Page 110 - Book of Abstracts

5 International Scientific Online Conference DOI: https://doi.org/10.15414/2021.9788055224015

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ANTIOXIDANT ACTIVITY OF TRANSGENIC PETUNIA HYBRIDA VILM. WITH
HETEROLOGOUS ZRNASE II GENE AFTER TOBACCO MOSAIC VIRUS INFECTION
Andrii Potrokhov, Olga Ovcharenko
Institute of Cell Biology and Genetic Engineering, Kyiv, Ukraine;
E-mail.: AlexGSMster@gmail.com
Petunia hybrida Vilm. is gradually becoming a new model for research in virology and
genetics. Petunia plants with ZRNase II gene encoding endoribonuclease were obtained earlier.
Transgenic plants were characterized by increased resistance to the Tobacco mosaic virus
(TMV). This study aimed to evaluate the degree of stress reactions in transgenic petunia plants
with the ZRNase II gene after their infection with TMV. Lipid peroxidation and antioxidant
activity were used as indicators of stress reactions.
Independent transgenic Petunia hybrida lines were obtained based on two separate
commercial clones. Lines TM1.1 and TM1.2 are derivatives M1 clone, while lines TP5.1, TP5.2,
and TP5.3 belong to P5. Plants were transformed previously with a genetic vector carrying the
heterologous ZRNaseII gene from Zinnia elegance. The plants with confirmed transgenic nature
were propagated in vitro and used for further ex vitro investigations. The experimental plants
were grown in neutral peat substrate TS1 (Klasman, Latvia) at a temperature of +24 °C and 16-
hour light. The transgenic and control plants were infected with virus (TMV) containing
material by mechanical inoculation of leaf blades. Lipid peroxidation was evaluated by
measuring diene conjugates. Plant material (leaves) was triturated in 0.1 M phosphate buffer
(pH 7.4), and the pellet was resuspended. A mixture of heptane with isopropyl alcohol 1:1 was
added. The resulting mixture was centrifuged for 10 minutes at 4000 g and 1/10 volume of
distilled water was added, the solution was stirred, 1 ml of heptane phase was taken, 5 ml of
ethyl alcohol was added. The optical density was measured at 233 nm. To determine malonic
dialdehyde, the resulting homogenate was precipitated with 5 % trichloroacetic acid and
centrifuged for 10 minutes at 4000 g. The supernatant was transferred to test tubes and 0.8 %
thiobarbituric acid was added and heated. The optical density was measured on a
spectrophotometer at 532 nm. To determine the total antioxidant activity, 0.2 ml of 10 mM 2-
deoxyribose solution, 0.2 ml of 0.1 mM Fe2 + / EDTA solution and 0.2 ml of 30 % H2O2 were
added to the homogenate. The resulting solution was adjusted with phosphate buffer to a
volume of 2 ml. The mixture was incubated for 4 hours. at + 37 C. After incubation, 1 ml of 2.8
0
% trichloroacetic acid solution and 1 ml of 1 % thiobarbituric acid solution in 50 mm NaOH
solution were added. The mixture was heated for 10 minutes at 100 °C. Optical density
measurements were performed at a wavelength of 532 nm.
In the transgenic lines obtained based on M1 clone, the level of diene conjugates (DC)
didn’t overrun the control, while in the line TM1.2 it was even lower than control. It was found
that the content of DC in the transgenic lines obtained from P5 petunia clone exceeded that in
the control. There was no significant difference in the content of MD in transgenic and control
lines of M1 petunia clone. While in transgenic plants of P5 petunia clone the amount of MD was
significantly higher than that in control. After infection with TMV, it was shown that antioxidant
activity in the transgenic M1 lines was at the same level as control or even exceeded it in the
case of TM1.2 line. In all infected transgenic plants of the P5 petunia clone, it was observed a
significant increase of AOA compared to control. The largest difference with control was in lines
TP5.2, TP5.3 and exceeded up to 50–53 %. Moreover, previously the line TP5.3 was determined
as the most resistant to viral damage.
The development of stress reactions in infected transgenic petunia plants with the ZRNase
II gene was studied. As a result, it was found that transgenic lines TP5.2, TP5.3, both with TM1.2
line were much more resistant to the biotic stress caused by TMV infection than control wild-
type lines.

Keywords: Petunia hybrida, ZRNase II gene, Tobacco mosaic virus, antioxidant activity.

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