In order to study the possibility of creating new plant forms resistant to phytopathogens, a collection of transgenic plants of model tobacco culture with new different plant protective genes was obtained by the agrobacterial transformation method. First an addition of a collection with serine proteinase inhibitor BWI-1a ( ISP ) from buckwheat with fragments of a spidroin gene as putable enhancer by vector constructions different designs was done. Secondly, transgenic plants with an antimicrobial peptide from sinthetic wheat Triticum kiharae and with defensine from Stellaria media . Comparative study of physiological characteristics of transgenic plants in biotests in vivo (with isolated leaves) and in vitro (with well biotests) was carried out. Regardless of the design of the vector construction, the target genes were expressed to a different extent in the tissues of all transgenic plants and their seed and vegetative progenies and gave their tissues antibacterial activity, indicating the synthesis of the functional protein. The introduction to the tobacco tissues of the heterologic plant protective genes of different nature that plants use in different defense mechanisms led to a similar increase in antibacterial activity of the transgenic tobacco tissues.

In this paper, the results of long-term screening of independently derived transgenic tobacco plants carrying the synthetic BWI-1a gene of serine proteases inhibitor from buckwheat are presented. For several years periodic spot checks of persistence and expression of the heterologous protective genes in vegetatively cloned collections and seeds of transgenic plants were conducted. The persistence of expression of the target gene after ten years of passage of plants in aseptic culture without selective pressure in their seed progeny for at least three generations and derived callus was shown. Extracts of tissues of all the variants of transgenic plants inhibited the growth of phytopathogenic bacteria and the germination of spores of fungi. The degree of the suppression of a pathogen in this case was hardly reduced. In the second seed generation, the number of defective seeds increased and there was a sharp decline of germination ability of the seeds even in nonselective conditions.

A novel plant hairpin-like defense polypeptide named EcAMP3 was isolated from latent barnyard grass (Echinochloa crusgalli L.) seeds. The native peptide and its recombinant analogue were characterized. EcAMP3 displays antifungal and antibacterial activity in vitro. The gene family encoding EcAMPs precursor protein was also characterized; the genes and pseudogenes of this family show 97-100% homology. Every member of EcAMPs precursor family contains seven identical cysteine motifs: C1XXXC2(11-13)C3XXXC4. One of those motifs corresponds to the isolated peptide. EcAMP3 is the first member of the plant hairpin-like peptide family that inhibits the growth of phytopathogenic bacteria. Obtained results can explain the nature of the complex resistance of barnyard grass to a variety of pathogenic microorganisms.

DNA samples obtained from the populations of the Red Data Book species Bellevalia sarmatica (Georgi) Worovow from Volgograd oblast were examined using RAPD and AFLP analyses. DNA marking revealed considerable differences in the levels of interpopulation and intraspecific polymorphisms the Bellevalia. Furthermore, RAPD analysis, despite of lower levels of interpopulation polymorphism identified, made it possible to obtain clearer data on population subdivision upon statistical treatment of the results. The results of this study can be used in developing conservation strategies for maintenance of the species abundance and genetic diversity, as well as in elaborating the criteria for construction genetic collections.

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.

Проведен RAPD и AFLP анализ ДНК популяций занесенной в Красную книгу бельвалии, или гиацинта сарматского (Bellevalia sarmatica (Georgi) Woronow), Волгоградской области. ДНК-маркирование продемонстрировало значительные различия в уровнях как межпопуляционного, так и внутривидового полиморфизма бельвалии. При этом данные RAPD анализа, несмотря на более низкие значения межпопуляционного полиморфизма, при статистической обработке позволили получить более четкое деление популяций на группы. На основании полученных данных можно составить план поддержания численности и генетического разнообразия вида и разрабатывать критерии формирования генетических коллекций.

We have analyzed genetic effects of heterologous plant genes insertion on genome functioning of higher plants, belonging to different systematic groups (tobacco, Arabidopsis). Plants of different species were responding differently to the insertion of the same transgene, which is likely to be associated with the location of alien DNA insertion and could manifest in morphological changes spectrum and target gene expression level.

The possibility of using RAPD and AFLP methods for genetic monitoring of populations of Matthiola fragrans (Bunge), a species included in the Red Book of the USSR, was shown for the first time. An analysis of inter- and intrapopulation and interspecies genome polymorphism was performed. Differences in the genetic structure of Matthiola populations from various geographical collection points were revealed. A simple method of performing RAPD analysis and the great number of unique markers found in each population compared with the AFLP analysis, as well as the good division of populations under statistical treatment, allow us to draw the conclusion that using the RAPD method in genetic monitoring of rare and insufficiently studied species is well founded.

The stability of marker nptII and target gene of serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds after agrobacterial transformation in the new foreign genome environment has been investigated. Different vegetative and seed progeny of self-pollinated transgenic tobacco was studied after propagation at selective and nonselective in vitro conditions. For the majority of transgene lines among random sample of independent transgenic plants stable and coupled PCR confirmed inheritance and expression of marker and target genes has been demonstrated after prolonged microcutting and repeated regeneration cycles at unselective in vitro conditions. The most of the seed T1 progeny of the studied lines has maintained Km-resistance too. However all transgenic lines had significant disturbances at the microsporogenesis process. Two lines (C5 and C12) after 4-year microcutting lacked Km-resistance and at the same time lacked their antibacterial activity. Two other clones revealed variability in Km-resistance (line C7) and seedlings size (line C22) that might be a result of more than 1 transgene insertion or chimaeric nature of transgene plants so it require an additional study.

