, Volume 32, Issue 6, pp 1489–1504 | Cite as

Improved germination conditions for Norway spruce somatic cotyledonary embryos increased survival and height growth of emblings

  • Mikko TikkinenEmail author
  • Saila Varis
  • Heli Peltola
  • Tuija Aronen
Original Article
Part of the following topical collections:
  1. Seed Biology and Micropropagation


Norway spruce is one of the most cultivated tree species in Nordic countries. However, intermittent shortages of improved seeds occur. As a powerful vegetative propagation technology, somatic embryogenesis (SE) could provide an alternative solution for this problem and also shorten the time required to obtain breeding gains. However, there are still large bottlenecks in SE, e.g. in the germination and acclimatization phases, which greatly affect the final outcome of somatic embryo plants (emblings). In this work, we examined the effects of in vitro embryo storage and germination treatments and ex vitro growing techniques on the survival and growth of emblings. The study comprised 32 genotypes from 18 full-sib families in four experiments, testing two different cold storage methods, three durations of in vitro germination, lower inorganic nitrogen content in the germination medium, and two plant-growing techniques. The best treatment combination—cold storage on filter paper, lower nitrogen content in the germination medium and one-week in vitro germination—resulted in an 88% higher survival and 28% higher growth compared to the poorest, reference treatment in the same test year. These emblings could be planted after a nursery period one year sooner than that of the control emblings. The results indicate that Norway spruce emblings germinated for one week in vitro can be transplanted and grown in nurseries without any additional treatments or environmental control differing from seedlings, which is a prerequisite to reach standards for forest regeneration material.


Somatic embryogenesis Picea abies Cold storage Germination Nitrogen Acclimatization Forest biotechnology 



The authors would like to acknowledge the editor and referees for their dedication to improve the manuscript with valuable comments.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

468_2018_1728_MOESM1_ESM.pdf (42 kb)
Appendix 1 Average length of shoot (positive scale) and root (negative scale) after in vitro germination treatments (1w-gel, 3w-gel and 5w-gel) in different genotypes in Experiment II. Average values for root and shoot length after in vitro germination are presented with standard error. The genotypes are organized in the same order as in Fig. 3 except the three additional genotypes included in Experiment II are added in the right (PDF 42 KB)
468_2018_1728_MOESM2_ESM.pdf (20 kb)
Appendix 2 Logistic regression models used for analyzing binary response (living or dead) in the nursery testing of emblings after first growth season. In the models, e1 is a design variable for embryo length before germination, g1 to g5 are design variables for germination treatments, c1 is a design variable for preservation method of embryogenic cell mass, and a1a21 are design variables for genotypes (PDF 20 KB)
468_2018_1728_MOESM3_ESM.pdf (27 kb)
Appendix 3 Mean height and survival of genotypes in different treatments in Experiment III. Average values are presented with standard error (PDF 27 KB)


