Summary• It has been shown previously that height growth and bud phenology are influenced by the temperature during zygotic embryogenesis in Picea abies .• To test whether this phenomenon operates within individual plants, clones produced through somatic embryogenesis were used. Seeds were from a full-sib family produced in both a cold (outdoor) and a warm (inside a glasshouse) environment. Embryogenic clones derived from mature zygotic embryos from both crossing environments were cultured at 18, 23 and 28 ° C during the proliferation and embryo maturation steps.• After the second growing season in a glasshouse, plants from the warm seed production environment were taller and had significantly later bud set. For the first time, it is also shown that plants are influenced by the in vitro temperature during somatic embryo development. The warmer the temperature, the later the plants formed terminal buds. The differences were similar to those produced by a provenance separation of 4 -6 degrees of latitude.• The results indicate that there exists a mechanism in P. abies that operates during embryo development and adjusts the timing of bud set in accordance with the temperature conditions in which the mother tree lives. This in turn counteracts negative effects of gene flow among populations located along altitudinal and latitudinal gradients.

Pathogen colonization and transcript levels of three host chitinases, putatively representing classes I, II, and IV, were monitored with real-time PCR after wounding and bark infection by Heterobasidion annosum in 32-year-old trees of Norway spruce (Picea abies) with low (clone 409) or high (clone 589) resistance to this pathogen. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. At 14 days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for clone 589 but had progressed further into the host tissue in clone 409. Transcript levels of the class II and IV chitinases increased after wounding or inoculation, but the transcript level of the class I chitinase declined after these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in clone 589 than in similar sites in clone 409 3 days after inoculation. This difference was even more pronounced 2 to 6 mm away from the inoculation point, where no infection was yet established, and suggests that the clones differ in the rate of chitinase-related signal perception or transduction. At 14 days after inoculation, these transcript levels were higher in clone 409 than in clone 589, suggesting that the massive upregulation of class II and IV chitinases after the establishment of infection comes too late to reduce or prevent pathogen colonization.

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.Determining the colonization rate of pathogenic fungi and other microbes in host tissues is central to phytopathological studies. One way to monitor fungal hyphae within the host is by microscopic examination of host tissue stained with fungusspecific dyes (15), but this procedure is tedious and difficult to apply in a quantitative manner. Therefore, indirect methods have been developed and applied for estimation of fungal biomass in planta.Ergosterol (6, 14)-based assays commonly have been used to determine fungal biomass within host tissues. Ergosterol is a membrane-associated sterol that occurs almost exclusively in fungi and is degraded in dying mycelium, so it correlates well with markers for metabolic activity (22). Thus, ergosterol assays are commonly used to estimate the amount of living fungal biomass (for examples, see reference 6). In all indirect methods for estimating fungal biomass, a conversion factor (i.e., the relationship between the quantity of the measured component and the total weight of the organism) must be established. The validity of the ergosterol assay for this purpose has been questioned, as the concentration of this component may vary due to nutrient availability and colony age (3). The ergosterol method also lacks specificity and is best suited for laboratory studies, where the presence of multiple fungi can be excluded and will not interfere with the results.PCR-based techniques can be used for sensitive and specific monitoring of phytopathogenic microbes in their natural substrates. Real-time PCR is currently a promising PCR method for quantifying DNA. The measurements are performed during the exponential phase of the reaction, when PCR efficiency is not...

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An efficient Biolistic® transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

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We studied how light from different light sources influences germination and postgerminative growth of plants from somatic embryos and seeds of Norway spruce (Picea abies [L.] Karst). Somatic embryos of three spruce genotypes and seeds were subjected to light from commercially available light sources: Philips TLD Blue 18W/18 (BL), Osram Fluora (FL), Philips Cool White TL 50W/33 (CW), Osram Warm White 18W/30 (WW), Philips Yellow 36W/16 (YE) and Philips TLD Red 36W/15 (RE), 18 h a day, with a photon flux (PAR) at 30 gmol m 2 s-l. After 6 wk the germination frequencies of the somatic embryo-derived plantlets were 50% under BL and 98% under RE. The corresponding mean root lengths were 6.7 and 15.4 mm. In somatic embryo-derived plantlets cultured under BL, FL, CW and WW, both roots and hypocotyls turned brown, presumably due to production of phenolic substances. Browning was less severe in somatic embryo-derived plantlets cultured under RE and YE. Under RE, the epicotyl elongated in 37% of the plantlets after 6 wk, compared with 70% under the other light sources. Seed germination and postgerminative seedling growth was modestly influenced by light from these light sources. RE and WW initially delayed germination as compared with BL, FL and CW, but after 2 wk, more than 90% of the seeds had germinated under all light sources. In conclusion, germination and postgerminative growth of somatic embryos of spruce is sensitive to differences in light quality, whereas seed germination and seedling growth is not.