Transgenic plants creation methodology developed for several decades has gained significant advances. However, problems of unanticipated effects of transgenosis, stability of GMO characteristics and establishing criteria of their safety evaluation remain unresolved. The analysis of different approaches to assessing the impact of plant genetic transformation is presented. It is concluded that the profound studies on the physiology of plant-agrobacterial symbiosis as a methodological basis of plants genetic engineering can answer many unresolved issues of genetic engineering.

The transgenic and non-transgenic tobacco cell cultures were analyzed for resistance to abiotic and biotic stress. The different physiological reaction of cell culture depending on T-DNA structure (or transgen structure) was observed. The cell culture transformed by disarmed Agrobacterium tumefaciense A699 with pCNL 65 nptII demonstrated the same stress-resistance as non-transgenic control cell culture. The cell culture transformed by Agrobacterium tumefaciense LBA 4400 pBiCaMV nptII + hsp101 showed a raised stress-resistance to high temperature, high KF concentration, and to the action of Clavibacter michiganensis ssp sepidonicus. Obviously, the expression of transferred arabodopsis gene hsp101 provides protection properties of transgenic cell culture under the influence of various stress factors. Moreover, that agrobacterial transformation as previous stress-factor is supposed to make a contribution to formation of transgenic cell culture cross-resistance.

The gene technology using in plant investigations came to the better understanding of plant physiological processes. At the same time unclear knowledge about transgenic plant physiology may occur a source of incorrect interpretation of obtained results and, consequently, wrong conclusions. In addition, the causes and mechanisms of pleiotropic effects associated with transgenic insertion and gene silencing are remaining unexplained. To solve the problem of transgenic plant physiology it is necessary to pay a close attention to physiological and biochemical peculiarities of plant-agrobacterium symbiosis, because it is a base of plant transformation. It was assumed earlier that agrobacterial transformation is a complex biotic stressing factor and transgenic plant is a long-term stressed organism. We suppose that physiological consequences of plant transformation are determined not only by foreign gene insertion, but largely by stress reaction of plant cells on agrobacterium transformation. Foreign DNA insertion to the plant recipient results in cascade of response reactions remarkably changing metabolism. The degree of such response is supposed to be in dependence on phylogenetic relations of gene donor and recipient. Cell cultures were obtained from tobacco plants (Nicotiana tabacum cv. ’Samsung’) transformed by following Agrobacterium tumefaciens strains: disarmed 669 one and LBA4400 one with hsp 101 in sense or antisense orientation. These cell cultures were used for investigations of the stress-reactions on biotic (bacterial infection agent Clavibacter michiganensis subsp. Sepedonicus) and abiotic (high temperature, potassium fluoride) factors. It was revealed that “sense” culture was superior to normal and “699” ones in tolerance to pointed stressing factors. Similar results were obtained for “antisense” culture, nevertheless it was a priori not expected to be tolerant. So, to assess the transformation consequence is necessary to take into account that observed effects may not result from action of the invected gene only. Conclusions: 1. To consider the transgenesis to be completed solely if expression of transferred gene is present – is methodologically incorrect way. The transferred genes could be silenced because of the response defense reaction likely as under a “pathogen attack”. So the absence of transferred gene expression doesn’t mean the absence of transformation as fact. Moreover the deletion of inserted construction could take place but physiological trace of the insertion nevertheless can be noticeable.2. The assessment of physiological consequences of transgenesis when using the plants transformed by disarmed constructions and the plants transformed by constructions including foreign geterological genes should be carried out carefully because of these systems are different. The process of transformation by disarmed constructions is very similar with natural agrobacterial infection where plants and bacteria have been coadapted during evolution, so the transformation by insertion of foreign genes leads to forming much more unstable systems.

The question how long transgenesis invected alterations are demonstrated in a succeeding generations remains of great interest. In this study we describe the development of T 1-T5 generations of Nicotiana tabacum L. transformed by Agrobacterium tumefacience strain 699 with disarmed plasmid. Tobacco plants were grown in the same environmental conditions. The characteristics of vegetative and generated parts had been assessed. Transgenic plants were superior to normal ones in leaf area and stem length, had earlier flowering and internodes development but not differ in a number and size of flowers. Growth activation is suggested to be a result of biotic stress induced by transformation.

