Transgenic Aspen Research Articles

Plant growth, biomass partitioning and soil carbon formation in response to altered lignin biosynthesis in Populus tremuloides

We conducted a glasshouse mesocosm study that combined (13)C isotope techniques with wild-type and transgenic aspen (Populus tremuloides) in order to examine how altered lignin biosynthesis affects plant production and soil carbon formation. Our transgenic aspen lines expressed low stem lignin concentration but normal cellulose concentration, low lignin stem concentration with high cellulose concentration or an increased stem syringyl to guaiacyl lignin ratio. Large differences in stem lignin concentration observed across lines were not observed in leaves or fine roots. Nonetheless, low lignin lines accumulated 15-17% less root C and 33-43% less new soil C than the control line. Compared with the control line, transformed aspen expressing high syringyl lignin accumulated 30% less total plant C - a result of greatly reduced total leaf area - and 70% less new soil C. These findings suggest that altered stem lignin biosynthesis in Populus may have little effect on the chemistry of fine roots or leaves, but can still have large effects on plant growth, biomass partitioning and soil C formation.

Insect Herbivory on Low-Lignin Transgenic Aspen

Ecological effects of genetically modified plants cannot always be predicted based on knowledge of the plant species or transgene. We studied the effects of transgenic aspen (Populus tremuloides Michaux) with reduced lignin and altered growth phenotypes on the feeding performance of gypsy moth larvae (Lymantria dispar L.) and forest tent caterpillars (Malacosoma disstria Hubner). Developmental trials were conducted using one control line and four separate transgenic lines of aspen. Gypsy moth larvae showed a significant reduction in survival on one high-lignin reduction transgenic tree line relative to all other lines, but weights of surviving larvae were similar across tree lines. Forest tent caterpillars showed similar survival and weights on all tree lines. Trials were also conducted to evaluate whether gypsy moth larvae preferred feeding on high-lignin reduction transgenic aspen lines or control trees. While gypsy moth larvae showed no significant preference between the control line and the transgenic line that caused significant reductions in larval survival during developmental trials, they did strongly prefer transgenic leaves causing no such reductions in larval survival. Because effects on feeding larvae varied among tree lines, we concluded that any potential phytochemical alterations in the transgenic lines could not be directly linked to lignin reduction. Because only one transgenic tree line had a negative effect on the herbivores, we propose that this may be an indirect consequence of transgenic manipulation resulting from the insertion point of the antisense Pt4CL gene in the genome, rather than 4CL suppression or lignin reduction.

Increasing cellulose production and transgenic plant growth in forest tree species

Cellulose is one of many important polymers in plants. Cellulose is made of repeat units of the monomer glucose. Cellulose is a major industrial biopolymer in the forest products, textile, and chemical industries. It also forms a large portion of the biomass useful in the generation of energy. Moreover, cellulose-based biomass is a renewable energy source that can be used for the generation of ethanol as a fuel. Cellulose is synthesized by a variety of living organisms such as plants and algae. It is the major component of plant cell walls with secondary cell walls having a much higher content of cellulose. The relationship between cellulose and lignin biosynthesis is complicated, but it is confirmed that inhibition of lignin biosynthesis in transgenic trees will increase cellulose biosynthesis and plant growth. Cellulose accumulation may be increased by down-regulating 4-coumarate:coenzyme A ligase (4CL, EC 6.2.1.12) as shown in transgenic aspen. There is no similar reports on down-regulating 4CL in transgenic conifers. Based on our establishedAgrobacterium tumefaciens-mediated transformation system in loblolly pine, we are able to produce antisense 4-CL transgenic loblolly pine which is predicted to have increasing cellulose accumulation. The overall objective of this project is to genetically engineer forest tree species such as loblolly pine with reduced amount of lignin and increased cellulose content. The research strategy includes: (1) isolate the 4-coumarate:coenzyme A ligase gene from loblolly pine seedlings by reverse transcription-polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends-Polymerase Chain Reaction (RACE-PCR) techniques from the cDNA library; (2) construct binary expression vectors with antisense 4CL coding sequences and introduce antisense constructs of the 4-coumarate:coenzyme A ligase gene cloned from loblolly pine into the loblolly pine to down regulate the 4-coumarate:coenzyme A ligase gene expression; (3) study the effect of the antisense transgene expression on lignin content, cellulose accumulation, and loblolly pine biomass; and (4) select fast growth and high cellulose accumulation transgenic loblolly pine lines for future commercial application.

