Optimization of Shoot Regeneration and Application of CRISPR/Cas9 Gene Editing to Cultivated Strawberry (Fragaria×ananassa)

Abstract Efficient methods of plant transformation and tissue culture are essential to CRISPR/Cas-based gene editing of crops, but neither is well established in cultivated octoploid strawberry ( F × ananassa ). In the present study, a method for shoot regeneration was established and optimized for two strawberry cultivars commercially grown in Florida, Sweet Sensation® ‘Florida 127’ FL127) and ‘Florida Brilliance’ (FB). Runner segments at the tip, node, and petiole obtained from greenhouse-grown plants were used as explants for comparisons of shoot regeneration rate. FL127 showed the highest frequency of shoot regeneration to a basal Murashige and Skoog media (MS) containing 1 mg·L-1 of TDZ, 0.05 mg·L-1 of BA, and 0.05 mg·L-1 of 2,4-D, while FB showed the best response to a lower concentration of BA (0.01 mg·L-1) in the same media type. The average conversion frequencies of somatic embryos into shoot regenerations from the runner tips (RT) were 42.8% in FL127 and 56.9% in FB, respectively, with RT being the most prolific in shoot generation for both cultivars. Using these optimized tissue conditions, Agrobacterium -mediated CRISPR/Cas9 gene editing was conducted to evaluate the efficiency of transformation and knockout mutations in the phytoene desaturase ( FaPDS ) gene of FL127. A total of 234 explants treated with Agrobacterium resulted in an 80.3% regeneration efficiency, with 13.3% of regenerated plants exhibiting partial or complete albino phenotypes. Amplicon sequencing of edited progeny revealed substitutions, insertions, and deletions at the gRNA target sites or flanking regions of all FaPDS homoeologous copies. Our results provide effective methods of tissue culture and transformation for the efficient application of CRISPR-mediated gene editing in cultivated strawberry.