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

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. Key words : transposable elements, excision site, transgenic wheat callus, particle bombardment, Ac/Ds.

Unstable transmission and frequent rearrangement of two closely linked transposed Ac elements in transgenic tomato

We are examining the behavior of the maize transposable element Ac in transgenic tomato with the goal of developing an efficient insertional mutagenesis system. Among the self progeny of a transgenic tomato plant containing an active Ac element, we identified six plants that contained the same germinally transposed Ac. In one of these plants, we found a second Ac element inserted in the same orientation and approximately 2 kb to the 5′ side of the original Ac insertion. Transmission of this composite structure was significantly reduced with less than one-quarter of the self progeny inheriting Ac either in the form of the intact parental allele (two neighboring Ac's) or derivatives of it. The derivative alleles that arose were complex in structure and could not be explained solely on the basis of the excision of one or the other Ac element. These results illustrate the potential of transposable elements to cause genetic instabilities and complex chromosomal rearrangements.Key words: Ac element, rearrangements, tomato, transposable element, transposon tagging.