Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers.
Novel 'super-binary' vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for beta-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47% GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co-transformation was relatively low.
Komari
T
T
Plant Breeding and Genetics Research Laboratory, Japan Tobacco Inc, Shizuoka, Japan.
Hiei
Y
Y
Saito
Y
Y
Murai
N
N
Kumashiro
T
T
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D012275:Oryza / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D010947:Plants, Toxic; D010957:Plasmids / Q000235:genetics; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; C036483:T-DNA; D005966:Glucuronidase