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Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx. (muscadine grape)

  • Genetic Transformation and Hybridization
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Abstract

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l−1 in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l−1, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.

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Abbreviations

BA:

Benzyladenine

GFP:

Green fluorescent protein

NAA:

Napthyleneacetic acid

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Acknowledgments

This research was supported by the Florida Agricultural Experiment Station and the Florida Department of Agriculture and Consumer Services’ Viticulture Trust Fund. D.J. Gray and Z.T. Li own stock in Florida Genetics LLC, which may license resulting patents and, as such, may benefit financially as a result of the outcome of research reported in this publication.

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Author notes

  1. M. Dutt

    Present address: Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

Authors and Affiliations

  1. Mid-Florida Research and Education Center, University of Florida/IFAS, 2725 Binion Road, Apopka, FL, 32703-8504, USA

    S. A. Dhekney, Z. T. Li, M. Dutt & D. J. Gray

Authors
  1. S. A. Dhekney
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  2. Z. T. Li
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  3. M. Dutt
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  4. D. J. Gray
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Correspondence to D. J. Gray.

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Communicated by K. Kamo.

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Dhekney, S.A., Li, Z.T., Dutt, M. et al. Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx. (muscadine grape). Plant Cell Rep 27, 865–872 (2008). https://doi.org/10.1007/s00299-008-0512-2

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  • DOI: https://doi.org/10.1007/s00299-008-0512-2

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