Electrolyte Leakage Test Research Articles

Bi-environmental evaluation of oat (Avena sativa L.) genotypes for yield and nutritional traits under cold stress conditions using multivariate analysis

AbstractOat being a rabi/winter crop in Kashmir, experiences extremely low temperatures which has detrimental effects on its growth and development. Therefore, this study was designed to evaluate a set of 130 oat genotypes in multi-location trials across temperate conditions of Kashmir valley from 2018 to 2022. From the preliminary data of 56 genotypes, including five checks, were selected and evaluated for nutritional and yield attributing traits under cold stress conditions at two locations. The results demonstrated significant genetic variation and high heritability for majority of traits, except for days to 50% flowering, days to maturity and dry fodder. Positive correlations were observed between green fodder yield and other traits, indicating their potential for enhancing yield. Principal component analysis identified four principal components that accounted for 69.87% of the total variation. Cluster analysis categorized the genotypes into two main clusters and six sub-clusters. Frost damage assessment was conducted at tillering stage after the snow melted in late January 2021 and 2022 using cold tolerance rating scale and subsequently tested for chilling injury through an electrolyte leakage test. From field and lab data analysis, five most promising cold tolerant, nutritious and high-yielding genotypes were identified. These genotypes have significant potential for utilization in future breeding programmes to improve cold tolerance in cultivated oats within the Kashmir valley thus promoting agricultural productivity and sustainability. The outcomes also provide valuable insights into the genetic variation, heritability, genotype-by-environment interactions, correlations and cold tolerance of oat genotypes in Kashmir.

Pomegranate freezing tolerance evaluation and its relationship with biochemical parameters

Low temperature is one of the most significant environmental factors that threaten the survival of subtropical and tropical plant species. By conducting a study, which was arranged in a completely randomized design with three replicates, the relative freezing tolerance (FT) of four Iranian pomegranate cultivars, including ‘Alak Torsh’, ‘Tabestaneh Torsh’, ‘Poost Sefid’, and ‘Poost Syah’, as well as its correlation with some biochemical indices, were investigated. From each cultivar, pieces of one-year-old shoot samples were treated with controlled freezing temperatures (−11, −14, and −17 ℃) to determine lethal temperatures (LT50) based on survival percentage, electrolyte leakage, phenolic leakage, and tetrazolium staining test (TST) methods. Results showed that FT was higher in the second year with a lower minimum temperature and a higher concentration of cryoprotectants. The stronger correlation of electrolyte leakage with survival percentage (r = 0.93***) compared to the other three indices explained that this index could be the most reliable injury index in determining the pomegranate FT to investigate freezing effects. Of all four cultivars, ‘Poost Syah’ was the hardest by presenting a higher FT than ~ −14 ℃ in mid-winter. Accordingly, this pomegranate cultivar seems to be promising to grow in regions with a higher risk of freezing and to be involved in breeding programs to develop novel commercial cultivars.

Frost hardiness of 10 olive cultivars after natural and controlled freezing

Selecting winter-hardy cultivars is an effective way to avoid freezing damage in olive trees. In this study, we evaluated the cold hardiness of 10 olive cultivars in the field after a natural freezing event (-12°C in December 2016, Sorkhe, Semnan, Iran) and treatment with controlled freezing temperatures ranging from 0°C to -21°C. The 50% lethal temperature (LT50) based on electrolyte leakage (LT50-EL) and tetrazolium tests (LT50-TZ) before, during, and after dormancy were determined in each cultivar's shoots. The proline and soluble carbohydrate content of leaves and shoots also were measured during dormancy. The LT50-EL had a mean of -9.5°C and ranged from -16.5°C to -4.8°C. The LT50-TZ had a mean of -10.2°C and ranged from -16.3°C to -6.4°C. We classified the olive cultivars into five groups based on their lab-freezing evaluations using cluster analysis. 'Manzanilla', 'Mission', and 'Rashid' were very sensitive to freezing, while 'Roughani' and 'Zrad' were the hardiest cultivars. In Field evaluation, the recovery ability and yield of each cultivar were evaluated six years after freezing damage. Visual scoring indicated that 'Roghani' and 'Zard' had the least amount of trunk damage. However, 'Zard' showed low regrowth power in the field after freezing damage despite its high freezing tolerance in lab and field experiments. 'Roughani' had the maximum fruit yield among the 10 olive cultivars six years after freezing damage (P < 0.01). Our finding indicates that laboratory techniques be combined with field assessments to more accurately evaluate the cold hardiness of olive trees.

