SOMATIC EMBRYOGENESIS IN CLONAL NEEM, AZADIRACHTA INDICA A. JUSS. AND ANALYSIS FOR IN VITRO AZADIRACHTIN PRODUCTION

An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens × P. grandidentada and P. tremuloides × P. davidiana). Callus was induced from in vitro leaf explants on modified Murashige and Skoog medium (MSA) and woody plant medium (WPM) containing four different combinations of cytokinins and auxins. Callus tissues regenerated into shoots on WPM medium supplemented with 2.0 mg l-1 (9.12 (mu)M) zeatin or 0.01 mg l-1 (0.045 (mu)M) thidiazuron. P. canescens × P. grandidentata exhibited the higher callus and shoot production. In vitro leaf explants from the two hybrid clones were cocultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uidA gene encoding for (beta)glucuronidase (GUS) and the nptII gene encoding for neomycin phosphotransferase II. Transformation was confirmed by GUS assays, polymerase chain reaction, and Southern blot analyses. Agrobacterium concentration, acetosyringone, and pH of the cocultivation medium were evaluated for enhancing transformation efficiency with the clone P. canescens × P. grandidentata.