Supercooling ability of Rhododendron flower buds in relation to cooling rate and cold hardiness

The freezing process and supercooling ability in flower budsof 11 native Rhododendron species were examined with referenceto the cooling rate and cold hardiness by differential thermalanalysis. The freezing patterns of the excised whole buds variedwith the season: in autumn, buds froze as whole units, whilein winter, freezing was initiated in the scales and propagatedto each floret. The supercooling ability of florets was enhancedduring winter. The freezing patterns in winter buds were stronglyinfluenced by the cooling rate (1 to 30°C/hr). Althoughthe first exotherm in scales occurred at –5 to –10°Gand was rate-independent, the occurrence of several floret exothermsshifted considerably to lower subzero temperatures at slowerrates. The most reliable cooling rate for testing maximum supercoolingability was l°C/hr. The exotherm in florets of hardier speciesoccurred at –20 to –25°C and at –7 to–20°C for less hardy ones, and were well correlatedwith their killing temperatures. Water relations within budtissues in response to freezing are briefly discussed. (Received June 26, 1980; )     

Freezing Avoidance Mechanisms by Supercooling in Some Rhododendron flower Buds with Reference to Water Relations

Excised florets of some hardy Rhododendron species did not toleratefreezing at –5°C when ice-inoculated due to intracellularfreezing. Florets in intact December buds, however, could besupercooled to about –30°C. When flower buds of R.japonicum were slowly cooled with daily decrements of 5°Cto temperatures ranging from 0 to –20°C, the exothermtemperatures of the florets drastically decreased. This wasaccompanied by a decrease in water content of florets and peduncleand an increase in that of scales. The water in florets andthe peduncle is thought to migrate to scales and other tissuesduring the early stages of freezing; the dehydrated floret hasa lower freezing point which enhances its supercooling abilityand the dehydrated peduncle helps to maintain the supercooledstate of the florets. This hypothesis would explain the dependenceon the cooling rate of supercooling in Rhododendron flower buds.Water migration within flower buds was observed in other hardyRhododendron species with some variation in ice formation siteand the quantity of migrated water. The exotherm temperatureof excised florets was inversely proportional to their watercontent. Dehydration of flower buds by wind at 0°C alsoenhanced their supercooling ability. Mechanisms of freezingavoidance by supercooling in Rhododendron flower buds and therelationship of supercooling to freezing tolerance are discussed. ¹ Contribution No. 2254 from the Institute of Low TemperatureScience ² This is a revised form of the master's thesis of the seniorauthor (M.I.) which is cited in the present and previous papers(Sakai 1979a, b, etc.). (Received August 11, 1980; Accepted June 1, 1981)     

Supercooling Ability, Water Content and Hardiness of Rhododendron Flower Buds during Cold Acclimation and Deacclimation

The relationship between supercooling ability and water contentand killing temperature of flower buds during cold acclimationand deacclimation were studied using R. kiusianum and R. x akebono.The occurrence of multiple floret exotherms and their shiftto a narrow range at lower subzero temperatures, as well asthe marked decrease of florets water content, were observedas the symptoms of cold acclimation occuring in flower budsfrom fall to winter, and vice versa in spring buds during deacclimation.In R. kiusianum, the fully acclimated period was from Novemberto March and two months longer than that of R. x akebono. Thesupercooling ability of the former was about –25°Cand about –20°C in the latter. Although the watermigration within bud tissues during the freezing process wasdetermined in the acclimated and deacclimated buds for R. xakebono, no significant water changes could be observed, evenin the acclimated buds. Thus, it is conceivable that deep supercoolingin florets may result not necessarily from water migration fromflorets and bud axes to scales in response to freezing, butfrom low water content in situ of cold-acclimated or artificiallydehydrated flower buds. (Received July 29, 1981; Accepted October 12, 1981)     

