A cotyledon double inoculation technique for evaluating resistance to anthracnose (Colletotrichum orbiculare) and scab (Cladosporium cucumerinum) in cucumber

A technique for simultaneous inoculation of cucumber cotyledons with Colletotrichum orbiculare race 1 and Cladosporium cucumerinum has been developed. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 24 h after emergence with Colletotrichum orbiculare (200 spores in 2 μ1 of water) and Cladosporium cucumerinum (1000 spores in 5 μ1 of water) followed by 48 h of incubation in the dark at 20°C and 100% r.h., and 48 h in a 20°C lighted growth chamber. Seedlings were then moved to a growth chamber at 21°C at night and at 26°C during the day for 4 days and plants were rated as resistant or susceptible 8 days after inoculation. No interference in the expression of resistance or susceptibility of cultivars to either pathogen was detected in simultaneous inoculations.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

Effects of Temperature and Leaf Weness on the Latent Period of Rbynchosporium Secalis (Leaf Blotch) on Leaves of Winter Barley

Effects of temperature and leaf wetness on the latent period of Rhynchosporium secaits (leaf blotch) on winter barley were examined in controlled environment experiments. At 100% relative humidity (continuous leaf wetness) the mean length of the latent period was c.24 days at 5°C, c. 19 days at 10°C, c. 16 days at l5°C and c. 13 days at 20°C. The mean number of days between the appearance of the first and the last lesions was c. 13 days at 5°C, c. 6 days at 10°C, c. 5 days at 15°C and c. 3 days at 20°C. A negative curvilinear regression of latent period on temperature accounted for 99% of the variance. The mean area of lesions per leaf was 38 mm² at 5°C, 46 mm² at 10°C, 24 mm² at 15°C and 24 mm² at 20°C. At 10°C, after a 48 h wet infection period, the interruption of leaf wetness for 5 or more days at any time during the next 15 days of the latent period did not decrease subsequent lesion area. However, absence of leaf wetness after these 15 days, at the onset of sporuiation, did decrease the area of lesions which developed.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Vernalization,photoperiod and GA3 interact to affect flowering of Japanese radish (Raphanus sativus Chinese Radish Jumbo Scarlet)

Raphanus sativus L. Chinese Radish Jumbo Scarlet has characteristics that make it an excellent plant model for vernalization studies. This study further characterizes flower induction of R. sativus Chinese Radish Jumbo Scarlet. Seed were imbibed in distilled water containing 0, 10⁻⁵ M or 10⁻³ M GA₃ for 24 h and were then exposed to 6 ± 0.5°C (vernalized) for 0, 2, 4, 6, or 8 days. Seedlings were then grown under a short- (8 h) or long-day photoperiod (8 h with or without a 4-h night interruption; 2200–0200 h). Of unvernalized plants grown under long- and short-day conditions, 45 and 3% flowered, respectively. Saturation of the vernalization response occurred after a 4- or 8-day vernalization treatment when plants were placed under long- or short-days, respectively. Basal leaf number and days to anthesis decreased when seeds were cooled for 2 or 4 days and were imbibed with 10⁻³ M GA₃ compared to distilled water only. These data indicate that R. sativus Chinese Jumbo Scarlet has principally an obligate vernalization requirement when grown under short-days. GA₃ application only facilitated flowering when the length of the vernalization treatment was marginal. Taken together, these data support the use of this plant as a model plant for identifying vernalization responses under short-day conditions.     

