| Summary: | Cell division was obtained from cultured microspores of Douglas-fir on medium supplemented with auxin, cytokinin, and sucrose, but without medium salts. Embryogenic callus was initiated from excised mature and immature zygotic embryos of Douglas-fir on media supplemented with cytokinin and auxin. Precotyledonary embryos produced most of the embryogenic calli in the cultures. Secondary embryogenic callus production, and subsequent subculturing, were required for the establishment of stable embryogenic callus lines for both mature and immature zygotic embryos. Somatic embryos at the precotyledonary stage were obtained in high frequency when Douglas-fir embryogenic callus was transferred onto hormone-free medium supplemented with 1% activated charcoal, while some cotyledonary somatic embryos were obtained from hormone-free medium supplemented with low ABA levels (0-10 uM). The level of ABA in the maturation medium significantly affected the quality of the somatic embryos produced. Cell suspensions were established from embryogenic calli and have been maintained for over one year. Protoplasts were isolated from suspension, cell colonies and calli were regenerated from protoplasts. GUS and CAT genes were successfully introduced into protoplasts of Douglas-fir via electroporation, and their transient expression was obtained 2-4 days after electroporation. The results so far indicate that the production of somatic embryos via embryogenesis in vitro is obtainable, and the application of direct gene transfer via electroporation for genetic engineering of trees in this species is promising. === Forestry, Faculty of === Graduate
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