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Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA‐ and cytochrome P450 gene‐mediated selection
Author(s) -
Koprek Thomas,
McElroy David,
Louwerse Jeanine,
WilliamsCarrier Rosalind,
Lemaux Peggy G.
Publication year - 1999
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1999.00557.x
Subject(s) - biology , selectable marker , hordeum vulgare , gene , negative selection , genetics , transgene , selection (genetic algorithm) , genetically modified crops , cytochrome p450 , botany , biochemistry , enzyme , poaceae , genome , artificial intelligence , computer science
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 cod A 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 cod A‐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. T 1 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.