Flowering requirements in Bromus inermis, a short-long-day plant

Smooth bromegrass plants ( Bromus inermis Leyss.) have a dual photoperiodic requirement for flowering. At temperatures ranging from 6 to 24°C, short days (SD) are necessary for primary induction while a transition to long days (LD) is required for initiation of flower primordia, culm elongation and flower development (secondary induction). Critical photoperiods for primary induction (50% flowering) were 13.5 h (15°C) and 12 h (24°C) in the American cv. Manchar and 14.5 and 13 h, respectively, in the Norwegian cv. Löfar. For the secondary induction the respective critical photoperiods were 14 and 15 h in 'Manchar' and 16 and 17.5 h in 'Löar', which also appeared to be better adapted to low temperatures. Low temperature vernalization in LD for up to 16 weeks at 3°C was unable to cause primary induction and temperatures below 12°C also strongly reduced the SD effect. At optimum temperature (15-2TC) 4 to 6 weeks of 8-10 h SD treatments were needed for optimal primary induction effect. A minimum of 8 LD cycles of 24 h were required for complete secondary induction in 'Manchar', while more than 16 cycles were needed in 'Löfar'. Seedlings grown in SD developed a rosette type of growth with shoots growing in a decumbent position, while those in LD grew upright and formed elongated vegetative culms. Rate of leaf initiation was enhanced by about 60% by LD while tillering was promoted by SD.     

A comparison of low temperature growth vs low temperature shifts to induce resistance to photoinhibition in spinach (Spinacia oleracea)

Plants of Spinacia oleracea L. cv. Savoy grown under cold-hardening (5°C) and nonhardening (16°C) conditions were exposed to a photoinhibitory irradiance of 1300 μmol rrr^: m-² S-¹ 5°C for 12 h. Plants grown at 5°C exhibited a greater resistance to photoinhibition at low temperature in comparison to plants grown at 16°C as measured by the photochemical efficiency of photosyslem II. In contrast, tuily expanded leaves of plants grown at 16°C and then shifted to 5°C for 10 days did not exhibit increased resistance to photoinhibition. This was observed irrespective of the phoioperiod experienced during the shift to a lower temperature. Furthermore, spinach grown at 16°C and subsequently exposed to a stepped, daily decrease in temperature from 16 to 1°C over 10 days w ith a concomitant reduction in photoperiod. also did not exhibit any change in susceptibility to photoinhibition. Thus, a decrease in photoperiod accompanied by either an abrupt or stepped low temperature shift cannot induce increased resistance to photoinhibition. This confirms the hypothesis that growth and development at cold-hardening temperature are absolute requirements for the acquisition of resistance to photoinhibition at low temperature.     

Respiratory Q10 of marigold (Tagetes patula) in response to long-term temperature differences and its relationship to growth and maintenance respiration

Acclimation of respiration to temperature is not well understood. To determine whether whole plant respiration responses to long-term temperature treatments can be described using the Q₁₀ concept, the CO₂ exchange rate of marigolds ( Tagetes patula L. 'Queen Sophia'), grown at 20°C or 30°C, was measured for 62 days. When plants of the same age were compared, plants grown at 20°C consistently had a higher specific respiration (R_spc) than plants grown at 30°C (long-term Q₁₀= 0.71–0.97). This was due to a combination of greater dry mass at 30°C and a decrease in R_spc with increasing mass. When plants of the same dry mass were compared, the long-term Q₁₀ was 1.35–1.55; i.e. R_spc was higher at 30°C than at 20°C. Whole plant respiration could be accurately described by dividing respiration into growth and maintenance components. The maintenance respiration coefficient was higher at 30°C than at 20°C, while the growth respiration coefficient was lower at 30°C, partly because of temperature-dependent changes in plant composition. These results suggest difficulties with interpreting temperature effects on whole plant respiration, because conclusions depend greatly on whether plants of the same age or mass are compared. These difficulties can be minimized by describing whole plant respiration on the basis of growth and maintenance components.     

