FAIR-CT96-5014 Product formation in tissue cultures of neem (Azadirachta indica) for the control of insect pests

Contacts




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FAIR Area 2.2 - Bioprocessing : FAIR Marie Curie Research Training Grants : Integrated Crop Protection & Biological Control

Objectives:

This project evaluated the potential of neem tissue cultures to produce azadirachtin and other bioactive compounds on a year round basis for the control of insect pests.

Activities and results:

Characterisation and growth of, as well as product formation in scaled up cultures of neem hairy roots has been successfully achieved. It was found that using 21 flasks, the exponential growth phase lasted three weeks with a doubling time for biomass of 0.95 weeks. This is faster than for the cultures grown in 250 ml flasks where the doubling time was 1.02 weeks. Azadirachtin production peaked at the end of the exponential growth of the cultures which occurred after three weeks of culture. The yield was around 0.0043 mg per g of dry weight (mg/gDW). This yield of azadirachtin per unit biomass was lower than in 250 ml flasks but if the rate of growth is taken into consideration in terms of the increased biomass and the decreased culture time in the 21. flasks then the resultant yield is higher in the scaled up culture system than the 250 ml flasks system (0.0095 mg/gDW/week and 0.007 mg/gDW/week respectively).

The development of improved high performance liquid chromatographic (HPLC) methods (water and acetonitrile as system solvents gradient) allowed good separation of the different compounds throughout the chromatographic run except for azadirachtin and 3- tigloylazadirachtol which co-eluted. To overcome this problem isocratic HPLC conditions were applied with water (57%) and acetonitrile as eluents enabling efficient quantification of both azadirachtin and 3-tigloylazadirachtol.

Six week old hairy root cultures (grown in 250 ml flasks) were fractionated using HPLC with fractions ranging from polar (F 1) to non polar compounds (F5). The individual fractions were tested for their anti-feeding effect on the desert locust. High anti-feeding levels were observed in fractions (F) 2. 3 and 4 whilst F I and F5 did not have a significant effect. Interestingly F3 did not contain any of the well known neem chemicals while F2 contained azadirachtin and 3- tigloylazadiractol and F4 nimbin and salannin. The effects of supplying exogenous auxins in the culture medium as well as changing the C/N concentration ratio on culture growth and azadirachtin production were also investigated. Hairy root extracts were also tested for their potential to control the peach potato aphid on oilseed rape plants in greenhouse experiments.

Keywords: azadirachtin, antifeedant

Contacts

Scientific Supervisor

• Anne MORDUE / University of Aberdeen Department of Zoology / UNITED KINGDOM