Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 98, Issue 2, pp 165–178 | Cite as

Plant regeneration in Stone pine (Pinus pinea L.) by somatic embryogenesis

  • E. Carneros
  • C. Celestino
  • K. Klimaszewska
  • Y.-S. Park
  • M. Toribio
  • J. M. Bonga
Original Paper


Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families, the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections) were obtained on a modified Litvay’s medium with 9 μM 2,4-D and 4.5 μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However, up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven converted into somatic seedlings.


Conifer Embryonal mass Genetic effect Half-sib families Somatic embryogenesis Somatic seedling Tree breeding 



2,4-Dichlorophenoxyacetic acid


Abscisic acid


Activated charcoal




Embryonal mass


Fresh weight


Multi-varietal forestry




Plant growth regulators


Photosynthetic photon flux density


Somatic embryogenesis


Standard error



The authors gratefully thank N. Cleto and Y. Vinuesa for their technical assistance. Funds were provided by projects AGL2002-00867 and AGL2005-07585, and IMIDRA and INIA grants to E. Carneros. We wish to thank the National Forest Breeding Centre “Puerta de Hierro” (Madrid) of the Spanish Ministry of Environment, and Dr. Mutke for all their help in collecting plant material. We thank the Canadian Forest Service for hosting E. Carneros at its laboratory in Fredericton.


