Somatic Embryogenesis in Selected Conifer Trees Pinus nigra Arn. and Abies Hybrids.

Abstract

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.