The production of the insect antifeedant azadirachtin and related metabolites by plant cell and tissue culture of neem (Azadirachta indica)
The production of azadirachtin and other related limonoids were investigated using plant cell culture systems of Neem (Azadirachta indica). The production of antifeedant compounds in callus lines was monitored by no-choice bioassays with Schistocerca gregaria with instar nymphs. Growth of two callus lines produced from leaves of a Ghanaian neem tree were monitored and the growth patterns displayed the typical lag, stationary and exponential phases. Growth and product formation, as measured by antifeedant bioassays with S.gregaria, showed non-growth associated product formation. Azadirachtin was isolated from callus derived from a Ghanaian neem tree by Prof. E.D. Morgan, University Keele using the standard procedure of solvent partitioning and column chromatography. Biological activity of the partitioning fractions was measured with antifeedant tests. Azadirachtin was identified by chromatography on three independent systems (Supercritical Fluid Chromatography (SFC), High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography). The yield of azadirachtin was 0.0007% based on dry weight of callus. Neem leaf explants collected from wild neem trees could be successfully surface sterilised by treating with 0.1% HgCl2 for 5 min followed by 10% Bleech (Care Products, Sri Lanka) or NaOCl (Sigma, UK) for 10 min. New callus lines were initiated from leaves collected from wild neem trees by incubating on Maintenance Medium (Kearney et al., 1994) at 25°C in the dark. Fifteen callus lines thus initiated were extracted with ethanol under reflux, quantified and screened for the production of azadirachtin, nimbin and salannin by reversed phase HPLC. There was no correlation between azadirachtin, nimbin and salannin yields of callus lines and those of seeds collected from same neem trees. The genetic variation of callus lines was examined by iso-enzyme electrophoresis using three enzymes, malate dehydrogenase, alcohol dehydrogenase and diaphorase and no variation was observed among the callus lines examined.