Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis.
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.
Galbiati
M
M
Yale University, Department of Molecular, Cellular & Developmental Biology, New Haven, CT 06520-8104, USA.
Moreno
M A
MA
Nadzan
G
G
Zourelidou
M
M
Dellaporta
S L
SL
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000613:Aminobutyrates / Q000494:pharmacology; D017360:Arabidopsis / Q000235:genetics*; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D015321:Gene Rearrangement; D008040:Genetic Linkage; D005822:Genetic Vectors; D018745:Genome, Plant; D006540:Herbicides / Q000494:pharmacology; D016254:Mutagenesis, Insertional / Q000379:methods*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics; D012639:Seeds / Q000235:genetics; D013302:Streptomyces / Q000235:genetics; D014170:Transformation, Genetic
D000613:Aminobutyrates; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D006540:Herbicides; C036483:T-DNA; C003121:phosphinothricin