Using CRISPR/Cas9 to produce haploid inducers of carrot through targeted mutations of centromeric histone H3 (CENH3)

Abstract

The generation of haploids is one of the most powerful means to accelerate the plant breeding process. Based on recent results published for Arabidopsis thaliana, manipulating the centromeres of the chromosomes has been proposed as a novel breeding tool for the production of haploid crop plants. By this way, haploids can be generated through cross-fertilizations after manipulating a single centromere protein, the centromere-specific histone H3 variant CENH3, in one of the parents designated as ‘haploid inducer’. In our carrot research, we are using the CRISPR/Cas9 technique in combination with an Agrobacterium rhizogenes-mediated transformation system to induce mutations in the carrot CENH3 gene. We are investigating two different approaches for CENH3-based uniparental genome elimination called as ‘1-Step’ and ‘2-Step’ strategies. In the ‘1-Step’ approach we are testing regenerated carrot plants carrying non-lethal mutations within the endogenous CENH3 gene for putative haploid inducer effects. The ‘2-Step’ strategy is based on a fully knocked-out carrot CENH3 gene which is rescued by an alien CENH3 gene isolated from a related plant species. Co-transformations of CRISPR/Cas9-based knockout constructs together with a ginseng (Panax ginseng) CENH3 gene were performed by using A. rhizogenes. We show that we could successfully target the carrot CENH3 gene and that the ginseng CENH3 protein is accumulated inside the kinetochore region. We also discuss preliminary results from crosses with putative haploid inducer genotypes developed by the ‘2-step’ approach.