Generation and Characterization of Arabidopsis T-DNA Insertion Mutants

Abstract

Transfer DNA (T-DNA) insertion mutants are often used in forward and reverse genetics to reveal the molecular mechanisms of a particular biological process in plants. To generate T-DNA insertion mutants, T-DNA must be inserted randomly in the genome through transformation mediated by Agrobacterium tumefaciens. During generation of a T-DNA insertion mutant, Agrobacterium competent cells are first prepared and plasmids containing the T-DNA introduced into Agrobacterium cells. Agrobacterium containing T-DNA vectors are then used to transform T-DNA into Arabidopsis. After screening and identifying T-DNA insertion mutants with interesting phenotypes, genomic DNA is extracted from the mutants and used to isolate the T-DNA flanking sequences. To finally determine the mutated genes causing the specific phenotype in the T-DNA insertion mutants, cosegregation analysis and complementation or recapitulation analysis are needed. In this chapter, we describe detailed protocols for generation and characterization of T-DNA insertion mutants.