Site-Directed Mutagenesis in Barley by Expression of TALE Nuclease in Embryogenic Pollen

In this chapter, a detailed protocol for the induction of targeted mutations in barley using customized transcription activator-like effector nucleases (TALENs) is provided. As explant for the introduction of TALEN expression constructs, embryogenic pollen cultures are used which consist primarily of haploid cells able to be converted via pollen embryogenesis and genome duplication into fertile doubled haploid plants. Thanks to the haploid nature of these target cells, a mutation induced by TALE nucleases in just one gfp allele can be readily detected in vitro and thus genetically fixed primary mutant plants obtained in one generation.