Low temperature exotherms of winter buds of hardy conifers

Differential thermal analysis (DTA) of winter buds and the excisedprimordial shoots of sub-alpine or sub-cold firs revealed thatthese buds had all low temperature exotherms around –30?C.However, no low temperature exotherm below –15?C was detectedin the spring buds. In the winter bud of Abies firma, a temperatefir native to Japan, a low temperature exotherm was detectedaround –20?C, which is higher by 10?C than that of sub-alpineor sub-cold firs. The low temperature exotherms of these firsoccurred at nearly the same temperatures that result in thedeath of these primordial shoots. On the other hand, littleor no low temperature exotherm was detected in the winter budsof sub-cold spruces. In larch winter buds, numerous small exothermswere observed, which are probably due to the many leaf primordiain the buds. Unlike many temperate deciduous broad-leaved trees,no low temperature exotherm was detected below –15?C inwinter twig xylem of conifers such as Abies, Picea, Pinus, Larixand Pseudotsuga. Thus, very hardy coniferous twigs can tolerateextracellular freezing to –70?C. ¹ Contribution No. 1907 from the Institute of Low TemperatureScience. (Received June 8, 1978; )     

Effects of Temperature on Cold Acclimation and Deacclimation in Flower Buds of Evergreen Azaleas

The effects of various storage temperature/duration combinations(5, 10 and 17°/4, 8, 12 and 16 weeks) on cold acclimationand deacclimation of flower buds were studied in four speciesof evergreen azaleas having different natural distribution andcold hardiness. The freezing process and the exotherm temperaturedistribution of florets in excised whole buds determined bydifferential thermal analysis were used as the diagnostics todetermine the degree of bud acclimation and deacclimation. Theacclimation in buds lasted for as long as 12 to 16 weeks at5°C storage, and from 8 to 12 weeks at 10°C, and itappeared to be maintained after the chilling requirement forbreaking bud dormancy had been satisfied. Therefore, bud acclimationseems to be maintained independently from bud dormancy. Thedehardening effect on acclimated buds occurred as a result ofshort exposures to higher temperatures or long exposures tolower temperatures, and there was no relation between the rateof deacclimation and the degree of hardiness in each species.Among three storage temperatures examined, 5°C was the mosteffective for the maintenance of cold acclimation in flowerbuds and the small difference of floret water contents at 5and 10°C storage is not significant. (Received August 28, 1982; Accepted February 4, 1983)     

Extraorgan freezing in wintering flower buds of Cornus officinalis Sieb. et Zucc.

Abstract. Extraorgan freezing as a mechanism for increasing cold hardiness was shown using flower buds of Cornus officinalis Sieb. et Zucc. Differential thermal analysis (DTA) revealed that florets in flower buds of C. officinalis owed their cold hardiness to deep supercooling and also that slower cooling rates increased the supercooling ability of florets. During slow stepwise cooling (5°C h⁻¹), the water content of florets decreased and that of scales (involucral bracts) increased, which resulted in accumulation of ice within the scales. This was more extensive in early winter and early spring buds than mid-winter ones. Flower buds with silicone oil in the space between florets and scales also showed a similar decrease in water content of florets and an increase in that of scales. This indicated that water migration from the florets to the scales probably took place by way of the peduncles and the receptacle, possibly through their vascular traces, and not directly from the surface of the florets to the ice sink in the form of vapour. Possible mechanisms of extraorgan freezing are postulated along with this finding.     

The Freezing Response of an Arctic Cushion Plant, Saxifraga caespitosa L.: Acclimation, Freezing Tolerance and Ice Nucleation

Cold hardiness in actively growing plants of Saxifraga caespitosaL., an arctic and subarctic cushion plant, was examined. Plantscollected from subarctic and arctic sites were cultivated ina phytotron at temperatures of 3, 9, 12 and 21 °C undera 24-h photoperiod, and examined for freezing tolerance usingcontrolled freezing at a cooling rate of 3–4 °C eitherin air or in moist sand. Post-freezing injury was assessed byvisual inspection and with chlorophyll fluorescence, which appearedto be well suited for the evaluation of injury in Saxifragaleaves. Freezing of excised leaves in moist sand distinguishedwell among the various treatments, but the differences werepartly masked by significant supercooling when the tissue wasfrozen in air. Excised leaves, meristems, stem tissue and flowerssupercooled to –9 to –15 °C, but in rosettesand in intact plants ice nucleation was initiated at –4to –7 °C. The arctic plants tended to be more coldhardy than the subarctic plants, but in plants from both locationscold hardiness increased significantly with decreasing growthtemperature. Plants grown at 12 °C or less developed resistanceto freezing, and excised leaves of arctic Saxifraga grown at3 °C survived temperatures down to about –20 °C.Exposure to –3 °C temperature for up to 5 d did notsignificantly enhance the hardiness obtained at 3 °C. Whenwhole plants of arctic Saxifraga were frozen, with roots protectedfrom freezing, they survived –15 °C and –25°C when cultivated at 12 and 3 °C, respectively, althougha high percentage of the leaves were killed. The basal levelof freezing tolerance maintained in these plants throughoutperiods of active growth may have adaptive significance in subarcticand arctic environments. Saxifraga caespitosa L., arctic, chlorophyll fluorescence, cold acclimation, cushion plant, freezing stress, freezing tolerance, ice nucleation, supercooling     

