Relationship between Prehardening, Photosynthetic Activity at Cold Acclimation Temperatures and Frost Tolerance in Winter Rape (Brassica napus var. oleifera). The Consequences for the Reliability of Frost Resistance Estimation under Controlled Conditions

The effect of prehardening (early stage of growth at moderate low temperatures (+12°C) during the day) on the reliability of frost resistance estimation in a controlled environment has been studied on three winter oilseed rape cultivars differing markedly in their field survival rate (Leo, Górczański and Idol). It has been also examined the relationship between the photosynthetic activity during the first stage of cold acclimation and the level of frost resistance observed in investigated cultivars. Presented results demonstrated that prehardening, which increases to a significant degree the effectiveness of the cold acclimation process, also increases the differences in cold hardiness between cultivars, and limits the magnitude of experimental errors made during frost resistance estimation under controlled conditions. In all studied cultivars, prehardening increases significantly the photosynthetic activity during cold acclimation. On the other hand, both in the prehardened and in the non-prehardened plants, no relation has been found between either gas exchange rates or chlorophyll 'a' fluorescence characteristics at low temperatures and cultivar ability for acclimation. Neither the existing differences in photosynthetic activity, nor the degree of photosynthetic apparatus acclimation to cold, which occurs during prehardening, are the factors responsible for the frost resistance variation observed between studied cultivars.     

Physiological Effects of Winter Rape (Brassica napus var. oleifera) Prehardening to Frost. II. Growth, Energy Partitioning and Water Status during Cold Acclimation

Prehardening of winter rape, i.e. its early growth in a reduced day temperature (+12°C) from emergence to the beginning of cold acclimation at chilling temperatures (the 1^st stage of cold acclimation), has a beneficial influence on frost resistance. In earlier studies it has been demonstrated that during prehardening, plants formed leaf rosettes and increased the photosynthetic efficiency at chilling temperatures. In the present study investigations were carried out on the effect of prehardening on the growth rate of the plants during the 1^st stage of cold acclimation, and the progress of selected physiological processes occurring during this stage and resulting in increased frost resistance. It has been demonstrated that a greater inflow of photoassimilates during the 1^st stage of cold acclimation results in greater increments of the dry mass of prehardened plants. These plants show also a distinct inhibition both of the elongation growth and the rate of expanding new leaves. The acquired energy is thus spent to a greater extent on the processes associated with increasing frost resistance, and not on growth. In prehardened seedlings, during the 1^st stage of cold acclimation, there have been observed in leaves a higher rate of both water content decrease and drop in the osmotic potential of the cell sap and water potential in the tissues. Also an increased accumulation of soluble sugars and free proline was noticed. However, the beginning of these processes was not observed during the prehardening period. Prehardening stimulated the effectiveness of the 1^st stage indirectly through changes leading to the increased amount of available energy and enabling the utilisation of the greater part of acquired energy in the cold acclimation process.     

Vegetative Performance of Tropical Highland Maize (Zea mays L.) in the Field

An attempt has been made to work out a simple and reliable method of fast frost resistance evaluation of winter oilseed rape using in vitro cultures. In winter rape cv. Górczański, there was investigated cold acclimation ability of hypocotyle sections from 5-days old seedlings and also of callus tissue formed on these sections after subsequent 4 weeks growth on induction medium. It has been found that hypocotyle sections are unable to cold-acclimate. Winter rape calli acclimated well and optimum conditions for acclimation is fortnight's growth at +2°C. Exposure to light during hardening was not necessary for acquiring maximum resistance. On five winter rape cultivars freezing tests were performed using the best cold acclimation conditions. The differences in resistance between hypocotyle sections did not match the differences in field survival or frost resistance of whole plants. As distinct from hypocotyle sections callus tissue appeared to be suitable for evaluation of frost resistance. However, to ensure the objectiveness of assessment in this method is not so easy. The testing temperature must be chosen carefully, because the results can be reliable only at sufficiently low temperature. For correct estimation of the frost resistance level, it is possible to use both the decrease of triphenyltetrazolium chloride reduction rate during freezing and the increase of callus dry weight during 14 days after freezing.
(Abbreviations: BAP — 6-Benzylaminopurine; 2,4-D — 2,4-dichlorophenoxyacetic acid; LT50 — temperature at which 50 % of the plants (or plant material) has been frost killed; MS(0.5;2) — Moorashige and Skoog Basic Medium with addition of 0.5 mg 2.4-D and 2 mg BAP; PPFD — photosynthetic photon flux density; TTC — 2,3,5 triphenyltetrazolium chloride)     

Comparison of Vernalization Requirements and Frost Resistance of Winter Rape Lines Derived from Double Haploids

