Frost Treatment Research Articles

Determining Seed Performance of Frost‐Damaged Maize Seed Lots

Seed quality of maize (Zea mays L.) can be negatively impacted by a fall frost event. It is important for the seed industry to detect frost damage early and to make marketing decisions before the seed lots are conditioned for sale. This study compared several seed quality tests (standard germination [WG], accelerated aging [AA], saturated cold [SC], and soak tests) for their ability to quantify frost damage. Additionally, these tests were used to predict field emergence under poor and average to good field conditions. Two genotypes (B73 × IRF311 and Mo17 × IRF311) were harvested at three moisture contents (300–350, 400–450, and 500–550 g H2O kg−1 fresh weight). An artificial frost treatment was applied to the seed and damage was determined by testing seed after approximately 0, 1.5, 3, 4.5, and 6 mo of storage. The artificial frost treatment significantly decreased viability and vigor of all the seed lots except Mo17 × IRF311 harvested at 300 to 350 g H2O kg−1 fresh weight. As seeds matured, the damage associated with frost treatment decreased. Laboratory tests did not accurately predict field emergence of frost‐damaged seed under poor field conditions; however, AA at 0 mo, SC at approximately 0, 1.5, and 3 mo, and soak at approximately 0, 1.5, and 3 mo had strong relationships to field emergence under average to good field conditions. These results indicate that frost damage in seed lots is quantifiable using the SC and soak tests during approximately the first 3 mo of storage and both tests accurately predict field emergence.

Implications of delayed soil thawing on trees: A case study of a Picea abies stand

The effects of experimentally delayed soil thawing were studied on a 32-year-old stand of Norway spruce [Picea abies (L.) Karst.] located in the eastern part of Finland. While the soil temperature at depths of 10 and 50 cm rose above 0°C at the beginning of May in the control treatment (CTRL), it stayed close to 0°C until mid-July in the delayed soil-thawing treatment (FROST). Budburst was delayed by a few days by the FROST treatment. Treatment affected the physiology of previous- and current-year needles, i.e. their electrical impedance, potential efficiency of photosystem II (F v/F m), and chlorophyll a and b content. At the end of the growing season, in November, both the area of the central cylinder and all of the measured dimensions inside the cylinder in the current needles and number of sieve cells in the previous year's needles were smaller in the FROST than the CTRL trees. At the same time, the fully developed dormant buds showed differences only in their side shoots, the width of the buds being smaller in the FROST than the CTRL trees. No effect was found on the onset of wood formation. In contrast, the radial increment tended to be faster in the delayed soil-thawing treatment during late summer.

Genotypic variation in cold tolerance influences the yield of Miscanthus

AbstractWhen grown in Europe, Miscanthus genotypes often produce yields lower than their potential due to late emergence of shoots in the spring or to damage from late frosts when shoots emerge too early. Here, we investigate genotypic variation in the base temperature (Tb) for shoot emergence and in the lethal temperature for shoots (LT50) in four Miscanthus genotypes. In all genotypes, lowering temperature increased the time to shoot emergence, with Tb ranging from 8.6°C in Sac‐5 to 6°C in Sin‐H9. Frost treatments below −8°C resulted in a marked reduction in growth in all four genotypes. Sin‐H9 was the most frost tolerant with an LT50 of −9.3°C. There was little variation found in leaf osmotic potential, but leaf moisture content was significantly lower in Sin‐H9 than in the other genotypes. The lower thermal requirement for emergence and lower LT50 seen in Sin‐H9 was incorporated into a model of Miscanthus production. The model showed an extended growing season that was predicted to increase yields by up to 25%.

Variability in the Relationship between Frost Temperature and Injury Level for Some Cultivated Prunus Species

The influence of the species in spring frost sensibility was determined for the Prunus species peach (P. persica (L.) Batsch), sweet cherry (P. avium L.), almond (P. dulcis (Mill.) Webb/P. amygdalus Batsch), japanese plum (P. salicina Lindl.), and blackthorn (P. spinosa L.). The confidence intervals for lethal temperatures of 10% (LT10) and 90% (LT90) bud injury were also determined. In 2000 and 2001, seven frost treatments were made for each one of the phenological stages comprised between B (first swell) and I (jacket split) in two cultivars per each species. The relationships between frost temperature and the proportion of frost damaged buds for each cultivar, year, and phenological stage were adjusted to linear regression models. The 95% confidence intervals were also calculated. The spring frost hardiness order of the species, from the least to most hardy, was as follows: sweet cherry, almond, peach, japanese plum, and blackthorn. Despite the highly homogeneous nature of the frost and bud characteristics, the temperature range for a given injury degree was quite broad, since the confidence interval's breadth for LT10 was as high as about 3 °C and as high as about 6 °C for LT90. Consequently, when critical temperatures are used in making decisions as to when to begin active frost protection, a prudent measure would be to take the temperature references from the upper limits in the confidence intervals.

Photosynthesis of birch (Betula pendula) is sensitive to springtime frost and ozone

Impacts of springtime frost and ozone enrichment, alone and in combination, on six birch (Betula pendula Roth) genotypes regenerated from a naturally occurring birch stand in southeastern Finland were studied. The seedlings were exposed to 65 ppb ozone (AOT40 (accumulated over a threshold of 40 ppb) exposure of 10.7 ppm·h) over 62 d in climate chambers, simulating spring conditions, and to –2 °C over two consecutive nights 33 d after the start of the experiment. The plants were measured for net photosynthesis, stomatal conductance, and concentrations of photosynthetic pigments, Rubisco, soluble proteins, carbohydrates, and macronutrients. Frost treatment caused a rapid 60%–77% decline in net photosynthesis and stomatal conductance rates. Recovery of net photosynthesis from frost was not complete during the subsequent 14 d, mainly because of impaired light capture through significant pigment loss and structural injuries. Concomitant ozone enrichment exacerbated the negative effect of frost on pigments and stomatal conductance. Both frost and ozone caused nutrient imbalance and increase in soluble proteins in leaves, whereas metabolism of carbohydrates was disturbed only when ozone was present. Responses to ozone and frost varied greatly among the genotypes, suggesting that there is a high capacity within the birch population to adapt to climate change through "preadapted" individuals.

EVALUATION OF FROST RESISTANCE OF TRADITIONAL AND NEWLY BRED HUNGARIAN WIN-GRAPE CULTIVARS

The frost hardiness of 102 traditional and newly bred Hungarian Vitis vinifera and interspecific cultivars were tested using artificial frost treatments in climatic chambers. The sugar content of treated buds was analysed for sucrose, glucose, fructose and raffinose by over pressure layer chromatography. The traditional Hungarian cultivars belonging to convar. Pontica, are regarded as tender cultivars, but some of these varieties which can only be found in gene banks showed fairly good frost tolerance. In the new V. vinifera varieties, ‘Cserszegi fűszeres’ and ‘Generosa’ were the most hardy. Among the interspecific cultivars ‘2426’ (Franco-American origin), ‘Amadeus’, ‘Orpheus’, ‘Odysseus’ and ‘Pannon frankos’ (V. amurensis origin) showed excellent frost resistance.

Plant responses of quinoa ( Chenopodium quinoa Willd.) to frost at various phenological stages

Frost is one of the principal limiting factors for agricultural production in the high Andean region. One of the most important grain crops in that region, quinoa ( Chenopodium quinoa Willd.), is generally less affected by frost than most other crop species, but little is known about its specific mechanisms for frost resistance. This study was undertaken to help understand quinoa’s response to various intensities and durations of frost under different levels of relative humidity (RH). The effect of frost on seed yield and plant death rate was studied, and content of soluble sugars, proteins, and free proline, was analyzed, in order to develop criteria for the selection of cultivars with improved resistance to frost. On the basis of greenhouse and phytotron experiments, it was concluded that at the two-leaf stage, cultivars from the altiplano of Peru, 3800 m above sea level, tolerated −8 °C for 4 h, whereas a cultivar from the Andean valleys tolerated the same temperature for only 2 h. At −4 °C, plant death rate increased from 25% at high relative humidity to 56% at low RH After a frost treatment of −4 °C applied at the two-leaf stage, final seed yield was reduced by 9% compared to control plants not exposed to frost. For the same treatment applied at the 12-leaf and flowering stages, yield reductions were 51 and 66%, respectively, indicating that frost for 2 h or more during anthesis caused significant damage to the plants. In general, an increased level of soluble sugars implied a greater tolerance to frost, resulting in higher yields.

Plant responses of quinoa (Chenopodium quinoa Willd.) to frost at various phenological stages

Frost is one of the principal limiting factors for agricultural production in the high Andean region. One of the most important grain crops in that region, quinoa (Chenopodium quinoa Willd.), is generally less affected by frost than most other crop species, but little is known about its specific mechanisms for frost resistance. This study was undertaken to help understand quinoa’s response to various intensities and durations of frost under different levels of relative humidity (RH). The effect of frost on seed yield and plant death rate was studied, and content of soluble sugars, proteins, and free proline, was analyzed, in order to develop criteria for the selection of cultivars with improved resistance to frost. On the basis of greenhouse and phytotron experiments, it was concluded that at the two-leaf stage, cultivars from the altiplano of Peru, 3800 m above sea level, tolerated −8 °C for 4 h, whereas a cultivar from the Andean valleys tolerated the same temperature for only 2 h. At −4 °C, plant death rate increased from 25% at high relative humidity to 56% at low RH After a frost treatment of −4 °C applied at the two-leaf stage, final seed yield was reduced by 9% compared to control plants not exposed to frost. For the same treatment applied at the 12-leaf and flowering stages, yield reductions were 51 and 66%, respectively, indicating that frost for 2 h or more during anthesis caused significant damage to the plants. In general, an increased level of soluble sugars implied a greater tolerance to frost, resulting in higher yields.

National Forgetting and Remembering in the Poetry of Robert Frost

Forgetting/7 wrote the French historian Ernest Renan, a crucial factor in the creation of a Indeed, he continues, historical enquiry brings to light deeds of violence which took place at the origin of all political formations, even of those whose consequences have been altogether beneficial. Unity is always effected by means of (11). In the case of the United States, the conquest of Native Americans exemplifies the violence that according to Renan must always be forgotten in the formation of a nation. A number of Robert Frost's poems reflect the necessary forgetting that Renan describes, but many of them engage in acts of remembering that honor the past without subverting any particular ideology. As a poet, Frost is both settled and unsettling, a writer who composes without resorting to simplistic moral categories or the easy romanticization of Indians as noble savages. At the same time that his poems testify to their conflicting positions within the Joycean nightmare of history, Frost himself distrusted progressive models . . . and was apt to see certain of his inheritances as natural and unchangeable (Rotella, 242). In his thinking about national history and empire, Frost adopts a Virgilian perspective, assuming that tears are in the nature of things and that in the long-term perspective of human history, the European conquest of the Americas merely gave rise to the world's most recent empire, which in its turn, too, would someday fall. In particular, Frost's treatment of the theme of the American Indian shows that despite the willed forgetting entailed by national narratives, the memory of the brutality that founds the nation persists in the imagination of European Americans. Many of Frost's poems show the ways in which that memory can haunt otherwise confident expressions of patriotism, troubling complacent formulations of American history as a straightforward progress toward freedom and equality.

Interactive effect of springtime frost and elevated ozone on early growth, foliar injuries and leaf structure of birch (Betula pendula).

• Impacts of ozone and late frost on six birch (Betula pendula) genotypes from south-eastern Finland were studied in an 8-wk chamber experiment. • The plants were measured for bud burst, growth, visible foliar injuries caused by ozone and frost, structural leaf properties and changes in chloroplasts. • Ozone delayed bud burst but stimulated subsequent growth. Acute frost injuries were compensated by increased leaf production. Early bud burst predisposed to frost damage, whereas late bud burst increased the vulnerability to ozone. In combined ozone + frost treatment, freezing reduced visible ozone injuries, counteracted ozone-induced growth enhancement and stomatal changes, and exacerbated ozone-caused reduction in palisade cell, chloroplast and starch grain size. Rapid changes in epidermal cell differentiation towards stomata and/or glandular trichomes occurred to enhance ozone/frost tolerance. • The results showed large genetic variation within birch population in response to frost and ozone. Generally, birch seem to recover from acute frost occurrence efficiently through compensating leaf production, but co-occurring ozone enhancement may disturb the recovery processes mechanistically through structural damage in photosynthetic tissue, especially in chloroplasts.

Characterizing the frost sensitivity of black spruce photosynthesis during cold acclimation.

We used photosynthetic light response curves to measure and model the responses of two provenances of 3-year-old black spruce (Picea mariana (Mill.) BSP) seedlings to severe artificial frost treatments applied at 2-week intervals during cold acclimation. Black spruce seedlings responded to cold acclimation with long-term suppression of photosynthetic capacity (Amax) and apparent quantum-use efficiency (alpha'). Short-term reductions in both photosynthetic parameters following frost treatments were dependent on the extent of cold acclimation of the seedlings and the severity of the frost treatments. Large reductions in Amax in response to the frost treatments were observed in seedlings that had undergone little cold acclimation and these reductions were associated with an irreversible reduction in alpha'. Such seedlings recovered only partially during the subsequent 23 days, whereas seedlings in most other treatments showed complete recovery of Amax after 13 days. The impact of frost treatments on Amax and alpha' did not vary with seedling provenance. We propose an algorithm that predicts the combined effects of cold acclimation and severe freezing temperatures on the extent of the suppression of A(max) during autumn. The algorithm is based on (1) the maximum Amax observed during the growing season, (2) the accumulation of cold degree-days, based on a minimum nocturnal temperature < 5 degrees C, and (3) the severity of freezing temperatures during autumn. The parameters developed in the algorithm showed that cold acclimation of black spruce seedlings had a greater impact on the reduction of Amax in autumn than did the severe frost treatments. Mean Amax of seedlings subjected to artificial frosts showed a strong correlation with values predicted by the algorithm (r2 = 0.91).

Effects of hard frost and freeze-thaw cycles on decomposer communities and N mineralisation in boreal forest soil

Decomposition and mineralisation rates generally increase with increasing moisture and temperature. The expected global climate change may enhance precipitation and raise the temperatures at boreal latitudes, but absence of snow together with occasional low temperatures may cause disturbances in soil processes and faunal communities. To test the effects of disturbances such as hard frosts and freeze-thaw cycles on decomposer populations and N mineralisation, we performed two experiments. In the field experiment, carried out in a pine forest, we induced low soil temperatures by preventing snow covering the ground. In the laboratory test we established three “winter” temperature regimes: constant −2 °C, freeze-thaw cycles of −2° to +2 °C, and the latter with two hard frost periods (−16 °C). The microcosm experiment included two decomposer communities; the “simple” community included microbes, protozoa and nematodes, and the “complex” community microbes, protozoa, nematodes, enchytraeids and microarthropods. In the field experiment, the populations of enchytraeids and several microarthropod taxa, as well as microarthropod species richness, decreased in snowfree plots. In the microcosms, virtually all enchytraeids died and microarthropod populations and taxa decreased in the hard frost treatment, but increased in the freeze-thaw treatment. In the laboratory systems with simple decomposer community structure the amount of mineral nitrogen decreased in the freeze-thaw treatment. The experiments revealed that exceptionally low soil temperatures exert a stronger influence on soil fauna than constant benign temperatures or freeze-thaw cycles, but the rapid recovery of populations may counteract the detrimental effect of occasional frost periods.

Methods of Establishing Annual Ryegrass and Clover into Broomsedge‐Infested Pastures

Establishing cool‐season forages into broomsedge (Andropogon virginicus L.)–infested pastures would increase forage nutritive value and extend the grazing season. Experiments were established in southeastern Oklahoma in late summer at four locations in 1995 and at six locations in 1996 to determine the effects of three forage removal techniques for establishing clovers (Trifolium spp.) and annual ryegrass (Lolium multiflorum Lam.). The main plots used in both years included: (i) mowing plus vegetation removal in late summer, (ii) paraquat (1,1′‐dimethyl‐4,4′‐bipyridinium ion) plus burning 1 wk after treatment (WAT), and (iii) burning after frost. In 1995, two annual ryegrasses (‘Marshall’ and ‘TAM90’) and three clovers [‘Yuchi’ arrowleaf (T. vesiculosum Savi.), ‘Overton R18’ rose (T. hirtum All.), and ‘Ladino’ white (T. repens L.)] were broadcast‐seeded into the three seedbed preparations. In the 1995 planting, dry conditions in the fall resulted in no establishment of annual ryegrass and erratic establishment and production of clovers. In 1996, Marshall annual ryegrass and a clover mix [arrowleaf, red (T. pratense L.), rose, and white] were each broadcast‐seeded into tilled and nontilled subplots in the three seedbed preparations. Clover and annual ryegrass dry matter (DM) production was generally highest in paraquat plus burn treatment and lowest in the burn after frost treatment. Tillage generally did not improve clover or annual ryegrass production. The nontilled paraquat plus burn treatment produced the most forage and had the lowest production cost for both clover and annual ryegrass; therefore, it should be considered when establishing clover and annual ryegrass into permanent pastures.

Chlorophyll fluorescence and CO(2) assimilation of black spruce seedlings following frost in different temperature and light conditions.