Effect of Temperature on the Formation of Microsclerotia of Verticillium dahliae

Microsclerotium formation by six isolates of Verticillium dahliae was studied at different temperatures both in vitro and in Arabidopsis thaliana . In vitro mycelial growth was optimal at 25°C, but microsclerotium formation was greatest at 20°C (two isolates) or 15–20°C (one isolate). Seedlings of A. thaliana were root-dipped in a conidial suspension, planted, and either placed at 5, 10, 15, or 25°C, or left at 20°C until the onset of senescence, after which some of the plants were placed at 5, 10, 15, or 25°C. The amount of microsclerotia per unit of shoot weight was assessed in relation to isolate and temperature. The optimal temperature for production of microsclerotia was 15–25°C. Two isolates each produced about 10 times more microsclerotia than each of the other four isolates. For these isolates, high R ²_adj.-values of 0.77 and 0.66 were obtained, with temperature and its square as highly significant (P   < 0.001) independent variables. R ²_adj.-values for the other isolates varied between 0.28 and 0.39. Moving plants to different temperatures at the onset of senescence led to microsclerotial densities that were intermediate between densities on plants that had grown at constantly 20°C and plants grown at other temperatures. This suggests that vascular colonization rate and rate of microsclerotium formation are similarly affected by temperature. The senescence rate of plants appeared unimportant except for plants grown at 25°C, which showed the highest amounts of microsclerotia per unit of plant weight in the most rapidly senescing plants.     

Effects of photoperiod and temperature on the development and diapause of the bark beetle Ips typographus

Abstract:  Diapause was induced in a Central European population of Ips typographus grown at 20°C when the day length decreased below 16 h [50% diapause incidence occurred in the 14.7:9.3 h L:D (light:dark) regime]. The non-diapausing adults fed on days 2–6 and 10–14 after the ecdysis and swarmed after the second feeding bout with chorionated eggs in the ovaries and sperm in the spermiducts. Neither gonads nor the flight muscles matured and no swarming occurred in the diapausing adults. The development from egg to adult took about 34 days in both 18:6 h (no diapause) and 12:12 h L:D (diapause) regimes, but it was extended by up to 30% without diapause induction when only larvae or pupae were exposed to L:D 12:12 h. Diapause was induced in insects reared at L:D 12:12 h through the last larval and the pupal instars and/or in the adult stage. Temperature ≥ 23°C prevented diapause induction at L:D 12:12 h but diapause occurred at L:D 14:10 h associated with 26:6°C thermoperiod. The effect of thermoperiods on the developmental rate requires further research. Exposure of the non-diapausing adults to 5°C for several days blocked feeding and evoked a diapause-like state, whereas diapausing adults fed and their gonads slowly developed at this temperature. Diapausing adults exposed in forest to low night temperatures and transferred in October to 20°C readily reproduced at 18:6, but not 12:12 h L:D photoperiods. After 2-months at 5°C and darkness, they became insensitive to the photoperiod, matured and most of them also swarmed at 20°C in the 12:12 h L:D regime. In a Scandinavian population, diapause occurred at 18:6 h L:D and was terminated either by exposure to 5°C or by very long photoperiod (L:D 20:4 h) combined with high temperature (23°C).     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Potato cold hardiness development and abscisic acid. II. De novo synthesis of proteins is required for the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

During cold acclimation of potato plantlets ( Solanum commersonii Dun, PI 458317), there are two transitory increases in free ABA content corresponding to a three-fold increase on the 2nd day and a five-fold increase on the 6th day (Ryu and Li 1993). During this period, plantlets increased in cold hardiness from −5°C (killing temperature, control grown at 22/18°C, day/night) to −10°C by the 7th day of exposure to 4/2°C (day/night). This increase in free ABA was not found when cycloheximide (CHI), an inhibitor of cytoplasmic protein synthesis, was added to the culture medium 6 h before exposure to low temperatures. Plantlets treated with CHI did not acclimate to cold, maintaining a hardiness level (−5°C) similar to that of the 22/18°C-grown plantlets. When the CHI-treated plantlets were exposed to low temperatures for 3 days, transferred to CHI-free culture medium and grown at low temperatures, the plantlets showed a transitory increase in free ABA 2 days later. This increase was followed by the development of cold hardiness (−8°C). Application of CHI to the culture medium after 3 days of cold acclimation, when the first ABA peak and a partial development of cold hardiness (−8°C) had occurred, blocked the second transitory increase in free ABA and resulted in no further development of cold hardiness. These results suggest that de novo synthesis of proteins is required for these transitory increases in free ABA during cold acclimation of potato plantlets.     

