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Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes

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Abstract

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.

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Abbreviations

AS:

Acetosyringone

BAP:

6-Benzylaminopurine

CH:

Casein hydrolysate

(2,4-D):

2,4-Dichlorophenoxyacetic acid

SE:

Somatic embryogenesis

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Acknowledgements

Dr. Adi Rahmat from Angewandte Botanik und Arboretum, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany is gratefully acknowledged for the technical support in the transformation experiments. Thanks are also due to Catarina Estêvão from IBET (Oeiras, Portugal) for the technical assistance in cryopreservation of the transformed lines. This research was supported by Fundação para a Ciência e Tecnologia (FCT) and the III Framework Programme of the European Community, through a PhD grant SFRH/BD/1186/2000 to Susana Tereso. Estação Florestal Nacional (EFN) is acknowledged for making plant material available.

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Authors and Affiliations

  1. Forest Biotech, IBET/ITQB, Quinta do Marquês, 2784-905, Oeiras, Portugal

    Susana Tereso, Célia Miguel & M. Margarida Oliveira

  2. Departamento de Biologia Vegetal, Faculdade de Ciências, Universidadede Lisboa, Bloco C2 Piso 1, Campo Grande, 1749-016, Lisboa, Portugal

    M. Margarida Oliveira

  3. AG Angewandte Botanik und Arboretum, Institut für Biologie, Humboldt-Universität zu Berlin, Invalidenstraβe 42, D-10115, Berlin, Germany

    Kurt Zoglauer

  4. Departamento de Biología Molecular y Bioquímica, Faculdad de Ciências, Universidad de Málaga, Campus de Teatinos, 29071, Málaga, Spain

    Carolina Valle-Piquera

Authors
  1. Susana Tereso
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  2. Célia Miguel
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  3. Kurt Zoglauer
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  4. Carolina Valle-Piquera
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  5. M. Margarida Oliveira
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Correspondence to M. Margarida Oliveira.

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Tereso, S., Miguel, C., Zoglauer, K. et al. Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes. Plant Growth Regul 50, 57–68 (2006). https://doi.org/10.1007/s10725-006-9126-2

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  • DOI: https://doi.org/10.1007/s10725-006-9126-2

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