Melatonin different increasing the cryopreservation recovery rate of shoot tips of Chrysanthemum morifolium cv. Chuju by regulating the level of oxidative stress

Cryopreservation is vital to the preservation of genotypic diversity of plant species. In the present study, an efficient procedure for cryopreservation of Chrysanthemum cv. Chuju shoot tips was established for the first time. The adventitious shoot clusters of Chuju were transferred to MS medium fortified with 100 µM melatonin (MLT)and cultured at 4 °C for 4 days. Then, the shoot tips were excised and precultured on MS medium supplemented with 0.4 M sucrose for 4 days at 4 °C in the dark. After that, the shoot tips were loaded in 60 percent plant vitrification solution 2 (PVS2)for 20 min at 0 °C, followed by immersion in 100 percent PVS2 solution for 60 min. Then, put the shoot tips into liquid nitrogen (LN,???196 °C). After at least 1 h, the LN-stored shoot tips were rapidly rearmed for 2 min at 39 °C, and liquid MS medium containing 1.2 M sucrose was used as the unloading solution for 15 min. Finally, the cells were postcultured in recovery medium for shoot regeneration. Under these conditions, 65.7 percent of cryopreserved shoot tips survived and regenerated. Further biochemical and histological analysis showed that exogenous MLT could improve the survival rate by regulating the level of oxidative stress and maintaining the morphological structure of shoot tip cells, which provided a new method for exploring the mechanism of MLT in cryopreservation. Intersimple sequence repeat analysis did not reveal any polymorphic bands in regenerants recovered from the shoot tips of Chuju. We expect that our results will facilitate the successful application of MLT in the cryopreservation of rare and commercially important plant germplasm.