The Influence of Photoperiod and Temperature on the Development of Frost Hardiness in Seedlings of Pinus silvestris and Picea abies

The influence of short days and low temperature on the development of frost hardiness in seedlings of Scots pine (Pinus silvestris L.) and Norway spruce [Picea abies (L.) Karst.], grown for 6 months in glasshouses and climate chambers, was investigated. The degree of hardiness was estimated by freezing the shoots of the seedlings to predetermined temperatures. After 8 weeks in a glasshouse the viability of the seedlings was determined by establishing bud flushing. The most effective climate for the development of frost hardiness was short days (SD) and low temperature (2°C); the next most effective was SD and room temperature (20°C). However, long days (LD) and low temperature also had a marked effect on the development of hardiness. A combination of 3 weeks’treatment with SD and 20°C, and 3 weeks with SD and 2°C gave the same results as 6 weeks with SD and 2°C. The results clearly demonstrate the importance of the photoperiod prior to low temperature for the development of frost hardiness. In conclusion both short days and low temperature induce frost hardiness development. Probably this occurs by initiation of different processes in the two cases. The degree of frost hardiness development appears to depend on the sum of these different processes and on the timing between them.     

Assessing frost hardiness of Pinus bungeana shoots and needles by electrical impedance spectroscopy with and without freezing tests

Aims Nursery and forest operations require that frost hardiness results be produced faster than can be provided by controlled freezing tests. There is a great challenge to develop a rapid method for predicting frost hardiness that might not necessitate controlled freezing tests. The aim of this study was to examine the assessment of the frost hardiness of shoots and needles of Pinus bungeana by electrical impedance spectroscopy (EIS) with and without controlled exposure to freezing.Methods The frost hardiness of current-year shoots and needles of P. bungeana in an 8-year-old provenance field trial was measured at Shisanlin Nursery in Beijing, China, from September 2006 to January 2007 by means of EIS and conventional electrolyte leakage (EL). In the same plants, but without controlled freezing test, were monitored the EIS parameters in current-year shoots and needles.Important findings The results showed that (i) after controlled freezing tests, the frost hardiness estimated by EIS parameters (extracellular resistance, r e, and membrane time constant, τ m) was significantly correlated with the frost hardiness assessed by EL method (r = 0.95) and (ii) for the samples not exposed to controlled freezing treatment, the relaxation time τ 1 for shoots and β for needles had greater correlations with the frost hardiness estimated by EL after controlled freezing tests relative to the other parameters (r = ?0.90 for shoots and r = 0.84 for needles, respectively). The parameters r e of shoots and needles and τ m of needles might be applied for measuring frost hardiness of samples after exposed to controlled freezing tests. The frost hardiness results can be obtained within 48 h. The parameters τ 1 of shoots and β of needles could be used for estimating the frost hardiness of samples without using a controlled freezing test. The frost hardiness results can be obtained within 24 h.     

The relation between growth cessation and frost hardening in Scots pines of different origins

The cessation of shoot elongation, diameter growth and needle elongation were compared with the initiation of frost hardening of the stems and needles in an 8-year-old provenance trial of Scots pine (Pinus sylvestris L.) established in central Finland. The saplings were of six different origins ranging from Estonia to northern Finland, forming a latitudinal gradient of ca. 10°N. The frost hardiness of the stems of current-year shoots was assessed by electrical impedance analysis and that of current-year needles by electrolyte leakage and visual scoring of damage. Artificial freezing tests were used in the assessments. The pattern of growth cessation (shoot and needle elongation, diameter growth) tended to follow the latitude of origin, i.e. growth ceased in the northernmost provenance first and in the southernmost one last. Both stems and needles of the northern provenances hardened earlier than the southern ones, but the differences in hardiness disappeared as hardening progressed. Growth cessation and initial hardening to -15°C were clearly correlated at the provenance level, indicating that growth must cease prior to hardening, and that earlier cessation of growth predicts earlier frost hardening of stems and needles. No differences in frost hardiness of stems were found at the provenance level at the end of the growing period in August. At that time, the frost hardiness of needles of the northernmost provenance was higher than that of other origins. Within the provenance, the stems were less hardy than the needles.     

Changes in Frost Hardiness of Stem Cortical Tissues of Cornus stolonifera Michx. after Recovery from Water Stress

Moderate water stress increases frost hardiness in many woody plants but little attention has been given to changes in hardiness after recovery from water stress. Tests were carried out to examine how much water stress-induced frost hardiness remained when plants were rewatered under different day length regimes. Red osier dogwood plants (Cornus stolonifera Michx.) were water-stressed at normal growing temperatures in long day (LD) or short day (SD) conditions, exposed to 6 nights of freezing temperatures, and then returned to normal growing conditions with full water supply. Water-stressed plants gained an additional 8 to 10 C of hardiness. The amount of freeze-induced hardiness in both stressed and control plants was not significant (approximately 2 C) and was not affected by photoperiod. When plants were kept in or transferred to LD, they lost nearly all of their water stress-induced hardiness within 7 days after rewatering. Water-stressed plants in SD lost the least amount of hardiness (5 C) when rewatered. In dogwood, water stress is an effective way to increase hardiness temporarily, but the photoperiod has a large effect on the retention of the acquired hardiness.     

Induction of Frost Hardiness in Stem Cortical Tissues of Cornus stolonifera Michx. by Water Stress: I. Unfrozen Water in Cortical Tissues and Water Status in Plants and Soil

Water supply and day length were varied in cold hardiness studies of red osier dogwood plants (Cornus stolonifera Michx.). The frost killing temperature, the content and freezing of stem cortical tissue water along with soil moisture content and tension were evaluated. Seven days of water stress in long and short day photoperiod regimes caused a rapid decrease in soil moisture content and plant water potential. During the same period, the frost hardiness increased from −3 to −11 C. Further water stress treatment had little effect. Control plants in short days showed only a gradual decrease in plant water potential and only gradually increased in frost hardiness while control plants in long days were unchanged. Freezing studies using nuclear magnetic resonance showed that increased hardiness in water-stressed plants resulted from both an increased tolerance of freezing and an increased avoidance of freezing, the latter resulting from higher solute concentration in the tissue solutions. The short day controls also showed similar changes; however, the changes were smaller over the 21 days of the study.     

Preliminary analysis of root exudates of in vitro-micropropagated Alnus glutinosa clones

Biochemical analysis at Alnus glutinosa (L.) Gaertn. root exudates was carried out in order to investigate the probable role of these exudates in the establishment of the Alnus-Frankia symbiosis. Sufficient amounts of sterile root exudates were collected from plants originating from five in vitro-micropropagated A. glutinosa clones (AG1, AG2, AG3, AG4, AG8) grown under aerobic conditions in nutrient solution with Ballotini glass beads. Cytological controls showed good preservation of roots at the end of the collecting period. The total protein and sugar contents of crude root exudates were determined and related to the dry root weight. These results showed a provenance effect. Soluble high molecular-weight fractions (i.e. the mucilage) were isolated following rigorous purification of the crude root exudates and within this fraction proteins were characterized by isoelectric focusing. The protein pattern of the five clones tested showed major bands in common and some minor specific ones. These experimental data will serve as a useful basis for future physiological investigations.     

Intracellular resistance correlates with initial stage of frost hardening in willow (Salix viminalis)

Distributed model parameters of shoots of five clones of willow ( Salix viminalis ) were examined with electrical impedance analysis at the end of the growing season and with cold acclimation. The parameters were compared with regard to frost hardiness, linoleic (18:2) and linolenic (18:3) fatty acids, unsaturation/saturation ratio of fatty acids and dry weight content. The intracellular resistance (r_i) correlated best with changes in frost hardiness. The value of r_i rose from 1–2 Ω m in non-hardened to about 12 Ω m in hardened samples. In the initial stages of frost hardening, a linear relationship was found between r_i and frost hardiness and levels of 18:2 fatty acid, and an inverse relationship between r_i and levels of 18:3 fatty acid. The unsaturation/saturation ratio of fatty acids rose fairly rapidly in the initial stage of hardening. The dry weight content increased stepwise during the experimental period, and less steadily than r_i. In addition, equivalent circuit parameters changed in the prehardening phase, and were probably connected with cell differentiation and lignification. Frost hardiness by the visual method and by extracellular resistance, determined after controlled freezing tests, correlated well in the initial stages of hardening until about − 10°C but deviated upon further hardening.     

Effect of frost nights and day and night temperature during dormancy induction on frost hardiness, tolerance to cold storage and bud burst in seedlings of Norway spruce

For trees, the ability to obtain and maintain sufficient levels of frost hardiness in late autumn, winter and spring is crucial. We report that temperatures during dormancy induction influence bud set, frost hardiness, tolerance to cold storage, timing of bud burst and spring frost hardiness in seedlings of Norway spruce (Picea abies (L.) Karst.). Bud set occurred later in 12°C than in 21°C, and later in cool nights (7°C) than in constant temperature. One weekly frost night (−2.5°C) improved frost hardiness. Cool nights reduced frost hardiness early, but improved hardiness later during cold acclimation. Buds and stems were slightly hardier in 21°C than in 12°C, while needles were clearly hardier in 12°C. Cold daytime temperature, cool nights and one weekly frost night improved cold storability (0.7°C). Seedlings receiving high daytime temperatures burst buds later, and were less injured by light frost some days after bud burst.     

