Studies on the reduction of 2,3,5-triphenyltetrazolium chloride as a viability assay for plant tissue cultures

Towill, Leigh E.; Mazur, Péter

doi:10.1139/b75-129

Cited by 358 publications

(149 citation statements)

References 12 publications

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“…Three replicates were collected at 4, -2, -4, -6, and -8°C and transferred to 4°C for 24 h in the dark before estimating viability. Cell survival was then measured by the TTC reduction method (Towill and Mazur, 1974 …”

Section: Determination Of Freezing Tolerance By the Ttc Methodsmentioning

confidence: 99%

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism

et al. 1997

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A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/ Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase i n cell osmolality.I n contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment.To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure t o lower temperatures was observed i n both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. l h e best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.Many plants become more resistant to freezing temperatures when first exposed to low, nonfreezing temperatures, a process known as cold acclimation or cold hardening. Cell-suspension cultures derived from plants capable of cold acclimation can also be hardened to withstand freezing, but few data are available on low-temperatureinduced cold acclimation in cell suspensions derived from woody plants (Hellergren, 1983;Tremblay et al., 1992;Arora and Wisniewski, 1995;Leborgne et al., 1995b).Biochemical changes associated with cold acclimation include alterations in the concentrations of a wide range of metabolites, including sugars (Hallgren and Oquist, 1990 These complex changes indicate that cellular metabolism undergoes considerable modifications during acclimation, but the cause/effect relationships have not been clearly established. Carbohydrates that are compatible solutes with a demonstrated cryoprotectant activity are often reported to accumulate at low temperatures (acclimation). These include oligosaccharides such as Suc and raffinose, and polyols such as sorbitol and mannitol. Carbohydrates seem to function in two ways that are often difficult to distinguish: osmotic adjustment (a colligative effect) and cryoprotection of cellular structures.Eucalyptus, an evergreen woody plant exhibiting excellent productivity, good fiber quality, and high pulp yield, is grown extensively around the world, mostly for pulp making. However, a major limitation to the expansion of these plantations is the relatively high frost sensitivity of some species. For example, for Eucalyptus globulus, one...

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Section: Determination Of Freezing Tolerance By the Ttc Methodsmentioning

confidence: 99%

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism

et al. 1997

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“…Cells were treated with a final concentration of 75 uM ABA at either 3 or 23°C. Freezing resistance was measured as described by Chen and Gusta (4) and the LT50 (50% killing temperature) values were determined by the TTC (2,3,5-triphenyl tetrazolium chloride) reduction method (24). Cell cultures were routinely screened for bacterial and fungal contamination by microscopy and plating on 2% YPD medium (2% glucose, 2% bactopeptone, and 1% yeast extract).…”

Section: Methodsmentioning

confidence: 99%

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Robertson¹,

Gusta²,

Reaney³

et al. 1987

Plant Physiol.

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Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by I'4Cileucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3WC. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of I'4Cileucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of I'4Clleucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.Freezing resistance is induced in some plants (20,27) and plant suspension cultures (6) by exposure to low temperatures.Endogenous levels of ABA have been shown to increase during high and low temperature growth conditions (8). Following application of exogenous ABA, improved cold resistance has been observed in alfalfa seedlings (22), potatoes (5), and suspension cultures of some plant species (4,19) and callus cultures (14). A novel aspect of these studies is that ABA can substitute for the cold requirement in inducing freezing resistance (4, 5).Cycloheximide inhibits cold hardening in winter wheat (26), winter rape (12), and potato stem cultures (5), which suggests protein synthesis is required for the induction of freezing resistance. However, little is understood about the effects of low temperature or ABA on protein synthesis or gene expression. Electrophoretic analysis ofproteins synthesized during cold hardening of winter wheat, rye (7, 23), rape (13,17), and mulberry trees (28) has shown increased or decreased synthesis of certain proteins in hardier varieties. The relationship of most of these proteins to freezing resistance in plants is probably equivocal, since other complicating processes such as vernalization, dormancy, adjustment to low temperature growth and development occur concomitantly.We have used bromegrass suspension cultures as a model system to study cold acclimation and ABA induced freezing tolerance. Our approach has been to compare protein changes occurring during ABA induced hardening responses at 23°C with those during low temperature hardening. The correlation of specific protein changes occurring during ABA induced freezing tolerance at both low (3°C) an...

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“…Cell survival was then measured by the triphenyl tetrazolium chloride (Ph,TzCl) reduction assay (Towill and Mazur, 1974) or the fluorescein diacetate assay (Larkin, 1976).…”

Section: Cell Suspension Culturesmentioning

confidence: 99%

Introduction of Specific Carbohydrates into Eucalyptus gunnii Cells Increases their Freezing Tolerance

Leborgne

Teulières

Travert

et al. 1995

European Journal of Biochemistry

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The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long‐term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.

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Studies on the reduction of 2,3,5-triphenyltetrazolium chloride as a viability assay for plant tissue cultures

Cited by 358 publications

References 12 publications

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Introduction of Specific Carbohydrates into Eucalyptus gunnii Cells Increases their Freezing Tolerance

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