Oxidative status in Medicago truncatula Gaertn. non-embryogenic and embryogenic tissues with particular reference to somatic embryogenesis

Abiotic stress conditions (e.g., wounding, sterilization) are often together with plant growth regulators (e.g., 2,4-d), considered as one of the most important factors initiating plant somatic embryogenesis (SE). The first goal of this work was to answer whether leaf explants of the Medicago truncatula non-embryogenic (M9) line and its embryogenic variant (M9-10a) respond equally to mechanical and chemical stress by analyzing ROS (reactive oxygen species, e.g., O2•−, H2O2) localization, ROS scavenging enzyme activity and expression of genes encoding these enzymes. In explants of both lines, the stress response induced by wounding and chemical sterilization and the defense reaction during the 1st week of callus growth was similar. These defense mechanisms first involve an increase in SOD and CAT activity, later APX. 2,4-d, present at a low concentration (0.5 µM) during the induction phase (IP), is necessary for embryogenic callus formation and, consequently, for embryo development. This herbicide in higher concentrations causes an increase in O2•− accumulation and in antioxidant enzyme activity; however, it does not block the formation of callus and somatic embryos, though it disturbs these processes. Moreover, inhibition or blocking studied processes by DPI, an inhibitor of NADPH oxidase responsible for the production of O2•− and also lowering the expression of genes encoding the antioxidant enzymes leading to change in their activities, clearly indicate that a certain level of ROS is necessary to induce SE.