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We previously described the identification of a chromosomal deletion in  Arabidopsis thaliana  that resulted in the elimination of genomic DNA between two T-DNA insertions located ca. 25 kilobases apart on chromosome IV. The mechanism responsible for this deletion appears to have been recombination between the closely spaced T-DNA elements located in trans in a parent plant. In our original study, we observed one such deletion event after screening ca. 2,000 seedlings using a polymerase chain reaction (PCR) assay. Because a method for precisely deleting a selected region of the  Arabidopsis  genome would have significant value as a research tool, we were interested in determining the frequency with which this type of T-DNA-directed deletion occurs. To do this we designed a genetic screen that would allow us to phenotypically screen for deletions caused by recombination between T-DNA inserts. This screen involved crossing T-DNA single-mutant lines in order to produce F1 plants in which the two T-DNA insertions flanked a  FUSCA  ( FUS ) locus present in the genome. Loss-of-function mutations of  FUS  genes cause a distinctive developmental phenotype that can be easily scored visually in young seedlings. We used T-DNA lines flanking  FUS2 ,  FUS6 ,  FUS7 , and  FUS11  for this study. Recombination between the T-DNAs in an F1 parent should result in deletion of the  FUS  gene located between the T-DNAs. Because the deletion would be heterozygous in the F2 generation, we screened the F3 progeny of pooled F2 individuals to search for the  fus  loss-of-function phenotype. Using this strategy we were able to evaluate a total of 28,314 meioses for evidence of deletions caused by recombination between the T-DNA inserts. No seedlings displaying the  fus  phenotype were recovered, suggesting that deletions caused by recombination between T-DNA inserts are relatively rare events and may not be a useful tools for genome engineering in  Arabidopsis .

Recombination between T-DNA insertions to cause chromosomal deletions in Arabidopsis is a rare phenomenon