, Volume 28, Issue 6, pp 1767–1776 | Cite as

Somatic embryogenesis and cryostorage of eastern hemlock and Carolina hemlock for conservation and restoration

  • Scott A. Merkle
  • Paul M. Montello
  • Hannah M. Reece
  • Lisheng Kong
Original Paper
Part of the following topical collections:
  1. Seed Biology and Micropropagation


Key message

Embryogenic cultures of eastern and Carolina hemlocks could be initiated, and somatic embryos and plantlets produced using standard conifer protocols and media. Embryogenic hemlock cultures were cryostored and recovered.


Eastern hemlock (Tsuga canadenesis) and Carolina hemlock (Tsuga caroliniana) are threatened with extirpation from their native ranges in eastern North America by the introduction of the hemlock woolly adelgid (HWA; Adelges tsugae), an exotic insect pest that has already killed millions of hemlock trees. Efforts to conserve and restore these members of the Pinaceae could be greatly enhanced by the availability of an in vitro propagation system. We conducted experiments to initiate embryogenic cultures from eastern and Carolina hemlock zygotic embryos at different stages of development using three media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BA). Cone collection date, medium and source tree had significant effects on induction of embryogenic tissue from zygotic embryo explants of both species, which ranged as high as 52 % for eastern hemlock and 17 % for Carolina hemlock. Embryogenic hemlock cultures could be cryostored using a protocol employing sorbitol and DMSO, and recovered following several months of frozen storage. Transfer of embryogenic tissue from proliferation media containing 2, 4-D and BA to a Litvay medium with abscisic acid promoted the development of somatic embryos, which were stimulated to mature by slow drying under semi-permeable plastic film. Embryos moved to an imbibition-germination medium without plant growth regulators and incubated in the light elongated and subsequently germinated. A small number of germinated embryos survived transfer to ex vitro conditions and grew into somatic seedlings. The embryogenesis and cryostorage systems developed in the study are already being integrated with hemlock breeding efforts to develop clones with resistance or tolerance to HWA.


Tsuga canadensis Tsuga caroliniana Cryopreservation Forest restoration Hemlock woolly adelgid 


Author contribution statement

SAM designed culture initiation experiments and helped design somatic embryo production experiments, conducted data analysis, took photos and wrote all drafts of the manuscript; PMM conducted culture initiation, somatic embryo production and cryopreservation experiments and took photos; HMR conducted culture initiation experiments; LK designed and conducted somatic embryo and somatic seedling production experiments and took photos. All authors approved the final draft of the manuscript.


