Karyotype of Norway spruce by multicolor FISH

The chromosomes (2n = 2x = 24) of Norway spruce are very large since their size reflects the huge amount of genomic DNA (2C = 30 × 10⁹ bp). However, the identification of homologous pairs is hampered by their high degree of similarity at the morphological level. Data so far presented in the literature were not sufficient to solve all the ambiguities in chromosome identification. Several genomic Norway spruce DNA clones containing highly repetitive sequences have been identified and characterised in our laboratory. Three of them were selected for fluorescent in situ hybridization (FISH) experiments because of their strong signals and suitability for chromosome identification: PATR140 hybridized at the centromeric site of three chromosome pairs; PAF1 hybridized in six subtelomeric and two centromeric sites; 1PABCD6 co-localized with the subtelomeric sites identified by PAF1. The statistical analysis of microscopic measurements of chromosomes in combination with the FISH signals of these probes allowed the unambigous construction of Norway spruce karyotype. We also compared the karyotype of Norway spruce with that of other spruce species to infer the number and kind of rearrangements that have occurred during the evolution of these species.Communicated by D.B. Neale     

Short-term pigment changes in Norway spruce needles

The photosynthetic pigment contents and the chlorophyll fluorescence induction in Norway spruce [ Picea abies (L.) Karst.] needles were monitored at 3 h intervals over a 10 day period. This covered 6 days of high insolation, one day of low insolation, followed by three more days of high insolation. During the first six days only small changes in mean pigment contents were observed but we interpret them as suggesting a daily rhythm in total chlorophyll content with two maxima and minima. The day of low insolation was paralleled by larger pigment changes, mostly an increase in chlorophyll b along with a decrease in carotenoid content. With the resumption of high insolation, pigments gradually return to their previous levels. During the entire period chlorophyll a fluorescence induction remained relatively constant. These short-term responses may reflect features of thylakoid organization in relation to pigment content and suggest possible long-term adaptive mechanisms in non deciduous conifers.     

Development of somatic embryos in Norway spruce

Embryogenic cell lines of Norway spruce consist of a large number of somatic embryos. The cell lines have been divided into two groups, A and B. The group B embryos are developmentally blocked. Extracts of mature spruce seeds stimulate group B embryos to develop a morphology comparable to group A embryos. However, seed extract inhibits early embryo development. The active components in seed extract were shown probably to be proteins. Extracts of mature seeds contain chitinase-like proteins as recognized by an antibody towards chitinase 4 in sugar beet. Proteins of similar sizes were detected by the same antibody in the conditioned medium of group A, but not in group B. A chitinase 4-related chitinase and a nod factor had a stimulating effect on early embryo development, but did not influence the later stages of embryo development.Key words: Picea abies, somatic embryogenesis, embryo development, seed extract.      

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators (PGRs) stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.     

Photoinactivation of catalase in needles of Norway spruce

Catalase IEC 1.11.1.6) activity in Norway spruce ( Picea abies [L.] Karst.) needles was examined under different environmental conditions. When shade-adapted spruce trees were exposed to full sunlight or to high light intensities in growth chambers, the catalase activity decreased. Under continuous light the activity was reduced in comparison to the control grown in light/dark cycles. The reduction of the activity was not temperature dependent. Under field conditions the activity was higher in hranches oriented north than in those oriented south. A diurnal rhythm with a maximum in the night was delected. The reduction in catalase activity also occurred in young white needles of Picea ahies vur. argenteospica . which are free of chlorophyll. It is concluded that in Norway spruce needles there is a ligh:-dependent photoinactivation of catalase. which is not temperature-dependent.     

Peroxisome proliferation in Norway spruce induced by ozone

Summary Catalase (EC 1.11.1.6) activity (both total and specific activity) of particulate fractions of needles of Norway spruce [Picea abies (L.) Karst.] was elevated approximately 2-fold following exposure of trees to 60–70 g/m³ of ozone during the growing season compared to trees receiving charcoal filtered air (about 15 g/m³ ozone). Measurements were from homogenates fractionated into particulate and soluble (supernatent) activities. In contrast, the catalase activity of the supernatant was unchanged in response to ozone treatment. Catalase activity declined as the needles aged comparing current, 1-, and 2-year needles but the ozone-induced increment remained constant. Electron microscope cytochemistry using peroxidatic coupling with 3,3-diaminobenzidine carried out in parallel, revealed catalase-containing peroxisomes both in situ and in the particulate fractions analyzed for catalase activity. The tissue volume occupied by peroxisomes in response to needle age and ozone appeared to vary approximately in proportion to the measured catalase activity. Overall cytochemical reactivity for catalase declined with needle age, but, for all years, was greater in needles of trees receiving air supplemented with ozone compared to those of trees receiving charcoal filtered air.Abbreviations DAB 3,3-diaminobenzidine tetrahydrochloride     

