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

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 ce ll “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 (30-39 478 SE g-1) displayed significantly higher embryogenic yield (148-1343 SE g-1). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation 41 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