Enhanced bacoside content in polyamine treated in-vitro raised Bacopa monnieri (L.) Wettst

Abstract

The study was designed to evaluate the role of various plant polyamines (PAs) on in-vitro multiplication and regeneration in the Ayurvedic memory enhancer Bacopa monnieri (L.) Wettst. Natural populations of B. monnieri were collected, sterilized and micropropagated in Murashige and Skoog (MS) media supplemented with different concentration and combination of plant growth regulators (PGRs) and PAs. MS media supplemented with 6-benzylaminopurine (BAP) (2.0 mg/l) + spermidine (spd) (1 mM) and in another set, kinetin (kin) (2.0 mg/l) + spd (1 mM) were found to be the best multiplication media whereas MS+ indole-3-butyric acid (IBA) (1.0 mg/l) + spermine (spm) (1.0 mM) produced highest average number of roots per shoot. After 4–6 weeks of incubation, nodal explants cultured in MS media supplemented with various PGRs and PAs produced green compact or white friable nodular base calli (NBC). The NBC showed organogenic potential when subjected to MS media supplemented with cytokinins and PAs. Clonal fidelity of the regenerated plants was assessed using inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) markers. Out of the 60 clear and reproducible bands recorded, 54 were found to be monomorphic displaying high degree of monomorphism (90%) and lower polymorphic level (10%) between the mother plant and in-vitro regenerants. High-performance liquid chromatography (HPLC) revealed total bacoside content in B. monnieri grown in-vitro (PA-treated) and the mother plant (MP) grown in nature to be 27.89 and 25.46 mg/g respectively. The phytochemical analysis of the PA-treated micropropagated plants also revealed significantly higher levels of phenolics and flavonoids with comprehensively higher antioxidant potentials in PA-treated in-vitro grown plants (PATP) and the subsequently acclimatized plants (PATP-A) over the PA-untreated in-vitro grown plants (with/no PGR) (PAUP) and the mother plant (MP).