Marker-assisted characterization of frost tolerance in barley (Hordeum vulgare L.)

Five molecular markers associated to two frost tolerance QTLs ( Fr-H1 and Fr-H2 ) were tested both on nine Turkish accessions, classified by breeders as highly frost-tolerant, and on a previously described sample of 26 barleys, winter, facultative and spring. Accessions were characterized in terms of frost tolerance under both field conditions and artificial freezing test at −12°C. The Turkish lines resulted to be equal or superior to the most tolerant European genotypes tested, showing that they can be used to improve the frost tolerance of the EU barley germplasm. The marker Hv BM5A ( Vrn-H1 and Fr-H1 ) resulted to be the best predictor for assisted selection within this germplasm, because of its high correlation between allelic variation and phenotypic traits. Only Hv CBF4 of the three Hv CBF markers tested at Fr-H2 was associated to the trait, but at lower significance than HvBM5A . The PCR-based molecular marker of Vrn-H1 can thus be used in barley breeding not only for selection of facultative and winter types, but also for fast routine selection of frost tolerant genotypes.     

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 Biomass Yield of Field Grown Maize Genotypes and Seedling Traits under Controlled Conditions

Investigations were done on six maize inbred lines of different origin and their diallel set of crosses. Seedlings grown at 14°, 22°, 30° and 38°C until three leaves stage were studied for morphological, anatomical and photosynthetic traits of leaves. These traits were screened for correlation with shoot dry weight at harvest under field conditions i) in 1981 and 1982 in North Germany, cool climate (CS); ii) 1981 in Thailand (WS). The correlation between seedling traits under controlled conditions and shoot dry weight under field conditions markedly changed after anthesis. Some few seedling traits closely correlated with biomass yield, favourably at 14° and 38°C. They mostly belonged to the morphology and anatomy of the leaves (area first leaf at 38°C, r = 0.6—0.7, all locations; depth of stomata below the epidermal surface at 38°C, r =—0.5 at CS and —0.7 at WS). In spite of significant correlations between single seedling traits and biomass yield a relyable prediction of biomass yield should best be based on a carefully chosen set of seedling traits.     

Relationship Between Early Field Growth of Maize Genotypes and Seedling Traits under Controlled Conditions

Investigations were done on six maize inbred lines of different origin and their diallel set of crosses. Seedlings grown at 14°, 22°, 30° and 38°C until three leaves stage were studied for morphological, anatomical and photosynthetic traits of leaves. These traits were screened for correlation with shoot dry weight at the six leaves stage under field conditions i) in 1981 and 1982 in North Germany, cool climate (CS); ii) 1981 in Thailand (WS). Some few seedling traits correlated well enough, dependent on temperature and location. CS shoot weights were closely correlated with dry weight of third leaves (14°) or NADP malate dehydrogenase activity (30°) (r = 0.66—0.85), whereas WS shoot weight correlated best with the depth of stomata below the epidermal surface (30°C) (r = -0.77). This shows that it is possible to predict early growth performance in the field within a group of genotypes adapted to different latitudes.     

Screening for frost tolerance in wheat using the expression of dehydrine genes Wcs120 and Wdhn13 at 17°C

Cold induces expression of Cor/Lea genes, in particular of Wcs120 and Wdhn13 . The level of the expression at low temperatures was related to the frost tolerance (FT) of wheat cultivars. We studied the expression in five cultivars very different in the FT, at relatively high temperature of 17 and 25°C, respectively. Significant correlations at the 1% level were found at 17°C between the expression level and FT, characterized by correlation coefficients 0.92** and 0.97** for Wcs120 and Wdhn13 , respectively. These preliminary results could be used for screening of frost tolerant lines.     

苗期低温胁迫对扬麦16叶片抗氧化酶和渗透调节物质的影响

以扬麦16为试验材料, 利用人工气候箱模拟低温逆境, 研究-4℃、-6℃和-8℃低温胁迫对小麦苗期生长和叶片生理特性的影响。结果表明, 随着处理温度的降低、胁迫时间的延长, 小麦植株由叶尖萎蔫变黄的2、3级冻害加重至叶片全枯的4级冻害, 甚至大部分茎蘖冻死的5级冻害, –4℃和–6℃处理24 h没有发生4级及以上冻害。–8℃处理24 h和48 h后植株5级冻害比例显著增加, 植株死亡率分别为16.7%和39.6%。低温胁迫初期小麦叶片中SOD、POD和CAT活性均呈上升趋势, 随胁迫程度加重, SOD最先表现出下降趋势, POD和CAT变化稍缓, MDA含量增加。苗期低温胁迫使叶片中游离脯氨酸、可溶性蛋白和可溶性糖含量明显高于对照, 且随温度的降低和处理时间的延长呈上升趋势, 表现出对低温逆境的适应性。     

A selection tool for freezing tolerance in common wheat using the fast chlorophyll a fluorescence transient

The analysis of fast chlorophyll fluorescence induction kinetics in dark-adapted leaves (JIP-test) is proposed as a rapid method for evaluating freezing tolerance in wheat. Wheat leaves were detached from the plants of 40 cultivars grown in an open-field in Smolice, during December and March of winters 2004/2005 and 2005/2006, transferred to laboratory in Krakow and frozen in −15°C. Next, measurements of chlorophyll fluorescence were taken. The correlation coefficients between the JIP-test parameters with freezing tolerance data obtained in multiple field-laboratory studies, varied between 0.58 and 0.70 depending on sampling time and parameter measured. The chlorophyll fluorescence parameter, which can always be used for the screening of freezing tolerance in the method described here, is the performance index (PI), which characterizes the overall energy flow efficiency in photosystem II (PSII). The results indicate, that the technique proposed here can be useful for freezing tolerance screening in plant breeding programmes. However, this method seems to be a more reliable tool in the selection of freezing tolerant germplasm, than for discarding freezing susceptible materials.     

Leaf Anatomy of Maize Inbred Lines and Crosses at Extreme Temperatures for Active Growth

Inbred lines and crosses from cool temperate regions (C) and from warm regions (W) were grown at 14, 22, 30 and 38°C up to the same physiological age, the full expansion of the third leaf. The laminae of the second leaf were studied for anatomical traits. For all traits the genotypic variability was high over the whole temperature range, but the rank order was influenced by temperature. Leaf thickness and cross sectional area of epidermal cells were smallest at 22°C. Stomatal frequency increased in inbred lines from 14 to 38°C and in crosses from 30 to 38°C. In crosses stomatal length was low at 22°C and high at 38°C while inbred lines either did not react to temperature at all (C) or had low to very low values over the whole temperature range (W). Cross sectional area of chlorenchyma cells (mesophyll, bundle sheath) was high at 14°C. For mesophyll cells of inbred lines and generally for bundle sheath cells a second maximum occurred at 30°C. Cross sectional area of both chloroplast types was high at 14°C and low at 22 or 30°C. Positive heterosis was seldom observed but negative heterosis occurred for chlorenchyma cell size at 14°C. This might indicate an improved fitness for photosynthesis at suboptimal temperature because of small cells, especially as W-genotypes generally had large cells at this temperature. A negative heterosis for the stomatal frequency over the whole temperature range might stand for a good fitness of hybrids for photosynthesis, too.
The investigations have shown that the expression of some important anatomical traits is dependent on temperature as well as on origin and genetic structure of maize genotypes.     

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.     

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.     

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.     

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.     

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)     

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.     

