Translocation at 0{degrees} C in Helianthus annuus

Potassium uptake by Helianthus annuus plants growing in waterculture was found to be closely dependent upon the translocationof sugar to the roots. This relationship was used to determinethe effect of cooling on the rate of translocation. A Q₁₀ ofapproximately 3 over the range 0–25° C was obtainedbut tracer experiments showed that translocation was not stoppedat 0° C. A complete recovery in translocation rate appeared to occurin some experiments after prolonged cooling. It is concludedthat this can only be satisfactorily explained on the assumptionthat carbohydrates move in the sieve tubes by mass flow. Thedriving force of such a mass flow is considered to be locatedin each sieve element.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in the Distribution of 14C-Labelled Assimilates in Sugar-beet with Variation of Temperature

Plants were allowed to assimilate ¹⁴CO₂ for 30 min at 5, 15,25, and 35 °C. The changes in ¹⁴C content of a mature expandedleaf (Leaf 4), young apical leaves, and storage root, were sequentiallyfollowed over a subsequent period of 24 h in continuous light.In a second experiment plants were transferred after ¹⁴CO₂ assimilationto temperatures of 10, 18, 26, and 34 °C, and the partitionof ¹⁴C between the ethanol-soluble and ethanol-insoluble fractionsof the roots and leaves was followed over a period of 72 h. The specific activities of the apical leaves and of the storageroot increased to a maximum 2 h after labelling at 25 °C,4 h at 15 and 35 °C, and 6 h at 5 °C suggesting thatthe optimum temperature for translocation of photosynthate wasabout 25 °C. The ¹⁴C partition to ethanol-soluble and ethanol-insoluble fractionsof the roots and leaves was largely attained in. 9 h. Littlerepartition of ¹⁴C assimilate fractions occurred as a resultof temperature change or growth. Root ethanol-insoluble activity,however, did increase significantly over the 72-h period : possiblecauses of this slow incorporation and their relevance to themechanism of sugar storage are discussed.     

Effect of ion channel blockers and H<Superscript>+</Superscript>-ATPase inhibitors on generation of local electrical response in a cucumber leaf

Electric potential difference was measured with extracellular electrodes between the leaf surface of 2-week-old cucumber (Cucumis sativus L.) plants and soil solution. When the leaf region with a diameter of 5 mm was gradually cooled during a 105-s period to 8–9°C, the temperature drop induced a local (confined to the cooled area) nonpropagating pulse-wise electric activity. The cessation of cooling was followed by gradual (within 12–15 min) restoration of the initial potential difference. Two peaks of electric potential with amplitudes of 100–120 mV usually appeared upon cooling. The first depolarizing stage of the pulse activity was sensitive to inhibition of voltage-gated and mechanosensitive calcium channels of plasmalemma by lanthanum and gadolinium chlorides and to verapamil treatment. Furthermore, the inhibition of this stage by ruthenium red implies the release of calcium ions from intracellular stores. The initial slow depolarization was followed by a fast depolarizing shift, which was sensitive to La³⁺ and the anion channel inhibitor 4-acetamido-4′-isothiocyano-stilbene-2,2′-dilsulfonic acid. At the next stage repolarization developed, which was sensitive to potassium channel blockers, tetraethylammonium and quinine sulfate. The influence of ion channels blockers indicates that generation of local bioelectric response is based on fluxes of the same ion species that are involved in the action potential. The depolarization stage was due to the transient Ca²⁺ influx into the cytosol from the apoplast and intracellular stores, together with the anion efflux from the cell; the repolarization stage involved potassium ions. Both stages of electric pulse generation were retarded by the H⁺-ATPase inhibitors, sodium orthovanadate and dicyclohexylcarbodiimide, which implies the involvement of the proton pump in the origin of electric pulses examined.     

Time course of low temperature inhibition of sucrose translocation in sugar beets

Further studies are presented characterizing the time-course response of sucrose translocation in sugar beet (Beta vulgaris L. cv Klein Wanzleben) to low temperature inhibition. Only the temperature of a 2 cm zone of the source-leaf petiole was varied (1° vs 25°, approximately). The half-time of inhibition, defined as the time required for 50% inhibition of the control or pre-cooling rate, varied from 4 to 15 minutes, and the half-time of recovery from 30 to 100 minutes. Maximum inhibition varied from 68 to 92%. Possible uncertainties in evaluating these parameters are discussed. When the duration of the low temperature period was sufficient to permit essentially full recovery, subsequent re-warming of the petiole zone to 25° to 30° effected little or no increase in the translocation rate. It is evident that the interposition between source and sink of a 2 cm petiole zone maintained at a temperature generally inhibitory to physiological processes resulted in little or no impairment to the translocation process, after a suitable thermal adaptation period. Thermally adapted petiole systems de-adapted after periods as short as 1 hour at 25°.     

