Genetic and biochemical stability of in vitro raised and conventionally propagated Centella asiatica - A valuable medicinal herb

Abstract

An efficient micropropagation protocol was developed for C. asiatica, a commercially important herb having high medicinal and cosmetic properties. Shoot induction was observed on Murashige and Skoog (MS) medium fortified with various combinations of benzyladenine (BA) (2.22 - 22.2 µM) and naphthalene acetic acid (NAA) (1.34 - 8.06 µM). The maximum number of shoots (11.30 ± 0.68) per explant was initiated on MS media containing BA (6.66 µM) and NAA (2.69 µM). Maximum shoot proliferation was achieved on MS medium supplemented with BA (6.66 µM) and indole-3-acetic acid (IAA) (2.84 µM). The best rooting (24.69 ± 0.08) per explant was achieved on half-strength MS medium supplemented with indole-3-butyric acid (IBA) (4.92 µM). The in vitro regenerated plantlets were acclimatized and shifted to the field. Genetic stability of the micropropagated regenerants was evaluated using inter-simple sequence repeat (ISSR) markers and flow cytometry. Out of 28 primers tested, 9 primers resulted in 561 distinct and reproducible bands ranging from 200 to 1400 bp. Monomorphic banding profile was observed between the mother plant and tissue culture regenerants. The mean 2C DNA content of mother and micropropagated plants evaluated by flow cytometry were estimated to be 0.14 and 0.12 pg, thereby indicating no significant change in the 2C DNA content. Additionally, HPLC (High performance liquid chromatography) analysis did not show any significant differences in the content of triterpenes and phenolic compounds in both mother and micropropagated plants. Thus, this protocol could be implemented for large scale cultivation of elite germplasm of C. asiatica.