Skip to main content

Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation

Abstract

Fraser fir (Abies fraseri [Pursh] Poir.) is a coniferous species native to the Southern Appalachian Mountains of the eastern United States. The species has high economic and recreational value but is vulnerable to extinction due to introduced pests and global warming. Somatic embryogenesis technology may assist in the clonal production of desired lines of Christmas trees and safekeeping of rare and valuable germplasm via cryopreservation. We have developed a highly effective medium for initiation of embryogenic tissue from immature or mature seeds of Fraser fir that contains AL salts (Kvaalen et al., in Can J For Res 35:1053–1060, 2005), brassinolide, paclobutrazol and abscisic acid. Using dominant embryos attached to the female gametophyte placed on medium, the highest initiation percentages occurred with precotyledonary stage 3 embryos. When tested with 11 high-value open-pollinated families over 5 years, initiation tests for medium containing brassinolide and paclobutrazol averaged 6–62 % initiation. A maintenance medium was developed that contained AL salts and 1.1 mg L−1 BAP and was able to capture approximately 50 % of the initiations. A maturation medium was developed containing AL salts, maltose, polyethylene glycol 8000 and abscisic acid that produced cotyledonary embryos capable of germination to produce a root and shoot. Culture cryopreservation and retrieval was also demonstrated.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  • Aitken-Christie J, Parkes BD (1996) Improved embryogenesis process for initiation and maturation. International application under the patent cooperation treaty (PCT). WO 96/37096, international publication date: 28 November 1996

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

    CAS  Article  Google Scholar 

  • Aurich C, Rupps A, Zoglauer K (2014) Embryo maturation ability is subjected to line ageing—a way to assure the quality of somatic embryos of Nordmann fir. In: Park, YS, Bonga JM (eds) Proceedings of the 3rd international conference of the IUFRO unit 2.09.02 on “Woody plant production integrating genetic and vegetative propagation technologies.” September 8–12, 2014. Vitoria-Gasteiz, Spain, pp 127–128. Published online: http://www.iufro20902.org. Accessed 19 May 2015

  • Becwar MR, Pullman GS (1995) Somatic embryogenesis in loblolly pine (Pinus taeda L.). In: Mohan-Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, Vol. 3-Gymnosperms. Kluwer, The Netherlands, pp 287–301

  • Breton D, Harvengt L, Trontin JF, Bouvet A, Favre JM (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 

  • Cairney J, Xu N, Pullman GS, Ciavatta VT, Johns B (1999) Natural and somatic embryo development in loblolly pine: gene expression studies using differential display and cDNA arrays. Appl Biochem Biotech 77–79:5–17

    Article  Google Scholar 

  • Cairney J, Xu N, MacKay J, Pullman G (2000) Transcript profiling: a tool to assess the development of conifer embryos. In Vitro Cell Dev Biol Plant 36:155–162

    CAS  Article  Google Scholar 

  • De Silva V, Bostwick D, Burns KL, Oldham CD, Skryabina A, Sullards MC, Wu D, Zhang Y, May SW, Pullman GS (2008) Isolation and characterization of a molecule stimulatory to growth of early-stage somatic embryos from early-stage female gametophyte tissue of loblolly pine. Plant Cell Rep 27:633–646

    CAS  Article  PubMed  Google Scholar 

  • Egertsdotter U, Pullman GS (2003) Clonal propagation of Fraser fir through somatic embryogenesis. In: Sixth international christmas tree research & extension conference. Sept 14–19, 2003, Hendersonville, NC. Poster

  • Fenning TM, Walter C, Gartland KMA (2008) Forest biotech and climate change. Nature Biotech 26:615–617

    CAS  Article  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

    CAS  Article  Google Scholar 

  • Gupta PK, Durzan DJ (1985) Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep 4:177–179

    CAS  Article  PubMed  Google Scholar 

  • Gupta PK, Pullman GS (1991) Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation. US Patent 5036007, 30 July 1991

  • Gutierrez L, Van Wuytswinkel O, Castelain M, Bellini C (2007) Combined networks regulating seed maturation. Trends Plant Sci 12:294–300

    CAS  Article  PubMed  Google Scholar 

  • Handley L III (1997) Method for regeneration of coniferous plants by somatic embryogenesis in culture media containing abscisic acid. US Patent 5,677,185. October 14, 1997

