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Published on October 11, 2007; 10.1104/pp.107.108563

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Received August 31, 2007
Accepted October 1, 2007

The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation

Vera Thole , Barbara Worland , John W. Snape , and Philippe Vain *

John Innes Centre, Department of Crop Genetics, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK

* Corresponding author; email: philippe.vain{at}bbsrc.ac.uk.

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 harbour either a (i) pCLEAN-G and pSoup, (ii) pGreen and pCLEAN-S or (iii) 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 minimising 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 optimised left border sequence, a double left border sequence, restriction sites outside the borders and two independent T-DNAs. In addition, selectable and/or reporter gene(s) 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.






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