  1. Adams GW, Kunze HA, McCartney A, Millican S, Park YS (2016) An industrial perspective on the use of advanced reforestation stock technologies. In: P YS, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. Korea Forest Research Institute, Seoul, pp 323–334Google Scholar
  2. Belmonte MF, Yeung EC (2004) The effects of reduced and oxidized glutathione on white spruce somatic embryogenesis. In Vitro Cell Dev Biol Plant 40(1):61–66CrossRefGoogle Scholar
  3. Bergsten U (1989) Temperature tolerance of invigorated seeds of Pinus sylvestris L. and Picea abies (L.) Karst. using TTGP-test. For Suppl 62:107–115Google Scholar
  4. Bonga JM (2015) A comparative evaluation of the application of somatic embryogenesis, rooting of cuttings, and organogenesis of conifers. Can J For Res 45:379–383CrossRefGoogle Scholar
  5. 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
  6. Brouwer R (1962) Nutritive influences on the distribution of dry matter in the plant. Neth J Agric Sci 10:361–376Google Scholar
  7. Carneros E, Yakovlev I, Viejo M, Olsen JE, Fossdal CG (2017) The epigenetic memory of temperature during embryogenesis modifies the expression of bud burst-related genes in Norway spruce epitypes. Planta 246:553–566CrossRefGoogle Scholar
  8. Carson M, Carson S, Te Riini C (2015) Successful varietal forestry with radiata pine in New Zealand. N Z J For 60(1):8–11Google Scholar
  9. Cen Y-P, Bornman JF (1990) The response of bean plants to UV-B radiation under different irradiances of background visible light. J Exp Bot 41(11):1489–1495CrossRefGoogle Scholar
  10. Chalupa V (1985) Somatic embryogenesis and plantlet regeneration from cultured immature and mature embryos of Picea abies (L.) Karst. Commun Inst For Czech Repub 14:57–63Google Scholar
  11. Grossnickle SC, Cyr D, Polonenko DR (1996) Somatic embryogenesis tissue culture for the propagation of conifer seedlings: a technology comes of age. Tree Planters’ Notes 47(2):48–57Google Scholar
  12. Gruffman L, Ishida T, Nordin A, Näsholm T (2012) Cultivation of Norway spruce and Scots pine on organic nitrogen improves seedling morphology and field performance. For Ecol Manage 276:118–124CrossRefGoogle Scholar
  13. 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 38:53–59CrossRefGoogle Scholar
  14. Hazubska-Przybył T, Wawrzyniak M, Obarska A, Bojarczuk K (2015) Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce. Acta Physiol Plant 37:1–9CrossRefGoogle Scholar
  15. Heiskanen J (1993) Favourable Water and Aeration Conditions for Growth Media used in Containerized Tree Seedling Production: A Review. Scand J For Res 8:337–358CrossRefGoogle Scholar
  16. Högberg K-A (2003) Possibilities and limitations of vegetative propagation of Norway spruce. Acta Universitatis Agriculturae Sueciae, Silvestria 294 Dissertation, Uppsala: Swedish University of Agricultural SciencesGoogle Scholar
  17. Högberg K-A, Bozhkov PV, Grönroos R, von Arnold S (2001) Critical factors affecting ex vitro performance of somatic embryo plants of Picea abies. Scand J For Res 16:295–304CrossRefGoogle Scholar
  18. Högberg K-A, Bozhkov PV, von Arnold S (2003) Early selection improves clonal performance and reduces intraclonal variation of Norway spruce plants propagated by somatic embryogenesis. Tree Physiol 23:211–216CrossRefGoogle Scholar
  19. Ingestad T, Kähr M (1985) Nutrition and growth of coniferous seedlings at varied relative nitrogen addition rate. Physiol Plant 65:109–116CrossRefGoogle Scholar
  20. Jain SM, Gupta PK, Newton RJ (1995) Somatic embryogenesis in woody plants, Gymnosperms, vol 3. Kluwer Academic Publishers, DordrechtGoogle Scholar
  21. Jansson G, Hansen JK, Haapanen M, Kvaalen H, Steffenrem A (2017) The genetic and economic gains from forest tree breeding programmes in Scandinavia and Finland. Scand J For Res 32(4):273–286CrossRefGoogle Scholar
  22. Kaakinen S, Jolkkonen A, Iivonen S, Vapaavuori E (2004) Growth, allocation and tissue chemistry of Picea abies seedlings affected by nutrient supply during the second growing season. Tree Physiol 24:707–719CrossRefGoogle Scholar
