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Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

  • Genetic Transformation and Hybridization
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Abstract

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene—mGFP5-ER and eGFP—both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

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Acknowledgements

We thank Jim Haseloff for providing the mGFP5-ER construct, Steve Mabon for cloning eGFP, Brenda Kivela for assisting in this project and all our laboratory colleagues for their support. We thank the NASA HEDS program for funding this research.

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Authors and Affiliations

  1. Department of Plant Sciences and Landscape Systems, University of Tennessee, Rm 252 Ellington Plant Sciences, 2431 Center Drive, Knoxville, TN 37996-4561, USA

    V. Cardoza & C. N. Stewart Jr.

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  1. V. Cardoza
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  2. C. N. Stewart Jr.
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Correspondence to C. N. Stewart Jr..

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Communicated by M.C. Jordan

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Cardoza, V., Stewart, C.N. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep 21, 599–604 (2003). https://doi.org/10.1007/s00299-002-0560-y

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  • DOI: https://doi.org/10.1007/s00299-002-0560-y

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