Efficient Cold Tolerance Evaluation of Four Species of Liliaceae Plants through Cell Death Measurement and Lethal Temperature Prediction

Although Liliaceae are valuable resource plants with medicinal and edible uses, techniques for evaluating their tolerance to various abiotic stresses are very limited. This study evaluated the cold tolerance using visual inspection, electrolyte leakage, and Evan’s blue assay. Visual inspection of the responses to different temperatures, using a temperature range of 4 to −12 °C, showed that Scilla scilloides was receive the least damage. However, electrolyte leakage tests showed slightly different results from visual inspection. The median lethal temperature (LT50) was expected to be between −4 and −8 °C. The LT50 was considered a measure of damage due to electrolyte leakage in plant cold tolerance evaluation. As a result of predicting the lethal temperature using the logistic regression equation, the average LT50 of the four plants was −9.0 °C. The species with the lowest LT50 was measured for Hosta plantagines (−11.14°C), whereas the highest LT50 was measured for Hemerocallis fulva (−7.14°C). As a result of the Evan’s blue assay, it was found that cell necrosis occurred when the plants were exposed to low temperatures. Visual observation showed that more than 50% of the three plants’ cells, except for H. plantaginea, were stained blue even at 8 °C. From this result, H. plantaginea was judged to have strong low-temperature tolerance. At −12 °C, more than 50% of the four Liliaceae plants were colored blue, and the LT50 value was expected to be below −12 °C. The reducing sugar content, an indicator of plant cold tolerance, was the highest in H. plantaginea, followed by S. scilloide and H. longipes. Combining the three methods, H. plantaginea had the highest cold tolerance, followed by H. longipes, S. scilloides, and H. fulva. The results of this study will be widely used in selecting cold-tolerant useful resource plants.

Estimating Freezing Injury on Olive Trees: A Comparative Study of Computing Models Based on Electrolyte Leakage and Tetrazolium Tests

Winter frost injury is a major limiting factor for olive cultivation in temperate regions. The response of olive shoots to freezing stress can be used for selecting genotypes resistant to freezing. The electrolyte leakage (EL) and tetrazolium tests (TZ) are commonly used to evaluate dead tissues in cold stress studies. The temperature–response curve of dead tissues to lethal temperature (LT) is measured with models to calculate LT50 and LT90. In this study, we evaluated the accuracy and efficiency of eighteen nonlinear regression models (NLRs) in calculating LT50 and LT90 of freezing stress in different olive cultivars at various stages of dormancy. After evaluating the prediction performance of NLR models, it was found that only eight models were suitable for the purpose of this research out of the eighteen models examined. The 2p-logistic and Gompertz models were selected for modeling EL and TZ, respectively. Our research findings indicate that the Roughani, Kawi, and Zard varieties of olive trees exhibit the best performance under artificial temperature-controlled conditions. Our findings provide valuable insights into selecting frost-resistant cultivars and designing effective strategies for cold acclimation in olive cultivation.

Aquaphotomic, E-Nose and Electrolyte Leakage to Monitor Quality Changes during the Storage of Ready-to-Eat Rocket.

The consumption of ready-to-eat (RTE) leafy vegetables has increased rapidly due to changes in consumer diet. RTE products are perceived as fresh, high-quality, and health-promoting. The monitoring of the RTE quality is crucial in relation to safety issues. This study aimed to evaluate the maintenance of RTE rocket salad freshness packed under modified atmospheres. A portable E-nose, the electrolyte leakage test (which measures the index of leaf damage—ILD), and NIR spectroscopy and Aquaphotomics were employed. Two trials were carried out, using the following gas mixtures: (A) atmospheric air (21% O2, 78% N2); (B) 30% O2, 70% N2; (C) 10% CO2, 5% O2, 85% N2. Samples were stored at 4 °C and analyzed at 0, 1, 4, 7, 11, and 13 days. ANOVA, PCA, PLS were applied for data processing. E-nose and ILD results identified the B atmosphere as the best for maintaining product freshness. NIR spectroscopy was able to group the samples according to the storage time. Aquaphotomics proved to be able to detect changes in the water structure during storage. These preliminary data showed a good agreement NIR/ILD suggesting the use of NIR for non-destructive monitoring of the damage to the plant membranes of RTE rocket salad.

