Azadirachtin, a Novel Biopesticide from Cell Cultures of Azadirachta indica

Callus cultures of Azadirachta indica flower petals were established on MS medium supplemented with naphthalene acetic acid (1 mg/L), kinetin (0.5 mg/L) and sucrose $(3\%\;w/v)$. Cell cultures of Azadirachta indica were established and studied the growth and production kinetics. Half 85 medium supplemented with dicamba (2 mg/L), kinetin (1 mg/L) and sucrose $(3\%\;w/v)$ was found to be suitable for initiation and maintenance of cell cultures from the calli. MS medium supplemented with naphthalene acetic acid (NAA) (1 mg/L), kinetin (0.5 mg/L) and sucrose $(3\%\;w/v)$ was found to be suitable as production medium. Around $80\%\;(0.05\%\;w/v)$ of azadirachtin was found to be intracellular. The effect of various precursors, elicitors, permeabilizing agents and growth retardants in cell cultures was studied. The addition of precursors sodium acetate (10 mg/L), squalene (10 mg/L), isopentenyl pyrophosphate (1 mg/L) and geranyl pyrophosphate (1 mg/L) to the cell cultures on day 3 has shown significant increase in bioproduction of azadirachtin $(64.94{\pm}4.40\;mg/L,\;72.81{\pm}0.04\;mg/L,\;51.63{\pm}1.26\;mg/L\;and\;30.70{\pm}0.28\;mg/L\;respectively)$ over the control cultures $(4.70{\pm}0.27 mg/L)$. $5\%$ v/v cell extracts of Fusarium solani has shown moderate increase in the content of azadirachtin $(5.71{\pm}0.34\;mg/L)$ when compared to control cultures $(2.40{\pm}0.56\;mg/L)$. The addition of methyl jasmonate $(500\;{\mu}M/L)$ on day 3 has shown $\~4$ fold improvement in bioproduction of azadirachtln $(6.92{\pm}0.11\;mg/L)$ when compared to control cultures $(1.63{\pm}0.02\;mg/L)$. There was no significant effect of the studied growth retardants and permeabilizing agents on bioproduction of azadirachtin. Cells are cultivated in large volumes using the effective precursors.