Spruce Embryogenesis

  • Sara von Arnold
  • David Clapham
Part of the Methods In Molecular Biology™ book series (MIMB, volume 427)


Somatic embryogenesis, the process in which embryos, similar in morphology to their zygotic counterparts, are induced to develop in culture from somatic cells, is a suitable model system for investigating the regulation of embryo development. Through this process, a large number of embryos at defined stages of development can easily be obtained. Somatic embryogenesis in Norway spruce is comprised of a sequence of steps including initiation, proliferation, early embryo formation, embryo maturation, desiccation and germination. To execute this pathway, a number of critical physical and chemical treatments should be applied with proper timing. Embryogenic cell lines of Norway spruce are initiated from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs) in the presence of auxin and cytokinin. Early somatic embryos develop from PEMs after withdrawal of auxin and cytokinin. PEM to somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. The embryos develop further, to a stage corresponding to late embryogeny, in the presence of abscisic acid. Some cell lines deviate from normal pattern formation exhibiting developmental arrest at certain stages. These arrested cell lines, together with transgenic lines, are valuable tools for studying embryo development. Particle bombardment is routinely used to produce transgenic plants of Norway spruce.


Conifers cryopreservation embryogenesis gene transfer gymno-sperms Norway spruce 


  1. 1.
    Singh H. Embryology of gymnosperms, In: Zimmerman W, Carlquist Z, Ozenda P, Wullf HD, eds. Handbuch der Pflanzen anatomie. Berlin: Gebryder Borntraeger, 1978:187–241.Google Scholar
  2. 2.
    Filonova LH, Bozhkov PV, von Arnold S. Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-laps tracking. J Exp Bot 2000;343:249–264.CrossRefGoogle Scholar
  3. 3.
    Van Zyl L, Bozhkov PV, Clapham D, Sederoff R, von Arnold S. Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis. Gene Expr Patterns 2003;3:83–91.CrossRefPubMedGoogle Scholar
  4. 4.
    Stasolla C, Bozhkov PV, Chu T-M, van Zyl L, Egertsdotter U, Suárez MF, Craigh D, Wolfinger R, von Arnold S. Sederoff R. Variation in transcript abundance during somatic embryogenesis in gymnosperms. Tree Physiol 2004;24:1073–1085.PubMedGoogle Scholar
  5. 5.
    Filonova LH, Bozhkov PV, Brukhin V, Danierl G, Zhivotovsky B, von Arnold S. Developmental programmed cell death in plant embryogenesis: exploring a model system of Norway spruce somatic embryogenesis. J Cell Sci 2000; 113: 4399–4411.PubMedGoogle Scholar
  6. 6.
    Bozhkov PV, Filonova LH, Suáarez MF, Helmersson A, Smertenko AP, Zhivotovsky B, von Arnold S. VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation. Cell Death Differ 2004;11:175–182.CrossRefPubMedGoogle Scholar
  7. 7.
    Bozhkov PV, Suáarez MF, Filonova LH, Daniel G, Zamyatnin AA, Rodriguez-Nieto S, Zhivotovsky B, Smertenko AP. Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proc Natl Acad Sci USA 2005;102:14463–14468.CrossRefPubMedGoogle Scholar
  8. 8.
    Suáarez MF, Filonova L, Smertenko AP, Savenkov E, Clapham C, von Arnold S, Zhivotovsky B, Bozhkov PV. Metacaspase-dependent programmed cell death is essential for plant embryogenesis. Curr Biol 2004;14:R339–R340.CrossRefGoogle Scholar
  9. 9.
    Smertenko AP, Bozhkov PV, Filonova LH, von Arnold S Hussey P. Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos. Plant J 2003;33:813–824.CrossRefPubMedGoogle Scholar
  10. 10.
    Ingouff M, Farbos I, Lagercrantz U, von Arnold, S. PaHB1 is an evolutionary conserved HD-GL2 homeobox gene defining the protoderm during Norway spruce embryo development. Genesis 2001;30:220–230.CrossRefPubMedGoogle Scholar
  11. 11.
    Sabala I, Clapham D, Elfstrand M, von Arnold S. Tissue-specific experssion of Pa18, a putative lipid transfer protein gene, during embryo development in Norway spruce (Picea abies). Plant Mol Biol 2000;42:461–478.Google Scholar
