Efficient CRISPR/Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa)

Recently, an Agrobacterium -mediated CRISPR/Cas9 editing system was successfully applied in a gene function analysis, highlighting its great value for improving strawberry genetics. However, the resulting low transformation rates and long regeneration cycles have limited its extensive application. Based on the biological characteristics of crown branching, an Agrobacterium tumefaciens -mediated CRISPR/Cas9 gene editing system was developed to increase the transformation rate and decrease the regeneration time of cultivated strawberry. Two single guide (sg) RNAs were designed for the strawberry anthracnose-related transcription factor, WRKY (FxaC₁7g55530), and its alleles. These sgRNAs were inserted into pKSE401G using pCBC-DT1T2; sgRNAs for subtilisin-like protease (FxaC₂2g21540) were designed and cloned in a similar manner. After 10 days of co-cultivating plantlets (without media supply of carbon) and GV3101, 65 (61. 9%) and 72 (68. 6%) GFP-positive calluses for the two genes were respectively obtained from the crown of 105 plantlets. The positive calluses were removed from the crown and placed on Murashige and Skoog media containing 3 mg/L thidiazuron and 0. 2 mg/L indole-3-butyric acid. After 50–80 days, 3–5 positive shoots were obtained from different positive calluses for each gene. The three T0 lines for FxaC₁7g55530 and FxaC₂2g21540 were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2. Overall, a quick and effective CRISPR-Cas 9 gene editing system was developed for cultivated strawberry, highlighting the applicability of gene editing in breeding and gene function analysis. • GFP-positive callus from crowns were obtained with a positive rate of more than 60% for gene editing, after 10 days of plantlets and GV3101 co-cultivation. • After 50-80 days, 3-5 positive shoots were achieved from different positive callus based on Murashige and Skoog media containing 3 mg/L TDZ and 0. 2 mg/L IBA. • All three T0 lines of four pairs of alleles of WRKY transcription factor and subtilisin-like protease were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2, respectively.