The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation.
The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.
Thole
Vera
V
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
Worland
Barbara
B
Snape
John W
JW
Vain
Philippe
P
D004269:DNA, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors; D018745:Genome, Plant; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics* / Q000382:microbiology; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics* / Q000382:microbiology; D014170:Transformation, Genetic; D019076:Transgenes
D004269:DNA, Bacterial; C036483:T-DNA