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利用电镜细胞化学焦锑酸钾沉淀法及Ca2+荧光探针Fluo-3AM染色法,研究了玻璃化法超低温保存过程中预培养、装载、脱水和洗涤4个关键步骤对百子莲胚性愈伤组织细胞内Ca2+分布情况及浓度变化的影响。结果表明,在预培养后细胞液泡内Ca2+数量多于细胞质基质,Ca2+主要贴膜分布,且细胞内的Ca2+荧光强度降到最低值。经装载处理后,细胞内出现较多囊泡和淀粉粒,在线粒体和囊泡中观察到Ca2+数量增多,淀粉粒周围有较多的Ca2+分布,细胞内的荧光强度增加。经脱水处理后,细胞内的Ca2+荧光强度升高到最大值呈梯度分布,含有Ca2+的囊泡增多,细胞内淀粉粒周围的Ca2+数量较多。经洗涤处理后,随着细胞内玻璃化溶液的减少,细胞内Ca2+数量减少并且荧光强度降低,细胞质中Ca2+浓度回落到与对照相近的正常水平,淀粉粒数量增加但周围Ca2+分布较少。在超低温保存处理过程中,细胞经受包含低温、渗透、氧化等复合胁迫,Ca2+分布和浓度的变化可能对提高细胞低温抗性及抗氧化能力起到了一定作用。推测Ca2+的变化决定了植物细胞在超低温保存过程中对复合胁迫的不同适应能力,为优化百子莲胚性愈伤组织超低温保存体系提供了理论依据,为超低温保存过程中植物的细胞信号转导及生理调控机制提供一定的理论基础。
Using electron microscopy, potassium metabisustrate precipitation method and Ca2 + fluorescence probe Fluo-3AM staining, we studied the effects of pre-incubation, loading, dehydration and washing during the vitrification process on the embryogenic callus Effect of intracellular Ca2 + distribution and concentration changes. The results showed that after pre-culture, the number of Ca2 + in the cytoplasm was more than that in the cytoplasm, Ca2 + was mainly distributed in the membrane, and the intracellular Ca2 + fluorescence intensity was reduced to the lowest value. After loading treatment, more vesicles and starch granules appeared in the cells, more Ca2 + was observed in the mitochondria and vesicles, more Ca2 + distribution around the starch granules, and fluorescence intensity in the cells increased. After dehydration, the intracellular Ca2 + fluorescence intensity increased to a maximum gradient distribution, Ca2 + containing vesicles increased, intracellular starch granules around the large number of Ca2 +. After washing, the amount of intracellular Ca2 + decreased and the fluorescence intensity decreased with the decrease of intracellular vitrification solution. The Ca2 + concentration in cytoplasm returned to the normal level similar to that of the control, whereas the amount of starch granules increased but the surrounding Ca2 + distribution was less. During the process of cryopreservation, the changes of the distribution and concentration of Ca2 + may play an important role in improving cell hypothermia and antioxidant capacity under the conditions of complex stress including low temperature, osmosis and oxidation. It is speculated that the change of Ca2 + determines the different adaptation ability of plant cells to composite stress during cryopreservation, and provides a theoretical basis for optimizing Cryopreservation system of embryogenic callus of Bermudaryus. It provides a theoretical basis for plant cell signal transduction during cryopreservation and Physiological regulation provides a theoretical basis.