Kim Haeng-Hoon, Lee Joung-Kwan, Yoon Ju-Won, Ji Jae-Jun, Nam Sang-Sik, Hwang Hae-Sung, Cho Eun-Gi, Engelmann Florent
National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea.
Cryo Letters. 2006 May-Jun;27(3):143-53.
The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.
液滴玻璃化法，即液滴冷冻法与基于溶液的玻璃化法相结合，被用于冷冻保存大蒜珠芽原基。在含有0.1 - 0.3 M蔗糖的固体培养基上于10℃预培养2 - 4天，在含有2 M甘油 + 0.5 M蔗糖的液体培养基中装载50分钟，用PVS3玻璃化溶液脱水90 - 150分钟，通过将铝箔条浸入液氮中，将放置在铝箔条上的5微升PVS3玻璃化溶液液滴中的原基冷却，将箔条投入预热至（40℃）的0.8 M蔗糖溶液中30秒进行解冻，并在同一溶液中进一步孵育30分钟后，冷冻保存的原基获得了最高的存活率和再生率（分别为90.1%至95.0%和82.7%至85.0%）。优化后的液滴玻璃化法成功应用于来自不同国家的五个大蒜品种的珠芽原基以及两种无性繁殖葱属物种的未成熟珠芽，再生率在77.4% - 95.4%之间。
