The Effect of Explant Type on the Establishment of Sitka Spruce [Picea sitchensis (Bong.) Carr.] in Culture

Five different types of shoot tip explant from three-year-oldand 13-year-old Sitka spruce trees were established on a rangeof media and shoot growth was recorded. While Schenk and Hildebrandtmedium gave the greatest shoot elongation, Webb and Street mediumproduced the healthiest-looking shoots. Poorest growth was recordedfor the explants cultured without the crown or any vasculartissues from the previous year's growth. [¹⁴C]sucrose tracerstudies indicated that poor uptake was not responsible for thereduced growth and it was suggested that damage due to inadequatewater uptake may be responsible. Results demonstrated that peelingthe protective scales from resting buds and trimming the shoottissues below the crown to a wedge shape and excluding the barkwas a reliable method of explant preparation for establishingmature and juvenile Sitka spruce in culture. Picea sitchensis, Sitka spruce, mature, juvenile, crown, [¹⁴C]sucrose, micropropagation     

The Influence of Composition of the Basal Medium on the Growth and Morphogenesis of Cultured Sitka Spruce (Picea sitchensis) Tissues

In vitro culture of Picea sitchensis (Bong.) Carr. needle explantson a number of basal culture media indicated that a complexmixture of organic additives was neither essential nor stimulatory.Adventitious bud production occurred at inorganic nitrogen levelsof 15–60 x 10^–3M and 7.5–30 x 10^–3 Min the adventitious bud induction and elongation media, respectively,when a well-balanced ratio of NH₄⁺:NO₃^– was maintained.However, necrosis was increased at the highest level of inorganicnitrogen. Organogenesis was more sensitive to changes in theratio of NH₄⁺:NO₃^–. Complete replacement of NH₄⁺ withNO₃^– during the adventitious bud induction passage severelyinhibited organogenesis, indicating that a reduced form of nitrogenmay be essential for adventitious bud differentiation. Conversely,a high proportion of NH₄⁺ in either the adventitious bud inductionor elongation medium increased tissue necrosis and inhibitedbud induction, reflecting the potential toxicity of this ion.Explants from different individual trees were found to varyconsiderably in their morphogenetic responses, but NH₄⁺:NO₃^–ratios of 1:5 or 1:2 were o ptimal for all individual treeswhich developed adventitious buds from needle cultures. Picea sitchensis, Sitka spruce, tissue culture, nitrogen     

Critical Water Potential for Stomatal Closure in Sitka Spruce

Steady state rates of net photosynthesis and stomatal conductance at high water potentials were measured under controlled conditions in a leaf chamber on Sitka spruce [Picea sitchensis (Bong.) Carr.] shoots detached from the forest canopy or on seedlings. The water supply to the seedlings was terminated by excision and the shoot water potential (or critical water potential) and osmotic potential at the onset of stomatal closure measured. The turgor potential was calculated. The initial osmotic potential before insertion of the shoot into the chamber was also measured. Shoot water potential and osmotic potential at stomatal closure, and initial osmotic potential were significantly higher (less negative) in foliage from the lowest level in the canopy compared with foliage in the upper canopy, and higher in shoots of seedlings transferred to low light than in those at high light. Critical water potential also varied with season, being higher in July than in October and November. In all except one instance, turgor potential at the onset of stomatal closure was negative, possibly because of dilution of the cell sap by the extracellular water during the estimate of osmotic potential. Over all the experiments variation in critical water potential was correlated with variation in critical osmotic potential and, to a lesser extent, the initial osmotic potential. However, turgor potential at the critical potential varied from +0.6 to -4.6 bar. This suggests that difference in turgor between the guard cells and subsidiary cells, which controls stomatal aperture, is only loosely coupled with the bulk leaf turgor and hence that bulk leaf turgor is not a good index of the turbor relations of the guard cells.     