Improving of Winter Cold Hardiness by Glycine Betaine in Strawberry

One of the most important problems of strawberry cultivation in temperate regions is winter cold injuries. Current study investigated impacts of foliar application of glycine betaine (GB) at 0, 0.5, 1, 2 and 4 mM concentrations on the cold hardiness of strawberry. The plants were divided into two groups: one group for evaluation of cold hardiness at temperatures of -6, -9, -12, -15 and -18 °C; and the other for study of some biochemical characteristics. Results showed that GB treatment increased soluble carbohydrate and proline concentrations in both leaf and crown tissues, total protein concentration in leaf, and relative water content in leaf as compared to those in control. Based on LT50 calculated from electrolyte leakage and tetrazolium staining test, the GB application increased cold hardiness in strawberry plant based on its concentration. The highest cold hardiness was found in the 2 mM GB concentration based on electrolyte leakage and tetrazolium staining tests at -13.3 and -15.3 °C. Meanwhile, the lowest values of cold hardiness were observed in the control treatments based on electrolyte leakage and tetrazolium staining tests at -10.2 and -11.0 °C. Significant correlations were found between soluble carbohydrate and proline concentrations in leaf and crown, and total protein concentration with LT50 calculated from electrolyte leakage and tetrazolium staining test. We conclude that application of 2 mM GBhas the capacity to increase the freezing tolerance of strawberry and could be used as a prophylactic tool to reduce winter cold injury.

Comparison of Cold Hardiness Evaluation of Woody Species by ELLT and TTCLT

Cold hardiness evaluation is important for screening woody species in cold areas. We compared cold hardiness by estimating the 50% lethal temperature (LT50) using electrolyte leakage test (ELLT50) and triphenyltetrazolium chloride test (TTCLT50) for 26 woody species in the Bashang region of China. One-year-old shoots were collected in January and exposed to five subfreezing temperatures in a programmable temperature and humidity chamber. LT50 was estimated by fitting relative electrolyte leakage and percentage of dead tissue against test temperature. For all tested species, triphenyltetrazolium chloride (TTC) staining of the pith was weak and the cambium TTCLT50 was lower than the extreme minimum temperature (−37 °C) recorded in the region. The cambium TTCLT50 and the sd were lower than that for the phloem and xylem. The phloem TTCLT50 was lower than the xylem TTCLT50, and the two sds were similar. The ELLT50 showed no significant correlation with any TTCLT50. For most species, the ELLT50 was higher than the cambium and phloem TTCLT50 and was not significant different with the xylem TTCLT50. The ELLT50 showed higher sd than any tissue TTCLT50. Based on results obtained in this study, when choosing cold hardiness of single stem tissue as an indicator for screening woody species, the xylem should be considered first, followed by the phloem; the cambium and pith were unsuitable. The cold hardiness estimated by ELLT50 was more suitable as indicator for screening woody species than that of stem tissue in winter estimated by TTCLT50.

C-repeat binding transcription factors from Miscanthus × giganteus and their expression at a low temperature

Giant miscanthus (Miscanthus × giganteus) is a valuable plant for bioenergy production and carbon sequestration in low-input agriculture. The newly establishing plantations of miscanthus are, however, at risk during the winter because of the small size of the clones and limited ability of reserves storage in the rhizomes. Increased freezing tolerance of both shoots and rhizomes of giant miscant has been observed after cold acclimation, when plants are exposed to low, non-freezing temperatures. In plants cold acclimation is under complex genetic control that is orchestrated by C-repeat binding factors (CBFs). The present paper aimed the identification and characterisation of CBF-family transcription factors in miscanthus and determined their expression profiles at a low temperature. Three miscanthus genes, homologous to sorghum and maize CBF-family genes were identified. However, only one – MgCBF6 was expressed in the cold-acclimated leaves of miscanthus with an expression profile typical of a CBF-family gene. The ability of miscanthus to cold acclimate was also confirmed using an electrolyte leakage test and survival count. An increase in freezing tolerance was observed both in leaves and rhizomes but only when an increase in the freezing tolerance of plasma membranes was considered. It may be concluded that CBF-induced gene expression pathway may be induced in M. × giganteus while this species use the dormancy strategy for overwintering.

The use of field and artificial freezing studies to assess frost tolerance in natural populations of Pinus oocarpa

The susceptibility of Pinus oocarpa to freezing temperatures limits the commercial deployment of the highly productive Pinus patula × P. oocarpa hybrid in South Africa. Identifying P. oocarpa germplasm with increased frost tolerance is important. Twenty-three P. oocarpa provenances, originating from Mexico, Honduras, Guatemala and Nicaragua, were therefore assessed for their tolerance to freezing conditions by analysing field survival after frost events, subjecting needles to freezing temperatures and assessing damage using the electrolyte leakage test, and exposing young plants to freezing temperatures in a semi-controlled environment and scoring tissue damage based on a visual assessment. The performance of many of the provenances represented in the field and artificial freezing studies were similar and there was a strong correlation between provenance ranking in the whole plant freezing and electrolyte leakage test. We therefore support the use of these techniques as a means to assess cold tolerance in P. oocarpa at the provenance level. Provenances from north-western Mexico demonstrated more frost tolerance than those from southern Mexico. Provenances representing Honduras and Guatemala appear to be highly susceptible to frost.

Factors affecting resistance of winter rapeseed (Brassica napus L.) shoots to cold stress in vitro

Research was carried out at the Institute of Biology and Plant Biotechnology of Aleksandras Stulginskis University and at the Laboratory of Agrobiotechnology of the Joint Research Centre. The effect of cultivar, acclimation duration and exogenous L-glutamic acid on the changes of proline level and electrolyte leakage in shoots of winter rapeseed was evaluated. The shoots were cultivated in the MS medium supplemented with 0, 1, 2, 3 and 4.0 mM l–1 L-glutamic acid. The resistance of shoots to cold stress (at –8 °C for 24 h) was estimated by an electrolyte leakage test. It was found that the exogenous L-glutamic acid stimulates the synthesis of endogenous proline in non-acclimated as well as acclimated shoots. The highest increasing in cold resistance of winter rapeseed shoots has been obtained under the synergic effect of L-glutamic acid and acclimation. The shoots of cultivar ‘SY Kolumb’ were most cold-resistant independent of the acclimation duration and the concentration of L-glutamic acid in the medium.

Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis.

As a result of global warming, vegetation suffers from repeated freeze-thaw cycles caused by more frequent short-term low temperatures induced by hail, snow, or night frost. Therefore, short-term freezing stress of plants should be investigated particularly in light of the current climatic conditions. Alcohol dehydrogenase (ADH) plays a central role in the metabolism of alcohols and aldehydes and it is a key enzyme in anaerobic fermentation. ADH1 responds to plant growth and environmental stress; however, the function of ADH1 in the response to short-term freezing stress remains unknown. Using real-time quantitative fluorescence PCR, the expression level of ADH1 was analyzed at low temperature (4°C). The lethal temperature was calculated based on the electrolyte leakage tests for both ADH1 deletion mutants (adh1) and wild type (WT) plants. To further investigate the relationship between ADH1 and cold tolerance in plants, low-Mr polar metabolite analyses of Arabidopsis adh1 and WT were performed at cold temperatures using gas chromatography-mass spectrometry. This investigation focused on freezing treatments (cold acclimation group: −6°C for 2 h with prior 4°C for 7 d, cold shock group: −6°C for 2 h without cold acclimation) and recovery (23°C for 24 h) with respect to seedling growth at optimum temperature. The experimental results revealed a significant increase in ADH1 expression during low temperature treatment (4°C) and at a higher lethal temperature in adh1 compared to that in the WT. Retention time indices and specific mass fragments were used to monitor 263 variables and annotate 78 identified metabolites. From these analyses, differences in the degree of metabolite accumulation between adh1 and WT were detected, including soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine). In addition, the correlation-based network analysis highlighted some metabolites, e.g., melibiose, fumaric acid, succinic acid, glycolic acid, and xylose, which enhanced connectedness in adh1 network under cold chock. When considered collectively, the results showed that adh1 possessed a metabolic response to freezing stress and ADH1 played an important role in the cold stress response of a plant. These results expands our understanding of the short-term freeze response of ADH1 in plants.

Correlation between total lipids, linolenic acid and membrane injury under PEG-induced dehydration in leaves of Vigna genotypes differing in drought resistance

Membrane lipids contain trienoic fatty acids which enhance fluidity, an essential feature for optimal membrane performance. However highly unsaturated molecules are prone to lipoperoxidation processes. In V. unguiculata, linolenic acid (C18:3) accounts for more than 90% of the total fatty acids of galactolipids. High C18:3 contents causes galactolipids molecules, especially monogalactosyl-diacylglycerol (MGDG), to become more susceptible to drought-induced peroxidative or enzymatic degradation. In this work 8 Vigna genotypes differing in drought resistance in the field were compared to search for an eventual correlation between genotypes ability to resist to water stress and some characteristics of their membrane lipids in well-watered plants. Plants were grown in a greenhouse with semi-controlled environmental conditions, and kept fully hydrated. Leaves were collected from one month old plants to perform electrolyte leakage test under PEG-induced dehydration and lipid analysis. Results showed that both a reduced amount of total lipids, which reflects a small amount of membranes, and a reduced amount of C18:3 in MGDG, which indicates a low membrane fluidity, were strongly correlated with a high membrane resistance to dehydration, and therefore seem to be useful screening tools for drought-tolerant genotypes. Taking into account what is known concerning drought resistance of the genotypes under study, it was also concluded that the degree of membrane resistance to osmotic stress could be extrapolated to drought resistance in the field.

COLD ACCLIMATION PATTERNS OF ACTINIDIA ARGUTE CULTIVARS IN A TEMPERATE CLIMATE ZONE

The kiwi berry, Actinidia arguta Planch is gaining importance in temperate Europe. Warm transient intervals during the winter season can initiate cold deacclimation and bud break of woody plants. Most A. arguta cultivars are characterised by an early bud burst which may result in horticultural limitations due to a high sensitivity to spring frost of these young shoots/inflorescences. The evolution of bud dormancy was observed in two Actinidia arguta 'Weiki' and 'Geneva' cultivars. From September through mid-October the final 30-day shooting percentage (FS) was low. Between mid-October and mid-December a steep rise in FS indicates that bud endodormancy was broken. 'Weiki' appears to have a lesser need for cold exposure in order to release it from dormancy. The seasonal pattern of cold acclimation and deacclimation was determined for both cultivars using the electrolyte leakage test. Maximal cold tolerance was achieved in midwinter. In early spring cold tolerance of the buds declined again. 'Geneva' was more cold tolerant than 'Weiki'.

Frost hardiness of mycorrhizal and non-mycorrhizal Scots pine under two fertilization treatments.

Survival and functioning of mycorrhizal associations at low temperatures are not known well. In an earlier study, ectomycorrhizas did not affect the frost hardiness of Scots pine (Pinus sylvestris L.) roots, but here we studied whether differential nutrient availability would change the result and additionally, alter frost hardiness aboveground. The aim in this experiment was to compare the frost hardiness of roots and needles of mycorrhizal (Hebeloma sp.) and non-mycorrhizal Scots pine seedlings raised using two fertilization treatments and two cold-hardening regimes. The fertilization treatments were low (LF) and high (HF) application of a complete nutrient solution. Three hundred mycorrhizal and non-mycorrhizal seedlings were cultivated in growth chambers in four blocks for 16weeks. For the first 9weeks, the seedlings grew in long-day and high-temperature (LDHT) with low fertilization and then they were raised for 3weeks in LDHT with either low or high fertilization. After this, half of the plants in each treatment combination remained in LDHT, and half were transferred to short-day and low-temperature (SDLT) conditions to cold acclimatize. The frost hardiness of the roots and needles was assessed using controlled freezing tests followed by electrolyte leakage tests (REL). Mycorrhizal roots were slightly more frost hardy than non-mycorrhizal roots, but only in the growing-season conditions (LDHT) in low-nutrient treatment. In LDHT and LF, the frost hardiness of the non-mycorrhizal roots was about -9°C, and that of the non-mycorrhizal HF roots and the mycorrhizal roots in both fertilization levels was about -11°C. However, no difference was found in the roots within the SDLT regime, and in needles, there was no difference between mycorrhizal and fertilization treatments. The frost hardiness of needles increased by SDLT treatment, being -8.5 and -14.1°C in LDHT and SDLT, respectively. The dry mass of roots, stems, and needles was lower in LF than in HF and lower in SDLT than in LDHT. Mycorrhizal treatment did not affect the dry mass or its allocation. Although the mycorrhizal roots were slightly more frost hardy in the growing-season conditions, this is not likely to have significance in the field.

Pattern triggered immunity (PTI) in tobacco: isolation of activated genes suggests role of the phenylpropanoid pathway in inhibition of bacterial pathogens.

BackgroundPattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless form of plant resistance. Upon inoculation of a plant with non-pathogens or pathogenicity-mutant bacteria, the induced PTI will prevent bacterial proliferation. Developed PTI is also able to protect the plant from disease or HR (Hypersensitive Response) after a challenging infection with pathogenic bacteria. Our aim was to reveal those PTI-related genes of tobacco (Nicotiana tabacum) that could possibly play a role in the protection of the plant from disease.Methodology/Principal FindingsLeaves were infiltrated with Pseudomonas syringae pv. syringae hrcC- mutant bacteria to induce PTI, and samples were taken 6 and 48 hours later. Subtraction Suppressive Hybridization (SSH) resulted in 156 PTI-activated genes. A cDNA microarray was generated from the SSH clone library. Analysis of hybridization data showed that in the early (6 hpi) phase of PTI, among others, genes of peroxidases, signalling elements, heat shock proteins and secondary metabolites were upregulated, while at the late phase (48 hpi) the group of proteolysis genes was newly activated. Microarray data were verified by real time RT-PCR analysis. Almost all members of the phenyl-propanoid pathway (PPP) possibly leading to lignin biosynthesis were activated. Specific inhibition of cinnamic-acid-4-hydroxylase (C4H), rate limiting enzyme of the PPP, decreased the strength of PTI - as shown by the HR-inhibition and electrolyte leakage tests. Quantification of cinnamate and p-coumarate by thin-layer chromatography (TLC)-densitometry supported specific changes in the levels of these metabolites upon elicitation of PTI.Conclusions/SignificanceWe believe to provide first report on PTI-related changes in the levels of these PPP metabolites. Results implicated an actual role of the upregulation of the phenylpropanoid pathway in the inhibition of bacterial pathogenic activity during PTI.

Photosynthetic Apparatus Responses to Short‐term low‐temperature Flooding May Contribute to Freezing Tolerance Changes in Forage Grasses

AbstractWinter conditions are subjected to rapid climate changes. Increased precipitation and snow melting during warmer winters may result in low‐temperature flooding. These factors probably affect plant overwintering strategies. This study investigated the relationships between the photosynthetic adjustment and freezing tolerance following a short‐term low‐temperature flooding in two forage grasses (Lolium perenne and Festuca pratensis). The effect of flooding on the photosynthetic apparatus acclimation to cold was determined using chlorophyll a fluorescence measurements. Freezing tolerance was estimated using an electrolyte leakage test. It has been shown that genotypes activating non‐photochemical mechanisms of photosynthetic acclimation to cold during low‐temperature flooding may show increased freezing tolerance. Freezing tolerance reduction observed in Festuca pratensis was probably connected with decreased photochemical activity and photoinhibition of photosynthesis. It can be concluded that photosynthetic apparatus response to low‐temperature flooding may contribute to changes in the freezing tolerance. The direction of the changes is associated with different photosynthetic apparatus performance.

Modeling Methodology for Cold Tolerance Assessment of Pittosporum tobira

Abstract: This study was carried out to develop a simple, rapid and reliable assessment model to predict cold tolerance in Pittosporum tobira, a broad-leaved evergreen commonly used in the southern region of South Korea, which can minimize the possible experimental errors appeared in a electrolyte leakage test for cold tolerance assessment. The modeling procedure comprised of regrowth test and a electrolyte leakage test on the plants exposed to low temperature treatments. The lethal temperatures estimated from the methodological combinations of a electrolyte leakage test including tissue sampling, temperature treatment for potential el ectrical conductivity, and statistical analysis were compared to the results of the regrowth test. The highest temperature showing the survival rate lower than 50% obtained from the regrowth test was -10°C and the lethal was -10°C~-5°C. Based on the results of the regrowth test, several methodological combinations of electrolyte leakage tests were evaluated and the electrolyte leaka ge lethal temperatures estimated using leaf sample tissue and freeze-killing method were closest to the regrowth lethal temperatur e. Evaluating statistical analysis models, linear interpolation had a higher tendency to overestimate the cold tolerance than non- linear regression. Consequently, the optimal model for cold tolerance assessment of P. tobira is composed of evaluating electrol yte leakage from leaf sample tissue applying freeze-killing method for potential electrical conductivity and predicting lethal temp erature through non-linear regression analysis.Additional key words:

