ABSTRACT
Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement. We demonstrate use of CRISPR/Cas methodology in diploid Fragaria vesca ssp. vesca ‘Hawaii 4’ and octoploid F. x ananassa ‘Calypso’ by targeting the visible endogenous marker gene PDS (phytoene desaturase). Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of either the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% (‘Hawaii 4’) and 50% (‘Calypso’) putative transgenic shoot lines exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 55% (‘Calypso’) and 75% (‘Hawaii 4’) included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in ‘Calypso’ and clearly demonstrate that CRISPR/Cas9 can be used to edit single copy genes at high frequency within the genome of the diploid and the same target in octoploid strawberry.