Micropropagation of Ficus religiosa L. via leaf explants and comparative evaluation of acetylcholinesterase inhibitory activity in the micropropagated and conventionally grown plants

A high-frequency, season-independent, in vitro regeneration of Ficus religiosa was developed, followed by comparative acetylcholinesterase inhibitory (AChEI) activity assay of the in vitro raised and conventionally grown plants. The use of AChEI activity is the most accepted strategy for the treatment of Alzheimer disease. Fully expanded, mature leaves were cut into different segments to initiate the cultures. The middle section of the leaf in vertical orientation with cut portion inserted inside the medium was found most suitable for direct shoot regeneration. Leaf explants responded with nearly consistent frequency (60-66.67 %) throughout the year. To obtain high frequency response with enhanced shoot multiplication rate, 32 plant growth regulator regimes were screened amongst which benzylaminopurine at 5.0 mg/l was found most suitable, yielding 100 % response and maximum number of shoots per explant (7.93); same concentration was also most supportive for repeated multiplication (6.53 shoots). The quality of the shoots and multiplication rate could be significantly enhanced (24.35 shoots) when adenine sulphate, glutamine and phloroglucinol, in an optimised concentration, were additionally supplemented. The clonal nature of the micropropagated plants was confirmed by random amplified polymorphic DNA analysis. A comparative analysis of AChEI activity was carried out amongst the methanolic extracts of stem segments of the mother plant, randomly selected seedlings of different age (4 and 6 months old) of the same mother plant and randomly selected micropropagated plants of different age (3 and 6 months age). The mother plant sample showed effective AChEI activity, with IC50 of 66.46 mu g/ml while seedlings, of different age groups, performed poorly (6-month-old seedlings, Se-1(6M), yielded IC50 of 20,538.46 mu g/ml, while two randomly selected 4 months' aged seedlings, Se-2(4M) and Se-3(4M) exhibited IC50 of 19,341.03 and 24,281.70 mu g/ml). On the other hand, various micropropagated plants, 2 of 3 months (MiP-1(3M), MiP-2(3M)) and 2 of 6 months (MiP-3(6M) and MiP-4(6M)) age behaved like the mother plant, exhibiting IC50 values of 71.87, 72.91, 67.65 and 69.65 mu g/ml, respectively.