Skip to main content
SpringerLink
Log in

A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Non-freezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4–8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1a–j
Fig. 2
Fig. 3a–l
Fig. 4
Fig. 5
Fig. 6
Fig. 7a–c
Fig. 8

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

BA:

6-Benzyladenine

2,4-D:

2,4-Dichlorophenoxyacetic acid

DMSO:

Dimethyl sulfoxide

mLV:

Modified LV medium

PEG:

Polyethylene glycol

RAPD:

Random amplification of polymorphic DNA

References

  • Arakawa T, Carpenter JF, Kita YA, Crowe JH (1990) The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 27:401–415

    Article  CAS  Google Scholar 

  • Aronen TS, Krajnakova J, Häggman HM, Ryynänen LA (1999) Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica. Plant Sci 142:163–172

    Article  CAS  Google Scholar 

  • Attree SM, Fowke LC (1993) Embryogeny of gymnosperms: advances in synthetic seed technology of conifers. Plant Cell Tissue Organ Cult 35:1–35

    Article  CAS  Google Scholar 

  • Attree SM, Moore D, Sawhney VK, Fowke LC (1991) Enhanced maturation and desiccation tolerance of white spruce [Picea glauca (Moench) Voss] somatic embryos: effects of a non-plasmolysing water stress and abscisic acid. Ann Bot 68:519–525

    Google Scholar 

  • Bomal C, Tremblay F-M (2000) Dried cryopreserved somatic embryos of two Picea species provide suitable material for direct plantlet regeneration and germplasm storage. Ann Bot 86:177–183

    Article  Google Scholar 

  • Breton D, Harvengt L, Trontin J-F, Bouvet A, Favre J-M (2006) Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine. Plant Cell Tissue Organ Cult 87:95–108

    Article  Google Scholar 

  • Burritt DJ (2008) Efficient cryopreservation of adventitious shoots of Begonia × erythrophylla using encapsulation–dehydration requires pretreatment with both ABA and proline. Plant Cell Tissue Organ Cult 95:209–215

    Article  CAS  Google Scholar 

  • Chalupa V (1985) Somatic embryogenesis and plantlet regeneration from cultured immature and mature embryos of Picea abies (L.) Karst. Comm Inst For Čech 14:57–63

    Google Scholar 

  • Cyr DR (1999) Cryopreservation of embryogenic cultures of conifers and its application to clonal forestry. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol IV. Kluwer, Dordrecht, pp 239–262

    Google Scholar 

  • DeVerno LL, Park YS, Bonga JM, Barrett JD, Simpson C (1999) Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]. Plant Cell Rep 18:948–953

    Article  CAS  Google Scholar 

  • Finkle BJ, Ulrich JM (1982) Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells. Cryobiology 19:329–335

    Article  PubMed  CAS  Google Scholar 

  • Finkle BJ, Zavala ME, Ulrich JM (1985) Cryoprotective compounds in the viable freezing of plant tissues. In: Kartha KK (ed) Cryopreservation of plant cells and organs. CRC Press, Boca Raton, pp 75–113

    Google Scholar 

  • Gale S, John A, Benson EE (2007) Cryopreservation of Picea sitchensis (Sitka spruce) embryogenic suspensor masses. Cryo Letters 28:225–239

    PubMed  Google Scholar 

  • Gale S, John A, Harding K, Benson EE (2008) Developing cryopreservation for Picea sitchensis (Sitka spruce) somatic embryos: a comparison of vitrification protocols. Cryo Letters 29:135–144

    PubMed  Google Scholar 

  • Guevin TG, Kirby EG (1997) Induction of embryogenesis in cultured mature zygotic embryos of Abies fraseri (Pursh) Poir. Plant Cell Tissue Organ Cult 49:219–222

    Article  CAS  Google Scholar 

  • Guevin TG, Micah V, Kirby EG (1994) Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea. Plant Cell Tissue Organ Cult 37:205–208

    Article  CAS  Google Scholar 

  • Gupta PK, Durzan DJ (1987) Biotechnology of somatic polyembryogenesis and plantlet regeneration in loblolly pine. Bio/Technol 5:147–151

    Article  Google Scholar 

  • Häggman HM, Ryynänen LA, Aronen TS, Krajnakova J (1998) Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tissue Organ Cult 54:45–53

    Article  Google Scholar 

  • Häggman HM, Aronen TS, Ryynänen LA (2000) Cryopreservation of embryogenic cultures of conifers. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol VI. Kluwer, Dordrecht, pp 707–728

    Google Scholar 

  • Hakman I, Fowke LC, 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 38:53–59

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Klimaszewska K, Ward C, Cheliak WM (1992) Cryopreservation and plant regeneration from embryogenic cultures of larch (Larix × eurolepis) and black spruce (Picea mariana). J Exp Bot 43:73–79

    Article  CAS  Google Scholar 

  • Klimaszewska K, Park Y-S, Overton C, MacEacheron I, Bonga JM (2001) Optimized somatic embryogenesis in Pinus strobus L. In Vitro Cell Dev Biol Plant 37:392–399

    Article  Google Scholar 

  • Kong L, von Aderkas P (2007) Genotype effects on ABA consumption and somatic embryo maturation in interior spruce (Picea glauca x engelmanni). J Exp Bot 58:1525–1531

    Article  PubMed  CAS  Google Scholar 

  • Kong L, Yeung EC (1995) Effects of silver nitrate and polyethylene glycol on white spruce (Picea glauca) somatic embryo development: enhancing cotyledonary embryo formation and endogenous ABA content. Physiol Plant 93:298–304

