Plant regeneration and Agrobacterium-mediated transformation of two elite aspen hybrid clones from in vitro leaf tissues

An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens X P. grandidentada and P. tremuloides X 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 muM) zeatin or 0.01 mg l(-1) (0.045 muM) thidiazuron. P. canescens X 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 npt II 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 X P. grandidentata.