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BREAKTHROUGH TECHNOLOGIES

An Inducible Targeted Tagging System for Localized Saturation Mutagenesis in Arabidopsis^1,[w]

Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604 (B.N.); and Section of Plant Biology and Department of Agronomy, University of California, Davis, California 95616 (B.N., T.T., V.S.)

We describe a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis (Arabidopsis thaliana). In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock-inducible transposase fusion is utilized to control the levels of transposase gene expression, generating transpositions that can be subsequently stabilized without requiring crossing or segregation. We have mapped 40 single-copy lines by thermal asymmetric interlaced-PCR, which can be used as potential launch pads for heat shock mutagenesis. Using a starter line selected for detailed analysis, the efficiency of tagging over a 50-kb region in the genome was examined. Hits were obtained in the targeted genes with multiple alleles for most genes, with approximately equal numbers of hits detected in genes on either side of the T-DNA. These results establish the feasibility of our approach for localized saturation mutagenesis in Arabidopsis. This system is very efficient and much less laborious as compared to conventional crossing schemes and may be generally applicable to other plant species for which large-scale T-DNA tagging is not currently feasible.

² These authors contributed equally to the paper.

³ Present address: 202 Plant Science, Cornell University, Ithaca, NY 14850.

^[w] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.104.050633.

^* Corresponding author; e-mail sundar{at}ucdavis.edu; fax 530–752–5410.

Received July 29, 2004; returned for revision October 23, 2004; accepted October 23, 2004.

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