Regeneration and Agrobacterium-mediated transformation of three economically important strawberry cultivars Kurdistan, Camarosa and Paros

Abstract

Genetic transformation studies were carried out to standardize a protocol for Agrobacterium-mediated genetic transformation of three economically important strawberry (Fragaria x ananassa Duch) cultivars Kurdistan’, Camarosa’, and ‘Paros’. Shoot regeneration frequency 72, 65 and 30% was obtained on MS (1) basal medium supplemented with 2% glucose and 4 mg/l TDZ for Camarosa, Kurdistan and Paros, respectively. To optimize the concentration of kanamycin for these cultivars, we observed the responses of leaf explants to different concentration of kanamycin (0-100 mg/l) in the media. Our results showed that 75 mg/l was an effective concentration of kanamycin. For genetic transformation, Agrobacterium tumefaciens strain C58C1RifR (pGV2260) harbouring the binary vector pTJK136 containing uidA gene with intron along with kanamycin resistance gene (npt-II) was used. After five days pre-incubation and 72 h co-cultivation, transformed cells (explants) were able to grow on the selective regeneration medium containing 75 mg/l kanamycin and 500 mg/l cefotaxime, while, control explants failed to grow. The regenerated putative transgenic shoots were analyzed by histochemical GUS assay and PCR analysis. Transformation efficiency based on inoculated explants number was 2, 1 and 3% for ‘, Camarosa’, ‘Paros ’, and ‘Kurdistan’, respectively. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of these cultivars with important agronomic genes.