Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium

The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor tree (Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium, on embryogenesis efficiency was assessed. The embryogenesis frequency was improved from 16.29 to 93.27%, while the average number of somatic embryos per explant increased from 3 to 12.57. Activated charcoal (AC), medium renewal, basal medium, light conditions and sucrose concentration in culture medium were also evaluated for their effect on somatic embryogenesis. AC addition and 10-day medium renewal did not increase embryogenesis efficiency significantly, and Murashige and Skoog (MS) medium proved to be more beneficial for somatic embryo formation than others. No differences were found between embryogenesis frequencies when cultured in darkness or under light, but culturing under light yielded more embryos. After the sucrose solution pretreatment, high level concentration of sucrose in induction medium was not needed for somatic embryogenesis, for it had a negative effect on somatic embryo formation when the concentration of sucrose was higher than 50 g l⁻¹. The derived embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium. Low ratio formation of embryogenic callus was observed on the surface of somatic embryos both on induction and proliferation medium. Plantlets derived from somatic embryos grew vigorously with normal appearance similar to germinated zygotic embryos.