The research reported here was supported by a grant from the USDA Forest Service—Forest Health Protection. The authors would like to thank the USDA Forest Service, the Georgia Department of Natural Resources, Blue Ridge Outdoor Education Center, Camcore, Rusty Rhea, Jim Compton, Chuck Gregory, Danny Tatum, Greg Yates, Bill Dvorak, Robert Jetton and Josh Rood for help with collecting hemlock material, Dale Smith for technical advice and Christine Holtz for help with statistical analysis.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Becwar MR, Nagmani R, Wann SR (1990) Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J For Res 20:810–817CrossRefGoogle Scholar
  2. Bozhkov PV, von Arnold S (1998) Polyethylene glycol promotes maturation but inhibits further development of Picea abies somatic embryos. Physiol Plant 104:211–224CrossRefGoogle Scholar
  3. DeVerno LL, Park YS, Bonga JM, Barrett JD (1999) Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]. Plant Cell Rep 18:948–953CrossRefGoogle Scholar
  4. Ellison AM, Bank MS, Clinton BD, Colburn EA, Elliott K, Ford CR, Foster DR, Kloeppel BD, Knoepp JD, Lovett GM, Mohan J, Orwig DA, Rodenhouse NL, Sobczak WV, Stinson KA, Stone JK, Swan CM, Thompson J, Von Holle B, Webster JR (2005) Loss of foundation species: consequences for the structure and dynamics of forested ecosystems. Front Ecol Environ 3:479–486CrossRefGoogle Scholar
  5. Finer JJ, Kriebel HB, Becwar MR (1989) Initiation of embryogenic tissue and suspension cultures of eastern white pine (Pinus strobus L.). Plant Cell Rep 8:203–206PubMedCrossRefGoogle Scholar
  6. Gupta PK, Durzan DJ (1985) Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep 4:177–179PubMedCrossRefGoogle Scholar
  7. Hargreaves C, Smith DR (1992) Cryopreservation of Pinus radiata embryogenic tissue. Comb Proc Intl Plant Prop Soc 42:327–333Google Scholar
  8. Jetton RM, Whittier WA, Dvorak WS, Rhea JR (2013) Conserved ex situ genetic resources of eastern and Carolina hemlock: eastern North American conifers threatened by the hemlock woolly adelgid. Tree Plant Notes 56:59–71Google Scholar
  9. Kartha KK, Fowke LC, Leung NL, Caswell KL, Hakman I (1988) Induction of somatic embryos and plantlets from cryopreserved cell cultures of white spruce (Picea glauca). Plant Physiol 132:529–539CrossRefGoogle Scholar
  10. Klimaszewska K, Overton C, Stewart D, Rutledge RG (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 233:635–647PubMedCrossRefGoogle Scholar
  11. 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 58:1525–1531PubMedCrossRefGoogle Scholar
  12. Kong L, von Aderkas P (2011) A novel cryopreservation method for conifer immature somatic embryos without cryoprotectant. Plant Cell Tissue Organ Cult 106:115–125CrossRefGoogle Scholar
  13. 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 93:298–304CrossRefGoogle Scholar
  14. 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 4:325–328PubMedCrossRefGoogle Scholar
  15. McClure MS, Salom SM, Shields KS (2001) Hemlock woolly adelgid. USDA Forest Service Forest Health Enterprise Technology Team Report FHTET-2001-03, MorgantownGoogle Scholar
  16. Merkle SA, Montello PM, Xia X, Upchurch BL, Smith DR (2005) Light quality treatments enhance somatic seedling production in three southern pine species. Tree Physiol 26:187–194CrossRefGoogle Scholar
  17. Mo LH, von Arnold S (1991) Origin and development of embryogenic cultures from seedlings of Norway spruce (Picea abies). J Plant Physiol 138:223–230CrossRefGoogle Scholar
  18. Olson JS, Stearns FW, Nienstaedt H (1959) Eastern hemlock seeds and seedlings: response to photoperiod and temperature. Connecticut Agric Expt Stn Bulletin 620, New HavenGoogle Scholar
  19. Potter KM, Dvorak WS, Crane BS, Hipkins VD, Jetton RM, Whittier WA, Rhea R (2008) Allozyme variation and recent evolutionary history of eastern hemlock (Tsuga canadensis) in the southeastern United States. New For 35:131–145CrossRefGoogle Scholar
  20. SAS Institute Inc (2011) SAS/STAT 9.3 User’s Guide, Cary, NC: SAS Institute IncGoogle Scholar
  21. Smith DR (1996) Growth Medium. US Patent No. 5,565,355Google Scholar
  22. Tautorus TE, Fowke LC, Dunstan DI (1991) Somatic embryogenesis in conifers. Can J Bot 69:1873–1899CrossRefGoogle Scholar
  23. Touchell DH, Chiang VL, Tsai CJ (2002) Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep 21:118–124CrossRefGoogle Scholar
  24. Vose JM, Wear DN, Mayfield AE III, Nelson CD (2013) Hemlock woolly adelgid in the southern Appalachians: control strategies, ecological impacts, and potential management responses. For Ecol Manag 291:209–219CrossRefGoogle Scholar
  25. Walter C, Grace LJ, Wagner A, White DWR, Walden AR, Donaldson SS, Hinton H, Gardner RC, Smith DR (1998) Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don. Plant Cell Rep 17:460–468CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Scott A. Merkle
    • 1
  • Paul M. Montello
    • 1
  • Hannah M. Reece
    • 1
  • Lisheng Kong
    • 1
    • 2
  1. 1.Warnell School of Forestry and Natural ResourcesUniversity of GeorgiaAthensUSA
  2. 2.Centre for Forest BiologyUniversity of VictoriaVictoriaCanada

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