Searching for a Knockout Line for a Gene of Interest in Arabidopsis GOAL: Identify a target plant line among 60,480 individual T-DNA transformed lines Wisconsin Madison Facili ty Screen for 1 out of 30 super pools (2,025 T-DNA lines per super pool) Screen for 1 out of 9 DNA pools (225 T-DNA lines per DNA pool) Screen for 1 out of 25 seed pools (9 T-DNA lines per seed pool) Screen for 1 out of 9 seed lines f or the target gene tagged with T-DNA (1 T-DNA line per seed line) Determine genotype of each plant to find T-DNA target line by PCR Heterozygote (a/A) Homozygote (a/a) The target gene may be INVOLVED in seed development No Yes The target gene may Collect seeds for further or may not involved analysis in the next in seed development generation (duplicate gene??) § Examine plants for mut ant phenotype § Collect seeds for duplicat e gene crosses