  • Handley L III (1999) Method for regeneration of coniferous plants by somatic embryogenesis in culture media containing abscisic acid. US Patent 5,856,191. January 5, 1999

  • Hibbert-Frey H, Frampton J, Blazich FA, Hinesley LE (2010) Grafting Fraser fir (Abies fraseri): effect of grafting date, shade, and irrigation. HortSci 45:617–620

    Google Scholar 

  • Hibbert-Frey H, Frampton J, Blazich F, Hundley D, Hinesley E (2011) Grafting Fraser fir: effect of scion origin (crown position and branch order). HortSci 46:91–94

    Google Scholar 

  • Hinesley E, Frampton J (2002) Grafting Fraser fir onto rootstocks of selected Abies species. HortSci 37:815–818

    Google Scholar 

  • Hristoforoglu K, Schmidt J, Bolhar-Nordenkampf H (1995) Development and germination of Abies alba somatic embryos. Plant Cell, Tissue Organ Cult 40:277–284

    Article  Google Scholar 

  • IUCN (2009) IUCN red list of threatened species. Version 2014.3. www.iucnredlist.org. Accessed 19 May 2015

  • Jasik J, Salajova T, Kormutak A, Salaj J (1999) Somatic embryogenesis in hybrid firs. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 4. Kluwer Academic, The Netherlands, pp 505–523

    Chapter  Google Scholar 

  • Kapik RH, Dinus RJ, Dean JF (1995) Abscisic acid and zygotic embryogenesis in Pinus taeda. Tree Physiol 15:485–490

    CAS  Article  PubMed  Google Scholar 

  • Kim YW, Newton R, Frampton J, Han KH (2009) Embryogenic tissue initiation and somatic embryogenesis in Fraser fir (Abies fraseri [Pursh] Poir.). In Vitro Cell Dev Biol Plant 45:400–406

    Article  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: Mohan-Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 4. Kluwer, Dordrecht, pp 1–28

    Chapter  Google Scholar 

  • Korecky J, Vitamvas J (2011) Somatic embryogenesis of the hybrid Abies cilicica × Abies cephalonica. J For Sci 57:401–408

    CAS  Google Scholar 

  • Krajnakova J, Haggman H, Gomory D (2009) Effect of sucrose concentration, polyethylene glycol and activated charcoal on maturation and regeneration of Abies cephalonica somatic embryos. Plant Cell Tiss Organ Cult 96:251–262

    CAS  Article  Google Scholar 

  • Krajnakova J, Sutela S, Aronen T, Gomory D, Vianello A, Haggman H (2011) Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity. Cryobiology 63:17–25

    CAS  Article  PubMed  Google Scholar 

  • Kvaalen H, Daehlen OG, Rognstad AT, Gronstad B, Egertsdotter U (2005) Somatic embryogenesis for plant production of Abies lasiocarpa. Can J For Res 35:1053–1060

    Article  Google Scholar 

  • Liao YK, Juan I-P (2015) Improving the germination of somatic embryos of Picea morrisonicola Hayata: effects of cold storage and partial drying. J For Res. doi:10.1007/s10310-014-0445-2

    Google Scholar 

  • Ma X, Bucalo K, Determann RO, Cruse-Sanders JM, Pullman GS (2012) Somatic embryogenesis, plant regeneration and cryopreservation for Torreya taxifolia, a highly endangered coniferous species. In Vitro Cell Dev Biol Plant 48:324–334

    CAS  Article  Google Scholar 

  • Malabadi R, Nataraja K (2007) 24-Epibrassinolide induces somatic embryogenesis in Pinus wallichiana A.B. Jacks. J Plant Sci 2:171–178

    CAS  Article  Google Scholar 

  • McManamay RH, Resler LM, Campbell JB, McManamayl RA (2011) Assessing the impacts of balsam woolly adelgid (Adelge piceae Ratz.) and anthropogenic disturbance on the stand structure and mortality of Fraser fir [Abies fraseri (Pursh) Poir.] in the Black Mountains, North Carolina. Castanea 76:1–19

    Article  Google Scholar 

  • Misson JP, Druart P, Panis B, Watillon B (2006) Contribution to the study of the maintenance of somatic embryos of Abies nordmanniana LK: culture media and cryopreservation method. Propag Ornam Plants 6:17–23

    Google Scholar 

  • Nørgaard JV, Krogstrup P (1995) Somatic embryogenesis in Abies spp. In: Mohan JS, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 3. Kluwer, The Netherlands, pp 341–355

