Accumulation of volatile constituents in agar and bioreactor shoot cultures of Verbena officinalis L.

In vitro shoot cultures of common vervain (Verbena officinalis L., Verbenaceae) were established and evaluated for essential oil content. The shoots were maintained as stationary microshoot culture, grown on Schenk–Hildebrandt medium supplemented with 9.84 µM of 2-isopentenyladenine and 1.0 µM of thidiazuron. The shoots were further adapted to grow in two temporary immersion bioreactors (RITA and Plantform), using Schenk–Hildebrandt medium supplemented with 4.92 µM indole-3-butyric acid. The best growth, in terms of dry weight production, was recorded for agar culture (0.73 g l−1 day−1), followed by 35 days (day) Plantform (0.53 g l−1 day−1), 35 days RITA (0.42 g l−1 day−1) and 56 days Plantform culture (0.29 g l−1 day−1). The volatile fractions were obtained by simultaneous hydrodistillation-extraction of the plant materials using Likens–Nickerson apparatus. Aerial parts of V. officinalis intact plant and 56 d Plantform culture had the highest essential oil content (8.1 g kg−1) whereas the stationary agar culture excelled in essential oil productivity (5.0 mg l−1 day−1). The volatile oils isolated from in vitro shoots contained 1-octen-3-ol, benzyl alcohol, phenethyl alcohol, coumaran and hexadecenoic acid as major constituents. 1-Octen-3-ol was the predominant (> 50%) constituent of 35 day shoot cultures maintained in temporary immersion bioreactors whereas coumaran (19%) and hexadecanoid acid (21%) prevailed in stationary microshoot culture. The volatile fraction obtained from aerial parts of V. officinalis intact plant was composed mainly of hexadecanoic acid (> 20%), linalool (> 8%), anethole (> 5%) and carvone (> 3%). Microshoot cultures of Verbena officinalis, grown in temporary immersion bioreactors, were investigated in terms of essential oil content and composition of volatile fraction