Knowledge about pollen ultra-morphology, storage characteristics and germination rate are essential for directional plant breeding and plant improvement. The objective of this study was to determine a suitable medium for pollen germination in vitro of Viola dissecta and to evaluate the effect of different storage temperatures on its pollen longevity. The pollen ultra-morphology of V. dissecta was observed using scanning electron microscopy (SEM), the suitable medium for pollen germination in vitro was determined by orthogonal test. Dried pollen of V. dissecta was stored at different temperatures (room temperature, 4, -20 and -80°C) and different storage times (24, 40, 72, 120, 184, 264 and 365 d), the germination rate of the stored pollen and the activities of SOD, POD and CAT were investigated. Pollen grains of V. dissecta were medium-sized with three germination ditches. The surface ornamentation was smooth with small grains set on the surface, which was different from Viola spp. pollen. The most suitable medium for V. dissecta was composed of 285 g•L-1 sucrose, 6 g•L-1 agar, 50 mg•L-1 GA3, 250 mg•L-1 boric acid, and 200 mg•L-1 Ca(NO3)2, The best storage temperature of pollen was -80oC, after 365 d of storage, the germination rate was still 57.86%. During storage, the pollen germination rate decreased significantly after the peak of the activities of the three antioxidant enzymes. Correlation analysis showed that SOD was major factor affecting the germination rate of V. dissecta pollen, and it has a significant positive correlation with pollen germination rate, followed by CAT and POD. SOD was a sensitive antioxidant enzyme at room temperature, 4 and -80°C, whereas at -20°C, both SOD and CAT were sensitive antioxidant enzymes. © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers©