New strategies for the development of haploid crop plants via genome elimination

Abstract

The generation of haploids is one of the most powerful means to accelerate the plant breeding process. In most crop species, an efficient haploid technology is not yet available or only applicable to a limited set of genotypes. Based on recent results published for Arabidopsis thaliana, manipulating the centromeres of the chromosomes has been proposed as universal novel method for the production of haploid plants. By this way, haploids can be generated through manual cross-fertilizations after manipulating a single centromere protein, the centromere-specific histone H3 variant CENH3, in one of the parents designated as ‘haploid inducer’. Crosses with haploid inducer genotypes result in karyotypically unstable embryo cells, which have lost one of the parent-specific chromosome sets. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, functional CENH3 genes of several Daucus species and ginseng (Panax ginseng) were cloned and cytogenetically analyzed. Since our aim was to knockout and complement the endogenous carrot CENH3, a co-transformation of a CRISPR/Cas9-based carrot CENH3 knockout construct together with the ginseng CENH3 gene was performed by using a wild type Agrobacterium rhizogenes strain. Molecular analyses of regenerated hairy roots and carrot plants have shown that CRISPR/Cas9-based modifications within the carrot CENH3 gene have been achieved in some transgenic lines, and that the over-expressed ginseng CENH3 gene is functionally active. Additionally, ‘one-step’ approaches based on targeted induction of mutations within the endogenous CENH3 gene through CRISPR/Cas9 are tested for their use to develop carrot haploid inducer genotypes.