Fedorchuk V. The application of inhibitors of protein kinases and phosphatases for the improvement of Agrobacterium tumefaciens-mediated plant transormation

The thesis is dedicated to the study of the effect of various inhibitors of protein kinases and protein phosphatases on Agrobacterium-mediated transformation of tobacco. The author describes the results of the evaluation of Agrobacterium-mediated transformation frequency of Nicotiana tabacum after application of a set of inhibitors concentrations. Using this approach, the most effective concentrations of each of the studied inhibitors that significantly increase the transformation frequency for each were determined. The influence of trifluoperazine, an inhibitor of serine-threonine protein kinases, on the frequency of Agrobacterium-mediated transformation of angiosperms was evaluated in a wide concentrations range from 10 to 300 µM using tobacco as a model object. It was shown that trifluoperazine at a concentration of 10 µM increased the frequency of Agrobacterium - mediated transformation of tobacco leaf discs by 25%. It should be emphasized that the present work is the first systematically study of the effect of Ca2+-calmodulin-dependent protein kinase inhibitor trifluoperazine on the frequency of Agrobacterium-mediated transformation of tobacco up to now. The effect of different concentrations of the serine-threonine protein kinases inhibitor W7 (from 25 to 100 µM) on the frequency of stable Agrobacterium - mediated transformation was investigated. It was shown that W7 at a concentration of 25 µM did not increase the transformation frequency, but significantly stimulated the growth rate of plants regenerants. The impact of tyrosine kinase inhibitor genistein in concentrations range from 10 to 100 µM on the frequency of stable Agrobacterium - mediated transformation of tobacco was studied. The effect of genistein depends on the time of co-cultivation: 10% increase of transformation was observed after 24 hours, while 48 hours of co-cultivation resulted in only 5% increase. On the next stage, we performed a study of protein phosphatases inhibitors sodium ortovanadat and cantaridin in our experimental settings. A concentrations range from 10 to 250 µM of sodium ortovanadat was tested. 200 and 250 µM of the inhibitor after 24 hours of co-cultivation provoked an increase of the stable Agrobacterium - mediated transformation frequency by 10 and 19%, respectively, while 48 hours of co-cultivation caused 30 and 40% of increase, correspondingly. Cantaridin was involved in the study at the range from 0.1 to 1 µM. 24 hours of co-cultivation with 0.1 and 0.5 µM of cantaridin induced the transformation frequency upregulation by 7 and 4%, respectively, followed by the further increase to 11 and 5%, correspondingly, after 48 hours. To confirm the transgenic origin of the regenerated plants we have carried out the polymerase chain reaction (PCR) to detect the gus reporter gene. 632 bp band which corresponds to the positive control (amplicon size using the рGH217 vector), was revealed in tested samples. The results of PCR verified the transfer, integration and expression of a reporter gene after transformation. Altogether, obtained results demonstrate that inhibitors of protein phosphorylation and dephosphorylation play an important role in Agrobacterium-mediated transformation of plants and could be used as modulators of this process both upregulating and downregulating the frequency and efficiency of transformants generation and cultivation.