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Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g(-1) dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g(-1) showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g(-1), embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g(-1), lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g(-1)-in which the degree of vacuolation remained moderate-were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.

Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and <0.4 g g(-1), respectively. In contrast, glycerol cryoprotection, which promoted retention of generally normal ultrastructure and also counteracted any increase in the degree of vacuolation, was associated with 100% and 90% survival of embryos at the higher and lower water contents. After exposure to liquid nitrogen (LN), ultrastructural irregularities were minimal in rapidly cooled glycerol-cryoprotected embryos, at water content <0.4 g g(-1), which showed 70% survival after retrieval from cryogenic conditions. At the other extreme, no embryos survived LN exposure when sucrose cryoprotected. The study relates the cumulative effects of subcellular abnormality and declining viability, in relation to experimental parameters for cryopreservation.

This investigation assessed the extent of rehydration of dehydrated plant tissues during aqueous fixation in comparison with the fine structure revealed by freeze-substitution. Radicles from desiccation-tolerant pea (Pisum sativum L.), desiccation-sensitive jackfruit seeds (Artocarpus heterophyllus Lamk.), and leaves of the resurrection plant Eragrostis nindensis Ficalho & Hiern. were selected for their developmentally diverse characteristics. Following freeze-substitution, electron microscopy of dehydrated cells revealed variable wall infolding. Plasmalemmas had a trilaminar appearance and were continuous and closely appressed to cell walls, while the cytoplasm was compacted but ordered. Following aqueous fixation, separation of the plasmalemma and the cell wall, membrane vesiculation and distortion of cellular substructure were evident in all material studied. The sectional area enclosed by the cell wall in cortical cells of dehydrated pea and jackfruit radicles and mesophyll of E. nindensis increased after aqueous fixation by 55, 20, and 30%, respectively. Separation of the plasmalemma and the cell wall was attributed to the characteristics of aqueous fixatives, which limited the expansion of the plasmalemma and cellular contents but not that of the cell wall. It is proposed that severed plasmodesmatal connections, plasmalemma discontinuities, and membrane vesiculation that frequently accompany separation of walls and protoplasm are artefacts of aqueous fixation and should not be interpreted as evidence of desiccation damage or membrane recycling. Evidence suggests that, unlike aqueous fixation, freeze-substitution facilitates reliable preservation of tissues in the dehydrated state and is therefore essential for ultrastructural studies of desiccation.