Frost hardiness of mycorrhizal (Hebeloma sp.) and non-mycorrhizal Scots pine roots

The frost hardiness (FH) of mycorrhizal [ectomycorrhizal (ECM)] and non-mycorrhizal (NM) Scots pine (Pinus sylvestris) seedlings was studied to assess whether mycorrhizal symbiosis affected the roots' tolerance of below-zero temperatures. ECM (Hebeloma sp.) and NM seedlings were cultivated in a growth chamber for 18weeks. After 13weeks' growth in long-day and high-temperature (LDHT) conditions, a half of the ECM and NM seedlings were moved into a chamber with short-day and low-temperature (SDLT) conditions to cold acclimate. After exposures to a range of below-zero temperatures, the FH of the roots was assessed by means of the relative electrolyte leakage test. The FH was determined as the inflection point of the temperature-response curve. No significant difference was found between the FH of mycorrhizal and non-mycorrhizal roots in LDHT (-8.9 and -9.8°C) or SDLT (-7.5 and -6.8°C). The mycorrhizal treatment had no significant effect on the total dry mass, the allocation of dry mass among the roots and needles or nutrient accumulation. The mycorrhizal treatment with Hebeloma sp. did not affect the FH of Scots pine in this experimental setup. More information is needed on the extent to which mycorrhizas tolerate low temperatures, especially with different nutrient contents and different mycorrhiza fungi.

A novel system for in situ determination of heat tolerance of plants: first results on alpine dwarf shrubs.

BackgroundHeat stress and heat damage to plants gain globally increasing importance for crop production and plant survival in endangered habitats. Therefore the knowledge of heat tolerance of plants is of great interest. As many heat tolerance measurement procedures require detachment of plants and protocols expose samples to various heat temperatures in darkness, the ecological relevance of such results may be doubted. To overcome these constraints we designed a novel field compatible Heat Tolerance Testing System (HTTS) that opens the opportunity to induce controlled heat stress on plants in situ under full natural solar irradiation. Subsequently, heat tolerance can be evaluated by a variety of standard viability assays like the electrolyte leakage test, chlorophyll fluorescence measurements and visual assessment methods. Furthermore, recuperation can be studied under natural environmental conditions which is impossible when detached plant material is used. First results obtained on three alpine dwarf - shrubs are presented.ResultsWhen heat tolerance of Vaccinium gaultherioides Bigelow was tested with the HTTS in situ, the visual assessment of leaves showed 50% heat injury (LT50) at 48.3°C, while on detached leaves where heat exposure took place in small heat chambers this already happened at 45.8°C. Natural solar irradiation being applied during heat exposure in the HTTS had significantly protective effects: In Loiseleuria procumbens L. (Desv.), if heat exposure (in situ) took place in darkness, leaf heat tolerance was 50.6°C. In contrast, when heat exposure was conducted under full natural solar irradiation heat tolerance was increased to 53.1°C. In Rhododendron ferrugineum L. heat tolerance of leaves was 42.5°C if the exposure took place ex situ and in darkness, while it was significantly increased to 45.8°C when this happened in situ under natural solar irradiation.ConclusionsThe results obtained with the HTTS tested in the field indicate a mitigating effect of natural solar irradiation during heat exposure. Commonly used laboratory based measurement procedures expose samples in darkness and seem to underestimate leaf heat tolerance. Avoidance of detachment by the use of the HTTS allows studying heat tolerance and recuperation processes in the presence of interacting external abiotic, biotic and genetic factors under field conditions. The investigation of combined effects of heat exposure under full solar irradiation, of recuperation and repair processes but also of possible damage amplification into the results with the HTTS appears to be particularly useful as it allows determining heat tolerance of plants with a considerably high ecological significance.