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Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the ~29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.
One of the most significant findings revealed through analysis of genomes of multicellular organisms is the large number of genes for which no function is known or can be predicted (1). An essential tool for the functional analysis of these completely sequenced genomes is the ability to create loss-of-function mutations for all of the genes. Thus far, the creation of gene-indexed loss-of-function mutations on a whole-genome scale has been reported only for the unicellular budding yeast Saccharomyces cerevisiae (2-4). Although targeted gene replacement via homologous recombination is extremely facile in yeast, its efficiency in most multicellular eukaryotes does not yet allow for the creation of a set of genome-wide gene disruptions (5, 6). Gene silencing has recently been used to study the role of ~86% of the predicted genes of the Caenorhabditis elegans genome in several developmental processes (7, 8). The RNA interference (RNAi) method has, however, several drawbacks, including the lack of stable heritability of a phenotype, variable levels of residual gene activity (9-11), and the inability to simultaneously silence several unrelated genes (12).
By comparison, the creation of genomewide collections of sequence-indexed insertion mutants has several advantages (5). We selected Agrobacterium T-DNA to generate a large collection of sequence-indexed Arabidopsis insertion mutants. About 150,000 transformed plants (T1 plants) expressing a T-DNA-located kanamycin-resistance gene (NPTII) were selected and individually propagated (13). To estimate the number of unlinked T-DNA insertions per plant line, the segregation of antibiotic resistance was scored in the progeny of 100 T1 plants. The average number of T-DNA insertions per line was found to be ~1.5 [a number similar to other T-DNA collections (14)], and therefore, the entire collection was estimated to contain...