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Norman W. Pammenter - One of the best experts on this subject based on the ideXlab platform.
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Towards understanding the protection afforded by cryoprotectants commonly used in the cryopreservation of zygotic explants of recalcitrant seeds
'Elsevier BV', 2014Co-Authors: Ballesteros Bargues D, Patricia Berjak, Boby Varghese, S Naidoo, Norman W. PammenterAbstract:Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. The post-cryo success achieved with the excised zygotic explants used for cryopreservation is variable, and for some species dependent on the use of cryoprotectants and rapid drying. However, the efficacy of different cryoprotectants seems to be species dependent and there is a paucity of information on the mechanisms via which particular cryoprotectants protect zygotic embryos from recalcitrat seeds during partial drying and cryogenic cooling. The present study used Differential Scanning Calorimetry (DSC) and Cryo- Scanning Electron Microscopy (Cryo-SEM) to characterise the behaviour of selected cryoprotectants in zygotic embryos during drying and cooling to cryo- genic temperatures. These studies were carried out on a range ofrecalitrant-seeded species (Castanospermum australe, Trichilia dregeana, Amaryllis belladonna, Strychnos gerrardii) that show differential tolerance to cryoprotectants and cryo- preservation. Exposure to glycerol or sucrose did not change the behaviour of water in zygotic explants of most of the species studied. DSC revealed little to no difference between fresh samples and those cryoprotected, showing very sim- ilar enthalpy of melting transitions (#2;300 J/gH 2 O) and amount of unfrozen water (#2;0.30 gH 2 O/gdm); except for Atropa belladonna treated with glycerol that showed a slightly higher amount of unfrozen water (#2;0.45 gH 2 O/gdm). However, Cryo- SEM images showed important changes in the mechanical properties of the cryo- protected tissue during rapid drying. In general, glycerol allowed for less destruc- tive compaction of the zygotic tissues during desiccation, particularly in A. belladonna. Glycerol was also observed to help in membrane stabilization of Corynosoma australe, particularly during rehydration of the tissue. The results are discussed in relation to other studies on the mechanisms of cryoprotection in shoot tips and will inform the future cryopreservation protocols for zygotic germplasm of recalcitrant-seeded species
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the effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of Amaryllis belladonna
Protoplasma, 2012Co-Authors: Patricia Berjak, Norman W. Pammenter, James WesleysmithAbstract: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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effects of cryopreservation of recalcitrant Amaryllis belladonna zygotic embryos on vigor of recovered seedlings a case of stress hangover
Physiologia Plantarum, 2010Co-Authors: Patricia Berjak, Norman W. PammenterAbstract:Cryopreservation is the most promising long-term storage option for recalcitrant (i.e. desiccation-sensitive) seed germplasm; however, its effects on the vigor of recovered seedlings are unclear. This study looked at the vigor of seedlings recovered from partially dried (D) and cryopreserved (C) recalcitrant zygotic embryos (ZEs) of Amaryllis belladonna. Seedlings recovered from fresh (F), D- and C-embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water deficit (S), followed by 3 days of re-watering. Seedling vigor was assessed in terms of physiological and growth responses to the imposed water stress. Compared with F-embryos, partial dehydration and cryopreservation reduced the number of embryos that produced seedlings, as well as the subsequent in vitro biomass of these seedlings. DW- and CW-seedlings (i.e. seedlings recovered from dried and cryopreserved ZEs that were watered for 12 days) exhibited lower CO(2)-assimilation rates and abnormal root growth. Stomatal density was also lower in C-seedlings. DS- and CS-seedlings were exposed to persistent low leaf water and pressure potentials and unlike FS-seedlings, displayed signs of having incurred damage to their photosynthetic machinery. CS-seedlings were less efficient at adjusting leaf water potential to meet transpirational demands and more susceptible to persistent turgor loss than DS- and FS-seedlings. DS-seedlings performed slightly better than CS-seedlings but drought-induced seedling mortality in both these treatments was higher than FS-seedlings. These results suggest that seedlings recovered from partially dried and cryopreserved embryos were less vigorous and more susceptible to hydraulic failure than those from fresh ZEs.
Patricia Berjak - One of the best experts on this subject based on the ideXlab platform.
