不同冻存条件对甘蔗原生质体活力和再生能力的影响

Effects of different freezing conditions on vitality and regeneration ability of sugarcane protoplast

为了获得活力高和再生能力强的甘蔗原生质体,该文对甘蔗原生质体的冻存液浓度、冻存温度和冻存部位进行了研究。结果表明:(1)不同的冻存液、不同的冻存温度和不同的取材部位原生质体冻存后复苏对甘蔗原生质体的活力影响有显著差异性,三个冻存液组合比较,在组合2(70%培养基+20%血清+10%DMSO),冻存30 d后复苏活力最强,高达72%;冻存90 d内复苏,-196℃液氮和-80℃冰箱冻存,甘蔗原生质体的活力差异不显著,活力均在75%以上,但90 d冻存后复苏,-196℃液氮冻存后复苏比-80℃冰箱冻存冻存后复苏原生质活力强;不同取材部位比较,幼叶冻存30 d后复苏所得原生质体活力较高(达79. 2%),茎尖冻存30 d后复苏所得原生质体活力仅为42.7%。(2)不同的冻存液和不同的冻存温度,细胞第一次启动分裂和形成细胞团的时间差异不显著,一般培养5～6 d,细胞壁基本形成完整,培养6 d后,细胞启动分裂,培养15 d后形成细胞团。不同的材料部位相比较,茎尖酶解所得原生质体再生能力最强,较幼叶酶解原生质体,形成细胞壁的时间早3 d,第一次分裂时间早2 d。

Abstract:In order to obtain high vitality and regeneration ability of sugarcane protoplast, this experiment was done to study the protoplast of frozen storage liquid combination, frozen storage temperature, frozen storage time and recovery temperature. The results were as follows:(1) Different cryopreservations, different cryopreservation temperatures and different protoplast sources of different materials had significant differences on the vitality of the protoplast of sugarcane. Compared with the combination of three frozen liquid deposits, in Combination 2 (70% medium +20% serum +10% DMSO), the recovery activity was the strongest after 30 d, as high as 72%. Cryopreserved recovery within 90 d, liquid nitrogen -80 ℃ and -196 ℃ freezer, sugarcane protoplast energy difference was not significant, and the vigor was above 75%. But after 90 d of frozenstorage, the protoplast dynamic at -196 ℃ was stronger than -80 ℃ after recovery. For different materials, the protoplast dynamic of the young leaves frozen stored for 30 d was up to 79.2%, and the protoplast dynamic of stem tip frozen for 30 d was only 42.7%.(2) There was no significant difference in the time between the first initiation of division and the formation of cell mass in the treatments with different cryopreservation liquids and different cryopreservation temperatures. After the protoplasts were cultured for about 5-6 d, the cell wall was basically complete, and the cells began to divide after 6 d, and the cell masses formed after 15 d of culture. For different materials, the strongest regeneration ability was found in the protoplasts from stem tip by enzymatic hydrolysis, which cell wall formation showed 3 d earlier, and 2 d earlier in the first cell division than those from the juvenile leaves.