The buckwheat serine protease inhibitor (BWI-1) target gene (ISP) was expressed under the control of the constitutive 35S promoter of the cauliflower mosaic virus was expressed in transgenic tobacco plants and conferred antibacterial resistance. A stable and linked inheritance and expression of the marker nptII and target genes were observed in a random sample of independent transgenic tobacco plants after longterm propagation by nodal segments or multiple (for 1.5 years) regenerations under nonselective conditions; the transgene insert was preserved in the T1 seed progeny. Transgenic plants displayed numerous alterations in microsporogenesis. A loss of kanamycin (Km) resistance was accompanied by a loss of antibacterial activity in two lines. Segregation was observed for Km resistance in line C7 and for seedling size in line C22.

From seeds of Nigella sativa L. (Ranunculaceae), an endemic plant of Uzbekistan, two novel defensins named Ns-D1 and Ns-D2, were isolated and sequenced. The peptides differ by a single amino acid residue and show high sequence similarity to Raphanus sativus L. defensins Rs-AFP1 and Rs-AFP2. The Ns-D1 and Ns-D2 defensins display strong although divergent antifungal activity towards a number of phytopathogenic fungi. High antifungal activity of N. sativa defensins makes them promising candidates for engineering pathogen-resistant plants.

Two forms of a novel antimicrobial peptide (AMP), named WAMP-1a and WAMP-1b, that differ by a single C-terminal amino acid residue and belong to a new structural type of plant AMP were purified from seeds of Triticum kiharae Dorof. et Migusch. Although WAMP-1a and WAMP-1b share similarity with hevein-type peptides, they possess 10 cysteine residues arranged in a unique cysteine motif which is distinct from those described previously for plant AMPs, but is characteristic of the chitin-binding domains of cereal class I chitinases. An unusual substitution of a serine for a glycine residue in the chitin-binding domain was detected for the first time in hevein-like polypeptides. Recombinant WAMP-1a was successfully produced in Escherichia coli. This is the first case of high-yield production of a cysteine-rich plant AMP from a synthetic gene. Assays of recombinant WAMP-1a activity showed that the peptide possessed high broad-spectrum inhibitory activity against diverse chitin-containing and chitin-free pathogens, with IC50 values in the micromolar range. The discovery of a new type of AMP active against structurally dissimilar microorganisms implies divergent modes of action and discloses the complexity of plant-microbe interactions.

The possibility to use agrobacterial transformation of leaf discs to produce resistance to bacterial infections in tobacco and potato plants by introduction of a single gene encoding the serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds is shown. All studied PCR-positive transgenic plants exhibited antibacterial activity in biotests. It was shown that the presence of just a single gene of serine proteinase inhibitor provides sufficient protection at least against two bacterial phytopathogens, Pseudomonas syringae pv. tomato and Clavibacter michiganensis sbsp. michiganensis. The biotest including tobacco plant infection by the white wings butterfly in the green house has also demonstrated the existence of protective effect in transgenic tobacco plants. Significant genotypic variations in the protection efficiency were found between members of different genera of the same family (potato and tobacco) as well as between different lines of the same species. Northern blot analysis of four transgenic potato lines and three tobacco lines transformed by a vector plasmid containing the ISP gene of serine proteinases BWI-1a from buckwheat seeds has shown the presence of the expected size mRNA transcript.

The order was developed for selection of summer rape regenerants on selective nutrient media with nystatin. Isolated plants-regenerants were tested on resistance to nystatin taking into account microcutting's striking root and frequency of callusogenesis. In the field and laboratory conditions the entomological estimation of tolerance of isolated rape plants to injurious insects Pieris brassicae L. and Myzodes persicae Sulz. was made. It was shown that nystatin resistance in substantial number of clones remains at level of undifferentiated cells and tissues. It was established that clones isolated on selective nutrient media with nystatin have increased resistance to Pieris brassicae L. Obtained results suggest about undirected action of nystatin.

Molecular analysis of the genome was performed for 14 species of the genus Stachys. RAPD and ISSR analyses of the Stachys genome revealed 574 polymorphic fragments, including genus- and species-specific markers. Based on the patterns, UPGMA and the Jacquard coefficient were used to estimate the genetic distances between Stachys species and populations and to construct dendrograms reflecting the phylogenetic relationships among the Stachys species. Molecular analysis of the Stachys genome refined the phylogenetic positions of some species and revealed synonymous species.

Morphogenesis of different Stachys species introduced in in vitro culture have been compared. The frequency of altered forms have been demonstrated to be related to the plant genotype. All regenerants of S. sieboldii, which reproduces in vivo only vegetatively, are phenotypically normal, irrespective of the concentrations of plant growth regulators at which they have been obtained. Only changes in isozyme patterns have been observed in the regenerants grown in media containing at least 10 mg/l benzyl aminopurine (BAP); most of these changes are the absence of a particular component of the pattern. The cross-pollinating species Stachys ocymastrum, which typically reproduces by seeds, has yielded morphologically altered forms even in phytohormone-free media; its isozyme patterns often contained a new component. Analysis of the isoperoxidase patterns of regenerants of both Stachys species obtained with the use of high phytohormone concentrations has demonstrated qualitative and quantitative changes suggesting the appearance of somaclonal variants even in the course of plant regeneration directly from nodal segments, bypassing callus formation. Changes have also been found in Stachysplants regenerating from the callus tissue.