Effect of heat shock treatment on Hordeum vulgare protoplast transformation mediated by polyethylene glycol

The stable transformation of barley protoplasts has been observed in cv. Di ssa by PEG-mediated pGL2 plasmid DNA uptake containing hygromycin phosphotr ansferase (hph) gene under the control of 35S transcript of CaMV. Hygromyci n resistant colonies were obtained, when protoplasts were incubated with hy gromycin B (25 mug cm(-3)) at 3 - 4 cell stage. Heat shock treatment, prior to polyethylene glycol (PEG) treatment, markedly increased the protoplast transformation frequency. The presence of active hph gene product, and inte gration of hph gene was confirmed by hygromycin assay and Southern blotting , respectively. Analysis indicated that rearrangement had possibly occurred during the integration of plasmid DNA into genomic DNA of barley callus. T hese transformed calli showed no morphogenic response onto the regeneration medium.