The effect of cold and heat treatments on the anther culture response of diverse rye genotypes

The reduction of saturated fats in canola oil has recently been promoted as a goal for breeders for commercial and human health benefits. Currently, saturated fatty acids in Canadian produced canola oil are above the 7% level, and the objective of this study was to generate canola lines with reduced major saturates (palmitic and stearic), by several percent. Mutant embryos generated from direct ultraviolet radiation mutagenesis of microspores in vitro were subjected to heat during the maturation stage. Heat artificially elevated the saturate levels in developing mutant embryos, allowing efficient identification of those with reduced saturates within the expanded range using HPLC fatty acid analysis of the embryo cotyledons. Mutagenesis produced embryos with fatty acids altered in both directions. Major saturate levels in the cotyledons of heat-treated mutant embryos ranged from 3.3 to 16.4% (heated control ca. 6–9%) and 1.3–10% (heated control ca. 2–4%) for palmitic and stearic fatty acids, respectively. Doubled haploid seed derived from embryos grown at normal temperatures confirmed the reduction of major saturates. HPLC fatty acid analysis of DH seed identified saturate levels ranging from 3.9 to 6.5% (control ca. 5.5%) and 0.9–2.7% (control ca. 1.7%) for palmitic and stearic fatty acids, respectively. Various doubled haploids were identified with major saturate levels below 5.5%. Concomitant positive changes in the unsaturated fatty acids (18:1, 18:2, 18:3) among the mutant lines are also discussed.     

Improved Cold and Drought Tolerance of Doubled Haploid Maize Plants Selected for Resistance to Prooxidant tert‐Butyl Hydroperoxide

To improve the abiotic stress tolerance of maize (Zea mays L.), doubled haploid (DH) plants were produced by in vitro selection of microspores exposed to tert‐butyl hydroperoxide (t‐BuOOH) as a powerful prooxidant This study investigated the tolerance of the progenies of t‐BuOOH‐selected DH lines to oxidative stress, cold and drought in controlled environment pot experiments by analyses of photosynthetic electron transport and CO₂ assimilation processes, chlorophyll bleaching and lipid peroxidation of leaves. Our results demonstrated that the t‐BuOOH‐selected DH plants exhibited enhanced tolerance not only to oxidative stress‐induced by t‐BuOOH but also to cold and drought stresses. In addition, they showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase and glutathione S‐transferase when compared with the DH lines derived from microspores that were not exposed to t‐BuOOH and to the original hybrid plants. The results suggest that the simultaneous up‐regulation of several antioxidant enzymes may contribute to the oxidative and cold stress tolerance of the t‐BuOOH‐selected DH lines, and that the in vitro microspore selection represents a potential way to improve abiotic stress tolerance in maize.     

Relationship Between Cold Severity and Yield Loss in Chickpea (Cicer arietinum L.)*

Seed yield in chickpea (Cicer arietinum L.) is substantially increased by advancing sowing date from the traditional spring to early winter at low to medium elevation areas around the Mediterranean Sea. This shift, however, increases the probability of the exposure to subzero temperatures as low as -10 °C for up to 60 days in a year. These low temperatures often reduce seed yield of cold-susceptible cultivars. Yield losses from cold were estimated in two experiments conducted at Tel Hadya, Syria. In experiment 1, of 96 genotypes sown on nine dates ranging from autumn to spring during the 1981–82 season, those lacking tolerance to cold were killed and produced no yield in autumn sowing, whereas lines with cold tolerance produced nearly 4 t/ha which corresponds to a four-fold increase over spring sowing. Moderately cold-tolerant genotypes sown during early winter produced substantially more seed yield than the normal spring-sown crop. Seedlings were more cold tolerant than the plants in early or late vegetative stages. In experiment 2, in which yield loss due to cold in the field was estimated in 12 yield trials comprising 288 newly bred lines in the 1989–90 season, the regression of cold susceptibility on seed yield in each of the trials was highly significant and negative. On average, winter-sown trials produced 67 % more seed yield than spring-sown trials, but 125 out of 288 genotypes produced yield more than double in winter sowing. Early maturing lines suffered severe cold damage and many lines produced no seed.     

