Growth and rosmarinic acid accumulation in callus, cell suspension, and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 muM) and indole-3-acetic acid (IAA) (0 or 3 muM). For root culture, hairy roots were cultured in B5 medium containing 2.7 muM alpha-naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 muM TDZ and 3 muM IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/100 mg dry weight) was obtained with 4% (w/v) sucrose. Incorporation of 10 mg l(-1) phenylalanine in the medium increased RA yield in the roots to 4.68 mg/100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/100 mg dry weight, respectively.