Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

Regeneration of plantlets from in vitro cultured cotyledons of white spruce (Picea glauca (Moench) Voss)

Adventitious shoot production from seedling cotyledons was investigated for white spruce [Picea glauca (Moench) Voss]. The age of the seedling was found to be important for shoot induction response, the optimum seedling age being 7 to 8 days old. Prior to that age, although response was seen, the multiplication rate was lower. After 7 to 8 days, the capacity to produce shoots declined considerably. The optimum cytokinin (BA) concentration for bud induction was 2 M BA. The response to specific BA concentrations was independent of seedling age. The shoot regeneration presented here was highly reproducible and provided primary multiplication rates of approximately 100 to 150 shoots per seedling which had an average of 6 cotyledons. Approximately 30% of 40 regenerated shoots were induced to differentiate roots and all the rooted plantlets were successfully transplanted to soil.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid. NRCC No. 29142     

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.)

Summary Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of ¹⁴C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of ¹⁴C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.     

Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.)

Interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.) cotyledonary somatic embryos were encapsulated in sodium alginate. Somatic embryo viability was retained, but germination occurred at a reduced frequency compared with the equivalent zygotic embryos. The addition of 0.5% (w/v) activated charcoal to the alginate capsule significantly enhanced root development and germination for somatic embryos but not for zygotic embryos. The possibility of developing an artiflcal endosperm was also investigated, by addition of Litvay (Litvay et al. 1981) nutrients with or without 90 mM sucrose to the alginate-charcoal capsule. This treatment significantly enhanced root development for all embryo categories with the exception of black spruce somatic embryos. Encapsulated and non-encapsulated somatic embryos survived one month cold storage at 4 °C without reduction in germination frequency.NRCC No. 35895     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter--glucuronidase-nopaline synthase 3 fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the -glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.     

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

Summary The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (±)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.     

Understanding changes in black (Picea mariana) and white spruce (Picea glauca) foliage biomass and leaf area characteristics

Key message

Growth conditions related to inter-tree competition greatly influence black and white spruce foliage biomass and projected leaf area characteristics.

Abstract

Foliage characteristics such as biomass and area are important among other reasons because they can be related to tree growth. Despite their economic and ecologic importance, equations to characterize foliage biomass and projected area of black (Picea mariana (Miller) BSP) and white (Picea glauca (Moench) Voss) spruces are sparse. Total foliage biomass and projected leaf area, foliage biomass and leaf area density, and relative vertical distribution of black and white spruces foliage biomass and leaf area were modelled with linear and nonlinear mixed effect models. A total of 65 white spruces and 57 black spruces were destructively sampled at four different locations in Alberta, Québec, and Ontario, Canada. Our results show that for each species, total tree foliage biomass and projected leaf area is proportional to stem diameter, total height, and crown length. The addition of crown length in the equations improved the precision of the predictions of total foliage biomass for both species and diminishes greatly the site level random effect. An increase in DBH for black spruce and in the DBH to total height ratio for white spruce skewed the relative vertical foliage biomass distribution toward the base of the living crown. According to our results, growth conditions or tree development stage influence both foliage biomass and leaf area characteristics of black and white spruces. Our results emphasize the importance of inter-tree competition on foliage biomass characteristics.     

Influence of excess nitrogen deposition on a white spruce (Picea glauca) stand in southern Alaska

Excess N delivered to forest ecosystems has been shown to alterinternal ecosystem biogeochemical cycles, contribute to forestdecline, and negatively affect the health of receiving waters.In the vicinity of the Nikiski Industrial Complex, Kenai Peninsula,Alaska, there has been recent concern about the influence ofNH3 emissions that have occurred for over twodecades on local soils and vegetation. The study site representedan opportunity to examine the influence of elevated N depositionon a northern coniferous ecosystem in an area with a low backgroundof N deposition. Overstory vegetation in the area is dominated bywhite spruce (Picea glauca Moench. Voss) and paper birch (Betulapapyrifera Marsh.). Mortality of both species has occurred adjacent(<2 km) to the industrial complex. Average annual Ndeposition rates ranged from 0.7 to 21.0 kg ha-1 y-1in the area, with the highest rates closest to the complex. Sulfatedeposition at the site was low. Due to the high NH3deposition, precipitation near the complex was less acidic thanprecipitation in general; bulk precipitation pH ranged from 5.51to 7.06. Within 1.80 km of the facility there was an increase inKCl- and resin- extractable soil NH4+ andNO3- in the O horizon, and a decrease in soil pHcompared to soils further from the facility. Spruce near thefacility had chlorotic foliage and thinning crowns, higherconcentrations of N, but lower foliar Ca and Mg. Foliar Mglevels approached deficiency levels, but foliar Ca was wellabove reported deficiency levels at all sites. Both Mg:N andCa:N ratios, however, suggest nutrient imbalances in the highN deposition zone. Canopy death and fertilization by N appearto have encouraged growth of the native bluejoint grass. Thepresence of elevated NO3- in O horizon soilextracts, elevated NO3- in resin bags placed betweenthe O and E horizons, and nutritional imbalances in the foliagesuggest that N saturation may be occurring in soils adjacent to thefacility.     

