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Volume 71, Issue 1


Tissue regeneration of Abies embryogenic cell lines after 1 year storage in liquid nitrogen

Terezia Salaj
  • Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O.Box 39A, 950 07 Nitra, Slovak Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Radoslava Matusova
  • Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O.Box 39A, 950 07 Nitra, Slovak Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Rony Swennen
  • Laboratory of Tropical Crop Improvement, KU Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium
  • Bioversity International, Willem de Croylaan 42, 3001 Leuven, Belgium
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Bart Panis / Jan Salaj
  • Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O.Box 39A, 950 07 Nitra, Slovak Republic
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2016-02-25 | DOI: https://doi.org/10.1515/biolog-2016-0004


Embryogenic tissues of hybrid firs (Abies alba × A. cephalonica, Abies alba × A. numidica) have been cryopreserved using a slow-freezing method. The cryotolerance of six cell lines initiated from immature or mature zygotic embryos was tested. Following sorbitol (0.5 M) and DMSO (5%) pretreatments the samples were slowly frozen at a rate of 1°C/min, plunged into liquid nitrogen and stored for 1 year. Post-thaw regeneration ocurred in all the six tested cell lines with recovery frequencies ranging from 100% (cell lines AC1, AC2, AC78, AN72), 90% (cell line AC2) to 44.4% (cell line AC79). Fresh and dry mass accumulation of cryopreserved tissues evaluated three month after thawing was identical to that of control (non-cryopreserved tissues without pretreatment). The cryopreservation procedure resulted in disintegration of bipolar structure of somatic embryos. The long vacuolised suspensor cells almost completely disrupted and the meristematic embryonal cells survived cryopreservation. In the post-thaw period, repeated cell divisions of meristematic cells led to formation of new cell clusters and their vacuolisation resulted in polarisation and finally to the formation of bipolar structures and somatic embryos.

Key words: cryopreservation; forest biotechnology; hybrid firs; somatic embryo; structure


  • Alvarez J.M., Cortizo M. & Ordas R.J. 2012. Cryopreservation of somatic embryogenic cultures of Pinus pinaster: effects on regrowth and embryo maturation. CryoLetters 33: 476–484.Google Scholar

  • Aronen T., Krajnakova J., Häggman H. & Ryynänen L.A. 1999.Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica. Plant Sci. 142: 163–172.Google Scholar

  • Bonga J.M. 2004. The effect of various culture media on the formation of embryo-like structures in cultures derived from ex-plants taken from mature Larix deciduas. Plant Cell Tiss. Organ Cult. 77: 43–48.Google Scholar

  • Cyr D., Lazaroff W.R., Grimes S.M.A., Quan G., Bethune T.D., Dunstan D.I. & Roberts D.R. 1994. Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures. Plant Cell Rep. 13: 574–577.Google Scholar

  • de Verno L.L., Park Y.S., Bonga J.M. & Barrett J.D. (1999).,Somaclonal variation in cryopreserved embryogenic clones of white spruce (Picea glauca Moench. Voss.). Plant Cell Rep. 18: 948–963.Google Scholar

  • dos Santos A.L.W., Steiner N., Guerra M.P., Zoglauer K. & Moerschbacher B.M. 2008. Somatic embryogenesis in Araucaria angustifolia. Biol. Plantarum 52: 195–199.Google Scholar

  • Engelmann F. 2004. Plant cryopreservation: Progress and prospect. In Vitro Cell. Dev. Biol. Plant 40: 427–433.Google Scholar

  • Erdelský K. & Frič F. 1979. Practicum and Analytical Methods in Plant Physiology. Slovak Pedagogical Publishers, Bratislava, 624 pp. (In Slovak).Google Scholar

  • Find J.I., Kristensen M.M.H., Norgaard J.V. & Krogstrup P.1998. Effect of culture period and cell density on regrowth following cryopreservation of embryogenic suspension cultures of Norway spruce and Sitka spruce. Plant Cell Tiss. Organ Cult. 53: 27–33.Google Scholar

  • Gale S., John A. & Benson E.E. 2007. Cryopreservation of Picea sitchensis (sitka spruce) embryogenic suspensor masses. CryoLetters 28: 225–239.Google Scholar

  • Gupta P.K. & Durzan D.J. 1985. Shoot multiplication from mature trees of Douglas fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep. 4: 177–179.Google Scholar

  • Häggman H., Ryynänen L.A., Aronen T. & Krajnakova J. 1998.Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tiss. Organ Cult. 54: 45–53.Google Scholar

  • Hakman I., Fowke L.C., von Arnold S. & Eriksson T. 1985.The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Sci. 38: 53–60.Google Scholar

  • Hargreaves C.I., Grace L.J. & Holden D.G. 2002. Nurse culture for efficient recovery of cryopreserved Pinus radiata D. Don embryogenic cell lines. Plant Cell Rep. 21: 40–45.Google Scholar

  • Hazubska-Przybyl T., Chmielarz P., Michalak M. & Bojarczuk K. 2010. Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce). Plant Cell Tiss. Organ Cult. 102: 35–44.Google Scholar

  • Klimaszewska K. & Cyr D. 2002. Conifer somatic embryogenesis:I. Development. Dendrobiol. 48: 31–39.Google Scholar

  • Klimaszewska K., Overton C., Steward D. & Rutledge R.G. 2011.Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process. Planta 233: 635–647.Google Scholar

  • Kong L. & von Aderkas P. 2011. A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer. Plant Cell Tiss. Organ Cult. 106: 115–125.Google Scholar

  • Krajňáková J., Sutela S., Aronen T., Gömöry D., Vianello A.& Häggman H. 2011. Long-term cryopreservation of Greek fir embryogenic cell lines: Recovery, maturation and genetic fidelity. Cryobiology 63: 17–25.Google Scholar

