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Genetic transformation of Eucalyptus globulus using the vascular-specific EgCCR as an alternative to the constitutive CaMV35S promoter

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Abstract

Strong constitutive promoters, such as CaMV35S, are widely used for plant transformation, but undesirable phenotypic changes have been reported when used to drive biotic stress tolerance and/or for modifying lignin content. The promoter of the eucalyptus cinnamoyl CoA reductase (CCR), a key enzyme of the lignin biosynthetic pathway, was shown to be preferentially expressed in vascular tissues both in herbaceous and woody transgenic plants but not eucalyptus. In this work, we transformed Eucalyptus globulus with the EgCCR promoter governing both β-glucuronidase (GUS) and GFP activity patterns. No statistical differences were found between the survival rate and percentage of GUS positive shoots between eucalyptus transformed with either the constitutive CaMV35S or with the EgCCR promoter. The EgCCR transformed plantlets exhibited high GUS expression levels associated with the vascular tissues opening the possibility of targeting vascular-associated traits such as lignin content or vascular pathogen resistance in adult elite plants of eucalyptus while avoiding the undesirable pleiotropic effects caused by strong constitutive promoters.

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Acknowledgments

We wish to thank Mr. Pedro M. Pantín for excellent technical assistance, Dr. Mónica Goicoechea for her help with fluorometric assays, and Mr. Juan Oller (ENCE) for the kind gift of 0-05MN E. globulus clone. Also, we want to thank the research group of Dr. Juan Segura and Dr. Isabel Arrillaga for their kind help on genomic hybridization analysis. Financial support from the European Community (FEDER), Ministry of Science and Technology (contract No AGL-2003-05877-FOR) and Xunta de Galicia (PGIDIT-02-BTF-30102PR) is gratefully acknowledged.

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Author notes

  1. Francisco de la Torre

    Present address: Departamento de Fisiología Vegetal, Facultad de Biología, Universidad de Santiago de Compostela, 15782, Galicia, Santiago de Compostela, Spain

  2. Gago Jorge

    Present address: Departamento de Biología, Universidad de les Illes Balears, 07122, Palma, Spain

  3. Beatriz Villar

    Present address: Northampton Community College, Monroe Campus, Tannersville, PA, 18372, USA

Authors and Affiliations

  1. Applied Plant and Soil Biology, Faculty of Biology, University of Vigo, 363100, Vigo, Spain

    Francisco de la Torre, Ruth Rodríguez, Gago Jorge, Beatriz Villar & Pedro Pablo Gallego

  2. Biología Celular, Facultad de Biología, Universidad de Vigo, 363100, Vigo, Spain

    Rosa Álvarez-Otero

  3. Laboratoire de Recherche en Sciences Végétales (LRSV), UPS, CNRS, Université Toulouse III, BP 42617, Auzeville, 31326, Castanet Tolosan, France

    Jacqueline Grima-Pettenati

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  1. Francisco de la Torre
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Corresponding author

Correspondence to Pedro Pablo Gallego.

Additional information

Francisco de la Torre and Ruth Rodríguez have contributed equally to this work.

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11240_2013_422_MOESM1_ESM.tiff

PCR amplification of nptII (a), uidA (b) and virD2 (c) genes. Lanes 1-10: transformed Eucalyptus globulus explants. Lane 11: no DNA added; lane 12: untransformed Eucalyptus globulus DNA; lane 13, DNA extracted from Agrobacterium containing p35S GUS int; lane 14, molecular weight marker (Promega® 1 kB ladder). PCR was carried out one month after transformation. Primers were designed according to Gago et al. (2011). UidA and nptII genes were employed as an indication of successful transformation, while virD2 was used for ruling out residual contamination by agrobacteria (TIFF 340 kb)

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de la Torre, F., Rodríguez, R., Jorge, G. et al. Genetic transformation of Eucalyptus globulus using the vascular-specific EgCCR as an alternative to the constitutive CaMV35S promoter. Plant Cell Tiss Organ Cult 117, 77–84 (2014). https://doi.org/10.1007/s11240-013-0422-z

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