A Procedure for Agrobacterium tumefaciens Mediated Genetic Transformation of Sweet Potato

Abstract

Many sweet potato plants have been successfully transformed but the transgenic plants regenerated, however, have been limited to a few genotypes. Reported in this paper is a procedure in which several explants of three sweet potato genotypes from Papua New Guinea (PNG) were used to transform and regenerate transgenic plants. To achieve stable transformation, an efficient shoot regeneration system for different explants was developed. The shoot regeneration protocol developed enabled for a reproducible stable transformation mediated by Agrobacterium tumefaciens strain 1065. The plasmid pVDH65 contains the npt II gene for kanamycin (km) resistance, hpt gene for hygromycin resistance and Gus-intron reporter gene (GUS) for β-glucuronidase. Explants inoculated with the bacterial strain were co-cultured for 3, 5 and 7 days (d) in the dark on Murashige and Skoog (MS) medium without growth hormones. After co-cultivation, the explants were washed in liquid MS medium containing 500 mg L-1 cefotaxime, rinsed in sterile, deionised water for 10 mins and cultured on km selection medium containing 100 mg L-1 km followed by transfer of explants to 125 mg L-1 km after 14 d of culture. The km-resistant shoots selected on the former km concentration were transferred to the latter for double selection. Km-resistant shoots obtained at 125 mg L-1 were rooted on MS based medium also containing 0.008 mg L-1 IAA, 0.03 mg L-1 kinetin and 0.001 mg L-1 folic acid. This double selection method led to effective elimination of escapes (up to 75%) and successful recovery of transgenic plants from stem explants at more than 25%, leaf discs 10% and petioles 13.3% of each sweet potato cultivar. Polymerase chain reaction (PCR) analysis of the three km-resistant and GUS-positive plants revealed the presence of the expected fragment for npt II. This is the first report of successfully transforming sweet potato plants with bacterial strain 1065 and selection of transgenic plants at km concentrations higher than 100 mg L-1.