Plant & Animal Genome IX Conference

Workshop: Barley
W219

TRANSPOSABLE ELEMENTS AS EFFICIENT VEHICLES FOR TAGGING AND GENE DELIVERY IN BARLEY

Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102

To devise a method for function-based gene isolation and improved gene delivery, a set of plasmids were constructed for stable introduction into barley. One plasmid contained the maize activator (Ac) transposase gene (AcTPase) and a negative selection gene, codA; the other plasmid contained Dissociation (Ds) inverted-repeat ends surrounding the herbicide resistance gene, bar. Use of a transient assay with Ds-bar-interrupted gus (McElroy et al. , 1997) indicated that efficient excision of Ds occurs in barley. Crosses were made between stably transformed plants either containing Ds-Ubi-bar or plants expressing functional AcTPase under the transcriptional control of either the putative AcTPase promoter or the maize ubiquitin promoter (Ubi1) and first intron. In F1 plants, low somatic and germinal transposition frequencies were observed; however, in F2 progeny, from individual selfed F1 plants, up to 70% of plants showed evidence of Ds transposition. Further analyses of F3 plants revealed two mutant phenotypes in which the transposed Ds elements co-segregated with the new phenotype. The sequence surrounding one of the elements has high homology with an EST from maize (Koprek et al., 2000), showing that this system can be used to tag genes in barley. In addition to its utility for tagging genes, the system also provides an opportunity to segregate the gene of interest from plasmid and selection gene sequences, as well as providing an opportunity to generate large numbers of independent insertions from only two original transformants. The system also has a positive effect on transgene expression stability, a significant problem in barley transformation.