Feeding behaviour of Red Deer (Cervus elaphus) offered Sitka Spruce saplings (Picea sitchensis) grown under different light and nutrient regimes

1. In addition to exhibiting preferences for particular plant species, vertebrate herbivores select particular individuals of these species whilst leaving others undamaged. This pattern of diet selection may reflect differences in the chemical composition (and hence nutritional quality) between individual plants, and/or variability in the physical constraints on intake rate, such as plant structure.
2. An experiment was conducted to test the effects of environmental manipulations on the morphology and chemical composition of Sitka Spruce saplings, and to evaluate the consequences for herbivory by Red Deer. Fertilizing the trees increased tree height, branch span and leader length, twig width and needle width, and decreased the concentrations of total phenolics, condensed tannins, fibre and lignin but monoterpene content was not altered. Shading also reduced phenolic and tannin concentrations.
3. When the fertilized and shaded trees were offered to deer in feeding trials, the probability of a tree being visited by a deer and the biomass removed were influenced by tree morphological variables, as were the bite rate and intake rate of the deer. More biomass was removed from larger trees.
4. Once the effects of tree morphology had been taken into account, there was no effect of the fertilizer and shade treatments on deer browsing behaviour that could be attributed to changes in chemical composition of the trees. The relationship between intake rate and bite size at each tree varied between individual deer, but the functional response relationship between intake rate and bite size was not influenced by the treatments applied to the trees.
5. Tree morphology may have a larger influence on deer feeding behaviour than tree chemical composition.     

Somatic embryogenesis in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Embryonal-suspensor masses from immature embryos from cones of Sitka spruce (Picea sitchensis (Bong.) Carr.) proliferated on a modified Murashige & Skoog medium with N⁶-benzyl-aminopurine, kinetin, 2,4-dichlorophenoxyacetic acid and an organic nitrogen source. The slimy white embryonal-suspensor masses with proembryos were maintained on a solid proliferation medium with reduced amounts of growth regulators. Transfer of embryonal-suspensor masses to a non-woven polyester carrier with liquid maturation media containing ±2-cis-4-trans-abscisic acid and a reduced amount of inositol and organic nitrogen resulted in synchronized embryo formation. Further development was achieved on a medium without ±2-cis-4-trans-abscisic acid and organic nitrogen. Somatic embryos were successfully transferred ex vitrum.Abbreviations ABA ±2-cis-4-trans-abscisic acid - BAP N⁶-benzyl-aminopurine - ESM embryonal-suspensor masses - KIN kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Leaf Water Potential and Control of Water Loss in Droughted Sitka Spruce Seedlings

Sitka spruce seedlings were subjected to drought in experimentsin a growthroom, a greenhouse, and out of doors. The plantswere grown in a double chamber with the bulk of the roots inthe upper part where they dried out the soil when water waswithheld. A few new roots penetrated into the lower part inwhich the soil remained moist. The double chamber system enabledthe plant to attain a high water psotential by night and theshoot was only periodically under mild water stress. Measurementswere made on soil water potential (_solt), leaf water potential(₁), transpiration (E), and stomatal conductance (k_s). As soildecreased over a period of 4.5 d, E and k_s decreased progressively.The decline in E and k_s which indicated stomatal closure, occurredat a higher ₁ than has been reported for Sitka spruce. The behaviourof the stomata appeared to be modified by conditions at theroot, and it is proposed that differences in the response to₁,depend on Whether the latter is reduced by resistances in thexylem between root and leaf, as is known to occur in large treesin moist soil, or by stresses at the root itself.     

Effect of carbenicillin and cefotaxime on somatic embryogenesis of Sitka spruce (Picea sitchensis (Bong.) Carr.)

Carbenicillin prevented the development of mature somatic embryos,reduced early stage embryos by >90% and tissue growth by50%. Cefotaxime had no effect on overall tissue growth, butreduced the development of early and mature embryos by 20% and66–80%, respectively. Carbenicillin should not be usedfor transformation of Sitka spruce. Key words: Sitka spruce, somatic embryogenesis, antibiotic sensitivity     

Effects of simulated acid precipitation on the cycling of manganese under Sitka spruce (Picea sitchensis)

