Increasing of antioxidant and superoxide dismutase activity in chicory transgenic plants

Aim. Determination of the antioxidant activity (AOA) and superoxide dismutase (SOD) activity in transgenic chicory plants carrying the human interferon 2b target and nptII or bar selective genes. Methods. AOA was measured by a method based on the determination of kinetics of the reduced 2,6-dichlorophenolindophenol oxidation. SOD activity was assayed using the system consisting of methionine, riboflavin, and nitroblue tetrazolium. Results. Antioxidant activity of transformed plants extracts was more than 1,91–2,59 and 2,04–2,43 times over the activity of control non-transgenic plants (at nptII and bar gene presence respectively). SOD activity was higher in transgenic plants than in the control, and was 2,03 ± 0,46–3,33 ± 0,54 U/g weight (nptII gene) and 2,25 ± 0,46–2,68 ± 0,08 U/g weight (bar gene). Conclusions. Transgenic C. intybus plants have higher antioxidant and superoxide dismutase activity compared to non-transgenic plants. The increasing of AOA and SOD activity is a response of plants to transformation stress factor and integration of foreign genes in plant genome.

Introduction.Genetic transformation is used to create transgenic plants for scientific purpose (for instance, investigation of gene functioning) and for obtaining plants, synthesizing new substances.However, recently the safety issues of using plants with artificially modified genome have become rather urgent [1,2].Physiological and biochemical properties of plants may change after the transformation [3].
There are the data, testifying to the stress of the transformation process, Agrobacterium-mediated, in particular, for plants at each stage [4]: in vitro cultivation, injury, contact with microorganism, selection, transfer of the foreign gene to the genome of plants, synthesis of corresponding proteins, biological activity of the protein.One of the reactions to the activity of stress agents is known to be the activation of antioxidant systems of plant protection, namely, the increase in the number of low-molecular antioxidants and the activity of enzymes (superoxide dismutase (SOD), catalase, peroxidase, etc.) [5,6].In particular, reactive oxygen species are formed and accumulated at the effect of drought, temperature stress, etc. which leads to oxidative stress.These changes are accompanied by the increase in the activity of the antioxidant system [5].Taking the abovementioned into account it would be interesting to investigate whether there are any changes in the activity of the antioxidant system and its components, induced by the transfer of the gene (genes).The current work is aimed at determining the antioxidant activity (AOA) and the activity of SOD of transgenic C. intybus L. plants with human interferon-a2b (ifn-a2b) gene and different selective nptII and bar genes.
Materials and Methods.The material for the investigation was transgenic C. intybus var.foliosum Hegi plants with target ifn-a2b gene and selective nptII (four lines) and bar (three lines) genes, previously obtained by us [9,10].Regenerant plants were cultivated in Murashige and Skoog medium [11] with twice decreased concentration of macroelements for 30 days.AOA of plants was determined by the method, described in [12] with modifications, SOD activity -as described in [13].The experiment results were statistically processed using variance analysis of singlefactor experiment.
Results and Discussion.AOA of the extracts of transgenic chicory plants was considerably higher than that of non-transformed plants (Fig. 1, a, b).For instance, AOA of the extract of transgenic lines with ifn-a2b-nptII genes was 17.15 ± 0.66-23.20 ± 0.60 ml/l per one minute (control -8.97 ± 0.79), while that for extracts of plant lines with ifn-a2b-bar genes -15.63±0.89-18.6 ± 0.84 ml/l per one minute (controlonly 7.67 ± 0.84).Disperse Ff ratio (219.66 and 91.55 for plants with ifn-a2b-nptII and ifn-a2b-bar genes, respectively) was considerably higher than the critical point Fst (2.5 and 7.59, respectively, with 5 % significance point).Therefore, the differences obtained are statistically reliable at the level of reliable probability P 095 .
The activity of SOD extracts of all plants with genes ifn-a2b-nptII turned out to be higher compared to the control, respectively, from 2.3 ± 0.6 to 3.33 ± 0.54 and 0.94 ± 0.07 U/g of fresh weight (Fig. 2, a).At the same time, the disperse ratio Ff was 0.89 which is much less than Fst (5.99 with 5 % significance point).Therefore, the reliability of obtained differences in SOD activity is not confirmed.Here the comparison of factorial and residual dispersion testifies to the necessity of increasing the sample to confirm the reliability of present differences.
The activity of SOD extracts from plants with bar gene is 2.25 ± 0.46-2.68± 0.08 U/g of wet weight which is 2.17-2.58times higher that the activity in the control (Fig. 2, b).Disperse Ff ratio is 12.25 which is much higher than Fst (7.59 at P 095 ).The differences in SOD activity of transgenic and control plants are statistically reliable at the mentioned level of significance.Thus, transgenic chicory plants differ from the control by the increased level of the antioxidant activity and SOD.
In vitro cultivation may be excluded from possible reasons of changes in AOA and SOD activity in transgenic chicory plants as both transgenic and control plants were cultivated in vitro and they passed the regeneration stage.The contact with bacteria and selection are likely not to condition the variation in AOA and SOD activity in transgenic plants as the investigations were conducted two years after obtaining them.The changes in AOA and SOD activity occur in plants with genes nptII and bar.The type of the selective gene is not likely to cause any changes.
The analysis of obtained plants for the presence of transgenes demonstrated that the transfer of both target and selective genes occurred for all the investigated lines.Thus, the increase in AOA and SOD activity by transformed plants may be related to the presence of foreign genes.The transcription of ifn-a2b gene was revealed only in one line of plants with nptII gene and in all the plants with bar gene.Therefore, there are no reasons to state that the transcription of transgenes is the reason of the increase in AOA and SOD activity in transgenic plants.
The plants with the increased AOA level and SOD activity are more resistant to oxidative damage, caused by the effect of stress factors -drought, salinization, etc. [14,15].Therefore, the obtained transgenic chicory plants with high AOA level may be more resistant to the effect of the mentioned stress factors and thus used in biotechnology and selection.
Conclusions.Transgenic C. intybus plants are remarkable for the increased activity of the antioxidant system and the enzyme of superoxide dismutase compared to the control non-transformed plants, which may be the response of the plant organism to the effect of transformation as a stress factor.The stress state of transgenic chicory plants is probably related to the transfer of foreign genes to the genome of plants.