Freezing avoidance mechanism of primordial shoots of conifer buds

Excised winter buds of very hardy fir supercooled to —30or — 35?C, though primordial shoots excised from thesewinter buds (freezing point: about —5.5?C) supercooledonly to —12 to — 14?C. Also, excised primordialshoots did not tolerate freezing, but were rather resistantto desiccation. Differential thermal analysis (DTA) of primordialshoots revealed that the capability of supercooling increasedwith decreasing water content and that no exotherm could bedetected in the primordial shoots with a water content belowabout 20%. When excised whole buds were cooled very slowly,the exotherm temperature shifted markedly to a lower value andthe exotherm became much smaller. Also, masses of needle icewere observed, mainly beneath the crown of the primordial shoot.From these results, it may be concluded that most of the waterin primordial shoots gradually migrates out through the crownand freezes as the temperature decreases (extraorgan freezing),which enables primordial shoots to survive at very low temperatures.Winter buds of Abies balsamea held at — 20?C for 30 daysand then slowly cooled down to —50 or —60?C remainedalive. Thus, there seems to be no low temperature limit to thisfrost avoidance mechanism, if the primordial shoots can resistintensive freeze-dehydration. Low temperature exotherms wereobserved in all genera which belong to Abietoideae and Laricoideaeof Pinaceae, all of which have a crown in the primordial shoots,but not in other conifers. ¹ Contribution No. 2037 from the Institute of Low TemperatureScience. (Received June 25, 1979; )     

Freeze Desiccation: a Second Mechanism for the Survival of Hydrated Lettuce (Lactuca sativa L.) Seed at Sub-zero Temperatures

Differential Thermal Analysis of hydrated lettuce cv. GreatLakes achenes using a rapid cooling rate (20 °C h^–1)produced two exotherms per achene. Both exotherms representedthe freezing of supercooled water. The high temperature exothermoccurred at –93 °C and was produced by freezing ofwater inside the pericarp but exterior to the endosperm. Thetemperature at which it occurred could be altered by the additionof nucleating agents. The low temperature exotherm produced by freezing of the embryooccurred at –162 °C and marked the death of the seed.Its temperature was not changed by the addition of nucleatingagents but its occurrence required the structural integrityof the endosperm. At low cooling rates (1 and 2 °C h^–1)low temperature exotherms were not recorded and samples removedat –25 °C had high viability. Slow cooling causeda redistribution of water within the seed whereby ice formingoutside the endosperm caused desiccation of the embryo and preventedits freezing. A mechanism is proposed, in terms of established supercoolingand nucleation theory, to explain the observed results and thevalue of freeze tolerance to the species in its natural habitatis discussed. Cooling rate, differential thermal analysis, freezing avoidance, Lactuca sativa L., lettuce, seed, supercooling, water migration     

Winterfat (Eurotia lanata(Pursh) Moq.) Seedbed Ecology: Low Temperature Exotherms and Cold Hardiness in Hydrated Seeds as Influenced by Imbibition Temperature

