Abstract
Nymphaea minuta and Nymphaea micrantha are two species of waterlilies in Brachyceras subgenus. Both seeds are freezing and desiccation intolerant, and could not be stored by conventional method for long-term. Therefore, they were considered as non-orthodox seeds, which have to be conserved by cryopreservation. In this study, vitrification-based cryopreservation method was applied by using plant vitrification solution 3 (PVS3) to avoid lethal ice crystals formation. The 2hr and 3hr PVS3 pretreatment were most suitable for cryopreserved seeds of N. micrantha and N. minuta, separately. To further promote the efficacy of cryopreservation, partial dehydration was applied for enhance the efficiency of vitrification, and the antioxidant, glutathione (GSH) was added in PVS3 to prevent cryopreserved seeds from the oxidative damage and programmed cell death which was induced by reactive oxygen species (ROS). The changes of epigenetic states in cryopreserved seeds with different pretreatment were next investigated. The global DNA methylation rate of seeds increased after cryopreservation in both species. Therefore, it was considered that changing the DNA methylation rate might improve the viability of cryopreserved seeds. Then, the cryopreserved seeds of N. micrantha were treated with 5-azacytidine, a DNA-hypomethylating molecule, in recovery stage, and the germination rate increased.