Chlorophyll Fluorescence‐Based Studies of Frost Damage and the Tolerance for Cold‐Induced Photoinhibition in Freezing Tolerance Analysis of Triticale (×Triticosecale Wittmack)

Freezing tolerance of 60 breeding lines of winter hexaploid triticale (×Triticosecale Wittmack) was studied in the field‐laboratory experiment. The experiment was repeated over three winters. The survey was also carried out in plants grown and cold‐acclimated in the laboratory. In both the experiments, plant survival analysis and chlorophyll fluorescence‐based studies on energy flows in photosystem II (PSII) (JIP‐test) after freezing of detached leaves were performed. In the laboratory experiment, the temperature of 50 % electrolyte leakage from leaves and the resistance against cold‐induced photoinhibition were additionally investigated. In the case of plants’ cold‐acclimated in the field, determination of the freezing tolerance of PSII gave similar results as the determination of freezing tolerance of whole plants. Both traits were strongly affected by genotype–environmental interactions, but these effects were less visible for PSII characteristics. In the laboratory experiments, a strong correlation between freezing tolerance and the tolerance for cold‐induced photoinhibition of photosynthesis was observed. The possibility of the use of chlorophyll fluorescence‐based techniques for the determination of freezing tolerance in triticale is discussed.     

PEG模拟干旱胁迫下玉米胚芽鞘长与其抗旱性的关系分析

以6份抗旱性不同的玉米自交系为试材,用不同浓度的PEG-6000(polyethylene glycol,PEG)渗透溶液进行模拟干旱胁迫处理,分析玉米萌芽期中胚轴长、胚芽鞘长及苗长的变化,并将其与田间干旱胁迫处理下相应玉米自交系产量及产量相关性状的抗旱指数进行相关分析,以期揭示萌芽期玉米幼苗相关性状与其抗旱性的关系。结果表明:(1)萌芽期随着PEG浓度增加,玉米自交系胚芽鞘长、中胚轴长及苗长都呈下降趋势,其中苗长降低幅度最大,平均降幅为14%,中胚轴长次之,平均降幅为11%;(2)方差分析表明,不同玉米自交系幼苗的中胚轴长、胚芽鞘长在自交系间及PEG浓度间差异极显著,苗长在PEG浓度间差异极显著;(3)花期田间干旱胁迫下,各玉米自交系的千粒重、穗粒数及产量较正常灌溉明显下降,且其平均降幅分别为8%、14%、13%;(4)通过玉米田间产量及产量相关性状抗旱指数与萌芽期各性状相关性分析表明,中胚轴长与千粒重、穗粒数及产量的抗旱指数呈显著或极显著正相关。胚芽鞘长与千粒重、穗粒数及产量的抗旱指数呈极显著正相关,而在5%的PEG-6000渗透溶液模拟干旱胁迫下,胚芽鞘长与千粒重抗旱指数相关系数最高为0.832。因此,胚芽鞘较长的自交系其抗旱性较强,胚芽鞘长度可作为玉米早期抗旱性筛选的重要指标。     

Plant damage after freezing, and the frost resistance of varieties from the facultative and winter wheat observation nurseries

In the first experiments, studies were made on the survival % of fourwinter wheat varieties with good frost resistance and two with poor frostresistance, and on the degree of plant damage after freezing at –14 ^°C and –16 ^°C under phytotron conditions. In the secondexperiment the frost resistance of the varieties included in the 9^thFacultative and Winter Wheat Observation Nurseries (FAWWON) wasdetermined after freezing in the phytotron at – 15 ^°C. The scoresgave a good reflection of the variety ranking determined on the basis ofsurvival % and of the different effects of the two freezing temperatures. Onthe basis of the scores, the plants were divided into two groups for eachtreatment and each variety: plants which suffered frost damage (a score of2–3), and plants which suffered no frost damage (a score of 4–5).Twenty plants from each group were raised to maturity in pots. Theyield parameters of plants damaged by freezing at – 14 ^°C werereduced to a lesser extent than those frozen at –16 ^°C. Of the eightparameters tested, the reduction in the number of ears per plant andconsequently in the number of grains in the side ears, the grain mass andthe total grain yield, gave the clearest indication of the extent of plantdamage. The results of the second experiment indicated that in some casesthe frost resistance of the varieties could be predicted from the wheatproduction zone (e.g. varieties from Eastern Europe and Nebraska haveexcellent frost resistance), while in other zones winter hardiness and frostresistance depended rather on the country or on the breeding location.     

Effects of Temperature and Frost on Genotypes of Medicago truncatuh L. and Medicago aculeata L. from Contrasting Climatic Origins

The growth of M. truncatula and M. aculeata genotypes collected from sites of contrasting altitudes and winter temperatures was compared under different temperature regimes. Genotypes collected from mild winter environments produced more shoot dry matter, had higher leaf area and lower specific leaf weight than those from cold environments; however these effects were largely related to seed size for M. aculeata. Only genotypes from mild environments were responsive to temperature.
Frost tolerance of the genotypes was tested using a laboratory freezing test based on seedling survival. There was a relationship between frost tolerance and winter temperature at site of collection for M. aculeata , with the most frost tolerant genotypes coming from high altitudes. All genotypes of M. truncatula demonstrated low survival rates following frost damage. Genotypes from high altitudes represent a promising source for breeding for first tolerance with greater variation m M. aculeata than M. truncatula.     

Differential Drought Responses of Faba Bean (Vicia faba L.) Inbred Lines

Drought responses of 19 inbred faba bean lines of different origin were studied in the field under rain shelters with and without irrigation. Inbred lines differed significantly in response to drought (P < 0.01): those with a lower drought sensitivity index (SI) (more resistant) originated from the drought-prone regions characterized by smaller plant size (r = 0.93), and more pods and seeds per plant (r ≥ 0.90) regardless of seed size, while lines with higher SI (more sensitive) were those which mainly exhibited higher yield under favourable conditions accompanied by a greater biomass. In a greenhouse experiment under mild drought (−0.15 MPa soil water potential), comparisons between relatively drought-sensitive (Adriewaalse) and drought-resistant (L7) inbred lines showed that Adriewaalse used 38 % more water than L7 and also produced 40 % more biomass. There was a stress-induced decrease in osmotic potential (ψ_s) in both lines (by 0.72 and 0.50 MPa for Adriewaalse and L7, respectively) accompanied by decreased turgor in Adriewaalse and increased turgor in L7. The difference in drought-induced solute accumulation between lines was diminished when solute accumulation due to water loss and growth inhibition was considered, which indicates that solute accumulation was the result of a concentration effect. Similarly, lower SI in the field was not the result of osmotic adjustment, as the relationship between SI and drought-induced decrease in ψ_s was negative. The water use efficiency of both inbred lines increased markedly with increasing water deficit, though there was no difference between the lines. It was concluded that differences in drought resistance between these inbred faba bean lines were manifested through plant size-induced water demand (avoidance) but were not associated with osmotic adjustment (tolerance).     

非生物胁迫对玉米杂交种及其亲本自交系产量性状的影响

以抗逆性较强玉米杂交种郑单958及其亲本(郑58、昌7-2)和抗逆性较差的杂交种陕单902及其亲本(K22、K12)为材料, 在不同种植密度(45 000株 hm^-2和75 000株 hm^-2)、施氮量(112.5 kg hm^-2和337.5 kg hm^-2)和灌水量(正常灌水和前期干旱控水)条件下, 分析了2个杂交种及其亲本产量及相关生理特性的变化规律。结果表明, 在非生物胁迫条件下(高密度、低氮和前期干旱控水), 与陕单902相比, 品种郑单958叶面积指数、SPAD值、花后干物质积累量和产量的中亲优势值分别增加18%、9%、28%和22%; 与陕单902亲本(K22、K12)比, 郑单958亲本(郑58、昌7-2)叶面积指数、SPAD值、花后干物质积累量和产量的中亲值分别增加45%、36%、51%和45%; 郑单958产量的中亲值和中亲优势显著高于陕单902, 且中亲值增幅高于杂种优势值。玉米杂交种郑单958较陕单902增产的同时, 增强了对非生物逆境适应的能力。玉米杂交种的抗逆性来自亲本自交系。玉米杂交种抗逆性强在于增强了花后叶片光合能力(较高的LAI和SPAD值), 促进了花后干物质积累。