Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis

Funct Integr Genomics. 2000 May;1(1):25-34. doi: 10.1007/s101420000007.

Abstract

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening.

Publication types

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

MeSH terms

  • Agrobacterium tumefaciens / genetics
  • Aminobutyrates / pharmacology
  • Arabidopsis / genetics*
  • Chromosome Mapping
  • DNA, Bacterial / genetics*
  • DNA, Single-Stranded / genetics
  • Gene Rearrangement
  • Genetic Linkage
  • Genetic Vectors
  • Genome, Plant
  • Herbicides / pharmacology
  • Mutagenesis, Insertional / methods*
  • Plants, Genetically Modified / drug effects
  • Plants, Genetically Modified / genetics
  • Seeds / genetics
  • Streptomyces / genetics
  • Transformation, Genetic

Substances

  • Aminobutyrates
  • DNA, Bacterial
  • DNA, Single-Stranded
  • Herbicides
  • T-DNA
  • phosphinothricin