Plant Germplasm Conservation Research, School of Biological and Conservation Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa.
Protoplasma. 2012 Jan;249(1):155-69. doi: 10.1007/s00709-011-0274-5. Epub 2011 Apr 15.
Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and <0.4 g g(-1), respectively. In contrast, glycerol cryoprotection, which promoted retention of generally normal ultrastructure and also counteracted any increase in the degree of vacuolation, was associated with 100% and 90% survival of embryos at the higher and lower water contents. After exposure to liquid nitrogen (LN), ultrastructural irregularities were minimal in rapidly cooled glycerol-cryoprotected embryos, at water content <0.4 g g(-1), which showed 70% survival after retrieval from cryogenic conditions. At the other extreme, no embryos survived LN exposure when sucrose cryoprotected. The study relates the cumulative effects of subcellular abnormality and declining viability, in relation to experimental parameters for cryopreservation.
冷冻储存（通常在液氮中或之上）是目前保存产生顽拗性（脱水敏感）种子物种种质资源的唯一选择。本研究调查了从顽拗性孤挺花种子中分离出的合子胚在冷冻保存过程中涉及的连续步骤的超微结构反应。经过快速或缓慢冷却，对未经处理、预先用蔗糖（非渗透）或甘油（渗透）进行了冷冻保护的冻干胚胎进行了回收，然后评估其超微结构和活力反应。未经处理的胚胎 100%存活，其超微结构表明其处于活跃代谢状态。尽管核形态发生了变化，但在暴露于甘油或蔗糖后，胚胎的活力不受影响，但线粒体超微结构表明代谢活性增强，特别是在蔗糖处理后。在蔗糖冷冻保护后进行冻干时，在含水量>0.4 g g(-1)和<0.4 g g(-1)时，分别有 70%和 60%的胚胎存活率下降，与活力下降相伴的是液泡化程度显著增加、异常的质体超微结构和一些细胞壁异常。相比之下，甘油冷冻保护促进了通常正常的超微结构的保留，并抵消了液泡化程度的任何增加，在较高和较低的含水量下，胚胎的存活率分别为 100%和 90%。在暴露于液氮（LN）后，在快速冷却的甘油冷冻保护的胚胎中，超微结构不规则性在含水量<0.4 g g(-1)时最小，在从低温条件中恢复后显示 70%的存活率。在另一个极端，用蔗糖冷冻保护时，没有胚胎能在 LN 暴露后存活。该研究将与冷冻保存实验参数相关的亚细胞异常和活力下降的累积效应联系起来。
