THE EFFECT OF CARBOHYDRATE TYPE AND ILLUMINATION MODE ON PROLINE, CAFFEIC ACID AND ROSMARINIC ACID ACCUMULATION IN TWO LINES OF ZATARIA MULTIFLORA CALLUS TISSUES.

Zataria multiflora Boiss. (Labiatae) is a valuable medicinal plant in traditional Iranian medicine. Rosmarinic acid is one of the major active components of this plant for its antioxidant properties. Production of rosmarinic acid in plant tissue culture is an attractive goal and has been the subject of intensive research for several Labiatae species. In this study callus induction from seedling shoot of Zataria multiflora was obtained on MS medium with two different hormonal compositions (2,4-D 0.5 mg.L^-1 and kinetin 1 mg.L^-1 ; BAP 1 mg.L^-1). The two callus lines (C1 and C2 respectively) were significantly different in their growth rate with a higher level of growth for C2 rather than C1, evaluated on the basis of fresh and dry weights. Callus structures were also different between the two lines. These two lines were experimented and compared for their production of rosmarinic acid. For this purpose they were cultivated with sucrose (30, 60 g.L^-1) or glucose (60, 75 and 90 g.L^-1) and the cultures were incubated under total obscurity or under a 16 h light/8 h obscurity photoperiod regime. Accumulation of proline, caffeic acid and rosmarinic acid was measured after 45 days of culture in these conditions. The level of proline was measured spectrophotometrically and HPLC analysis was used for caffeic and rosmarinic acid determination. High quantities of rosmarinic acid were found in Zataria callus tissues. The highest level (15,6% of dry weight) was found in the tissues of C1 line cultivated on 90 g.L^-1 glucose with light photoperiod. Rosmarinic acid was more abundant in the C1 line and there appeared an inverse correlation between the rate of growth and the accumulation of rosmarinic acid in Zataria tissues. Rosmarinic acid production was improved by increasing the carbohydrate dose added to the medium and the best performance was recorded with glucose in a significant manner. At the high concentrations of glucose, the increase of rosmarinic acid was accompanied by an increase of proline content. The synthesis of caffeic acid in these tissues seemed to have an inverse correlation with rosmarinic accumulation. These results indicated that the hormonal treatment for callus induction, the type and dose of carbohydrate in the culture medium and the illumination play a significant role in the accumulation of proline, caffeic acid and rosmarinic acid in Zataria callus cultures.