Somatic embryogenesis in immature cotyledons of Manchurian ash (Fraxinus mandshurica Rupr.)

Somatic embryogenesis was obtained from immature cotyledon explants that were cultured on half-strength Murashige and Skoog (MS) salts and vitamins with 5.4 μM naphthaleneacetic acid (NAA) and 0.2 μM thidiazuron (TDZ) plus a 4 × 4 factorial combination of 0, 9.8, 24.6, or 49.2 μM indole-3-butyric acid (IBA) and 0, 8.9, 22.2, or 44.4 μM 6-benzyladenine (BA). The addition of 44.4 μM BA improved the percentage of cotyledon explants that produced somatic embryos to >20%, if 9.8 or 24.6 μM IBA was also present. Somatic embryogenesis was >30% when seeds were harvested on 31 July or 15 August. The addition of 50 or 70 g l⁻¹ sucrose enhanced embryogenesis. Histological examination showed that somatic embryos originated from epidermis cells of zygotic embryos. A peak germination rate (69%) was attained when somatic embryos were desiccated for 10 min before they produced green cotyledons and elongating shoot tips. Of the germinated embryos from this desiccation treatment, 65.6% also grew roots and therefore converted into plants. Chilling somatic embryos at 4°C for 15 days resulted in the highest germination rate (69.4%), which was significantly higher than those without chilling treatment (27.6%). However <10% of the chilled germinated embryos formed roots and grew into plants. Plantlets from somatic embryos were transplanted into a 2 vermiculite: 1 sphagnum peat medium, where they had a survival rate of 80.8%, and had no morphological abnormalities.