Differences in frost hardiness of two Norway spruce morphotypes growing at Mt. Brocken, Germany

Norway spruce (Picea abies (L.) Karst.) exhibits strong ecotypic variation along altitudinal gradients in morphological traits, e.g. slenderness of crowns or arrangement of second-order branches. We were interested whether montane and lowland morphotypes differ in a key trait for the survival in cold environments, i.e. frost hardiness, and asked: (i) are montane morphotypes more resistant to frost damage and (ii) do they have a lower risk of frost damage by late frosts in spring than lowland morphotypes?We used the electrolyte leakage-method to measure frost hardiness on a monthly basis from October 2006 to May 2007 in stands of the montane and lowland morphotypes at Mt. Brocken in the Harz Mountains, Germany.LT₅₀ (i.e. the temperature that results in 50% of maximum electrolyte leakage) was assessed by freezing treatments in a frost chamber and was significantly influenced by morphotype, month and minimum ambient temperatures. LT₅₀ was significantly lower in the montane than in the lowland morphotype, with −107 °C and −49 °C, respectively. However, the interactions between morphotype with minimum ambient temperature or month were not significant. Thus, as frost hardiness of the two morphotypes responded to temperature in the same way, both morphotypes can be supposed to be exposed to the same risk of frost damage during hardening in autumn and dehardening in spring.     

Causes of variation in cold hardiness among fast-growing willows (Salix spp.) with particular reference to their inherent rates of cold hardening

Causes of variation in cold hardiness in the autumn were assessed among closely related, fast‐growing clones of willow of northern/continental and southern/maritime origins, under controlled regimes and natural conditions. Cold hardiness was assessed by controlled freezing followed by injury analysis, based on measurements of chlorophyll fluorescence (stems) and electrolyte leakage (leaves). During growth at a given temperature, the cold hardiness of the clones' stems was negatively correlated with their rate of growth. This apparently phenotypic variation was independent of temperature and, hence, the absolute growth rate. At later stages, cold hardiness of stems varied mainly with respect to genetic differences in the timing and rate of cold hardening. Cold hardening began up to 7 weeks earlier in northern/continental clones, and their rates of hardening in cool temperature regimes were up to three times higher than in southern/maritime clones. Ranking of clones with respect to rates was essentially the same whether natural or abrupt reductions of day length were used to trigger cold hardening. Results closely agreed with those of a previous field trial. Comparisons of rates at cool and warm temperatures suggest that cold hardening became increasingly dependent on cool temperatures with time. Increasing sucrose‐to‐glucose ratios, and especially dry‐to‐fresh weight ratios, paralleled early cold hardening. Before leaves were shed in the autumn they underwent cold hardening in parallel with stems, eventually allowing them to tolerate temperatures down to ?10 °C.     

Does ice crystal formation in buds explain growth disturbances in boron-deficient Norway spruce?

Loss of apical dominance in boron-deficient trees has been suggested to be due to frost damage of terminal buds and leaders. Excessive nitrogen (N) supply can exacerbate boron (B) deficiency by the dilution-effect. N may also have direct effects on winter hardiness. We studied frost hardening of buds of Norway spruce (Picea abies L. Karst.) in healthy-looking trees and in trees with growth disturbances. The effect of B and N on frost hardiness was studied in a factorial fertilisation experiment during cold acclimation. Frost hardiness was determined by differential temperature analysis (DTA) and scoring of visual damage. In a DTA profile of apical buds with a piece of stem, low-temperature exotherm (LTE) predicted bud injury, while two of the observed high-temperature exotherms and two of the observed intermediate-temperature exotherms were non injurious. Appearance of LTE followed changes in air temperature. The risk of frost damage was not affected by fertilisation treatments or previously observed growth disturbances. However, when the bud structure was deformed by severe B deficiency, the supercooling ability disappeared. Such buds are probably killed by freezing in nature and therefore, frost damage may play a secondary role in the development of growth disturbances.     

Altitudinal and seasonal variation of frost resistance of Fagus crenata and Betula ermanii along the Pacific slope of Mt. Fuji, Japan

1 Frost resistance of Fagus crenata (Siebold's beech) and Betula ermanii (Japanese mountain birch) was investigated with respect to the species' altitudinal distribution on the Pacific slope of Mt. Fuji from 1996 to 1997. Flint's Index of Injury, which is based on electrolyte leakage from freeze-injured tissue, was used to assess frost hardiness of shoots produced in the previous growing season.
2 Fagus crenata is found on the lower slopes (700–1600 m a.s.l.). Mid- to late November hardening of shoots was enhanced, midwinter damage below −30 °C reduced and dehardening delayed nearer the upper limit. To here temperatures began to rise at least 3 weeks before dehardening began. Shade crown shoots were more susceptible to deep-freeze damage than light crown shoots. If the ultimate upper distribution limit was determined by frost hardiness, F. crenata would be expected to occur up to 1800 m altitude.
3 Betula ermanii is found between 1600 m and 2800 m, and intensive hardening occurred at all altitudes during the second half of October. Frost hardiness increased considerably with altitude up to the forest limit, where frost acclimation preceded the temperature decline by 2 weeks. Once maximum frost resistance had been attained freezing to −47 °C failed to cause tissue injury. Dehardening began slightly later at the tree line, but the time–course was the same at all altitudes. Main and lateral shoots did not differ in frost hardiness.
4 Comparison of monthly air temperature minima over the past 66 years with the course of frost resistance showed that F. crenata and B. ermanii found on the Pacific slope of Mt. Fuji were unlikely to suffer damage by frost.
5 The observed uppermost distribution limit for B. ermanii at 2800 m altitude on Mt. Fuji is considered both with our observations and with previous hypotheses.     

Genetic analysis of frost hardiness traits in tuber-bearingSolanum species

The inheritance of frost hardiness and cold acclimation potential traits was studied in three segregating populations derived from a cross betweenSolanum commersonii Dun. PI 243503 (cmm) andSolanum cardiophyllum Lindl., PI 184762 (cph), two parental genotypes with contrasting frost hardiness and cold acclimation potential. The levels of frost hardiness and cold acclimation potential were expressed as the LT₅₀, the temperature at which 50% of the cells in leaf discs were killed, as measured by the ion leakage method, following a controlled freeze test There was considerable variation in both frost hardiness and cold acclimation potential in all three segregating populations (F₁ F₁ xcmm, and F₁ xcph). Frost hardiness and cold acclimation potential were not correlated, suggesting that these two traits are under independent genetic control. The analysis of generation means indicated that the variation for both traits could be best explained by an additive-dominance model, with additive gene effects the most important Broad-sense heritability was 0.73 and 0.74 in the F₁ population, for frost hardiness and cold acclimation potential, respectively, and was 0.85 for either trait in the F₁ xcmm population, indicating that these two traits are highly inheritable. Our results suggest that it should be possible to incorporate the frost hardiness and cold acclimation traits from S.commersonii into cultivated potato species.     

Use of intraspecific variation in thermal responses for estimating an elevational cline in the timing of cold hardening in a sub‐boreal conifer

To avoid winter frost damage, evergreen coniferous species develop cold hardiness with suitable phenology for the local climate regime. Along the elevational gradient, a genetic cline in autumn phenology is often recognised among coniferous populations, but further quantification of evolutionary adaptation related to the local environment and its responsible signals generating the phenological variation are poorly understood. We evaluated the timing of cold hardening among populations of Abies sachalinensis, based on time series freezing tests using trees derived from four seed source populations × three planting sites. Furthermore, we constructed a model to estimate the development of hardening from field temperatures and the intraspecific variations occurring during this process. An elevational cline was detected such that high‐elevation populations developed cold hardiness earlier than low‐elevation populations, representing significant genetic control. Because development occurred earlier at high‐elevation planting sites, the genetic trend across elevation overlapped with the environmental trend. Based on the trade‐off between later hardening to lengthen the active growth period and earlier hardening to avoid frost damage, this genetic cline would be adaptive to the local climate. Our modelling approach estimated intraspecific variation in two model components: the threshold temperature, which was the criterion for determining whether the trees accumulated the thermal value, and the chilling requirement for trees to achieve adequate cold hardiness. A higher threshold temperature and a lower chilling requirement could be responsible for the earlier phenology of the high‐elevation population. These thermal responses may be one of the important factors driving the elevation‐dependent adaptation of A. sachalinensis.     

