A transposon tagging strategy with Ac on plant cell level in heterologous plant species

The maize transposable element Ac can have a ‘late’ excision time during leaf development in certain transgenic tobacco plants. This was visualized with an assay based on Ac-excision restoring GUS-expression. Leaves of the described plants contain over 10 3 small blue spots, each of these spots representing an independent excision event. Leaves showing this ‘late’ excision phenomenon may be used for transposon tagging experiments at plant cell level. Plants which display ‘late’ Ac-excision do not detectably express GUS during the preceding callus phase, thus allowing transformants to be preselected for a ‘late’ timing of excision. To examine the applicability of this phenomenon a phenotypic selection assay for excision of Ac was used. Transformed calli containing Ac within the hygromycin resistance gene were regenerated and protoplasts isolated from leaves of regenerated plants were selected on hygromycin. Up to 0.8% of these protoplasts displayed hygromycin resistance. The hygromycin resistant derivatives analyzed were shown to represent independent transposition events. Ac-insertions which can be generated in this way may be used for transposon tagging experiments at cell level.