Doubled haploidy as a tool for chimaera dissolution of talen-induced mutations in barley

Abstract

Site-specific genome engineering is a breakthrough technology that facilitates the functional validation of genes and offers versatile novel opportunities of crop improvement. In the approach described in this chapter, the two units of transcription activator-like effector nucleases (TALENs) required for site-directed cleavage activity are separately used to produce homozygous transgenic lines, each carrying only one unit of the TALEN pair. Crossings of these plants result in pairwise combination of both TALEN units, which causes their activation during early zygotic embryogenesis in the hybrid caryopses and facilitates site-directed mutagenesis with unprecedented efficiency in a Triticeae species. As revealed by sequencing of target-specific PCR amplicons, the individual primary mutants are not only heterozygous for the target gene but also chimaeric typically harbouring multiple mutant alleles. We demonstrate the highly efficient production of homozygous mutant lines from the chimaeric primary mutants using haploid microspores as embryogenic founder cells for the generation of doubled haploid plants.