Norway spruce galactoglucomannans exhibiting immunomodulating and radical-scavenging activities

Wood-derived naturally acetylated galactoglucomannans (AcGGM) can be recovered even in ton-scale at mechanical pulp mills using spruce as raw material. These cell wall polysaccharides have a great potential as hydrocolloids and bioactive polymers in food and pharmaceutical applications, or as starting material for production of functional polymers. The immunostimulatory activity of both AcGGM and its deacetylated form (GGM) was now in vitro tested. The biological response of both AcGGM and GGM in the lymphocyte transformation test was dose-dependent. The direct mitogenic as well as comitogenic activities of the AcGGM were comparable to those of the immunogenic corn cob xylan used as control, and GGM showed significantly higher biological responses also at lower doses. In contrast to GGM, AcGGM possessed also DPPH radical-scavenging activity. The results suggested that the spruce AcGGM and GGM are potentially important as additives with immuno-potentiating and antioxidant properties in food products and pharmaceutical formulations.     

The impact of spruce aphids on nutrient flows in the canopy of Norway spruce

1 This study investigated the effects of honeydew from aphids in the canopy of Norway spruce (Picea abies (L.) Karst.) on the nitrogen chemistry of throughfall using a rainfall simulation experiment. Throughfall collected beneath infested trees was compared with that from beneath uninfested trees, while standardizing the quality and quantity of the precipitation and plant age. 2 Honeydew excreted by Cinara pilicornis (Hartig) and C. costata (Zett.) significantly increased the concentrations of dissolved organic carbon (DOC) and hexose-C in throughfall. The average concentrations of nitrogenous compounds (NH₄-N, NO₃-N) in throughfall collected beneath infested trees were significantly lower than beneath uninfested trees. 3 Multiple regression analysis indicated that the amount of rain and NH₄-N concentrations were the best predictors of the concentrations of dissolved organic nitrogen (DON) in throughfall. Parameters that were closely associated with the level of infestation (DOC, hexose-C concentrations) did not have a direct relationship with DON. About 40% of the reduction in the concentration of DON in the throughfall was attributed to aphid–micro-organism interactions. 4 Particle amino nitrogen (PAN)-concentrations were highest under infested trees in July after aphid numbers had declined, indicating a concomitant decline in microbial biomass after honeydew becomes a limiting resource. 5 The comparison of the concentrations of different nitrogen compounds in throughfall of infested and uninfested trees indicated that aphids affect the carbon and nitrogen cycles in the phyllosphere by providing energy that fuels the metabolism of the micro-organisms. These processes seem to occur very rapidly. 6 We discuss the significance of the results and the prospects of linking the ecology of micro-organisms and herbivores with flows of nutrients through the canopy of trees.     

Some effects of long-term ozone fumigations on Norway spruce

Summary Potted cuttings of a 12-year-old, and grafts of an 80-year-old, Norway spruce (Picea abies (L.) Karst.) were subjected to 100 or 300 g O₃·m^–3 for 1215 h (45 h of daylight per week) during the growing season of 1985. At the end of the fumigation the plants did not exhibit any visible signs of injury. Whereas in the fumigation with 100g O₃·m^–3 we did not detect any significant change in gas exchange, 300 g O₃·m^–3 did alter the CO₂ uptake after 27 weeks, and in one clone transpiration was also altered. Stomatal reaction to a change of light suggested sluggishness, but the change was not statistically significant.     

Some effects of long-term ozone fumigation on Norway spruce

Summary Potted cuttings of a 12-year-old Picea abies tree were fumigated with ozone, 100 or 300 g O₃· m^–3 (50 or 150 ppb O₃) being added to charcoal-filtered air during the 1985 growing season for a total of 1215 h. The wax structure of ozone-fumigated needles was no different from that of controls. Because flattened wax structures and fused wax fibrils also occurred in controls, these phenomena could not serve as bioindications for the ozone concentrations applied. A smooth layer was found beneath the soluble wax layer and covered needle surface and stomatal openings of ozone-fumigated needles to a greater extent than in controls. Wax quantity was considerably reduced by fumigation with 300 g O₃ · m^–3. Leaf pigments (as extracted with the wax) were less abundant in needles treated with 300 gO₃; the smooth layer probably contributed to the impeded extraction of pigments.     

