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The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Müll. Arg)

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Abstract

An efficient genetic transformation procedure using a recombinant green fluorescent protein (GFP) has been developed in Hevea brasiliensis clone PB260. Transformation experiments have been performed using an Agrobacterium tumefaciens binary vector harbouring both uidA and S65T-GFP reporter genes in order to compare selection methods using glucuronidase assay (GUS activity) and paromomycin resistance, GFP activity and paromomycin resistance, or GFP activity only. At transient level, the number of spots showing GUS or GFP activities was similar for 4 and 5 days after coculture. After selection, stable transformation events were observed and led to the establishment of transgenic callus lines. A higher number of lines were generated with GFP selection compared to the GUS one. GFP selection is less time-consuming in terms of callus subculturing, and offers the possibility of producing antibiotic resistance marker-free transgenic plants.

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Abbreviations

ABA:

Abscisic acid

BAP:

Benzylaminopurine

MM:

Maintenance medium

PM:

Pre-culture medium

DM:

Decontamination medium

3,4-D:

3,4-Diclorophenoxyacetic acid

GUS:

β-Glucuronidase

GFP:

Green fluorescent protein

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Acknowledgments

This work was supported by the Institut Français pour le Caoutchouc. The authors thank Dr. J.-C. Breitler for providing the GFP-intron cassette and Dr. R. Jefferson (CAMBIA, Australia) for providing the pCAMBIA vectors and the EHA105 A. tumefaciens strain. The authors would also like to thank André Clément-Demange for the statistical analyses; and F.-C. Baurens, P. Biggins and T. Tranberger for critical reading of the manuscript.

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Authors and Affiliations

  1. UMR DAP, CIRAD, TA A-96/03, Avenue Agropolis, 34398, Montpellier Cedex 5, France

    J. Leclercq, L. Lardet, F. Martin, G. Oliver & P. Montoro

  2. UPR Systèmes de Pérennes, CIRAD, TA B-34/02 Avenue Agropolis, 34398, Montpellier Cedex 5, France

    T. Chapuset

Authors
  1. J. Leclercq
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  2. L. Lardet
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  3. F. Martin
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  4. T. Chapuset
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  5. G. Oliver
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  6. P. Montoro
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Corresponding author

Correspondence to P. Montoro.

Additional information

Communicated by L. Jouanin.

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Leclercq, J., Lardet, L., Martin, F. et al. The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Müll. Arg). Plant Cell Rep 29, 513–522 (2010). https://doi.org/10.1007/s00299-010-0840-x

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  • DOI: https://doi.org/10.1007/s00299-010-0840-x

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