A competitive PCR-based method to detect a single copy of T-DNA insertion in transformants

Abstract

Gene function studies benefit from the availability of mutants. In plants, Agrobacterium-mediated genetic transformation is widely used to create mutants. These mutants, also called transformants, contain one or several transfer-DNA (T-DNA) copies in the host genome. Quantifying the copy number of T-DNA in transformants is beneficial to assess the number of mutated genes. Here, we developed a competitive polymerase chain reaction (PCR)-based method to detect a single copy of a T-DNA insertion in transformants. The competitor line BHK^- -1 that contains a single copy of competitor BHK^- (BHK, Basta, Hygromycin, Kanamycin-resistant genes) was crossed with test transformants and the genomic DNA of F₁ plants was subjected to competitive PCR. By analyzing the gray ratio between two PCR products, we were able to determine whether or not the test transformants contained a single copy of T-DNA insertion. We also generated the control lines BHK^±1:1 and BHK^±2:1 , which contain the target (BHK⁺ ) and competitor (BHK^- ) in a ratio of 1:1 and 2:1, respectively. The ratios of their PCR products are useful references for quantitative analysis. Overall, this method is reliable and simple in experimental manipulations and can be used as a substitute for Southern-blot analysis to identify a single copy of T-DNA insertion in transformants.