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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

Abstract

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.

Keywords

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

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

ABA

Abscisic acid

AC

Activated charcoal

BAP

Benzylaminopurine

EM

Embryonal mass

FW

Fresh weight

MVF

Multi-varietal forestry

OP

Open-pollinated

PGR

Plant growth regulators

PPFD

Photosynthetic photon flux density

SE

Somatic embryogenesis

se

Standard error

Notes

Acknowledgments

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.

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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

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