FT-IR imaging and pyrolysis-molecular beam mass spectrometry: new tools to investigate wood tissues

Fourier transform infrared (FT-IR) microimaging spectroscopy and pyrolysis molecular beam mass spectrometry (py-MBMS) were used as rapid analysis tools to evaluate differences in the chemical composition of 1-year-old transgenic aspens. Multivariate analysis of the spectroscopic data sets was used to compare the cell wall composition of nontransformed control to transgenic aspen plants with GRP-iaaM gene and with GRP-iaaM/35S-ACCase gene. Principal component analysis (PCA) was applied to both the FT-IR and py-MBMS spectra, which revealed sample groupings due to differences in chemical composition. Evaluating the PCA loadings allows determination of the chemical features responsible for the groupings. The FT-IR microimaging data was also used to monitor changes in the chemical composition as a function of the distance from the pith to the bark using partial least squares (PLS) analysis. The analysis shows that the changes in the composition of the xylem that occur over one annual growth ring can be monitored with FT-IR microimaging.

Photoperiodic Regulationof Apical Growth Cessation in Northern Tree Species

Summary Tree species adapted to the climatic conditions of the northern boreal and subarctic vegetation zones have a capacity to develop a very high level of frost hardiness, even to survive the temperature of liquid nitrogen in midwinter. Proper timing of hardening, as well as of dehardening, is crucial for winter survival of these species. In northern tree species, cessation of apical elongation growth and bud set is a prerequisite for developmental and metabolic processes leading to hardening, and this chain of events is induced by photoperiod. The northern tree species are closely adapted to the local light climate and display photoperiodic ecotypes. The critical photoperiod is under genetic control and increases with increasing latitude of origin of the eco-type. The photoperiod is probably perceived by the phytochrome system, but the role of other pigment systems, like cryptochrome, has not been studied in woody plants. Phytochrome genes have been cloned from both conifers and deciduous species, but so far we do not have any information about possible differences between photoperiodic ecotypes at the phytochrome level. Northern and southern ecotypes have different responses to red:far red ratios, which could indicate differences in composition of their phytochrome systems, for example, the proportions of phytochrome A and B. Both phytochrome A and phytochrome B can be involved in photoperiodic responses. Experiments with transgenic hybrid aspen suggest that responses to photoperiod could be affected by the amount of phytochrome A present in plants. In deciduous species, the plant hormone gibberellin A1 (GA1) can completely substitute for a long photoperiod, and short day induced cessation of growth is preceded by a significant reduction of GA1 levels, particularly in the elongation zone. Photoperiodic control of GA metabolism is supported by several studies, but very little is known about the interaction between phytochrome and GA metabolism and/or responsiveness to GA1. Although our knowledge is still very fragmentary, available results suggest that cessation of growth and initiation of hardening in trees can be controlled both through the phytochrome and the GA mediated systems. Research with tree species is a tedious and slow process, but with the emerging new methods and approaches, we may expect exciting new results in the near future.

Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism.

A PCR-based suppression subtractive hybridization (SSH) technique was used to identify differentially expressed genes in developing tissues of control and transgenic aspen (Populus tremuloides Michx.) with down-regulated 4CL1 (4-coumarate:coenzyme A ligase) expression and enhanced growth. A total of 11,308 expressed sequence tags (ESTs) representing 5,028 non-redundant transcripts encoding 4,224 unique proteins was obtained from shoot apex, young stem, young leaf and root tip SSH libraries. Putative functions can be assigned to 60% of these transcripts. Approximately 14% of the ESTs are not represented among the 111,000 entries already present in Populus EST databases. In general, ESTs of the metabolism class occurred at a higher frequency in control- than transgenic-enriched libraries of all tissues, whereas protein synthesis and protein fate ESTs were over-represented in meristematic tissues of transgenics where 4CL1 was relatively strongly suppressed. Among all tissues, leaves yielded the highest percentage of ESTs with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. Of particular interest was a large number of ESTs (16%) associated with signal transduction in transgenic leaves. Among these were several leucine-rich-repeat receptor-like protein kinases with markedly elevated expression in transgenic leaves. We also identified homologs of transposable elements that were up-regulated in transgenic tissues, providing the first experimental data for active expression of DNA mobile elements in long-lived tree species.

