This paper is a review describing the now widely used method of insertional mutagenesis for obtaining a knockout mutation in each and every gene of the model plant, Arabidopsis thaliana. My laboratory published an earlier research paper (Krysan, Young, Tax and Sussman, PNAS 93:8145-8150, 1996) which laid out the basic methodology for the technique, and this review elaborated on the procedure and pointed out some of the pros and cons of the overall strategy. After the PNAS paper, we also received NSF funding to establish an international Arabidopsis knockout facility at the University of Wisconsin, and this paper is one of those we recommend people read to become familiar with the technique.

Yes, it describes an excellent and exhaustive means of performing reverse genetics in plants. Plants hate to do homologous recombination (unlike yeast and mammals) but since recent easy methods have been devised to obtain tens of thousands of random insertional mutations, this insertional mutagenesis approach is quite adequate.

The PCR method originally described in 1996 and in this review has now been complemented by using TAIL PCR from DNA isolated from individual T-DNA lines, which since the entire Arabidopsis genome has been sequenced, allows one to know definitively where the insertion has occurred in that seed. Thus, NSF is now funding several projects where tens and hundreds of thousands of such individual lines are being characterized and provided to academic scientists at no cost, just by contacting the Ohio State University Arabidopsis Seed and DNA Resource Facility. Thus, one can simply screen a database in silico to find out if a knockout mutant seed in your gene of interest exists in the public collection. However, one should be aware that small genes are very difficult to isolate in this random insertional mutagenesis procedure and, together with the large number of genes duplicated in tandem, represent a proportion of the genome (ca. 20%) from which knockout mutants will probably not be accessible using current screening procedures.

Reverse genetics is essential in understanding the function of each and every one of the 25,000 Arabidopsis genes. Our paper describes a method in which a plant can be identified in which only the one gene you are interested is deleted. In forward genetics, you first identify a phenotypically unusual plant and then you try to figure out which DNA sequence has been changed. In reverse genetics, we do the opposite, i.e., we first isolate a plant containing a mutant gene, and then we ask, what is the phenotype?