TRANSPOSITION AND BEHAVIOR OF THE MAIZE TRANSPOSABLE ELEMENT AC IN TRANSGENIC HAPLOID DATURA-INNOXIA MILL

To develop a transposon tagging system for haploid plants, the maize A c element was introduced into haploid protoplasts of Datura innoxia vi a PEG-mediated direct gene transfer. Different plasmids were used harb ouring this element in the untranslated leader region of the NPTII gen e, which allows the phenotypic assay for excision of the Ac element. K anamycin resistant clones (600) were regenerated, 40 randomly selected and further analysed. Biochemical NPTII assay demonstrated the presen ce of the NPTII gene product in 39 of them. In contrast, Southern blot analysis and PCR experiments revealed that about 25% of the plants ex pressing the NPTII gene showed transposon excision from the untranslat ed NPTII leader region. These findings may be due to events which occu r during illegitimate recombination, the mode of integration of exogen ous DNA in direct gene transfer. The continued somatic excision and re integration of the transposable element was also demonstrated. Sequenc e analysis of excision loci in the donor DNA reveals that the Ac eleme nt is able to excise, leaving behind an empty donor site comparable to that in maize. The number of integrated constructs ranged from two to more than ten copies. If multiple copies were present in plants with an empty donor site, somatic transposition as well as the existence of non-transposed elements could be detected within one individual plant . Jumping of the transposable element seems to be independent from the number of integrated copies of Ac. These results show that the maize transposable element Ac is able to transpose in the heterologous host D. innoxia and are essential for the development of a transposon taggi ng system in haploids of this species.