Mechanism of action of nitrous oxide gas applied as a polyploidizing agent during meiosis in lilies

Abstract

Nitrous oxide gas (N₂O) can be used to produce polyploid plants, but the mechanism of action is unknown. The actin and microtubule cytoskeleton was observed in N₂O-treated microsporocytes of Lilium spp ‘Asiatic hybrid lilies’ using fluorescence microscopy after staining with DAPI, FITC-conjugated tubulin antibody, and phalloidin-conjugated Alexa Fluor 546. Additionally, microsporocytes of L. longiflorum were observed with acetocarmine staining following N₂O treatment. A typical metaphase I microtubule distribution was observed in control microsporocytes. After treatment with N₂O for 24 h, microtubules were effectively depolymerized; this prevented chromosomes from moving to the poles, resulting in chromosome retention in the center of N₂O-treated cells. Cell plate formation took place without delay, however, yielding one daughter cell with a diploid genome and another daughter without chromosomes. In addition, N₂O treatment often induced micronuclei due to aberrant chromosome separation during cytokinesis. Actin filaments in microsporocytes are insensitive to N₂O. These findings indicate that N₂O mediates polyploidization by inhibiting microtubule polymerization, but not actin filament formation, during microsporocyte meiosis.