Molecular, chemical and biological stability of plants derived from artificial seeds of Centella asiatica (L.) Urban-An industrially important medicinal herb

Synthetic seed technology offers a viable option for large scale multiplication and in vitro conservation of endangered and elite plant genotypes with economy of space, time, and operational cost. In the present study an efficient method for the preparation, short-term conservation, and plantlet regeneration from synthetic seeds of Centella asiatica (L.) Urban, is described for the first time. Axillary buds/nodal segments excised from multiple shoot cultures were encapsulated in 4% (w/v) sodium alginate beads following complexation in 75 mM calcium chloride solution. The seeds were kept on moist filter paper in sealed petri plates and stored at 25 ± 3 °C temperature for 200 days. The seeds thus conserved recorded more than 85% germination and plantlet conversion frequency upon shifting onto a hormone-free Murashige and Skoog medium. The rooted plantlets recorded 85-90% establishment in soil and did not show any morphological variation when compared with mother plants. The genetic fidelity of the regenerated plants was further confirmed using random amplified polymorphic DNA (RAPD) analysis wherein 19 decamer primers produced a total of 60 distinct monomorphic bands. The HPLC analysis of leaf extracts of synthetic seed-derived plant progeny and the mother plant showed qualitative and quantitative uniformity in terms of four major bioactive centellosides namely, asiaticoside, madecassoside, asiatic acid, and madecassic acid. The leaf extracts of the regenerated plants depicted the same magnitude of wound healing activity in excision wound model of mice as that of the mother plant. The relevance of the developed synseed technology for short-term in vitro conservation, micro-cloning and exchange of selected germplasm of C. asiatica without affecting the chemical and biological activities is discussed.