SOMATIC EMBRYOGENESIS IN PINUS-PATULA

Embryogenic callus was initiated from cultured Pinus patula Scheide. et Deppe. megagametophyte explants containing immature zygotic embryos. An average of 2.6% of the gametophytes containing at least one embryo produced embryogenic tissue. Cones used for explant isolation were collected on a weekly basis during the summer months from December 1991 to February 1992. Embryogenic tissue was induced on both MSG and DCR (Douglas-fir Cotyledon Revised) basal media. The highest induction frequency was on a DCR1 medium supplemented with 0.5 mg L-1 BA and 3.0 mg L-1 2,4-D, using L-glutamine as the major nitrogen source. Histological examination showed that three distinct types of tissue were produced. A translucent to white mucilaginous tissue was observed proliferating at the micropylar end of the female gametophyte from the suspensor region of the embryo. This embryogenic tissue was induced in the latter half of the collection period, at a stage when cleavage polyembryony was manifest in the immature zygotic embryos, Both stage 1 (translucent suspensor) and stage 2 (opaque apex) somatic embryos were produced. Compact, crystalline non-embryogenic tissue developed from the surface of the female gametophyte throughout the collection period. The third type of tissue consisted of a mixture of embryogenic and non-embryogenic tissues, resulting in sphaeroblast production. This tissue type was usually limited to explants collected prior to the initiation of cleavage polyembryony. Anatomical studies showed the tissue types to be morphologically different, although ultrastructural differences were not as marked.