Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

Matthews, Peter R., Wang, Ming-Bo, Waterhouse, Peter M., Thornton, Sarah, Fieg, Sarah J., Gubler, Frank, & Jacobsen, John V. (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector. Molecular Breeding, 7(3), pp. 195-202.

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Abstract

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

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118 citations in Web of Science®

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ID Code: 65980
Item Type: Journal Article
Refereed: Yes
Additional URLs:
Keywords: Agrobacterium transformation, Barley, Co-transformation, Marker-free, Selectable marker, Transgene segregation, binary vector, DNA transfer, gene insertion, gene segregation, genetic line, genetic transformation, inheritance, marker gene, plant biotechnology, plant breeding, plant leaf, polymerase chain reaction, transgenic plant, Agrobacterium, Hordeum vulgare subsp. vulgare, Rhizobium
DOI: 10.1023/A:1011333321893
ISSN: 1380-3743
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > PLANT BIOLOGY (060700)
Divisions: Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2001 Springer Netherlands
Deposited On: 09 Jan 2014 07:43
Last Modified: 09 Jan 2014 07:45

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