  1. Becwar MR, Nagmani R, Wann SR (1990) Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can J Res 20:810–817. doi: 10.1139/x90-107 CrossRefGoogle Scholar
  2. Bonga JM (2004) The effect of various culture media on the formation of embryo-like structures in cultures derived from explants taken from mature Larix decidua. Plant Cell Tissue Organ Cult 77:43–48. doi: 10.1023/B:TICU.0000016488.79965.b7 CrossRefGoogle Scholar
  3. Breton D, Harvengt L, Trontin J-F et al (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. doi: 10.1007/s11240-006-9144-9 CrossRefGoogle Scholar
  4. Capuana M, Giannini R (1995) In vitro plantlet regeneration from embryonic explants of Pinus pinea L. In Vitro Cell Dev Biol Plant 31:202–206. doi: 10.1007/BF02632022 CrossRefGoogle Scholar
  5. Cheliak WM, Klimaszewska K (1991) Genetic variation in somatic embryogenic response in open-pollinated families of black spruce. Theor Appl Genet 82:185–190. doi: 10.1007/BF00226211 CrossRefGoogle Scholar
  6. Deb CR, Tandon P (2002) Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Pinus kesiya (Royle ex. Gord.). J Plant Biol 29:301–306Google Scholar
  7. Diamantoglou S, Panagopoulos I, Munoz-Ferriz A et al (1990) In vitro studies of embryo growth, callus formation and multiple bud induction of Pinus pinea L. J Plant Physiol 137:58–63Google Scholar
  8. Duchefa Biochemie BV (2003) Biochemicals, Plant cell and tissue culture, Plant molecular biochemicals. Catalogue 2003–2005. Haarlem, The NetherlandsGoogle Scholar
  9. El-Kassaby Y (2004) Feasibility and proposed outline of a global review of forest biotechnology. Forest Genetic Resources Working Paper FGR/77E: Forest Resources Development Service, Forest Resources Division. FAO, RomeGoogle Scholar
  10. Find J, Grace L, Krogstrup P (2002) Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana). Physiol Plant 116:231–237. doi: 10.1034/j.1399-3054.2002.1160213.x PubMedCrossRefGoogle Scholar
  11. Finer JJ, Kriebel HB, Becwar MR (1989) Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L). Plant Cell Rep 8:203–206. doi: 10.1007/BF00778532 CrossRefGoogle Scholar
  12. García-Férriz L, Serrano L, Pardos JA (1994) In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine. Plant Cell Tissue Organ Cult 36:135–140. doi: 10.1007/BF00048324 CrossRefGoogle Scholar
  13. Garin E, Isabel N, Plourde A (1998) Screening of large numbers of seed families of Pinus strobus L. for somatic embryogenesis from immature and mature zygotic embryos. Plant Cell Rep 18:37–43. doi: 10.1007/s002990050528 CrossRefGoogle Scholar
  14. González MV, Rey M, Tavazza R et al (1998) In vitro adventitious shoot formation on cotyledons of Pinus pinea. HortScience 33:749–750Google Scholar
  15. Gupta PK, Grob JA (1995) Somatic embryogenesis in conifers. In: Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 1. Kluwer Academic, Dordrecht, pp 81–98Google Scholar
  16. Klimaszewska K, Cyr DR (2002) Conifer somatic embryogenesis: I. Development. Dendrobiology 48:31–39Google Scholar
  17. Klimaszewska K, Smith D (1997) Maturation of somatic embryos of Pinus strobus is promoted by a high concentration of gellan gum. Physiol Plant 100:949–957. doi: 10.1111/j.1399-3054.1997.tb00022.x CrossRefGoogle Scholar
  18. Klimaszewska K, Park Y-S, Overton C et al (2001) Optimized somatic embryogenesis in Pinus strobus L. In Vitro Cell Dev Biol Plant 37:392–399. doi: 10.1007/s11627-001-0069-z CrossRefGoogle Scholar
  19. Kong L, Yeung EC (1994) Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation. Plant Sci 104:71–80. doi: 10.1016/0168-9452(94)90192-9 CrossRefGoogle Scholar
  20. Lelu MA, Bastien C, Drugeault A et al (1999) Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators. Physiol Plant 105:719–728. doi: 10.1034/j.1399-3054.1999.105417.x CrossRefGoogle Scholar
  21. Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2006) Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait). Plant Cell Rep 25:767–776. doi: 10.1007/s00299-006-0115-8 PubMedCrossRefGoogle Scholar
  22. Lelu-Walter MA, 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 92:31–45. doi: 10.1007/s11240-007-9300-x CrossRefGoogle Scholar
  23. Li X, Huang F, Gbur E (1998) Effect of basal medium, growth regulators and phytagel concentration of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L.). Plant Cell Rep 17:298–301. doi: 10.1007/s002990050396 CrossRefGoogle Scholar
  24. 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 4:325–328. doi: 10.1007/BF00269890 CrossRefGoogle Scholar
  25. MacKay JJ, Becwar MR, Park Y-S et al (2006) Genetic control of somatic embryogenesis initiation in loblolly pine and implications for breeding. Tree Genet Genomes 2:1–9. doi: 10.1007/s11295-005-0020-2 CrossRefGoogle Scholar
  26. Merkle SA, Nairn CJ (2005) Hardwood tree biotechnology. In Vitro Cell Dev Biol Plant 41:602–619. doi: 10.1079/IVP2005687 CrossRefGoogle Scholar
