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.
Similar content being viewed by others
References
Benson EE (2008) Cryopreservation theory. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 15–30
Benson EE, Bremner D (2004) Oxidative stress in the frozen plant: a free radical point of view. In: Fuller BJ, Lane N, Benson EE (eds) Life in the frozen state. CRC Press, Boca Raton, Florida, USA, pp 205–241
Benson EE, Noronha-Dutra AA (1988) Chemiluminescence in cryopreserved plant tissue cultures: the possible role of singlet oxygen in cryoinjury. Cryo Lett 9:120–131
Berjak P, Mycock DJ (2004) Calcium, with magnesium, is essential for normal seedling development from partially dehydrated recalcitrant axes: a study on Trichilia dregeana Sond. Seed Sci Res 14:217–231
Berjak P, Pammenter NW (2000) What ultrastructure has told us about recalcitrant seeds. Rev Bras Fisiol Veg 12:22–55
Berjak P, Pammenter NW (2004) Recalcitrant seeds. In: Benech-Arnold RL, Sánchez RA (eds) Handbook of seed physiology: applications to agriculture. Haworth Press, New York, pp 305–345
Berjak P, Pammenter NW (2008) From Avicennia to Zizania: seed recalcitrance in perspective. Ann Bot 101:213–228
Berjak P, Farrant JM, Pammenter NW (1989) The basis of recalcitrant seed behaviour. In: Taylorson RB (ed) Recent advances in the development and germination of seeds. Plenum Press, New York, pp 80–105
Berjak P, Walker M, Watt MP, Mycock DJ (1999) Experimental parameters underlying failure or success in plant germplasm cryopreservation: a case study on zygotic axes of Quercus robur L. Cryo Lett 20:252–262
Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097
Echlin P (1992) Low-temperature microscopy and analysis. Plenum Press, New York
Farrant JM, Pammenter NW, Berjak P (1989) Germination-associated events and the desiccation sensitivity of recalcitrant seeds—a case study on three unrelated species. Planta 178:89–98
Farrant JM, Pammenter NW, Berjak P (1992) Development of recalcitrant (homoiohydrous) seeds of Avicennia marina: anatomical, ultrastructural and biochemical events associated with development from histodifferentiation to maturation. Ann Bot 70:75–86
Farrant JM, Pammenter NW, Berjak P, Walters C (1997) Subcellular organization and metabolic activity during the development of seeds that attain different levels of desiccation tolerance. Seed Sci Res 7:135–144
Finkle BJ, Zavala ME, Ulrich JM (1985) Cryoprotective compounds in the viable freezing of plant tissues. In: Kartha KK (ed) Cryopreservation of plant cells and organs. CRC Press, Boca Raton, Florida, USA, pp 75–113
Fuller BJ (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Lett 25:375–388
Gotelli NJ, Entsminger GL (2009) EcoSim: Null models software for ecology. Version 7. Acquired Intelligence Inc. & Kesey-Bear, http://garyentsminger.com/ecosim.htm., Jericho, VT 05465. 77: 120–125
Kaczmarczyk A, Rutten T, Melzer M, Keller ERJ (2008) Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum L.) shoot tips. Cryo Lett 29:145–156
Lamb JM, Berjak P (1981) A unifying view of vacuolar ontogeny from studies on the root cap of Zea mays L. S Afr J Sci 77:120–125
Marty F (1999) Plant vacuoles. Plant Cell 11:587–599
Meryman HT, Williams RJ (1985) Basic principles of freezing injury to plant cells: natural tolerance and approaches to cryopreservation. In: Kartha KK (ed) Cryopreservation of plant cells and organs. CRC Press, Boca Raton, Florida, USA, pp 13–47
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497
Mycock DJ (1999) Addition of calcium and magnesium to a glycerol and sucrose cryoprotectant solution improves the quality of plant embryo recovery from cryostorage. Cryo Lett 20:77–82
Mycock DJ, Berjak P, Finch-Savage WE (2000) Effects of desiccation on the subcellular matrix of the embryonic axes of Quercus robur. In: Black M, Bradford KJ, Vázquez-Ramos J (eds) Seed biology: advances and applications. CABI Publishing, Wallingford, UK, pp 1197–1203
Pammenter NW, Berjak P (1999) A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Sci Res 9:13–37
Pammenter NW, Berjak P, Wesley-Smith J, Vander Willigen C (2002) Experimental aspects of drying and recovery. In: Black M, Pritchard H (eds) Desiccation and survival in plants: drying without dying. CAB International, Wallingford, Oxon, UK, pp 93–110
Pammenter NW, Greggains V, Kioko JI, Wesley-Smith J, Berjak P, Finch-Savage WE (1998) Effects of differential drying rates on viability retention of Ekebergia capensis. Seed Sci Res 8:463–471
Polge C, Smith AV, Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666
Popova AV, Busheva MR (2001) Cryoprotective effect of glycine betaine and glycerol is not based on a single mechanism. Cryo Lett 22:5–16
Sershen, Berjak P, Pammenter NW (2008) Desiccation sensitivity of excised embryonic axes of selected amaryllid species. Seed Sci Res 18:1–11
Sershen, Pammenter NW, Berjak P, Wesley-Smith J (2007) Cryopreservation of embryonic axes of selected amaryllid species. Cryo Lett 28:387–399
Staehelin LA (1997) The plant ER: a dynamic organelle composed of a large number of discrete functional domains. Plant J 11:1151–1165
Sussex IM (1952) Regeneration of the potato shoot apex. Nature 170:755–757
Walters C, Pammenter NW, Berjak P, Crane J (2001) Desiccation damage, accelerated aging and respiration in desiccation tolerant and sensitive seeds. Seed Sci Res 11:135–148
Walters C, Wesley-Smith J, Crane J, Hill LM, Chmielarz P, Pammenter NW, Berjak P (2008) Cryopreservation of recalcitrant (i.e. desiccation-sensitive) seeds. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, New York, pp 465–482
Webb MA, Arnott HJ (1982) Cell wall conformation in dry seeds in relation to preservation of structural integrity during desiccation. Am J Bot 69:1657–1668
Wen B, Song S-Q (2007) Acquision and loss of cryotolerance in Livistona chinensis embryos during seed development. Cryo Lett 28:291–302
Wesley-Smith J, Pammenter NW, Berjak P, Walters C (2001a) The effects of two drying rates on the desiccation tolerance of recalcitrant jackfruit (Artocarpus heterophyllus Lamk.) seeds. Ann Bot 88:653–664
Wesley-Smith J, Walters C, Pammenter NW, Berjak P (2001b) Interactions among water content, rapid (non-equilibrium) cooling to −196°C and survival of embryonic axes of Aesculus hippocastanum L. seeds. Cryobiology 42:196–206
Wesley-Smith J, Vertucci CW, Berjak P, Pammenter NW, Crane J (1992) Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and thermal properties of tissue water. J Plant Physiol 140:596–604
Wise RR (2006) The diversity of plastid form and function. In: Wise RR, Hoober JK (eds) The structure and function of plastids. Advances in photosynthesis and respiration, vol 23. Springer, Dordrecht, Netherlands, p 3
Acknowledgements
Financial support for the work and a bursary to the senior author, were provided by the National Research Foundation (NRF) South Africa.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Handling Editor: Peter Nick
Rights and permissions
About this article
Cite this article
Sershen, Berjak, P., Pammenter, N.W. et al. The effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of Amaryllis belladonna . Protoplasma 249, 155–169 (2012). https://doi.org/10.1007/s00709-011-0274-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00709-011-0274-5