Production of phenolic diterpenes in tissue culture of Rosmarinus officinalis L

Rosemary (Rosmarinus officinalis L.) is an herb native to the Mediterranean that has been used to prevent the oxidation of fats and oils in foods and cosmetics. The leaves of rosemary are a powerful source of natural antioxidants; the potent antioxidant properties of its extracts are mainly due to the phenolic diterpenes, carnosol and carnosic acid. At present, field-grown plants are the sole source of carnosic acid and carnosol. Rosemary is relatively easy to grow in many parts of world; however the variation of seasonal stresses such as temperature, solar irradiation, and rainfall can significantly alter the level and quality of phenolic diterpenes. An alternative method with more consistent results to obtain phenolic diterpenes from rosemary is a biotechnological approach, specifically, plant tissue culture. In this research, different culture types (callus cultures, suspension cultures, and shoots from in vitro propagated plants) of rosemary were established, and the effects of temperature, light intensity, and UV radiation for enhancing the synthesis of carnosic acid, carnosol, and related phenolic diterpenes in these cultures were evaluated. MS medium supplemented with either 10 mg/L indole-3-acetic acid (IAA), 4 mg/L benzylaminopurine (BAP) and 1 mg/L 6-furfurylaminopurine (kinetin) or 0.05 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg/L BAP at 18 and 25°C with 67.5 to 81.0 mumol m-2s-1 proved to be the best medium and conditions for developing and sustaining rosemary callus cultures.;Shoots from in vitro regenerated plants of rosemary accumulated various amounts of carnosic acid and carnosol, which were also present in callus culture, but the yields of these compounds were considerably lower than in regenerated shoots or in dried field grown leaves. Only carnosol and rosmanol, not carnosic acid, were detected in cell aggregates in suspension cultures, whereas spent medium contained only rosmanol. Exposure of the callus cultures to several stress conditions by increasing temperatures and light intensities, and UV radiation treatments enhanced the level of the phenolic diterpenes, carnosic acid and carnosol.