One-step Agrobacterium mediated transformation of eight genes essential for rhizobium symbiotic signaling using the novel binary vector system pHUGE

PLoS One. 2012;7(10):e47885. doi: 10.1371/journal.pone.0047885. Epub 2012 Oct 24.


Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics*
  • Cloning, Molecular / methods
  • DNA, Bacterial / genetics
  • Genetic Vectors / genetics*
  • Medicago truncatula / genetics
  • Medicago truncatula / microbiology
  • Plants / genetics*
  • Plants / microbiology
  • Plants, Genetically Modified / genetics*
  • Plants, Genetically Modified / microbiology
  • Rhizobium / physiology
  • Symbiosis
  • Transformation, Genetic*
  • Transgenes


  • DNA, Bacterial
  • T-DNA

Grant support

The financial support of the Netherlands Organisation for Scientific Research (NWO) (NWO-ALW 816.02.007 and NWO-VIDI 864.06.007) is gratefully acknowledged. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.