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Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) Embryogenic Cell Suspensions of Banana (Musa spp. Cavendish AAA and Lady finger AAB)

Khanna, Harjeet K., Becker, Douglas K., Kleidon, Jennifer, & Dale, James L. (2004) Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) Embryogenic Cell Suspensions of Banana (Musa spp. Cavendish AAA and Lady finger AAB). Molecular Breeding, 14(3), pp. 239-252.

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Abstract

Centrifugation-assisted Agrobacterium-mediated transformation CAAT protocol, developed using banana cultivars from two economically important genomic groups AAA and AAB of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group AAA cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars Cavendish and Lady Finger and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt hygromycin phosphotransferase and gusA -glucuronidase or nptII neomycin phosphotransferase and a modified gfp green fluorescent protein gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome.

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43 citations in Web of Science®

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ID Code: 7842
Item Type: Journal Article
Additional Information: For more information, please refer to the publisher's website (link above) or contact the author: h.khanna@qut.edu.au
Keywords: Agrobacterium, Banana, Cavendish, Grand Nain, Lady finger, Musa, Necrosis, Transformation
DOI: 10.1023/B:MOLB.0000047771.34186.e8
ISSN: 1572-9788
Subjects: Australian and New Zealand Standard Research Classification > TECHNOLOGY (100000) > MEDICAL BIOTECHNOLOGY (100400) > Medical Molecular Engineering of Nucleic Acids and Proteins (100403)
Divisions: Current > Research Centres > Centre for Tropical Crops and Biocommodities
Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2004 Springer
Copyright Statement: The original publication is available at SpringerLink http://www.springerlink.com
Deposited On: 23 May 2007
Last Modified: 29 Feb 2012 23:08

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