| Summary: | The octoploid-cultivated strawberry variety Benihope (<i>Fragaria</i> × <i>ananassa</i> Duch cv. Benihope) is an important commercial plant. It is highly susceptible to different diseases, which ultimately leads to a reduction in yield. Gene-editing methods, such as CRISPR/Cas9, demonstrate potential for improving disease resistance in the strawberry cv. Benihope. Establishing a plant regeneration system suitable for CRISPR/Cas9 gene editing is crucial for obtaining transgenic plants on a large scale. This research established a callus induction and plant regeneration system for <i>Agrobacterium</i>-mediated CRISPR/Cas9 gene editing in strawberry cv. Benihope by evaluating multiple types of explants and various plant growth regulators throughout the entire tissue culture process. The results showed that the efficiency of callus induction is strongly influenced by the type of explant and is highly sensitive to the combination of plant growth regulators. Among the different plant growth regulators employed, thidiazuron (TDZ), in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), effectively induced callus formation and plant regeneration from explants derived from nutrient tissues such as runner tips and crowns. In addition, the regeneration experiment demonstrated that the addition of polyvinylpyrrolidone (PVPP) to the shoot regeneration medium could inhibit tissue browning. The gene-edited plants in which some or all of the <i>Fvb7-1</i>, <i>Fvb7-2</i>, <i>Fvb7-3</i>, and <i>Fvb7-4</i> genes in the MLO (<i>Mildew resistance Locus O</i>) gene family were knocked out by CRISPR/Cas9 system were obtained by applying the plant regeneration system developed in this study.
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