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Propagation and cryopreservation of Ulleungdo hemlock (Tsuga ulleungensis) via somatic embryogenesis


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This study describes a method for the regeneration of Ulleungdo hemlock through somatic embryogenesis and a protocol for the cryopreservation of embryogenic cultures.


Ulleungdo hemlock (Tsuga ulleungensis) is a rare endemic forest species growing on Ulleung Island in Korea. Climate change and other factors such as insect pests and a low seed germination rate further endanger this species. Here, we established an in vitro propagation and cryopreservation system for Ulleungdo hemlock via somatic embryogenesis. Whole megagametophytes with zygotic embryos from immature Ulleungdo hemlock seeds were cultured on a modified Litvay medium supplemented with 9 µM 2,4-dichlorophenoxyacetic acid and 4.5 µM 6-benzylaminopurine. The cone collection date had a significant effect on the induction of embryogenic tissue (P < 0.001). Maturation medium containing Litvay salts, 50 g l−1 polyethylene glycol 4000, 30 g l−1 maltose, and abscisic acid (ABA) was used to test the effects of three different concentrations (30, 60, and 120 µM) of ABA on somatic embryo production. There were statistically significant effects of ABA concentration on somatic embryo production per 80 mg of embryogenic tissue (P < 0.001), and the most productive ABA concentration was 60 µM. We also conducted experiments to determine the effect of cryopreservation treatment on the maturation capacity of somatic embryos. Although the highest mean number of somatic embryo production per 80 mg of embryogenic tissue was obtained from a non-cryopreserved cell line (36.3 ± 3.5), no significant differences between the cryopreservation and non-cryopreservation treatment were observed (P = 0.093). Our results described here have great potential to contribute to the propagation and conservation of Ulleungdo hemlock in its native habitat.

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  • Ahn CH, Choi YE (2017) In vitro clonal propagation and stable cryopreservation system for Platycladus orientalis via somatic embryogenesis. Plant Cell Tissue Org Cult 131:513–523

    CAS  Article  Google Scholar 

  • Ahn CH, Tull R, Montello PM, Merkle SA (2017) A clonal propagation system for Atlantic white cedar (Chamaecyparis thyoides) via somatic embryogenesis without the use of plant growth regulators. Plant Cell Tissue Org Cult 130:91–101

    CAS  Article  Google Scholar 

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

    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 Lett 29:135–144

    Google Scholar 

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

    Article  Google Scholar 

  • Gupta PK, Holmstrom D (2005) Double staining technology for distinguishing embryogenic cultures. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, Dordrecht, pp 573–575

    Chapter  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 

  • Holman G, Tredici PD, Havill N, Lee NS, Cronn R, Cushman K, Mathews S, Raubeson L, Campbell CS (2017) A new species and introgression in Eastern Asian Hemlocks (Pinaceae: Tsuga). Syst Bot 42:733–746

    Article  Google Scholar 

  • IUCN (2001) IUCN red list categories and criteria. Version 3.1. IUCN Species Survival Commission, IUCN, Gland

    Google Scholar 

  • Klimaszewska K, Hargreaves CL, Lelu-Walter M-A, Trontin J-F (2016) Advances in conifer somatic embryogenesis since year 2000. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis in higher plants, methods in molecular biology, vol 1359. Springer, New York, pp 131–166

    Chapter  Google Scholar 

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

    CAS  Article  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

    CAS  Article  Google Scholar 

  • Label P, Lelu MA (2000) Exogenous abscisic acid fate during maturation of hybrid larch (Larix × leptoeuropaea) somatic embryos. Physiol Plant 109:456–462

    CAS  Article  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

    CAS  Article  Google Scholar 

  • Merkle SA, Montello PM, Reece HM, Kong L (2014) Somatic embryogenesis and cryostorage of eastern hemlock and Carolina hemlock for conservation and restoration. Trees-Struct Funct 28:1767–1776

    Article  Google Scholar 

  • Montgomery ME, Yao D, Wang H (2000) Chinese Coccinellidae for biological control of the hemlock woolly adelgid: description of native habitat. In: McManus KA, Shields KS, Souto DR (ed) Proceedings: symposium on sustainable management of hemlock ecosystems in eastern North America. General technical report NE-267. Newtown Square, PA: U.S. Department of Agriculture, Forest Service, Northeastern Forest Experiment Station, pp 97–102

  • Seidl R, Thom D, Kautz M, Martin-Benito D, Peltoniemi M, Vacchiano G, Wild J, Ascoli D, Petr M, Honkaniemi J, Lexer MJ, Trotsiuk V, Mairota P, Svoboda M, Fabrika M, Nagel TA, Reyer CPO (2017) Forest disturbances under climate change. Nat Clim Change 7:395–402

    Article  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

    CAS  Article  Google Scholar 

  • Tsuyama I, Nakao K, Higa M, Matsui T, Shichi K, Tanaka N (2003) What controls the distribution of the Japanese endemic hemlock, Tsuga diversifolia? Footprint of climate in the glacial period on current habitat occupancy. J For Res 19:154–165

    Article  Google Scholar 

  • Varis S, Ahola S, Jaakola L, Aronen T (2017) Reliable and practical methods for cryopreservation of embryogenic cultures and cold storage of somatic embryos of Norway spruce. Cryobiology 76:8–17

    CAS  Article  Google Scholar 

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This study was conducted with the support of ‘R&D Program for Forest Science Technology (Project no. FTIS 2018131B10-1820-BB01)’ provided by the Korea Forest Service (Korea Forestry Promotion Institute). This research was also supported by Grants from the Rural Development Administration, Republic of Korea [Next-Generation Bio-Green 21 Program (PJ01344401)]. We would like to thank Dr. Scott Merkle, Mr. Paul Montello, and Mr. Ryan Tull (University of Georgia, USA) for their technical assistance and advice.

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Correspondence to Yong-Eui Choi.

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The authors declare that they have no conflicts of interest to disclose.

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Communicated by Klimaszewska.

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Ahn, CH., Han, JY., Kim, YS. et al. Propagation and cryopreservation of Ulleungdo hemlock (Tsuga ulleungensis) via somatic embryogenesis. Trees 32, 1801–1808 (2018).

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  • Conifer
  • Endangered species
  • Embryogenic tissue initiation
  • Cryopreservation
  • Abscisic acid