Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv 'Bobwhite' were bombarded with plasmid pAHG11 containing the rice chitinase gene chill and the hm gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T-0) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal acid self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T-0 and T-1 transgenic plants. Mendelian segregation of herbicide resistance was observed in some T-1 progenies. Interestingly, a majority of the T-1 progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PET gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promotor is selectively inactivated in T-1 transgenic wheat plants.