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 electrical impedance spectroscopy of Scots pine (Pinus sylvestris L.) shoots in relation to cold acclimation

Electrical impedance spectroscopy (EIS) was applied to stems of Scots pine (Pinus sylvestris L.) in a provenance field trial during frost hardening to find an EIS parameter for assessing frost hardiness (FH) without a controlled freezing test. The FH of stems and needles assessed by controlled freezing tests was compared with the equivalent circuit EIS parameters of a distributed model of stems (not exposed to controlled freezing treatment) and with dry matter (DM) content of stems. Significant differences in the equivalent circuit parameters, FH and DM content were found between provenances. The relaxation time (tau(1)), describing the peak of the high frequency arc of the impedance spectrum, and the intracellular resistance (r(i)) of stems increased with increasing FH. According to the linear regression, the coefficient of determination (R(2)) between the FH of stems and needles with tau(1) of the stem was 0.87 and 0.89, and with r(i) of the stem 0.74 and 0.85, respectively. The relation between FH and tau(1) changed with the degree of hardiness. The highest coefficient of determination was 0.95 in September when the FH of needles, ranging from -10 degrees C to -25 degrees C, was predicted with an accuracy of +/-2.0 degrees C. The resistance parameter r(2), describing the width of the low frequency arc of the impedance spectrum, decreased prior to and during the initial hardening: significant differences were found between provenances. This indicates that r(2) was not related to frost hardening per se. It is concluded that it is possible to distinguish the hardening patterns of different provenances by tau(1) in the rapid phase of hardening without controlled freezing tests.     

Winter hardening in first-year black spruce (Picea mariana) seedlings

Winter hardening of first-year black spruce [ Picea mariana (Mill.) B.S.P.] seedlings was studied by assessing a number of morphological and physiological changes under three hardening regimes: 1) early removal (ER), in which seedlings were exposed to natural daylengths and low ambient temperatures outside. 2) extended greenhouse culture (EG), in which seedlings were exposed to natural daylengths and warm temperatures, and 3) short day (SD), in which seedlings were exposed to short daylengths and low ambient temperatures outside. Measurements included needle primordia initiation, embryonic shoot volume, terminal bud mitotic index, embryonic shoot average cell volume, and shoot tip frost hardiness. EG seedlings formed buds containing 4 times as many needle primordia as ER stock. Embryonic shoot volume increased with number of needle primordia initiated, until late in the hardening period, when significant reductions in meristem volumes of SD and EG stock were observed. Frost hardiness increased sooner in seedlings which set bud in response to short days, but SD treatment did not result in significantly greater frost hardiness at the end of the trial. Frost hardiness was correlated with mitotic index of the embryonic shoot. Cell size in the embryonic shoot declined in seedlings of all treatments during hardening, however, EG seedlings had significantly lower cell volumes by the end of the trial in comparison to ER and SD seedlings.     

The effects of long-term elevation of air temperature and CO on the frost hardiness of Scots pine

The frost hardiness of 20 to 25-year-old Scots pine (Pinus sylvestris L.) saplings was followed for 2 years in an experiment that attempted to simulate the predicted climatic conditions of the future, i.e. increased atmospheric CO₂ concentration and/or elevated air temperature. Frost hardiness was determined by an electrolyte leakage method and visual damage scoring on needles. Elevated temperatures caused needles to harden later and deharden earlier than the controls. In the first year, elevated CO₂ enhanced hardening at elevated temperatures, but this effect disappeared the next year. Dehardening was hastened by elevating CO₂ in both springs. The frost hardiness was high (相似文献   

The electrical impedance spectroscopy of Scots pine needles during cold acclimation

The electrical impedance spectroscopy (EIS) was applied to current-year needles of Scots pine ( Pinus sylvestris L.) in an 8-year provenance field trial in central Finland during frost hardening. The EIS analysis of the needles using a Model-A equivalent circuit indicated a sequence of events in the needles during their cold acclimation. Some of the EIS-parameters referred to maturation phenomena occurring during the pre-hardening phase at the end of the growing season, and some parameters displayed a clear coincidence with the frost hardening itself. Significant differences between provenances were found in several of the Model-A parameters. Extracellular resistance (r_e) and β -coefficient decreased in all provenances in the pre-hardening phase in August and until mid-September. In the same phase, both the intracellular resistance (r_i) and the cell membrane time constant (τ_m) first increased and then decreased. According to τ_m, r_e and β there was a clear gradation between provenances in the pre-hardening phase. From the end of September significant differences were found in the intracellular resistance between provenances, corresponding with the differences in their hardening pattern. The dry weight (DW) content of needles increased during the study period but no clear differences were found between the provenances.     

