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  • Title: Generation and characterization of Arabidopsis T-DNA insertion mutants.
    Author: Qu LJ, Qin G.
    Journal: Methods Mol Biol; 2014; 1062():241-58. PubMed ID: 24057370.
    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.
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