Complementary strategies for ploidy manipulations in gynogenic onion (Allium cepa L.)

Successful application of doubled-haploid (DH) technology in onion (Allium cepa L.) breeding programs requires production of large numbers of fertile DH plants. Only a small percentage of the onion plants recovered via gynogenesis are spontaneous DH, so induction of chromosome doubling is required. The chromosome doubling step is currently one of the major limitations in onion DH programs. To address this problem, three complementary strategies for increasing the frequency of DH onion plants obtained via gynogenesis were investigated. First, the doubling efficiencies of three anti-mitotic agents (amiprofos methyl (APM), colchicine, and oryzalin) on whole basal explants from in vitro haploid plant were tested. APM at 100 and 150 μM and colchicine at 750 and 1000 μM gave similar regeneration of explants (70–80%) and recovery of diploid plants (25–32%). Colchicine is highly toxic to mammalian cells, so the comparable efficacy of APM at much lower concentrations offers a safer approach. Some gynogenic plants remain haploid even after exposure to anti-mitotic agents. The second strategy recovered diploid plants from such haploid plants via spontaneous chromosome doubling in somatic shoots regenerated from haploid flowers cultured in vitro. About 60% of the somatic shoots formed on flowers from haploid plants were diploid. Addition of colchicine (12.5–50 μM) to the shoot regeneration medium did not increase the frequency of diploid plants recovered but raised the frequency of tetraploid somatic plants. The third strategy used a second cycle of gynogenesis to recover diploid plants from in vivo tetraploid and mixoploid plant materials. The diploid gynogenic plants recovered produced bulbs comparable to those of diploid control plants, averaging ca. 200 g. Use of the three strategies in combination can maximize the recovery of fecund DH plants in gynogenesis-derived populations for use in onion breeding programs.