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Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein

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Abstract

Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈108/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).

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Fig. 1
Fig. 2

Abbreviations

BES:

Brown eye spot

pp:

Protoplast

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Acknowledgments

Support for this research was provided by CNPq/NCSU/UFV/FAPEMIG. We thank Aydin Beseli, Andrew Mealin, Shante Bryant, and Jeff Gillikin for assistance with this work.

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Authors and Affiliations

  1. Departamento de Fitopatologia, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa, MG, 36570-000, Brasil

    André Gomes Coelho Souza & Luiz Antonio Maffia

  2. Department of Plant Biology, North Carolina State University, Raleigh, NC, 27695, USA

    Sonia Herrero & Margaret Elizabeth Daub

Authors
  1. André Gomes Coelho Souza
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  2. Sonia Herrero
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  3. Luiz Antonio Maffia
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  4. Margaret Elizabeth Daub
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Correspondence to André Gomes Coelho Souza.

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Souza, A.G.C., Herrero, S., Maffia, L.A. et al. Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein. Eur J Plant Pathol 139, 241–244 (2014). https://doi.org/10.1007/s10658-013-0301-9

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  • DOI: https://doi.org/10.1007/s10658-013-0301-9

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