    Chapter  Google Scholar 

  • Norgaard JV, Baldursson S, Krogstrup P (1993) Genotypic differences in the ability of embryogenic Abies nordmanniana cultures to survive cryopreservation. Silvae Genet 42:93–97

    Google Scholar 

  • North Carolina Department of Agriculture (2012) North Carolina Department of Agriculture and Consumer Service Marketing: Christmas Trees. http://www.ncagr.gov. Accessed 19 May 2015

  • Pullman GS, Bucalo K (2011) Pine somatic embryogenesis using zygotic embryos as explants. In: Thorpe T, Yeung E (eds) Plant embryo culture: methods and protocols. Humana Press, New York, pp 267–291

    Chapter  Google Scholar 

  • Pullman GS, Bucalo K (2014) Pine somatic embryogenesis: analyses of seed tissue and medium to improve protocol development. New For 45:353–377

    Article  Google Scholar 

  • Pullman GS, Johnson S (2002) Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation rates. Ann For Sci 59:663–668

    Article  Google Scholar 

  • Pullman GS, Johnson S (2009) Osmotic measurements in whole megagametophytes and embryos of loblolly pine (Pinus taeda L.) during seed development. Tree Physiol 29:819–827

    Article  PubMed  Google Scholar 

  • Pullman GS, Skryabina A (2007) Liquid medium and liquid overlays improve embryogenic tissue initiation in conifers. Plant Cell Rep 26:873–887

    CAS  Article  PubMed  Google Scholar 

  • Pullman GS, Webb DT (1994) An embryo staging system for comparison of zygotic and somatic embryo development. TAPPI R&D division biological sciences symposium. TAPPI Press, Atlanta, pp 31–34

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Pullman GS, Namjoshi K, Zhang Y (2003b) Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation with abscisic acid and silver nitrate. Plant Cell Rep 22:85–95

    CAS  Article  PubMed  Google Scholar 

  • Pullman GS, Zhang Y, Phan B (2003c) Brassinolide improves embryogenic tissue initiation in conifers and rice. Plant Cell Rep 22:96–104

    CAS  Article  PubMed  Google Scholar 

  • Pullman GS, Olson K, Egertsdotter U (2004) Somatic embryogenesis of Fraser fir (Abies fraseri). 2004 IUFRO joint conference of division 2—forest genetics and tree breeding in the age of genomics: progress and future, November 1–5, 2004, Charleston, SC, #27. Poster

  • Pullman GS, Johnson S, Van Tassel S, Zhang Y (2005a) Somatic embryogenesis in loblolly pine (Pinus taeda L.) and Douglas fir (Pseudotsuga menziesii): improving culture initiation and growth with MES pH buffer, biotin, and folic acid. Plant Cell Tissue Organ Cult 80:91–103

    CAS  Article  Google Scholar 

  • Pullman GS, Mein J, Johnson S, Zhang Y (2005b) Gibberellin inhibitors improve embryogenic tissue initiation in conifers. Plant Cell Rep 23:596–605

    CAS  Article  PubMed  Google Scholar 

  • Pullman GS, Chopra R, Chase K-M (2006) Loblolly pine (Pinus taeda L.) somatic embryogenesis: improvements in embryogenic tissue initiation by supplementation of medium with organic acids, Vitamins B12 and E. Plant Sci 170:648–658

    CAS  Article  Google Scholar 

  • Pullman GS, Johnson S, Bucalo K (2009) Douglas fir embryogenic tissue initiation. Plant Cell Tissue Organ Cult 96:75–84

    Article  Google Scholar 

  • Pullman GS, Zeng X, Copeland-Kemp B, Crockett J, Lucrezi J, May SW, Bucalo K (2015) Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents. Tree Physiol 35:209–224

    Article  PubMed  Google Scholar 

  • Rajbhandari N, Stomp A (1997) Embryogenic callus induction in Fraser fir. HortSci 32:737–738

    Google Scholar 

  • Rédei GP (1974) ‘Fructose effect’ in higher plants. Ann Bot 38:287–297

    Google Scholar 

  • Rosier CL, Frampton J, Goldfarb B, Blazich FA, Wise FC (2004) Growth stage, auxin type, and concentration influence rooting stem cuttings of Fraser fir. HortSci 39:1397–1402

