| Summary: | Most <i>Hieracium</i> subgenus <i>Pilosella</i> species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic <i>Hieracium</i> by targeting mutations in the endogenous <i>PHYTOENE DESATURASE</i> (<i>PDS</i>) gene using <i>Agrobacterium</i>-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of <i>PDS</i> knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify <i>PDS</i> gene edits. Up to 86% of seeds produced albino seedlings with complete <i>PDS</i> knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing <i>Cas9</i> segregation from the <i>PDS</i> target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic <i>Hieracium</i>, enabled development of the discussed strategies for future identification of causal apomixis genes.
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