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.
|Titolo:||Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis|
|Parole Chiave:||Arabidopsis thaliana ; Functional genomics ; Insertional mutagenesis ; T-DNA|
|Settore Scientifico Disciplinare:||Settore BIO/18 - Genetica|
|Data di pubblicazione:||2000|
|Digital Object Identifier (DOI):||10.1007/s101420000007|
|Appare nelle tipologie:||01 - Articolo su periodico|