Vernalization requirements and cold resistance of 13 lines of winter rape derived from doubled haploids obtained by androgenesis were investigated. The degree of vernalization requirements was examined in two parallel experiments. In the first experiment seedlings aged 2 weeks were vernalized during 63 and 35 days under controlled conditions, at 5 °C. In the second experiment the natural conditions of vernalization were differentiated by sowing the plants at four different dates in spring: March 15th, April 15th, May 15th, and at the latest date excluding vernalization, June 5th. The lines examined revealed differentiation of vernalization requirements that were similar in both experiments; all the lines required a period of exposure to cold before flowering. Cold resistance of lines was estimated in two successive series at −15 °C and −17 °C. Before testing the plants were kept in natural field conditions from the beginning of September until the middle of November. Testing of cold resistance was preceded by hardening under controlled conditions. Considerable differences in cold resistance was found in the forms investigated.
The results obtained do not show any relation between frost resistance and the degree of vernalization requirements, as the line characterized by the strongest winter habit of growth showed low cold resistance, whereas the lines which were closer to spring growth habit showed the highest level of cold resistance; the lines with the least vernalization requirements revealed the highest level of cold resistance. This conclusion can be confirmed by the significance of the correlation coefficients between the various indices defining the frost resistance and the indices of vernalization requirements.     

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.     

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.     

Effect of Cold Hardening on Growth and Essential Oil Content of Mentha piperita L.

Divisions of running rhizomes of peppermint plants were planted in loamy clay soil in the experimental farm of National Research Centre, Dokki, Cairo, Egypt. After 21 days, the plants were exposed to low temperature 4, 7°C for 6, 9 and 12 days in controlled chamber before transplanting to large pots in winter season. Three cuttings were taken from the plants of different treatments during the season after 4, 7 and 10 months from transplanting. Representative samples of 6 replicates from each treatment were taken for the determination of vegetative growth and oil content in the leaves and branches.
The results obtained indicated that the highest fresh and dry weight of leaves and branches was recorded in the second cutting, however, it markedly decreased in the third one. The highest favourable effect of cold hardening on vegetative growth was observed at 4°C for 12 days exposure in the second cutting. The percentage of volatile oil in the leaves recorded the highest values in treatment of 7°C for 6 days in the second sampling date. Cold hardening either at 4°C or 7°C resulted in pronounced increase in the essential oil yield produced by the plant with the superiority of 4°C for 12 days which recorded the highest value.
It can be concluded from the obtained results that hardening peppermint plants with low temperature, particularly 4°C for 12 days before transplanting improved plant growth and considerably increased the volatile oil content in plant leaves and branches.     

Photosynthetic Activity and Soluble Carbohydrate Content Induced by the Cold Acclimation Affect Frost Tolerance and Resistance to Microdochium nivale of Androgenic Festulolium Genotypes

The aim of the experiment was to verify whether changes in photosynthetic activity measured by means of chlorophyll fluorescence techniques and soluble carbohydrate level induced during pre-hardening and cold acclimation in androgenic forms of Festulolium are related to their winter hardiness and pathogen resistance. The study was performed on six androgenic genotypes of Festulolium, which differ in their degrees of winter hardiness. The chlorophyll fluorescence parameters and sugar content were measured in the control plants, pre-hardened at 12 °C and cold-acclimated at 2 °C. Cold acclimation at 2 °C, preceded by pre-hardening at 12 °C, induced a decline in maximum PSII quantum efficiency ( F _v/ F _m) and in non-photochemical quenching (NPQ). Exposure to 2 °C also resulted in reduced efficiency of energy conversion in photochemical processes ( q _p). The soluble carbohydrate amount increased in the susceptible genotypes and was associated with a decline in NPQ and current quantum yield of PSII (φ_PSII). In resistant plants, a decrease in the sugar content was observed, which could be explained by their consumption in metabolic processes initiated during cold acclimation. The changes in sugar content were associated with a decline in NPQ and q _p.     

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.     

Winter Hardiness, Frost Resistance and Vernalization Requirement of European Winter Oilseed Rape (Brassica napus var. oleifera) Cultivars within the Last 20 Years

Cultivars of European winter oilseed rape cultivated in the second half of the 1970s and in the mid-1990s were screened for their winter hardiness, frost resistance and vernalization requirement. A strong correlation between winter hardiness and frost resistance in both groups of rape has been noticed. Among oilseed rapes cultivated in the late 1970s, low erucic acid and particularly double zero cultivars were less winter hard than high erucic acid cultivars. Double zero cultivars were characterized by lower frost resistance and lower vernalization requirement. A significant correlation between vernalization requirement and both frost resistance and field survival has also been shown. Frost resistance of the 1990s (double zero) cultivars was higher than that of double low cultivars from the late 1970s. Their vernalization requirement was still small and did not correlate with either frost resistance or winter hardiness. It was concluded that reduction in the content of glucosinolates in the 1970s involved decrease in winter hardiness and vernalization requirement of cultivars. During the following 20 years winter hardiness of double low cultivars has been improved, but vernalization requirements have not changed. As a result no correlation between winter hardiness and vernalization requirement in contemporary canola cultivars has been observed.     

