Improving the Production of Indole Alkaloids from Madagascar Periwinkle (Catharanthus roseus L.) Using Stressful Tissue Cultures

The research work was conducted in the National Commission for Biotechnology, Damascus, during the period 2016 – 2021, in order to micro propagate periwinkle (Catharanthus roseus L.), and determine the optimum culture medium of such species, and to induce callus tissues from both leaves and stems of periwinkle in vitro using MS medium and its evaluation chemically and morphologically, in addition to study the somaclonal variation using ISSR technique. The study also included the assessment of the effect of both osmotic and NaCl-induced salinity stresses on the production of some important terpene indole alkaloids (vincristine and vinblastine) from the callus and the most important biochemical and physiological mechanism associated with tolerance. Experiments were laid according to complete randomized design with four replicates. Results revealed that the culture medium MS11 (2 mg.L-1 BA, 1 mg.L-1 NAA) was superior over the other culture media, where the number of branches and the number of leaves per plant were significantly higher. The growth regulators combination of the culture medium MS8 (2 mg.L-1 Kin, 1 mg.L-1 NAA) was found to be the most suitable for callus formation from the periwinkle plant, which gave the highest callus formation ratio from both leaves and stems. The two investigated abiotic stresses (osmotic and salinity) caused significant reduction in both the fresh and dry weight of callus, meanwhile they increased the percentage of solutes leakage, and the concentration of both malondialdehyde and proline compared with the control treatment. The activity of anti-oxidant enzymes (SOD, catalase and peroxidase) increased in response to abiotic stresses, but the enzymatic activity was significantly higher under salinity stress compared with osmotic stress. The linear phase of callus was superior in the fresh weight of callus over other phases and the concentration of Vincristine was significantly higher at the two ages 35 and 42 days, while the concentration of Vinblastine was significantly higher at the ages of 28, 35 and 42 days. The concentration of both Vincristine and Vinblastine was significantly higher in the NaCl-salinity level of 75 mM, but it was found that the addition of PEG-6000 caused a remarkable decline in these two effective compounds. The results of molecular study confirmed the effectiveness of the investigated primers (21 primer) in giving polymorphism among the studies lines. Cluster analysis revealed the separation of the in vitro grown plant from the callus which is produced from it.