Differential activity of the mannopine synthase and the CaMV 35S promoters during development of transgenic rapeseed plants

Abstract

Fusions of the promoter of the cauliflower mosaic virus 35S RNA transcript (CaMV 35S) and the mannopine synthase ( mas) gene to the β-glucuronidase (GUS) reporter gene have been introduced into various cultivars of Brassica napus via Agrobacterium-mediated transformation. Transgenic rapeseed plants have been also regenerated from winter cultivars (Santana, Arabella) by shoot induction from kanamycin-resistant callus tissues on the medium supplemented with AgNO 3. Transformation was confirmed by Southern hybridization of genomic DNA from primary transformants and PCR analysis of DNA from second generation seedlings. β-glucuronidase activity analyzed by fluorometric assay or histochemical staining indicated a differential expression pattern for the two promoters. Organogenesis from in vitro cultured callus tissues was coupled with a relative increase of CaMV 35S promoter activity and reduction of mas promoter function. In seedlings, the CaMV 35S promoter had maximum activity in the primary roots, while the mas promoter was the most active in the cotyledons. Etiolated seedlings cultured in dark, showed reduced activity of the mas promoter. At rosette stage, both promoters were more active in elder plant parts than in younger ones. The highest activity values were recorded in cotyledons. After bolting, reduced promoter function was detected in upper parts of the transformed plants. Histological staining showed that the CaMV 35S promoter was active in the cortex, the phloem and the vascular cambium, while the mas promoter directed gene expression in the phloem. In conclusion, both promoters were found to be functional in majority of the studied organs of transgenic rapeseed plants, however the promoter activity varied considerably between organs and tissues at various developmental stages.