Thermal analyses of freezing events in hydrated lettuce (LactucasativaL.) seeds show a correlation between low temperature exotherms(LTEs) (evidence of ice crystal formation) and seed death. Yet,weather patterns common to the Northern Great Plains of NorthAmerica regularly create conditions where non-dormant seedsof native plants hydrate with snow melt and are subsequentlyexposed to -30 °C or colder conditions. To determine ifsuch weather patterns decimate dispersed seeds, we measuredthe effects of freezing on fully hydrated winterfat (Eurotialanata(Pursh) Moq.) seeds harvested from the Northern Plainsat two USA and one Canadian location. Survival of hydrated seedsto -30 °C at a cooling rate of 2.5 °C h^-1was similarto that of seeds not subjected to cooling, even though botha high temperature exotherm (HTE) and an LTE were observed.Although the LTE was not related to winterfat seed survival,freeze-stressed seeds had reduced germination rates and reducedseedling vigour, particularly for the collection with the lightestseeds. The temperature of LTEs was similar among seed collectionswith a mean of -17.6 °C, but was warmer when the seeds wereimbibed at 0 °C compared to 5, 10 or 20 °C. We founda significant correlation between the HTE and LTE temperatures.The difference and the correlation may be due to the highermoisture content of seeds imbibed at 0 °C. After pericarpremoval, only one exotherm in the range of the LTE was observed.This was also true for the naked embryo. We conclude that anLTE indicates ice formation in the embryo, but that it doesnot signal the death of a winterfat seed.Copyright 1998 Annalsof Botany Company Eurotia lanata(Pursh) Moq.,Krascheninnikovia, Ceratoides,winterfat, exotherm, freezing tolerance, freezing avoidance, seedbed ecology, germination, D50, seedling vigour, seed collection     

Influence of Temperature and Floret Age on Nectar Secretion in Trifolium repens L.

The influence of temperature on nectar secretion in non-pollinatedflorets of Trifolium repens was investigated in growth chambersat 10, 14, 18 and 22°C. The effect of temperature on therate of nectar secretion was significant in all clones. Theoptimum temperature for secretion in three clones varied from10°C for a clone of Icelandic origin, to 18°C in a cloneselected from a Danish variety. Similarly, the average nectaryield varied significantly among clones of different geographicalorigin. One clone secreted two to four times more than othersat 10°C. The optimum day temperature for nectar secretionwas higher when the plants were exposed to low night temperature,presumably a result of decreased night respiration. Nectar accumulatedat the floret base until senescence. Evidence for reabsorptionof nectar was obtained in four clones. Sucrose, fructose andglucose were identified as the major sugars in the nectar. Highnight temperatures led to decreased sucrose percentage in favourof glucose and fructose. The frequency of new florets openingper day was not influenced by temperatures between 10 and 22°Cin one clone, whereas low temperatures significantly decreasedthe number of new florets in another. Few or no modified stomatawere observed in the epidermis of the nectary. The high variationwith respect to nectar secretion at low temperatures, alongwith the high heritability of this quality, suggests that breedingfor high nectar production at low temperature is plausible.The significance of nectar yield in pollination biology is discussed.Copyright1994, 1999 Academic Press Trifolium repens, white clover, nectar, temperature, floret age, flowering, nectary     

Pigment Production by Cultured Florets of Chrysanthemum morifolium

Individual florets (4–5 mm long) of a purple cultivar(Fandango) of the horticultural chrysanthemum (Chrysanthemummorifolium Ramat) were taken from flower buds just prior toopening and cultured in a sterile liquid medium (containinginorganic salts and sucrose) at 15 °C under a 12-h day.For the first 14 days increase in wet weight was exponential.Anthocyanin appeared on the third day and was then synthesizedrapidly. Chlorophyll and carotenoid were present initially:carotenoid levels rose quickly while chlorophyll remained almostconstant. Highest pigment content and most growth were foundwhen the florets were grown on 3 per cent sucrose. However,the highest anthocyanin concentration was found with 4 per centsucrose, the highest carotenoid concentration with 0.6 per centsucrose. No anthocyanin was produced when the florets were grownat 6 or 30 °C; maximum yield was at 15 °C. Most carotenoidwas formed at 30 °C and most chlorophyll was found at 20–5°C. All florets from 1 to 7 mm long could be cultured. Theseresults are discussed in relation to flower colour and pigmentformation in vivo.     

Estimation of the Freezing Injury in Flower Buds of Evergreen Azaleas by Water Proton Nuclear Magnetic Resonance Relaxation Times

Temperature dependence of longitudinal relaxation times (T₁)of water protons in flower buds of six azalea species differingin cold hardiness and ecological distribution was investigatedby pulse nuclear magnetic resonance spectroscopy. Thermal hysteresiswas observed for T₁ following a slow freeze-thaw cycle. TheT₁ ratio (the ratio obtained from the difference between theoriginal T₁ value in an unfrozen sample and the final T₁ aftera freeze-thaw treatment, both at 20C, divided by the originalT₁) was closely correlated with the viability of florets innon-acclimated buds of R. kiusianum. If the buds were frozento a lethal temperature and then thawed to 20C, the T₁ ratioincreased. The T₁ ratios of acclimated winter buds for the sixspecies used were correlated with the level of cold hardiness(supercooling ability of florets determined by differentialthermal analysis). The T₁ ratio of deacclimated spring buds,especially those from hardier species, markedly increased uponcooling to a lethal temperature. Species differences observedin acclimated winter buds disappeared upon deacclimation. TheT₁ ratio appears to be related to the viability of florets andthe degree of freezing damage (membrane disruption) in florets. (Received December 28, 1984; Accepted May 24, 1985)     

Freezing Tolerance in Hydrated Lactuca sativa (L) Seed: A Model to Explain Observed Variation Between Seed Lots

Low temperature tolerance was investigated in the imbibed seedof 15 seed lots compnsmg seven cultivars of Lactuca sativa L.During rapid cooling (20 °C h^–1) some seeds of allseed lots survived to –16 °C but none to –20°C. The majority of seed lots retained over 50 per centviability above –14 °C due to isolation of the embryofrom external ice by the endosperm, and subsequent embryo super-cooling.Certain seed lots, including all three seed lots of cv. TomThumb, showed high mortality at temperatures above –10°C. Correlation of mortality with the formation of externalice suggested that the endosperm is not an effective nucleationbarrier in these seed lots. Survival to –20 °C was increased at slower coolingrates (6 to 1 °C h^–1) due to freeze desiccation ofthe embryo, but seed lots varied considerably in their toleranceof specific cooling rates. A model to explain this variationwas developed incorporatmg (1) seed lot super-cooling limittemperature, (2) the rate at which freeze dehydration of thesupercooled embryo took place, (3) the moisture content at whichnucleation (at –20 °C) was no longer certain and (4)the.initial equilibrium moisture content of the fully imbibedseed. Factors (1), (2) and (3) were found to be relatively constant,but low (or artificially reduced) seed moisture content wasclosely correlated with high survival at natural cooling rates.Seed size fractions of similar moisture content from a singlecultivar showed that more small seeds survive cooling at 3 °Ch^–1 to –20 °C than larger seed. Seed with pierced endosperms or ineffective nucleation barrierswere capable of surviving to at least –10 °C if cooledslowly (1 °C h^–1) but were killed by rapid (20 °Ch^–1) cooling. Lactuca sativa L, lettuce, seed germination freezing tolerance, super-cooling     

Supercooling of xylem ray parenchyma cells in tropical and subtropical hardwood species

 The freezing behavior of xylem ray parenchyma cells in several woody species, Ficus elastica, F. microcarpa, Mangifera indica, Hibiscus Rosa-sinensis, and Schefflera arboricola, that are native to non-frost tropical and subtropical zones, was investigated by differential thermal analysis (DTA), cryo-scanning electron microscopy (cryo-SEM) and freeze-replica electron microscopy. Although profiles after DTA did not exhibit clear evidence of supercooling in the xylem ray parenchyma cells, electron microscopy revealed that the majority of xylem ray parenchyma cells in all of the woody species examined were supercooled to around –10°C upon freezing temperatures and were not frozen extracellularly. It seems likely that DTA failed to reveal the low temperature exotherm (LTE), that is produced by breakdown of supercooling in the xylem ray parenchyma cells as a consequence of the overlap between the high temperature exotherm and the LTE in each case. The xylem ray parenchyma cells in these woody species were very sensitive to dehydration, and supercooling had, to some extent, a protective effect against freezing injury. It is suggested that the capacity for supercooling of xylem ray parenchyma cells of tropical and subtropical woody species might be the result of inherent structural characteristics, such as rigid cell walls and compact xylem tissues, rather than the result of positive adaptation to freezing temperatures. The present and previous results together indicate that the responses of xylem ray parenchyma cells in a wide variety of hardwood species to freezing temperatures can be explained as a continuum, the specifics of which depend upon the temperatures of the growing conditions. Received: 24 January 1997 / Accepted: 13 May 1997     

Effects of Irradiance and Temperature on Flowering of Chrysanthemum morifolium Ramat. in Continuous Light

