Identification of Arabidopsis Knockout Lines for Genes of Interest

Determining gene function through reverse genetics has been an important experimental approach in the field of flower development. The method largely relies on the availability of knockout lines for the gene of interest. Insertional mutagenesis can be performed using either T-DNA or transposable elements, but the former has been more frequently employed in Arabidopsis. A primary concern for working with insertional mutant lines is whether the respective insertion results in a complete or rather a partial loss of gene function. The effect of the insertion largely depends on its position with respect to the structure of the gene. In order to quickly identify and obtain knockout lines for genes of interest in Arabidopsis, more than 325,000 mapped insertion lines have been catalogued on indexed libraries and made publicly available to researchers. Online accessible databases provide information regarding the site of insertion, whether a mutant line is available in a homozygous or hemizygous state, and outline technical aspects for plant identification, such as primer design tools used for genotyping. In this chapter, we describe the procedure for isolating knockout lines for genes of interest in Arabidopsis.