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The in vivo and in vitro interactions between tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) and the bacteria Pseudomonas fluorescens, Acinetobacter baumannii, Rhodococcus erythropolis,Pseudomonas aureofaciens, Pseudomonas putida, Methylovorus mays have been studied. These microorganisms were shown to be in stable associations with plants. The colonized plants were characterized by more rapid growth, a higher yield, and better adaptation to in vivo conditions. The colonized plants were more resistant to bacterial phytopathogens Erwinia carotovora and Pseudomonas syringae. Plants colonized by naphthalene-resistant bacteria can grow steadily on a medium containing this compound. The results demonstrate the prospects of the use of beneficial associative microorganisms in the development of technologies for plant protection against biotic and abiotic stressors.

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system.

The method of producing of water extract from transgenic Kalanchoe pinnata L. plants expressing cecropin P1 gene has been developed and adapted for pharmaceutical production. Chlorophorm has been shown to be an efficient preservative for this extract. The antibiotic activity of the extract has kept well even after 1-h boiling. In addition, the prolonged antimicrobial activity of the extract has been confirmed by the “accelerated aging” method. Being added to culture medium, the leaf extract from transgenic K. pinnata plants has provided more intensive callus growth and improved rhizogenesis in ice plant (Mesembryanthemum crystallinum). The obtained results demonstrate an increased biological activity of the extract from kalanchoe plants producing antimicrobial cecropin P1 peptide and good prospects for its use in pharmacology.

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.

A gene of antimicrobial peptide cecropin P1 (CP1) was inserted into the vector plasmid pPCV91 under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) containing four enhancer sequences CaMV 35S and nontranslated leader sequence Ω RNA of tobacco mosaic virus. The recombinant vector obtained was used for agrobacterial transformation of tobacco plants (Nicotiana tabacum L., variety Samsun) with the polymerase chain reaction (PCR)-based method. The presence of gene CP1 in the genome of plants was proven by western-blot analysis and testing the antibiotic activity of plant extracts. In different plant lines, the level of cecropin P1 synthesis amounted 0.02–0.2% of total soluble plant leaf protein. The transgenic plants, unlike the control ones, displayed enhanced tolerance to phytopathogenic microorganisms and oxidative stress. It was established that the ability of the transgenic plants to express cecropin P1 is transmitted to progeny.

Using molecular and isotopic mass spectrometry, we investigated the toxic effect of naphthalene as a representative of polycyclic aromatic hydrocarbons (PAHs) on plants growing under sterile conditions and plants inoculated with microorganisms capable and incapable of naphthalene degradation. Tobacco plants of the Samsun variety were grown in a closed gas-nutrient system on a mineral medium with sucrose as a carbon source. Naphthalene used as a toxicant at a concentration of 5.2 × 10−4% contained 13C isotope whose amount was characterized by the value δ13C = +281.4 ± 0.6‰ relative to the PDB standard and differed from that of sucrose, the main source of carbon (δ13C = −12.0 ± 0.1‰). Degradation of naphthalene was determined by the inclusion of its carbon in metabolic CO2 and plant tissues (the root, stem, leaves). The effect of naphthalene on plants was indicated by the rates of O2 production and CO2 uptake during the light period as compared with the dark period of exposure. A decrease of the toxic effect of naphthalene on plants was observed only at the inoculation of plants with Pseudomonas aureofaciens BS1393 rhizosphere bacteria bearing plasmid pBS216, which controls the naphthalene biodegradation ability. The occurrence of other heterotrophic microorganisms incapable of naphthalene degradation had no similar protective effect.

Transgenic rapeseed (Brassica napus L.) plants carrying an artificial gene for the antimicrobial peptide cecropin P1 (cecP1) were obtained and characterized. The agrobacterial transformation was done by vacuum infiltration of seeds with agrobacterium GV3101(pMP90RK) containing a binary vector pGA482::cecP1. The cec1 gene expression was analyzed by Western blotting and confirmed by antimicrobial activity measurements of plant extracts. The obtained plants showed the resistance to the bacterial and fungal pathogens Erwinia carotovora and Fusarium sporotrichioides. The photosynthetic activities of control and transgenic plants under biotic stress conditions of E. carotovora infection were comparatively studied. The higher tolerance of the cecP1 plants to the oxidative stress caused by paraquat was shown. The results obtained point to the possibility of incorporation of the cecropin P1 gene into the integral stress protection system of plants.

The review considers the basic strategies used to produce biologically safe marker-free transgenic plants and analyzes their advantages and disadvantages. The systems of positive and negative selection as safer approaches for transformant identification are briefly described. The application of co-transformation, transposition, and site-specific recombination for production of marker-free plants is described. Special attention is paid to novel approaches to create marker-free plants initially containing no selective genes in their genomes.

