Spatial pattern of cauliflower mosaic virus 35S promoter-luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging

A protocol has been developed for efficiently transforming and regenerating the hybrid aspenPopulus tremula x P. tremuloides. Stem segments were co-cultivated with a strain ofAgrobacterium tumefaciens carrying a disarmed binary vector conferring resistance to kanamycin or hygromycin. The respective vectors also carried a fused bacterialluxF2 gene expressed from the cauliflower mosaic virus 35S promoter. All transformants had a normal phenotype. Genetic tranformation and stable integration of the heterologous DNA was confirmed by Southern hybridization and luciferase expression. The latter was measured by destructive enzymatic assay throughout the transformatnt and by non-destructive image analysis in leaves left attached to intact plants. Both measurement techniques detected marked within- and between-organ variation in luciferase expression. However, the spatial patterns detected by each technique in the leaves were similar. The results indicate thatin vivo imaging of light emission can be used to measure repeatedly the expression of a promoter-luciferase gene fusion in a particular leaf over an extended time period. It was also demonstrated that enzymatically assayed luciferase activity in leaves was notably lowere in transgenic hybrid aspen plants than in tobacco plants transformed with the same vector. This was not due to a difference in luciferase enzyme activity between the two species, and therefore indicated that the 35S promoter is not as active in hybrid aspen as in tobacco.