Transformation Of Embryogenic Suspension Cultures And Efficient Regeneration Of Transgenic Plants In Sweetpotato (Ipomoea Batatas (L.) Lam.)

An Agrobacterium tumefaciens-mediated transformation system of sweetpotato, Ipomoea batatas (L.) Lam., was established by using embryogenic suspension cultures of the cultivar Lizixiang. A.tumefaciens strain EHA101 harboring a binary vector pROA93 with p-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes was used in the present study. Embryogenic suspension cultures of 3 d after subculture were cocultivated with EHA101 (OD600nm=0.5) for 4 days. After cocultivation, the infected suspension cultures were first cultured for 1 week in MS medium with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/L carbencillin but without kanamycin and then transferred into MS medium with 2 mg/L 2,4-D, 50 mg/L kanamycin and 300 mg/L carbencillin for the selection culture. 2~4 weeks after selection, 2453 kanamycin-resistant cell aggregates about 1 mm in size from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/L 2,4-D, 50 mg/L kanamycin and 100 mg/L carbencillin. and after 8 ~ 20 weeks of selection formed 46 kanamycin-resistant embryogenic calluses. After transfer to MS medium supplemented with 1 mg/L abscisic acid (ABA), 50 mg/L kanamycin and 100 mg/L carbencillin, these embryogenic calluses formed 138 plantlets via somatic embryogenesis. PCR and PCR-Southern blot analysis indicated that 104 regenerated plants (75.4%) were transgenic plants.Using such gene transfer system, the transfer of OCI gene to sweetpotato cv. Lizixiang was also conducted. A.tumefaciens strain LBA4404 harboring a binary vector pBinh with OCI and neomycin phosphotransferase (NPT II) genes was used in the study. Embryogenic suspension cultures of 3 d after subculture were cocultivated with LBA4404 (OD600nm=0.5) for 4 days. After cocultivation, the infected suspension cultures were first cultured for 1 week in MS medium with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/L carbencillin but without kanamycin and then transferred into MS medium with 2 mg/L 2,4-D, 50 mg/L kanamycin and 300 mg/L carbencillin for the selection culture. 2-4 weeks after selection, 1847 kanamycin-resistant cell aggregates about 1 mm in size from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/L 2,4-D, 50 mg/L kanamycin and 100 mg/L carbencillin. and after 8-20 weeks of selection formed 19 kanamycin-resistant embryogenic calluses. After transfer to MS medium supplemented with 1 mg/L abscisic acid (ABA), 50 mg/L kanamycin and 100 mg/L carbencillin, these embryogenic calluses formed 31 plantlets via somatic embryogenesis. PCR and PCR-Southern blot analysis showed that 16 regenerated plants (51.6%) were transgenic plants. SDS-PEG analysis of OCI from transgenic plants with OCI negative control indicated that accumulation was found in transgenic plants with OCI gene.These transgenic plants with gusA or OCI gene were transferred to greenhouse. No obvious morphological variations were observed in most of transgenic plants. However, some variations in vein base color and stem color were observed in part of transgenic plants with gusA or OCI gene. Also, one transgenic plaint with gusA gene showed variation in plant type. It needs further study if these morhphological variations are due to the transformation or the somatic mutations.