclonal variants regenerated from callus culture are presented and described. Among the tested genetically stable lines the somaclonal variant SE-7 was the most interesting due to its acquired important agricultural traits, such as a higher seed and biomass yield, rapid seed germination at lower temperatures, shortening of the main plant development stages. Data analysis shows that somaclonal variability can be a source to obtain initial material for further selection of new finger millet varieties.

The approaches for new marker genes usage in selection of transformed plant cells, which are based on using mutant tubulin genes from natural plant biotypes and, in perspective, from induced plant mutants have been considered. The results of investigations of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action on molecular and cellular levels have been summarized. The reports dealing with study the transferring and the expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and to trifluralin (dinitroaniline herbicide) from corresponding N. plumbaginifolia mutants into related and remote plant species by somatic hybridisation methods have been analyzed. The results of experiments on monocotyledonous and dicotyledonous. plant transformation by mutant alpha-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its using as a marker gene with obtaining, at the same time, a dinitroaniline-resistant plants.

An analysis of Nicotiana sylvestris mutant plants resistant to isopropyl-N-phenylcarbamate (IPC), one of the representative from phenylcarbamate herbicides, was carried out. The plants were characterized by pollen semi-sterility caused by disruptions during microsporogenesis and cytoplasm synthesis in microspores. Low seed productivity of mutant plans was caused by the high pollen sterility and partially by the low vital activity of seed-buds. Obtained data testify that mutations inducing IPC-resistance lead to cytogenetical and morphological abnormalities in male generative system development, that results in decreasing of seed productivity in N. sylvestris mutants. Character of discovered cytological disruptions testifies about possible defects in organization and functioning of microtubules in mutant plants that can correlate with mutation(s) in one or several microtubule protein genes.

Embryogenic potential of 19 soybean cultivars originated from different eco-geographic regions (Europe, China, America and Far East) has been established and analyzed. Cultivars TSZ-14, Amurskaja-111, Gribovskaja mestnaja were emphasized as highly embryogenic genotypes. Cultivar Mantherova biela vel. revealed the highest embryogenic potential (80%). The comparison of embryogenic potential of the Ukrainian cultivars with given data indicated various parameters which could affect the induction of soybean somatic embryogenesis. It had also been supposed that cultivars from the Far East and Europe could have the potential for further screening of new highly embryogenic genotypes. The data obtained enlarge the data base pool of the soybean somatic embryogenesis research and enable using the most appropriate cultivars for further genetic improvement.

Seven soybean (Glycine max (L.) Merr.) cultivars zoned in forest-steppe and marshy woodlands of Ukraine: "Chernuatka", "Vasylkivska", "Kyivska-91", "Kyivska-27", "Marjana", "Chernobura" and "Altair" were established in vitro for somatic embryogenesis inducing. Cultivars "Marjana" (88%) and "Vasylkivska" (86%) demonstrated the highest embryogenic capacity among all the tested cultivars. During the further plant regeneration cultivars "Marjana" and "Kyivska-91" showed the best capacity to form adult plants despite the fact that embryogenic capability of the cultivar "Kyivska-91" was 71%. According to the obtained data three genotypes were selected for the further investigation of effective methods of biolictic transformation of ukrainian cultivars.

The perspectives of restricted plant genome transfer by means of fusion of isolated microprotoplasts containing one or few chromosomes with recipient protoplasts are considered. Efficiency of the usage of antimicrotubular compounds such as phosphoroamidates (amiprophosmethyl and cremart), dinitroanilines (oryzalin) as well as propizamide and microtubule organising center disrupters (griseofulvin and phenylcarbamates) for micronucleation are analyzed. Advantages of the combined usage of antimicrotubular drugs and cytochalasin B as disrupter of actin filaments for enhanced yield of microprotoplasts are considered. Results of subsequent establishment of microprotoplast-mediated chromosome transfer technique is summarized. The examples of successful microprotoplast-mediated transfer of individual chromosomes from a donor potato line into mesophyl protoplasts of Nicotiana tabacum and into recipient cells of wild tomato (Lycopersicon peruvianum) with subsequent selection and regeneration of hybrids are considered. Development of alternative methods of partial genome transfer using microprotoplasts produced from plant gametes (microspores) is presented. The advantages of microprotoplast-mediated transfer of chromosomes as an efficient technique of parasexual crossing in comparison with traditional somatic hybridization are discussed.