Soluble sugar content of white spruce (Picea glauca) seeds during and after germination

In white spruce ( Picea glauca [Moench.] Voss.) seeds, the raffinose family oligosaccharides (RFOs) provide carbon reserves for the early stages of germination prior to radicle protrusion. Some seedlots contain seeds that are dormant, failing to complete germination under optimal conditions. Since dormancy may be imposed through a metabolic block in reserve mobilization, the goal of this project was to identify any impediment to RFO mobilization in dormant relative to nondormant seeds. Desiccated seeds contain primarily, and in order of abundance on a molar basis, sucrose and the first 3 members of the RFOs, raffinose, stachyose and verbascose. Upon radicle protrusion at 25°C, the contents of RFOs decreased to low amounts in all seed parts, regardless of prior dormancy status and sucrose was metabolized to glucose and fructose, which increased in seed parts. During moist chilling at 4°C, RFO content initially decreased before stabilizing and then increasing. In seeds that did not complete germination, the synthesis of RFOs at 4°C favored verbascose, so that at the end of 14 (nondormant) or 35 (dormant) weeks, verbascose contents in megagametophytes exceeded the amount initially present in the desiccated seed. This was also true in the embryos of the dormant seedlot. In seed parts from both seedlots after months of moist chilling, stachyose amounts exceeded raffinose amounts. Upon radicle protrusion at 4°C, RFO contents decreased to amounts most similar to those present in seeds that completed germination at 25°C. Hence, the RFOs are utilized as a source of energy, regardless of the temperature at which white spruce seeds complete germination. Based on the similarity of sugar contents in seed parts between dormant and nondormant seeds that did not complete germination, differences in sugar metabolism are probably not the basis of dormancy in white spruce seeds.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

Phylogeography of white spruce (Picea glauca) in eastern North America reveals contrasting ecological trajectories

Aim The main objective of this study was to investigate the origin of white spruce (Picea glauca) range discontinuities in eastern North America. A first analysis aimed at uncovering the glacial origin and post-glacial migration route of white spruce in the eastern part of the range. A second analysis aimed at evaluating whether disjunct white spruce populations in central Québec result from a recent expansion or are remnant stands following a northern range contraction. Location The first analysis covered the North American transcontinental boreal distribution of white spruce, and the second focused on the eastern part of its range. Methods A total of 589 P. glauca individuals from 51 populations, 24 of which were from a previous published source, were analysed for variation in three chloroplast DNA regions (trnT/L, trnL/F and ndhK/C). At the continental scale, haplotypic diversity, zones of genetic discontinuities, clustering analysis and analysis of molecular variance (AMOVA) were employed. In the eastern part of the range, haplotypic diversity was assessed with a permutation procedure, demographic history with mismatch analyses, and genetic structure with AMOVA. Results At the continental scale, various lines of evidence (genetic-diversity hotspots, endemic haplotypes, zones of genetic discontinuities, and pattern of molecular variation) show that different lineages migrated from three glacial refugia (Beringian, Mississippian, east Appalachian). Strong genetic discontinuities, low diversity and lack of evidence for demographic expansion were found among stands from isolated high hills in central Québec. Main conclusions White spruce in the eastern part of its range has an east Appalachian origin, a lineage genetically distinct from populations west of the Great Lakes. Previous studies have shown that the northward migration of white spruce is still ongoing as the northernmost populations expand into the tundra. In contrast, mixed white spruce and balsam fir (Abies balsamea) stands from isolated high hills in central Québec have already reached their Holocene maxima and are currently declining. This range contraction is probably a result of increased wildfire disturbance, which gradually creates a pattern of regional white spruce isolation and replacement by fire-prone black spruce (Picea mariana) forest.