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

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Abstract

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.

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Acknowledgements

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.

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Authors and Affiliations

  1. INRA, UR 0588, Amélioration, Génétique et Physiologie Forestière, 2163 Avenue de la Pomme de pin, CS 40 001 Ardon, 45075, Orléans Cedex 2, France

    Marie-Anne Lelu-Walter, Florian Gautier, Leopoldo Sanchez, Caroline Teyssier & Claire Le Metté

  2. Université de Limoges, Laboratoire de Chimie des Substances Naturelles, 123 Avenue Albert Thomas, 87000, Limoges, France

    Florian Gautier

  3. Institute of Experimental Botany CAS, Rozvojová 263, 165 02, Praha 6-Lysolaje, Czech Republic

    Kateřina Eliášová

  4. Université de Bordeaux, Centre de Génomique Fonctionnelle, Plateforme Protéome, 33000, Bordeaux, France

    Anne-Marie Lomenech

  5. Scion, Private Bag 3020, Rotorua, 3046, New Zealand

    Cathy Hargreaves & Cathie Reeves

  6. FCBA, Pôle Biotechnologie et Sylviculture Avancée, Campus Forêt-Bois de Pierroton, 71 Route d’Arcachon, 33610, Cestas, France

    Jean-François Trontin

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  1. Marie-Anne Lelu-Walter
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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.

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Correspondence to Marie-Anne Lelu-Walter.

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Communicated by Sergio J. Ochatt.

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Lelu-Walter, MA., Gautier, F., Eliášová, K. et al. High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in Pseudotsuga menziesii [Mirb.]. Plant Cell Tiss Organ Cult 132, 137–155 (2018). https://doi.org/10.1007/s11240-017-1318-0

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