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Towards understanding the protection afforded by cryoprotectants commonly used in the cryopreservation of zygotic explants of recalcitrant seeds
'Elsevier BV', 2014Co-Authors: Ballesteros Bargues D, Patricia Berjak, Boby Varghese, S Naidoo, Norman W. PammenterAbstract:Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. The post-cryo success achieved with the excised zygotic explants used for cryopreservation is variable, and for some species dependent on the use of cryoprotectants and rapid drying. However, the efficacy of different cryoprotectants seems to be species dependent and there is a paucity of information on the mechanisms via which particular cryoprotectants protect zygotic embryos from recalcitrat seeds during partial drying and cryogenic cooling. The present study used Differential Scanning Calorimetry (DSC) and Cryo- Scanning Electron Microscopy (Cryo-SEM) to characterise the behaviour of selected cryoprotectants in zygotic embryos during drying and cooling to cryo- genic temperatures. These studies were carried out on a range ofrecalitrant-seeded species (Castanospermum australe, Trichilia dregeana, Amaryllis belladonna, Strychnos gerrardii) that show differential tolerance to cryoprotectants and cryo- preservation. Exposure to glycerol or sucrose did not change the behaviour of water in zygotic explants of most of the species studied. DSC revealed little to no difference between fresh samples and those cryoprotected, showing very sim- ilar enthalpy of melting transitions (#2;300 J/gH 2 O) and amount of unfrozen water (#2;0.30 gH 2 O/gdm); except for Atropa belladonna treated with glycerol that showed a slightly higher amount of unfrozen water (#2;0.45 gH 2 O/gdm). However, Cryo- SEM images showed important changes in the mechanical properties of the cryo- protected tissue during rapid drying. In general, glycerol allowed for less destruc- tive compaction of the zygotic tissues during desiccation, particularly in A. belladonna. Glycerol was also observed to help in membrane stabilization of Corynosoma australe, particularly during rehydration of the tissue. The results are discussed in relation to other studies on the mechanisms of cryoprotection in shoot tips and will inform the future cryopreservation protocols for zygotic germplasm of recalcitrant-seeded species
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the effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of Amaryllis belladonna
Protoplasma, 2012Co-Authors: Patricia Berjak, Norman W. Pammenter, James WesleysmithAbstract: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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effects of cryopreservation of recalcitrant Amaryllis belladonna zygotic embryos on vigor of recovered seedlings a case of stress hangover
Physiologia Plantarum, 2010Co-Authors: Patricia Berjak, Norman W. PammenterAbstract:Cryopreservation is the most promising long-term storage option for recalcitrant (i.e. desiccation-sensitive) seed germplasm; however, its effects on the vigor of recovered seedlings are unclear. This study looked at the vigor of seedlings recovered from partially dried (D) and cryopreserved (C) recalcitrant zygotic embryos (ZEs) of Amaryllis belladonna. Seedlings recovered from fresh (F), D- and C-embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water deficit (S), followed by 3 days of re-watering. Seedling vigor was assessed in terms of physiological and growth responses to the imposed water stress. Compared with F-embryos, partial dehydration and cryopreservation reduced the number of embryos that produced seedlings, as well as the subsequent in vitro biomass of these seedlings. DW- and CW-seedlings (i.e. seedlings recovered from dried and cryopreserved ZEs that were watered for 12 days) exhibited lower CO(2)-assimilation rates and abnormal root growth. Stomatal density was also lower in C-seedlings. DS- and CS-seedlings were exposed to persistent low leaf water and pressure potentials and unlike FS-seedlings, displayed signs of having incurred damage to their photosynthetic machinery. CS-seedlings were less efficient at adjusting leaf water potential to meet transpirational demands and more susceptible to persistent turgor loss than DS- and FS-seedlings. DS-seedlings performed slightly better than CS-seedlings but drought-induced seedling mortality in both these treatments was higher than FS-seedlings. These results suggest that seedlings recovered from partially dried and cryopreserved embryos were less vigorous and more susceptible to hydraulic failure than those from fresh ZEs.
Ramon Jordan - One of the best experts on this subject based on the ideXlab platform.
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First Report of Nerine yellow stripe virus in Amaryllis in the United States.
Plant Disease, 2013Co-Authors: Mary Ann Guaragna, D. Groth-helms, S. Juszczak, Dimitre Mollov, Benham E Lockhart, T. Van Schadewijk, Julia Lamborn, J. Hammond, Ramon JordanAbstract:Ornamental flower bulbs (including true bulbs, bulbils, corms, tubers, and rhizomes) are increasingly important floriculture crops. Amaryllis is a small genus of flowering bulbs, with two species. The South African native, Amaryllis belladonna, also known as belladonna lily, Jersey lily, naked lady, Amarillo, or March lily, is one of numerous ornamental species with the common name “lily” due to their flower shape and growth habit. Amaryllis are popular for their 6- to 10-inch trumpet shaped colorful flowers that are borne on 1- to 2-foot stalks. In January, 2011, a home gardener in California observed mosaic symptoms on the leaves of A. belladonna growing in her garden. Leaf samples were sent to Agdia Inc. for testing. Samples tested positive for the presence of Potyvirus in a reverse transcription (RT)-PCR screen using universal potyvirus primers (2) yielding the expected ∼1,600-bp product corresponding to the partial nuclear inclusion body (NIb) gene, full-length coat protein (CP) gene, and 3′ end untr...