    Article  CAS  Google Scholar 

  • Kong L, Attree SM, Fowke LC (1997) Changes of endogenous hormone levels in developing seeds, zygotic embryos and megagametophytes in Picea glauca. Physiol Plant 101:23–30

    Article  CAS  Google Scholar 

  • Kong L, Attree SM, Fowke LC (1998) Effects of polyethylene glycol and methylglyoxal bis(guanylhydrazone) on endogenous polyamine levels and somatic embryo maturation in white spruce (Picea glauca). Plant Sci 133:211–220

    Article  CAS  Google Scholar 

  • Kong L, Attree SM, Evans DE, Binarova P, Yeung EC, Fowke LC (1999) Somatic embryogenesis in white spruce: studies of embryo development and cell biology. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol IV. Kluwer, Dordrecht, pp 1–28

    Google Scholar 

  • Litvay JD, Verma DC, Johnson MA (1985) Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328

    Article  CAS  Google Scholar 

  • Martín C, González-Benito ME (2005) Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation–dehydration. Cryobiology 51:281–289

    Article  PubMed  Google Scholar 

  • Nagmani R, Bonga JM (1985) Embryogenesis in subcultured callus of Larix decidua. Can J For Res 15:1088–1091

    Article  Google Scholar 

  • Newton RJ, Marek-Swize KA, Magallanes-Cedeno ME, Dong N, Sen S, Jain SM (1995) Somatic embryogenesis in slash pine (Pinus elliottii Engelm.). In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, III. Gynosperms. Kluwer, Dordrecht, pp 183–195

    Google Scholar 

  • Park YS, Lelu-Walter MA, Harvengt L, Trontin JF, MacEacheron I, Klimaszewska K, Bonga JM (2006) Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France. Plant Cell Tissue Organ Cult 86:87–101

    Article  Google Scholar 

  • Popova EV, Lee E-J, Wu C-H, Hahn E-J, Paek K-Y (2009) A simple method for cryopreservation of Ginkgo biloba callus. Plant Cell Tissue Organ Cult 97:337–343

    Article  CAS  Google Scholar 

  • Pullman GS, Johnson S, Peter G, Cairney J, Xu N (2003) Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination, and gene expression. Plant Cell Rep 21:747–758

    PubMed  CAS  Google Scholar 

  • Roberts DR, Flinn BS, Webb DT, Webster FB, Sutton BCS (1989) Characterization of immature embryos of interior spruce by SDS-PAGE and microscopy in relation to their competence for somatic embryogenesis. Plant Cell Rep 8:285–288

    Article  CAS  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. Cryo Letters 31:206–217

    PubMed  CAS  Google Scholar 

  • Stasolla C, Yeung EC (2003) Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult 74:15–35

    Article  CAS  Google Scholar 

  • Stasolla C, Kong L, Yeung EC, Thorpe TA (2002) Maturation of somatic embryos in conifers: morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol Plant 38:93–105

    Article  CAS  Google Scholar 

  • Stasolla C, van Zyl L, Egertsdotter U, Craig D, Liu W, Sederoff RR (2003) The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos. Plant Physiol 131:49–60

    Article  PubMed  CAS  Google Scholar 

  • Tamminen I, Mäkelä P, Heino P, Palva ET (2001) Ectopic expression of ABI3 gene enhances freezing tolerance in response to abscisic acid and low temperature in Arabidopsis thaliana. Plant J 25:1–8

    Article  PubMed  CAS  Google Scholar 

  • Tessereau H, Florin B, Meschine MC, Thierry C, Pétiard V (1994) Cryopreservation of somatic embryos: a tool for germplasm storage and commercial delivery of selected plants. Ann Bot 74:547–555

    Article  Google Scholar 

  • Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann Rev Plant Physiol Plant Mol Biol 50:571–599

    Article  CAS  Google Scholar 

  • Touchell DH, Chiang VL, Tsai CJ (2002) Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep 21:118–124

    Article  CAS  Google Scholar 

  • Verhagen SA, Wann SR (1989) Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos. Plant Cell Tissue Organ Cult 16:103–111

    Article  Google Scholar 

  • von Aderkas P, Bonga JM (2000) Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol 20:921–928

    Google Scholar 

  • von Aderkas P, Kong L, Hawkins B, Rohr R (2007) Effects of non-freezing low temperatures on quality and cold tolerance of mature somatic embryos of interior spruce (Picea glauca (Moench) Voss × P. engelmannii Parry ex. Engelm.). Propag Ornam Plants 7:112–121

    Google Scholar 

  • Yamazaki H, Ayabe K, Ishii R, Kuriyama A (2009) Desiccation and cryopreservation of actively-growing cultured plant cells and protoplasts. Plant Cell Tissue Organ Cult 97:151–158

    Article  Google Scholar 

  • Yin M-H, Hong S-R (2010) A simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation–vitrification. Plant Cell Tissue Organ Cult 101:349–358

    Article  Google Scholar 

Download references

Acknowledgments

This work was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to PvA. The authors would like to thank Barry Jaquish (BC Ministry of Forests and Range) for providing interior spruce seeds from the seed orchard at Kalamalka Research Station (Vernon, BC, Canada).

Author information

Authors and Affiliations

  1. Centre for Forest Biology, Department of Biology, University of Victoria, 3800 Finnerty Rd., Victoria, BC, V8W 3N5, Canada

    Lisheng Kong & Patrick von Aderkas

Authors
  1. Lisheng Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patrick von Aderkas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lisheng Kong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kong, L., von Aderkas, P. A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer. Plant Cell Tiss Organ Cult 106, 115–125 (2011). https://doi.org/10.1007/s11240-010-9899-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-010-9899-x

Keywords

Search

Navigation