A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots.
There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.
Firek
S
S
Department of Botany, University of Leicester, UK.
Ozcan
S
S
Warner
S A
SA
Draper
J
J
D016960:Agrobacterium tumefaciens / Q000235:genetics; D001483:Base Sequence; D005786:Gene Expression Regulation; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006651:Histocytochemistry; D019868:Kanamycin Kinase; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D009024:Morphogenesis; D010770:Phosphotransferases / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012641:Selection, Genetic; D014170:Transformation, Genetic
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C053376:pathogenesis-related proteins, plant; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D005966:Glucuronidase