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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections

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

item Volk, Gayle
item Jenderek, Maria
item Staats, Elise
item Shepherd, Ashley
item Bonnart, Remi
item LEADO, ANA - US Department Of Agriculture (USDA)
item Ayala-Silva, Tomas

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2018
Publication Date: 2/28/2019
Citation: Volk, G.M., Jenderek, M.M., Staats, E.R., Shepherd, A.N., Bonnart, R.M., Leado, A., Ayala Silva, T. 2019. Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation. Acta Horticulturae. 1234:335-342.

Interpretive Summary: The USDA-ARS National Plant Germplasm System (NPGS) sugarcane (Saccharum) collection is a field collection of clonally propagated plants maintained in Miami, Florida and is vulnerable to environmental disasters, including hurricanes. We sought to develop a cryopreserved collection of sugarcane as back-up to the field collection, and expected that protocols reported by other labs could be readily implemented at the National Laboratory for Genetic Resources Preservation (NLGRP). We did not achieve comparable levels of regrowth reported by other labs when we tested diverse Saccharum species and cultivars. Efforts to improve regrowth levels after cryopreservation focused on the addition of antioxidants, choice of cryopreservation method (droplet vitrification, encapsulation dehydration, or V-plate vitrification), choice of vitrification solution (PVS2 vs. PVS3), and length of cryoprotectant exposure. Although regrowth levels were as high as 50% for one cultivar after liquid nitrogen exposure, the variation in regrowth levels across cultivars caused us to conclude that sugarcane cryopreservation is not yet ready for implementation at NLGRP.

Technical 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.