Optimizing -Mediated Transformation and CRISPR-Cas9 Gene Editing in the Rice Variety Presidio

Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice ( L.) has highly efficient transformation protocols, along with reasonably efficient protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both -mediated and biolistic transformation of the high-yielding U.S. cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the () and () genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with led to a 33% regeneration efficiency. The targets had ambiguous phenotypes, but 60% of regenerated plants for showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of rice varieties.