Developing Regeneration System for Cryopreservation in Sugarcane (Saccharum officinarum L.)

A regeneration system for cryopreservation of sugarcane (Saccharum officinarum L.), commercial cultivar 'U-Thong 5', has been established. Somatic embryogenesis has been done using immature inflorescence segments as explants. The highest induction percentage of the initial embryogenic callus was obtained in Murashige and Skoog media supplemented with 10-15 mu M 2,4-dichlorophenoxyacetic acid, 50 ml/L coconut water, 500 mg/L casein hydrolysate, and 6% (w/v) sucrose in dark condition. Embryogenic callus developed to somatic embryos and plantlets on MS media containing 50 ml/L coconut water, 0.5 gm/L orchid fertilizer (21-21-21) and 6% (w/v) sucrose. Shoots were able to root on these media. For cryopreservation, a vitrification protocol using different plant vitrification solutions was employed, PVS2, PVS3, and variants of PVS3 (various concentrations of glycerol and sucrose). Somatic embryos in the early stages have been precultured on MS media containing 50 ml/L coconut water, 2 mu M abscisic acid, 0.3 M sucrose for 24 h. After 24 h, the somatic embryos have been cultured on the same media with 0.5 M for 24 h. Subsequently, the somatic embryos have been treated with loading solution comprising of 0.5 M sucrose and 2 M glycerol in MS media for 30 min. Then, somatic embryos were dehydrated with different types of PVSs for 30 min at 5 degrees C prior to being immersed in liquid nitrogen for 7 days. After freezing in liquid nitrogen, regeneration percentages of the somatic embryos treated with PVS3 and variants of PVS3 were 34-42%. In contrast using PVS 2 during dehydration step, lower survival and regeneration percentages of the somatic embryos were obtained, when compared to those of PVS3.