Comparison of two PVS2-based procedures for cryopreservation of commercial sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp.) germplasm and confirmation of genetic stability after cryopreservation using ISSR markers

Conservation of Saccharum spp. germplasm as ex situ collections of plants has a high cost, and in natural conditions, the plants remain exposed to pests, pathogens, and natural disasters. Long-term preservation of plant germplasm is important for agricultural biodiversity and food safety, so the aim of this study was to develop a cryogenic procedure for cryopreservation of sugarcane germplasm. The first study compared droplet vitrification and encapsulation-vitrification techniques for cryopreservation of in vitro shoot tips of Saccharum spp. variety Halaii. The best regeneration rate (70.9%) was obtained from 45-min PVS2 vitrification solution-treated shoot tips via the droplet vitrification technique. This technique was tested on two other Saccharum sp. varieties, and the best regeneration rates for varieties NG 57-024 and H 83-6179 were 63.3 and 76.3%, respectively. Shoots derived from cryopreserved shoot tip buds developed well-formed roots, and were easily acclimated to greenhouse conditions. The second study evaluated genetic stability of the cryopreserved varieties using ten inter-simple sequence repeat primers. A total of 211 (Halaii), 198 (H83-6179), and 201 (NG 57-024) reproducible bands, ranging from 125 to 5500 bp, were scored with this technique. One hundred genetic stability was detected from Halaii and H 83-6179 whereas 98.5% genetic stability was detected from varieties of NG 57-024. The PCR reactions showed that there was no crucial variation on genetic stability for all cryopreserved varieties.