  23. Klimaszewska K, Smith DR (1997) Maturation of somatic embryos of Pinus strobus is promoted by a high concentration of gellan gum. Physiol Plant 100:949–957CrossRefGoogle Scholar
  24. Klimaszewska K, Lachance D, Pelletier G, Lelu A-M, Seguin A (2001a) Regeneration of transgenic Picea glauca, P. mariana. and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens. In Vitro Cell Dev Biol Plant 37:748–755CrossRefGoogle Scholar
  25. Klimaszewska K, Park Y-S, Overton C, Maceacheron I, Bonga JM (2001b) Optimized somatic embryogenesis in Pinus strobus L. Vitro Cell Dev Biol Plant 37:392–399. CrossRefGoogle Scholar
  26. Klimaszewska K, Trontin J-F, Becwar MR, DeVillard C, Park Y-S, Lelu-Walter M-A (2007) Recent progress in somatic embryogenesis of four Pinus spp. Tree For Sci Biotechnol 1:11–25Google Scholar
  27. Kozlowski TT, Pallardy SG (1984) Effect of flooding on water, carbohydrate, and mineral relations. Flooding and Plant growth. Kozlowski T (ed), T. Academic Press, Orlando, pp 165–193Google Scholar
  28. Kvaalen H, Johnsen O (2008) Timing of bud set in Picea abies is regulated by a memory of temperature during zygotic and somatic embryogenesis. New Phytol 177:49–59PubMedGoogle Scholar
  29. Lamhamedi MS, Chamberland H, Tremblay FM (2003) Epidermal transpiration, ultrastructural characteristics and net photosynthesis of white spruce somatic seedlings in response to in vitro acclimatization. Physiol Plant 118:554–561. CrossRefGoogle Scholar
  30. Landis TD, Dumroese RK, Haase D (2010a) The Container Tree nursery manual. Containers and Growing Media. Agricultural handbook, vol 2. U.S. Department of Agriculture, Forest Service, Washington, DC, p 674Google Scholar
  31. Landis TD, Dumroese RK, Haase D (2010b) The Container Tree nursery manual. Atmospheric environment. Agricultural handbook, vol 3. U.S. Department of Agriculture, Forest Service, Washington, DC. p 674Google Scholar
  32. Landis TD, Dumroese RK, Haase D (2010c) The Container Tree nursery manual. Seedling Nutrition and Irrigation. Agricultural handbook, vol 4. U.S. Department of Agriculture, Forest Service, Washington, DC, p 674Google Scholar
  33. Landis TD, Dumroese RK, Haase D (2010d) The Container Tree nursery manual. Seedling Propagation. Agricultural handbook, vol 6. U.S. Department of Agriculture, Forest Service, Washington, DC, p 674Google Scholar
  34. Leinonen K, Nygren M, Rita H (1993) Temperature control of germination in the seeds of Norway spruce (Picea abies (L.) Karst.). Scand J For 8:107–117CrossRefGoogle Scholar
  35. Lelu M-A, Bastien C, Klimaszewska K, Charest PJ (1994) An improved method for somatic plantlet production in hybrid larch (Larix½leptoeuropaea): part 2. Control of germination and plantlet development. Plant Cell Tissue Organ Cult 36:117–127CrossRefGoogle Scholar
  36. Lelu-Walter M-A, Bernier-Cardou M, Klimaszewska K (2008) Clonal plant production from self- and cross-pollinated seed families of Pinus sylvestris (L.) through somatic embryogenesis. Plant Cell Tissue Organ Cult 92:31–45CrossRefGoogle Scholar
  37. Lelu-Walter M-A, Thompson D, Harvengt L, Sanchez L, Toribio M, Pâques LE (2013) Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genet Genomes 9:883–899CrossRefGoogle Scholar
  38. Lelu-Walter M-A, Klimaszewska K, Miguel C, Aronen T, Hargreaves C, Teyssier C, Trontin J-F (2016) Somatic embryogenesis for more effective breeding and deployment of improved varieties in Pinus sp.: bottlenecks and recent advances. In: Loyola-Vargas VM, Ochoa-Alejo N (eds), Somatic embryogenesis—fundamental aspects and applications, Chap 19. Springer, New York, pp 319–365. CrossRefGoogle Scholar
  39. Libby WJ, Jund E (1962) Variance associated with cloning. Heredity 17:533–540CrossRefGoogle Scholar
  40. 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 wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328CrossRefGoogle Scholar
  41. Llebrés MT, Avila C, Cánovas FM, Klimaszewska K (2018a) Root growth of somatic plants of hybrid Pinus strobus (L.) and P. wallichiana (A. B. Jacks.) is affected by the nitrogen composition of the somatic embryo germination medium. Trees 32(2):471–484. CrossRefGoogle Scholar