    CAS  Google Scholar 

  • Rosier C, Frampton J, Goldfarb B, Blazich FA, Wise FC (2005) Effects of stumping height, auxin and crown position on the rooting of Fraser fir cuttings. HortSci 40:771–777

    Google Scholar 

  • Salaj T, Salaj J (2003/2004) Somatic embryo formation on mature Abies alba × Abies cephalonica zygotic embryo explants. Biologia Plant 47:7–11

  • Salaj T, Vookova B, Salaj J (2005) Protocols for somatic embryogenesis in hybrid firs. In: Jain SM, Gupta PK (eds) Protocols for somatic embryogenesis in woody plants. Springer, The Netherlands, pp 483–496

    Chapter  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Salajova T, Jasik J, Kormutak A, Salaj J, Hakman I (1996) Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba × Abies cephalonica, and Abies alba × Abies numidica). Plant Cell Rep 15:527–530

    CAS  PubMed  Google Scholar 

  • Schuller A, Reuther G, Geier T (1989) Somatic embryogenesis from seed explants of Abies alba. Plant Cell Tissue Organ Cult 17:53–58

    Google Scholar 

  • Silva AMN, Kong XL, Parkin MC, Cammack R, Hider RC (2009) Iron(III) citrate speciation in aqueous solution. Dalton Trans 2009:8616–8625

    Article  Google Scholar 

  • Timmis R (1998) Bioprocessing for tree production in the forest industry: conifer somatic embryogenesis. Biotechnol Prog 14:156–166

    CAS  Article  Google Scholar 

  • Van Winkle SC, Pullman GS (2003) The combined impact of pH and activated carbon on the elemental composition of a liquid conifer embryogenic tissue initiation medium. Plant Cell Rep 22:303–311

    Article  PubMed  Google Scholar 

  • Vondrakova Z, Eliasova K, Fischerova L, Vagner M (2011) The role of auxins in somatic embryogenesis of Abies alba. Cent Eur J Biol 6:587–596. doi:10.2478/s11535-011-0035-7

    CAS  Google Scholar 

  • Vookova B, Komutak A (2001) Effect of sucrose concentration, charcoal, and indole-3-butyric acid on germination of Abies numidica somatic embryos. Biologia Plant 44:181–184

    CAS  Article  Google Scholar 

  • Vookova B, Kormutak A (2009) Improved plantlet regeneration from open-pollinated families of Abies alba trees of Dobroc primeval forest and adjoining managed stand via somatic embryogenesis. Biologia 64:1136–1140

    Article  Google Scholar 

  • Webster FB, Roberts DR, McInnis SM, Sutton BCS (1990) Propagation of interior spruce by somatic embryogenesis. Can J For Res 20:1759–1765

    Article  Google Scholar 

  • Welty DE (2000) Method for storing and improving the survival rate of conifer somatic embryo germinants. US Patent 6134830, issued 24 October, 2000

  • Wu D, Sullards MC, Oldham DD, Gelbaum L, Pullman GS, May SW (2012) Myo-inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early-stage somatic embryos. New Phytolo 193:313–326

    CAS  Article  Google Scholar 

  • Xu N, Johns B, Pullman GS, Cairney J (1997) Rapid and reliable differential display from minute amounts of tissue: mass cloning and characterization of differentially expressed genes from loblolly pine embryos. Plant Mol Biol Rep 15:377–391

    CAS  Article  Google Scholar 

Download references

Acknowledgments

We thank Jerry Moody, Avery County Extension Agent, NCSU, Ken Roeder, Forest Geneticist, North Carolina Division of Forest Resources, and Bryan Davis, formerly NCSU Cooperative Extension Technician, for cone collections. We also thank the NC Christmas Tree Association for financial support and the Institute of Paper Science and Technology (Renewable Bioproducts Institute) and Georgia Institute of Technology for providing materials and supplies for undergraduate research in support of this project. We are grateful for the help of K-M Chase, E. Clark, J. Dregar, J. Grabowski, R. Gray, R. Gupta, C. Umejiego, and Y. Zhang.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gerald S. Pullman.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pullman, G.S., Olson, K., Fischer, T. et al. Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation. New Forests 47, 453–480 (2016). https://doi.org/10.1007/s11056-016-9525-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11056-016-9525-9

Keywords

  • Abies fraseri
  • Embryogenic tissue initiation
  • Conifer
  • Fraser fir
  • Somatic embryogenesis