Somatic embryogenesis of Pinus patula Scheide et Deppe.
Immature, green female cones of Pinus patula Scheide et Deppe. were collected on a weekly basis during the South African summer months from December 1991 to February 1992 and from December 1992 to March 1993. Embryogenic tissue was initiated from excised megagametophyte explants containing immature zygotic embryos. Embryogenic induction was achieved using both MSG (BECWAR, NAGMANI & WANN 1990) and DCR (GUPTA & DURZAN 1985) media. The highest induction frequency was obtained on DCR1 (Douglas-fir Cotyledon Revised) medium supplemented with 0.5 mg 1 ¯¹ BA and 3.0 mg 1 ¯¹ 2,4-D, using L-glutamine as the major nitrogen source. Embryogenic tissue was translucent-to- white and mucilaginous in nature, composed of elongated, suspensor-like cells. The tissue was extruded from the micropylar end of the female gametophyte. In comparison, nonembryogenic tissue was produced from the gametophytic tissue itself and consisted of small, compact, spherical cells, crystalline in nature. Anatomical studies of developing patula seed demonstrated that the production of embryogenic tissue from the immature explants co-incided with the period, approximately two weeks after fertilization and with the occurrence of cleavage polyembryony in the developing zygotic embryos. Embryogenic tissue was maintained in culture by a recapitulation of the cleavage process. Transfer of the embryogenic tissue to DCR2 medium containing 1.3 mg 1 ¯¹ ABA resulted in tissue maturation and in the subsequent development of somatic embryos. Presence of ABA in the culture medium stimulated the development of cotyledonary initials in the apical region of the embryos. Elongated embryos, possessing small cotyledons, were rooted (50 to 60 %) on MSG6 medium containing no plant growth regulators. Somatic plantlets were successfully hardened-off under greenhouse conditions. Liquid culture methods were found to be a useful means of rapidly increasing the volume of embryogenic suspensor masses. Maturation , in terms of somatic embryo development and the production of cotyledonary initials, though , was not obtained in suspension. Reestablishment onto agar-solidified medium (DCR2) was required before maturation could occur. ABA is also responsible for stimulating reserve deposition and mobilization. In this regard, lipid accumulation in the developing somatic embryos was quantified and found to be significantly lower than in developing zygotic embryos. Similarly, non-matured embryogenic tissue contained less lipid deposits than matured (ABA-treated) tissue, indicating the requirement for ABA during maturation. Quantification of the lipids deposits is useful in determining the potential for somatic embryos to acclimatize to ex vitro conditions since their further growth and development is based on their ability to accumulate storage reserves. Somatic embryogenesis was found to be a useful method of propagation, producing plantlets with seedling-like qualities. This development has important consequences for the production of clonal plantlets in the Forestry Industry.