包埋玻璃化法超低温保存条斑紫菜O3D自由丝状体的研究

【作者】 张岚翠；

【导师】 王起华；

【作者基本信息】 辽宁师范大学 ， 植物生物技术， 2006， 硕士

【摘要】 本文采用包埋-玻璃化法超低温保存条斑紫菜自由丝状体。将1.5g／10mL(鲜重)材料包埋于含30‰的NaCl的3％的褐藻酸钙胶球中，在暗处恢复培养1d后用植物玻璃化溶液(plant vitrification solution，PVS)(5％(v／v)二甲基亚砜+30％(v／v)甘油+15％(v／v)乙二醇+0.4mol／L蔗糖溶液)在不同的条件下处理不同的时间。根据胶球内叶绿素的含量计算存活率。本文主要研究了PVS处理时间，装载条件，处理温度等因素对存活率的影响，以及在脱水过程和冰冻前后丙二醛(malondialdehyde，MDA)含量以及超氧化物歧化酶(superoxide dismutas，SOD)活性的变化。结果如下： 1 PVS处理时间对存活率的影响 将30个胶球放入100％的PVS溶液中在4℃下分别处理(min)0，2，5，10，20。结果发现，在PVS处理5min后冻存的材料存活率最高为28.1％。 2．装载时间对条斑紫菜冰冻存活率的影响 在用100％PVS脱水处理之前，用25％的PVS溶液对条斑紫菜丝状体在4℃下进行装载处理(min)0，2，5，10，20，结果显示，最佳装载时间为10min，经此过程冰冻后的材料的存活率由未经装载的28.7％提高到了42.3％。 3 处理条件对条斑紫菜冰冻后存活率的影响 在0℃条件下处理，条斑紫菜自由丝状体冰冻之后存活率从36.4％(25℃)增加到47.9％，提高了10个百分点。 4 化冻条件对条斑紫菜冰冻存活率的影响 研究发现：在40℃的化冻温度下，条斑紫菜丝状体的存活率显著高于0℃-20℃下的存活率，为41.9％。但是化冻温度超过40℃以后，虽然进一步提高了化冻速率，但存活率却有所下降。 5 脱水和冰冻过程中MDA含量的变化 冰冻后材料的MDA含量均高于未冰冻材料的MDA含量，同时经过装载处理的材料冰冻后的MDA含量低于未经装载的材料冰冻后的MDA含量，说明装载过程可以减少冰冻过程中对膜的伤害。 6 脱水和冰冻过程中SOD活性的变化 冰冻后材料的SOD活性均高于未冰冻材料的SOD活性，同时经过装载处理的材料冰冻后的SOD活性低于未经装载的材料冰冻后的SOD活性。更多还原

【Abstract】 Porphyra yezoensis Ueda is an eatable seaweed which has great commercial value. The free-living conchocelis is an important form for phycoculture of Porphya yezoensis. Encapsulation-vitrification, which combines the advantages of encapsulation and vitrification is a new technology of cryopreservation. So far there is no work which has been published on cryopreservation of conchocelis of Porphyra by encapsulation- vitrification.The free-living conchocelis of Porphya yezoensis Ueda 03D was encapsulated in 3% ca-alginate beads with 30‰ NaCl. After loading and dehydration treatment by the plant vitrification solution (PVS), the beads were plunged into liquid nitrogen (LN) for 24h, and then they were thawed, washed and cultured before determining the viability of the conchocelis and the malondialdehyde (MDA), superoxide dismutas (SOD) content in the sample. The main results were as follows:1 Beads were treated with 100% PVS for 2,5,10,15 and 20 min at 4℃. The results showed that the highest rate of viability was obtained with PVS treatment for 5 min, which was 28.1%.2 Beads were loaded with 25% PVS for 0, 2, 5, 10 and 20 min at 4℃, before they were treated with 100% PVS for 5 min and plunged into LN. The loading for 5min gave the best result, which was 42.3%.3 When the beads were treated at 0℃ the viability, which was 47.9%, increased greatly compared with the treatment at 25℃.4 The evaluation of thawing temperature showed that the best thawing temperature was 40℃5 The MDA and SOD content after loading and dehydrating the beads was not changed, while it increased dramatically after storing the beads in LN for 24h.更多还原