扁桃茎尖包埋玻璃化超低温保存条件研究

采用包埋玻璃化法对莎车18号扁桃茎尖超低温保存进行了研究。主要探讨低温锻炼、预处理浓度和预处理时间、装载时间和PVS2处理时间对莎车18号扁桃茎尖超低温保存后存活率的影响。结果表明,将低温锻炼4周的莎车18号扁桃茎尖切2 mm长用3%海藻酸钠包埋后,在含有0.3 mg/L蔗糖＋5%DMSO（二甲基亚砜）的MS培养基预培养1 d,装载液处理20min,在0℃条件下用PVS2处理50 min后迅速投入液氮,24 h后取出,放入40℃水浴锅中化冻1～2 min,用1.2 mg/L蔗糖的MS洗涤液洗涤2次,每次10 min,接种于含6-BA0.3 mg/L和IAA 0.3 mg/L的MS培养基上进行培养,暗培养15 d,转移至正常光下,存活率高达52%。

Studies on Cryopreservation of Almond Shoot Tips by Encapsulation-Vitrification

A procedure had been studied on cryopreservation of almond cultivars Shache18 shoot tips in vitro by encapsulation-vitrification. In the experiment,five factors,that is to say cold-hardening time,pre-culture time,sucrose concentration,loading time and exposure time to PVS2,were considered and a good protocol was achieved. The result showed the shoot tips of almond cultivars Shache18 were cold acclimatized at a low temperature（ 4 ℃） for 4 weeks; The shoot tips of almond cultivars Shache18 were cuted about 2 mm in length and embedded by 3% sodium alginate（ SA）,then fixed by 0. 1 mol /LCaCl2solution of 0. 4 mol / L sucrose before forming alginate-coated shoot tips. the alginate-coated shoot tips of almond cultivars Shache18 were precultured for one day on MS medium supplemented with a mixture of 0. 3 mol /L sucrose plus 5% DMSO,After preculture,the alginate-coated shoot tips were loaded for 20 minutes on MS medium supplemented with a mixture of 2 mol /L glycerol plus 0. 4 mol /L sucrose,then changed PVS2solution for 50 min at 0 ℃,which was a highly concentrated vitrification solution of contain 30%（ w /v） glycerol,15%（ w /v） ethylene glycol,and 15% dimethyl sulfoxide prepared in liquid MS,and then stored under liquid nitrogen（ LN） for 1 day. After rapid thawing for 1 ～ 2minutes at 40 ℃,the alginate-coated shoot tips of almond cultivars Shache18 were washed two times of 10 minutes each on the MS medium containing 1. 2mol /L sucrose and then cultured on MS medium with 0. 3mg /L6-BA and 0. 3 mg /L IAA in dark for fifteen days prior to exposure to the light. Survival rate of the alginate-coated shoot tips of almond cultivars Shache18 reached to 52%.