Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA- and cytochrome P450 gene-mediated selection

Efficient negative selection systems are increasingly needed for numerous a pplications in plant biology. In recent years, various counter-selectable g enes have been tested in six dicotyledonous species, whereas there are no d ata available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of t wo different conditional negative selection systems in transgenic barley. T he bacterial codA gene encoding cytosine deaminase, which converts the non- toxic 5-fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU), was use d for in vitro selection of germinating seedlings. Development of codA-expr essing seedlings was strongly inhibited by germinating the seeds in the pre sence of 5-FC. For selecting plants in the greenhouse, a bacterial cytochro me P450 mono-oxygenase gene, the product of which catalyses the dealkylatio n of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was use d. T-1 plants expressing the selectable marker gene showed striking morphol ogical differences from the nontransgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as p redicted from the physiological appearance of the plants under selection, w as confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenab le to large-scale screening under greenhouse or field conditions.