Effects of artificial frosts on light-saturated photosynthesis (A(max)) and ground, maximal and variable fluorescence variables (F(o), F(m), and F(v) and F(v)/F(m)) were monitored on 1-year-old foliage of black spruce seedlings (Picea mariana (Mill.) BSP) grown at high (25 degrees C), moderate (15 degrees C) and low (5 degrees C) temperatures and moderate (240 &mgr;mol m(-2) s(-1)) and low (80 &mgr;mol m(-2) s(-1)) irradiances. Photoinhibition of 1-year-old foliage was greater in seedlings grown in moderate light than in seedlings grown in low light. Photoinhibition increased with decreasing growth chamber temperature at both irradiances. Most changes in F(v)/F(m) were caused by changes in F(v). Exposure to -4 degrees C decreased both F(v)/F(m) and A(max) compared with control values. The effect of the -4 degrees C frost treatment was greater in seedlings grown in low light than in seedlings grown in moderate light, probably because seedlings grown in moderate light were already partially photoinhibited before the frost treatment. Following -4 degrees C treatment, neither F(v)/F(m) nor A(max) recovered in seedlings grown in low light. Light-saturated photosynthesis decreased with decreasing growth chamber temperature. Light-saturated photosynthesis was more sensitive to the -3 and -4 degrees C frost treatments in seedlings grown at 25 degrees C than in seedlings grown at 15 and 5 degrees C. The A(max) of seedlings grown at 15 degrees C was sensitive only to the -4 degrees C frost treatment, whereas A(max) of seedlings grown at 5 degrees C was not sensitive to any of the frost treatments. Recovery of A(max) following frost took longer in seedlings grown at high temperatures than in seedlings grown at low temperatures. For seedlings grown at the same temperature but under different irradiances, both A(max) and F(v)/F(m) reflected damage to the photosynthetic system following a moderate frost. However, for seedlings grown at the same irradiance but different temperatures, A(max) provided a more sensitive indicator of frost damage to the photosynthetic system than F(v)/F(m) ratio.

Readiness to frost harden during the dehardening period measured in situ in leaves of Rhododendron ferrugineum L. at the alpine timberline

Summary The readiness to frost harden during the dehardening period can be crucial to the frost survival of Rhododendron ferrugineum leaves. When already dehardened plants occasionally fall snow free in late winter or spring still heavy night frosts can occur in the subalpine environment. R. ferrugineum shrubs were treated in situ using a newly developed field portable freezing chamber. They were exposed to controlled night frosts of close to the lowest temperature sustained without frost damage (LT 0 ) to determine the potential frost hardening response under otherwise completely natural conditions. There was a lag period of 3 days during which no significant increase of frost resistance was observed. After three days frost resistance increased suddenly by 5.9°C within 24 hours. High daytime leaf temperatures (+19°C) combined with night frosts further retarded the rate of frost hardening. The in situ frost treatment generally yielded frost resistances (LT 10 ) approaching the highest ever measured in that season. In early spring the in situ frost hardening response was three times greater than early reports for detached twigs with a total hardiness gain between 7.8°C and 9.2°C. It is suggested that the limiting step in frost hardening in leaves of Rhododendron ferrugineum in spring is not the extent of frost resistance achieveable but rather the slow rate of frost hardening.

ARTIFICIAL FROST TREATMENT METHODS OF STONE FRUITS

Routine application of artificial frost treatment needs a detailed, exact technology, which would ensure the reliability of the results obtained. Buds being on spurs loose their frost resistance sooner because of their quick development. According to these results to examine samples containing both types of buds can lead to significant mistakes. Considering our experiences it seems that a sample containing 200 buds provides the reliable correctness. Further increasing of sample size decreases the value of deviation but it is not proportional to the work needed for the experiment. The following treatments are suggested: deep dormancy phase: -24 - -26°C, directly after deep dormancy:-21 - -24°C. beginning of February: -19 - -21°C, two weeks before blossoming -11- -12°C. two -days before blossoming: -4 - -6°C.

