The effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of <Emphasis Type="Italic">Amaryllis belladonna</Emphasis> |
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Authors: | Sershen Patricia Berjak N W Pammenter James Wesley-Smith |
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Institution: | (1) Plant Germplasm Conservation Research, School of Biological and Conservation Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001, South Africa;(2) Electron Microscope Unit, University of KwaZulu-Natal (Westville Campus), Durban, 4001, South Africa; |
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Abstract: | 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. |
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