Cold-Inhibited Phloem Translocation in Sugar Beet: IV. ANALYSIS OF THE COOLING-INDUCED REPARTITIONING HYPOTHESIS

Systems identification techniques were used to calculate photo-assimilatetransport characteristics in Beta vulgaris and Pisum sativum,before and after the application of localized perturbationsto the transport path. Changes in photo-assimilate partitioningto various monitored sinks were found to be promoted by slowcool (25 ?C to 1 ?C in 40 min), quick cool (25 ?C to 1 ?C in2 min)/quick warm, and apoplastic osmotic treatments of a localizedregion of the source leaf petiole in Beta. Photo-assimilatepartitioning into both intact and surgically modified (embryo-less)ovules of Pisum was also observed to change following quickcool/quick warm treatments applied to a 2.0 cm region of thepeduncle leading to the monitored pod. No changes in transportproperties were observed through the treated petiole regionof Beta during or following the slow cool treatment (i.e. transittime or system gain). High apoplastic osmolality (addition of1 000 mol m^–3 sorbitol) reduced the transit time of tracermovement through the treated petiole of Beta, while enhancingtracer washout from the bathed tissue region. Our modellingtechniques have shown that the physical or physiological basisfor the sink partitioning changes must be due to alterationsdownstream of the treated pathway zone, suggesting the involvementof physical signals transmitted from the treated region. Theseresults support the previously presented cooling-induced repartitioninghypothesis (Grusak and Lucas, 1986) and demonstrate that pathway-originatedstimuli can alter source-sink photo-assimilate partitioning. Key words: Photo-assimilate partitioning, phloem translocation, Beta vulgaris, Pisum sativum     

Activity of enzymatic antioxidant defense systems in chilled and heat shocked cucumber seedling radicles

Chilling whole cucumber seedlings that had 10‐mm long radicles for 4 days at 2.5°C significantly inhibited subsequent radicle growth both by increasing the time it took the seedlings to recover from chilling and attain a linear rate of radicle growth, and by decreasing the subsequent rate of linear growth. Exposing cucumber seedlings to 45°C for up to 20 min had no effect on subsequent radicle growth, while longer exposures produced reductions in growth. A heat shock at 45°C for 10 min induced the optimal protection to 4 days of chilling at 2.5°C by reducing chilling inhibition from 60 to 42%. Two hours after being chilled, heat shocked or heat shocked and then chilled, there was no difference in protein content of the apical 1 cm of the seedling radicle among these treatments and the non‐heat shocked, non‐chilled control. Two days after treatment, the protein content was still similar in tissue that had been heat shocked or heat shocked and chilled, while it was significantly reduced in tissue that had been chilled. In general, 2 h after treatment, the activity of the 5 antioxidant enzymes examined in this study [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2)] were reduced by chilling and unaffected or increased by heat shock. When heat shock was followed by chilling, there was a consistent effect of the heat shock treatment on preventing the loss of enzyme activity following chilling. This protective effect of the heat shock treatment was even more pronounced after 2 days of recovery at 25°C for SOD, CAT and APX. In contrast, the activity of GR and GPX was substantially higher in chilled tissue than in tissue that had been heat shocked before being chilled. Elevated levels of GR and GPX therefore appear to be correlated with the development of chilling injury, while elevated levels of SOD, CAT and APX appear to be correlated with the development of heat shock‐induced chilling tolerance.     

Carbon Translocation in the Tomato: Effects of Fruit Temperature on Carbon Metabolism and the Rate of Translocation

The effects of fruit temperature on the rate of carbon translocationand on the contents of the major carbon metabolites in the experimentalfruits were investigated. Carbon import, which was inverselyproportional to fruit size, was inhibited by fruit cooling (5°Cand enhanced by fruit warming (35°C) when compared withcontrols (25°C); furthermore, there was net export of carbonfrom the largest cooled fruits. The carbon translocation rateswere related both to the rates of accumulation or depletionof starch and insoluble residue and to the concentration ofsucrose in the fruit. The possible dependence of the rate oftranslocation on a gradient of sucrose concentration betweensource and sink is discussed.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Activation of plasmalemmal NADPH oxidase in etiolated maize seedlings exposed to chilling temperatures

Five-day-old etiolated seedlings of maize (Zea mays L.) were used to study the kinetics of hydrogen peroxide formation upon lowering growth temperature from 25 to 6°C. The total content of hydrogen peroxide in root and shoot tissues increased by 30–40% after 2-h cooling compared to the control level but returned to the initial level or decreased even lower after 24-h cooling. In order to prove the involvement of plasma membrane NADPH oxidase in changes of hydrogen peroxide content upon cooling, isolated plasma membranes were obtained from untreated plants and from seedlings chilled at 6°C for 2 and 24 h. The NADPH-dependent generation of superoxide anion radical in isolated plasma membranes was quantified by measuring the rate of formazan production from the tetrazolium salt XTT. The activity of plasma membrane NADPH oxidase in shoots was 50 ± 9 nmol O₂/(mg protein min), which was 1.5 times higher than the activity in roots. The enzyme activity in plasma membranes was inhibited by low concentrations of diphenyleneiodonium. The effective concentration EC₅₀ was 5.10 μM for shoots and 9.05 μM for roots. The activity of plasma membrane NADPH oxidase increased after 2-h cooling of seedlings but reversed to the control level after 24-h cooling. This transient activation of NADPH oxidase upon cooling was similar to the pattern of hydrogen peroxide formation in shoots and roots. Analysis of NADPH oxidase activity of plasma membrane proteins after their separation in denaturing conditions followed by subsequent renaturation revealed four diphenyleneiodonium-sensitive bands with mol wt of 130, 88, 51, and 48 kD. Western blot analysis of the reaction with antibodies against the catalytic domain of phagocyte NADPH oxidase revealed the proteins with mol wt of only 88 and 48 kD. The properties of molecular organization of plasma membrane NADPH oxidase are discussed in terms of its role in cell signaling.     

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers. 1. The Possible Role of Cell Death

The ability of a cell-free secretion from germinating conidia(germination fluid) and heat-killed conidia of Botrytis cinereaPers. ex Pers. to induce resistance in exposed carrot root tissueswas unaffected during freezing to –25 °C and rapidthawing, and by heating to 50 °C for 10 min. Activity waslost by boiling for 10 min or at room temperature after 24 h. There was less cell death (Evans blue and neutral red staining)in the surface tissue of slices pretreated with heat-killedconidia and subsequently exposed to infection than in similartissue of untreated, inoculated slices. Both inducing preparationsalone caused maceration and cell death in one or two cell layersof the slice surface. Healthy tissue killed by freezing to –25°Cfor 2 h and rapid thawing, released a factor which, when similarlytested on slices, resulted in germ-tube inhibition and reducedvisual symptoms following inoculation with live spores. Thepossibility that cell death triggered the release of a hostelicitor which caused enhanced accumulation of phytoalexinsand suberin deposition is discussed. Botrytis cinerea, carrot, induced resistance, cell death, vital staining     

Mechanism of inhibition of translocation by localized chilling

Arrhenius plots of translocation velocity as a function of petiole temperature show a marked increase in temperature dependence below 10 C in bean (a chilling-sensitive species) but not in sugar beet (chilling-resistant). The increased temperature dependence below 10 C was not observed for cytoplasmic streaming or oxygen uptake in bean. Bean petioles were served to release pressure in order to determine whether sieve tubes are obstructed in cold-treated petioles. The resulting pressure release caused serious displacement of the crystalline protein bodies in the sieve tubes of petioles at 25 C, but in those locally cooled to 0 C for 30 minutes little displacement occurred, indicating obstruction in the latter. An ultrastructural study of sieve tubes in tissue frozen rapidly in situ and dehydrated by freeze substitution revealed that treatment at 0 C for 30 minutes caused structural alteration and displacement of the cytoplasmic material lining the sieve tube wall resulting in occlusion of sieve plates. The sieve plates of the control petioles at 25 C were generally clear of obstructions. The results indicate that inhibition of translocation by chilling in chilling-sensitive plants results from physical blockage of sieve plates rather than from direct inhibition of a metabolic process which drives translocation.     

Two-step accelerating freezing protocol yields a better motility,membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.     

Cryopreservation of horse-chestnut (Aesculus hippocastanum L. ) somatic embryos using three different freezing methods

Cryopreservation of somatic embryos of Aesculus hippocastanum L. cultured on nutritive media containing abscisic acid (ABA) at concentrations of 0.75 μM, 7.5 μM and 75.0 μM was evaluated for three cooling methods: (i) slow freezing with cryoprotectants, (ii) fast freezing with cryoprotectants, and (iii) fast freezing with desiccation techniques. The ‘cryoprotectant’ freezing techniques included the embryo pretreatment on ABA containing medium for 4 days, followed by cryoprotective treatment in liquid medium containing 0.5 M dimethylsulfoxide, 0.5 M glycerol, 1.0 M sucrose, and cooled at slow, and rapid rates. Embryos pretreated on a medium containing 0.75 μM ABA, and cooled to −35 °C at 1°C /min, held for 30 min at this transfer temperature and then immersed in liquid nitrogen (LN) had the best embryo recovery (43%). The ‘desiccation’ method involved an air drying step of similar ABA-pretreated, non-cryoprotected embryos followed by rapid cooling. Embryos precultured on 0.75 μM ABA, then subjected to a 4 h period of air desiccation (water content reduction to 13%) showed about the same level of survival (46%) as found with the ‘cryoprotectant’ slow freezing technique. The air-dry ‘desiccation’ method is easier to apply than the more complicated ‘cryoprotectant’ method. This revised version was published online in June 2006 with corrections to the Cover Date.     