Harnessing mutant donor plants for microspore culture in Indian mustard [Brassica juncea (L.) Czern and Coss]

Haploid mutagenesis offers several advantages over conventional (seed) approach. However, its potential has not been utilised for Brassica juncea, an important oilseed. In this study, mutant donor plants of three Indian B. juncea genotypes, generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU), were used for microspore culture. The response of mutant donor plants was about 100 times lower than non-mutant controls; a total of 9,411 embryos were produced from the EMS treated donor plants, while microspores isolated from ENU treated donors did not yield any embryos. The lethality of induced mutations demonstrated itself mainly as the induction of abnormal embryos (80%), failure of germination (70%) and failure of plantlet development (70%). Nine doubled haploid (DH) mutant lines and three non-mutant DH lines obtained through this approach were tested for agronomic and biochemical variation over two growing seasons. High variability was observed and stable mutants were recovered for reduced height (125 vs. 168 cm for the control), appressed pod character, altered fatty acid composition higher protein proportion in de-oiled meal (48%) and a lower glucosinolate content in de-oiled meal (59.5 μM/g) relative to controls. The approach demonstrates that despite severe reduction in efficiency of the DH line production, valuable mutants can be recovered from mutated donor plants.     

Assessment of Cold and Heat Tolerance of Winter-grown Canola (Brassica napus L.) Cultivars by Pollen-based Parameters

Winter‐grown canola (Brassica napus L.) production is limited mostly by frost and winter kill in the southern canola‐growing regions of the United States. Tolerance to cold and heat were assessed by studying percentage of pollen viability (PV), in vitro pollen germination (PG) and pollen tube length (PTL) for 12 field‐grown cultivars. Freshly collected pollen from all cultivars were incubated on artificial solid growth media at a constant temperature ranging from 10 to 35 °C at 5 °C interval for 30 h to determine PG and PTL. A modified bilinear model best described the temperature response functions of PG and PTL. Canola cultivars showed significant variability (P < 0.001) for PV (61.3 % to 89.7 %), PG (29.0 % to 48.2 %) and PTL (463 to 931 μm). The average cardinal temperatures, T_min, T_opt and T_max, for PG and PTL were 6.4, 24.3 and 33.7 °C, respectively. Principal component analysis revealed that maximum PG, PTL, T_min and T_opt of both PG and PTL were the most important factors in determining cold tolerance, whereas T_max of PG and PTL, and maximum PG and PTL were more responsible in separating the cultivars for heat tolerance. The canola cultivar, KS3077, was the most cold tolerant with the lowest T_min and the widest temperature adaptability range, and the cultivar Kadore was the most heat tolerant with the highest T_max for the PG. The identified cold‐ and heat‐tolerant cultivars may be useful in canola‐breeding programmes to develop cultivars suitable for a niche environment.     

Biochemical markers for cabbage seedpod weevil (<Emphasis Type="Italic">Ceutorhynchus obstrictus</Emphasis> (Marsham)) resistance in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

In the last decade, the cabbage seedpod weevil (Ceutorhynchus obstrictus (Marsham)) has become a major insect pest of canola (Brassica napus L.) in Canada reducing seed yields up to 35%. Therefore, the benefits of developing weevil resistant germplasm to canola breeders and the industry would reduce input costs, pesticide use, environmental degradation and increase yield. Yellow mustard (Sinapis alba L.) is resistant to C. obstrictus (CSPW), although the exact mechanism is not known (McCaffrey et al. 1999). A unique canola population was generated at the University of Guelph from a cross between B. napus and S. alba through embryo rescue and backcrossed to canola several times prior to double haploid (DH) production. Approximately one-half of this DH population had canola quality glucosinolate concentration (<16 μmol/g) and was used for further breeding. The hypothesis was that some DH progeny from this cross inherited resistance to CSPW from S. alba. Weevil infestation levels were assessed for the B. napus × S. alba BC2 and BC3 DH populations in the field over 7 years in Alberta where weevil pressure is strong to establish the resistant or susceptible status of these lines. The basic objectives for this study were to confirm field resistance in the B. napus × S. alba germplasm in Ontario and to identify any biochemical markers associated with resistance/susceptibility. Canola doubled haploid lines derived from BC2 or BC3 families were field screened for resistance (R) followed by chemical analysis of glucosinolates to detect biochemical polymorphisms correlated with CSPW resistance using High Performance Liquid Chromatography (HPLC). Two polymorphic peaks were found, one each, from extracts of upper cauline leaves and Stage 3 pod seed, with retention times of ~23 and 19 min, respectively. These HPLC peaks consistently correlated with larval infestation data and the peak differences between R and S DH lines were significant. Therefore, these two peaks can be considered as biochemical markers in this breeding germplasm and may play a role in rapid and early detection of CSPW resistance.     

Genome-wide association study for frost tolerance in rapeseed/canola (Brassica napus) under simulating freezing conditions

Rapeseed/canola seedlings can be easily damaged by spring frost, which can rupture the cells and kill the plant. Genetic variations for frost tolerance have known to exist within rapeseed/canola gene pool. A genome-wide association study (GWAS) was conducted using 231 diverged rapeseed/canola germplasm to find the significant markers of the freezing tolerance traits. The genotypes were obtained from 21 countries and comprised of spring, winter and semi-winter growth types. The genotypes were evaluated in plant growth chamber under simulated freezing conditions. Highly significant genotypic variation was observed for the freezing tolerance. The best three freezing tolerant germplasms (Rubin, KSU-10, and AR91004) were winter type, while the four most freezing susceptible germplasms (Polo Canada, Prota, Drakkar, and BO-63) were all spring type. No geographical or growth habit type clusters were identified by structure analysis in this mixed population. One QTL was identified that was located on chromosome A02. Six freezing/abiotic stress tolerance genes have been identified in this study.     

Response of maize inbred lines to a defoliation treatment inducing tolerance to cold at germination

Defoliation during maize (Zea mays L.) kernel development has been observed to induce tolerance to cold of germinating seeds in responsive genotypes. The objectives of this study were to evaluate the response to defoliation of immature embryo and mature seed germinability at cold and to verify if the response was influenced by the developmental stage at which the treatment was applied. In three environments, six inbred lines (B73, IABO78, Lo1016, Lo964, Mo17, Os420) were defoliated (D) approximately 20 days after pollination (DAP) or not defoliated (ND). Immature embryos were excised three days after defoliation and germinated in vitro at 9 or 25 ^∘C. At maturation, kernel germination was tested at the same temperatures. Defoliation improved cold tolerance and mean time to germination (MTG) at 9 ^∘C of both embryos and kernels of Lo1016. To study the effect of kernel developmental stage on response to defoliation, plants of B73, Lo1016 and Lo964 were defoliated at 15, 18, 21, 24, 27, 30, 33, 36, and 39 DAP, or not defoliated. At the same DAP, immature grains were analyzed for dry weight, water and abscisic acid (ABA) content. In Lo1016, low amounts of kernel ABA were detected at all stages, while in Lo964 and B73 ABA increased during development. Lo1016 mature kernels showed an improvement of cold tolerance due to defoliation at all times, while the other genotypes did not. In conclusion inbred lines showed variability for mature seed and immature embryo tolerance to cold at germination and for the ability to acquire tolerance after defoliation.     

Glycine max and Glycine soja are capable of cold acclimation

Soybean has been considered a cold intolerant species; based largely upon seed germination and soil emergent evaluations. This study reports a distinct acquisition of cold tolerance, in seedlings, following short acclimation periods. Diversity in cold responses was assessed in eight cultivars of Glycine max and six accessions of G. soja. All varieties of soybean significantly increased in freezing tolerance following acclimation. This study indicates soybean seedlings are indeed capable of sensing cold and acquiring cold tolerance. Germination rates after cold imbibition were negatively correlated with maturity group, but positively correlated with cold acclimation potential in G. soja. Seed fatty acid composition was varied between the species, with Glycine soja accessions containing about 2‐times more linolenic acid (18:3) than G. max. Furthermore, high levels of linoleic acid (18:2) in seeds were positively correlated with germination rates following cold imbibition in G. soja only. We suggest that domestication has not impacted the overall ability of soybean to cold acclimate at the seedling stage and that there is little variation within the domesticated species for ability to cold acclimate. Thus, this brief comparative study reduces the enthusiasm for the “wild” species as an additional source of genetic diversity for cold tolerance.     