Simulated acid rain at pH 3.3, 4.3 and 5.3 has been applied by overheadspraying to reconstructed soil profiles and young Sitka spruce (Piceasitchensis) trees in pots to assess the effect of rainfall acidityonthe fate and recycling efficiency of Mn from ⁵⁴Mn-labelled Sitkaspruce litter. The concentration of ⁵⁴Mn in throughfall wassignificantly increased by acidification of the rain, but ⁵⁴Mnactivity in drainage water remained low and was not significantly affected. Thefact that only < 1.5% of ⁵⁴Mn was lost in drainage water suggeststhat forest ecosystems efficiently retain Mn, at least in the short term. Thebulk of ⁵⁴Mn (approximately 60 to 70%) was retained in the litterlayer. Compared with rain at pH 4.3, rain at pH 3.3 acidified L/F and H/Ahorizon soils. The amount of ⁵⁴Mn found in the different soilhorizons was only influenced significantly by rainfall acidity in the E horizonsoil, however, where ⁵⁴Mn activity was increased by the most acidtreatment. The activity of ⁵⁴Mn in Sitka spruce needles did notdiffer significantly with treatments.     

Divergent selection and heterogeneous migration rates across the range of Sitka spruce (Picea sitchensis)

Gene flow and effective population size (N(e)) should depend on a population's position within its range: those near the edges are expected to have smaller N(e) and lower relative emigration rates, whereas those nearer the centre should have larger N(e) and higher relative emigration rates. In species with continuous ranges, this phenomenon may limit the ability of peripheral populations to respond to divergent selection. Here, we employ Sitka spruce as a model to test these predictions. We previously genotyped 339 single nucleotide polymorphisms (SNPs) in 410 individuals from 13 populations, and used these data to identify putative targets of divergent selection, as well as to explore the extent to which central-peripheral structure may impede adaptation. Fourteen SNPs had outlier F(ST) estimates suggestive of divergent selection, of which nine were previously associated with phenotypic variation in adaptive traits (timing of autumn budset and cold hardiness). Using coalescent simulations, we show that populations from near the centre of the range have higher effective populations sizes than those from the edges, and that central populations contribute more migrants to marginal populations than the reverse. Our results suggest that while divergent selection appears to have shaped allele frequencies among populations, asymmetrical movement of alleles from the centre to the edges of the species range may affect the adaptive capacity of peripheral populations. In southern peripheral populations, the movement of cold-adapted alleles from the north represents a significant impediment to adaptation under climate change, while in the north, movement of warm-adapted alleles from the south may enhance adaptation.     

Localised impact of Sitka spruce (Picea sitchensis (Bong.) Carr.) on soil permeability

A typical upland soil catena afforested with Sitka spruce (Picea sitchensis (Bong.) Carr.) was chosen to examine the localised effect of trees on soil permeability. A borehole permeameter was used to measure soil permeability at 0.2 m and approximately 2 m distance from the stem of 20 trees at a fixed measurement depth of 0.25 to 0.45 m. In the case of the near-tree measurements, this corresponded to soil beneath the main root plate of each conifer. Two principal elements of the soil catena: the ferric podzol of the mid-slope and histosol soil of the foot-slope were investigated.The preliminary data set shows that within the ferric podzol element, the permeability of the soil beneath individual conifers was a factor of 5.4 less than that of the adjacent soil. In contrast, within the histosol sub-tree permeabilities could not be distinguished from those of soil 2 m away from each tree. The decrease in sub-tree permeability within the podzol may be caused by sensitivity of the Bsl horizon to consolidation by tree weight or by enhanced illuviation resulting from changes in local soil chemistry. The histosol may be less sensitive to such processes. The results of a consolidation test applied to the Rawls and Brakensiek model of soil permeability supported the possible role of consolidation in the reduction of soil permeability beneath conifers in podzolic soil. Additional data on soil bulk density, porosity and texture are required to corroborate either the consolidation or illuviation hypotheses. As the Bsl horizon of ferric podzol soil is typically slowly permeable, a further decline may (i) restrict root development and thus, increase windthrow hazard, and (ii) increase the lateral flow of water within podzolic Eag horizons and thus affect stream acidification. Deep ploughing of a site prior to afforestation may mitigate such impacts.     

Metabolic dynamics during autumn cold acclimation within and among populations of Sitka spruce (Picea sitchensis)

? Autumnal cold acclimation in conifers is a complex process, the timing and extent of which vary widely along latitudinal gradients for many tree species and reflect local adaptation to climate. Although previous studies have detailed some aspects of the metabolic remodelling that accompanies cold acclimation in conifers, little is known about global metabolic dynamics, or how these changes vary among phenotypically divergent populations. ? Using untargeted GC-MS metabolite profiling, we monitored metabolic dynamics during autumnal cold acclimation in three populations of Sitka spruce from the southern, central, and northern portions of the species range, which differ in both the timing and extent of cold acclimation. ? Latitudinal variation was evident in the nature, intensity, and timing of metabolic events. Early development of strong freezing tolerance in the northern population was associated with a transient accumulation of amino acids. By late autumn, metabolic profiles were highly similar between the northern and central populations, whereas profiles for the southern population were relatively distinct. ? Our results provide insight into the metabolic architecture of latitudinal adaptive variation in autumn acclimation and show that different mechanisms are the basis of early October cold hardiness and autumn-acclimated cold hardiness.     

The effect of wind exposure on the tree aerial architecture and biomechanics of Sitka spruce (Picea sitchensis, Pinaceae)

This paper reports on the effect of wind loading below damaging strength on tree mechanical and physical properties. In a wind-exposed Sitka spruce stand in western Scotland, 60 trees at four different levels of wind exposure (10 m, 30 m, 50 m, 90 m from edge) were characterized for stem and crown size and shape and mechanical properties, including structural Young's modulus (E(struct)), natural frequency, and damping ratio. E(struct) increased from the stand edge to the mid-forest, but with a large inter-tree variation. Swaying frequency and damping ratio of the trees also increased with distance from edge. Wind-exposed edge trees grew shorter, but more tapered with an overall lower E(struct), allowing for greater flexural stiffness at the stem base due to the larger diameter and for higher flexibility in the crown region of the stem. The trees at the middle of the stand compensated for their increased slenderness with a higher E(struct). Thus, for the different requirements for wind-firmness at stand edge and mid-forest, an adapted combination of tree form and mechanical properties allows the best withstanding of wind loads. The results show the requirement to understand the different strategies of trees to adapt to environmental constraints and the heterogeneity of their growth reactions in response to these strategies.     

Graft Formation in Sitka Spruce: A Scanning Electron Microscope Study

Low-temperature and conventional scanning electron microscopyhave been used to examine the callus formed at the graft interfacein Picea sitchensis (Bong.) Carr. Callus cells are producedby both cambium and ray parenchyma dedifferentiation and redifferentiationin scion and stock. Adhesion between the cells derived fromscion and rootstock is thought to be my means of pectinaceousbeads at the surface of the callus cells, preceding a more generalfusion of cell walls. The cambia of the two graft componentsare prevented from growing towards each other by the presenceof callus. Instead, the differentiation of new cambium withinthe callus, in the vicinity of the cambia exposed at the preparedsurfaces of the scion and rootstock, links them to form a continuouscambial layer around the combined stem. Callus, cambium, differentiation, grafting, Picea sitchensis     

Leaf Conductance as Related to Xylem Water Potential and Carbon Dioxide Concentration in Sitka Spruce

Current year shoots of Sitka spruce [Picea sitchensis Bong. (Carr.)] from the forest canopy were equilibrated in a leaf chamber. The shoots were excised in air, and removed at differing times in order to establish a relationship between stomatal conductance and xylem water potential. The experiment was repeated at five ambient CO₂ concentrations. A second set of excised forest shoots, and shoots excised from 2-year- old nursery seedlings were allowed to evaporate freely in a controlled environment wind tunnel until a constant rate of transpiration was measured, to establish a relationship between cuticular conductance and xylem water potential. Cuticular conductance was estimated to be 0.012 cm s^-1 at high water potential and declined linearly to 0.007 cm s^-1 at ?3.5 MPa. The implication of this decline in the subsequent calculation of stomatal and mesophyll conductance is considered. Stomatal conductance remained constant at water potentials above ?1.4 MPa and was not affected by ambient carbon dioxide concentrations between 20 and 600 cm^-3. At lower water potentials, stomatal conductance declined and approached zero at ?2.5 to ?2.6 MPa. The results suggest that stomatal aperture is not controlled by either ambient or intercellular space carbon dioxide concentration, and that stomatal closure at low water potential is unlikely to be mediated by carbon dioxide.     

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl₂. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent M_r approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent M_r approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed. Received: 9 January 1997 / Accepted: 14 March 1997     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.