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本文研究建立了大苞鞘石斛(Dendrobium wardianum Warner)原球茎玻璃化法超低温保存的技术体系。结果发现,预处理和玻璃化溶液(plant vitrification solution 2,PVS2)装载脱水是影响大苞鞘石斛原球茎相对存活率的两个关键步骤,高渗与低温-高渗两种预处理方法测定的相对存活率具有显著性差异;玻璃化溶液的种类以及脱水时间对冻后存活率具有重要的影响。基于此,建立了大苞鞘石斛原球茎的超低温保存体系,即:以继代培养60 d的大苞鞘石斛原球茎为材料,1/2MS+0.8 mol/L蔗糖的培养基上4℃低温预处理6 d后,转至1/2 MS+2 mol/L甘油+0.4 mol/L蔗糖的装载液中室温下装载40 min,在0℃下装载PVS2脱水40 min,然后转入装有新鲜PVS2冷冻管中并迅速投入液氮。在液氮保存1 h后放在40℃水浴中快速解冻1 min,利用含1.2 mol/L蔗糖的1/2MS培养液洗涤3次,每次间隔10 min;待恢复培养30 d后统计存活率,可使大苞鞘石斛原球茎超低温保存后存活率达到20.0%。
In this paper, a technical system for the cryopreservation of protocorms of Dendrobium wardianum Warner was established. The results showed that pretreatment and PVS2 loading dewatering were the two key steps affecting the relative viability of protocorm-like bodies of Dendrocalamus dahurica. Hypertonic and hypothermia-hyperosmotic two pretreatment methods determined Relative survival rate has significant difference; the type of vitrification solution and dehydration time has important effect on the survival rate after freezing. Based on this, the cryopreservation system of protocorm-like bodies of Dendrobium baicalensis was established, that is, the protocorm-like bodies of D. baicalensis were cultured for 60 days in subculture, and the medium of 1/2 MS + 0.8 mol / L sucrose at 4 ℃ After pretreatment for 6 d, the solution was transferred to 1/2 MS + 2 mol / L glycerol + 0.4 mol / L sucrose for 40 min at room temperature, dehydrated at 0 ° C for 40 min and then transferred to fresh water PVS2 cryovials and quickly put liquid nitrogen. After being stored in liquid nitrogen for 1 h, they were thawed in a water bath at 40 ° C for 1 min and washed twice with 1/2 MS medium containing 1.2 mol / L sucrose for 10 min each. After 30 days of recovery, the statistical survival rate , So that the protocorm-like protocorm-like body after cryopreservation survival rate of 20.0%.