In vitro Conservation and GC-MS-based Metabolite Profiling of Tissue Culture-raised Ehretia aquatica (Lour.) Gottschling & Hilger. at Different Growth Stages

Abstract

Ehretia aquatica (Lour.) Gottschling & Hilger is an important medicinal plant belonging to the family Boraginaceae. An in vitro culture method was developed for the rapid multiplication of E. aquatica from nodal explants, and GC-MS-based chemical profiling was used to compare tissue culture-raised plants with the mother plant. Surface-sterilised nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of BAP (0.5, 1.0, 2.5, 5.0, and 10.0 mg/l), ascorbic acid (100 mg/l), and citric acid (25 mg/l). The highest shoot multiplication response was obtained on MS medium containing BAP (5.0 mg/l), kinetin (1.0 mg/l), ascorbic acid (100 mg/l), and citric acid (25 mg/l). The induced multiple shoots were subcultured at 5-week intervals. Roots developed from isolated shoots on half-strength MS medium supplemented with IBA and NAA. The highest number of roots was obtained with IBA (1.0 mg/l) and NAA (0.1 mg/l). Samples from the mother plant, in vitro plants, and hardened plants aged 3, 6, and 9 months were analysed by GC-MS to compare their secondary-metabolite profiles. Tissue culture-raised and hardened plants showed a greater number of detected phytochemicals at different growth stages than the mother plant, indicating variation in their chemical profiles. This study reports an in vitro regeneration protocol and a comparative GC-MS profile of the mother plant, in vitro plants, and hardened plants at 3, 6, and 9 months.