D. J. Durzan

Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana)

Opening of apical and axillary buds of mature Douglas-fir and sugar pine trees was obtained on a newly formulated basal medium (DCR) without growth regulators. Elongation of buds was observed on 1/2 strength DCR with 0.3% activated charcoal (DCR-1). In sugar pine, multiple shoots were obtained when explants on DCR with 0.5 mg/1 BAP for 5–6 weeks were transferred to DCR-1 medium. On subculture, axillary buds again developed when shoots were cultured on DCR with 0.2 mg/1 BAP for Douglas-fir and 0.5 mg/1 BAP for sugar pine. These buds were again elongated on DCR-1 medium. By subculturing 7–10 shoots of Douglas-fir and 2–3 shoots of sugar pine, over 100 shoots can be obtained in a year.

Tissue culture in forestry

1. Introduction.- 2. Tissue Culture Techniques.- 3. Cell and Tissue Culture in Forest Industry.- 4. In Vitro Propagation of Gymnosperms.- 5. Vegetative Propagation of Dicotyledonous Trees.- 6. Vegetative Propagation of Eucalyptus.- 7. Vegetative Propagation of Palm Trees.- 8. Phytopathology and Tissue Culture Alliances.- 9. Action of Growth Regulators.- 10. Nitrogen Metabolism and Vegetative Propagation of Forest Trees.- 11. Carbohydrate Utilization and Metabolism.- 12. The Use of inVitro Techniques for Genetic Modification of Forest Trees.- 13. Vegetative Propagation in Relation to Juvenility, Maturity, and Rejuvenation.- 14. Tree Species Index.- 15. General Index.

Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures

Abstract A proliferating embryonal-suspensor mass (ESM) was initiated from immature embryos of Douglas-fir ( Pseudotsuga menziesii (Mirb.) Franco), 4–5 weeks after fertilization, on modified MS medium with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and N 6 -benzylaminopurine (BAP). ESMs were maintained for over 6 months as cell suspension cultures on modified DCR media with low 2,4-D and with kinetin and BAP. The development of individual somatic embryos was initiated in suspension culture by the gradual reduction of plant growth regulators and by addition of abscisic acid. The early stages of zygotic embryogenesis in Douglas-fir are unique among conifers and cleavage polyembroyogenesis is unknown. In somatic embryogenesis, characteristic stages of zygotic embryonic development were recapitulated and complete embryos were recovered by inhibiting cleavage polyembryony with abscisic acid and culturing individual embryos without growth regulators. Histological examination confirmed bipolar organization of somatic embryos. While conversion is low, plantlets with multiple cotyledons have been transferred to soil and continue to grow with production of a new shoot.

Tannin inclusions in cell suspension cultures of white spruce

SummaryTannins were detected cytochemically in cell suspension cultures of white spruce (Picea glauca [Moench] Voss) and were studied by electron microscopy. Tannin inclusions originated within cytoplasmic vacuoles, possibly derived from the endoplasmic reticulum, and accumulated in the central vacuole through enlargement and coalescence of those cytoplasmic vacuoles. Structural information supported the suggested metabolic relationship between starch and tannin, although tannins did not develop within plastids. Membranous material, resembling myelinlike bodies, was often observed in close association with tannins.

Loblolly Pine ( Pinus taeda L.)

Loblolly pine (Pinus taeda L.) is the leading commercial timber species in the southern United States. Carolus Linnaeus gave loblolly pine its scientific name, Pinus taeda, over 225 years ago. Taeda is the ancient name for resinous pines. It comes from the Latin, meaning torch. In Roman times, taeda was applied to several of the hard, pitchy pines (Wahlenberg 1960).

Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

SummaryEmbryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N6-benzyladenine (BAP) each (20×10−6M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10−6M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10−6M) and 2,4-D (5×10−6M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10−6M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability.

Development of somatic embryos from cell suspension cultures of Norway spruce (Picea abies Karst.)

Cell suspension cultures were established from embryonal-suspensor masses derived from mature seeds. Transfer of cell masses on to a medium containing abscisic acid stimulated development of already established individual embryos. Somatic embryos developed shoots when supported by cheese cloth in liquid medium in Petri dishes. The percentage of well-developed roots remained low even though all embryos had root meristems. We have recovered an average of 25 plantlets from an initial PCV of ca 1 g fresh wt per 10 ml.

Somatic proembryo formation and transient expression of a luciferase gene in Douglas fir and loblolly pine protoplasts

Somatic pro-embryos were regenerated from morphogenic protoplasts of cell suspension cultures of Douglas fir and loblolly pine. Morphogenic protoplasts were obtained from suspension cultures of embryonal cells grown in a modified basal medium high in myo-inositol. After 11–12 weeks, microcolonies of embryonal-suspensor masses (ESMs) were recovered from agarose-embedded protoplasts. Somatic pro-embryos were regenerated after 18–20 weeks by somatic polyembryogenesis from new ESMs on agar plates. The luciferase (luc) gene was successfully introduced into fir and pine protoplasts by electroporation. While viability of protoplasts was reduced from 90% to 45–55% by electroporation, the transient expression of the luc gene was detected in protoplasts surviving 36 h after electroporation. Gene expression was improved by the addition of polythylene glycol (PEG) to the electroporation mixture.

Rapid multiplication of adventitious somatic embryos in peach and nectarine by secondary embryogenesis

Somatic embryos were multiplied by secondary embryogenesis in cotyledonary cultures of peach and nectarine (Prunus persica L.) using a simplified culture medium for immature seeds. A three-stage process with an initial callus phase was established in darkness on a medium containing basal salts (modified MS) supplemented with 2,4-D (5 mg/l), Kn (2 mg/l) and BAP (2 mg/l) and casein hydrolysate (500 mg/l). This was followed by a growth regulator-free medium with activated charcoal for the adventitious and direct multiplication of somatic embryos under continuous light. Somatic embryos (10–15) originated from the epidermal layer of primary somatic embryos of 4–6 mm size. The incidence of morphologically abnormal embryos was reduced by subculturing every 20 days. Calli which were isolated and grown on a 2,4-D medium were more embryogenic than those on NAA. These embryos multiplied continuously for more than 10 months by a repetitive somatic embryogenic process. A third stage medium, supplemented with BAP (2 mg/l), was required for axis elongation, germination and transfer to soil.

Effects of Environmental Changes on Sugars, Tannins, and Organized Growth in Cell Suspension Cultures of White Spruce

SummaryCallus from hypocotyls of white spruce (Picea glauca [Moench] Voss) was grown on agar under defined conditions with high levels of calcium nitrate. Transfer of callus to liquid suspension cultures and maintenance of suspensions either under a regime of constant temperature and light or under alternating conditions similar to those of a late spring day, affected the content of free sugars, tannins, and aldehydes. Under the alternating conditions the levels of these substances increased greatly compared to those under the constant environment. By contrast, vascularization of cell clumps, which was comparable to the differentiation of hypocotyls in seedlings, was obtained only under constant conditions. Cells at the centre of the clumps developed secondary wall thickenings and bordered pits, and were surrounded by cambial-like initials.