Hygromycin-resistant calli generated by activation and excision of maize Ac/Ds transposable elements in diploid and hexaploid wheat cultured cell lines

Abstract

To investigate the activation and transposition of maize transposable elements in wheat cultured cells, plasmid DNAs containing the maize Ac/Ds elements located between the CaMV 35S promoter and a hygromycin B resistance gene (hph) were introduced into two wheat (Triticum aestivum and Triticum monococcum) cultured cell lines by microprojectile bombardment. In the first experiment, hph was activated by excision of the Ac element, which encodes transposase, in the two wheat cell lines. In the second experiment, the Ds element was excised by a stabilized Ac element, lacking inverted repeats of the Ac element and located on another plasmid, and therefore leading to activation of hph. After selection of bombarded cells by hygromycin B, many resistant calli were recovered in both wheat cell lines. The integration of hph and the Ac transposase gene was confirmed by PCR and genomic Southern analysis. The stable expression of hph and the transposase gene was also assessed by Northern blot and reverse transcriptase PCR analysis, respectively. Moreover, characteristic sequence alterations were found at Ac/Ds excision sites. These findings indicate that the maize Ac/Ds transposable elements are activated and excised by expression of the Ac transposase gene in both diploid and hexaploid wheat cells.