Papaya ringspot virus resistance in <Emphasis Type="Italic">Carica papaya</Emphasis> via introgression from <Emphasis Type="Italic">Vasconcellea quercifolia</Emphasis>

We report the first successful production of PRSV-P resistant backcross (BC) papaya plants following intergeneric hybridisation between C. papaya and a Vasconcellea species after 50 years of reports on unsuccessful attempts. This follows our previous reports of PRSV-P resistant F₁ hybrids developed by intergeneric hybridisation between C. papaya and V. quercifolia. One PRSV-P resistant BC 1 (BC₁) plant was produced after 114,839 seeds were dissected from 940 fruits. The seeds yielded 1,011 embryos and 733 germinated in vitro from which 700 developed into plantlets that were screened in a glasshouse and in the field under high disease pressure and exposure to inoculation by viruliferous aphids. From the PRSV-P resistant backcross 1 (BC₁) male plant, 1465 plants [137 BC₂, 546 SbC₂ (BC2 sib-crosses), 147 BC₃, 379 SbC₃ and 256 BC₄] were grown from seed and inoculated with PRSV-P and virus resistant BC₃ and BC₄ plants were selected from these generations. Presence or absence of virus was confirmed by ELISA serological tests. BC plants generally developed mild symptoms of PRSV-P after periods ranging from 5 to 18 months in the field but many showed the ability to produce new growth free of symptoms. All control plants developed severe symptoms after 3 months in the field. Some BC₃ and BC₄ plants were free from viral infection after 18 months in the field. Subsequently they developed very mild symptoms on their leaves and a few ringspots on their fruit. They continued to grow vigorously and produce fruit for 3 years under high disease pressure provided by the infected controls and other susceptible plants. Good quality marketable fruit were produced on these plants. Application of these results should lead to restoration of the papaya industry in virus-infested regions of the Philippines and worldwide.     

Effects of high oleic acid soybean on seed yield,protein and oil contents,and seed germination revealed by near‐isogeneic lines

Typical soybean oil is composed of palmitic, stearic, oleic, linoleic and linolenic acids. High oleic acid content in soybean seed is a key compositional trait that improves oxidative stability and increases oil functionality and shelf life. Using a marker‐assisted selection method, near‐isogenic lines (NILs) of G00‐3213 for the high oleic trait were developed and yield tested. These NILs have various combinations of FAD2‐1A and FAD2‐1B alleles that were derived from the same backcrossing populations. The results indicated that G00‐3213 NILs with both homozygous mutant FAD2‐1A and FAD2‐1B alleles produced an average of 788 g/kg oleic acid content. The results also demonstrated that possessing these mutant alleles did not cause a yield reduction. Furthermore, seed germination tests across 12 temperatures (12.8–32.0°C) showed that modified seed composition for oleic acid in general did not have a major impact on seed germination. However, there was a possible reduction in seed germination vigour when high oleic seeds are planted in cold soil. The mutant FAD2‐1A and FAD2‐1B alleles did not hinder either seed or plant development.     

Variability in Osmotic Stress Tolerance of Starch Potato Genotypes (Solanum tuberosum L.) as Revealed by an In Vitro Screening: Role of Proline,Osmotic Adjustment and Drought Response in Pot Trials

Solanum tuberosum (potato) as a drought sensitive plant is also one of the most promising plants to meet the demands for food and starch of a growing population. Distinguishing genotypes into tolerant and susceptible is therefore of utmost interest. We subjected eighteen potato genotypes and two wild species, S. tarijense and S. chacoense, to osmotic stress applied in vitro by addition of 0.2 m sorbitol to a solid medium. Here, we report that a ratio of root:shoot dry mass (DM) together with the SSI (stress susceptibility index, equivalent to drought susceptibility index by Fischer and Maurer, Aust. J. Agron. Res., 29, 1978) of shoot DM were found to be relevant parameters to characterize genotypes in vitro for their osmotic stress tolerance. Drought stress data from pot trials in a rainout shelter (2013 and 2015) correlated poorly with the data obtained in in vitro experiments. However, the most tolerant and most sensitive genotypes in vitro were also categorized to be more tolerant or sensitive than the average to drought stress in vivo. Both, under in vitro and in vivo conditions, proline displayed an increase under osmotic stress conditions in nearly all potatoes tested, but no direct correlations were found to stress tolerance. However, a genotype classified as tolerant displayed earlier proline accumulation. Proline is thought of as one factor for plants to withstand stressful conditions, but cannot be used to distinguish potato genotypes for their stress tolerance to osmotic stress in vitro. Analysis of the osmotic potential of in vitro and in vivo stressed plants displayed a general increase compared to the control.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. I. Production of F1 hybrids through embryo rescue

Summary Triazine resistant Brassica napus ssp. oleifera and ssp. rapifera were hybridized to cultivars of B. oleracea ssp. italica, ssp. botrytis, ssp. capitata and ssp. fimbriata. The interspecific embryos did not survive in vivo but could be rescued in vitro using a culture medium developed by Monnier (1973). The embryos did not grow directly into normal plants but were successfully regenerated using the protocol developed by Keller (1984). Hybridization efficiency ranged from 0 to 2.64 hybrids per pollination. Interspecific embryo abortion may be related to abnormal endosperm development.     

Factors affecting seed set in the crosses between <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> (Ramat.) Kitamura and its wild species

Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and seriously result in low seed set, consequently reducing breeding efficiency. For the purpose of revealing the factors leading to low seed set, we investigated pollen viability, germination behavior of pollen grains on stigmas and embryo development in the crosses between D. grandiflorum and three wild species, D. nankingense (Nakai) Tzvel., D. indicum (L.) Des Moul. and D. zawadskii (Herb.) Tzvel. using technique of paraffin section, and light, fluorescence and scanning electron microscopy. The results indicated pollen viability of three wild species ranged from 20 to 25%. In the cross between D. grandiflorum and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in 12% ovaries at 8 days after pollination and thereafter all the embryos aborted. In other two crosses, many pollen grains germinated on stigmas and pollen tubes penetrated stigmas normally after pollination. Moreover, normal embryos were observed in over 50% ovaries from 8 to 15 days after pollination in the cross between D. grandiflorum and D. indicum, and seed set was 59%. In the cross between D. grandiflorum and D. zawadskii, normal embryos were observed in 52% ovaries at 8 days after pollination. After that, however, most embryos degenerated and seed set was only 9%. These data suggest that pollen viability has no significant effects on seed set of the three crosses. Very few germinated pollen grains on stigmas and abnormal growth of most pollen tubes before fertilization, and embryo abortion are the main factors causing failure of the cross between D. grandiflorum and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between D. grandiflorum and D. zawadskii and no barriers occur in the cross between D. grandiflorum and D. indicum.     

Genetic Variation for Heat Tolerance During the Reproductive Phase in Brassica rapa

We investigated heat tolerance at the reproductive stage in six spring‐type B. rapa accessions and one B. juncea accession as a control. Plants were subjected to two temperature treatments for seven days in controlled environmental rooms, beginning one day before the first open flower on the main stem inflorescence. The high‐temperature treatment ranged from 25 °C to 35 °C during 16 h light and 25 °C during 8 h dark. The control temperature treatment was set at 23 °C during 16 h light and 15 °C during 8 h dark. Soil moisture was maintained at close to field capacity to avoid drought stress. Main stem buds that emerged during the treatment period were tagged, and pod and seed production was recorded at each reproductive node. Leaf temperature depression and leaf conductance increased in the high‐temperature treatment which indicated that plants were not drought stressed. A leafy vegetable type of B. rapa from Indonesia was the most tolerant to high temperature, as defined by its ability to set seed equally well in the control and high‐temperature treatments, followed by an oilseed type from Pakistan. Pollen viability remained above 87 % in all accessions and treatments. We conclude that bud number and length, and pod number produced under high temperatures, might provide a useful preliminary screen for high‐temperature tolerance and that B. rapa may be a valuable source of heat tolerance in canola (B. napus).     

Exogenous Abscisic Acid Application During Grain Filling in Winter Wheat Improves Cold Tolerance of Offspring's Seedlings

Low temperature seriously depresses seed germination and seedling growth in winter wheat (Triticum aestivum L.). In this study, wheat plants were sprayed with abscisic acid (ABA) and fluridone (inhibitor of ABA biosynthesis) at 19 days after anthesis (DAA) and repeated at 26 DAA. The seeds of those plants were harvested, and seed germination and offspring's seedling growth under low temperature were evaluated. The results showed that exogenous ABA application decreased seed weight and slightly reduced seed set and seed number per spike. Under low temperature, seeds from ABA‐treated plants showed reduced germination rate, germination index, growth of radicle and coleoptile, amylase activity and depressed starch degradation as compared with seeds from non‐ABA‐treated plants; however, activities of the antioxidant enzymes in both germinating seeds and seedling were enhanced from those exposed to exogenous ABA, resulting in much lowered malondialdehyde (MDA) and H₂O₂ concentrations and production rate. In addition, the maximum quantum efficiency of photosystem II was also enhanced in ABA‐treated offspring's seedlings. It is concluded that exogenous ABA treatment at later grain‐filling stage could be an effective approach to improve cold tolerance of the offspring during seed germinating and seedlings establishment in winter wheat.     

In vitro culture of immature seed for rapid generation advancement in tomato

The importance of fast-trackt generation advancement in developing superior germplasm has been recognized in breeding of many crop species. To address this issue in tomato, immature seeds were excised from fruit at different maturity stages and transferred to culture medium. The best culture medium was modified full strength Moorashige–Skoog (MS) salts supplemented with 0.1 mg l⁻¹ IAA, 0.5 mg l⁻¹ IBA, 0.5 mg l⁻¹ GA₃ and 2% sucrose. If the excised seeds were able to grow, most showed shoot formation after a week. Seeds extracted as early as 10 days after pollination were successfully cultured provided they were transferred aseptically and without injury. No morphological or physiological changes in regenerated plants and their fruit relative to the parent were detected. Germination from immature seeds of tomato is a simpler alternative to in vitro culture of immature embryos or callus, as it can be undertaken in comparatively less stringent laboratory conditions. Using this approach, five generations can be produced in a year in contrast to a maximum of three generations with conventional methods. This offers an opportunity for rapid generation advancement aimed towards population development when coupled with marker assisted selection in tomato breeding for biotic and abiotic stress tolerance.     

Effect of genetic background on the target fatty acids of acyl-ACP thioesterase transgenes in Brassica napus

Acyl‐acyl carrier protein (ACP) thioesterase (TE) is involved in the biosynthetic fatty acid pathway of plants. Conventional canola lines transformed individually with the bay‐TE (Uc FatB1), elm‐TE (Ua FatB1), nutmeg‐TE (Mf FatB1) or Cuphea‐TE transgene (Ch FatB1), produce seed oil with modified fatty acid compositions. This study assessed the effects of genetic background, cytoplasm, maternal parent, and interaction of different TE transgenes, on the target fatty acids using F1 seeds and double haploid (DH) lines. The F₁ seeds were produced by crossing four TE transgenic parental lines and three non‐transgenic cultivars with distinct fatty acid compositions. The DH lines were developed from microspores of F₁ plants. DH lines from different crosses showed that genetic background does not have an effect on the relative levels of the target fatty acids of the same TE, indicating the stability of the substrate specificity of the TE within canola. However, significant effects of genetic background on the content of the major target fatty acids, lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, were observed. Expression of the TE in low erucic acid (C22:1) genotypes resulted in higher target fatty acid levels than expression in high C22:1 genotypes. Reciprocal crosses showed maternal effects, but not cytoplasmic effects. In addition, co‐expression of two different TE transgenes in the same seeds was observed. These results indicate the importance of selection for appropriate genetic backgrounds in order to maximize the expression of the target fatty acids of TE transgenes, and also indicate potential uses of TE co‐expression in modifying canola seed oil.     

Increasing pre‐acclimation temperature reduces the freezing tolerance of winter‐type faba bean (Vicia faba L.)

Autumn‐sown winter‐type faba bean (Vicia faba L.) has been shown to have a yield advantage over spring sowing. Still, adoption of this overwintered pulse crop remains limited in temperate locations, due to inadequate winter hardiness. This research sought to understand how the prevailing temperature during emergence and seedling development, that is pre‐acclimation, influences freezing tolerance. Seedlings grown under a controlled “warm” 17/12°C (day/night) pre‐acclimation environment were initially less freezing tolerant than those grown under a “cold” 12/5°C temperature treatment. Stem and particularly root tissues were primarily responsible for slower cold acclimation, and there was a genotype specific response of above‐ground tissues to pre‐acclimation treatment. Both above and below‐ground tissues should be tested across a range of pre‐acclimation temperatures when screening faba bean germplasm for freezing tolerance.     

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.