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Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit

Nature Biotechnology volume 19, pages 870–875 (2001)Cite this article

Abstract

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to ∼50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.

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Figure 1: Construction of chloroplast transformation vectors containing polylinkers for cloning of passenger genes.
Figure 2: Generation of tomato plants with transgenic plastids.
Figure 3: RFLP analysis to identify homoplasmic transplastomic tomato lines.
Figure 4: Example of a test for maternal inheritance of the spectinomycin resistance trait in the F1 progeny of transplastomic tomato plants.
Figure 5: Foreign protein accumulation in leaves, green and ripe red fruits of transplastomic tomato plants.
Figure 6: Molecular analyses of F1 progeny resulting from a cross of a transplastomic plant with a wild-type plant.

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Acknowledgements

We thank Dr. Walter J. Siqueira (Centro de Genética, IAC-Instituto Agronomico de Campinas, Brazil) for tomato seeds. We are grateful to Dr. Jean-David Rochaix (University of Geneva, Switzerland) for providing us with an AadA antibody and Dr. Pal Maliga (Rutgers University, New Jersey) for a chimeric aadA gene. We thank Dr. Jeff Staub (Monsanto Co., St. Louis, MO) for communicating results before publication. This research was supported by a grant in the PROBRAL Program of the Deutscher Akademischer Austauschdienst (DAAD) and the Coordenação Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to H.C. and R.B.

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Authors and Affiliations

  1. Institut für Biologie III, Universität Freiburg, Schänzlestraße 1, Freiburg, D-79104, Germany

    Stephanie Ruf, Marita Hermann & Ralph Bock

  2. Institut für Biochemie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, Münster, D-48143, Germany

    Stephanie Ruf & Ralph Bock

  3. Centro de Biotecnologia Agrícola, ESALQ, Universidade de São Paulo, Av., Pádua Dias 11, Piracicaba, 13418-900, SP, Brazil

    Irving J. Berger & Helaine Carrer

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Correspondence to Ralph Bock.

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Ruf, S., Hermann, M., Berger, I. et al. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19, 870–875 (2001). https://doi.org/10.1038/nbt0901-870

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