GROWTH MEDIUM EFFECTS ON VITRIFICATION, TOTAL PHENOLICS, CHLOROPHYLL, AND WATER CONTENT OF IN VITRO PROPAGATED OREGANO CLONES

A tissue culture system developed for the in vitro micro propagation of oregano clones (Origanum vulgare) involved axillary bud induction from shoot apex explants on Murashige and Skoog (MS) media supplemented with 1 mg/L 6-benzylaminopurine (BAP) and incubated at 24°C, 16 h light cycle with a light intensity of 40 μMol·M^-2.-1. Greater than 90% occurrence of vitrification on both MS-BAP and one-half MS hormone-free (HF) media led to the investigation of the effect of media factors on vitrification, plantlet morphology, total phenolics, chlorophyll, and water content. Media factors analyzed by addition to a ½MS medium included various levels of ions (Ca⁺⁺0-10 mM; K⁺0–100 mM; Na⁺0–20 mM), gelling agent (Phytagel®0.3%–1.2%), sucrose (0–6%), and yeast extract (0–200 mg/l). Vitrified plantlets had elongated stems with light green, translucent, and brittle leaves and stems while unvitrified plantlets had shorter internodes and opaque, dark green, flexible stems and leaves. Ion supplemented ½MS media was not found to have an effect on vitrification, chlorophyll, phenolics, and water content. Yeast extract decreased the occurrence of vitrification but increased the rate of necrosis and inhibited growth rate. The greatest decrease in the occurrence of vitrification was seen with elevated levels of gelling agent. No change in phenolic content was found in tissues over the range of gelling agent levels, but a stimulation occurred with yeast extract. Elevated phenolic levels correlated with increased sucrose, but no effect was seen on vitrification. Unvitrified tissues had the highest level of chlorophyll. Slightly higher water content was observed in vitrified tissues of controls compared with unvitrified tissues produced by elevated Phytagel® levels or yeast extract addition.