Generation and characterization of Arabidopsis T-DNA insertion mutants.
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.
State Key Laboratory of Protein and Plant Gene Research, Center for Life Sciences, College of Life Sciences, Peking University, Beijing, People's Republic of China.
Li-Jia
LJ
Qu
Genji
G
Qin
D016960:Agrobacterium tumefaciens / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D018274:Electroporation; D005818:Genetic Engineering; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional / Q000379:methods; D030821:Plants, Genetically Modified / Q000235:genetics; D016133:Polymerase Chain Reaction; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA