Planta

, 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
Article

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.

Key words

Brassica Embryogenesis Firefly luciferase Microprojectile bombardment 

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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References

  1. Bilang R, Shigeru I, Peterhans A, Potrykus I, Paszkowski J (1991) The 3′-terminal region of the hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotiana tabaccum. Gene 100: 247–250Google Scholar
  2. Bilang R, Zhang S, Leduc N, Iglesias VA, Gisel A, Simmonds J, Potrykus I, Sautter C (1993) Transient gene expression in vegetative shoot apical meristems of wheat after ballistic micro-targeting. Plant J 4: 735–744Google Scholar
  3. Christou P (1992) Genetic transformation of crop plants using microprojectile bombardment. Plant J 2: 275–281Google Scholar
  4. Cone KC, Burr FA, Burr B (1986) Molecular analysis of the maize anthocyanin regulatory locus C1. Proc Natl Acad Sci USA 83: 9631–9635Google Scholar
  5. deJong AJ, Schmidt EDL, deVries SC (1993) Early events in higherplant embryogenesis. Plant Mol Biol 22: 367–377Google Scholar
  6. deWet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 725–737Google Scholar
  7. Finer JJ, Vain P, Jones MW, McMullen MD (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11: 323–328Google Scholar
  8. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151–158PubMedGoogle Scholar
  9. Goff SA, Klein TM, Roth BA, Fromm ME, Cone KC, Radicella JP, Chandler VL (1990) Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J 9: 2517–2522Google Scholar
  10. Iglesias VA, Gisel A, Bilang R, Leduc N, Potrykus I, Sautter C (1994) Transient expression of visible marker genes in meristem cells of wheat embryos after ballistic micro-targeting. Planta 192: 84–91Google Scholar
  11. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405Google Scholar
  12. Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83: 8447–8451PubMedGoogle Scholar
  13. Jürgens G, Mayer U, Torres Ruiz RA, Berleth T, Miséra S (1991) Genetic analysis of pattern formation in the Arabidopsis embryo. Development Suppl 1: 27–38Google Scholar
  14. Klein TM, Gradziel T, Fromm ME, Sanford JC (1988) Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. Bio Technology 6: 559–563Google Scholar
  15. Klein TM, Arentzen R, Lewis PA, Fitzpatrick-McElligott S (1992) Transformation of microbes, plants and animals by particle bombardment. Bio Technology 10: 286–291Google Scholar
  16. Koncz C, Langridge WHR, Olsson O, Schell J, Szalay AA (1990) Bacterial and firefly luciferase genes in transgenic plants: advantages and disadvantages of a reporter gene. Dev Genet 11: 224–232Google Scholar
  17. Kost B, Potrykus I, Neuhaus G (1992) Regeneration of fertile plants from excised immature zygotic embryos of Arabidopsis thaliana. Plant Cell Rep 12: 50–54Google Scholar
  18. Kost B, Schnorf M, Potrykus I, Neuhaus G (1995) Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and optimized assay. Plant J, in pressGoogle Scholar
  19. Leduc N, Iglesias VA, Bilang R, Gisel A, Potrykus I, Sautter C (1994) Gene transfer to inflorescence and flower meristems using ballistic micro-targeting. Sex Plant Reprod 7: 135–143Google Scholar
  20. Lindsey K, Topping JF (1993) Embryogenesis: a question of pattern. J Exp Bot 44: 359–374Google Scholar
  21. Liu C-M, Xu Z-H, Chua N-H (1993a) Proembryo culture: In vitro development of early globular-stage zygotic embryos from Brassica juncea. Plant J 3: 291–300Google Scholar
  22. Liu C-M, Xu Z-H, Chua N-H (1993b) Auxin polar transport is essential for the establishment of bilateral symmetry during early plant embryogenesis. Plant Cell 5: 621–630Google Scholar
  23. Lloyd AM, Walbot V, Davis RW (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258: 1773–1775Google Scholar
  24. Ludwig SR, Habera LF, Dellaporta SL, Wessler S (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86: 7092–7096Google Scholar
  25. Lusardi MC, Neuhaus-Url G, Potrykus I, Neuhaus G (1994) An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems. Plant J 5: 571–582Google Scholar
  26. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  27. Mansfield SG, Briarty LG (1991) Early embryogenesis in Arabidopsis thaliana: II. The developing embryo. Can J Bot 69: 461–476Google Scholar
  28. Mayer U, Torres Ruiz RA, Berleth T, Miséra S, Jürgens G (1991) Mutations affecting body organization in the Arabidopsis embryo. Nature 353: 402–407Google Scholar
  29. Meinke D W (1991) Embryonic mutants of Arabidopsis thaliana. Dev Genet 12: 382–392Google Scholar
  30. Mendel RR, Müller B, Schulze J, Kolesnikov V, Zelenin A (1989) Delivery of foreign genes to intact barley cells by high-velocity microprojectiles. Theor Appl Genet 78: 31–34Google Scholar
  31. Millar AJ, Short SR, Hiratsuka K, Chua N-H, Kay SA (1992) Firefly luciferase as a reporter of regulated gene expression in higher plants. Plant Mol Biol Rep 10: 324–337Google Scholar
  32. Monnier M (1990) Zygotic embryo culture. In: Bhojwani SS (ed) Plant tissue culture: applications and limitations. Elsevier, Amsterdam Oxford New York Tokyo, pp 366–393Google Scholar
  33. Perl A, Kless H, Blumenthal A, Galili G, Galun E (1992) Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures. Mol Gen Genet 235: 279–284Google Scholar
  34. Sautter C, Waldner H, Neuhaus-Url G, Galli A, Neuhaus G, Potrykus I (1991) Micro-targeting: high efficiency gene transfer using a novel approach for the acceleration of micro-projectiles. Bio Technology 9: 1080–1085Google Scholar
  35. Schulz R, Jensen WA (1968a) Capsella embryogenesis: the early embryo. J Ultrastruct Res 22: 376–392Google Scholar
  36. Schulz R, Jensen WA (1968b) Capsella embryogenesis: the egg, zygote, and young embryo. Am J Bot 55: 807–819Google Scholar
  37. Shevell DE, Leu W-M, Gillmor CS, Xia G, Feldmann KA, Chua N-H (1994) EMB30 is essential for normal cell division, cell expansion and cell adhesion in Arabidopsis and encodes a protein that has similarity to Sec7. Cell 77: 1051–1062Google Scholar
  38. Tykarska T (1976) Rape embryogenesis: I. The proembryo development. Acta Soc Bot Pol 45: 3–16Google Scholar
  39. Tykarska T (1979) Rape embryogenesis: II. Development of embryo proper. Acta Soc Bot Pol 48: 391–421Google Scholar
  40. Tykarska T (1980) Rape embryogenesis: III. Embryo development in time. Acta Soc Bot Pol 49: 369–385Google Scholar
  41. West MAL, Harada JJ (1993) Embryogenesis in higher plants: an overview. Plant Cell 5: 1361–1369Google Scholar
  42. Wu Y, Haberland G, Zhou C, Koop H-U (1992) Somatic embryogenesis, formation of morphogenetic callus and normal development in zygotic embryos of Arabidopsis thaliana in vitro. Protoplasma 169: 89–96Google Scholar
  43. Ye GN, Daniell H, Sanford JC (1990) Optimization of delivery of foreign DNA into higher-plants chloroplasts. Plant Mol Biol 15: 809–819Google Scholar

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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