Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis |
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Authors: | Massimo Galbiati Maria A Moreno Gregory Nadzan Melina Zourelidou Stephen L Dellaporta |
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Institution: | (1) Yale University, Department of Molecular, Cellular & Developmental Biology, New Haven, CT 06520-8104, USA,;(2) Present address: Dipartimento di Genetica e Biologia Microrganismi, Universita' degli Studi di Milano, Milan, Italy,;(3) Present address: Mendel Biotechnology, Inc., 21375 Cabot Boulevard, Hayward, CA 94545, USA, |
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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.
Electronic Publication |
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Keywords: | Arabidopsis thaliana Insertional mutagenesis T-DNA Functional genomics |
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