Full Length Research Paper

Callus and azadirachtin related limonoids production through in vitro culture of neem (Azadirachta indica A. Juss)

Muhammad Rafiq* and Muhammad Umar Dahot

Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro, Pakistan.

*Corresponding author. E-mail: mrafiqmb@yahoo.co.uk. Tel: +92-22-9239024. Fax: +92-22-2771372.

Abbreviations: IAA, indoleacetic acid; NAA, naphthylacetic acid;  2,4–D, 2,4-dichlorophenoxyacetic acid; BAP, benzylaminopurine; DCW, callus dry weight.

Accepted 27 November, 2009

   Abstract

A protocol was established for the induction of callus and suspension cultures for azadirachtin production from neem explants. Different concentrations and combinations of plant growth regulators (2,4-D, NAA, IAA and BAP) were supplemented in MS medium. Immature flowers, nodular stem sections, leaves immature embryos and mature seeds were used as explants. The highest callus development (78%) was observed when immature flowers were inoculated on MS basal medium with addition of 1.0 mg/l 2,4-D, 1.0 mg/l BAP, 0.2 mg/l NAA and 3% sucrose. The azadirachtin containing liminoids were determined from calli obtained from different explants. Effect of sucrose, glucose, NH₄NO₃, KNO₃ and urea on cell suspension cultures and azadirachtin contents were also investigated. The dry cell weight and azadirachtin contents increased to 373.1 and 359.2 mg/ 50 ml when 0.25 and 0.5 g/l NH₄NO₃ was added in MS liquid media and supplemented with 1.0 mg/l 2,4-D, 0.2 mg/l BAP and 3% sucrose.

Key words: Neem (Azadirachta indica A. Juss), in vitro, indoleacetic acid (IAA), naphthylacetic acid (NAA), 2,4-Dichlorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP).