The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1⁻¹ 3,6-dichloro-o-anisic acid, 0.1 mg 1⁻¹ naphthaleneacetic acid, 2.0 mg l⁻¹ 6-benzylaminopurine, 80.0 mg 1⁻¹ adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.     

The production of auxin by autolysing tissues

Summary Autolysing plant tissues are known to produce auxin when extracted with ether. It has been shown that autolysing plant, yeast and rat liver tissues produce auxin in vitro; this suggests that relatively unspecific mechanisms are involved. Furthermore, sterile plant and animal tissues which have been killed by freezing and thawing induce nodules of differentiated cells in a previously undifferentiated callus of Phaseolus vulgaris. The callus tissue is known to differentiate in response to applied gradients of auxin. Plant and animal tissues killed by boiling were considerably less effective in inducing differentiation in the tissue. The evidence indicates that auxin is a normal product of autolysing cells. It is suggested that dying cells are an important source of auxin in the plant.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

ANAEROBIC SUBSTRATE TOLERANCE IN SPOROBOLUS VIRGINICUS (L.) KUNTH.

The purpose of this study was to determine if and how the two genetically distinct forms, marsh and dune, of Sporobolus virginicus (L.) Kunth. tolerate anaerobic substrates. The treatments in the hydroponic study, conducted in the greenhouse for approximately 6 months, involved growing the marsh and dune forms in aerobic, anaerobic, and alternating aeration treatments. Plants were examined for morphological and physiological responses to the aeration treatments. In response to the continuous anaerobic treatment, the dune form of S. virginicus exhibited increased stolon biomass, but no difference of total biomass or rhizome aerenchyma when compared with the aerobic treatment. In response to alternating aeration, rhizome aerenchyma increased, total biomass decreased, and stolon biomass remained constant. Belowground transport of oxygen enabled the root tissue in all of the aeration treatments to maintain aerobic respiration. The marsh form grown in the alternating aeration treatment had the same total biomass but more rhizome aerenchyma when compared to the aerobic treatment. Growth in the continuous anaerobic treatment resulted in a reduction of total biomass and increased rhizome arenchyma. Marsh form roots did not appear to be respiring anaerobically or producing ethanol or additional malate at the time of harvest; however, root respiration was higher in the anaerobic and alternating treatments. The marsh and dune forms of S. virginicus were able to adjust morphologically or physiologically or to use existing morphological features to tolerate anaerobic substrates. Thus, it appears that the distribution of the two forms of S. virginicus found in coastal sand dunes and in salt marshes is not limited by differences in ability to tolerate waterlogged soils.     

Thermal adaptation of heterotrophic soil respiration in laboratory microcosms

Respiration of heterotrophic microorganisms decomposing soil organic carbon releases carbon dioxide from soils to the atmosphere. In the short term, soil microbial respiration is strongly dependent on temperature. In the long term, the response of heterotrophic soil respiration to temperature is uncertain. However, following established evolutionary trade‐offs, mass‐specific respiration (R_mass) rates of heterotrophic soil microbes should decrease in response to sustained increases in temperature (and vice‐versa). Using a laboratory microcosm approach, we tested the potential for the R_mass of the microbial biomass in six different soils to adapt to three, experimentally imposed, thermal regimes (constant 10, 20 or 30 °C). To determine R_mass rates of the heterotrophic soil microbial biomass across the temperature range of the imposed thermal regimes, we periodically assayed soil subsamples using similar approaches to those used in plant, animal and microbial thermal adaptation studies. As would be expected given trade‐offs between maximum catalytic rates and the stability of the binding structure of enzymes, after 77 days of incubation R_mass rates across the range of assay temperatures were greatest for the 10 °C experimentally incubated soils and lowest for the 30 °C soils, with the 20 °C incubated soils intermediate. The relative magnitude of the difference in R_mass rates between the different incubation temperature treatments was unaffected by assay temperature, suggesting that maximum activities and not Q₁₀ were the characteristics involved in thermal adaptation. The time taken for changes in R_mass to manifest (77 days) suggests they likely resulted from population or species shifts during the experimental incubations; we discuss alternate mechanistic explanations for those results we observed. A future research priority is to evaluate the role that thermal adaptation plays in regulating heterotrophic respiration rates from field soils in response to changing temperature, whether seasonally or through climate change.     

Seasonal patterns and environmental control of carbon dioxide and water vapour exchange in an ecotonal boreal forest

Carbon dioxide, water vapour, and sensible heat fluxes were measured above and within a spruce dominated forest near the southern ecotone of the boreal forest in Maine, USA. Summer, mid-day carbon dioxide uptake was higher than at other boreal coniferous forests, averaging about – 13 μmol CO₂ m^–2 s^–1. Nocturnal summer ecosystem respiration averaged ≈ 6 μmol CO₂ m^–2 s^–1 at a mean temperature of ≈ 15 °C. Significant ecosystem C uptake began with the thawing of the soil in early April and was abruptly reduced by the first autumn frost in early October. Half-hourly forest CO₂ exchange was regulated mostly by the incident photosynthetically active photon flux density (PPFD). In addition to the threshold effects of freezing temperatures, there were seasonal effects on the inferred photosynthetic parameters of the forest canopy. The functional response of this forest to environmental variation was similar to that of other spruce forests. In contrast to reports of carbon loss from northerly boreal forest sites, in 1996 the Howland forest was a strong carbon sink, storing about 2.1 t C ha^–1.     

Direct and indirect effects of elevated CO2 on leaf respiration in a forest ecosystem

We measured the short‐term direct and long‐term indirect effects of elevated CO₂ on leaf dark respiration of loblolly pine (Pinus taeda) and sweetgum (Liquidambar styraciflua) in an intact forest ecosystem. Trees were exposed to ambient or ambient + 200 µmol mol^?1 atmospheric CO₂ using free‐air carbon dioxide enrichment (FACE) technology. After correcting for measurement artefacts, a short‐term 200 µmol mol^?1 increase in CO₂ reduced leaf respiration by 7–14% for sweetgum and had essentially no effect on loblolly pine. This direct suppression of respiration was independent of the CO₂ concentration under which the trees were grown. Growth under elevated CO₂ did not appear to have any long‐term indirect effects on leaf maintenance respiration rates or the response of respiration to changes in temperature (Q₁₀, R₀). Also, we found no relationship between mass‐based respiration rates and leaf total nitrogen concentrations. Leaf construction costs were unaffected by growth CO₂ concentration, although leaf construction respiration decreased at elevated CO₂ in both species for leaves at the top of the canopy. We conclude that elevated CO₂ has little effect on leaf tissue respiration, and that the influence of elevated CO₂ on plant respiratory carbon flux is primarily through increased biomass.     

Coupling of respiration, nitrogen, and sugars underlies convergent temperature acclimation in Pinus banksiana across wide-ranging sites and populations

Patterns and mechanisms of short‐term temperature acclimation and long‐term climatic adaptation of respiration among intraspecific populations are poorly understood, but both are potentially important in constraining respiratory carbon flux to climate warming across large geographic scales, as well as influencing the metabolic fitness of populations. Herein we report on leaf dark respiration of 33‐year‐old trees of jack pine (Pinus banksiana Lamb.) grown in three contrasting North American common gardens (0.9, 4.6, and 7.9 °C, mean annual temperature) comprised of identical populations of wide‐ranging geographic origins. We tested whether respiration rates in this evergreen conifer acclimate to prevailing ambient air temperatures and differ among populations. At each of the common gardens, observed population differences in respiration rates measured at a standard temperature (20 °C) were comparatively small and largely unrelated to climate of seed‐source origin. In contrast, respiration in all populations exhibited seasonal acclimation at all sites. Specific respiration rates at 20 °C inversely tracked seasonal variation in ambient air temperature, increasing with cooler temperatures in fall and declining with warmer temperatures in spring and summer. Such responses were similar among populations and sites, thus providing a general predictive equation regarding temperature acclimation of respiration for the species. Temperature acclimation was associated with variation in nitrogen (N) and soluble carbohydrate concentrations, supporting a joint enzyme and substrate‐based model of respiratory acclimation. Regression analyses revealed convergent relationships between respiration and the combination of needle N and soluble carbohydrate concentrations and between N‐based respiration (R_N, μmol mol N^{? 1} s^{? 1}) and soluble carbohydrate concentrations, providing evidence for general predictive relationships across geographically diverse populations, seasons, and sites. Overall, these findings demonstrate that seasonal acclimation of respiration modulates rates of foliar respiratory carbon flux in a widely distributed evergreen species, and does so in a predictable way. Genetic differences in specific respiration rate appear less important than temperature acclimation in downregulating respiratory carbon fluxes with climate warming across wide‐ranging sites.     

Responses of Respiration to Increases in Carbon Dioxide Concentration and Temperature in Three Soybean Cultivars

The purpose of this experiment was to determine how respirationof soybeans may respond to potential increases in atmosphericcarbon dioxide concentration and growth temperature. Three cultivarsof soybeans (Glycine max L. Merr.), from maturity groups 00,IV, and VIII, were grown at 370, 555 and 740cm³m^-3carbon dioxideconcentrations at 20/15, 25/20, and 31/26°C day/night temperatures.Rates of carbon dioxide efflux in the dark were measured forwhole plants several times during exponential growth. Thesemeasurements were made at the night temperature and the carbondioxide concentration at which the plants were grown. For thelowest and highest temperature treatments, the short term responseof respiration rate to measurement at the three growth carbondioxide concentrations was also determined. Elemental analysisof the tissue was used to estimate the growth conversion efficiency.This was combined with the observed relative growth rates toestimate growth respiration. Maintenance respiration was estimatedas the difference between growth respiration and total respiration.Respiration rates were generally sensitive to short term changesin the measurement carbon dioxide concentration for plants grownat the lowest, but not the highest carbon dioxide concentration.At all temperatures, growth at elevated carbon dioxide concentrationsdecreased total respiration measured at the growth concentration,with no significant differences among cultivars. Total respirationincreased very little with increasing growth temperature, despitean increase in relative growth rate. Growth respiration wasnot affected by carbon dioxide treatment at any temperature,but increased with temperature because of the increase in relativegrowth rate. Values calculated for maintenance respiration decreasedwith increasing carbon dioxide concentration and also decreasedwith increasing temperature. Calculated values of maintenancerespiration were sometimes zero or negative at the warmer temperatures.This suggests that respiration rates measured in the dark maynot have reflected average 24-h rates of energy use. The resultsindicate that increasing atmospheric carbon dioxide concentrationmay reduce respiration in soybeans, and respiration may be insensitiveto climate warming. Glycine max L. (Merr.); carbon dioxide; respiration; temperature; climate change     

Effect of freezing and cold storage on phospholipids in developing soybean cotyledons

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. “Harosoy 63” were frozen with liquid N₂, dry ice, or stored in a freezer (−20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at −20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with ³H, and fatty acids with ¹⁴C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.     

The cryopreservation of Chlorella

The temperature at which Chlorella 211/8h was grown determined the response to a subsequent stress of freezing to and thawing from-196°C. Cells cultured at 20°C were the most sensitive to freezing injury; at both higher and lower growth temperatures resistance to damage induced by freezing developed. At all culture temperatures examined the freezing tolerance varied with the age of culture.     

Experimental cryosurgery investigations in vivo

Cryosurgery is the use of freezing temperatures to elicit an ablative response in a targeted tissue. This review provides a global overview of experimentation in vivo which has been the basis of advancement of this widely applied therapeutic option. The cellular and tissue-related events that underlie the mechanisms of destruction, including direct cell injury (cryolysis), vascular stasis, apoptosis and necrosis, are described and are related to the optimal methods of technique of freezing to achieve efficacious therapy. In vivo experiments with major organs, including wound healing, the putative immunological response following thawing, and the use of cryoadjunctive strategies to enhance cancer cell sensitivity to freezing, are described.     

A Cryomicroscopic Study of Cylindrocystis brebissonii De Bary and Two Species of Micrasterias Ralfs (Conjugatophyceae, Chiorophyta) during Freezing and Thawing

The changes in morphology of the unicellular algae Cylindrocystisbrebissonii and two species of Micrasterias during freezingand thawing were observed on a light microscope fitted witha temperature controlled stage. At slow rates of cooling extensiveshrinkage of the protoplast was observed. The response of thecell wall varied with cell-type. In C. brebissonii plasmolysiswas not observed and the cell wall and protoplast shrank together.In Micrasterias the cell wall did not contract and a distinctplasmolysis was observed. Following freezing to and thawingfrom –25?C cells of C. brebissonii were non-viable butremained osmotically responsive. Cooling at faster rates inducedintracellular ice formation in all cell-types. The criticalrate of cooling varied with cell-type and was determined bycell volume and suface area. Intracellular gas bubbles wereobserved during thawing following both rapid and slow cooling. Following cooling in dimethylsulphoxide cells of C. brebissoniiwere protected against freezing injury. The recovery on thawingfrom –196?C being determined by the rate of cooling, anoptimum rate of 1?C min^–1 was observed. During slow ratesof cooling (<2?C min^–1) cells remained unshrunken,at faster rates (10?C min^–1) the loss of cell viabilitywas related to osmotic shrinkage during cooling rather thanto nucleation of intracellular ice. Intracellular ice formationwas observed only following significantly faster rates of cooling(>20?C min^–1). Key words: Cylindrocystis, Micrasterias, cryomicroscopy, freezing injury     

CHARACTERIZATION OF TISSUE CULTURE INITIATION AND PLANT REGENERATION IN SPOROBOLUS VIRGINICUS (GRAMINEAE)

Tissue cultures of the halophytic saltmarsh grass Sporobolus virginicus were initiated from unemerged immature inflorescence tissue. Typical graminaceous embryogenic and nonembryogenic callus and cell types were noted. Embryogenic callus was compact golden yellow. Histological evidence indicated that proliferation of the ovary tissue of the immature pistil was the source for embryogenic callus. Plants regenerated after first reducing and then eliminating auxin from the culture medium. Regeneration was observed both through the concerted development of bipolar meristems from somatic embryos and by the formation of multiple shoot meristems that were either connected through callus tissue to root meristems or which later adventitiously rooted. The main mode of regeneration appeared to be somatic embryogenesis with additional multiple shoot formation probably due to precocious germination of somatic embryos. Plants recovered from culture were acclimated to soil, grown up in a greenhouse, and planted in field plots with saline irrigation to ensure stability of salt tolerance.     

Environmental effects on CO2 efflux from riparian tundra in the northern foothills of the Brooks Range,Alaska, USA

Summary Carbon dioxide efflux and soil microenvironmental factors were measured diurnally in Carex aquatilus-and Eriophorum angustifolium-dominated riparian tundra communities to determine the relative importance of soil environmental factors controlling ecosystem carbon dioxide exchange with the atmosphere. Measurements were made weekly between 18 June and 24 July 1990. Diurnal patterns in carbon dioxide efflux were best explained by changes in soil temperature, while seasonal changes in efflux were correlated with changes in depth to water table, depth to frozen soil and soil moisture. Carbon dioxide efflux rates were lowest early in the growing season when high water tables and low soil temperatures limited microbial and root activity. Individual rainfall events that raised the water table were found to strongly reduce carbon dioxide efflux. As the growing season progressed, rainfall was low and depth to water table and soil temperatures increased. In response, carbon dioxide efflux increased strongly, attaining rates late in the season of approximately 10 g CO₂ m^–2 day^–1. These rates are as high as maxima recorded for other arctic sites. A mathematical model is developed which demonstrates that soil temperature and depth to water table may be used as efficient predictors of ecosystem CO₂ efflux in this habitat. In parallel with the field measurements of CO₂ efflux, microbial respiration was studied in the laboratory as a function of temperature and water content. Estimates of microbial respiration per square meter under field conditions were made by adjusting for potential respiring soil volume as water table changed and using measured soil temperatures. The results indicate that the effect of these factors on microbial respiration may explain a large part of the diurnal and seasonal variation observed in CO₂ efflux. As in coastal tundra sites, environmental changes that alter water table depth in riparian tundra communities will have large effects on ecosystem CO₂ efflux and carbon balance.     