The in vivo and in vitro interactions between tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill.), cabbage (Brassica oleracea var. capitata L.), rape (B. napus L.), and the common ice plant (Mesembryanthemum crystallinum L.) and bacteria Pseudomonas aureofaciens, P. putida, and Methylovorus mays were studied. Stable associations of these microorganisms with plants are demonstrated. Colonized plants were characterized by accelerated growth, more efficient rooting, better adaptation to in vivo conditions, and enhanced resistance to bacterial and fungal phytopathogens (Erwinia carotovora, Sclerotinia sclerotiorum and Phytophthora infestans). Plants colonized by bacteria resistant to kanamycin and naphthalene can grow steadily on the medium containing these compounds. The results obtained indicate a promising usage of beneficial associative microorganisms for the development of technologies for plant protection against biotic and abiotic stressors.

Effects of colonization of micropropagated potato (Solanum tuberosum L.) and strawberry (Fragaria L.) plants by the rhizosphere bacterium Pseudomonas aureofaciens strain BS1393 (VKM B-2188 D) on plant growth and resistance to bacterial and fungal phytopathogens were studied. Pseudomonad colonization improved the physiological characteristics of plants and enhanced their adaptation to in vivo conditions. The presence of P. aureofaciens cells in various plant tissues (leaves, stems, and roots) in vitro was demonstrated on the background of plant cocolonization by two associative strains—P. aureofaciens strain BS1393 (VKM-B-2188 D) and Methylovorus mays (VKM-B-2221). The colonized plants displayed an increased resistance to the phytopathogens Erwinia carotovora, Sclerotinia sclerotiorum, and Phytophthora infestans. These results demonstrate that pseudomonades are promising for practical application in the microbial protection of plants against phytopathogens.

DNA methylation is an integral part of the mechanism of a remodeling and modification of the chromatin structure. The global complex net of chromatin modification and remodeling reactions is still to be determined, and studies of the mechanisms controlling the epigenetic processes of histone modification and DNA methylation are in their infancy. Cytosine methylation occurs predominantly in CpG sequences of the eukaryotic genome, and it also takes place at symmetric CpHpG and nonsymmetric CpHpH sites (where H is A, T, or C). The modification efficiency of the three types of DNA methylation sites depends on their genomic localization. Different regions of the eukaryotic genome are remarkable for their methylation features: CpG-islands, CpG-island shores, differentially methylated regions of imprinted genes, and regions of nonalternative site-specific modification. The three canonical sites (CpG, CpHpG, and CpHpH) differ in DNA methylation efficiency depending on their nucleotide context. An epigenetic code of DNA methylation can be assumed with context differences playing a specific functional role. The review summarizes the main up-to-date data on the structural and functional features of site-specific cytosine methylation in eukaryotic genomes. Pathogenesis-related alterations in the methylation pattern of the eukaryotic genome are considered.

The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or self-modified genes. Plants are considered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism.

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01-0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

143 Selection genes conferring resistance to antibiotics and herbicides [1] or reporter genes [2] are conventionally used for selection of transgenic plants. Currently used commercial transgenic plants resistant to pests and herbicides pose a considerable potential biological and environmental hazard associated with their adverse effect on useful organisms of agrobiocenoses. In view of this, the development of methods for obtaining newgeneration transgenic plants without genetic garbage (such as selection genes conferring resistance to antibiotics and herbicides and other marker genes) is a topical problem.

Infection of plants by soil bacterium Agrobacterium tumefaciens induces tumors referred to as crown galls. Tumor development is determined by the introduction of agrobacterial genes governing phytohormone (auxin and cytokinin) production into the plant genome. The most important of these genes are iaaM and ipt. Development of transgenic plants inhibiting the expression of these genes allows a raise of varieties resistant to crown gall disease. For this purpose, single and double tobacco transformants with antisense copies of iaaM and ipt fused with single and double promoters for the 35S RNA of the cauliflower mosaic virus (CaMV 35S and CaMV 35SS) were obtained. Inoculation of transgenic plants harboring the antisense oncogene copies with virulent A. tumefaciens strains C58 (pTiC58) and A6 (pTiA6) revealed significant, but still incomplete, inhibition of these genes. Agrobacterium-mediated transformation of transgenic plants gave rise to weakened tumors, which varied in morphology and allowed regeneration of whole plants. Analysis of the inhibition of the iaaM and ipt expression in tumor cells demonstrated that the RNA interference strategy is promising for developing plant varieties resistant to agrobacterial infection.