Integration of multiple copies of a foreign sequence into the Ti plasmid of Agrobacterium tumefaciens

Eric R. Ward, Wayne M. Barnes

Research output: Contribution to journalArticlepeer-review


A method for constructing Ti plasmids bearing multiple copies of a sequence integrated in tandem is described. A small plasmid that confers tetracycline resistance (TcR), contains homology to a Ti plasmid, and is unable to replicate in Agrobacterium tumefaciens, was mobilized from Escherichia coli to A. tumefaciens. Ti plasmids of exconjugants selected for resistance to 12-14 μg Tc/ml all contained multiple tandem repeats of the integrative plasmid. Tc-sensitive variants with fewer integrated copies arose spontaneously at low frequency in the absence of Tc selection, or could be enriched for by selection on Tc in combination with the bactericidal antibiotic augmentin. Variants having an increased number of integrated copies were obtained by growth on high Tc concentrations. Tandem repeats integrated between border sequences provide, in principle, a way to reproducibly introduce many linked copies of any foreign gene into plants.

Original languageEnglish
Pages (from-to)305-314
Number of pages10
Issue number2
StatePublished - Feb 20 1989


  • Copy number
  • T-DNA
  • gene amplification
  • plant genetics
  • recombination
  • tandem repeat


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