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

Abstract

Summary Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years various counter‐selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non‐toxic 5‐fluorocytosine (5‐FC) into the toxic 5‐fluorouracil (5‐FU), was used for in vitro selection of germinating seedlings. Development of codA‐expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5‐FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono‐oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T1 plants expressing the selectable marker gene showed striking morphological differences from the non‐transgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large‐scale screening under greenhouse or field conditions.

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice

“…To date, only the bar gene was proven to work efficiently in rice as a transposon marker (Chin et al 1999;Greco et al 2001b), while the effectiveness of su1 is still controversial. The functionality of su1 in monocot species was previously reported by Koprek et al (1999), which demonstrated its potential applicability in barley for large-scale greenhouse screenings. In rice, Chin et al (1999) described the use of this marker in their original T-DNA, but did not report about its efficiency.…”

Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke

“…Our experiments indicate that selection is easily achieved in vitro by including herbicide in the growth medium or via leaf treatment of soil-grown plants with commercial phenylurea formulations. The only selection system using a P450 gene described so far is a negative selection system using a bacterial P450 gene that is also effective in vitro and in soil (O'Keefe et al, 1994;Koprek et al, 1999). CYP76B1 has the advantage over other positive selection systems in that it is a plant gene conferring resistance to a whole class of generic herbicides and also leading to their detoxification.…”

Crop Improvement through Modification of the Plant's Own Genome

“…Alternative negative selection markers may include a bacterial cytochrome P450 monooxygenase gene (Koprek et al, 1999), the Xanthobacter dhlA gene (Naested et al, 1999), and various genes involved in the activation of apoptotic cell death.…”