Efficient Agrobacterium tumefaciens-mediated transformation of sweet potato (Ipomoea batatas (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method

To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for beta-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l(-1) of Km for 6 wk and then transfer to 30 mg l(-1) of Hyg for 6-16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 muM 4-fluorophenoxyacetic acid and 250 mg l(-1) cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 muM abscisic acid and 2.89 muM gibberellic acid under a 16 h photoperiod of 30 mumol m(-2) s(-1). The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf discs 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.