Cryopreservation is the best way for long-term in vitro preservation of plant germplasm resources. The preliminary studies found that reactive oxygen species (ROS) induced oxidative stress and ice-induced membrane damage are the fundamental causes of cell death in cryopreserved samples. How to improve plant cryopreservation survival rate is an important scientific issue in the cryobiology field. This study found that the survival rate was significantly improved by adding single-wall carbon nanotubes (SWCNTs) to plant vitrification solution (PVS) in cryopreservation of Agapanthus praecox embryogenic callus (EC), and analyzed the oxidative response of cells during the control and SWCNTs-added cryopreservation protocol. The SWCNTs entered EC at the step of dehydration and mainly located around the cell wall and in the vesicles, and most of SWCNTs moved out of EC during the dilution step. Combination with physiological index and gene quantitative expression results, SWCNTs affect the ROS signal transduction and antioxidant system response during plant cryopreservation. The EC treated by SWCNTs had higher antioxidant levels, like POD, CAT, and GSH than the control group EC. The EC mainly depended on the AsA-GSH and GPX cycle to scavenge H2O2 in the control cryopreservation, but depended on CAT in the SWCNTs-added cryopreservation which lead to low levels of H2O2 and MDA. The elevated antioxidant level in dehydration by adding SWCNTs enhanced cells resistance to injury during cryopreservation. The ROS signals of EC were balanced and stable in the SWCNTs-added cryopreservation. The SWCNTs regulated oxidative stress responses of EC during the process and controlled oxidative damages by the maintenance of ROS homeostasis to achieve a high survival rate after cryopreservation. This study is the first to systematically describe the role of carbon nanomaterial in the regulation of plant oxidative stress response, and provided a novel insight into the application of nanomaterials in the field of cryobiology.

中文翻译：

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单壁碳纳米管提高百子莲胚性愈伤组织冷冻保存细胞存活率并减少氧化损伤

冷冻保存是植物种质资源体外长期保存的最佳方式。初步研究发现，活性氧（ROS）诱导的氧化应激和冰诱导的膜损伤是冷冻保存样品中细胞死亡的根本原因。如何提高植物低温保存成活率是低温生物学领域的一个重要科学问题。本研究发现将单壁碳纳米管 (SWCNTs) 添加到植物玻璃化溶液 (PVS) 中冷冻保存的 Agapanthus praecox 胚性愈伤组织 (EC) 的存活率显着提高，并分析了对照和 SWCNTs 期间细胞的氧化反应-添加了冷冻保存协议。SWCNTs 在脱水阶段进入 EC 并主要位于细胞壁周围和囊泡中，并且大多数 SWCNT 在稀释步骤中移出 EC。结合生理指标和基因定量表达结果，SWCNTs影响植物冷冻保存过程中的ROS信号转导和抗氧化系统反应。与对照组 EC 相比，SWCNTs 处理的 EC 具有更高的抗氧化水平，如 POD、CAT 和 GSH。EC在对照冷冻保存中主要依靠AsA-GSH和GPX循环清除H2O2，但在添加SWCNTs的冷冻保存中依赖CAT，导致H2O2和MDA含量低。通过添加单壁碳纳米管提高脱水中的抗氧化水平可增强细胞在冷冻保存过程中对损伤的抵抗力。在添加 SWCNTs 的冷冻保存中，EC 的 ROS 信号平衡且稳定。SWCNT在此过程中调节EC的氧化应激反应，并通过维持ROS稳态来控制氧化损伤，从而在冷冻保存后实现高存活率。本研究首次系统描述了碳纳米材料在调控植物氧化应激反应中的作用，为纳米材料在低温生物学领域的应用提供了新的视角。