title: Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.
abstract: An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers. Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin. A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks. The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis. Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture. The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR.
authors: Graduate School of Agriculture, Hokkaido University, 060-8589 Sapporo, Japan. masakoa@affrc.go.jp
authors: M
authors: M
authors: Akutsu
authors: T
authors: T
authors: Ishizaki
authors: H
authors: H
authors: Sato
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D031402:Alstroemeria / Q000235:genetics*; D017077:Culture Media, Conditioned; D046508:Culture Techniques; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics; D019868:Kanamycin Kinase / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D012038:Regeneration; D014170:Transformation, Genetic
chemical_list: D017077:Culture Media, Conditioned; D019868:Kanamycin Kinase; D005966:Glucuronidase