Carbohydrate status during somatic embryo maturation in Norway spruce

Summary The development of Norway spruce (Picea abies (L.) Karst.) somatic embryos on a maturation medium was accompanied by changes in nonstructural carbohydrate status. During embryo maturation, the content of total soluble sugars in the embryonal suspensor mass decreased and the partitioning between sucrose and hexoses changed considerably in favor of sucrose. Developing somatic embryos were mainly responsible for these changes. Osmotic stress caused by the presence of 3.75% polyethylene glycol (PEG) in the maturation medium (decrease in osmotic potential by 52.5 kPa) resulted in dramatic changes in the content of endogenous saccharides. There was a lower total carbohydrate content in the embryonal suspensor mass grown on the medium containing PEG in comparison with the untreated control. Isolated embryos from later stages of embryo development contained mainly sucrose with a small amount (20%) of fructose and nearly no glucose. A further increase in PEG concentration in the medium (7.5%; decrease in osmotic potential by 112.5 kPa compared to the maturation medium) led to a large increase in the total endogenous sugar content. This increase in sugars was a result of the enhanced content of sucrose, fructose, and glucose. The increased glucose content was in contrast to embryos grown on the medium with lower or no PEG content.     

Phenolics of mycorrhizas and non-mycorrhizal roots of Norway spruce

The occurrence and amount of soluble and insoluble phenolics in mycorrhizal and non-mycorrhizal roots of Picea abies (L.) Karst, were investigated, p-Hydroxybenzoic acid glucoside, picein, piceatannol and its glucoside, isorhapontin, catechin and ferulic acid could be identified by high-performance liquid chromatography in mycorrhizas of Picea abies-Lactarius deterrimus and Picea abies-Laccaria amethystea. Both types were collected from axenic cultures and the latter also from a spruce stand. The same phenolics occurred in non-mycorrhizal short roots from sterile cultures. However, the amounts of p-hydroxybenzoic acid glucoside, picein, catechin and cell wall-bound ferulic acid were considerably reduced in mycorrhizas from axenic culture, whereas the hydroxystilbenes piceatannol, its glucoside and worhapontin were not significantly reduced. Pure mycelia of Laccaria amethystea (Bull.) Murr, and Lactarius deterrimus Gröger were also analysed for phenolic compounds. Both fungal species contained none of the identified phenolics. The results are discussed with respect to mycorrhization in different mycorrhizal types.We are grateful to the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Physiologie der Bäume) and the Fonds der Chemischen Industrie for financal support. We thank Dr. V. Wray (GBF, Braunschweig, FRG) for linguistic advice.     

Revitalization experiments in magnesium deficient Norway spruce stands in Austria

Amelioration of degraded forest ecosystems on acidic substrates showing the new type of forest decline is a major goal of forest management. A number of experiments show positive effects of Mg-application to systems suffering from Mg-deficiencies. The current paper compares experiments conducted in the Austrian part of the Bohemian Massif, where both effects on soil solution chemistry and effects on plant nutrition, vitality and growth were investigated. It turned out that any type of Mg-source is able to improve Mg-nutrition of trees; both a neutral salt like KIESERITE as well as alkaline reacting magnesite and dolomite derived materials. A positive reaction of vitality and growth could however only be induced with dolomitic lime or magnesite. Using mineral NPK fertilizers, even with high Mg-content, induced Mg-deficiencies and led to nutritional imbalances. In addition significant NO₃ ^--leaching occured. On the other hand an organic slow release fertilizer (BACTOSOL*) amended with magnesite derived fertilizers (BIOMAG**) led to balanced nutrition and a fast recovery of tree health status, as judged by crown transparency, vitality index and growth rates. In both cases, when either magnesite derived compounds or combinations with the organic slow release fertilizer were applied, NO₃ ^--leaching occured only during the first three years after fertilization. The leaching rates declined afterwards to values comparable to unfertilized plots, while Mg-content of the soil solution could be elevated compared to the CONTROL, showing the sustainability of proper fertilization.