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Development of a pollen-mediated transformation method forNicotiana glutinosa

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Abstract

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2′-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.

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Authors and Affiliations

  1. Department of Developmental Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), PO Box 15, 6700 AA, Wageningen, Netherlands

    Leonne M. van der Leede-Plegt, Bernadette C. E. van de Ven, Mirjam Schilder, John Franken & Arjen J. van Tunen

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  1. Leonne M. van der Leede-Plegt
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  2. Bernadette C. E. van de Ven
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  3. Mirjam Schilder
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  4. John Franken
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  5. Arjen J. van Tunen
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van der Leede-Plegt, L.M., van de Ven, B.C.E., Schilder, M. et al. Development of a pollen-mediated transformation method forNicotiana glutinosa . Transgenic Research 4, 77–86 (1995). https://doi.org/10.1007/BF01969410

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  • DOI: https://doi.org/10.1007/BF01969410

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