Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F. v. semperflorens

Abstract

By identifying antibiotics that eliminated Agrobacterium tumefaciens with the least phytotoxic effects, we were able to select appropriate A. tumefaciens strains for a more efficient transformation of seasonal Fragaria vesca and everbearing F. v. semperflorens. An efficient and reproducible method of shoot regeneration from leaf discs was developed with optimal shoot regeneration obtained on medium supplemented with 0.25 mg l^–1 indole-3-butyric acid and 3 mg l^–1 benzyladenine for both F. vesca forms. Petiole sections were also effective for F. v. semperflorens regeneration, and culture vessel capacity was found to influence shooting from both explant types and F. vesca forms. Of a range of antibiotics commonly used for Agrobacterium elimination, carbenicillin had the least inhibitory effect on shoot regeneration and was found to effectively control strain LBA4404. Both F. vesca forms showed a high sensitivity to kanamycin and, therefore, a selection regime ramped from 10 mg l^–1 to 25 mg l^–1 kanamycin over a period of 8 weeks was developed. Optimal transformation efficiency (15%) was achieved by the appropriate use of explant type and age, leaf-disc orientation, Agrobacterium density and inoculation time and phenolic compounds for bacterial virulence induction. Southern analysis confirmed the integration of the β-glucuronidase reporter gene into the genomic DNA of both F. vesca L. forms.