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 <span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">-mediated genetic transformation of three economically important </span></span>strawberry (<em><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;"><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;">Fragaria </span></span></em><span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">x </span></span><em><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;"><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;">ananassa </span></span></em><span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">Duch) cultivars Kurdistan', Camarosa', and ‘Paros'. </span></span>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, <em><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;"><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;">Agrobacterium tumefaciens </span></span></em><span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">strain C58C1RifR (pGV2260) </span></span>harbouring the binary vector pTJK136 containing <em><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;"><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;">uidA </span></span></em><span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">gene with intron along with </span></span>kanamycin resistance gene (<em><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;"><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;">npt-II</span></span></em><span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">) was used. After five days pre-incubation and 72 h </span></span>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 <em><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;"><span style="font-family: TimesNewRomanPS-ItalicMT; font-size: small;">Agrobacterium</span></span></em><span style="font-family: TimesNewRomanPSMT; font-size: small;"><span style="font-family: TimesNewRomanPSMT; font-size: small;">-mediated </span></span>genetic transformation of these cultivars with important agronomic genes.