Abstract: The emergence and implementation of Cas endonuclease technology has undoubtedly taken plant research and biotechnology to a higher level. After the development of transformation vectors harbouring cas9 and gRNA expression units compatible for monocotyledonous plants, transient expression tests relying on biolistic DNA transfer into epidermal cells or PEG-mediated transformation of protoplasts were established to prevalidate candidate target gene-specific constructs to be used for site-directed mutagenesis in stable transgenic plants. Agronomically relevant traits like row-type (VRS1) and malting quality (LOX1) were considered among the first of our approaches taking advantages of this new technology. Transgenic barley plants of the model cultivar ‘Golden Promise’ ectopically expressing target gene-specific endonucleases were generated, and by simultaneously targeting different positions within the target region, a wide variety of mutations was obtained. Beside indels in the target motifs, also deletions between pairs of guide-RNA target sites were detected. The typically multiple mutant alleles present in primary transgenics were efficiently resolved and fixed in doubled haploids generated via embryogenic pollen cultures. In the LOX1 approach, knockout experiments were extended to elite malting barley background. Doubled haploid lox1 mutant plants were subsequently used for micro malting experiments and showed differences in some malting parameters. Finally, it was demonstrated that 2-rowed barley can be readily converted to 6-rowed barley by the targeted deletion of just a single nucleotide of the VRS1 gene.
Bio: Goetz Hensel was trained as a molecular biologist recently becoming head of the Centre of Plant Genome Engineering at Heinrich-Heine-University Dusseldorf. Previously he was working as senior scientist in the Plant Reproductive Biology group of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). During his scientific career he was crucially involved in the improvement of methods of Agrobacterium-mediated gene transfer to cereals (barley and wheat) and in a new principle of RNA-mediated downregulation of genes called host-induced gene silencing (HIGS). Recently he focused on designer endonuclease-mediated gene targeting (CRISPR/Cas), molecular mechanisms underlying the initiation of pollen embryogenesis, the telomere-mediated truncation of chromosomes as well as genes involved in the spike architecture and domestication of barley.