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lines produced bands of varying intensity but always weaker than the band produced by the single copy gene, spi1. When the PCR was repeated, line K1 7.4- 1 was also positive. All eight lines were also found positive at Skogforsk. Later when the DNA extraction was repeated for the same lines at KVL, no bands for nptII were produced by PCR, and extraction of the same lines using the DNeasy kit only identified nptII from K1 7.4 16. However, spi1 could be easily amplified after every extraction, leaving no reason to believe that inhibitory conditions had been present during the amplification. Of the remaining 17 lines tested at KVL, only 2 lines were found to be positive for nptII after repeated DNA extractions. Neomycin-phosphotransferase protein was detected by ELISA only in 5 out of 48 lines tested (Table 2). Discussion Amplification of nptII yielded bands of varying intensity in the different lines, indicating that the nptII-gene might be present in a different number of copies in the different lines. The different embryogenic lines may also contain both transformed and non-transformed cells, in that case the transgenic lines are chimaeras. The single-copy gene spi1 yields stronger bands than the nptII gene. This may indicate an average of less that one copy of spi1 per cell, although this difference could also be explained by a better amplification efficiency of spi1 than of nptII. The presence of chimaeras may to some extent explain why repeated extractions of DNA from the same lines led to different PCR-results (presence or absence of nptII), especially if transgenic cells have been rare in the transgenic lines. Npt II protein was detected by ELISA only in 5 out of 48 lines. This implies that either few transgenic cells have been present in the selected lines or that nptII has been silenced. A few transgenic Norway spruce lines carrying the nptII gene, but without detectable amounts of nptII protein were identified by Walter et 12 al. (1999). Though not finally confirmed, we suspect the transgenic lines to be chimaeras. As cell aggregates, not single cells, were bombarded, we expect that not all the cells were hit. The non-transformed cells should have died during selection with geneticin, but a shielding effect from transformed neighbour cells might have let non-transformed cells escape the antibiotics, giving rise to chimaeras. A shielding effect seems likely as the gene product of the antibiotic resistance gene nptII is secreted out of the cell, as predicted by the program SignalP 3.0, Technical University of Denmark, www.cbs.dtu.dk/services/SignalP/ which finds a signal peptide comprising the first 15 nucleotides or 19 nucletides of the nptII gene depending of the model used. NptII is derived from E. coli, but the signal peptide is predicted to be present also when SignalP are trained (run) on eukaryotes, indicating that the nptII signal peptide functions also in Norway spruce. Selection for transgenic cells was carried out only on solid media. A better effect of selection might have been obtained, if the transgenic cell aggregates appearing on the solid medium had been transferred to liquid medium containing geneticin for a better exposure to the antibiotic. In general, the occurrence of chimaras is difficult to avoid. After transformation of Pinus radiata with pCW122 followed by selection with geneticin (15 mg/l), a screening for nptII in the transgenic lines produced bands of very different intensity. Band intensity could not be correlated with copy number as estimated after southern analysis, and the presence of chimaeras, though stated to be unlikely, could not be rejected (Walter et al. 1998). The GUS assay is useful to screen for the presence of transgene activity. This was done with success by Walter et al. (1999), Norway spruce plantlets turning bright blue upon GUS induction were obtained. In the
current study we removed the GUS reporter gene from pCW122 to reduce the size of the pCW122 (8174 bp) and thereby ease the transformation into E. coli. Even without uidA pCW122 was fairly large (6273 bp) increasing to 7641 bp after insertion of PSS1. We now consider the removal of uidA a mistake. Knowing the difficulties met to screen for the presence of nptII, and the problems to interpret the test results, a simple stain for β-galactosidase activity to indicate the presence of non-transgenic (or silenced) tissue would have been very useful. Despite the problems met during PCR-testing, a comparison of DNA analysis test results obtained at KVL and at Skogforsk makes it very unlikely that no transgenic embryogenic lines of Norway spruce has been produced. PCR-results at KVL and at Skogforsk for the cell line 11703-B63 support each other, and suggest that several transgenic lines of the types K1 and K4 have been produced. These lines are marked with a‘*’ in Table 2. Unfortunately, few of these seem to produce the NptII protein. Embryogenic lines produced after bombardment of the cell line 186-3C were found to be transgenic only at Skogforsk, leaving uncertainty about these results. The lines were tested to be transgenic for nptII, and may also be transgenic for the pinosylvin synthase gene PSS1 as this gene is carried on the same vector. A test for PSS1 is needed to prove this. Suitable primers would amplify an 805 bp band from non-transformed Scots pine, whereas no bands should be detected from nontransformed Norway spruce. Fifty Norway spruce seedlings were produced successfully from the cell line 11703-B63, transformed with plasmids carrying pinosylvin synthase both in sense and anti-sense directions. The cell line 186-3C proved more difficult to regenerate plants from and only 3 seedlings were produced. The seedlings had a normal appearance. Unfortunately they died during cold storage before any testing of the plants had been carried out. The authors believe the transformation technique applied needs improvement, before further regeneration of transgenic seedlings is worthwhile. A transformation system where only single cells instead of cell aggregated are bombarded with plasmid would eliminate the possibility of non-transgenic escapes, especially in combination with high efficient selection techniques. No results about the effect of modified stilbene production in Norway spruce on spruce pathogens could be obtained from this study. References Brignolas, F., Lieutier, F., Sauvard, D., Yart, A., Drouet, A. and Claudot, A. C. 1995a. Changes in soluble phenol content of Norway spruce (Picea abies) phloem in response to wounding and inoculation with Ophiostoma polonicum. European journal of forest pathology 25: 253-265. Brignolas, F., Lacroix, B., Lieutier, F., Sauvard, D., Drouet, A., Claudot, A.-C., Yart, A., Berryman A. A. and Christiansen, E. 1995b. Induced responses in phenolic metabolism in two Norway spruce clones after wounding and inoculation with Ophiostoma polonicum, a bark beetle-associated fungus. Plant Physiology 109: 821-827. Celimene, C. C., Micales, J. A., Ferge, L., Young, R. A. 1999. Efficacy of pinosylvins against white-rot and brown-rot fungi. Holzforschung 53(5): 491-497. Chiron, H., Drouet, A., Lieutier, F., Payer, H. D., Ernst, D. and Sandermann, H. 2000. Gene induction of stilbene biosynthesis in Scots pine in response to ozone treatment, wounding, and fungal infection. Plant physiology 124(2): 865-872. Clapham, D., Demel, P., Elfstrand, M., Koop, H.-U., Sabala, I. and von Arnold, S. 2000. Gene transfer by 13
Page 1 and 2: Transformation of Norway spruce wit
Page 3 and 4: Figure 1. Pathways for stilbenoid a
Page 5 and 6: as vector for transformation into N
Page 7 and 8: g/1) added to solidify the gel. The
Page 9 and 10: Measurement of neomycin-phosphotran
Page 11: Bombardment Cell line B63 K8 11703-
Page 15 and 16: Krogstrup, P. 1986. Embryo-like str

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