Nitrite reductase silencing as a tool for selecting spontaneous haploid plants

Using tobacco as a model species, we have developed a simple procedure for the selection of spontaneous haploid plants under horticultural conditions, which does not require the use of any selective agent. One transgenic tobacco plant, homozygous for an antisense transgene able to silence the expression of nitrite reductase host genes, and encoding the second enzyme of the nitrate assimilation pathway, was used to pollinate two different cultivars of wild type tobacco plants. Seeds were sown at high density in the greenhouse and watered with a nutrient solution containing nitrate. Green plants able to develop normally emerged at a frequency of 5.10(-4) in a mass of chlorotic retarded plants. Phenotypic and genetic analysis, chloroplast counting in stomatal guard cells and molecular hybridizations revealed that 22% of these plants were gynogenetic haploid plants exhibiting the maternal phenotype whereas the remaining 78% were true diploid plants that have lost the antisense transgene. These results demonstrate that a transgene able to silence the expression of a housekeeping gene can be utilized as a counter-selectionable marker for the rapid and easy selection of spontaneous haploid plants in transformable species.