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.     

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.     

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.     

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)     

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.     

Leaf Photosynthesis During Grain Filling Under Mediterranean Environments: Are Barley or Traditional Wheat More Efficient Than Modern Wheats?

Barley is one of the most popular crops in dryland agricultural systems of Mediterranean areas, where it is assumed that barley, or traditional wheat cultivars, performs better than modern wheat under low‐yielding conditions. It was tested whether variations in net leaf photosynthetic rate (P_N) during grain filling provide any basis for the potential better performance of barley and traditional wheat compared to modern wheats in Mediterranean areas. Two groups of field experiments were conducted in Agramunt (NE Spain) during 2005/06 (06) and 2006/07 (07) growing seasons combining low and high nitrogen (N) availabilities under rain‐fed and irrigated conditions. Cultivars used in the first group of experiments were a traditional (Anza) and a modern (Soissons) wheat, whilst in a second group of experiments, a wheat (Soissons) and a barley (Sunrise) modern cultivars were used. Both wheat cultivars showed a similar P_N during grain filling but higher than that of the modern barley cultivar. Differences between species in P_N were maximized under high‐yielding conditions. There were no differences between cultivars in instantaneous water‐use efficiency. The barley cultivar showed a higher specific leaf area, but lower N content per unit of leaf area, than wheat. Photosynthetic nitrogen‐use efficiency was similar between the traditional and the modern cultivar but lower than barley. Decreases in P_N after anthesis were not exactly observable in SPAD measurements. In conclusion, we found no consistent differences between cultivars in terms of post‐anthesis photosynthetic activity to support the assumption of better performance under Mediterranean farm conditions of traditional wheat or barley against modern wheat.     

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.     

Allelopathic Effects of Black Mustard (Brassica nigra) on Germination and Growth of Wild Barley (Hordeum spontaneum)

Black mustard [Brassica nigra (L.) Koch.] contains water‐soluble allelochemicals that inhibit the germination and growth of other species. This characteristic could be used in weed management programmes. Greenhouse and laboratory experiments were conducted to determine the effects on wild barley (Hordeum spontaneum Koch.) germination and seedling growth of (i) preceding crops, (ii) fresh black mustard residue incorporation, and (iii) black mustard leaf, stem, flower and root water extract concentrations. Growth of wild barley, as indicated by plant height and weight, was significantly reduced when grown in soil previously cropped to black mustard compared with that cropped to wild barley. Soil incorporation of fresh black mustard roots and both roots and shoots reduced wild barley germination, plant height and weight when compared with a no‐residue control. In bioassays, black mustard extracts reduced wild barley hypocotyl length, hypocotyl weight, radicle weight, seed germination, and radicle length by as much as 44, 55, 57, 63 and 75 %, respectively, when compared with a water control. Increasing the water extract concentrations from 4 to 20 g per 100 ml of water of all black mustard parts significantly increased the inhibition of wild barley germination, seedling length and weight. Based on 8‐day‐old wild barley radicle length, averaged across all extract concentrations, the degree of toxicity of different black mustard plant parts can be ranked in the following order of inhibition: leaves > flowers > mixture of all plant parts > stems > roots.     

Improved frost tolerance and winter hardiness in proline overaccumulating winter wheat mutants obtained by in vitro-selection is associated with increased carbohydrate,soluble protein and abscisic acid (ABA) levels

In previous studies in vitro-selection of proline overaccumulating lines of winter wheat (Triticum sativum L. cv. Jo 3063) with increased frost tolerance was reported. These traits were found to be genetically stable. In the present study the improvement of frost tolerance (winter hardiness) under field conditions is confirmed for F₇ progenies of the mutants. Moreover, the mutants accumulated higher levels of glucose and fructose, soluble protein and abscisic acid (ABA) in addition to proline than the wild type under cold hardening conditions in a growth chamber as well as under cold hardening field conditions. ABA and proline levels peaked when the temperature decreased, whereas carbohydrate levels increased more slowly at decreasing temperature. Soluble protein levels also increased during cold hardening, but in addition showed sharp declines during frost periods. Increased carbohydrate levels of the mutants were associated with lower osmotic potential values. The differences in carbohydrate, protein and ABA levels between the mutants and the wild type are probably due to pleiotropic effects of the mutation.     

Barley mutants with enhanced level of resistance to Swedish isolates of Bipolaris sorokiniana,casual agent of spot blotch

Spot blotch, caused by Bipolaris sorokiniana, has become an increasingly important disease on barley in Sweden. In order to enhance our knowledge of this disease, eight Swedish B. sorokiniana isolates were assessed for their virulence pathotype on 30 barley genotypes including eight lines of mutant origin, designated B. sorokiniana tolerant (bst). A leaf screen assay revealed the presence of virulence groups 1 and 7 in Sweden. Several mutant genotypes demonstrated enhanced resistance responses (P = 0.0001), of which bst1:3 had the lowest leaf scoring value to the most virulent isolate, Swe2 (virulence group 7). To evaluate the host root responses, two contrasting isolates Swe2 and Swe4 (virulence group 7 and 1) were used to inoculate a set of eight differential lines, including four promising mutant lines from the leaf screen. Mutant bst1:6 was significantly less susceptible (P = 0.001) compared with the other barley genotypes tested, independent of the two isolates used. Mutants bst1, bst1:3 and bst1:4, which displayed less or no spots, are also considered promising genotypes for further breeding attempts.     

Cold acclimation in white clover subjected to chilling and frost: Changes in water and carbohydrates status

The effect of cold acclimation on the water status and the fate of carbohydrates was studied in white clover (Trifolium repens L., cv Huia) subjected to chilling and/or freeze-thaw cycles. Treatments were applied in a controlled environment, with a constant photoperiod, for 6 weeks to plants either acclimated or non-acclimated to low temperature.

Cold acclimated plants had a higher concentration of starch in the stolons at the end of the acclimation period than non-acclimated plants (54 vs. 15 mg g⁻¹ DW). During the experiment, the leaves of cold acclimated plants subjected to frosts maintained a higher relative water content (RWC) than did leaves of plants not acclimated for which we observed a strong dehydration of 80%.

Under chilling, cold acclimated plants demonstrated an osmotic adjustment. We showed that only 30–40% of the variation of the osmotic potential was explained by variation in free sugar concentration, suggesting that compounds other than carbohydrates participated in the osmotic regulation.

Chilling increased the carbohydrate content and frost decreased the starch concentration in non-acclimated plants only.

We showed in white clover that the cold acclimation contributed to frost and chilling tolerance by the maintenance of the hydration of tissues. We demonstrated that osmotic adjustment was not completely explain by the accumulation of free sugars.     

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.     

Mapping of genes involved in glutathione, carbohydrate and COR14b cold induced protein accumulation during cold hardening in wheat

Using some of the chromosome substitution lines developed from thecrosses of the donor Cheyenne to Chinese Spring we showed that theaccumulation of water soluble carbohydrates during different stages ofhardening was time dependent. Moreover there was a significantcorrelation between the rate of carbohydrate accumulation and the frosttolerance. The expression and regulation of a wheat gene homologous tothe barley cold regulated cor14b gene was compared in frost sensitiveand frost tolerant wheat genotypes at different temperatures. Studies madewith chromosome substitution lines showed that the threshold inductiontemperature polymorphism of the cor14b wheat homologous genewas controlled by loci located on chromosome 5A of wheat, while cor14b gene was mapped, in Triticum monococcum, onto the longarm of chromosome 2A^m. Our study on the effect of cold hardeningon glutathione (GSH) metabolism showed that chromosome 5A of wheathas an influence on the GSH accumulation and on the ratio of reduced andoxidised glutathione as part of a complex regulatory function during coldhardening. In addition, the level of increase in GSH content duringhardening may indicate the degree of the frost tolerance of wheat.     

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.     

The Impact of Underseeding Barley (Hordeum vulgare L.) on Timothy (Phleum pratense L.)-Clover (Trifolium pratense L.; Trifolium hybridum L.) Forage Production in a Cool Maritime Climate

Growing barley (Hordeum vulgare L.) in the year of forage establishment is a common agronomic practice in marginal growing regions, but is often not recommended to growers. We examined the effect of silage barley production over an establishing timothy (Phleum pratense L.)‐clover (Trifolium pratense L.; Trifolium hybridum L.) forage sward in a 4‐year study near St. John's, Newfoundland, Canada. The experiment compared two barley varieties differing in plant height (semidwarf Chapais and Leger), three barley seeding rates and the effect of a forage understorey on forage (barley) production in the establishment year and forage (timothy‐clover) production in the subsequent year. The taller Leger yielded similar forage biomass to Chapais in the year of forage establishment, which (when planted at 375 plants m^?2) was roughly twice that of a pure‐stand timothy‐clover mix. Increasing the barley seeding rate from 125 to 375 plants m^?2 resulted in a linear increase in forage yield in the year of planting. The production of barley forage in the establishment year resulted in reduced timothy‐clover forage yield in the year following barley planted at 125 and 375 seeds m^?2, but not for barley planted at 250 seeds m^?2. Companion planting also altered forage species composition in that higher barley seeding rates resulted in 12–18 % less timothy and 2–4 % lower fibre levels in the year following planting. Barley seeded at rates of 250–375 seeds m^?2 and undersown with a timothy‐clover mixture (harvested at mid‐milk) resulted in greater forage yield of poorer quality than pure‐stand timothy‐clover in the planting year, and a barley seeding rate of 250 seeds m^?2 did not impede forage production in the subsequent year.     

