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 c onditions, which does not require the use of any selective agent. One transgenic tobacco plant, homozygous for an antisense transgene able t o silence the expression of nitrite reductase host genes, and encoding the second enzyme of the nitrate assimilation pathway, was used to po llinate two different cultivars of wild type tobacco plants. Seeds wer e sown at high density in the greenhouse and watered with a nutrient s olution containing nitrate. Green plants able to develop normally emer ged at a frequency of 5.10(-4) in a mass of chlorotic retarded plants. Phenotypic and genetic analysis, chloroplast counting in stomatal gua rd cells and molecular hybridizations revealed that 22% of these plant s were gynogenetic haploid plants exhibiting the maternal phenotype wh ereas the remaining 78% were true diploid plants that have lost the an tisense transgene. These results demonstrate that a transgene able to silence the expression of a housekeeping gene can be utilized as a cou nter-selectionable marker for the rapid and easy selection of spontane ous haploid plants in transformable species.