Effect of defoliation on tracheid production and the level of indole-3-acetic acid in Abies balsamea shoots

To simulate feeding by the spruce budworm ( Choristoneura fumiferana Clem.), the apical current-year shoots on 1-year-old branches in the uppermost whorl of 6-year-old balsam fir [ Abies balsamea (L.) Mill.] trees were either removed completely by debudding before the start of the growing season or defoliated 0, 50, 90 or 100% shortly after budbreak. Debudded branches were treated at the apical end with 0, 0.1 or 1.0 mg of indole-3-acetic acid (IAA) (g lanolin)⁻¹. Ninety % of the 1-year-old needles were also removed from some of the experimental branches. After ca 4 weeks of growth, the radial width of new xylem and the level of IAA were determined in the 1-year-old internode. The IAA content was measured by radioimmunoassay.
The removal or defoliation of current-year shoots inhibited tracheid production and decreased the IAA level. Exogenous IAA stimulated tracheid production and increased the IAA level in debudded branches. Current-year shoot defoliation also inhibited current-year shoot elongation. The inhibitory effect of current-year needle removal on all parameters generally increased with increasing intensity of defoliation. The removal of 1-year-old needles did not affect the IAA level or current-year shoot elongation, nor did it influence tracheid production in branches with current-year shoots. However, removal of 1-year-old needles inhibited tracheid production in debudded branches supplied with exogenous IAA. The results indicate that (1) IAA is involved in the control of tracheid production in the 1-year-old internode, (2) IAA is supplied primarily by current-year shoots, and (3) defoliation by the spruce budworm inhibits tracheid production partly by decreasing the supply of IAA.     

Changing Environmental Effects on Frost Hardiness of Scots Pine During Dehardening

The effects of raised temperature and extended photoperiod onthe dehardening of quiescent and winter-hardy Scots pine saplingswere examined in an open-top-chamber experiment. The saplingswere exposed during winter to natural, square-curve fluctuating(between 1 and 11 °C with a 14 d interval), and constant(6 °C) temperatures with a natural and an extended (17 h)photoperiod. Frost hardiness of needles was determined by controlledfreezing tests and visual damage scoring. The constant 6 °Ctemperature treatment caused a gradual dehardening of needleswhereas under fluctuating temperatures the level of frost hardinessfluctuated. Trees exposed to extended photoperiods were lesshardy than under natural photoperiods after the initiation ofshoot elongation, but before this there were no clear differencesin frost hardiness between different photoperiodic treatments.The results indicate that the frost hardening competence ofScots pine changes during quiescence. Climate change; frost hardiness; hardening competence; photoperiod; Pinus sylvestris, Scots pine; temperature     

Photoperiodic reactions in conifer species

In Picea abies seedlings the critical night length for bud-set was determined for provenances from different latitudes, longitudes and altitudes within the natural range of the species. The clinal variation of this character was demonstrated. Inheritance studies indicated that this character is controlled by many genes with predominantly additive effects. In seedlings of Pinus sylvestris and Pinus contorta, growth cessation and bud-set took place in all light regimes, thus, even under continuous illumination. A photoperiodic optimum for height growth was determined. The photoperiodic influence on such characters as recurrent flushing of shoots, needle growth, dry matter production and frost resistance was demonstrated for northern and southern populations of the two Pinus species. Shorter nights were needed to induce a particular photoperiodic response in the northern populations as compared with those from the south. The importance of reliable phenological characters for assessing frost hardiness in provenance and progeny trials by means of early tests, is discussed.     

