Development of an efficient transposon tagging system in Arabidopsis thaliana

We are currently developing a transposon tagging system in Arabidopsis thaliana using the maize transposable elements Ac and Ds. In order to make the system as efficient as possible, four different antibiotic resistance markers have been tested for their usefulness in monitoring excision and reinsertion of transposons in the Arabidopsis genome. Owing to the low transposition frequency of wild-type Ac in Arabidopsis we have also tested a number of modifications to the Ac element. Deletion of the CpG-rich region in the transposase 5' untranslated leader was found to significantly increase the activity of Ac in Arabidopsis. In our first non-targeted tagging experiment, 200 individuals with an inherited transposed Ac element have been collected and their progeny are being screened in families of twelve for segregation of mutant phenotypes. A two-element system, using Ac and Ds, is also being developed and a way of stabilising the Ds-induced mutations by counter selecting plants carrying the transposase source is being investigated. The Agrobacterium T-DNA insertions carrying the different transposons are currently being mapped onto the Arabidopsis RFLP map. Inverse polymerase chain reaction (IPCR) is being used to generate flanking DNA probes, that will be used on the RFLP blots to map the T-DNA relative to the other known markers. Once the first few have been mapped the first targeted tagging experiment will be initiated.