The objective of the present work was to develop a simple and reliable protocol for chromosome doubling in haploid onion plants. Research on gynogenic plants obtained from different genotypes of onion was carried out over two years. In the first year, diploidization was examined in vivo with 2.5 mM colchicine for 24 h. In the next year, in vitro tests using 0.25 mM and 1.25 mM colchicine or 1.00 muM and 5.00 muM trifluralin were carried out. Both antimitotic agents were applied for 72 h at 14?C or 23?C. The obtained results showed that colchicine applied in vivo caused twice as high loss of then plant material than diploidization at in vitro conditions. Antimitotic agents added to culture media decreased the plant capability to regenerate. Colchicine was found to be less toxic than trifluralin. The use of colchicine allowed for producing more diploids than trifluralin.