Agrobacterium rhizogenes mediated genetic transformation in Ficus religiosa L. and optimization of acetylcholinesterase inhibitory activity in hairy roots

Abstract

The use of decoctions prepared from stem bark to treat neurodegenerative problems is amongst the widespread applications of Ficus religiosa L., a tree of great ethno-medicinal importance in the Indian sub-continent. The stem bark of the tree has been reported to have significant acetylcholinesterase inhibitory activity (AChEI). Latter is one of the most effective modalities used for the treatment of Alzheimer's disease (AD). The development of any commercial treatment modality for AD from this tree faces serious challenges for sample consistency in terms of quality (being strongly influenced by environmental and developmental stages) and quantity. Hairy root (HR) cultures are an excellent choice for such studies. Since no study has been reported for HR culture systems for F. religiosa , the present study was targeted to develop one. Three different HR lines were induced and cultured on different media. Root extracts, obtained from HRs on different media, were subjected to an AChEI assay to select the best conditions where HRs exerted higher AChEI. The wild-type strain of A. rhizogenes was used to induce hairy roots in different explants. The method of genetic transformation was optimised by changing concentrations of bacterial suspension, acetosyringone and calcium chloride in the infection and co-cultivation medium. The assay for measuring AChEI activity was modified from the assays described by Ellman et al. (1961) and Ingkaninan et al. (2000) . These methods recommend the use of IC 50 (half maximal inhibitory concentration) value for comparing AChEI activities in different samples. We developed a protocol for induction and long-term maintenance of HR cultures of F. religiosa L. In vitro conditions were optimized where hairy root extracts exerted higher AChEI activity (IC 50 value of 0.16 µg/ml) compared to that in the stem segments of the mother plants (IC 50 value of 0.49 µg/ml). The present work has been granted a patent by the Indian Patent Office, Government of India (Patent No360050, Date of grant 02/03/2021) (Siwach and Gill, 2021). The present study confirms the utility of F. religiosa in AD treatment and provides a standardized protocol for large-scale production of HR cultures (75ml medium in Erlenmeyer flask of 250 ml volume) that have higher AChEI activity; these cultures offer excellent platforms for future characterization and commercial preparation.