Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.
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.
Akutsu
M
M
Graduate School of Agriculture, Hokkaido University, 060-8589 Sapporo, Japan. masakoa@affrc.go.jp
Ishizaki
T
T
Sato
H
H
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
D017077:Culture Media, Conditioned; D019868:Kanamycin Kinase; D005966:Glucuronidase