Optimization of Shoot Regeneration and Agrobacterium-mediated Transformation in Strawberry (Fragaria ×ananassa)

Efficient methods of plant transformation and tissue culture are essential for the genetic engineering and advanced molecular breeding of plants, but neither is well established in cultivated octoploid strawberry ( Fragaria × ananassa ). In the present study, a method for shoot regeneration was established and optimized for two genetically different strawberry cultivars, 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 in the optimized condition, whereas ‘FB’ showed the best response to a lower concentration of N6 -benzyladenine (BA) (0.01 mg/L) in the same media type. The average conversion frequencies of somatic embryos into shoot regenerations from the runner tips (RTs) were 42.8% in ‘FL127’ and 56.9% in ‘FB’, respectively. Using these optimized tissue culture conditions, Agrobacterium -mediated CRISPR/Cas9 gene editing was conducted to examine the efficiency of transformation and target gene editing for the phytoene desaturase , FaPDS , in the cultivar, FL127. A total of 234 explants inoculated with Agrobacterium harboring Cas9-FaPDS resulted in an 80.3% callus induction efficiency, with 13.3% of regenerated plants exhibiting partial or complete albino phenotypes. Amplicon sequencing of edited progeny showed mutations (substitutions, insertions, and deletions) at the guide RNA (gRNA) target sites or flanking regions of all FaPDS homoeologous copies. Our results provide effective tissue culture and transformation methods for strawberry functional genomics research and gene editing guided cultivar improvement.