The freezing characteristics of wheat at ear emergence

Wheat is occasionally exposed to freezing temperatures during ear emergence and can suffer severe frost damage. Few studies have attempted to understand the characteristics of freezing and frost damage to wheat during late development stages.

It was clearly shown that wheat appears to have an inherent frost resistance to temperatures down to −5 °C but is extensively damaged below this temperature. Acclimation, whilst increasing the frost resistance of winter wheat in a vegetative state was incapable of increasing frost resistance of plants at ear emergence. It is proposed that the ability to upregulate frost resistance is lost once vernalisation requirement is fulfilled.

Culms and ears of wheat were able to escape frost damage at temperatures below −5 °C by supercooling even to as low as −15 °C and evidence collected by infrared thermography suggested that individual culms on a plant froze as independent units during freezing with little or no cross ice-nucleation strategies to protect wheat from frost damage in the field appear to revolve around avoiding ice nucleation.     

Frost Resistance of Somaclones Derived from Triticum aestivum L. Winter Wheat Calli

Frost resistance was studied in SC₄ seedlings generated by self pollination from 31 (SC₄) plants of ‘GK Csongor’ winter wheat variety derived from resistance than ‘GK Csongor’. With respect to percentage survival, one family possessed significantly higher frost resistance as compared to the control at a temperature of -13°C In the case of regrowth analysis, 22 of the 31 families showed less growing capacity and 5 proved to be significantly better than ‘GK Csongor’. According to both testing methods, one family showed significantly higher frost resistance than the control.     

A Genetic Study of Frost Resistance in Wheat Callus Culture

The genetic control of frost resistance was studied in callus cultures using some of the chromosome substitution lines of the variety ‘Cheyenne’ into ‘Chinese Spring’. The survival of the calli derived from immature embryos was studied with triphenyltet-razolium chloride (TTC) and fluorescein diacetate (5DA) methods after hardening and freezing at a temperatures of -7 °C, -9 °C, -11 °C, -13 °C, and –15 °C. The donor ‘Cheyenne’ and the substitution lines 5A and 5D proved to be more frost resistant than the recipient ‘Chinese Spring1. These results are well correlated with the previously published studies when seedlings were tested under controlled conditions. Based on these results the tissue culture technique seems to be useful for testing varieties and lines for different levels of frost resistance and even for mutant selection.     

Assessment of Sensitivity to Frost in Ochrus chickling (Lathyrus ochrus (L.) D.C.) by Chlorophyll Fluorescence Analyses

Ochrus chickling ( Lathyrus ochrus (L.) D.C.) is a promising feed legume for rain-fed farming systems in the Mediterranean environments of West Asia and North Africa (WANA) because of its drought tolerance and resistance to broom rape ( Orobanche crenata Forsk.). However, it is highly susceptible to cold and improving Its resistance would further improve its adaptation.
The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in seedlings of five Lathyrus ochrus genotypes and one each of Vicia ervilia (L.) Wild and V. sativa L. was assessed by measuring the rate of rise in induced or variable chlorophyll fluorescence of fully developed leaflets after cold hardening in the open and subsequent exposure to freezing. The method differentiated the highly frost-susceptible L. ochrus genotypes from the more tolerant Vicia species. Testing the technique with nine genotypes of L. ochrus in which the plants were established, cold-hardened and frost-stressed under controlled conditions confirmed its sensitivity even to reveal the narrow inter plant differences that generally exist within a cultivar or genotype to frost. It is therefore considered as a useful screening technique for cold/frost tolerance in L. ochrus.     

