Heritable improvement of frost tolerance in winter wheat by in vitro-selection of hydroxyproline-resistant proline overproducing mutants

In a previous study (Dörffling et al., J. Plant Physiol. 142, 222–225, 1993) in vitro-selection and regeneration of hydroxyproline (Hyp)-resistant lines of winter wheat (Triticum aestivum cv. Jo 3063) with increased frost tolerance and increased proline contents was reported. In this study the heritability of these traits was investigated. The F ₁progenies derived from the pollination of regenerated plants with pollen from wild type plants developed higher mean frost tolerance (lower LT ₅₀values) and higher proline levels compared with in vitro-controls and wild type plants. In the F ₂generation, which was obtained by self-pollination, segregation of the Hyp-lines in a 3:1 phenotypic ratio with regard to the traits frost tolerance and proline content was observed. Subsequent selection of extremes in the F ₂yielded homozygous plants in the F ₃generation. Lower LT ₅₀values and increased proline contents in those F ₃plants were significantly correlated. Furthermore, improved frost tolerance of one of the selected mutants was also observed in studies with whole plants in the F ₄generation. These results, which are similar to results of parallel studies on winter barley, provide strong evidence for the heritability of the traits 'increased frost tolerance' and 'increased proline content' obtained by this in vitro-selection procedure. The mutation seems to be due to a single incompletely dominant gene. A comparison of frost tolerance values from F ₃Hyp-progenies with those of seven standard varieties differing in frost tolerance indicates a considerable improvement of frost tolerance by the described in vitro-selection method.     

Improved frost tolerance and winter survival in winter barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by <Emphasis Type="Italic">in vitro</Emphasis> selection of proline overaccumulating lines

Embryogenic calli derived from anther cultures of the two-rowed winter barley cultivar Igri were plated on solid L3 medium containing the proline analogue hydroxyproline (Hyp), 10–20 mmol l^–1. Exposure to Hyp caused severe degeneration of most of the calli. Hyp resistant calli, distinguishable by their lighter colour and higher growth rate, and control calli not exposed to Hyp were plated on L3 regeneration medium. From 22,500 anthers exposed to Hyp 46 Hyp resistant regenerates were obtained, which were transferred to soil. After cultivation for 5–10 weeks at normal growth conditions they were cold hardened at 2 C under short day conditions together with control regenerates. Frost tolerance assays with segments of fully grown leaves of unhardened and cold hardened plants revealed that Hyp resistant regenerants were significantly more frost tolerant than the control regenerants. Improved frost tolerance was found also in the progenies R₁ to R₉, and genotypic segregation in the R₁ generation in a 1:2:1 ratio was indicated. Increased proline content was observed in the R₂ generation and in subsequent generations and was significantly (P 0.001) correlated with increased frost tolerance in the Hyp lines. Comparative studies of R₉ progenies from homozygous R₂ plants with the wild type Igri under field conditions in winter at three locations in Europe as well as crossing experiments confirmed the heritable improvement of frost tolerance and winter survival, respectively, in the Hyp lines. The results support the hypothesis that proline accumulation in cold acclimated winter barley plants is causally related to the acquisition of frost tolerance. Moreover, the described biotechnological procedure may be applicable in breeding programs for improved winter hardiness and possibly also for other stress tolerances.     

Chilling Tolerance, Cold Hardening and Freezing Tolerance of Low-temperature-tolerant Mutants of Dendranthema × grandiflorum (Ramat.) Kitamura

Two low-temperature-tolerant mutants of the Dendranthema × grandiflorum (Ramat.) Kitamura cultivar ‘Puck’ derived from an x-radiated tissue culture system were compared, as vegetatively growing plants, to ‘Puck’ ssfor several physiological properties under low-temperature conditions. So far, the low-temperature tolerance of the mutants has been defined as the capacity to flower earlier under low temperature conditions (8—15°C) the wild type. All three lines were not chilling sensitive in the vegetative state and were able to acclimatize and to develop freezing tolerance. Differences between ‘Puck’ and the mutants were found but could not be related to the low-temperature tolerance of the mutants in the generative phase. Levels of the amino acid proline increased during the hardening process, reaching the highest level in the cultivar ‘Puck’. No evidence could be obtained that proline is causally involved in the cold-hardening process. Freezing-induced membrane injury was found to decrease with cold hardening (0—28 days) but the two mutants did not show a greater capacity to acclimatize and cold harden than the original cultivar ‘Puck’.     

Abscisic Acid and Proline Levels in Cold Hardened Winter Wheat Leaves in Relation to Variety-Specific Differences in Freezing Resistance

Changes in dry weight, osmotic potential, abscisic acid and free proline contents were measured during cold hardening of nine winter wheat varieties differing in freezing resistance. During the first weeks of cold hardening dry weight and proline levels increased and the osmotic potential decreased parallel to the development of freezing resistance. Dry weight reached a broad maximum between the seventh and tenth week of hardening. ABA levels had a sharp maximum around the seventh week of hardening, when the dry weight increase began to cease. Maximal levels of proline were observed seven to ten weeks after the start of cold hardening. The mean and final dry weights, the mean, maximal and final proline contents, as well as the maximal ABA contents of the nine varieties correlated significantly with freezing resistance measured at the end of the hardening period.
The possible use of proline and of ABA as biochemical markers for freezing resistance in winter wheat breeding is discussed.     

Involvement of Chromosomes 5A and 5D in Cold-Induced Abscisic Acid Accumulation in and Frost Tolerance of Wheat Calli

The relationship between frost tolerance and abscisic acid (ABA) accumulation was studied in callus cultures of three wheat cultivars differing in the level of frost-tolerance, and of the 5A and 5D chromosome substitution lines from the frost-tolerant variety ‘Cheyenne’ into frost-sensitive ‘Chinese Spring’. Following cold hardening, the increase in ABA level in the calli of the two frost tolerant cultivars was significantly higher than in those of the frost-susceptible cultivar. Similarly, in 5A and 5D substitution lines, significantly higher ABA levels were detected than in the recipient ‘Chinese Spring’. One week-long ABA treatment at 26 °C induced a significantly higher level of frost tolerance than that achieved by cold hardening, irrespective of the frost sensitivity of the examined genotypes.     

Estimating winter survival of winter wheat by simulations of plant frost tolerance

Based on soil temperature, snow depth and the grown cultivar's maximum attainable level of frost tolerance (LT_50c), the FROSTOL model simulates development of frost tolerance (LT₅₀) and winter damage, thereby enabling risk calculations for winter wheat survival. To explore the accuracy of this model, four winter wheat cultivars were sown in a field experiment in Uppsala, Sweden in 2013 and 2014. The LT₅₀ was determined by tests of frost tolerance in November, and the cultivars’ LT_50c was estimated. Further, recorded winter survival from 20 winter wheat field variety trials in Sweden and Norway was collected from two winter seasons with substantial winter damages. FROSTOL simulations were run for selected cultivars at each location. According to percentage of winter damage, the cultivar survival was classified as “survived,” “intermediate” or “killed.” Mean correspondence between recorded and simulated class of winter survival was 75% and 37% for the locations in Sweden and Norway, respectively. Stress factors that were not accounted for in FROSTOL might explain the poorer accuracy at the Norwegian locations. The accuracy was poorest for cultivars with intermediate LT_50c levels. When low temperature was the main cause of damage, as at the Swedish locations, the model accuracy was satisfying.     

Studies for assessing the influence of hardening on cold tolerance of barley genotypes

Summary Frost tolerance of 30 barley (Hordeum vulgare L.) cultivars have been field evaluated in North Italy during the 1990/1991 winter season that was characterized by exceptionally low temperatures without snow cover. The results showed a significant correlation between cold injury and grain yield loss (r=0.61^**). Five cultivars chosen for their varying degree of frost tolerance were further evaluated using laboratory tests. Measurements of survival rate and membrane damage were used to assess the influence of hardening on frost resistance. The reliability of the tests is shown by the high correlation to the field data. For both the laboratory temperature regimes and field conditions, the tested cultivars showed the same order of classification. The effect of a rise in temperature at the end of the hardening treatment on frost tolerance is also reported. The laboratory tests here proposed can be integrated in a breeding programme for improving frost tolerance in barley.     

Use of chlorophyll fluorescence to evaluate the cold acclimation and freezing tolerance of winter and spring oats

The efficiency of the excitation capture by open Photosystem II (PSII) reaction centres was measured by the F_v/F_m ratios in a collection of winter and spring oats in order to assess the effects of hardening and freezing on the functionality of PSII and also the suitability of a chlorophyll fluorescence‐based method to screen oat cultivars for frost tolerance. A significant reversible decrease in F_v/F_m was found in all genotypes during acclimation to low, non‐freezing temperatures. F_v/F_m analysis appears to be an attractive test for the evaluation of frost tolerance in oats, being rapid, non‐invasive and capable of monitoring a trait related to a crucial stage in the acquisition of frost tolerance. It is more sensitive and precise than other standard methods and highly correlated with field‐evaluated frost damage. The measurements made during recovery 1 or 2 days after stress when the visual symptoms are not yet expressed, were especially advantageous because of the large variability in genotype response. The r‐values (close to 0.8) were reduced due to the non‐standard behaviour of the winter cultivar ‘Aintree’. The cold acclimation response of this genotype has been analysed in detail and the limits of artificial freezing tests are discussed.     

Overwintering of winter cereals in Hungary in the case of global warming

Summary Under phytotronic conditions investigations were made on the effect of important environmental factors, such as temperature, water and an increasing concentration of atmospheric CO₂, on the hardening of young cereal plants.In all the varieties derived from the major wheat growing regions of the world the hardening process was favourably influenced by a doubling of atmospheric CO₂ content, so that a significantly larger number of plants survived the frost test than for plants of the same variety raised under normal conditions.A reduction in freezing temperature and an increase in soil moisture content caused a slight reduction in survival % for varieties with excellent frost resistance and a great reduction for those with medium or poor frost resistance.Predictions suggest that in Central Europe, as the result of global climatic changes, there will be a reduction in the quantity of winter precipitation, a considerable rise in winter temperatures and an increase in atmospheric CO₂ concentration. Judging by the experimental results, these changes could improve the overwintering of winter cereals; at the same time, however, a number of factors (mainly the reduction of precipitation) leading to yield losses must be expected during the vegetation period.     

Plant age and in vitro or in vivo propagation considerably affect cold tolerance of Miscanthus × giganteus

The aims of the study were to determine why young Miscanthus × giganteus plants are more frost sensitive during the first winter than older plants, to compare cold tolerance of plants propagated in in vivo and in vitro conditions, and to select plants with higher cold tolerance. The study was performed in three experiments in which plants were prehardened at 12 °C for 2 weeks, hardened at 5 °C for 3 weeks and next chilled at 0 °C or ?3 °C for 3 or 14 days. Afterwards shoot regrowth from rhizomes was evaluated. In Experiment 1 frost tolerance of young plants obtained from a horticultural farm and plants that had already survived the first winter in the field was compared on the basis of LT₅₀ coefficient. In Experiment 2 frost tolerance of plants obtained in vivo and in vitro was compared. Experiment 3 was performed on four groups of plants: in vivo and in vitro obtained plants which were twice selected in cold, as well as in vivo and in vitro obtained plants which were cold treated once. Plants of all these groups were analysed with respect to their frost tolerance. They were prehardened, hardened and subjected to a temperature of 0 °C or ?3 °C for 14 days. The changes in processes accompanying cold acclimation occurring in the rhizomes or leaves of these plants were investigated. The content of abscisic acid, low-molecular antioxidants and phenolics, as well as catalase and non-specific peroxidase activities were analysed.Young commercially obtained plants were more frost sensitive than plants which had survived the first winter in the field. This effect could be caused by a small amount of storage compounds accumulated in finely divided rhizomes produced in a horticultural farm. Prehardening temperature of 12 °C caused more considerable changes in cold acclimation processes in Miscanthus rhizomes than hardening temperature of 5 °C. Plants propagated by in vitro culture were more cold tolerant but only in the first vegetative season compared to plants obtained in vivo. Plants chilled twice demonstrated a higher low-molecular antioxidant level, as well as a greater capability of phenolic accumulation compared to plants which were once cold stressed. Regardless of the recurrence of cold acclimation, ABA level was significantly increased in leaves by prehardening and in rhizomes by hardening. Each repetition of cold acclimation increased cold tolerance and shoot regeneration ability of M. × giganteus rhizomes.     

Breeding winter hardy grasses

Summary Perennial grasses are vital for Norwegian agricultural production. The nature and extent of winter damage on grasslands is highly dependent on climatic conditions, and determines both persistency and yield. Physical stresses such as frost and ice encasement predominate in coastal regions with an unstable winter climate, while biotic stresses such as low temperature fungi are more common in the inland regions. Development of hardening depends on plant adaptation and climatic conditions during autumn and winter. New winter-hardy cultivars should be bred for wide adaptation to winter stresses. The genetic background for the most important character, freezing tolerance, seems to be of polygenic nature with mainly additive gene action. Selection for increased freezing tolerance has been effective over generations in grasses, and in most grass species ample variation still exists to be exploited by breeding. However, in some species like perennial ryegrass, modern biotechnological methods should be used to improve freezing tolerance and winter hardiness.     

Relationship between Frost Tolerance and Cold-Induced Resistance of Spring Barley, Meadow Fescue and Winter Oilseed Rape to Fungal Pathogens

Plants exposed to one stress factor may become more tolerant to another. Cold is the most often documented factor inducing plant resistance to pathogens. The aim of this work was to investigate whether resistance of spring barley and meadow fescue to Bipolaris sorokiniana and resistance of winter oilseed rape to Phoma lingam induced at 5 °C for 2, 4 or 6 weeks are associated with frost tolerance, water potential and soluble carbohydrate content. Cold‐acclimated plants of each species showed increased resistance to the studied pathogens. Barley, fescue and rape plants demonstrated higher frost tolerance after hardening, but only in the case of fescue a correlation between resistance to frost and resistance to B. sorokiniana was found. A significant decrease in the water potential of leaf cells was observed in cold‐acclimated barley and fescue. In these two species, water potential greatly affected resistance to B. sorokiniana. However, only in barley did accumulation of fructose, glucose and sucrose correlate as well with changes in water potential as with cold‐induced resistance to the pathogen. In the case of hardened rape, no correlation between the studied parameters was found. The results obtained indicated that the temperature of 5 °C used during cold acclimation was not favourable for hardening of this plant species.     

Photosynthetic Acclimation to Cold as a Potential Physiological Marker of Winter Barley Freezing Tolerance Assessed under Variable Winter Environment

Winter‐hardiness is a complex trait limiting cultivation of winter barley (Hordeum vulgare ssp. vulgare) with respect to the regions of temperate climate. In the present studies, we verified whether inexpensive and fast physiological markers characterizing photosynthetic acclimation to cold may provide robust characteristics of winter barley genotypes for improved frost resistance. Freezing tolerance of 28 winter barley varieties and advanced breeding lines were tested for three winters in field‐laboratory experiment and under fully controlled conditions. To increase the environmental variability of freezing tolerance, a part of the plants were also de‐acclimated under semi‐controlled conditions and re‐acclimated in laboratory before freezing tests. After controlled cold acclimation, apparent quantum yield of photosystem II (F_v/F_m) as well as photochemical (q_P) and non‐photochemical (NPQ) coefficients of chlorophyll fluorescence quenching were studied. Field‐laboratory method assessment of freezing tolerance gives distinct and even opposite results in subsequent years. Also de‐acclimation interacted with growth conditions in the field, giving different rankings of genotypes each year. The results obtained suggest that high level of freezing tolerance measured in laboratory, which is connected with photosynthetic acclimation to cold may be not sufficient for the expression of field resistance, especially when winter conditions are not favourable for cold acclimation.     

Changes in abscisic acid, salicylic acid and phenylpropanoid concentrations during cold acclimation of androgenic forms of Festulolium (Festuca pratensis × Lolium multiflorum) in relation to resistance to pink snow mould (Microdochium nivale)

We investigated changes in concentrations of abscisic (ABA) and salicylic acid (SA), phenolic compounds and phenylalanine ammonia-lyase (PAL) activity in relation to cold-induced tolerance of four androgenic genotypes of Festulolium ( Festuca  ×  Lolium hybrids ) to frost and to the snow mould fungus Microdochium nivale . Cold acclimation increased frost tolerance and resistance to snow mould. Resistant genotypes were characterized by higher ABA concentrations during the first 54 h of cold acclimation and lower concentrations of SA than susceptible genotypes. After cold acclimation, the content of phenolics was significantly lower in genotypes tolerant to frost and M. nivale infection than in susceptible genotypes, while PAL activity was significantly higher. Signalling networks controlling cold acclimation to frost (abiotic) and mould infection (biotic) appears to involve increases in foliar concentrations of ABA and decreases in the SA level during successful cold acclimation. Higher PAL activity and lower concentrations of phenolic compounds also appear to be associated with enhanced tolerance to frost and fungal attack.     

Genetic variation for shoot elongation before winter and its correlation with vernalization requirement in winter oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Changing climatic conditions in north-western Europe are accompanied by occasional extreme weather conditions. This requires breeding of winter oilseed rape cultivars which are resilient to diverse abiotic stress factors, e.g. frost, drought and heat. The degree of vernalization requirement of winter oilseed rape has been found to be related to frost tolerance and winter hardiness. Shoot elongation before winter in particular has been identified as one decisive factor for frost tolerance in winter oilseed rape. However, the relationship between vernalization requirement and shoot elongation before winter is not known. In the present study the genetic variation for shoot elongation before winter and vernalization requirement of 19 genetic diverse breeding lines and cultivars were analyzed. Autumn and spring sown field experiments in multiple environments were performed to determine shoot elongation before winter and vernalization requirement, respectively. In spring sown field experiments, genotypes with a low vernalization requirement were characterized by the occurrence of long bolting plants with flower buds. Large and significant genotypic variation was found for shoot length in the autumn sown and spring sown environment. Broad sense heritability was quite high for shoot length in the spring sown environment (h²?=?97%), whereas it was only of medium size for shoot length before winter (h²?=?62%). Although the correlation between shoot length before winter and shoot length in the spring sown environment was positive (Spearman’s rank r_S?=?0.48*), a number of genotypes with reduced shoot elongation before winter and low vernalization requirement were identified. Results indicate that genotypes with a reduced shoot elongation before winter independent of their vernalization requirement can be selected in breeding programs.     

Variation in cold tolerance and spring growth among Italian white clover populations

Seven populations collected at different altitudes in northern Italy, two Ladino breeding populations and two control cultivars (AberHerald and Grasslands Huia) of white clover (Trifolium repens L.) were included in a series of experiments analysing: (i) levels of cold tolerance using artificial and field-based methods; (ii) relationships between these measures of cold tolerance; (iii) components of spring yield, various physiological traits, and their relationships with cold tolerance. Rates of seedling and growing point mortality in the populations over winter, assessed in separate field experiments, were closely related (r = 0.85). Grasslands Huia showed the highest death rates, and material originating from high altitudes the lowest. The LT₅₀ value, i.e., the temperature at which 50% of the growing points would die, estimated by an artificial freezing test, was significantly correlated with field-based measures of seedling (r = 0.64) and growing point (r = 0.84) mortality. The existence of these correlations is of potential interest for the development of indirect selection criteria for complex and expensive-to-evaluate traits such as winter survival in field plots. Besides being reliable, in this study the artificial assessment was also sensitive, providing a greater degree of separation of the populations means than field-based measures. Of the several physiological traits (water content,concentrations of water soluble and total non-structural carbohydrate, and water soluble protein content of stolons) measured at a mid-winter sampling date under field conditions, the only character showing significant variation between populations was soluble protein content. There was a slight trend for material with a higher protein content to exhibit greater field-based values of cold tolerance. High altitude populations tended to have low spring yields. The highest spring yield was found in one of the Ladino populations. The study identified two populations which combined, to differing extents, cold tolerance and spring yield characteristics that would be of potential use in breeding for specified agronomic/climatic zones. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chilling Tolerance of Maize Seedlings in the Field during Cold Periods in Spring is Related to Chilling-Induced Increase in Abscisic Acid Level

Recent studies on chilling tolerance mechanisms in maize revealed a significant positive correlation between genotypic chilling tolerance and chilling‐induced accumulation of the stress hormone abscisic acid (ABA) under controlled growth chamber conditions. Based on this and other results, the hypothesis was developed that chilling tolerance in maize is related to the ability to accumulate large amounts of ABA rapidly, as a protection against chilling injury. The objective of the present study was to test this hypothesis under field conditions during natural cold weather periods in spring, which often cause severe chilling injury in maize fields. In two experiments at two locations in Europe with contrasting climates, eight maize genotypes with different genotypic chilling tolerance were cultivated in spring in the field according to agronomic practice for maize. Before and at the end of cold weather periods, ABA levels and water contents were determined in the third leaves. It was found that chilling‐tolerant genotypes accumulated higher amounts of ABA during the chilling period than chilling‐sensitive genotypes. A significant positive correlation between chilling tolerance and the levels of ABA in the leaves was found. These results support the above‐mentioned hypothesis. In contrast to earlier studies performed under growth chamber conditions, the water content in chilled leaves was mostly higher than in non‐chilled leaves. The increase in ABA is therefore not attributable to chilling‐induced water deficit, but probably to the low temperature itself.     