C A Chang - One of the best experts on this subject based on the ideXlab platform.
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first report of capsicum chlorosis virus infecting Amaryllis and blood lily in taiwan
Plant Disease, 2009Co-Authors: C C Chen, C H Huang, Y H Cheng, Tsungchi Chen, Shyidong Yeh, C A ChangAbstract:Capsicum chlorosis virus (CaCV), a thrips-transmitted, tentative species in the genus Tospovirus, family Bunyaviridae, was first identified in solanaceous crops, but also infects several ornamental crops such as orchid (4), gloxinia (3), and calla lily (1). From 2005 to 2007, virus-like yellow ringspots were observed on the leaves of Amaryllis (Hippeastrum hybridum Hort.) and blood lily (Haemanthus multiflorus Martyn.) plants cultured in screenhouses and a private garden, respectively. Three of several hundred Amaryllis plants in screenhouses from two places were observed as showing yellow ringspot symptoms and one of six blood lily plants was observed as showing similar yellow ringspot symptoms. Sap extracts from symptomatic leaves were inoculated to Chenopodium quinoa Willd. and the resulting local lesions were passaged three successive times to C. quinoa for virus isolation. Using the tospovirus genus-specific primers gL3637 and gL4435c designed from the conserved region in the L RNA (2), DNA fragments of the expected size of 800 bp were amplified by reverse transcription (RT)-PCR from field samples and local lesions from C. quinoa. Extracts from the diseased plants and local lesions of C. quinoa reacted strongly with antiserum against the nucleocapsid (N) protein of CaCV in ELISA and western blotting. To confirm the identity of this virus, we amplified the N gene from three Amaryllis and one blood lily source using primer pair WN2328 and WN3534 designed from the S RNA of Watermelon silver mottle virus (1), and these products were cloned and sequenced. The sequence from each virus isolate was determined from three independent clones. The nucleotide and deduced amino acid sequences of N genes for the blood lily isolate (GenBank Accession No. EF101344) and three Amaryllis isolates (GenBank Accession Nos. EF101343, EF137177, and FJ185170) had identities greater than 97% with that of a CaCV isolate infecting Capsicum spp. found in Australia (GenBank Accession No. AY036057). Phylogenetic analysis using maximum parsimony showed that these sequences clustered with CaCV. These results show that the virus identified from Amaryllis and blood lily that were expressing yellow ringspot symptoms are isolates of CaCV. To our knowledge, this is the first report of CaCV naturally infecting Amaryllis and blood lily and it could become an important threat to ornamental production in Taiwan. References: (1) C. C. Chen et al. Plant Dis. 91:1201, 2007. (2) F. H. Chu et al. Phytopathology 91:361, 2001. (3) H. T. Hsu et al. J. Gen. Plant Pathol. 66:167, 2000. (4) Y. X. Zheng et al. Eur. J. Plant Pathol. 120:199, 2008.
Colin D Reynolds - One of the best experts on this subject based on the ideXlab platform.
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x ray structure solution of Amaryllis lectin by molecular replacement with only 4 of the total diffracting matter
Acta Crystallographica Section D-biological Crystallography, 1996Co-Authors: L Chantalat, S D Wood, Pierre J Rizkallah, Colin D ReynoldsAbstract:It is often the case that analogous proteins from different species crystallize in a different form. These structures can usually be easily solved by the molecular-replacement (MR) technique, as the protein folding is very often conserved. However, the results from MR become more uncertain as the proportion of diffracting matter decreases as a result of multimericity and/or absence of some of the atoms in the model. In this paper results are presented on the structure solution of Amaryllis lectin (109 residues per monomer) containing two protein molecules in the asymmetric unit. The structure was solved by MR using the Calpha coordinates of one monomer from snowdrop lectin which has 85% amino-acid sequence identity to Amaryllis lectin. This represents only 6% of the non-H atoms of the protein molecule to be used for structure determination and it is a major improvement on previous reports. Further calculations were carried out in order to establish the minimum number of atoms which could be included in the model before a clear solution to the MR problem was revealed. This study showed that the structure of Amaryllis lectin could still have been solved easily with 3.85% of the model, which even in the most favourable cases, will probably constitute a minimum for molecular-replacement structure solution.