Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation

Abstract

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.

Cryopreservation of Sugarcane Species by Droplet-Vitrification

“…For variety Q42431, the highest level (76.3%) was observed after dehydration with PVS2 for 30 min. Some factors play a significant role in the success of the sugarcane cryopreservation, and the age and quality of the in vitro cultures should be considered [17]. The results obtained in this work emphasize the high level of specificity in the behavior of sugarcane varieties when submitted to cryogenic procedures.…”

“…For other hand, Rafique et al ( 2015) [14] precultured shoot tips in the presence of 0.3 M sucrose. Preculture on medium with high sucrose concentration was important to induce tolerance to vitrification solutions [7,17].…”

“…In this state, the formation of ice crystals is inhibited or minimized. This cryopreservation technique has been the target of studies with variations in the results as a function of the pre-culture medium, vitrification solution, exposure time, among other factors [13,14,15,16,17]. Most of these results were obtained for S. officinarum varieties and responses from other species of the genus Saccharum are scarce in the literature.…”

“…Most of these results were obtained for S. officinarum varieties and responses from other species of the genus Saccharum are scarce in the literature. In addition, variations in the responses between species and genotypes of the same species have been reported as a difficulty in applying a standard cryopreservation protocol for the Saccharum genus [17,18].…”

The Tip of the Iceberg: Cryopreservation Needs for Meeting the Challenge of Exceptional Plant Conservation

“…In comparing this search with the List of Exceptional Plants there were 74 families in common, or 67% of the exceptional families. The Orchidaceae and the Poaceae, both in the top 25 families with exceptional species, had over 100 articles each, likely reflecting the commercial importance of these two families, and providing a good basis for work with additional taxa in these groups [31,32]. However, comparing the two lists at the level of genus revealed only 92 genera in common, representing only 25% of the currently known exceptional genera.…”