Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium composition and substrate feeding

Oleandrin has been identified as the most potent antitumor ingredient of the Mediterranean herb Nerium oleander L. A strategy for optimization of medium compositions and conditions was developed for enhanced oleandrin production in suspension cultures from leaf-origin explants of Nerium oleander. The cell suspension cultures were grown in various modifications of MS medium as a basal medium. The effects of different natural extracts, plant growth substances, carbon and nitrogen sources and phosphate on the growth and oleandrin accumulation were investigated as well as effect of light, pH, shaking speed and substrate feeding. The highest oleandrin yield was obtained when the nitrogen concentration was lowered to two-thirds and the phosphate concentration increased by two-thirds of that specified in the MS medium in the presence of 3% sucrose, coconut milk, indolebutyric acid and benzyladenine in concentrations of 1 and 2mg l-1, respectively. Lower pH and faster shaking speed favored oleandrin accumulation. Chemical feeding of progesterone and cholesterol boosted the oleandrin concentration to higher levels reaching 8.230.05mg g-1 dry weight. This was about 10-fold higher than that detected in field-grown plants using the same extraction and analytic conditions, and about 24-fold higher than that determined in control cultures with regular MS medium and without precursor feeding.