Role of Hydrogen Peroxide on Viability, Morphology and Antioxidant Enzyme Activity in Callus Cells of Catharanthus roseus L.

Hydrogen peroxide (H2O2) is a representative reactive oxygen species (ROS), molecules that readily damage biological molecules including DNA and proteins, and which can eventually lead to apoptotic or necrotic cell death. In this experiment, the influence of H2O2 on viability, morphology and antioxidant enzyme activity in cell suspension culture of Catharanthus roseus was investigated. For callus induction, leaves of Catharanthus roseus seedlings were incubated on Murashige and Skoog (MS) medium. Following callus culture, the liquid medium was supplemented with sucrose, vitamins such as thiamin, pyridoxine, nicotine and macro and micro elements and hormones including 2-4-D, Kin, IAA. In addition, the calluses were treated with different concentrations of H2O2 (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μm) for 1, 3 or 6 days. The viability of these cells was investigated by a method using trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Additions of 1, 5 or 10 μm H2O2 at day 6 were selected as the treatments most suitable for further research. Callus morphology was studied using coloring Hoechst and acridine orange. Furthermore, antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were measured. Results indicated that H2O2 leads to a significant decrease in viability of treated Catharanthus roseus calluses compared to a control. Activity of the antioxidant enzymes SOD, POD and CAT showed a significant increase in treated cells when compared to the control and were related to H2O2concentration. Furthermore, oxidative stresses caused generation of H2O2 and resulted in significant enhancement of antioxidant enzyme activity and intensified intracellular H2O2content as well.