Consequences of agrobacterial transformation have resemblance with plant stress response. So agrobacterial transformation is being considered as a complex multilevel biotic stress factor including reactions on the wounding, contact with pathogen, culturing in vitro and T-DNA insertion. Methodical approaches need developing to distinguish the effects of the transgenesis and associated stresses.

Consequences of agrobacterial transformation have resemblance with plant stress response. So agrobacterial transformation is being considered as a complex multilevel biotic stress factor including reactions on the wounding, contact with pathogen, culturing in vitro and T-DNA insertion. Methodical approaches need developing to distinguish the effects of the transgenesis and associated stresses.

The synthetic chimeric gene TBI-HBS encoding the synthesis of immunogenic ENV and GAC epitopes of HIV-1 (immunogenes of T- and B-lymphocytes) and of the surface protein (HBsAg) of the hepatitis B virus was introduced into tomato plants var. Ventura by agrobacterial vector pBIN35TBI-HBS; transgenic tomato plants with the integrated gene TBI-HBS were generated. The integration of the TBI-HBS target gene was confirmed by PCR. The synthesis of antigenic proteins of TBI and HBsAg in fruits of transgenic tomato plants was displayed by immunoassay. The fruits of transgenic tomato plants were fed to experimental mice with a 1-week interval. On days 14 and 28, there was discovered a sufficiently high content of antibodies to the antigenic proteins of HBV and HIV-1 in serum of experimental animals. Antibodies were found in feces of experimental mice; no antibodies were found in the control group of mice. Hence, it was established that the TBI (HIV-1) and HBsAg (HBV) antigens were synthesized in transgenic tomato fruits due to the integrated construction of pBINNp35TBI-HBS in an amount that was enough to induce the immunogenic response in mice to the oral delivery of edible vaccine.

D-Tryptophan (DTry) accumulating in wilted plant tissues and seeds may be used simultaneously in IAA biosynthesis and in the formation of N-malonyl-D-tryptophan (MDTry). MDTry synthesis may be considered as a drought inducible step in the regulation of IAA biosynthesis; MDTry synthesis results in a temporary reserve for the inactivated precursor when cells lose water. The stereochemical compartmentation of the indole precursor in the form of DTry or MDTry offers the possibility to use Try only for the IAA biosynthesis but not for the synthesis of proteins.

To study the hormonal control of germination, the contents of Trp, MTrp and IAA were determined in coleoptiles, scutellum and roots of wheat seedlings (Triticum aestivum L.) on the 3 and 5 days of growth in the darkness. The concentration of Trp high at the 3rd day increased 1.15 and 2.5 times to the 5th day of germination for root and scutellum and coleoptile respectively. The MTrp and IAA contents decreased with seedling growth from the 3rd to the 5th day. The highest concentration of MTrp was in the upper 3 mm of coleoptile apex. The IAA concentration was high in the apex and in the elongation zone of coleoptile on the 3rd day of germination. The ratio MTrp/IAA was several times lower than the Trp/IAA ratio in almost all parts of seedlings, indicating that Trp is not a limiting factor for IAA bio-synthesis. The activity of tryptophan racemase (EC 5.1.1.10) in etioplasts fraction was higher in the direction LTrp → DTrp on the 3rd day of germination. Both cytosol and etioplasts tryptophan racemase diminished with time and in the coleoptile at the 7th day the activity was less than a tenth. The activities of both L-TAT (EC 2.6.1.27) and D-TAT (EC 2.6.1.21) were also present in etioplasts fractions from upper part and roots of etiolated seedlings. Both activities were sensitive to the addition of cofactors Na pyruvate and NAD +. Thus it was suggested that DTrp and MTrp participated in IAA biosynthesis during wheat germination and growth of seedlings and the direction LTrp → DTrp of tryptophan racemase played a key role during first days of heterotrophic growth. Abbreviations: DAO – D-amino acid oxidase, ME – β-mercaptoethanol, MTrp – N-malonyl-D-tryptophan, PMSF – phenylmethylsulphanylflouride, PLP – pyridoxal phosphate, L-(D-) TAT – L-(D-) tryptophan aminotransfer-ase, Trp – L-(D-) tryptophan