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    Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts


    Nugent, Gregory D. and Coyne, Seamus and Nguyen, Thi T. and Kavanagh, Tony A. and Dix, Philip (2006) Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant Science, 170 (1). pp. 135-142. ISSN 0168-9452

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    Abstract

    Most plastid transformation studies with tobacco, and all reports for other species (except tomato [G.D. Nugent, M. ten Have, A. van der Gulik, P.J. Dix, B.A. Uijtewaal, A.P. Mordhorst, Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep. 24 (2005) 341–349]), have used biolistics for plastid transformation. However, nuclear transformation via biolistics has not been reported for any vegetable Brassica species so we used protoplast culture and PEG-mediated DNA uptake, to examine both nuclear and plastid transformation of cauliflower, an important vegetable Brassica. Avector containing genes for hygromycin resistance and b-glucuronidase activity (pGUS-HYG) was used for nuclear transformation, while plastid transformation utilised a vector (pZB1) containing accD–rbcL plastome targeting regions cloned from Brassica napus (oil seed rape), and the selectable marker gene aadA, conferring resistance to spectinomycin. Protoplasts were embedded in agarose and selected on media containing hygromycin or spectinomycin. From five experiments, a single plastid transformant of the commercial cultivar Thalassa was obtained, whereas nuclear transformants were obtained at an absolute transformation frequency up to 1.3 x 10ˉ⁵. No spontaneous spectinomycin resistant mutants were observed in any plastid transformation experiments. PCR and Southern blot analysis confirmed the transgenic status of plants regenerated from the protoplast-derived calli.

    Item Type: Article
    Keywords: Cauliflower; Plastid transformation; Nuclear transformation; Protoplast culture;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 7370
    Identification Number: https://doi.org/10.1016/j.plantsci.2005.08.020
    Depositing User: Prof. Philip J. Dix
    Date Deposited: 23 Aug 2016 15:35
    Journal or Publication Title: Plant Science
    Publisher: Elsevier
    Refereed: Yes
    URI:

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