CRYOPRESERVATION OF SWEET POTATO SHOOT TIPS USING A DROPLET-VITRIFICATION PROCEDURE

摘要

BACKGROUND: Sweet potato is a staple food worldwide, but a problematic species in terms of long term storage, as it is not suitable for germplasm conservation. OBJECTIVE: This study aimed to develop cryopreservation protocols for sweet potato shoot tips based on a droplet-vitrification procedure. METHODS: As a standard procedure, sweet potato shoot tips were precultured in a liquid MS medium supplemented with 10% sucrose (S-10%) and 17.5% sucrose (S-17.5%) for 31 and 17 h, respectively. They were then osmoprotected with C4-35% (17.5% glycerol + 17.5% sucrose) for 50 min and cryoprotected with PVS3 (50% glycerol + 50% sucrose) for 60 min. A set of experiments was designed to investigate critical factors, i.e. stepwise sucrose preculture, osmoprotection, cryoprotection with PVS2- and PVS3-based vitrification solutions, and their combinational effect, as well as temperature alteration through placement in a cooling/rewarming container. RESULTS: Sucrose preculture was determined to be necessary for the adaptation of sweet potato shoot tips to cryoprotection with PVS3, and the highest post-thaw (LN) regeneration rate was observed in a preculture with S-10% for 31 h -> S-17.5% for 17 h (19.0%). The effect of one-step or two-step osmoprotection was not significant on survival or regeneration of either the cryoprotected-control (LNC) or LN shoot tips. Responses of sweet potato shoot tips to osmoprotection and cryoprotection were linked to the level of sucrose preculture. The use of alumimium foil strips (droplet-vitrification) resulted in significantly higher LN survival (89.8%) and regeneration (19.0%), compared to those using cryovials (vitrification, 67.2% and 0%, respectively). LN regeneration increased by 67.5% when cryopreserved shoot tips were transferred to a new postculture medium. CONCLUSIONS: This study demonstrates that the combination of stepwise sucrose preculture with a higher final concentration (up to 17.5%), cryoprotection with PVS3 and cooling with foil strip is crucial to the regeneration of LN sweet potato shoot tips.