Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture

Biotechnol Prog. May-Jun 2004;20(3):890-6. doi: 10.1021/bp034306w.

Abstract

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Agrobacterium tumefaciens / classification
  • Agrobacterium tumefaciens / genetics*
  • Agrobacterium tumefaciens / isolation & purification
  • Agrobacterium tumefaciens / metabolism
  • Cells, Cultured
  • Coculture Techniques / methods
  • Feasibility Studies
  • Gene Transfer Techniques*
  • Genes, Reporter / genetics
  • Genetic Engineering / methods*
  • Genetic Vectors / genetics
  • Glucuronidase / biosynthesis
  • Glucuronidase / genetics
  • Plants, Genetically Modified / genetics*
  • Plants, Genetically Modified / metabolism
  • Plants, Genetically Modified / microbiology*
  • Recombinant Proteins / biosynthesis
  • Species Specificity
  • Tobacco / genetics*
  • Tobacco / growth & development
  • Tobacco / metabolism
  • Tobacco / microbiology*

Substances

  • Recombinant Proteins
  • Glucuronidase