Cryopreservation of Hybrid Pinus elliottii × P. caribaea

  • Liliana MarumEmail author
  • Sandra Nunes
  • Tânia Almeida
  • Vanessa Tolentino Pereira
  • Nelson Farinha
  • Maria Celeste Dias
  • Conceição Santos
Part of the Forestry Sciences book series (FOSC, volume 84)


The cryopreservation of embryogenic cultures within operational forestry proves to be a crucial tool, to store clones without loss of juvenility, while field tests, to identify genotypes with significant genetic gain, are being conducted. Besides several conifers species have been cryopreserved till now, only few studies have been reported for hybrid species. The interspecific pine hybrid, P. elliottii var. elliottii x P. caribaea var. hondurensis has a major economic importance mainly in South America, West Africa and Australia. The hybrid superiority appears to be derived from a complementary recombination of traits from the two parental species–growth rate and high yield of resin production from P. caribaea var. hondurensis, combined with wind-firmness, adaptability to wet sites, high wood-density and stem straightness of P. elliottii var. elliottii. This chapter describes a complete cryopreservation procedure for embryonal mass of the hybrid P.elliottii var. elliottii x P. caribaea var. hondurensis, and a methodology to analyse the ploidy stability by flow cytometry.


Cryopreservation Cryoprotectors Pine hybrid P. elliottii × P. caribaea Genome stability Ploidy level 



2,4-dichlorophenoxyacetic acid


Abscisic acid




Dimethyl sulfoxide


Embryogenic cell line


Embryonal mass


Modified Litvay’s medium


Multi-varietal forestry


Modified Litvay medium


Polyethylene glycol


Propidium iodide


Plant growth regulators


Somatic embryogenesis


Pinus elliottii var. elliottii


Pinus caribaea var. hondurensis



This research was suported by CENTRO-07-0202-FEDER-018579- “I & D em tecnologias e técnicas de clonagem ‘in vitro’, micropropagação e clonagem de plantas e genótipos”, co-funded by QREN, “Programa Mais Centro-Operacional Regional do Centro”, EU through European Regional Development Fund (FEDER), and FCT/MEC and FEDER, PT2020 Partnership Agreement & COMPETE 2020; POCI/01/0145/FEDER/007265, UID/QUI/50006/2013, UID/BIA/04004/2013, UID/QUI/00062/2013. F.C.T. funded M.C. Dias fellowship SFRH/BPD/100865/2014. Authors thank to Eng. Cláudio Pinheiro from Brazilian company Resisul for the supply of seeds and to Klon’s technicians D. Sousa, J. Figueiredo and N. Mano for technical support.


  1. Cappa EP, Marcó M, Garth Nikles D, Last IS (2013) Performance of Pinus elliottii, Pinus caribaea, their F1, F2 and backcross hybrids and Pinus taeda to 10 years in the Mesopotamia Region, Argentina. New For 44(2):197–218CrossRefGoogle Scholar
  2. Dieters M, Brawner J (2007) Productivity of Pinus elliottii, P. caribaea and Their F1 and F2 hybrids to 15 years in Queensland, Australia. Ann For Sci 64(7):691–698CrossRefGoogle Scholar
  3. Doležel J, Bartoš JAN (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95(1):99–110CrossRefPubMedPubMedCentralGoogle Scholar
  4. Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plant 85(4):625–631CrossRefGoogle Scholar
  5. Gauchat M, Rodríguez G, Belaber E, Bischoff D (2005) Pinus elliottii var. elliottii × P. caribaea var. honduresnsis. Hîbridos de Alta Productividad Combinando Crecimiento Y Forma. IDIA XXI Forestales 8:162–164Google Scholar
  6. Häggman HM, Ryynänen LA, Aronen TS, Krajnakova J (1998) Cryopreservation of embryogenic cultures of Scots pine. Plant Cell, Tissue Organ Cult 54(1):45–53CrossRefGoogle Scholar
  7. Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. Cryo Lett 25(1):3–22Google Scholar
  8. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43(2–3):179–188CrossRefPubMedGoogle Scholar
  9. Klimaszewska K, Hargreaves C, Lelu-Walter M-A, Trontin J-F (2016) Advances in Conifer somatic embryogenesis since year 2000. Methods in Mol Biol (Clifton, NJ) 1359:131–166Google Scholar
  10. Klimaszewska K, Park Y-S, Overton C, Maceacheron I, Bonga JM (2001) Optimized somatic embryogenesis in Pinus strobus L. In Vitro Cell Dev Biol—Plant 37(3):392–399CrossRefGoogle Scholar
  11. Krajňáková J, Sutela S, Aronen T, Gömöry D, Vianello A, Häggman H (2011) Long-term cryopreservation of Greek Fir embryogenic cell lines: recovery, maturation and genetic fidelity. Cryobiology 63(1):17–25CrossRefPubMedGoogle Scholar
  12. Latutrie M, Aronen T (2013) Long-term cryopreservation of embryogenic Pinus sylvestris cultures. Scand J For Res 28(2):103–109CrossRefGoogle Scholar
  13. 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 spp.: bottlenecks and recent advances. In: Somatic embryogenesis: fundamental aspects and applications. Springer International Publishing, Cham, pp 319–365CrossRefGoogle 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(6):325–328CrossRefPubMedGoogle Scholar
  15. Loureiro J, Rodriguez E, Dolezel J, Santos C (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100(4):875–888CrossRefPubMedPubMedCentralGoogle Scholar
  16. Martinez-Montero M, Harding K (2015) Cryobionomics: evaluating the concept in plant cryopreservation. In: Barh D, Khan MS, Davies E (eds) PlantOmics: the Omics of plant science. Springer India, New Delhi, pp 655–682Google Scholar
  17. Miguel C, Marum L (2011) An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J Exp Bot 62(11):3713–3725CrossRefPubMedGoogle Scholar
  18. Miura A, Yonebayashi S, Watanabe K, Toyama T, Shimada H, Kakutani T (2001) Mobilization of transposons by a mutation abolishing full DNA methylation in arabidopsis. Nature 411(6834):212–214CrossRefPubMedGoogle Scholar
  19. Nikles D (2000) Experience with some pinus hybrids in Queensland, Australia. In: Dungey H, Dieters M, Nikles D (eds.) Proceedings of QFRI/CRC-SPF symposium: hybrid breeding and genetics of forest trees, held 2000 at Noosa, Queensland, Australia. Department of Primary Industries, Brisbane, pp 27–43Google Scholar
  20. Nunes S, Marum L, Farinha N, Pereira VT, Almeida T, Dias MC, Santos C (2017a) Plant regeneration from ploidy-stable cryopreserved embryogenic lines of the hybrid Pinus elliottii × P. caribaea. Ind Crops Prod 105:215–224CrossRefGoogle Scholar
  21. Nunes S, Marum L, Farinha N, Pereira VT, Almeida T, Sousa D, Mano N, Figueiredo J, Dias MC, Santos C (2017b) Somatic embryogenesis of hybrid Pinus elliottii var. elliottii × P. caribaea var. hondurensis and ploidy assessment of somatic plants. Plant Cell, Tissue and Organ Cult (PCTOC) 132:71–84CrossRefGoogle Scholar
  22. Ozudogru EA, Lambardi M (2016) Cryotechniques for the long-term conservation of embryogenic cultures from woody plants. Methods in Mol Biol (Clifton, NJ) 1359:537–550Google Scholar
  23. Park Y-S, Beauliau J, Bousquet J (2016) Multi-varietal forestry integrating genomic selection and somatic embryogenesis. In: Park Y-S, Bonga J, Moon H (eds) Vegetative propagation of forest trees, held 2016 at Seoul, South Korea. National Institute of Forest Science (NIFoS), pp 302–322Google Scholar
  24. Park YS, Barrett JD, Bonga JM (1998) Application of somatic embryogenesis in high-value clonal forestry: deployment, genetic control, and stability of cryopreserved clones. In Vitro Cell Dev Biol—Plant 34(3):231–239CrossRefGoogle Scholar
  25. Peredo EL, Arroyo-García R, Reed BM, Revilla MÁ (2008) Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.). Cryobiology 57(3):234–241CrossRefPubMedGoogle Scholar
  26. Roux J, Eisenberg B, Kanzler A, Nel A, Coetzee V, Kietzka E, Wingfield MJ (2007) Testing of selected South African Pinus hybrids and families for tolerance to the pitch canker pathogen, Fusarium circinatum. New For 33(2):109–123Google Scholar
  27. Salaj T, Matušíková I, Fráterová L, Piršelová B, Salaj J (2011) Regrowth of embryogenic tissues of Pinus nigra following cryopreservation. Plant Cell, Tissue and Organ Cult (PCTOC) 106(1):55–61CrossRefGoogle Scholar
  28. Schellenbaum P, Mohler V, Wenzel G, Walter B (2008) Variation in DNA methylation patterns of grapevine somaclones (Vitis Vinifera L.). BMC Plant Biol 8(1):78CrossRefPubMedPubMedCentralGoogle Scholar
  29. Schenone, R.A. and Pezzutti, R. V. (2003) ‘Productividad de Progenies de Pinus elliottii × Pinus caribaea var. hondurensis’. In: XII Congreso Forestal Mundial, held 2003 at Quebéc City, Canada, 1Google Scholar
  30. Shepherd M, Cross M, Dieters M, Henry R (2002) Branch architecture QTL for Pinus elliottii var. elliottii × Pinus caribaea var. hondurensis Hybrids. Ann For Sci 59(5–6):617–625Google Scholar
  31. Shepherd M, Cross M, Dieters M, Toon P, Harding K, Nikles D, Henry R, Haines R (1999) Genetic mapping of wood properties in Pinus elliottii var. elliottii × P. Caribaea var. hondurensis hybrids. In: Proceedings of the 25th Biennial Southern forest tree improvement conferenceGoogle Scholar
  32. Slee MU (1970) Crossability values within the Slash-caribbean pinus species complex. Euphytica 19(2):184–189CrossRefGoogle Scholar
  33. Trueman SJ (2006) Clonal propagation and storage of subtropical pines in Queensland, Australia. South African For J 208(1):49–52Google Scholar
  34. van der Sijde HA, Roelofsen JW (2010) The potential of pine hybrids in South Africa. South African For J 136(1):5–14; 1986Google Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Liliana Marum
    • 1
    • 5
    • 6
    Email author
  • Sandra Nunes
    • 1
  • Tânia Almeida
    • 1
  • Vanessa Tolentino Pereira
    • 1
  • Nelson Farinha
    • 1
  • Maria Celeste Dias
    • 2
    • 3
  • Conceição Santos
    • 4
  1. 1.KLÓN—Innovative Technologies from CloningCantanhedePortugal
  2. 2.Department of Life ScienceCentre for Functional Ecology (CFE), University of CoimbraCoimbraPortugal
  3. 3.Department of Chemistry and QOPNAUniversity of AveiroAveiroPortugal
  4. 4.Department of Biology & LAQV/REQUIMTE, Faculty of SciencesUniversity of PortoPortoPortugal
  5. 5.Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL), Instituto Politécnico de Beja (IPBeja)BejaPortugal
  6. 6.Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de ÉvoraÉvoraPortugal

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