Inger Hakman

The development of somatic embryos in tissue-cultures initiated from immature embryos of Picea abies (Norway spruce)

Embryos of Picea abies at various developmental stages were cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (10−5 M) and N6-benzyladenine (BA) (5×10−6 M). The immature embryos gave rise to a highly friable and embryogenic callus which could be maintained by subculture and contained polarized and organized structures (somatic embryos) consisting of long highly vacuolated cells at one end (suspensor) and a group of small meristematic cells at the other (embryonal end). These structures closely resembled the early stages of normal zygotic embryogeny. Upon further culture these structures formed a bipolar shoot-root axis with an independent and closed vascular system. In many instances either the shoot or the root meristems failed to differentiate. Embryogenic tissues obtained on agar media could be transferred to liquid media and maintained by subculture for at least 6 months. The development of somatic embryos was observed in the liquid cultures also.

Regulation of somatic embryo development in Picea abies by abscisic acid (ABA)

Summary Embryogenic callus was derived from immature and mature zygotic embryos of Picea abies cultured in the dark on half strength LP medium containing 30 mM sucrose, 10 μM NAA and 5 μM BA. The embryogenic callus was composed of small somatic embryos which consisted of small, densely cytoplasmic cells subtended by a suspensor of long highly vacuolated cells. For further development of the somatic embryos it was essential to transfer the callus to other growth regimes. The highest yield of plandets was obtained if the calli were cultured on half strength LP medium containing 90 mM sucrose and 7.6 μM ABA for one month. During this treatment the somatic embryos accumulated lipids and also became firm and developed a smooth surface. After the ABA treatment the calli were transferred to full strength LP medium containing 60 mM sucrose. On this medium plandets started to regenerate within three weeks. As the somatic embryos developed cotyledons they were isolated and cultured individually. Plants produced according to this protocol continued to grow when placed under greenhouse conditions.

Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce)

Summary Embryogenic callus was produced from immature zygotic embryos of Picea abies cultured on a defined medium supplemented with 2,4-dichlorophenoxyacetic acid (10-5M) and a cytokinin (10-6-10-5 M). Subsequently numerous somatic embryos developed from the callus. Upon subculture the somatic embryos could be stimulated to develop further into plantlets with cotyledons, a hypocotyl and a root. Under suitable conditions 24% of the calli produced plantlets, and up to 25 plantlets were formed in a single callus, in addition to numerous somatic embryos of smaller sizes. Plantlet formation was followed both in living and sectioned materials and showed close similarity to zygotic embryogeny.

Induction of Somatic Embryos and Plantlets from Cryopreserved Cell Cultures of White Spruce (Picea glauca)

Summary A cell suspension culture of Picea glauca with potential for sustained somatic embryo production has been successfully cryopreserved retaining very high levels of viability even after 1 year storage in liquid nitrogen. The following cryobiological components were identified as optimal for the recovery of viable cells postcryopreservation: 1) preculturing the cells for 24 h prior to freezing, in liquid nutrient medium enriched with 0.4 M sorbitol, 2) treating the cells after preculture with a cryoprotectant combination of 0.4 M sorbitol + 5 % DMSO, and 3) controlled freezing at a cooling rate of 0.3 °C min −1 to −35 °C followed by storage in liquid nitrogen. Dimethylsulfoxide alone at concentrations ranging from 5 to 20 %, in addition to being cytotoxic, was ineffective as a cryoprotectant. The callus re-established from cryopreserved cells maintained embryogenic potential identical to the unfrozen controls. Light and scanning electron microscopic examination of somatic embryos revealed structural and organizational conformity with those induced from unfrozen controls. Calli regrown from cryopreserved cells were induced to differentiate into plantlets through embryogenesis, and into shoots by organogenesis.

Effect of Sucrose on Initiation of Embryogenic Callus Cultures from Mature Zygotic Embryos of Picea abies (L.) Karst. (Norway Spruce)

Summary Embryogenic callus was produced from mature embryos of Picea abies. The sucrose concentration was critical.

Isolation and growth of protoplasts from cell suspensions of Pinus contorta dougl. ex loud.

Cell suspensions were initiated from embryo derived calli of Pinus contorta. Some of these cell lines could be maintained in culture for at least one year without reduced growth.A high yield of protoplasts was obtained from the cell suspensions. The protoplasts started to divide after two days and cell clusters could be observed after about two weeks. The growth phase of the cell suspensions was very important for the division of protoplasts. Only protoplasts isolated from suspensions in an actively dividing phase were able to divide with a high frequency and to give rise to cell clusters.

Plantlet Regeneration in vitro via Adventitious Buds and Somatic Embryos in Norway Spruce (Picea Abies)

Methods are described for plant regeneration via adventitious bud formation and somatic embryogenesis from mature zygotic embryos of Picea abies. Adventitious buds are formed on embryos pulse-treated with cytokinin. A few epidermal and subepidermal cells adjacent to the stomata are stimulated to give rise to meristemoids which subsequently develop further into adventitious buds. Embryogenic callus is formed when the embryos are cultured on medium containing both auxin and cytokinin. The somatic embryos arise from the region between the hypocotyl and the cotyledons. The different developmental stages during initiation and development of adventitious buds and somatic embryos are described.