Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection

Plant Cell Rep. 2003 Jun;21(10):1010-19. doi: 10.1007/s00299-003-0617-6. Epub 2003 Apr 12.

Abstract

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

MeSH terms

  • Biolistics
  • Blotting, Southern
  • Gene Expression
  • Glycine / analogs & derivatives*
  • Glycine / pharmacology*
  • Glyphosate
  • Herbicides / pharmacology
  • Plants, Genetically Modified
  • Regeneration
  • Rhizobium / genetics*
  • Selection, Genetic
  • Transformation, Genetic*
  • Transgenes / genetics
  • Triticum / drug effects*
  • Triticum / genetics*

Substances

  • Herbicides
  • Glycine