Closely related freshwater macrophyte species,Ceratophyllum demersum and C. submersum,differ in temperature response

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Waterlogging responses of Sporobolus virginicus (L.) Kunth.

Summary Flooding responses in Sporobolus virginicus (L.) Kunth., a perennial C₄ grass, propagated from plants collected on the fringes of a mangrove swamp, were examined in a glasshouse study over 42 days. Flooding significantly reduced soil redox potential, induced adventitious root development, shifted resource allocation from below- to above-ground components without affecting total biomass accumulation and significantly decreased below-ground/above-ground biomass ratios. Although soil waterlogging significantly increased alcohol dehydrogenase activity (ADH) after 30 h, significant increase in central air space by 45–50% of the cross-sectional stem area eliminated root hypoxia, and ADH activity decreased to levels equivalent to drained controls after 42 days. In addition, flooded plants exhibited significantly higher carbon dioxide assimilation rates but similar relative growth rates (RGR) to drained controls. The results indicate that S. virginicus responds to water-logging by a combination of metabolic, morphological and anatomical mechanisms, which may account for its widespread distribution in coastal lagoons, estuaries and marshes.     

Cryopreservation and storage of embryogenic callus cultures of several Citrus species and cultivars

Nucellus-derived embryogenic callus cultures of Salustiana sweet orange were subjected to cryoconservation assays. Cryoprotection with 10%(vol/vol) dimethylsulfoxide, freezing by slow cooling and thawing by fast warming was suitable to recover viable growing cultures and whole plants through embryogenesis. Evaluation of liquid phase R ₁ and solid phase R ₂ cooling rates using a programmable freezing unit indicated that 100% of embryogenic cultures survived when frozen using a range of cooling rates (R ₁ not above 0.5°C min^–1 and R ₂ not above 1°C min ¹) and thawed by fast warming. Storage up to 2 years in liquid nitrogen did not affect the growth of the cryopreserved cultures and the recovery of whole plants. Cultures of four cultivars of sweet orange (C. sinensis Osb.), three cultivars of grapefruit (C. paradisi Macf.), and one cultivar each of lemon [C. limon (L.) Burm. f.], Cleopatra mandarin (C. reshni Hort. ex Tan.), sour orange (C. aurantium L.) and Mexican lime [C. aurantifolia (Christm.) Swing.] have been successfully cryopreserved. Problems using a viability assessment using fluorescein diacetate staining are discussed. Received: 15 April 1996 / Revision received: 22 July 1996 / Accepted: 6 August 1996     

Leaf respiration is differentially affected by leaf vs. stand-level night-time warming

Plant respiration is an important physiological process in the global carbon cycle serving as a major carbon flux from the biosphere to the atmosphere. Respiration is sensitive to temperature providing a link between environmental variability, climate change and the global carbon cycle. We measured leaf respiration in Populus deltoides after manipulating the air temperature surrounding part of a single leaf, and compared this to the temperature response of the same leaves after manipulating the temperature of the stand. The short‐term temperature response of respiration (Q₁₀– change in the respiration rate with a 10 °C increase in leaf temperature) was 1.7 when the leaf temperature was manipulated, but 2.1 when the stand‐level temperature was changed. As a result, total night‐time carbon release during the five‐day experiment was 21% lower when using the Q₁₀ estimates from the tradition leaf manipulation compared to the stand‐level manipulation. We conclude that the temperature response of leaf respiration is related to whole plant carbon and energy demands, and that appropriate experimental procedures are required in examining respiratory CO₂ release under variable temperature conditions.