  • Krajňáková J., Bertolini A., Gömöry D., Vianello A. & Häggman H. 2013. Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell line. In Vitro Cell. Develop. Biol. – Plant 49: 560–571.Google Scholar

  • Kristensen M.M.H., Find J.I., Floto F., Moller J.D., Norgaard J.V. & Krogstrup P. 1994. The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture of Picea sitchensis. Protoplasma 182: 65–70.Google Scholar

  • Latutrie M. & Aronen T. 2013. Long-term cryopreservation of embryogenic Pinus sylvestris cultures. Scandin. J. Forest Res. 28: 103–109.Google Scholar

  • Lelu-Walter M.-A., Bernier-Cardou M. & Klimaszewska K. 2006.Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.). Plant Cell Rep. 25: 767–776.Google Scholar

  • Marum L., Estevao C., Oliveira M.M., Amancio S., Rodrigues L. & Miguel C. 2004. Recovery of cryopreserved embryogenic cultures of maritime pine – effect of cryoprotectant and suspension density. CryoLetters 25: 363–374.Google Scholar

  • Mathur G., Alkutkar V.A. & Nadgauda R.S. 2003. Cryopreservation of embryogenic culture of Pinus roxburghii. Biol. Plant. 46: 205–210.Google Scholar

  • Montalbán I.A., De Diego N. & Moncaleán P. 2012. Enhancing initiation and proliferation in radiata pine (Pinus radiata D. Don.) somatic embryogenesis through seed family screening, zygotic embryo staging and media adjustments. Acta Physiol. Plant. 34: 451–460.Google Scholar

  • Nörgaard J.V., Baldursson S. & Krogstrup P. 1993. Genotypic differences in ability of embryogenic Abies nordmanniana cultures to survive cryopreservation. Silvae Gen. 42: 93–97.Google Scholar

  • Panis B. & Lambardi M. 2005. Status of cryopreservation technologies in plants (crops and forest trees), pp. 43–54. In: The Role of Biotechnology, Vila Gualino, March 5–7, Turin, Italy.Google Scholar

  • Panta A., Panis B., Ynouye C., Swennen R. & Roca W. 2014. Development of a PVS2 droplet vitrification method for potato cryopreservation. CryoLetters 35: 255–266.Google Scholar

  • Park S.Y., Klimaszewska K., Park J.Y. & Mansfield S.D. 2010.Lodgepole pine: the first evidence of seed based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees. Tree Physiol. 30: 1469– 1478.Google Scholar

  • Reinhoud P. J., van Iren F. & Kijne J.W. 2000. Cryopreservation of undifferentiated plant cells, pp. 91–102. In: Engelmann F. & Takagi H. (eds), Cryopreservation of Tropical Germplasm: Current Research Progress and Application, IPGRI, Roma.Google Scholar

  • Ruaud J.N., Bercetche J. & Paques M. 1992. First evidence of somatic embryogenesis from needles of 1–year–old Picea abies plants. Plant Cell Rep. 11: 563–566.Google Scholar

  • Runions C.J. & Owens J.N. 1999. Sexual reproduction of interior spruce (Pinaceae). II. Fertilisation to early embryo formation. Int. J. Plant Sci. 160: 641–652.Google Scholar

  • Salaj T. & Salaj J. 2003. Somatic embryo formation on mature Abies alba x Abies cephalonica zygotic embryo explants. Biol. Plant. 47: 7–11.Google Scholar

  • Salaj T., Panis B., Swennen R. & Salaj J. 2007. Cryopreservation of embryogenic tissues of Pinus nigra Arn. by a slow freezing method. CryoLetters 28: 69–76.Google Scholar

  • Salaj T., Matusikova I., Panis B., Swennen R. & Salaj J. 2010.Recovery and characterisation of hybrid firs (Abies alba × A.cephalonica, Abies alba × A. numidica) embryogenic tissues after cryopreservation. CryoLetters 31: 206–217.Google Scholar

  • Salaj T., Matušíková I., Fráterová L., Piršelová B. & Salaj J.2011. Regrowth of embryogenic tissues of Pinus nigra following cryopreservation. Plant Cell Tiss. Organ Cult. 106: 55–61.Google Scholar

  • Salaj T., Matusiková I., Swennen R., Panis B. & Salaj J. 2012.Long-term maintenance of Pinus nigra embryogenic cultures through cryopreservation. Acta Physiol. Plant. 34: 227–233.Google Scholar

  • Salajova T. & Salaj J. 2001. Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs. J. Plant Physiol. 158: 747–755.Google Scholar

  • Suzuki T., Kaneko M. & Harada T. 1997. Increase in freezing resistance of excised shoot tips of Asparagus officinalis L. by preculture on sugar-rich media. Cryobiology 34: 264–275.Google Scholar

  • Vondrakova Z., Cvikrova M., Eliasova K., Martincova O. &Vagner M. 2010. Cryotolerance in Norway spruce and its assotiation with growth rates, anatomical features and polyamines of embryogenic cultures. Tree Physiol. 30: 1335– 1348.Google Scholar

  • Vookova B. & Kormutak A. 2009. Improved plantlet regeneration from open-pollinated families of Abies alba trees of Dobro primeval forest and adjoing managed stand via somatic embryogenesis. Biologia 64: 1136–1140.Google Scholar

  • Widholm J.M. 1972. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47: 189–194.Google Scholar

About the article

Received: 2015-06-04

Accepted: 2015-12-02

Published Online: 2016-02-25

Published in Print: 2016-01-01

Citation Information: Biologia, Volume 71, Issue 1, Pages 93–99, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.1515/biolog-2016-0004.

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