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This project evaluates the potential of neem (Azadirachta indica) tissue cultures to produce azadirachtin and other bioactive compounds for the control of insect pests. Two hairy root lines of the neem tree were screened for azadirachtin production. Azadirachtin production peaked at 6 weeks of culture, as measured by SFC with the L line being more productive than the S line(0.0036% and 0.0027% of dry weight respectively). The L line was selected for more detailed analysis. Studies of the effect of the culture medium on biomass and azadirachtin production revealed that sucrose was the critical factor on both biomass and azadirachtin production while the level of inorganic supplements (as low as half the amounts that are normally used) of the nutrient media didn't have any effect. Azadirachtin production from the hairy roots was confirmed by on-line HPLC-MS analysis and NMR proton spectra. Extracts from 6 week old hairy roots were HPLC fractionated by collecting fractions at 5 min intervals (from 7 to 32 min after injection with chromatographic run of 35 min) ranging from polar to non polar compounds and tested against the desert locust (Schistocerca gregaria) in choice feeding bioassays. Fractions (F) 2, 3 and 4 were highly antifeedant (62.25%, 56.1% and 23.9% at a concentration of 0.1 mg dry weight/ml respectively) whilst F1 and F5 were not significantly antifeedant at the same concentration. Interestingly F3 did not contain any of the well known neem chemicals while F2 contained azadirachtin and F4 nimbin and salannin. Scale-up of neem hairy root cultures has been successfully achieved in 2 l flasks. Azadirachtin peak production occurred after 3 weeks (as measured by HPLC) of culture with yield of 0.0045 mg/g of dry weight. Current research indicates that hairy root extracts are efficient in controlling important such European insect pests as Spodoptera littoralis and Myzus persicae in laboratory and greenhouse trials.
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