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Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

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Abstract

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus.

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Author notes

  1. M. Tor

    Present address: Akdeniz Universitesi, Ziraat Fakultesi, Bitki Koruma Bolumu, Antalya, Turkey)

Authors and Affiliations

  1. Unit for Advanced Propagation Systems Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent, TN25 5AH, UK

    M. Tor, C.T. Twyford & S. H. Mantell

  2. Laboratoire d'Amelioration des Plantes et de Biotechnologies, Ecole National Superieur d'Horticulture - Ecole Nationale d'IngenieursTechniques Horticoles et du Paysage, 2 rue le Notre, 49045, Angers Cedex, France

    I. Funes & J. Boccon-Gibod

  3. Plant Molecular Biology Laboratory, Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent, TN25 5AH, UK

    C. C. Ainsworth

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  1. M. Tor
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  4. J. Boccon-Gibod
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  6. S. H. Mantell
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Tor, M., Twyford, C., Funes, I. et al. Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies. Plant Cell, Tissue and Organ Culture 53, 113–126 (1998). https://doi.org/10.1023/A:1006028406641

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