71. Cryopreservation of in vitroshoot-tips of a sweet potato Famaillá 6 clone (Ipomoea batatas (L.) Lam.) by droplet-vitrification technique

Sweet potato varieties constitute a valuable germplasm as a source of carbohydrates, vitamins, fiber and minerals, with excellent functional properties in the prevention of diseases because of the presence of anthocyanins and carotenoids (Martí, 2008; CIP, 2009). The maintenance of germplasm in the field are under risk of losing material, high labor force, time and space consuming (Engelmann and Engels, 2002). The in vitro conservation entails high costs of maintenance, risks of losses through accidental contaminations, human errors and somaclonal variations (Niino et al., 2000). The cryopreservation techniques are promising alternatives for the long-term conservation of germplasm. At temperatures of liquid nitrogen, −196°C, all cellular divisions and metabolic processes are detained ensuring unaltered storage for theoretically unlimited periods of time, with minimum space and maintenance (Engelmann, 1997; Pennycooke and Towill, 2000, 2001). In order to implement long-term conservation of sweet potato germplasm, the droplet-vitrification technique was applied to shoot-tips of micropropagated in vitro plants of Famaillá 6 clone, conserved at the Base Germplasm Bank in INTA Castelar. Shoot-tips were precultured in sucrose liquid solutions. The liquid solutions were the following: (A) 0.3M 24h; (B) 0.3M 24h+0.5M 24h; (C) 0.3M 24h+0.5M 24h+0.75M 24h. Subsequently, they were osmoprotected with loading solution (LS: 2M glycerol, 0.4M sucrose) for 20, 30, 40, 50 and 60min at 25°C and immediately transferred to plant vitrification solution 2 (PVS2: 30% glycerol, 15% Me₂SO, 15% ethylene glycol, 0.4M sucrose) for 8, 16 and 24min at 0°C. Following, shoot-tips were placed into small drops of PVS2 on an aluminum foil and introduced directly into liquid nitrogen. After 24h, shoot-tips were rewarmed in unloading solution (1.2M sucrose) for 20min at 25°C. The percentage of regenerated shoots tips was recorded after 2months of culture in normal medium (solidified medium MS supplemented with 3% sucrose, 0.4mg/l Kinetin and 20mg/l GA₃) at 25°C±2°C, first in darkness, then dim light and finally at 50μmol/mseg². Two replicates of 8–10 shoot-tips were used in each treatment. The average percentage of regenerated shoot tips did not show statistically differences between the preculture treatments. Considering the loading solutions treatments, the average percentage of the regenerated shoot tips did not show statistically differences between (A) and (B) preculture treatments at all LS exposure times, varying from 65.42% to 87.5%; (C) preculture treatment showed the lowest regeneration percentage at all LS exposure times, and was not considered in further experiments. After the dehydration step with PVS2 solution, the highest regeneration values (87.5%) were observed after 16min of dehydration, when shoot-tips were precultured in 0.3M sucrose for 24h and osmoprotected with 30 and 40min in LS. These combinations showed low survival after cryopreservation with no shoot formation. According to these results it is necessary to incorporate modifications in the technique to obtain better responses and then extend the analyses to different genotypes in order to be able to use this technique for the long-term conservation of sweet potato germplasm.