In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

  • Chang Ho Ahn
  • Kweon Heo
  • Hyeong Soo Park
  • Yong Eui ChoiEmail author
Plant Tissue Culture


Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.


Cupressaceae Endangered conifer Embryogenic tissue Cryostorage Genetic fidelity 



This research was carried out with the support of ‘R&D Program for Forest Science Technology (Project No. FTIS 2018131B10-1820-BB01) provided by Korea Forest Service (Korea Forestry Promotion Institute). We would like to thank the National Park Research Institute of Korea and Korea National Arboretum. We also thank to Dr. Hyo-In Lim (Korea Forest Research Institute) for the assistance.

Author contributions

YEC and CHA designed all experiments and wrote all drafts of the manuscript. CHA conducted the experiments and data analysis. KH carried out cytological analysis. HSP collected KAV cones for culture initiation and provided some photos.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11627_2019_9989_MOESM1_ESM.docx (15 kb)
Table S1 (DOCX 14 kb)
11627_2019_9989_MOESM2_ESM.docx (15 kb)
Table S2 (DOCX 15 kb)


  1. Ahn CH, Choi YE (2017) In vitro clonal propagation and stable cryopreservation system for Platycladus orientalis via somatic embryogenesis. Plant Cell Tissue Org Cult 131:513–523CrossRefGoogle Scholar
  2. Ahn CH, Han JY, Kim YS, Choi YE (2019) Propagation and cryopreservation of Ulleungdo hemlock (Tsuga ulleungensis) via somatic embryogenesis. Trees-Struct Funct 32:1801–1808CrossRefGoogle Scholar
  3. Ahn CH, Kim YS, Lim S, Yi JS, Choi YE (2011) Random amplified polymorphic DNA (RAPD) analysis and RAPD-derived sequence characterized amplified regions (SCAR) marker development to identify Chinese and Korean ginseng. J Med Plant Res 5:4487–4492Google Scholar
  4. 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 Organ Cult 130:91–101CrossRefGoogle Scholar
  5. Aronen TS, Krajnakova J, Häggman HM, Ryynänen LA (1999) Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica. Plant Sci 142:163–172Google Scholar
  6. 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 87:95–108CrossRefGoogle Scholar
  7. 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 18:948–953Google Scholar
  8. Fernandes P, Rodriguez E, Pinto G, Roldán-Ruiz I, Loose MD, Santos C (2008) Cryopreservation of Quercus suber somatic embryos by encapsulation-dehydration and evaluation of genetic stability. Tree Physiol 28:1841–1850CrossRefGoogle Scholar
  9. Gupta PK, Durzan DJ (1985) Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep 4:177–179CrossRefGoogle Scholar
  10. Gupta PK, Durzan DJ (1987) Biotechnology of somatic polyembryogenesis and plantlet regeneration in loblolly pine. Nat Biotechnol 5:147–151CrossRefGoogle Scholar
  11. Harry IS, Thompson MR, Lu CY, Thorpe TA (1987) In vitro plantlet formation from embryonic explants of eastern white cedar (Thuja occidentalis L.). Tree Physiol 3:273–283CrossRefGoogle Scholar
  12. Hazubska-Przybył T, Chmielarz P, Michalak M, Bojarczuk K (2010) Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce). Plant Cell Tissue Organ Cult 102:35–44CrossRefGoogle Scholar
  13. Hu R, Sun Y, Wu B, Duan H, Zheng H, Hu D, Lim H, Tong Z, Xu J, Li Y (2017) Somatic embryogenesis of immature Cunninghamia lanceolata (Lamb.) hook zygotic embryos. Sci Rep 7:56CrossRefGoogle Scholar
  14. IUCN (2011) IUCN red list categories and criteria. Version 3.1. IUCN Species Survival Commission. IUCN, Gland, Switzerland and Cambridge, U. K.Google Scholar
  15. Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-Walter M (2009) Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.). In Vitro Cell Dev Biol-Plant 45:20–33Google Scholar
  16. Krajňáová J, Sutela S, Aronen T, Gӧmӧry D, Vianello A, Hӓggman H (2011) Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity. Cryobiology 63:17–26CrossRefGoogle Scholar
