Sara von Arnold

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.

Improved Efficiency of Somatic Embryogenesis in Mature Embryos of Picea abies (L.) Karst.

Summary Embryogenic callus was formed from mature zygotic embryos of Picea abies cultured in darkness on media containing 5 to 20 μM 2,4-D or NAA and 5 μM BA. The yield of embryogenic callus was higher on half strength basal medium than on full strength. On full strength medium only low sucrose concentration (30 mM) was efficient, while on half strength basal medium it was possible to increase it to 90 mM without a reduction in the formation in embryo. genic callus. By culturing embryos on media containing high concentrations of sorbitol, it was shown that the higher sensitivity for increased sucrose concentrations on full strength basal medium was not due to increased osmotic pressure. The optimal concentration of NH 2 NO 3 varied depending on the sucrose concentration. In medium containing 30 mM sucrose 15 mM NH 4 NO 3 was optimal, while in medium containing 90 mM sucrose the NH 4 NO 3 concentration could be varied from 7.5 to 30 mM. Proline addition up to 15 mM did not influence the formation of embryogenic callus. At higher proline concentrations the yield decreased. When appropriately cultured, 50% of the embryos produced embryogenic callus, and 15% of these calli produced plantlets.

Effect of agar concentration on growth and anatomy of adventitious shoots of Picea abies (L.) Karst

The development of adventitious shoots of Picea abies was affected by the agar concentration in the culture medium. Increasing the agar concentration from 0.5 to 2.0% decreased vitrification, but at the same time reduced shoot growth and rooting potential. Slightly vitrified plantlets could become acclimatized to greenhouse conditions. The mesophyll of needles developed in vitro was interspersed with large air spaces; the lower the agar concentration, the larger the air spaces. After transfer to the greenhouse, the new needles from the acclimatized plantlets had an anatomy approaching that of plants growing in field.

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.

Factors influencing formation, development and rooting of adventitious shoots from embryos of picea abies (L.) Karst

Abstract A method is described for multiple shoot and plantlet formation on embryos of Picea abies (L.) Karst. Adventitious bud primordia were induced by culturing newly isolated embryos on media containing cytokinin. Further development of the bud primordia required transfer to cytokinin-free media. Adventitious shoots with a distinct stem were isolated from the embryos. After a couple of months the shoots were given a root-inducing treatment. When the roots had started to elongate the shoots were transferred to unsterile conditions. The capacity for bud production varied depending on the background of the seeds. The results showed that the method used for induction of adventitious buds strongly affected the ability of the induced buds to develop into vigorous adventitious shoots which could be rooted. The most critical factors which are discussed are: basal media concentration, cytokinin concentration, choice of cytokinins, and photoperiod.

Bud induction on isolated needles of Norway spruce (Picea abies L. Karst.) grown in vitro

Isolated needles of Picea abies L., Karst. were induced to form adventitious bud primordia when cultured on media containing a relatively high cytokinin: auxin ratio. In order to receive a high frequency of needles forming bud primordia it was necessary to collect the needles from buds just after flushing. Short needles (1–3 mm) more readily formed bud primordia than long needles (3–5 mm). Development of primordia into buds occurred when needles bearing primordia were transferred to media either lacking growth substances or with a reduced level. A higher yield of buds was obtained if the needles were incubated at lower temperature (10–16°C) for the first days after isolation and then transferred to 20°C. Buds with 3–5 mm-long needles were excised from the initial tissue and planted individually on culture media.

The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies

Resting vegetative buds of Picea abies collected from phytotron-grown rooted cuttings of 24-year-old trees or a 12-year-old hedge were tested for their capacity to form adventitious buds after various cytokinin treatments. The most effective method for obtaining a high yield of adventitious buds within 8 weeks was to pulse treat the buds in 250 μM BA for 3 h and then culture them on medium containing 5 μM each of BA and kinetin for 1 week. The developmental pattern for adventitious bud production, with the formation of 10 to 20 adventitious buds per bud, was similar for all tested genotypes, although the number of buds giving rise to adventitious buds varied significantly. The capability of some clones to form adventitious buds was correlated to endogenous cytokinin content. The clone which contained most endogenous cytokinin in its resting bud had the highest potential for adventitious bud formation.

Indole-3-acetic acid content in buds of five willow genotypes

Summary Free IAA content in vegetative axillary buds from non-growing shoots of five willow genotypes was measured using RIA. The buds were analysed either immediately after removal of the bud scales or after several weeks culture in vitro with or without BA pre-treatment. Noncultured buds had lower IAA levels than buds cultured in vitro . Genotypic differences in the IAA level were found in buds cultured in vitro but not in non-cultured buds. BA pre-treatment resulted in decreased IAA levels in some clones but not in others. Specific morphogenic genotype differences could not be correlated to IAA levels.