Wesley-Smith James, Walters Christina, Pammenter N W, Berjak Patricia
Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA.
Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA
Ann Bot. 2015 May;115(6):991-1000. doi: 10.1093/aob/mcv009. Epub 2015 Mar 25.
Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than -180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.
Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g(-1) dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s(-1) or programmed cooling at 3·3 °C s(-1). Samples were thawed rapidly (177 °C s(-1)) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.
Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.
Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2-0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.
顽拗性种子所具有的遗传多样性可通过冷冻保存来实现，即把切下的胚轴（若可能，为胚）进行处理后，使用液氮在低于-180°C的温度下保存。此前研究表明，糖槭（银枫）快速冷却的胚轴中会形成细胞内冰，但当冰晶较小时，这不一定是致命的。本研究旨在了解细胞内冰造成损伤的性质和程度，以及存活组织的恢复和再生长过程。
未经过脱水或冷冻保护处理（含水量为1·9 g H₂O g⁻¹干重）的糖槭胚轴，采用两种方法冷却至液氮温度：投入液氮冻浆以达到97°C s⁻¹的冷却速率，或程序降温至3·3°C s⁻¹。样品快速解冻（177°C s⁻¹），立即在显微镜下检查细胞结构，并在体外培养长达72小时的间隔时间内进行检查。体外培养4周后评估存活率。胚轴按常规方法处理用于光学显微镜和超微结构检查。
低温暴露后解冻后立即观察，胚轴细胞在超微结构水平上未显示损伤迹象。体外培养数小时后，细胞受损的迹象明显，表现为自噬分解。在存活组织中，死细胞脱落，存活细胞团是再生长的起源。在根中，再生长发生于基本分生组织和原形成层，而非远端分生组织，远端分生组织已受到致命损伤。芽的再生长发生于外围和髓分生组织中存活细胞的孤立细胞团。这些细胞团的大小可能决定再生长的可能性、程度和活力。
低温暴露后细胞的自噬降解以及最终的自溶，以及小（0·2 - 0·4 µm）细胞内冰晶的形成，对当前认为冰会立即对细胞造成物理损伤的观点提出了挑战。相反，冷冻应激可能诱导程序性细胞死亡（PCD）信号。冷却过程中形成更多冰晶的细胞具有更快的PCD反应。