Fatty acid desaturation in monogalactosyldiacylglycerol of Brassica napus leaves during low temperature acclimation

Brassica napus plants grown at 5°C have the potential to desaturate fatty acids in the major membrane diacylglycerols of leaves at rates much higher than those of plants grown at 30°C. This low temperature-induced desaturation (LTD) is rapidly deactivated if plants grown at 5°C are transferred to 30°C for several hours. By exposure to ¹⁴CO₂ and a computer simulation program, we estimated the rate of desaturation of monogalactosyldiacylglycerol by (ω9-, ω6- and ω3-desaturases of plants grown at 5, 10, 20 and 30°C. Data show that LTD can be induced in mature leaves of plants grown at 20 and 30°C after transfer to 5°C. Full activation of LTD takes several weeks and occurs more rapidly in plants grown at 20°C than 30°C. This activation is largely due to the increased activity of ω9- and ω6-desaturases on C16 fatty acids and ω6-desaturase on C18 fatty acids.     

AN INCREASE OF ORNITHINE δ-AMINOTRANSFERASE-MEDIATED PROLINE SYNTHESIS IN RELATION TO HIGH-TEMPERATURE INJURY IN GRACILARIA TENUISTIPITATA (GIGARTINALES, RHODOPHYTA)

Changes of proline biosynthesis in relation to high-temperature (35° C) injury were investigated in Gracilaria tenuistipitata var. liui Zhang et Xia. On exposure to 35° C, the specific growth rate decreased after 5 days while free proline levels increased gradually after 2 days and reached the maximal level on days 4–6 but decreased at day 7. The repair ability of thalli treated at 35° C by measuring the growth rate after transfer to 25° C for another 5 days decreased in thalli that had been grown at 35° C for more than 2 days, and the extent increased as treatment at 35° C was prolonged. After 4 days of treatment at 35° C, the activities of both ornithine δ-aminotransferase (δ-OAT; EC 2.6.11.3) and Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) increased, but that of γ-glutamyl kinase (γ-GK; EC 2.7.2.11) remained unchanged, and that of glutamate-5-semialdehyde dehydrogenase (GSAd; EC 1.4.1.3) decreased. The application of 10 μM gabaculine, an irreversible inhibitor of δ-OAT, at 35° C recovered the growth ability but inhibited the increase of both δ-OAT activity and free proline level; its effects were reversed by 1 mM proline. G. saliconia, which is relatively tolerant to high temperature, showed a decrease of both δ-OAT activity and free proline level at 35° C. It seems that a stimulation of proline synthesis from the ornithine pathway via an increase in both δ-OAT and P5CR activities might be associated with high-temperature injury in G. tenuistipitata.     

Elevation of the heat resistance of Salmonella typhimurium by sublethal heat shock

The survival of Salmonella typhimurium after a standard heat challenge at 55°C for 25 min increased by several orders of magnitude when cells grown at 37°C were pre-incubated at 42°, 45° or 48°C before heating at the higher temperature. Heat resistance increased rapidly after the temperature shift, reaching near maximum levels within 30 min. Elevated heat resistance persisted for at least 10 h. Preincubation of cells at 48°C for 30 min increased their resistance to subsequent heating at 50°, 52°, 55°, 57° or 59°C. Survival curves of resistant cells were curvilinear. Estimated times for a '7D' inactivation increased by 2.6- to 20-fold compared with cells not pre-incubated before heat challenge.     

Flowering strategies of the high-arctic and high-alpine snow bed grass species Phippsia algida

Flowering requirements of the high-arctic and high-alpine snow bed grass species Phippsia algida (Sol.) R. Br. have been studied in controlled environments. Seedlings flowered rapidly in continuous long days (LD) at temperatures ranging from 9 to 21°C. They also initiated inflorescence primordia at the same temperatures in continuous short days (SD), whereas LD were required for heading and anthesis. The plant thus has the characteristics of a regular long day plant, although the daylength requirement is associated with floral development only. The critical daylength for the LD response was about 17 h at 21°C and 19 h at 9°C. A single LD cycle was enough to trigger inflorescence development, while 5 cycles were required for the full response. Anthesis was reached within a week of LD treatment at 21°C in SD grown plants with preformed inflorescence primordia. The advantages of these versatile flowering responses are discussed in relation to the extreme climatic regime of late snow bed sites.     

