Anti-oxidant enzyme responses during in vitro embryogenesis in Catharanthus roseus

The present study was carried out to analyse the activities of several anti-oxidant enzymes at various stages of somatic embryogenesis in Catharanthus roseus. The hypothesis was that anti-oxidant enzymes accumulated as part of a cellular defence mechanism in response to stress. We therefore measured superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities in various in vitro-grown tissues such as embryogenic and non-embryogenic calli, and in embryos at various stages. SOD activity increased gradually from the early embryogenic stage to heart-shaped stage embryos, but declined in the later stages (i.e., torpedo-shaped and cotyledonary embryos). In contrast, APX activity was high in non-embryogenic callus and decreased rapidly during the stage of embryo initiation. This pattern was the same for CAT. The maximum CAT activity was observed in non-embryogenic callus, then it declined almost linearly at the embryonic and post-embryonic developmental stages. The effect of exogenous hydrogen peroxide (H2O2) on in vitro embryogenesis was also evaluated. Lower H2O2 levels (0.025 mM) promoted embryo formation, whereas higher levels (0.10 mM) inhibited embryogenesis in C. roseus. Finally, higher soluble protein, free amino acid, and proline contents were found in embryogenic calli compared to non-embryogenic calli.