  42. Llebrés MT, Pascual MB, Debille S, Trontin J-F, Harvengt L, Avila C, Cánovas FM (2018b) The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.). Tree Physiol 38(3):371–381. CrossRefGoogle Scholar
  43. Majada JP, Centeno ML, Feito I, Fernandez B, Sanchez-Tames R (1998) Stomatal and cuticular traits on carnation tissue culture under different ventilation conditions. Plant Growth Regul 25:113–121CrossRefGoogle Scholar
  44. Majada JP, Sierra MI, Sanchez-Tames R (2001) Air exchange rate affects the in vitro developed leaf cuticle of carnation. Sci Hort 87:121–130CrossRefGoogle Scholar
  45. Morel A, Trontin J-F, Corbineau F, Lomenech A-M, Beaufour M, Reymond I, Le Metté C, Ader K, Harvengt L, Cadène M, Label P, Teyssier C, Lelu-Walter M-A (2014) Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses. Planta 240(5):1075–1095. ( CrossRefPubMedGoogle Scholar
  46. Nygren M (2003) Metsäpuiden siemenopas [Seedling guide for forest trees]. Metsäntutkimuslaitoksen tiedonantoja 882, p 144. ISBN 951-40-1869-9, ISSN 0358-4283Google Scholar
  47. Rikala R (2012) Metsäpuiden paakkutaimien kasvatusopas [Container Seedling Growing Manual for Forest Trees]. The Finnish Forest Research Institute, Suonenjoki, p 247Google Scholar
  48. Ritchie GA, Short KC, Davey MR (1991) In vitro acclimatization of Chrysanthemum and sugar beet plantlets by treatment with paclobutrazol and exposure to reduced humidity. J Exp Bot 42:1557–1563CrossRefGoogle Scholar
  49. Rook DA (1991) Seedling development and physiology in relation to mineral nutrition. In: Mineral nutrition of conifer seedlings / Eds. Van den Driessche. CRC Press, Boca Raton, Ann Arbor, Boston, pp 85–112Google Scholar
  50. Spittlehouse DL, Steward RB (2003) Adaptation to climate change in forest management. BC J Ecosyst Manag 4(1):1–11Google Scholar
  51. Stasolla C, Yeung EC (2003) Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult 74:15–35CrossRefGoogle Scholar
  52. Sutter E, Langhans RW (1979) Epicuticular wax formation on carnation plantlets regenerated from shoot tip culture. J Am Soc Hort Sci 104:493–496Google Scholar
  53. Svobodová H, Albrechtová J, Kumstýřová L, Lipavská H, Vágner M, Vondráková Z (1999) Somatic embryogenesis in Norway spruce: anatomical study of embryo development and influence of polyethylene glycol on maturation process. Plant Physiol Biochem 37(3):209–221CrossRefGoogle Scholar
  54. Thompson D (2013) Development of improved Sitka spruce for Ireland. Ir For 70:(1):2): 104–118Google Scholar
  55. Tikkinen M, Varis S, Peltola H, Aronen T (2017) Norway spruce emblings as cutting donors for tree breeding and production. Scand J For Res 33(3):207–214. CrossRefGoogle Scholar
  56. Turtola S, Sallas L, Holopainen JK, Julkunen-Tiitto R, Kainulainen P (2006) Long-term exposure to enhanced UV-B radiation has no significant effects on growth or secondary compounds of outdoor-grown Scots pine and Norway spruce seedlings. Environ Pollut 144:166–171CrossRefGoogle Scholar
  57. Varis S, Heiska S, Aronen T (2014) Kuusen solukkolisäys [Somatic embryogenesis of Norway spruce]. Working Papers of the Finnish Forest Research Institute, vol 310, p 50. Internet publication available at: Accessed 28 June 2018
  58. 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 76:8–17CrossRefGoogle Scholar
  59. von Arnold S, Hakman I (1988) Regulation of somatic embryo development in Picea abies by abscisic acid (ABA). J Plant Physiol 132:164–169CrossRefGoogle Scholar
  60. von Aderkas P, Kong L, Prior NA (2016) In vitro techniques for conifer embryogenesis. In: Park YS, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. Korea Forest Research Institute, Seoul, pp 335–350Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Natural Resources Institute Finland (Luke), Production SystemsPunkaharjuFinland
  2. 2.Faculty of Science and Forestry, School of Forest SciencesUniversity of Eastern Finland (UEF)JoensuuFinland

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