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.

Blue sky’s the limit? Somatic embryogenesis as a means of propagating recalcitrant blue spruce (Picea pungens) cultivar Hoopsii

“…The model conifer Norway spruce (Picea abies) successfully produces ET from mature somatic embryos (Bojarczuk et al 2007), but we found that the optimal explant source was the precotyledonary immature zygotic embryo, collected from ~June 20 to ~July 10 (a develop-mental window of approximately two weeks) in eastern Ontario. Work in black pine (Pinus nigra) has demonstrated that the use of immature zygotic embryos enclosed within the intact megagametophytes is effective in producing ET that differentiate into viable somatic embryos for propagation (Salaj et al 2019). Hoopsii megagametophytes containing small embryos collected prior to this window did not produce ET, nor did ovuliferous scales containing mature embryos collected after this window.…”

“…After three to five weeks on induction culture media, translucent and filamentous tissue emerged from the radicle end of the megagametophyte. Embryo production was characterized by the appearance of embryonal 'heads' (composed of meristematic cells (Salaj et al 2019)) connected by vacuolated 'suspensors' emerging from the polyembryonic cell mass (Fig. 1c).…”

Propagation of Juniper Species by Plant Tissue Culture: A Mini-Review

“…The leaf explants of mature J. oxycedrus trees, which were cultivated on modified SH medium with the addition of 1.33 or 2.21 mg L −1 2,4-D or picloram, were used as explants. In most coniferous species, the successful induction of somatic embryogenesis is dependent on the presence of both auxins and cytokinins in the medium [73][74][75]. However, the results showed that in this juniper species, the induction of a callus was possible only in the presence of auxins.…”

Seed Dormancy Breaking of an Endangered Medicinal Tree Species (Taxus Baccata L.) Using Embryo Culture

“…As Salaj et al (2019) found that DCR medium (Gupta and Durzan, 1985) was a better nutrient medium for conifer tree embryo culture. In the present study, DCR medium was used as a basal medium for embryo germination.…”