冬小麦品种抗霜冻力的影响因素分析

应用移动式人工霜箱,研究了冬小麦幼穗抗霜冻力随发育期的变化规律。结果表明,随发育期进程,幼穗抗霜冻力呈下降趋势,从药隔前期开始进入低温敏感期。利用室内人工霜箱,在药隔前期对21个品种进行了抗霜冻力鉴定,进而分析了不同品种、冬春性、越冬抗冻力以及叶片可溶性糖含量等对冬小麦抗霜冻力的影响。结果表明,除少数品种有一定抗霜冻力外,多数品种抗霜冻力无显著差异。品种抗霜冻力与其冬春性和越冬抗冻力均无显著相关性,与叶片可溶性糖含量之间亦无显著相关性。     

白菜型冬油菜叶片结构和光合特性对冬前低温的响应

为明确白菜型冬油菜在冬前低温下叶片结构特征、光合作用特性及其抗寒性,本研究在0℃和–7.6℃自然低温条件下,选用白菜型冬油菜品种陇油7号(超强抗寒)和天油4号(弱抗寒),测定并比较其叶片气孔性状、解剖结构和光合、荧光参数的日变化等指标。结果表明,随着冬前温度下降,2个白菜型冬油菜叶片气孔密度、气孔面积、气孔周长、栅栏组织和海绵组织厚度均变小,细胞间隙变大,叶片变薄;P_n日变化呈"单峰"型曲线,无光合"午休"现象;叶片的P_n、G_s、T_r和CE均降低,而C_i均升高,说明是非气孔限制引起P_n降低。白菜型冬油菜在冬前低温条件下发生了光抑制现象,表现为F_m和F_v/F_m下降,F_o上升。与超强抗寒品种陇油7号相比,弱抗寒品种天油4号叶片气孔密度和气孔面积均较大,气孔总周长较长,叶片较厚,P_n、F_m和F_v/F_m均较高,说明冬前低温条件下,天油4号光合能力较强,光抑制程度较弱。白菜型冬油菜在冬前低温条件下的叶片气孔密度越大、气孔面积越大、气孔周长越长、叶片及栅栏组织和海绵组织越厚,光合能力越强,地上部生长越旺盛,品种抗寒性越差。本研究为冬油菜抗寒种质创新和育种提供了部分理论支撑。     

Induction of Generative Development of Winter Rape (Brassica napus L. var. oleifera) in Relation to Vernalization Conditions and Age of Vernalized Plants

Germinating seeds and young winter rape plants were vernalized 56–63 days at 5 or 2°C under nine-hour days or in darkness. The highest percentage of generative plants and the most rapid flowering were obtained following the vernalization of young seedlings and germinating seed under conditions of nine-hour day, at 5°C. The least effective induction of generative development followed the vernalization at 2°C in continuous darkness. The vegetation period from the end of vernalization till the beginning of flowering was the shortest when four-week-old plants were vernalized under conditions of nine-hour day, yet the vegetation period from the beginning of germination to flowering was the shortest when seed germinating under conditions of nine-hour day were vernalized. The period was extending as older plants were being vernalized. Data indicating that the optimal temperature for vernalization of older plants is higher than for germinating seed and young seedlings were obtained.     

Frost resistance of 'Chinese Spring'/'Cheyenne' chromosome substitution lines under short- and long-day hardening conditions

Investigations were carried out under phytotronic conditions to study the effect of daylength on the frost resistance of a ‘Chinese Spring’/‘Cheyenne’ chromosome substitution series. The frost resistance of the 5A, 7A, 4B, 5B, 1D and 5D lines was significantly better when raised and hardened with long-day (16 h) rather than short-day (8h) illumination. The frost resistance-increasing effect of daylength could be demonstrated after freezing lines 5A and 5D at both - 10°C and -12°C. An increase in the duration of illumination in the course of preliminary growth and hardening promoted the development of a higher level of frost resistance. This positive effect was most pronounced for chromosome substitution lines, where the frost resistance was significantly better than that of ‘Chinese Spring’ even with normal daylengths.     

A Rapid Method for Measuring Freezing Resistance in Crop Plants

The objective of this study was to develop a technique based on chlorophyll fluorescence to assess freezing injury and resistance of leaves. Optimization was done with faba bean leaves and applicability to other crops was examined at winter and spring with types of barley, oats, rape and faba beans. Selected leaves from young hardened beans were subjected to standardized freezing tests with different minimum temperatures ( T _min) and fluorescence was monitored. After a dark period basic fluorescence ( F _O was induced by 0.2 μmol m⁻² s⁻¹ pulsed red light and maximum fluorescence ( F _m) was assayed at different light intensities. 1500 μmol m⁻² s⁻¹ rendered to give the maximum possible output of Fm and best differentiation of differently damaged leaves by F _n= F _m - F _O. Leaf temperature during measurement and during a short storage (± 2 h) should be kept at about 0°C to avoid biases between differently damaged leaves. The measuring spot on the leaf must be standardized since fluorescence response differed at the tip and base of a leaflet, but not between the two leaflets of a faba bean leaf. The applicability of F _rr (ratio of F _r of stressed to unstressed leaves) as a measure of resistance was demonstrated by comparison of winter hardiness of cultivars with freezing resistance calculated from the relationship of F _vr and the T _min used in freezing tests.     

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.