  17. Label P, Lelu M-A (1994) Influence of exogenous abscisic acid on germination and plantlet conversion frequencies of hybrid larch somatic embryos (Larix × leptoeuropaea). Plant Growth Regul 15:175–182CrossRefGoogle Scholar
  18. Lambardi M, Harry IS, Menabeni D, Thorpe TA (1995) Organogenesis and somatic embryogenesis in Cupressus sempervirens. Plant Cell Tissue Organ Cult 40:179–182CrossRefGoogle Scholar
  19. Litvay JD, Verma DC, Johnson MA (1985) Influence of 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 4:325–328CrossRefGoogle Scholar
  20. Loureiro J, Capelo A, Brito G, Rodriguez E, Silva S, Pinto G, Santos C (2007) Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry. Biol Plant 51:7–14CrossRefGoogle Scholar
  21. 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 48:324–334Google Scholar
  22. Marum L, Rocheta M, Maroco J, Oliveira MM, Miquel C (2009) Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster). Plant Cell Rep 28:673–682CrossRefGoogle Scholar
  23. Maruyama E, Tanaka T, Hosoi Y, Ishii K, Morohoshi N (2000) Embryogenic cell culture, protoplast regeneration, cryopreservation, biolistic gene transfer and plant regeneration in Japanese cedar (Cryptomeria japonica D. Don). Plant Biotechnol 17:281–296CrossRefGoogle Scholar
  24. Maruyama E, Hosoi Y, Ishii K (2002) Somatic embryogenesis in Sawara cypress (Chamaecyparis pisifera Sieb. et Zucc.) for stable and efficient plant regeneration, propagation and protoplast culture. J For Res 7:23–34CrossRefGoogle Scholar
  25. Maruyama E, Ishii K, Hosoi Y (2005) Efficient plant regeneration of Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. J For Res 10:73–77CrossRefGoogle Scholar
  26. 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 28:1767–1776CrossRefGoogle Scholar
  27. Mikuła A, Olas M, Sliwinska E, Rybczyński JJ (2008) Cryopreservation by encapsulation of Gentiana spp. cell suspensions maintains regrowth, embryogenic competence and DNA content. Cryo-Letters 29:409–418Google Scholar
  28. Nour KA, Thorpe TA (1993) In vitro shoot multiplication of eastern white cedar (Thuja occidentalis). In Vitro Cell Dev Biol-Plant 29:65–71Google Scholar
  29. Popova EV, Lee EJ, Wu CH, Hahn EJ, Paek KY (2009) A simple method for cryopreservation of Ginkgo biloba callus. Plant Cell Tissue Organ Cult 97:337–343CrossRefGoogle Scholar
  30. Pullman GS, Bucalo K (2014) Pine somatic embryogenesis: analyses of seed tissue and medium to improve protocol development. New For 45:353–377CrossRefGoogle Scholar
  31. Pullman GS, Olson K, Fischer T, Egertsdotter U, Frampton J, Bucalo K (2016) Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation. New For 47:453–480CrossRefGoogle Scholar
  32. Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Paques M (2001) Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos. In Vitro Cell Dev Biol-Plant 37:29–34Google Scholar
  33. Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204CrossRefGoogle Scholar
  34. Song JH, Lee JJ, Koo YB, Lee KY, Han SD, Yang BH (2006) Propagation by cutting method of Korea rare endemic Thuja koraiensis Nak. J Korean For Soc 95:393–397Google Scholar
  35. Urbanová M, Čellárová E, Kimáková K (2002) Chromosome number stability and mitotic activity of cryopreserved Hypericum perforatum L. meristems. Plant Cell Rep 20:1082–1086CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Chang Ho Ahn
    • 1
  • Kweon Heo
    • 2
  • Hyeong Soo Park
    • 3
  • Yong Eui Choi
    • 1
    Email author
  1. 1.Devision of Forest Resources, College of Forest and Environmental SciencesKangwon National UniversityChuncheonRepublic of Korea
  2. 2.Department of Applied Plant ScienceKangwon National UniversityChuncheonRepublic of Korea
  3. 3.Seoraksan National Park Office, Korea National Park ServiceSokchoRepublic of Korea

Personalised recommendations