PubMed · 1995 · 14 citations · 0 references
Life SciencesBiotechnologyGeneticsGenetic EngineeringMolecular BiotechnologyRecombinant Dna TechnologyAgrobacterium-mediated TransformationCell Culture TechnologyHorizontal Gene TransferMolecular Cloning WorkflowsPlant TransformationPlasmid BiologyAgrobacterium TumefaciensAgrobacterium-mediated Transformation ConstructsPlant Transformation EfficiencyPlasmid Vector ConstructionRecovery TimeStorageTransformation Efficiency OptimizationTransformation Protocol Optimization
A method for successful introduction of Ti plasmid vector ( > 10 kb) into Agrobacterium tumefaciens is described. Competent A.tumefaciens cells were prepared with 50 mmol/L CaC1(2) at room temperature and introduction was done on ice followed by a 28 degrees C heat pulse. The efficiency of transformation was 10(-4)-10(-5) transformants per total recipient population or 10(6) transformants per microgram DNA. The effects of growth phase of A. tumefaciens cells, concentration of CaC1(2) solution, temperature, liquid N2, heat pulse, recovery time, and storage time of competent cells at 4 degrees C or -20 degrees C (in 15% glycerin) on transformation were studied.