Parameters interacting with mannose selection employed for the production of transgenic sugar beet

The mannose selection system employs the phosphomannose isomerase (PMI) gen e as selectable gene and mannose, converted to mannose-6-phosphate by endog enous hexokinase, as slective agent. The transgenic PMI-expressing cells ha ve acquired the ability to convert mannose-6-phosphate to fructose-6-phosph ate, while the non-transgenic cells accumulate mannose-6-phosphate with a c oncomitant consumption of the intracellular poets of phosphate and ATP, Thu s, certain steps of mannose selection depend on the cells' own metabolism,w hich may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens-mediated gene transfer to sugar beet cotyl edonary explants, Four frequently employed saccharides (sucrose, glucose, f ructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being a ble to counteract nearly 100% of an almost complete mannose-induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly th e mannose-induced growth inhibition, but mere 4-, 5-, and 7-fold less poten t than glucose, respectively (calculated as hexose equivalents). The transf ormation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highe st transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non-transgenic escapes were found over a range of concentrations. The effect of the light intensity mas also investi gated and the transformation frequencies mere positively correlated to ligh t intensity, although the relative impact of light on growth in the presenc e of mannose appeared not to be dependent on the mannose concentration. Add itional phosphate in the selection media had a strong positive effect on th e transformation frequencies, suggesting phosphate limitation during select ion. The mannose selection system was found to be relatively genotype-indep endent, provided a slight optimization of the mannose concentrations during selection. Analysis of F-1-offspring showed that all studied primary trans formants resulted in PMI-expressing plantlets and that the segregational pa tterns were in accordance with expectations in at least 50%, of the transfo rmants, confirming the stable and active inheritance of the PMI-gene.