Equilibrium,quasi-equilibrium,and nonequilibrium freezing of mammalian embryos

The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about −70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.     

Effect of Temperature on Photosynthetic Activities of Senescing Detached Wheat Leaves

Changes in various components of photosynthetic activity duringthe dark induced senescence of detached wheat leaves, maintainedat 25°C (control) and 35°C (mildly elevated temperaturetreatment), were examined. Senescence-associated decline measuredup to 96 h, in photosynthetic activity was appreciably hastenedat 35°C, than at 25°C as evident by the relative higherlosses of chlorophyll, photosystem (PS) II and PS I catalyzedphotochemical activities and ribulose-1,5-bisphosphate (RuBP)carboxylase activity. In addition, a comparatively higher risein light scattering profile of isolated chloroplasts was notedat 35°C than at 25°C. Senescence-induced degradationof chlorophyll was faster at 35°C than at 25°C; on theother hand, the degradation of carotenoids was faster at 25°Cthan at 35°C. Furthermore, the ratio of carotenoids to chlorophyllincreased with senescence up to 96 hours, higher ratio beingobtained at 35°C than at 25°C. Both PS II and PS I activitiesshowed a transient rise in the beginning phase of dark incubation,whereas loss in chlorophyll was continuous throughout the periodof senescence. The initial rise observed in photochemical activitieswas attributable to the uncoupling of electron transport fromphotophosphorylation. Elevated temperature treatment resultedin greater inactivation of RuBP carboxylase than control. Itappears that during senescence the loss in chlorophyll and RuBPcarboxylase activity are triggered simultaneously. (Received June 7, 1985; Accepted October 30, 1985)     

Acclimation of different strains of the olive fly, Dacus oleae, to low temperatures

Male and female D. oleae have similar powers of acclimation when exposed to low temperatures. Their torpor thresholds depend upon the temperature to which they have been acclimatised. During slow cooling (i.e. less than 1°C per min) they are capable of some rapid acclimation which enables them to lower their torpor threshold by almost 1°C degree, as compared with when they are chilled quickly. After abrupt transfer from 25°C to a different temperature, acclimation takes some time to be accomplished. At 15°C and above it occurs within 10 days but at temperatures below this, progressive acclimation lowers the torpor thresholds to the very low levels typical of flies overwintering under natural conditions. During this long term acclimation torpor thresholds may change by almost 0.5°C per 1°C change of acclimation temperature.No differences were observed in the ability of either flies from northern and southern Greece, or normal and γ-irradiated laboratory reared flies to acclimate to winter conditions in the field. In all cases, torpor thresholds were progressively lowered in advance of the decline in weekly minimum temperatures.     

Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.     

31P-NMR Study of the Physiological Conditions in Intact Root Cells of Mung Bean Seedlings under Low-Temperature Stress

Intracellular pH and levels of ATP in intact root-tip cellsof mung bean (Vigna mungo [L.] Hepper) under low-temperaturestress were investigated in vivo by ³¹P nuclear magnetic resonance(³¹P-NMR) spectroscopy. Root-tips of 3 mm in length were excisedfrom seedlings of mung bean that had been chilled at 0°Cafter grown at 30°C. Chilling for longer than 12 h causedchanges in the intracellular pH and decreased levels of ATPin the seedlings. The level of ATP recovered within 30 min butlittle change in pH was observed when samples were rewarmedto 20° C after chilling at 5°C. However, after chillingfor longer than 48 h, neither the intracellular pH nor the levelof ATP was restored. These results suggest that a decline in the activity of tonoplastH⁺-ATPase, induced by chillings, might be a significant earlyevent in cold-induced injury that leads to cell damage. (Received October 27, 1994; Accepted May 10, 1995)     

Germination as an Unreliable Indicator of the Effectiveness of Cryopreservative Procedures for Imbibed Seeds

Tomato seeds of 49 per cent moisture content have been thawedfrom liquid nitrogen (—196 °C) with no loss of germinationfollowing both slow (12 °C min^–1) and rapid (850 °Cmin^–1) cooling, using 35 per cent dimethyl sulphoxide(DMSO) as a cryoprotectant. Lower concentrations of DMSO resultedin reduced germination. High proportions of seedlings from frozen/thawedseed failed to survive transplanting into soil, especially ifthey had been cooled rapidly. Seedlings grown to maturity afterthe slow cooling treatment showed a greater proportion of morphologicalabnormalities than a control population or those that had beenrapidly cooled. It is suggested that if cryopreservative techniques are to beevaluated for the storage of recalcitrant seeds, false impressionsof efficacy might be gained by reliance solely on germinationtesting. Cryopreservation, germination, recalcitrant seed