We evaluated the stability of trangene expression in a hybrid poplar (Populus tremula $\times$ P. alba) clone transformed with constructs carrying a reporter gene (uidA) under the control of either a constitutive (35S) or a vascular-specific promoter. Analyses of transgene expression by GUS fluorometry and histochemistry was performed on several hundreds of trees, originating from 44 different transgenic lines, grown under in vitro, greenhouse and field conditions. While important variations in expression levels occurred, the transgene appeared to be stably expressed throughout a 6-year period. Only one silenced transgenic line was detected under in vitro conditions: molecular analyses indicated that this line contained an elevated number of transgene copies and was probably silenced from the beginning, at the post-transcriptional level. Overall, these results suggest that transgene expression in perennial species such as trees remains stable over an extended period.

A transformation procedure was developed for hybrid larch embryogenic tissue using Agrobacterium tumefaciens. The cocultivation procedure yielded one to two transformation events per 100 cocultivated masses. The addition of 100 µM coniferyl alcohol increased the yield. This improved procedure was successfully applied to three other genotypes. After 3 months on selective medium, the transgenic tissue remained embryogenic, which allowed production of transgenic plants in the greenhouse. Stable integration of the transgene was confirmed by PCR and Southern hybridisation on transformed tissues and acclimatised plants.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the last step in the biosynthesis of the lignin precursors, which is the conversion of cinnamaldehydes into cinnamyl alcohols. A poplar (P. trichocarpa x P. deltoides) CAD cDNA was introduced in sense and in antisense orientation into P. tremula x P. alba via an Agrobacterium-mediated transformation. The CAD activity was reduced by 70% in the xylem tissue of three antisense and two co-suppressed lines. A red coloration of the xylem was detected in all poplar lines with a residual CAD activity of less than 60%. The lignin content and the lignin composition (syringyl/guaiacyl) were similar in co-suppressed, antisense CAD and control lines. However, chemical properties of the cell wall were different. First, a phloroglucinol staining showed a different coloration in the xylem of the down-regulated CAD poplars, being red-brown as compared to the typical pink staining of the control xylem. Second, after NaOH treatment, lignin could be precipitated by acidification of the alkali-soluble fraction of the cell wall of the red poplars but not of that of the white poplars. A spectral characterisation of the alkali fraction from red poplars showed an increased level of vanillin and syringaldehyde. Third, pulping experiments showed less residual lignin in the pulp of CAD down-regulated poplars.

SummaryLignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bi‐specific caffeic acid/5‐hydroxyferulic acid O‐methyltransferase (COMT) catalyses the O‐methylation of caffeic acid and 5‐hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa x P. deltoides) in a P. tremula x P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transformants, the COMT activity was reduced by 95%. In the latter case, the syringyl/ guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5‐hydroxyguaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild‐type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown‐midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls.