苹果砧木离体茎尖小滴玻璃化超低温保存体系的优化

Droplet-vitrification cryopreservation of in vitro-cultured shoot tips of apple rootstock

本文以苹果砧木‘54-118’组培苗为材料,采用正交试验对离体茎尖小滴玻璃化超低温保存体系进行优化。结果表明,低温锻炼和植物玻璃化保护溶液2(PVS2)处理时间是影响小滴玻璃化超低温保存后苹果砧木茎尖成活率和再生率的关键因素。优化后的苹果砧木离体茎尖小滴玻璃化超低温保存体系为:继代生长30 d的组培苗在4°C黑暗条件下低温锻炼10 d,取0.5~1.0 mm的茎尖,预培养2 d,加载液(LS)处理20min,PVS2脱水处理60 min,处理后的茎尖包裹在小液滴中,液氮冷冻保存;冷冻茎尖在卸载液(ULS)中常温处理20 min,转入茎尖再生培养基进行恢复培养,暗培养7 d后,转入正常光照条件下培养,获得再生苗。简单重复系列区间(ISSR)检测表明小滴玻璃化超低温保存未改变材料的遗传稳性。优化后的小滴玻璃化超低温保存体系成功用于其他3个苹果砧木品种的超低温保存,4个苹果砧木品种超低温保存后的平均茎尖成活率和再生率分别为74.58%和64.81%。

The orthogonal experiment was designed to obtain the optimal cryopreservation system for in vitro-cultured shoot tips of apple rootstock'54-118'based on the main factors involved in the process of cryopreservation by droplet-vitrification.The results showed that cold acclimation and the treatment time of plant vitrification protection solution 2(Pvs2)were key factors affecting the survival rate and regeneration rate of cryopreserved shoot tips of apple rootstock.An optimized droplet-vitrification cryopreservation system was developed with the following parameters:use of 30 d in vitro plants,10 d acclimatization at 4℃,2 d preculture,20 min LS treatment,60 min PVS2 treatment,exposure to liquid nitrogen by droplet-vitrification.Frozen shoot-tips were treated in unloading solution(ULS)for 20 min(room temperature)and then post-cultured on a shoot tip regeneration medium for shoot regrowth under the normal culture condition after 7 d in the dark.Inter-simple sequence repeats(ISsR)analyses showed no any polymorphic loci in the plants from cryopreserved shoot tips compared to the original cultures.This protocol was successfully applied to three other apple rootstocks and the average survival rate and regeneration rate of the four apple rootstocks were 74.58%and 64.81%,respectively.