Determination of changes in mtr-miR159 and mtr-miR393 gene levels of CaO NPs' and graphene oxide (GO) appli̇ed to callus of some local alfalfa (Medicago sativa L.) ecotypes exposed to drought stress under in vi̇tro conditions

Drought stress affects fertile soils in the World, It causes important changes in the physiological, biochemical, and molecular mechanisms of plants and these changes adversely affect agricultural areas. Nanotechnology offers new opportunities to solve the negative effects of environmental factors. In this study, two different alfalfa ecotypes subjected to drought stress were tested in response to physiological, biochemical, and gene level changes applying CaO NPs and GO under tissue culture conditions. Firstly, seeds of different local ecotypes collected from Erzurum and Konya regions were regenerated in MS medium, and the alfalfa leaves formed as a result of regeneration ensured callus formation in 2,4-D and Kinetin medium. These callus samples were kept for 1 month in media containing different concentrations of mannitol (50 and 100 mM), CaO NPs and GO (0,5 and 1,5 ppm). It was observed that the dry/wet weight decreased as the mannitol concentration increased in the callus tissues and increased in CaO NPs and GO treatment. Proline, DNSA, MDA, and H2O2 increased in direct proportion to drought stress, while CaO NPs, and GO decreased. According to the results of physiological and biochemical analyses, the most optimum medium conditions for Erzurum were 50 mM mannitol/2 CaO NPs/0,5 ppm GO, while the optimum medium conditions were 50 mM mannitol/0,5 ppm GO for Konya. The changes in mtr-miR159 and mtr-miR393 gene levels were up-regulated as drought stress increased and down-regulated in CaO NPs and GO treatments. Ca2+ accumulation in alfalfa tissue was confirmed using SEM and CLSM. In conclusion, CaO NPs and GO treatment significantly reduced the negative effects of drought stress on alfalfa callus under tissue culture conditions.