The short-day plant Chrysanthemum morifolium cv. Polaris initiatedflower buds in all irradiances of continuous light from 7.5to 120 W m^–2. As the irradiance increased, the transitionto reproductive development began earlier and the number ofleaves initiated before the flower bud was reduced. The autumn-floweringcultivars Polaris and Bright Golden Anne, and the summer-floweringGolden Stardust were also grown in continuous light at differenttemperatures; all initiated flower buds at temperatures from10 to 28 °C but only the buds of Golden Stardust developedto anthesis and then only at 10 and 16°C. Flower initiationbegan earliest at 16–22 °C, and the number of leavesformed before the flower bud was increased at 28°C. GoldenStardust was exceptional in that the number of leaves formedwas also increased at 10 °C. Axillary meristems adjacentto the terminal meristem initiated flower buds rapidly at 10°C but not at 28 °C in all three cultivars. These resultsare discussed in relation to the autonomous induction of flowerinitiation and the effects of the natural environment on floweringof chrysanthemum. Chrysanthemum morifolium Ramat, flowering, irradiance, temperature     

Supercooling in overwintering azalea flower buds

Differential thermal analysis and nuclear magnetic resonance spectroscopy experiments on whole flower buds and excised floral primordia of azalea (Rhododendron kosterianum, Schneid.) proved that supercooling is the mode of freezing resistance (avoidance) of azalea flower primordia. Increase in the linewidth of nuclear magnetic resonance spectra for water upon thawing supports the view that injury to the primordia occurs at the moment of freezing. Nonliving primordia freeze at the same temperatures as living primordia, indicating that morphological features of primordial tissues are a key factor in freezing avoidance of dormant azalea flower primordia. Differential thermal analyses was used to study the relationship of cooling rate to the freezing points of floral primordia in whole flower buds. At a cooling rate of 8.5 C per hour, primordia in whole buds froze at about the same subfreezing temperatures as did excised primordia cooled at 37 C per hour. At more rapid cooling rates primordia in intact buds froze at higher temperatures.     

The Effect of High Temperature at Different Stages of Ripening on Grain Set, Grain Weight and Grain Dimensions in the Semi-dwarf Wheat 'Banks'

Grain number in the wheat cultivar Banks was reduced by up to11 % with a rise in temperature from 21/16 °C to 30/25 °Cover a 10-d period immediately following first anthesis in general,the upper ‘d’ and ‘c’ florets were moreaffected by high temperature than the basal ‘a’and ‘b’ florets within a spikelet and florets fromthe upper spikelets were more sensitive than those lower onthe ear Grain weight and grain length at maturity were most affectedby a 10 d period of high temperature commencing 7–10 dafter anthesis However, if dry-matter accumulation between thestart of a treatment and grain maturity was used as a base forcomparison, the response was more uniform throughout development,with a peak in sensitivity 25 d after anthesis Although grainposition within an ear did not have a large effect on the responseto temperature, grains from the basal spikelets were more sensitivethan those from the apex, and the upper floret grains of a spikeletwere more sensitive to high temperature than those at the base There is a need to obtain, for a range of cultivars, more comprehensivedata on the effect of the timing and degree of temperature stressfollowing anthesis, for use in interpreting the response torising temperatures late in the development of the crop in thefield Triticum aestivum L, wheat, temperature, grain development     

Relationship of Nuclear Magnetic Resonance Relaxation Time to Water Content and Cold Hardiness in Flower Buds of Evergreen Azalea

The longitudinal relaxation time (T₁) of water protons in floretsof R. ? akebono flower buds was measured with a pulse NMR spectrometerto determine the relationship of T₁ to water content and coldhardiness (supercooling ability). Seasonal changes of T₁ inflorets were closely correlated with water content and supercoolingability of florets. T₁ of florets was short for acclimated budshaving a low water content and long for non-acclimated budshaving a high water content. Flower buds collected in Novemberand stored at 0 and 5?C for 4 weeks had shorter T₁ values thanbuds stored at 10?C even though the floret water content andsupercooling ability were similar. Thus, the short T₁ of coldacclimated buds hardened naturally or by storage at low temperaturesis due to a combination of both reduced water content and temperature. (Received August 27, 1983; Accepted May 26, 1984)     

The Formation and Distribution of Ice within Forsythia Flower Buds

Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0°C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between −10 and −25°C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze-fixed at −5°C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil.