Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 132, Issue 1, pp 137–155 | Cite as

High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in Pseudotsuga menziesii [Mirb.]

  • Marie-Anne Lelu-WalterEmail author
  • Florian Gautier
  • Kateřina Eliášová
  • Leopoldo Sanchez
  • Caroline Teyssier
  • Anne-Marie Lomenech
  • Claire Le Metté
  • Cathy Hargreaves
  • Jean-François Trontin
  • Cathie Reeves
Original Article


Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L−1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g−1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g−1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g−1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg−1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg−1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.


Cell density Cleavage polyembryony Douglas-fir Embryogenic potential Protein pattern Vegetative propagation 



This research was partially funded by Future Forests Research Limited and a grant from the French Ministry of Foreign Affairs and the French Ministry of Higher Education and Research, and Technology Support Programme and Core funding provided by The Ministry of Business, Innovation and Employment in New Zealand through the France/New Zealand Science Cooperation Programme Dumont d’Urville (No. 25815PH). We would like to acknowledge the support of the University of Limoges, and The Chair of Excellence Forest Resources and Wood Uses, for the grant to Florian Gautier.

Author contributions

MALW designed and coordinated the study, carried out somatic embryogenesis and drafted the manuscript. FG participated in somatic embryogenesis and helped to draft the manuscript. KE performed histological and microscopic analyses and drafted the manuscript. LS performed the statistical analyses and drafted the manuscript. CT performed protein analysis and drafted the manuscript. AML carried out mass spectrometric analysis and helped to draft the manuscript. CLM carried out somatic embryogenesis and collected the material. CH participated in the design of the study and helped to draft the manuscript. JFT participated in the design of the study and drafted the manuscript. CR performed English editing and drafted the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2017_1318_MOESM1_ESM.docx (45 kb)
Supplementary material 1 (DOCX 44 KB)
11240_2017_1318_MOESM2_ESM.docx (2.2 mb)
Supplementary material 2 (DOCX 2299 KB)


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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Marie-Anne Lelu-Walter
    • 1
    Email author
  • Florian Gautier
    • 1
    • 2
  • Kateřina Eliášová
    • 3
  • Leopoldo Sanchez
    • 1
  • Caroline Teyssier
    • 1
  • Anne-Marie Lomenech
    • 4
  • Claire Le Metté
    • 1
  • Cathy Hargreaves
    • 5
  • Jean-François Trontin
    • 6
  • Cathie Reeves
    • 5
  1. 1.INRA, UR 0588, Amélioration, Génétique et Physiologie ForestièreOrléans Cedex 2France
  2. 2.Université de Limoges, Laboratoire de Chimie des Substances NaturellesLimogesFrance
  3. 3.Institute of Experimental Botany CASPraha 6-LysolajeCzech Republic
  4. 4.Université de Bordeaux, Centre de Génomique Fonctionnelle, Plateforme ProtéomeBordeauxFrance
  5. 5.ScionRotoruaNew Zealand
  6. 6.FCBA, Pôle Biotechnologie et Sylviculture AvancéeCestasFrance

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