Summary
The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN2). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN2. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN2. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.
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Abbreviations
- EC:
-
embryogenic cells
- ECC:
-
embryogenic cell clusters
- FDA:
-
fluorescein diacetate
- GMA:
-
glycol methacrylate
- LN2 :
-
liquid nitrogen (−196°C)
- NEC:
-
non-embryogenic cells
References
Attree SM, Fowke LC (1993) Embryogeny of gymnosperms: advances in synthetic seed technology of conifers. Plant Cell Tissue Organ Cult 35: 1–35
Becwar SM, Wann SR, Johnson MA, Verhagen SA, Feirer RP, Nagmani R (1988) Development and characterization of in vitro embryogenic systems in conifers. In: Ahuja MR (ed) Somatic cell genetics of woody plants. Kluwer, London, pp 1–18
Bellarosa R, Mo HK, von Arnold S (1992) The influence of auxin and cytokinin on proliferation and morphology of somatic embryos ofPicea abies (L.) Karst. Ann Bot 70: 199–206
Cornu D, Geoffrion C (1990) Aspects de l'embryogènese somatique le mélèze. Bull Soc Bot France 137: 25–34
Dogra PD (1978) Morphology, development, and nomenclature of conifer embryo. Phytomorphology 28: 307–322
Egersdotter U, von Arnold S (1992) Classification of embryogenic cell-lines ofPicea abies as regards protoplast isolation and culture. J Plant Physiol 141: 222–229
Find JI, Floto F, Krogstrup P, Møller JD, Nørgaard JV, Kristensen MMH (1993) Cryopreservation of an embryogenic suspension culture of sitka spruce (Picea sitchensis). Scand J For Res 8: 156–162
George EF (1993) Plant propagation by tissue culture. Part 1: The technology. Exegetics, Westbury, Wiltshire
Hakman I, Fowke LC (1987) An embryogenic cell suspension culture ofPicea glauca (white spruce). Plant Cell Rep 6: 20–22
—, Rennie P, Fowke L (1987) A light and electron microscope study ofPicea glauca (white spruce) somatic embryos. Protoplasma 140: 100–109
Hartmann S, Lang H, Reuter G (1992) Differentiation of somatic embryos from protoplast isolated from suspension cultures ofAbies alba L. Plant Cell Rep 11: 554–557
de Jong AJ, Schmidt EDL, de Vries SC (1993) Early events in higherplant embryogenesis. Plant Mol Biol 22: 367–377
Klimaszewska K (1989) Recovery of somatic embryos and plantlets from protoplast cultures ofLarix×eurolepis. Plant Cell Rep 8: 440–444
Komamine A, Kawahara R, Matsumoto M, Sunabori S, Toya T, Fujiwara A, Tsukahara M, Smith J, Ito M, Fukuda H, Fujimura T (1992) Mechanisms of somatic embryogenesis in cell cultures: physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol 28: 11–14
Krogstrup P (1990) Effect of culture densities on cell proliferation and regeneration from embryonic cell suspensions ofPicea sitchensis. Plant Sci 72: 115–123
—, Eriksen EN, Møller JD, Roulund H (1988) Somatic embryogenesis in sitka spruce [Picea sitchensis Bong. (Carr.)]. Plant Cell Rep 7: 594–597
Laine E, David A (1990) Somatic embryogenesis in immature embryos and protoplasts ofPinus caribaea. Plant Sci 69: 215–224
Maheswari P (1950) An introduction to the embryology of angiosperms. McGraw-Hill, New York
Mo LH (1993) Somatic embryogenesis in Norway spruce (Picea abies). PhD thesis. Department of Forest Genetics, Uppsala Genetic Center, Swedish University of Agricultural Sciences, Uppsala, Sweden, Research Notes 49
Nagmani R, Becwar MR, Wann SR (1987) Single-cell origin and development of somatic embryos inPicea abies (L.) Karst. (Norway spruce) andP. glauca (Moench) Voss (white spruce). Plant Cell Rep 6: 157–159
O'Brien TP, McCully ME (1981) The study of plant structure: principles and selected methods. Termarcarphi Pty, Melbourne
Tautorus TE, Attree SM, Fowke LC, Dunstan DI (1990) Somatic embryogenesis from immature and mature zygotic embryos and embryo regeneration from protoplasts in Black spruce (Picea mariana Mill.). Plant Sci 67: 115–124
Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Tech 47: 189–194
Wilde HD, Nelson SW, Hubert B, de Vries SC, Thomas TL (1988) Gene-expression programs in embryogenic and non-embryogenic carrot cultures. Planta 176: 205–211
Wurtele ES, Keller GL, Nikolau BJ, Ulrich TH (1988) Quantification of starch and ADP-glucose pyrophosphorylase in non-embryogenic cells and embroygenic cell clusters from carrot suspension cultures. J Plant Physiol 132: 683–689
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Kristensen, M.M.H., Find, J.I., Floto, F. et al. The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis . Protoplasma 182, 65–70 (1994). https://doi.org/10.1007/BF01403690
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DOI: https://doi.org/10.1007/BF01403690