Genetic Transformation and Hybridization

Plant Cell Reports

, Volume 25, Issue 8, pp 815-820

Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation

  • Mette LangeAffiliated withDepartment of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg Email author 
  • , Eva VinczeAffiliated withDepartment of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg
  • , Marianne G. MøllerAffiliated withDepartment of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg
  • , Preben B. HolmAffiliated withDepartment of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg

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Abstract

We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T1 plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos.

Keywords

Barley transformation pVec8-GFP T-DNA integration Vector backbone integration