Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers

The improvement of perennial fruit trees through traditional breeding methods is a long-term effort because of their long generation time. The production of haploid and doubled haploid plants should offer new possibilities for genetic studies and breeding work. In this study, haploid clones of pear were treated in vitro by oryzalin for chromosome doubling; the level of ploidy was assessed by flow cytometry. Oryzalin appeared to be an efficient agent for chromosome doubling, the optimal concentrations range from 200 to 300 muM. For homozygosity assessment, analyses of isozyme markers were carried out, together with microsatellite markers PCR-amplified with primers initially developed for apple. The use of isozyme markers confirmed homozygosity of all the doubled haploid clones except for one. The microsatellite markers can be used earlier than isozymes for checking homozygosity during the programme of haploid and doubled haploid clones production. True doubled haploid clones of pear were obtained in less than one year and their acclimatisation in greenhouse has already started.