  27. Miguel C, Gonçalves S, Tereso S et al (2004) Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine. Plant Cell Tissue Organ Cult 76:121–130. doi: 10.1023/B:TICU.0000007253.91771.e3 CrossRefGoogle Scholar
  28. Mutke S, Gordo J, Gil L (2000) The stone pine (Pinus pinea L.) breeding programme in Castile-Leon (Central Spain). FAO-CIHEAM NUCIS-Newsletter 9:50–55Google Scholar
  29. Nehra NS, Becwar MR, Rottman WH et al (2005) Forest biotechnology: Innovative methods, emerging opportunities. In Vitro Cell Dev Biol Plant 41:701–717. doi: 10.1079/IVP2005691 CrossRefGoogle Scholar
  30. Niskanen AM, Lu J, Seitz S et al (2004) Effect of parent genotype on somatic embryogenesis in Scots pine (Pinus sylvestris). Tree Physiol 24:1259–1265PubMedGoogle Scholar
  31. Oliveira P, Barriga J, Cavaleiro C et al (2003) Sustained in vitro root development obtained in Pinus pinea L inoculated with ectomycorrhizal fungi. Forestry 76:579–587. doi: 10.1093/forestry/76.5.579 CrossRefGoogle Scholar
  32. Park Y-S (2002) Implementation of conifer somatic embryogenesis in clonal forestry: technical requirements and deployment considerations. Ann Sci 59:651–656. doi: 10.1051/forest:2002051 CrossRefGoogle Scholar
  33. Park Y-S, Pond SE, Bonga JM (1993) Initiation of somatic embryogenesis in white spruce (Picea glauca): genetic control, culture treatment effects, and implications for tree breeding. Theor Appl Genet 86:427–436. doi: 10.1007/BF00838557 CrossRefGoogle Scholar
  34. Park Y-S, Pond SE, Bonga JM (1994) Somatic embryogenesis in white spruce (Picea glauca): genetic control in somatic embryos exposed to storage, maturation treatments, germination, and cryopreservation. Theor Appl Genet 89:742–750. doi: 10.1007/BF00223714 CrossRefGoogle Scholar
  35. Park Y-S, Lelu-Walter MA, Harvengt L et al (2006) Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster and P. sylvestris at three laboratories in Canada and France. Plant Cell Tissue Organ Cult 86:87–101. doi: 10.1007/s11240-006-9101-7 CrossRefGoogle Scholar
  36. Percy RE, Klimaszewska K, Cyr DR (2000) Evaluation of somatic embryogenesis for clonal propagation of western white pine. Can J Res 30:1867–1876. doi: 10.1139/cjfr-30-12-1867 CrossRefGoogle Scholar
  37. Pullman GS, Johnson S (2002) Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation rates. Ann Sci 59:663–668. doi: 10.1051/forest:2002053 CrossRefGoogle Scholar
  38. Radojevic L, Álvarez C, Fraga MF et al (1999) Somatic embryogenic tissue establishment from mature Pinus nigra Arn. Ssp. Salzmannii embryos. In Vitro Cell Dev Biol Plant 35:206–209. doi: 10.1007/s11627-999-0078-x CrossRefGoogle Scholar
  39. Ramarosandratana A, Harvengt L, Bouvet A et al (2001a) Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos. Plant Sci 160:473–479. doi: 10.1016/S0168-9452(00)00410-6 PubMedCrossRefGoogle Scholar
  40. Ramarosandratana A, Harvengt L, Bouvet A et al (2001b) 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 37:29–34. doi: 10.1007/s11627-001-0006-1 CrossRefGoogle Scholar
  41. Salajová T, Salaj J (2005) Somatic embryogenesis in Pinus nigra: embryogenic tissue initiation, maturation and regeneration ability of established cell lines. Biol Plant 49:333–339. doi: 10.1007/s10535-005-0003-z CrossRefGoogle Scholar
  42. Tang W, Guo Z, Ouyang F (2001) Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos. In Vitro Cell Dev Biol Plant 37:558–563. doi: 10.1007/s11627-001-0097-8 CrossRefGoogle Scholar
  43. Tautorus TE, Fowke L, Dunstan DI (1991) Somatic embryogenesis in conifers. Can J Bot 69:1873–1899. doi: 10.1139/b91-237 CrossRefGoogle Scholar
  44. Valdés AE, Ordás RJ, Fernández B et al (2001) Relationships between hormonal contents and the organogenic response in Pinus pinea cotyledons. Plant Physiol Biochem 39:377–384. doi: 10.1016/S0981-9428(01)01253-0 CrossRefGoogle Scholar
  45. von Arnold S, Sabala I, Bozhkov P et al (2002) Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult 69:233–249. doi: 10.1023/A:1015673200621 CrossRefGoogle Scholar
  46. Zoglauer K, Behrendt U, Rahmat A (2003) Somatic embryogenesis—the gate to biotechnology in conifers. In: Laimer M, Rücker W et al (eds) Plant tissue culture—100 years since Gottlieb Haberlandt. Springer, Heidelberg, pp 175–202Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • E. Carneros
    • 1
  • C. Celestino
    • 1
  • K. Klimaszewska
    • 2
  • Y.-S. Park
    • 3
  • M. Toribio
    • 1
  • J. M. Bonga
    • 3
  1. 1.Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA)MadridSpain
  2. 2.Natural Resources Canada, Canadian Forest Service, Laurentian Forestry CentreQuébecCanada
  3. 3.Natural Resources Canada, Canadian Forest Service, Canadian Wood Fibre CentreFrederictonCanada

Personalised recommendations