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The current application of genome editing to crop plants is limited to cultivars that are amenable to  in vitro  culture and regeneration. Here, we report an  in planta  genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target  TaGASR7  were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T 1  plants identified plants homozygous for the three homeologous genes, which were all derived from one T 0  plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve  in planta  genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.

Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat