Establishing a high-efficiency <i>in vitro</i> regeneration system for <i>Agrobacterium</i>-mediated transformation in <i>Fragaria nilgerrensis</i>

Abstract

<italic>Fragaria nilgerrensis</italic> Schlechtendal ex J. Gay, a diploid wild strawberry, has many excellent characteristics such as fruit with white color and peach fragrance, and strong disease resistance. However, the lack of an efficient and stable regeneration and genetic transformation system for <italic>F. nilgerrensis</italic> has largely limited the functional studies of related genes governing excellent traits. In this study, a regeneration system for <italic>F. nilgerrensis</italic> leaf discs was developed by optimizing factors such as different hormone combinations, dark culture times and casein hydrolysate (CH) concentrations, with an average regeneration rate of 97.3% at 45 d of culture. By paraffin section observation of callus with different colors induced from the leaf disc regeneration, light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Based on this, an average transformation percentage of 8.67% was achieved by screening kanamycin concentration and referring to transformation procedures described by predecessors. PCR-positive transformants were obtained within 4-5 months by confirmation of PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. The establishment of efficient regeneration system provided a feasible platform for genetic transformation, which laid the foundation for further gene functional studies in <italic>F. nilgerrensis</italic>.