THE ROLE OF RESERVES IN LEAVES,BRANCHES, STEMS,AND ROOTS ON SHOOT GROWTH OF RED PINE

By defoliation, girdling, and various combinations of these before the growing season began, the role of foods in old needles, branches, main stems, and roots on shoot growth of 8-yr-old red pine (Pinus resinosa) trees was studied in northern Wisconsin. Defoliation and girdling, alone or in combination, reduced shoot growth but defoliation reduced shoot dry weight more than it reduced shoot elongation. Reductions in shoot growth due to treatment were in the following decreasing order: branch girdling + needle removal > girdling at stem base + needle removal > needle removal > branch girdling > base girdling. The old needles were the major source of food for shoot growth and accounted for four-fifths or more of all shoot growth. The combined reserves in the branches, main stem, and roots accounted for less than 15% of shoot growth. The contribution of reserves from tissues other than old leaves was in the following order: branches > main stem > roots. Defoliation weakened apical dominance relations. In defoliated trees, many secondary axes elongated more than the terminal leader, and secondary axes in lower whorls often grew more than those in upper ones. The data suggest an important role of nutritional factors in correlative growth inhibition.     

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.     

The consequences of freezing temperatures followed by high irradiance on in vivo chlorophyll fluorescence and growth in Picea abies

Picea abies (L.) Karst. plants, propagated by cuttings, were subjected to one night of freezing temperatures (-5°C), high irradiance (1 200 or 1 800 μmol m⁻² s⁻¹), or freezing temperatures followed by high irradiance. The treatments were applied at bud burst, at time of shoot elongation, and when the shoots had ceased to elongate. The maximum quantum yield of photosynthesis, F_v/F_m, dry weight of branches and needles, and length and survival of shoots were measured. F_v/F_m and growth decreased after a night of freezing temperatures followed by high irradiance, at the time of bud burst and shoot elongation. High irradiance alone influenced F_v/F_m, but not growth. Freezing temperatures affected F_v/F_m, and growth at the time of shoot elongation. F₀ increased after a night of freezing temperatures and decreased with age of the current-year needles. It was concluded that the use of short-term measurements of chlorophyll fluorescence induction to predict changes in growth after a night of frost and subsequent high light was not a reliable method.     

水杨酸对大叶黄杨茎抗寒性和电阻抗图谱参数的影响

在抗寒锻炼前,对当年生大叶黄杨(Euonymus japonicus)扦插苗喷施不同浓度水杨酸,用电阻抗图谱(EIS)法和电导(EL)法估测茎的抗寒性,以探明抗寒锻炼期间水杨酸对大叶黄杨抗寒性和电阻抗图谱参数的影响,找到适合不经冷冻处理估测大叶黄杨茎抗寒性和经冷冻处理后测定其抗寒性的EIS参数。结果表明,水杨酸处理能够提高大叶黄杨茎的抗寒性,最适浓度为5.0mmol.L-1;不经冷冻处理茎的EIS参数电阻率r和r1、胞外电阻率re、胞内电阻率ri、弛豫时间τ、弛豫时间分布系数ψ与EL法测定的抗寒性有较高的相关性(r=0.70~0.87),说明不经冷冻处理样本用以上参数估测大叶黄杨茎抗寒性是可行的,r1为最佳参数;冷冻处理后茎的re、τ、ri求得的抗寒性与EL法测定的抗寒性有较高的相关性(r=0.85~0.94),说明冷冻处理后re、τ、ri可以作为测定大叶黄杨茎抗寒性的参数,re为最佳参数。     

Effects of freeze-thaw injury on parameters of distributed electrical circuits of stems and needles of Scots pine seedlings at different stages of acclimation

Frost hardening and frost injury of both the stems and needlesof Scots pine (Pinus sylvestris L.) seedlings were studied byelectrical impedance analysis. This impedance analysis was basedon equivalent circuits with distributed circuit elements (DCE).A double-DCE model for stems and two single-DCE models for needlesprovided excellent fit to the experimental impedance data. However,the single-DCE model for needles which takes into account theasymmetry of the impedance arc proved more appropriate thanthe symmetric model. Several parameters changed in proportionto frost injury. In stems, extracellular resistance and onerelaxation time decreased with increasing damage, whereas intracellularresistance remained relatively unchanged. In needles, the overallpattern for extracellular resistance and relaxation time wassimilar to that in the stem. Intracellular resistance remainedapproximately constant in the case of the symmetric DCE model.During frost hardening, both extracellular and intracellularresistance increased in stems. In needles, extracellular resistanceincreased but relaxation time decreased with hardening. Theskewness of the impedance spectra in the Cole-Cole plot forneedles increased with hardening. The coefficient for distributionof the relaxation time(s) did not change in either stems orneedles with frost hardening but some changes were found withfrost damage. Key words: Pinus sylvestris L, electrical impedance, equivalent circuit, frost hardening, frost injury     

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     

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.     