  12. 12.
    Hjortswang H, Sundras A, Bharathan G, Bozhkov PV, von Arnold S, Vahala T. KNOTTED 1-like homeobox genes of a gymnosperm, Norway spruce, expressed during somatic embryogenesis. Plant Physiol Biochem 2002;40:837–843.CrossRefGoogle Scholar
  13. 13.
    Ciavatta V, Egertsdotter U, Clapham C, von Arnold S, Cairney J. A promoter from the loblolly pine PtNIP1;1 gene directs expression in an early-embryogenesis and suspensor-specific fashion. Planta 2002;215:694–698.CrossRefPubMedGoogle Scholar
  14. 14.
    Ingouff M, Farbos M, Wiweger M, von Arnold S. The molecular characterization of PaHB2, a homeobox gene of the HD-GL2 family expressed during embryo development in Norway spruce. J Exp Bot 2003;54:1343–1350.CrossRefPubMedGoogle Scholar
  15. 15.
    Footitt S, Ingouff M, Clapham D, von Arnold S. Expression of the viviparous 1(Pavp1) and p32 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies). J Exp Bot 2003;54:1711–1719.CrossRefPubMedGoogle Scholar
  16. 16.
    von Arnold S, Eriksson T. A revised medium for growth of pea mesophyll protoplasts. Plant Physiol 1977;39:257–260.CrossRefGoogle Scholar
  17. 17.
    Bozhkov PV, von Arnold S. Polyethylene glycol promotes maturation but inhibits further development of Picea abies somatic embryos. Physiol Plant 1998;104: 211–224.CrossRefGoogle Scholar
  18. 18.
    Krogstrup P. Embryo-like structures from cotyledons and ripe embryos of Norway spruce (Picea abies). Can J For Res 1986;16:164–168.CrossRefGoogle Scholar
  19. 19.
    Schenk U, Hildebrandt AC. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 1972;50: 199–204.CrossRefGoogle Scholar
  20. 20.
    Mo LH, von Arnold S. Origin and development of embryogenic cultures from seedlings of Norway spruce (Picea abies). J Plant Physiol 1991;138:223–230.Google Scholar
  21. 21.
    Wiweger M, Farbos I, Ingouff M, Lagercrantz U, von Arnold, S. Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. J Exp Bot 2003;54:2691–2699.CrossRefPubMedGoogle Scholar
  22. 22.
    Egertsdotter U, von Arnold S. Importance of arabinogalactan proteins for the development of somatic embryos of Picea abies. Physiol Plant 1995;93:334–345.CrossRefGoogle Scholar
  23. 23.
    Dyachok J, Wiwegwe M, Kenne L, von Arnold, S. Endogenous Nod-factor-like signal molecules suppress cell death and promote early somatic embryo development in Norway spruce. Plant Physiol 2002;128:523–533.CrossRefPubMedGoogle Scholar
  24. 24.
    Bozhkov PV, Filonova LH, von Arnold S. A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification. Biotechnol Bioeng 2002;77:658–667.CrossRefPubMedGoogle Scholar
  25. 25.
    Nörgaard JV, Duran V, Johnsen Ö, Krogstrup P, Baldursson S, von Arnold S. Variations in cryotolerance of embryogenic Pice abies cell lines and association to genetic, morphological and physiological factors. Can J For Res 1993;23: 2560–2567.CrossRefGoogle Scholar
  26. 26.
    Clapham DH, Häggman H, Elfstrand M, Aronen T, von Arnold S. Transformation of Norway spruce (Picea abies) by particle bombardment. In: Jackson JF, Linskens HF, Inman RB, eds. Molecular Methods of Plant Analysis. Berlin Heidelberg Springer-Verlag, vol. 23, 2003:127–146.Google Scholar
  27. 27.
    Clapham D, Demel P, Elfstrand M, Koop H-U, Sabala I, von Arnold S. Effective biolistic gene transfer to embryogenic cultures of Picea abies and the production of transgenic plantlets. Scand J For Res 2000;15:151–160.CrossRefGoogle Scholar
  28. 28.
    Clapham D, Newton R, Sen S, von Arnold, S. Transformation of Picea species. In: Jain M, Minocha SC eds. Molecular Biology of Woody Plants. Dordrecht: Kluwer, vol. 2. 2000:105–118.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Sara von Arnold
    • 1
  • David Clapham
    • 1
  1. 1.Department of Plant Biology and Forest GeneticsSwedish University of Agricultural SciencesUppsalaSweden

Personalised recommendations