Physiological Effects of Winter Rape (Brassica napus var. oleifera) Prehardening to Frost. I. Frost Resistance and Photosynthesis during Cold Acclimation

The objective of the research was to define the changes in photosynthetic activity induced by prehardening and to determine their involvement in frost tolerance of winter rape.
Prehardening of winter rape, consisting of keeping the plants at + 12°C during the light periods from sprouting until the beginning of the 1st stage of cold hardening, contributed to increasing its effectiveness. After 42 days of hardening at + 2°C the resistance of the prehardened plants equalled that attained by winter rape in the most favourable seasons of vegetation in the field. Prehardening stimulated the efficiency of photosynthesis at chill temperatures (+ 2–5°C). Differences in photosynthetic efficiency, like those in frost resistance, increase with successive weeks of hardening. They also concern the leaves already developed at the hardening temperature. A prehardened photosynthetic apparatus is less susceptible to the progress of photoinactivation taking place when the seedlings are kept at + 2°C. It also demonstrated greater activity even during the first hour of hardening or in the newly expanding leaves, and also at higher temperatures, most probably because of the more efficient progress of the dark, processes. The described changes in the functioning of the photosynthetic apparatus induced by prehardening were thus qualitatively very similar to those observed during long-term growth at + 5°C. already described in the literature.     

Production of mutants with high cold tolerance in spring canola (<Emphasis Type="Italic">Brassica napus</Emphasis>)

A major factor affecting spring canola (Brassica napus) production in Canada is killing frosts during seedling development in the spring and seed maturation in the fall. The objective of this study was to explore the possibility of producing spring canola lines with mutations that have altered biochemical pathways that increase cold tolerance. The approach was to generate UV point mutations in cultured microspores followed by chemical in vitro selection of individual mutant microspores or embryos resulting in measurable alterations to various biochemical pathways with elevated levels of key defense signaling molecules such as, salicylic acid (SA), p-Fluoro-d,l-Phenyl Alanine (FPA), and jasmonic acid (JA). In addition, since proline (Pro) is known to protect plant tissues in the cold-induced osmotic stress pathway, mutants that overproduce Pro were selected in vitro by using three Pro analogues: hydroxyproline (HP), azetidine-2-carboxylate (A2C); and, 3,4-dehydro-d,l-proline (DP). Of the 329 in vitro selected mutant embryos produced, 74 were identified with significant cold tolerance compared to their donor parents through indoor freezer tests at −6°C, and 19 had better winter field survival than winter canola checks. All chemically selected mutant doubled haploids with increased cold tolerance compared well with parent lines for all seed quality and agronomic parameters. Development of increased frost tolerant cultivars should allow for spring canola to be produced in western Canada without compromising seed quality.     

Cold acclimation of winter and spring peas: carbon partitioning as affected by light intensity

Like most plants, pea (Pisum sativum L.) becomes tolerant to frost if it is first exposed to low non-freezing temperatures, a process known as cold acclimation. Cold acclimation is a complex process involving many physiological and metabolic changes. Two spring dry peas, two winter dry peas and one winter forage line were exposed to cold temperature in a controlled environment in two experiments, one using low light intensity and the other regular light intensity. Plants were harvested throughout the experiment and dry matter accumulation, water content, soluble and insoluble sugar concentrations were determined from shoot and root samples. Cold acclimation did not occur when temperatures were low if light intensity was low, even in winter peas. In contrast, with regular light intensity, the winter peas acquired more freezing tolerance than spring peas and a close relationship was found between the soluble sugar concentration of leaves just before the frost and the degree of freezing tolerance obtained by the different genotypes. Relationships between freezing tolerance and carbon partitioning between shoot and roots are discussed.