, Volume 198, Issue 2, pp 211–220 | Cite as

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

  • Benedikt Kost
  • Nathalie Leduc
  • Christof Sautter
  • Ingo Potrykus
  • Gunther Neuhaus


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.

Key words

Brassica Embryogenesis Firefly luciferase Microprojectile bombardment 



cauliflower mosaic virus


embryo culture medium

LUC (Luc)

firefly luciferase (gene)




particle inflow gun


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Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • Benedikt Kost
    • 1
  • Nathalie Leduc
    • 1
  • Christof Sautter
    • 1
  • Ingo Potrykus
    • 1
  • Gunther Neuhaus
    • 1
  1. 1.Institute for Plant Sciences, Swiss Federal Institute of TechnologyZürichSwitzerland

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