Laboratory protocols: CIMMYT applied genetic engineering laboratory

Abstract

This manual provides the theoretical and technical basis required for the successful establishment of maize and wheat somatic cell tissue cultures that are suitable for in vitro genetic manipulation through the use of biolistic-based and Agrobacterium-mediated gene transfer systems for maize and wheat. Achieving effective transfer integration, and expression of transgenes using appropriate gene constructs promotes understanding of the process of gene transfer and the nature of transgenic plants. In addition the manual introduces readers to the basics of plant genome analysis and its application to the analysis of both maize and wheat transgenic plants. Also covered in this manual are the isolation, digestion, electrophoresis, and transfer of DNA to blots; the non-radioactive hybridization and detection technology in use at CIMMYT, and the application of polymerase chain reaction (PCR) technology to the analysis of transgenic plants. To complete the coverage of the topic, information on phenotypic screening of transgenic plants in the biosafety greenhouses is also presented.