Identification of transgenes from wood of genetically transformed poplar trees

We have extracted total DNA from different fractions of fresh wood as well as from cold-stored and air-dried wood harvested from transgenic aspen grown in the field. The highest amounts of DNA were obtained from bark/cambium tissue; the DNA quality, however, was poor. Best results in PCR and Southern blot analyses were obtained from DNA extracted from early wood. Using appropriate primer pairs, amplification products were obtained from both the foreign gene (transgene) and aspen genomic sequences. In Southern blot analyses transgene-specific hybridisation signals were obtained. This is the first report on the detection of foreign genes in wood sampled from genetically modified trees.

Antifungal activity of stilbenes in in vitro bioassays and in transgenic Populus expressing a gene encoding pinosylvin synthase.

The effect of two stilbene compounds, pinosylvin and resveratrol, on the growth of several fungi was evaluated in plate tests. Wood decay tests were carried out with birch and aspen samples impregnated with the two stilbenes. In plate experiments, resveratrol had an enhancing effect on growth at concentrations where pinosylvin was already enough to prevent the growth of most fungi studied. Pinosylvin impregnated at 0.2% (w/w) concentration significantly reduced the decay caused by all fungi except Phellinus tremulae. In contrast, a resveratrol content of 0.8%, did not protect the wood from decay. A pinosylvin-synthase-encoding gene from Pinus sylvestris was transferred into aspen ( Populus tremula) and two hybrid aspen clones ( Populus tremulax tremuloides) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants accumulated pinosylvin synthase-specific mRNA and showed stilbene synthase enzyme activity in vitro. Transgenic aspen line H4 showed increased resistance to Phellinus tremulae, while two hybrid aspen transformants decayed faster than the control trees. However, we were unable to detect the accumulation of stilbenes in the transgenic plantlets.

Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics.

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for beta-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen ( Populus sieboldii x P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.

Vegetative and generative dispersal capacity of field released transgenic aspen trees

Transfer of genes by pollen or wind-dispersed seed is considered a main potential risk when field release experiments with transgenic trees are initiated. In Germany, the first release experiment with genetically transformed trees was initiated in 1996. To ensure that the transgenic trees remained in the vegetative phase, the duration of the experiment was limited to 5 years. In total, 457 1-year-old trees including eight transgenic aspen lines carrying either the 35S-rolC or the rbcS-rolC gene construct, and three control clones were transferred to the field. In 1998 and 2000, 12 plants of transgenic lines all carrying the 35S-rolC gene construct formed female flower buds. Furthermore, one young aspen plant identified as a root sucker was observed in 1999 followed by an increasing number of root suckers derived from transgenic and non-transgenic trees in 2000 and 2001. In 2001, the last year of the field trial, 15 root suckers were detected outside the field. In total, 234 root suckers were harvested in 2000 and 2001 and analysed for their transgenic status. More than half of the roots suckers investigated showed the presence of the rbcS-rolC gene construct. We concluded that in addition to the widely accepted generative propagation, vegetative dispersal capacity of transgenic perennial plants is also important and must be included in risk assessment studies.

Changes in gene expression in the wood-forming tissue of transgenic hybrid aspen with increased secondary growth.

Transgenic lines of hybrid aspen with elevated levels of gibberellin (GA) show greatly increased numbers of xylem fibres and increases in xylem fibre length. These plants therefore provide excellent models for studying secondary growth. We have used cDNA microarry analysis to investigate how gene transcription in the developing xylem is affected by GA-induced growth. A recent investigation has shown that genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under developmental-stage-specific transcriptional control. The present study shows that the highest transcript changes in our transgenic trees occurs in genes generally restricted to the early stages of xylogenesis, including cell division, early expansion and late expansion. The results reveal genes among those arrayed that are up-regulated with an increased xylem production, thus indicating key components in the production of wood.

Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration.

To obtain insight into the mechanism of transferred DNA (T-DNA) integration in a long-lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T-DNA/plant junctions, 20 left T-DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T-DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T-DNA left and right ends are treated mechanistically differently during the T-DNA integration process. Comparison of the genomic target sites prior to integration to the T-DNA revealed that the T-DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T-DNA. Short sequence similarities between recombining strands near break points, in particular for the left T-DNA end, were observed in most of the lines analysed. These results confirm the well-accepted T-DNA integration model based on single-stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T-DNA integration was also identified in nine transgenic lines, in which the right border of the T-DNA was partly truncated. Such integration events are described via a model for the repair of genomic double-strand breaks in somatic plant cells based on synthesis-dependent strand-annealing. This report in a long-lived tree system provides major insight into the mechanism of transgene integration.

Gene Stability in Transgenic Aspen‐Populus ‐ III. T‐DNA Repeats Influence Transgene Expression Differentially among Different Transgenic Lines

Abstract: Transgene silencing mechanisms have been confirmed in many crop plants, however, few reports are currently available for long‐lived tree species. Three transgenic aspen lines carrying the rolC gene of Agrobacterium rhizogenes, under control of the 35S promoter of the cauliflower mosaic virus, but showing very different types of reversion from rolC‐phenotype to wild type are investigated in detail. A complete suppression of rolC phenotype was observed after one year of in vitro culturing in one transgenic line, whereas the second transgenic line showed gradual decrease in rolC expression, and the plants were comparable to control plants after three to four years in the greenhouse. In the third transgenic line, however, transgene inactivation was observed only in some shoots of the greenhouse‐grown plants which reverted to the rolC phenotype in subsequent years. Southern blotting experiments using a rolC probe revealed the integration of one or two tandem copies of the foreign genes into the host genome. All three transgenic lines contain in/complete T‐DNA repeats, as indicated by reverse primer (rp) PCR. In the reverted tissues of two transgenic lines loss of rolC protein was observed despite the presence of the rolC transcript. The third transgenic line, showing a completely suppressed rolC phenotype, was found positive for the rolC transcript and protein. On the basis of the molecular data presented for the three unstable lines, including Southern, Northern and Western blotting experiments, as well as a survey of transgenic methylation patterns, new insights were obtained with respect to structure and inactivation of transgenes following their transfer in a host tree species.

Mycorrhizal colonization of transgenic aspen in a field trial.

Mycorrhizal colonization of genetically modified hybrid aspen (Populus tremula x P. tremuloides Michx.) was investigated over 15 months in a field experiment. The aspen carried the rolC gene from Agrobacterium rhizogenes under control of either the constitutive cauliflower mosaic virus 35S promoter or the light-inducible rbcS promoter. Arbuscular mycorrhizas (AMs) were rare in all root samples, while fully developed ectomycorrhizas (EMs) were found in all samples. No significant differences in the degree of mycorrhizal colonization between aspen lines were seen with either AMs or EMs. The EM community on the release area was dominated by four fungal species that formed more than 90% of all mycorrhizas, while eleven EM types were found occasionally. Mycorrhizal diversity did not differ between transgenic and non-transgenic trees. The structure of mycorrhizal communities was similar for most aspen lines. The sole significant difference was found in the abundance and development of one of the four common EM morphotypes, which was rare and poorly developed on roots from the transgenic aspen line Esch5:35S-rolC-#5 compared with non-transgenic controls. This effect is clone specific as the formation of this EM type was not affected by the transgene expression in the other transgenic line, Esch5:35S-rolC-#1. This is the first demonstration of a clonal effect influencing the ability of a transgenic plant to form a mycorrhizal symbiosis with a potential fungal partner.

Differentiation of xylem cells in rolC transgenic aspen trees - a study of secondary cell wall development

Xylem cell differentiation of 35S-rolC transgenic aspen was analyzed and compared with wild type trees. The transgenic trees, main- ly characterized by dwarfism and physiological alterations, did not show distinctive differences in the structure and chemical composition of xy- lem cell walls as compared with the wild type trees. However, there was a difference in the dynamics of differentiation. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to a few rows. PATAg and KMnO4 staining in electron microscopy as well as UV-microspectrophotometry revealed that in the transgenics during secondary wall formation polysaccharides and lignin were simulta- neously deposited close to the cambium. Immunocytochemical techniques have visualized the first deposition of condensed lignin in cell corners and of non-condensed lignin in secondary walls near cell corners. Because of altered formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful to study factors controlling the differentiation continuum. Populus / rolC / wood formation / lignification / electron microscopy / immunogoldlabelling / UV-microspectrophotometry

Asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species.

We describe here a practical system for generating selectable marker-free transgenic woody plants independent of sexual crossing. We previously reported that the GST-MAT vector system could produce marker-free transgenic tobacco plants containing a single-copy transgene at high frequency. The GST-MAT vector system consists of a DNA excision cassette of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the isopentenyltransferase gene from Agrobacterium tumefaciens has been inserted. In this study, we applied this new GST-MAT vector to hybrid aspen (Populus Sieboldii X Populus grandidentata), a model of vegetatively propagated plant species, to produce selectable marker-free transgenic woody plants. In the new GST-MAT vector, the chimeric ipt gene fused with a light-inducible rbcS promoter efficiently produced transgenic ipt-shooty with GUS activity from 38.0% of infected stems. Upon excision of the R and ipt genes between RS sites, regulated by the inducible promoter of the maize glutathione-S-transferase (GST-II-27) gene, 3 (21.4%) transgenic hybrid aspen plants with marker-free and normal phenotype were generated from 14 ipt-shooty lines within 2 months after cutting induction. These results suggest that the MAT-vector system might be useful for removing a selectable marker gene independent of sexual crossing in vegetatively propagated species.

Morphology, wood structure and cell wall composition of rolC transgenic and non-transformed aspen trees

Morphology, wood structure and cell wall composition of 35S-rolC transgenic hybrid aspen (P. tremula×tremuloides) were compared with non-transformed control trees. The transgenics are characterised by stunted growth, altered physiological parameters and light green leaves of reduced size. Histometric measurements revealed thinner fibre walls as compared to the controls. UV microspectrophotometry of individual wall layers did not reveal distinctive differences in the lignification of xylem cells, but in the extremely thin-walled fibres of the transgenics the secondary walls were less lignified as revealed by KMnO4 staining in transmission electron microscopy. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to only a few rows. Immunocytochemical analyses revealed the deposition of lignins in less differentiated xylem cells as compared to the controls. The first labelling of condensed lignin appeared in cell corners and of non-condensed lignin in secondary walls near cell corners during the deposition of S1 polysaccharides. Because of alterations in the formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful for studies on molecular factors controlling the differentiation continuum.

CLONAL STABILITY, SEASONAL PERIODICITY AND TRANSGENES: THE LESSON FROM LONG-TERM MICROPROPAGATION OF TRANSGENIC AND NON-TRANSFORMED POPULUS TREMULA L. PLANTS

Annual and diurnal rhythms of plant development are manifested as ordered changes in growth and regeneration, in physiological processes and metabolism, and in molecular events. This periodic behavior may reflect either external (environmental) stimuli, and/or endogenous (intrinsic) biological clocks of various types. Although exogenous stimuli can be controlled in vitro, the tissue also exhibits rhythmic behavior under environmentally controlled in vitro culture. A combination of in vitro culture and gene insertion via transformation could, therefore, be used to elucidate some of the potential interactions between culture growth rhythms and stability, the regeneration potential, and molecular control mechanisms such as silencing. These and related aspects were examined in our laboratory, using long-term cultures of transgenic and non-transformed aspen plants (Populus tremula L.). Stem segments have been routinely subcultured every 4 to 8 weeks under standard conditions, without any hormones, since 1990, resulting currently in plantlets representing about 80 to 120 passages in culture. In addition, two types of transgenic plants have been generated: (1) expressing the rol-T-DNA fragment from Agrobacterium rhizogenes LBA9402, carrying a Ri vir, and (2) expressing the water stress-regulated BspA cDNA. Seasonal periodicity in non-transformed aspen development was clearly evident with respect to adventitious root formation, shoot elongation and axillary bud break. Generally, these growth parameters were at a maximum when explants were subcultured in the summer, and extremely low during the winter. This was also reflected in the considerably higher in vitro propagation coefficient in the summer vs. winter. This recorded pattern of development has persisted for over more than 10 years since the primary explants were excised from the mother plant and cloned in vitro and no other phenotypic changes have been detected. The in vitro-cultured transformed lines, transgenic for rol-T-DNA and expressing the rolB and rolC genes, were characterized by accelerated regeneration and growth of roots and shoots, and by the loss of a seasonal growth rhythm. Seasonal fluctuation in the expression of the gene encoding an aspen water-stress-responsive boiling-stable protein (BspA) at the RNA and protein levels as also detected in the same plant material. The persistence of growth periodicity in long-term cultures suggests that the actual rate of phenotypic polymorphism in plants is often far less frequent than DNA and protein band polymorphism. It is proposed that specific existing and induced mechanisms contribute to clonal stability in tissue culture, and that these are at least as important as the suggested mechanisms of variation induction in vitro.

486 Expression of β-glucuronidase Gene in Aspen under Control of CaMV35S, Heat Shock and RolC Promoters

Transgenic hybrid aspens (Populus tremuloides × P. tremula) were produced by Agrobacterium-mediated transformation and confirmed by polymerase chain reaction. Three promoters (CaMV 35S, Heat shock, and Rol C) were used to drive transcription of chimeric genes -glucuronidase (GUS), npt-II, and rol B. Stem sections in ≈100 mm thick, leaf blades, and root tips of transgenic aspen were treated with X-Gluc solution for 2 to 12 h in a 37 °C incubator and fixed in a solution containing 5% formaldehyde, 5% acetic acid, and 20% ethanol (FAA) for 10 min. After washing with 50% ethanol twice and clearing with absolute ethanol until free of chlorophyll, the GUS expression (localization and intensity of blue staining) in leaf, stem, and root at different growth stages were evaluated and photographed under the light microscope. When CaMV35S and rol C were used as promoters, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in phloem. The heat shock promoter gave rise to very strong expression only in epidermis and phloem. In the young stem, GUS activity was detected in epidermis, parenchyma, vascular cambium, and primary xylem in CaMV35S-GUS transformed aspen shoots. The rol C promoter produced GUS gene expression in all stem tissues. When the heat shock promoter was used, the GUS gene expressed in a more tissue-specific manner, especially in mature stems, with activity mainly in parenchyma. In young leaf tissues, the GUS activity was primarily located in veins and mesophyll. In the mature leaves, no blue staining was found in the main vein. In root tip, the GUS gene driven by CaMV35S and heat shock promoters were expressed in the columella, vascular, and root apical meristem with very strong expression in the root apical meristem. Aspen plants transformed by rol C-Gus construct showed less or no expression in the columella.

Determination of transgene repeat formation and promoter methylation in transgenic plants.

The integration of transgenes into a plant host genome following Agrobacterium tumefaciens-mediated or direct transformation may occur as a single copy or in the form of tandem repeats. The latter has been associated with promoter methylation and silencing of transgenes. Thus, the early screening of such transgenic plants is desirable for ruling out future repeat-dependent transgene instability. We developed a simple PCR-based method in which primer pairs were specifically designed so that amplifications could only be obtained if the transgene was present in the form of multiple inserts in a transgenic line. The method was established using 35S-rolC transgenic aspen lines showing morphologically visible transgenic silencing. Later, it was possible to screen independent transgenic lines showing no visible marker gene expression. Furthermore, a method was developed in which positive PCR amplification was indicative of promoter methylation. The results were consistent and reproducible across different independent transgenic lines. The methods were quick, reliable, consistent and reproducible, and can be useful for routine screening of transgene silencing in lines derived from many different systems.