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Transient marker-gene expression during zygotic in-vitro embryogenesis of Brassica juncea (Indian mustard) following particle bombardment

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Abstract

A method has been established that allows the transfer of genes into single cells of excised globular-stage zygotic Brassica juncea L. embryos. The fate of single, genetically marked cells was followed during in-vitro embryogenesis. A simple and defined embryo culture medium has been designed on which zygotic B. juncea embryos, excised at the globular or at later stages, develop normally into mature, fully grown embryos. The smallest embryos which can be efficiently cultured are 30 μm long (embryo proper without suspensor) and are comprised of less than 20 cells. The embryos grow on the surface of solid medium without embedding and are freely accessible to microprojectile bombardment. Shooting at globular, transition and early heart-shaped embryos using both a particle inflow gun and a micro-targeting particle accelerator resulted in transient expression of genes encoding visible markers. For both particle-acceleration devices the shooting conditions have been optimised based on transient β-glucuronidase (GUS) expression. Bombarding embryos under optimal conditions had no deleterious effects on in-vitro embryogenesis. Multicellular GUS-expressing sectors were obtained, showing that cells can survive receiving a particle and resume normal development. The examination of these sectors has provided new information about the cell division patterns characterising early B. juncea embryogenesis. To be able to follow the development of particular genetically marked sectors, we tried to identify reporter genes that, in contrast to the uidA gene (which encodes GUS), can be non-destructively assayed in embryonic cells. Preliminary data has shown that expression of the firefly luciferase gene (Luc) can be detected in bombarded embryos without affecting their viability.

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Abbreviations

CaMV:

cauliflower mosaic virus

EMB:

embryo culture medium

LUC (Luc):

firefly luciferase (gene)

GUS:

β-glucuronidase

PIG:

particle inflow gun

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Authors and Affiliations

  1. Institute for Plant Sciences, Swiss Federal Institute of Technology, Universitätsstrasse 2, CH-8092, Zürich, Switzerland

    Benedikt Kost, Nathalie Leduc, Christof Sautter, Ingo Potrykus & Gunther Neuhaus

Authors
  1. Benedikt Kost
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  2. Nathalie Leduc
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  3. Christof Sautter
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  4. Ingo Potrykus
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  5. Gunther Neuhaus
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Additional information

We would like to thank Victor Iglesias (Institute of Molecular Biology, Austrian Academy of Science, Salzburg, Austria), Nam-Hai Chua (Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY, USA) and Chun-Ming Liu (Department of Applied Genetics, John Innes Institute, Norwich, UK) for very fruitful discussion as well as Diane Shevell (Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY, USA) for helpful comments on the manuscript and language correction. We are also grateful to Victor Iglesias and Heinz Waldner (Institute for Plant Science, Swiss Federal Institute of Technology, Zürich, Switzerland) for constructing the PIG as well as to Ted Klein (DuPont, Wilmington, Del., USA), Susan Wessler ( Department of Botany, University of Georgia, Athens, Ga., USA), Udo Wienand (Institut für Allgemeine Botanik, University of Hamburg, Hamburg, Germany) and Nam-Hai Chua for providing plasmids. B. K. was supported by a grant of the Swiss Federal Institute of Technology.

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Kost, B., Leduc, N., Sautter, C. et al. Transient marker-gene expression during zygotic in-vitro embryogenesis of Brassica juncea (Indian mustard) following particle bombardment. Planta 198, 211–220 (1996). https://doi.org/10.1007/BF00206246

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  • DOI: https://doi.org/10.1007/BF00206246

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