Abstract Recalcitrant seeds are shed at relatively high water contents and are metabolically active, initiating germination without exogenous provision of water. There is an urgency for these seeds to be conserved in light of the rapidly diminishing numbers of indigenous species and associated genetic diversity. This may be achieved by long term storage of whole seeds or seed germplasm. This study centred on the development of a cryopreservation protocol for embryonic axes (explants). Indicators of stress i.e. superoxide (·O2−), hydrogen peroxide (H2O2) and total aqueous antioxidants (TAA) levels were measured, as well as explant viability and shoot and root ultrastructure in response to exogenously applied cathodic water after each cryopreparative step. Explant viability gradually decreased after each cryopreparative step indicating the cumulative effect of the stresses imposed by cryopreservation. In conjunction with reduced viability, the levels of ·O2− and H2O2 increased gradually after each of these steps. Total aqueous antioxidant levels remained relatively unchanged throughout the cryopreservation process. The ROS levels in cathodic water treated explants were significantly lower compared with those that were not treated with cathodic water. A small number of explants from seeds obtained in Port Elizabeth (30%) survived after cryopreservation, while none of the explants from seeds obtained in St Lucia did. Root ultrastructure across explants from seeds of both provenances was well preserved after each cryopreparative step. Organelles were well developed and the ultrastructure was typical of metabolically active tissue. However, gradual ultrastructural deterioration was observed in the shoot meristem after each cryopreparative step. Typical of a stress response, increased vacuolation and a lack of visible organelles were noted. Root and shoot production (or lack thereof) was supported by ultrastructural observations. This study reports that the explants of PE origin were able to survive cryopreservation while those of St Lucia did not. The key findings of this study support the role of cathodic water in regulating ROS and indirectly preserving explant ultrastructure and survival, and its exogenous application during cryopreservation of recalcitrant seeded germplasm is recommended.

中文翻译：

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不同种源的白头翁顽固种子对冷冻保存的响应

摘要 顽固的种子在相对较高的水分含量下脱落，并且具有代谢活性，在没有外源水供应的情况下启动发芽。鉴于本地物种的数量和相关的遗传多样性迅速减少，迫切需要保护这些种子。这可以通过长期储存完整种子或种子种质来实现。这项研究的中心是胚胎轴（外植体）冷冻保存方案的开发。测量压力指标，即超氧化物 (·O2-)、过氧化氢 (H2O2) 和总水性抗氧化剂 (TAA) 水平，以及在每个冷冻制备步骤后响应外源施加阴极水的外植体活力和芽和根超微结构。每个冷冻制备步骤后外植体活力逐渐降低，表明冷冻保存施加的压力的累积效应。随着生存能力的降低，·O2- 和H2O2 的水平在每个步骤之后逐渐增加。在整个冷冻保存过程中，总水性抗氧化剂水平保持相对不变。与未用阴极水处理的外植体相比，阴极水处理的外植体中的 ROS 水平显着降低。少数来自伊丽莎白港种子的外植体（30%）在冷冻保存后存活下来，而来自圣卢西亚种子的外植体没有一个存活下来。在每个冷冻制备步骤后，来自两种来源的种子的外植体的根超微结构都得到了很好的保存。细胞器发育良好，超微结构是代谢活跃组织的典型特征。然而，在每个冷冻制备步骤后，在芽分生组织中观察到逐渐的超微结构退化。注意到典型的应激反应、增加的空泡和缺乏可见的细胞器。超微结构观察支持根和芽的产生（或缺乏）。这项研究报告说，PE 来源的外植体能够在冷冻保存中存活下来，而圣卢西亚的外植体则不能。本研究的主要发现支持阴极水在调节 ROS 和间接保存外植体超微结构和存活中的作用，并建议在顽固种子种质的冷冻保存过程中外源应用。在每个冷冻制备步骤后，在芽分生组织中观察到逐渐的超微结构退化。注意到典型的应激反应、增加的空泡和缺乏可见的细胞器。超微结构观察支持根和芽的产生（或缺乏）。这项研究报告说，PE 来源的外植体能够在冷冻保存中存活下来，而圣卢西亚的外植体则不能。本研究的主要发现支持阴极水在调节 ROS 和间接保存外植体超微结构和存活中的作用，并建议在顽固种子种质的冷冻保存过程中外源应用。在每个冷冻制备步骤后，在芽分生组织中观察到逐渐的超微结构退化。注意到典型的应激反应、增加的空泡和缺乏可见的细胞器。超微结构观察支持根和芽的产生（或缺乏）。这项研究报告说，PE 来源的外植体能够在冷冻保存中存活下来，而圣卢西亚的外植体则不能。本研究的主要发现支持阴极水在调节 ROS 和间接保存外植体超微结构和存活中的作用，并建议在顽固种子种质的冷冻保存过程中外源应用。超微结构观察支持根和芽的产生（或缺乏）。这项研究报告说，PE 来源的外植体能够在冷冻保存中存活下来，而圣卢西亚的外植体则不能。本研究的主要发现支持阴极水在调节 ROS 和间接保存外植体超微结构和存活中的作用，并建议在顽固种子种质的冷冻保存过程中外源应用。超微结构观察支持根和芽的产生（或缺乏）。这项研究报告说，PE 来源的外植体能够在冷冻保存中存活下来，而圣卢西亚的外植体则不能。本研究的主要发现支持阴极水在调节 ROS 和间接保存外植体超微结构和存活中的作用，并建议在顽固种子种质的冷冻保存过程中外源应用。