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The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water were sufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure using either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation-dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.