Genetic Resources Center, National Agriculture and Food Research Organization, Tsukuba, Japan.
Gene Research Center, University of Tsukuba, Tsukuba, Japan.
Adv Exp Med Biol. 2018;1081:355-369. doi: 10.1007/978-981-13-1244-1_19.
Cryopreservation encompasses several interconnect disciplines including physiology and cryophysics. This chapter reviews the current techniques for cryopreservation of plant genetic resources (PGRs). Vitrification is an effective ice crystal avoidance mechanism for hydrated cells and tissues. With any cryopreservation method, whole or partial parts of specimens which are sufficiently dehydrated can be vitrified by rapid cooling in liquid nitrogen (LN). Techniques discussed are the vitrification protocol, encapsulation-vitrification protocol, droplet vitrification protocol (DV), vitrification protocol using cryo-plates (V cryo-plate), and air dehydration protocol using cryo-plates (D cryo-plate). In these DV, V, and D cryo-plate protocols, specimens to be cryopreserved are immersed directly into LN on aluminum foil strips or cryo-plates; removal from LN to rewarming solution results in a high level of plant regrowth with ultrarapid cooling and warming. The protocols were applied to a wide array of plant species including wild and multi-ploid species, although fine tuning of the protocols was required for successful application to specific plant species and lines. These three protocols efficiently complement each other and appear highly promising to facilitate large-scale cryobanking of PGRs in genebanks. Cryo-scanning electron microscopy makes it possible to examine the cellular and water behavior in plant tissues when immersed in LN. It has been verified that tissues cryopreserved by the process of vitrification and the cryo-plate protocols are cryopreservation methods for reliable long-term preservation of PGRs.
冷冻保存涵盖了几个相互关联的学科，包括生理学和低温物理学。本章回顾了植物遗传资源（PGR）冷冻保存的当前技术。玻璃化是水合细胞和组织避免冰晶形成的有效机制。对于任何冷冻保存方法，经过充分脱水的标本的整体或部分都可以通过在液氮（LN）中快速冷却而玻璃化。讨论的技术包括玻璃化方案、包埋-玻璃化方案、液滴玻璃化方案（DV）、使用 cryo-plate 的玻璃化方案（V cryo-plate）和使用 cryo-plate 的空气脱水方案（D cryo-plate）。在这些 DV、V 和 D cryo-plate 方案中，要冷冻保存的标本直接浸入 LN 中的铝箔条或 cryo-plate 上；从 LN 中取出并放入再温溶液中会导致植物再生率非常高，因为采用了超快速冷却和升温。这些方案已应用于广泛的植物物种，包括野生和多倍体物种，尽管需要对特定植物物种和系进行微调才能成功应用。这三个方案相互补充，对于在基因库中大规模冷冻保存 PGR 非常有前景。冷冻扫描电子显微镜使得在 LN 中浸泡时检查植物组织的细胞和水行为成为可能。已经验证了通过玻璃化和 cryo-plate 方案冷冻保存的组织是 PGR 可靠长期保存的冷冻保存方法。
