N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N(2)O) gas to obtain 2n male gametes. N(2)O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N(2)O treatment restores fertility in sterile hybrids. To establish optimal N(2)O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N(2)O gas at different anther developmental stages from fertile and sterile hybrid lilies. N(2)O treatment using the optimal 1-4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N(2)O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC(1) plants. Thus N(2)O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.