We aim to provide a theoretical basis for improving the cold tolerance of Miscanthus spp., a widely recognized C4 perennial bioenergy crop, and extending its application in the industry. This study evaluated its cold tolerance by measuring the relative electrical conductivity (REC) of detached leaves. We calculated the half-lethal temperature (LT₅₀) during non-acclimation and acclimation treatments in the 12 wild genotypes of M. sacchariflorus and M. lutarioriparius from different regions of China. In this study, five temperature treatments were carried out to simulate the natural early spring cold process, with temperatures of 0 °C, −4 °C, −8 °C, −12 °C, and−16 °C. We compared the REC and LT₅₀ during the non-acclimation and acclimation treatments, and the results show that the REC of the 12 genotypes increased with a decrease in the treatment temperature, forming an S-shaped curve, which was significantly negatively correlated with the corresponding temperature. Under non-cold acclimation, the M. sacchariflorus B0111 from Jiamusi, Heilongjiang Province, had the lowest LT₅₀ of −9.49 °C, showing extraordinarily strong cold tolerance. However, M. lutarioriparius A0630 from Shaoxing, Zhejiang Province, had the highest LT₅₀ of −6.43 °C, demonstrating the weakest cold tolerance. After 21 days of cold acclimation, B0111 still exhibited the most substantial cold tolerance. While A0630 showed an enhanced cold tolerance, it remains the weakest in this study. The cold acclimation abilities of the 12 genotypes varied from −0.016 to 0.666 °C. Additionally, we found that the tolerance abilities of Miscanthus were enhanced after acclimation. Furthermore, its tolerance capacity was found to vary according to the geographic factor of its original location, which shows the significance of the correlation with latitude. However, there was no correlation found with altitude or longitude.

Seed storage is critical for preserving genetic resources, but optimal long-term storage conditions for Miscanthus seeds have not been established. In this five-year study, we evaluated storage protocols by comparing seed germination after four and five years, along with field establishment performance. The results demonstrated that genotype, storage conditions, and the storage duration all significantly influenced germination percentage and vigor index of Miscanthus seeds. Low temperature storage yielded the highest germination percentage (59.44%) and vigor index (132.06) in the 4th year, while low temperature with desiccant gave the highest germination percentage (42.41%) in the 5th year. The field performance after direct sowing was also significantly influenced by genotype and storage conditions, with the highest seedling survival (7.80%) observed under low temperature with desiccant. The seeds stored under low temperature exhibited minor structural damage, with the intact cell membranes, the small intercellular gaps, and the orderly cell arrangement. Through comprehensive evaluation, storage at −18 °C with desiccant was determined to be optimal. Based on these results, we strongly recommend storing Miscanthus seeds at −18 °C with desiccant. This protocol offers a reliable and effective solution for farmers, seed producers, and storage facilities to ensure long-term seed viability.