PCR rescue and analysis of transgene sequences directly from crude extracts of transgenic embryos and plants

Abstract

We report the application of a PCR-based method in conjunction with automated sequencing for the reliable detection and verification of transgenes in crude extracts of leaf and callus tissue from different plant species. Transformed tissue can be identified easily at any stage of the regeneration process, whether it is via embryogenesis or organogenesis. This allows researchers to focus their attention and resources on truly transformed tissues and avoid unwittingly culturing untransformed tissues. This protocol can also be used to rescue relatively large PCR products as well as duplexing the detection of transgenes. Direct sequencing of the PCR products allows confirmation of the integrity of the transgenein planta.