The herbaceous species Catharanthus roseus (L.) G. Don commonly known as Madagascar Periwinkle, belongs to the family Apocynaceae that native to tropical and subtropical region such as southern india, Indonesia and Madagascar but it is distributed worldwide now. This plant accumulates more than 130 terpenoid indole alkaloids (TIAs), of which two dimeric alkaloids Vinblastine and Vincristine have antineoplastic activity and useful for the treatment of various cancers. Although these drugs are demanded highly, the amount of these alkaloids are very low in the intact plant which is still the only source for them. Complexity and multiple step of biosynthetic pathway of these two alkaloids and existence of multiple chiral centers, spesific production of some biosynthetic pathway procedures of them in specialized tissue, less effectiveness of semi-synthetic medicine in comparsion with natural ones, and high demand for these two medicines are the most important reasons which draw researcher’s attention to use biotechnological methods and plant tissue culture in order to increase the production of these important alkaloids in plant. In this research to indicate the best media for micropropagation and increase in amount of anti-cancer alkaloids Vinblastine and Vincristine in Periwinkle plant, 25 plant hormones combination to produce callus from leaf explant, 7 treatments for regeneration of calluses and 30 media supplemented with various concentration of plant hormones and activated charcoal for direct regeneration of node explant are considered. Then the content of Vinblastine and Vincristine alkaloids in regenerated plants are extracted by Hollow fiber-based liquid-phase microextraction (HF-LPME) method and analyzed by ion-pair liquid chromatography. Results showed that indirectly regeneration of leaf explant is not a suitable method to micropropagation of Periwinkle plant due to hard regeneration of callus, probability of mutation and lower number of produced branches compared with direct regeneration. But using BAP in culture media containing 1 g/l activated charcoal, showed the best direct regeneration of node explants and shoot proliferation. Also increasing of BAP concentration and decreasing of activated charcoal, the amont of Vinblastine and Vincristine increased. Therefore, although due to exist of many phenolic compounds in Periwinkle plant, using of activated charcoal in culture media is necessary for plant growth, its use in media is important because it has negative effect on adsorption of hormones and resulted in reducing the shoot proliferation and alkaloids production, so seems that concentration of 1 g/l activated charcoal is a appropriate amount to make shoot proliferation media.
