Necessity of a two-stage process for the production of azadirachtin-related limonoids in suspension cultures of Azadirachta indica.

The effect of major nutrients on growth and azadirachtin-related limonoids (AZRL) production in plant cell culture of Azadirachta indica (neem) was studied with the objective to increase the yield of AZRL, one of the major group of pesticidal compounds found in intact neem trees. We report the novel online monitoring of plant cell respiration activities in a new parallel shake flask measuring device. Results obtained using three standard plant cell culture media showed non-growth-associated production characteristics for AZRL. These findings were supported by the oxygen uptake rate data. Further investigations on AZRL production in a modified MS medium with different concentrations of nitrogen and phosphorus sources resulted in 0.25 mg.g(-1) dry weight of AZRL, compared to no detectable AZRL production in standard MS media. These characteristics suggest the necessity of a two-stage process for the production of AZRL in plant cell culture. Compared to the single-stage process, an almost twofold increase in the volumetric productivity of AZRL was achieved using the two-stage process.