Relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet.

Abstract

The relationship between the expression level of the selectable marker gene and transformation frequency was investigated in transgenic sugar beets with five different promoters, a modified cauliflower mosaic virus 35S RNA promoter (E35S), an enhanced nopaline synthase promoter (ENOS), a modified mannopine synthase promoter (SMAS), a heat shock protein promoter (HSP80) and a chlorophyll a/b-binding protein promoter (CAB3), to drive the expression of the selectable marker gene. The selection system employed was based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable marker gene and mannose as selective agent. The selected transgenic shoots were analysed for PMI activity and the average activity for each promoter was found to be 5.9 (HSP80), 31 (SMAS), 38 (E35S), 49 (ENOS) and 61 (CAB3) mU/mg. The weakest promoter, HSP80, resulted in the lowest transformation frequency (0.30%), suggesting that this promoter was too weak to confer sufficient resistance to mannose. On the other hand, the strongest promoters, ENOS and CAB3, only gave intermediate transformation frequencies, 0.44% and 0.47% respectively, while the somewhat weaker SMAS promoter produced the highest transformation frequency, 0.89%. Thus, these data suggest that the activity of the selectable PMI gene should be above a certain threshold level; however, above this level, no simple correlation between the PMI activities, calculated as averages, and transformation frequencies could be deduced. However, extended data analysis by dividing the transgenic shoots into 4 groups according to their PMI activities (low( 100 mU/mg) expressers) revealed a significant positive correlation between the relative number of shoots having medium levels of expression and transformation frequency. This indicated that promoters that predominanthly give rise to intermediate expression levels of the selectable PMI gene result in high transformation frequencies.