Random Insertional Mutagenesis in Arabidopsis

Abstract

Arabidopsis thaliana is now recognized as a model organism for research in plant biology, especially genetics and molecular biology, and is used for the functional analyses of various genes. However, accumulation of genomic and expressed sequence tag (EST) DNA sequence data (Lin et al., 1999; Mayer et al.,1999; Hofte et al.,1993; Newman et al.,1994) revealed a large number of Arabidopsis genes remaining functionally uncharacterized. After determination of the Arabidopsis whole genome sequence, we will obtain all the Arabidopsis genes, but many of the genes will be only annotated as ‘hypothetical’ or ‘putative’. This means that a reverse-genetical approach will remain important to characterize gene functions. One of the best strategies for reverse genetics is based on insertional mutagenesis. The most effective method for insertional mutagenesis is targeted gene disruption. In budding yeast (Saccharomyces cerevisiae),this technique is commonly used because of the high frequency of homologous recombination, and the systematic approach to the discovery of gene function is progressing (Oliver, 1996). However, in other organisms including higher plants, this method is still laborious and time consuming, although a few Arabidopsis genes have been successfully disrupted by this technique (Miao and Lam, 1995; Kempin et al., 1997). As an alternative method, the random insertional mutagenesis approach using a transposon or T-DNA [an element of Ti (tumor inducing)-plasmid of Agrobacterium] as a mutagen offers a viable method for obtaining insertion mutants for genes of interest. The inserted foreign DNA not only introduces a mutation but also ’tags’ the responsible gene. Since this strategy depends on random insertion in the genome, a huge number of transposon or T-DNA insertions has to be prepared for saturated mutagenesis. After construction of this huge insertion library, an effective screening method for isolation of genes of interest has to be developed. These two steps are critical for random insertional mutagenesis.