Does the lack of vernalization requirement interfere with winter survival of oilseed rape plants?

Recent studies (Rapacz 1999) have shown that cultivars of spring-type oilseed rape are able to cold-acclimate to the level comparable with winter cultivars, but only after prehardening which results both in the increase of photosynthetic activity and in growth cessation. It is commonly known that under field conditions spring-type cultivars could not survive winter. Present studies were undertaken to explain the reasons for low winter hardiness of spring type rape plants. Six cultivars of spring and two of winter rape were sown in the open-air vegetation room at the end of August. The obtained results indicate that the degree of frost damage in spring-type plants increased in the course of winter and this increase was parallel to elongation of generative shoots observed after periods of warming. Each spring cultivar was completely killed by frost just after its generative shoot reached 15–20 cm, irrespective of its frost resistance level, determined previously under laboratory conditions. In the case of winter cultivars survival rate was consistent with laboratory-estimated frost resistance. It is suggested that spring rape could not survive winter because of its limited ability to prevent shoot elongation during winter at temperatures slightly above 0 °C. It was also found that less efficient photosynthetic electron transport in autumn was observed in these spring cultivars in which the elongation of generative shoots was observed already during the first warm break in winter.     

Frost hardiness in bark and needles of Norway spruce in southern Sweden

Bark necrosis and resin flows in Norway spruce have increased in southern Sweden over the last few decades. Frost damage late in spring has been suggested as a possible cause, but other factors besides the climate may have contributed to the damage. The nutrient status influences the hardening processes and plants with poor nutritional conditions have an increased sensitivity to frost. In this study the sensitivity to frost of bark and the hardiness status of needles of Norway spruce were compared with the nutrient status at two sites with different soil fertility. The trees were 30-40 years old. The hardiness status of the bark and needles was negatively affected by low concentrations of P and Mg.     

Hardiness dependent accumulation of phospholipids in leaves of wheat cultivars

Phospholipid content and composition in the leaves of thirteen cultivars of wheat differing in frost hardiness, were compared before and after hardening in order to see whether phospholipids play a role in surviving at low temperatures. The content of phospholipids in the leaves at the end of the hardening could be related to the aquired hardiness, and the relationship between the two phenomena could be described by the regression equation y = (0.174 ± 0.0016) x + 1311. Accumulation of phospholipids represents a hardiness dependent augmentation of membranes. Phosphatidylcholine appeared to be preferentially accumulated in hardy cultivars during the hardening process. As there are no significant differences in phospholipid fatty acid compositions investigated earlier, these results suggest the importance of the polar head group composition of membranes in avoiding frost injury.     

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.     

A Simulation Model for the Annual Frost Hardiness and Freeze Damage of Scots Pine

The changes in the frost hardiness of Scots pine were modelledby a dynamic model where the input variables were temperatureand photoperiod and the phase of annual development. The damagecaused by freezing was described by the sigmoidal relationshipbetween the relative needle damage and freezing temperature.The model simulations were carried out using temperature datafrom two sites in central Finland—Suonenjoki and Tampere.The validity of the frost hardiness model was tested with measuredfrost hardiness data from Suonenjoki. The effects of climaticwarming were also simulated by increasing temperature of thelong-term climatic data. Genotypic differences in chilling requirement,which determines the timing of the reduction of hardening competence,were included in the simulations. The simulated needle damageincreased as a result of climatic warming, and the differencesin the chilling requirement had a stronger effect on the amountof damage in the warmed climate than in the present climate.A large variation between years was found in the level of damage. Annual development; climatic change; dynamic model; freeze damage; frost hardiness,Pinus sylvestris ; Scots pine