Resistance of barley genotypes to brown leaf spot

Resistance of 59 barley cultivars to three distinct isolates of Bipolaris sorokiniana, causing brown leaf spot was tested on seedlings and adult plants in a screen house and in a growth room, by determination of disease incidence, disease severity, and rate of lesion expansion. The disease reaction and severity at the seedling stage differed among cultivars. The cultivars MNS Brite, NDB 112, Kindred, Parkland, and Oderbrucke were moderately resistant to brown leaf spot, but the resistance was partial to all three isolates. None genotype with complete resistance was found. The cultivars CI 9539, Svanhals, and Chevron, considered as sources of resistance, were classified as moderately susceptible, and susceptible, respectively. Disease severity, disease reaction, and incidence of B. sorokiniana in grains also differed among the cultivars at the adult plant stage. Nevertheless, none of the genotypes was resistant to B. sorokiniana. The rate of lesion expansion differed among cultivars BR 2, MN 698, Kitchin, Svanhals, and NDB 112, with NDB 112 having the slowest expansion rate (0.194–0.205 mm²/day).     

Management of Italian and Perennial Ryegrasses for Seed and Forage Production in Crop Rotations

Italian ryegrass (Lolium multiflorum Lam.) and perennial ryegrass (L. perenne L.) can be grown for seed and forage in cold winter regions provided the stand persists well over winter. Seed yield and plant characteristics during primary growth, and forage yield during regrowth, were determined for two Italian and one perennial ryegrass cultivars in Atlantic Canada. Establishment methods and dates included sowing ryegrass in cultivated soil alone or with barley in mid‐May and, after harvesting the barley crop, by sowing ryegrass following conventional or reduced cultivation and by no‐till drilling into barley stubble in mid‐August and early September. Despite some winterkill, particularly in Italian ryegrass, seed and forage yields were adequate in post‐establishment growing seasons. Seed yield for Italian ryegrass was greatest (1270 kg ha^?1) when it was sown into cultivated soil in mid‐August and least (890 kg ha^?1) when sown alone in May. Italian ryegrass yielded 15–17 % more seed when plots were established in mid‐August rather than in mid‐May or early September. Italian ryegrass cv. Lemtal had a greater density of fertile tillers (1030 m^?2) in the sward than cv. Ajax (860 m^?2) and its tiller density was greater when seeded into cultivated soil in September than in mid‐August. There were fewer spikelets per seed head for sowing Italian ryegrass with barley in May than for the other methods of establishment. Forage yield in regrowth was greater for Italian ryegrass cv. Ajax (2770 kg ha^?1) than for cv. Lemtal (2480 kg ha^?1). Seed yield of perennial ryegrass was greater when seeded in mid‐May than in mid‐August or early September. The seed yield of perennial ryegrass was greater when it was sown with barley in May and harvested for grain, than when it was sown alone or with barley harvested at late milk stage. The establishment methods for mid‐August and early September sowing had little effect on seed yield. However, the no‐till and reduced tillage methods resulted in a greater tiller density than sowing into the cultivated seedbed. Fertile tillers tended to be denser under reduced cultivation for sowing in August. Forage yield of perennial ryegrass regrowth was not influenced by the sowing method and timing. In conclusion, Italian and perennial ryegrasses produce adequate seed and forage regrowth under different establishment methods and timing. However, the poor persistence of Italian ryegrass may limit commercial production after the establishment year in Atlantic Canada.     

Genome-wide association studies in a barley (Hordeum vulgare) diversity set reveal a limited number of loci for resistance to spot blotch (Bipolaris sorokiniana)

Spot blotch caused by Bipolaris sorokiniana is an important disease in barley worldwide, causing considerable yield losses and reduced grain quality. In order to identify QTL conferring resistance to spot blotch, a highly diverse worldwide barley set comprising 449 accessions was phenotyped for seedling resistance with three isolates (No 31, SH 15 and SB 61) and for adult plant resistance at two locations (Russia and Australia) in two years. Genotyping with the 50 k iSelect barley SNP genotyping chip yielded 33,818 informative markers. Genome-wide association studies (GWAS) using a compressed mixed linear model, including population structure and kinship, revealed 38 significant marker-trait associations (MTA) for spot blotch resistance. The MTA corresponded to two major QTL on chromosomes 1H and 7H and a putative new minor QTL on chromosome 7H explaining between 2.79% and 13.67% of the phenotypic variance. A total of 10 and 14 high-confidence genes were identified in the respective major QTL regions, seven of which have a predicted involvement in pathogen recognition or defence.