Direct organogenesis from leaf and internode explants of in vitro raised wintergreen plant (Gaultheria fragrantissima)

Protocol for rapid regeneration of the important medicinal plant Gaultheria fragrantissima Wall. (Ericaceae) through direct organogenesis from leaf, internode (stem without axillary bud), and root explants of in vitro raised seedlings has been developed. Different cytokinins (thidiazuron, 6-benzyladenine, kinetin, and 2-isopentenyl adenine) at a range of various concentrations in modified rhododendron medium have been investigated for organogenesis. Among the four cytokinin investigated, only thidiazuron (TDZ) was effective for direct shoot regeneration from leaf and internode explants but root explants failed to respond. Percentage of response in shoot bud induction increases as the concentration of TDZ increases. After 6 weeks culture on TDZ supplemented medium the shoots were elongated on medium devoid of TDZ. Although the percentage of response was larger at higher concentration of TDZ, 1-2 mg/l TDZ was the optimum concentration for obtaining normal, healthy shoots. The shoots developed from shoot bud induced at higher concentration of TDZ were stunted and hyperhydric. Explant culture on induction medium containing TDZ for more than 6 weeks has a detrimental effect on shoot development. Supplement of 2iP (5, 8 mg/l) or Kin (4, 6 mg/l) on the development medium enhanced the number of shoot and shoot length. Half strength modified rhododendron medium supplemented with 2iP (5 mg/l) was the ideal shoot development medium, producing 12-16 shoots from single explant within 8 weeks. Significant carry over effect of TDZ on root inhibition of microshoots was observed. Two month old rooted plantlets were transferred from lab to land and successfully established.