Highly efficient and genotype-independent barley gene-editing based on anther culture

Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops. In barley, immature embryos of Golden Promise have been mainly used as explants for transformation. However, the genotype-dependent approach limits the genetic modification in commercial varieties. Here, we develop an anther culture-based system that effectively creates transgenic and gene-edited plants from commercial barley varieties. The protocol was tested in four Australian varieties and Golden Promise with different phenology, callus induction and green plant regeneration responses. Agrobacterium-mediated transformation was performed on microspore-derived callus when targeting the HvPDS gene, and T0 albinos with targeted mutations were successfully obtained from commercial varieties. Further editing of three targets was achieved with an average mutation rate of 53% in the five varieties. In the 51 analysed T0 individuals, Cas9 induced about 69% of single-base insertion/deletions and two-base deletions in targeting sites, with variable mutation rates among targets and varieties. Both on-target and off-target activities were detected in T1 progenies. Compared with immature embryo protocols, this genotype-independent platform can deliver a high editing efficiency and more regenerants within a similar time frame. It is promising for functional genomics and application of CRISPR technologies for precise improvement in commercial varieties.