黑果腺肋花楸枝条的深超冷与抗寒性

差热分析法测得黑果腺肋花楸枝条有深超冷。黑果腺肋花楸低温放热峰的起始温度和组织褐变法测得的致死温度相吻合。以黑果腺肋花楸低温放热峰的起始温度作为枝条的抗寒性指标,抗寒性的季节变化表现为：晚秋至１２月上旬为抗寒性驯化阶段,１２月上旬至次年２月上旬为最强抗寒性维持阶段,２月上旬以后开始脱锻炼。初冬后的锻炼过程和早春的脱锻炼过程十分迅速并与环境温度密切相关。     

Cold-acclimation of Hibiscus rosa-sinensis L. and Hibiscus syriacus L. in natural and controlled environments

Abstract Seasonal cold-acclimation patterns and the effects of photoperiod and temperature on cold-hardiness of Hibiscus rosa-sinensis L. and Hibiscus syriacus L. were determined. Field-grown H. rosasinensis consistently failed to survive freezing at - 2°C. Two genotypes of field- and container-grown H. syriacus initiated cold-acclimation in mid September, in response to decreasing daylength, and continued to an ultimate midwinter hardiness level of - 27°C in early February. Controlled environment experiments using combinations of short days (SD) and cool day/night temperatures were unable to induce even minimal cold acclimation of H. rosasinensis. In controlled environments, H. syriacus attained a moderate amount of cold tolerance at warm temperatures and long days (LD). Low night temperature combined with LD, warm day produced the same degree of cold-acclimation as the SD treatments. While not essential, SD enhanced H. syriacus cold-acclimation in controlled environments. A - 5°C frost treatment of intact plants did not enhance cold-hardiness of H. syriacus.     

Vegetative growth and frost hardiness of cloudberry (Rubus chamaemorus) as affected by temperature and photoperiod

The effect of photoperiod and temperature on growth and induction and development of frost hardiness in cloudberry ( Rubus chamaemorus L.) was examined in two experiments. The photoperiods were 8, 12 or 24 h and the temperatures were 18, 15, 12, 9, 4, 3, –3 or –4°C depending on the experiment. The level of hardiness was expressed as LT₆₆ or LT₅₀ (the lethal temperature for 66 or 50% of the plant material) for percentage of bud break and for the degree of coloring by triphenyltetrazolium chloride for rhizomes. The vegetative growth was clearly affected by daylength; petiole elongation, leaf growth, shoot dry weight and number of shoots per plant were all reduced under short days compared with long days. However, the photoperiod had no significant effect on hardening of buds or rhizomes. Hardening increased with successively decreasing temperatures. To get the maximum hardiness, plants had to be exposed to freezing temperatures.     

昆虫耐寒性研究

昆虫是变温动物,气候变化是造成种群季节消长的基本原因之一。尤其在不良的低温环境中,昆虫耐寒力的高低是其种群存在与发展的种要前提,昆虫对低温的适应能力及其机理也因而成为昆虫生态学和生物进化研究中的一个深受重视的问题,本文论述了与耐寒性直接相关的过冷却点昆虫的抗寒对策,明确了昆虫耐寒性的一些基本概念,一方面从环境影响昆虫的角度对耐寒性的一般规律,如季节性变化,地理变异快速冷驯化的作用等做了简要的概念括,另一方面阐述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等做了简要的概括,另一方面简单述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等。强调昆虫与环境相互作用过程中的生态生理适应,并指出昆虫耐寒性应当与生活史中别的因素联系起来,这样才能对耐寒性有一个更加全面的理解。     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Effects of Hormones on Morphogenesis and Cold Resistance in Berseem Clover (Trifolium alexandrinum L.)

Plants of berseem clover (Trifolium alexandrinum L.) cv. Taborwere raised under conditions inhibiting the acquisition of coldhardiness (non-hardened) or inducing cold hardiness (hardened).All non-hardened plants developed an elongated shoot and exhibitedconsiderable frost sensitivity, as measured by the extent ofthe reduction in yield of variable chlorophyll fluorescenceafter exposure to sub-zero temperature. Hardened plants developeda shorter shoot, with fewer leaves and a greater percentageof dry matter in the root system. These parameters were associatedwith a marked increase in frost resistance. Exogenous applicationof ABA to plants effected similar morphological modificationsin both hardening and non-hardening temperature regimes; plantsdeveloped a shorter primary shoot axis and leaves exhibiteda marked increase in frost hardiness. In berseem clover ABAcan thus substitute, at least partially, for the low temperaturetreatment required to induce cold hardiness. Spraying plantsraised under hardening conditions with gibberellic acid reversedthe effects of the hardening treatment, since they developedan elongated shoot and exhibited frost sensitivity comparableto non-treated plants grown under non-hardening conditions.It is concluded that these endogenous hormones are directlyinvolved in triggering changes in morphogenesis which accompanyphysiological and metabolic events associated with the inductionof plant cold hardiness. Key words: Frost resistance, morphogenesis, abscisic acid, giberellic acid, Trifolium alexandrinum