Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Davidson, A L; Newcomb, William W.

doi:10.1007/bf01280505

Cited by 6 publications

(4 citation statements)

References 51 publications

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“…Electron microscopy revealed that the plasmalemma maintains its trilaminar structure, continuity, and apposition against cell walls even at the very low water content of 0.08 g of water per g of dry mass. This is in agreement with previous freeze-fracture studies (e.g., Platt et al 1994) and TEM studies using truly anhydrous tissue processing (Browning and Gunning 1977;Opik 1980Opik , 1985Kiss et al 1990;Tiwari et al 1990;Smith 1991;Platt et al 1997;Thomson and Platt 1997;Davidson and Newcomb 2000) but different from observations following exposure to aqueous media during processing (Webster and Leopold 1977, Thomson 1979, Fincher Chabot and Leopold 1982, Yatsu 1983). Nuclear magnetic resonance and X-ray studies of orthodox seeds have also shown that membrane integrity is maintained even in the dry state and that membranes retain a lamellar configuration (Seewaldt et al 1981).…”

Section: Discussionsupporting

confidence: 88%

“…6 d). The superior ultrastructural preservation of plant structures attained by cryotechniques is well documented (Staehelin and Chapman 1987, Craig and Staehelin 1988, Kiss et al 1990, Davidson and Newcomb 2000; for a review, see Gilkey and Staehelin 1986), which enhances the reliability of observations made on tissues prepared by such methods.…”

Section: Discussionmentioning

confidence: 94%

“…It involves the replacement of solidified water in the sample by an anhydrous solvent at temperatures precluding ice crystal growth. Following optimal cryofixation, FS can overcome most shortcomings of other anhydrous protocols, facilitating superior morphological preservation of hydrated plant tissues (Browning and Gunning 1977, Mersey and McCully 1978, McCully and Canny 1985, Lancelle et al 1986, Kiss et al 1990, Lancelle and Hepler 1992, Kellenberger 1991, Davidson and Newcomb 2000.…”

Section: Introductionmentioning

confidence: 99%

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Freeze-substitution of dehydrated plant tissues: Artefacts of aqueous fixation revisited

Wesley-Smith

2001

Protoplasma

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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.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

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Freeze-substitution of dehydrated plant tissues: Artefacts of aqueous fixation revisited

Wesley-Smith

2001

Protoplasma

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“…The effective cooling rate of nitrogen may be approximately doubled by the use of slush nitrogen (SN 2 ), a mixture of liquid and solid nitrogen (63 K). Melting propane was also used in this study to achieve even higher cooling rates (30,31).…”

Section: Introductionmentioning

confidence: 99%

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et al. 2004

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Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.

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Cytology of fungal pathogens and plant—host interactions

Howard

2001

Current Opinion in Microbiology

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Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Cited by 6 publications

References 51 publications

Freeze-substitution of dehydrated plant tissues: Artefacts of aqueous fixation revisited

Freeze-substitution of dehydrated plant tissues: Artefacts of aqueous fixation revisited

Untitled

Cytology of fungal pathogens and plant—host interactions

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