Robert Frost's Arthropods

ARTHROPODS HAVEA SPECIAL PLACE IN THE poetry of Robert Frost (18741963). In Frost's world, commonplace things, especially natural ones, take on cosmic meanings. He uses stone walls, birch trees, brooks, snowy woods, forks in a road, and a diversity of animals to raise universal questions about life and the nature of time and space. Frost uses arthropods as vehicles for raising philosophical issues. With arthropods as central characters, he constructs little scenes or natural history vignettes with themes that, when listed, look like a sylla bus of general entomology-plantinsect interactions, biological control/predation, cryptic coloration, resource utilization, effects of plant secondary compounds, and instinct and learning among many others. Although several dozen poems mention arthropods, only 17 of Frost's 347 poems are focused on insects, mites, or spiders 1, This fact, in itself, does not recommend Frost as poet laureate of entomology. However, humor and richness of those poems, their accuracy of detail, their thought-provoking themes as well as affection and respect with which he treats arthropods all recommend this great American poet's works to anyone who loves insects and their kin. For those of us who have chosen to dedicate our lives to arthropods, it is reinforcing to see insects and their kin respectfully and lovingly depicted and interpreted by artists, photographers, and poets. In this vein, I long have found Frost's treatment of arthropods a rewarding and thought-provoking experience that grows richer every time Ire-read works that I discuss here.

The effects of radiation frost on freezing damage and apical abortion in calabrese (Brassica oleracea var. italica) transplants

SummaryFreezing damage has been reported to be associated with apical abortion (blindness) in calabrese, preventing the formation of a marketable head. In the past, laboratory based experiments studying the freezing of whole plants using convective freezing have resulted in death of the plants while failing to induce blindness. Using a unique radiation freezing facility, capable of reproducibly simulating glasshouse frost conditions, we investigated the effect of hardening and freezing duration on calabrese transplants. Four cultivars were raised and subjected at four different growth stages (3, 5, 7 and 9 leaves) to a factorial combination of frost (0-4 d of frost between 22 to 248C) and hardening regimes (0-6 d at 48C prior to freezing). Following each frost/hardening treatment, half the sample was dissected and the remainder potted up and grown on. Light and transmission electron microscopy of the dissected plants revealed that the apical region of all the varieties at each of the growth stages remained intact following all frost treatments applied. No significant apical abortion was recorded. No blind plants were recorded following a repeat of the experiment using commercially grown plants (cv. Marathon). It was observed that the apical region of the calabrese transplants has a high degree of frost tolerance and that freezing conditions alone are not responsible for the induction of apical abortion.

Development of freezing tolerance in roots and shoots of Scots pine seedlings at nonfreezing temperatures

The hardening of hydroponically cultured Scots pine (Pinus sylvestris L.) seedlings and their recovery after freezing was studied at the end of the second growing season (LD), after 3 weeks of short day treatment (SD), after a gradual decrease in temperature to 5°C over 4 weeks (H1), and after 4 weeks at 5°C (H2). Frost hardiness was determined by several methods and the recovery as survival of the seedlings. The highest frost hardiness was achieved in the distal parts of needles (-21 to -27°C) and in the proximal parts of needles (-18 to -25°C), followed by woody roots (-7 to -9°C), the 1-year-old and current stem (-8°C), and the fine roots (-5°C), all at the end of H2. Hardening of needles was induced by SD, but the stem and woody roots started to harden later, as a response to low temperature. As a result of frost treatment during LD and SD, potential plasma membrane roman H+-ATPase activity of roots decreased as electrolyte leakage increased and extracellular resistance decreased, but this relationship was lost during H1 and H2. The present study demonstrates the lack of hardening capacity in the fine roots of Scots pine seedlings at nonfreezing temperatures and the increased mortality of the young seedlings having frost damage on roots.

Frost damage and recovery of Scots pine seedlings at the end of the growing season

Freeze-thaw injury and recovery were studied in unhardened seedlings of Scots pine (Pinus sylvestris L.) at the end of the second growing season. Visual damage scoring, microscopy, gas exchange, and plasma membrane H + -ATPase activity were used to determine the degree of damage to the needles. The measurements were performed immediately after the frost treatments and after a 21-day recovery period under favorable conditions. The first signs of injuries were found by light microscopy immediately after the frost treatments in the mesophyll cells of needles exposed to -2°C. Low H + -ATPase activity indicated that injuries occurred at -3.5°C and gas exchange at -5°C. The seedlings exposed to -6.5°C or below lost their needles and died. The seedlings exposed to -5°C showed incomplete recovery and irreversible damage after 21 days when assessed structurally and by gas exchange and visual scoring. At the microscopic level, recovery was complete in the needles exposed to -3.5 and -2°C. The needles subjected to -3.5°C showed high H + -ATPase activity, indicating ongoing repair. Accordingly, the temperature range for cellular damage to unhardened needles was between -2 and -5°C, depending