Contrasting photo- and thermoperiod-induced changes in abscisic acid and lipid contents in leaves of mungbean seedlings

Seedlings of Phaseolus aureus ROXB were grown under 12/12 h light/dark cycles with the light period at 32.5°C and darkness at 10°C (normal conditions N) or with light at 10°C and darkness at 32.5°C (inverse conditions, I). I-conditions affected the level of chlorophyll and carotenoids (very low), monogalactosyldiacylgycerol (low) and phosphatidylinositol (high) in the leaves. Leaves of I-seedlings showed a sharp and durable decline of relative water content during the low temperature phase. For the N-seedlings, loss of water was restricted to the end of this period. The loss of water was accompanied by visible symptoms of wilting at specific times of day. Although the pigment content remained nearly unchanged, ABA content of leaves of both N-and I-seedlings increased during water stress. Upon return to the warm period, ABA level continued to increase after the leaves had regained turgor, this 'after stress'increase being more pronounced in the leaves of I-seedlings. Exogenous application of ABA induced a slight increase in the content of phospholipids in N- and I-leaves and a decrease in free fatty acids, whereas monogalactosyldiacylglycerol content was significantly reduced in N-leaves after application of ABA. Upon transfer of I-plants to 20°C for 12 h during the light period, pigment and chloroplastic lipid content increased rapidly whereas upon a further exposure to 10°C in light, pigments and especially monogalactosyldiacylglycerol were lost. The control of pigment and lipid metabolism and the role of ABA during chilling stress are discussed.     

Hardly Increased Oxidative Stress After Exposure to Low Temperature in Chilling-Acclimated and Non-Acclimated Maize Leaves

Abstract: Seedlings of Zea mays L. were grown at optimal (25 °C) and suboptimal (15 °C) temperature and then exposed to severe chilling temperature (6 °C) at their growth light intensity (450 ìmol quanta m⁻² s⁻¹) for 4 d. Photosynthetic parameters, hydrogen peroxide, antioxidant contents, and activity of scavenging enzymes were investigated before, during, and after chilling stress. This stress caused a stronger reduction in photosynthetic activity, maximum quantum efficiency of photosystem II primary photochemistry ( F _v/ F _m), and catalase activity in plants which had been grown at 25 °C rather than at 15 °C. Maize plants grown at suboptimal temperature de-epoxidized their xanthophyll cycle pool to a greater extent and exhibited a faster recovery from chilling stress than plants which had not been acclimated to chilling. Antioxidant content, activity of scavenging enzymes, with the exception of catalase, hydrogen peroxide formation, and the size of the xanthophyll cycle pool were hardly affected by chilling stress. However, chilling induced a temporary increase in the glutathione content and triggered the synthesis of á-tocopherol during the phase of recovery at 25 °C. The results indicate that leaves respond to chilling stress by down-regulation of photosystem II accompanied by de-epoxidation of the xanthophyll cycle pool, probably to prevent enhanced formation of superoxide radicals at photosystem I and, consequently, other reactive oxygen species.     

Oxygen requirements of embryos and larvae of the largemouth bass, Micropterus salmoides (Lacépède)

Starting with the second day after fertilization, largemouth bass embryos and larvae became increasingly sensitive to oxygen deficiency until the sixth day of life. On this day few survived exposure to 1 mg O₂/l for 3 h at 20°C; many were damaged at 2 mg/1, but not at 2.5. Death was by asphyxiation or by starvation resulting from an apparent inability to close the lower jaw. On the seventh day the larvae became more resistant again, possibly because opercular movement began on that day. At 25°C the effects of reduced oxygen concentration were intensified, and even a concentration of 2.5 mg/1 became lethal in 3 h. At 3 mg O₂/l and 20° the normally quiescent yolk-sac larvae became very active and swam vertically to at least 5 or 6 cm above the substrate; 4 and 5 mg/1 had this effect at 23–24°. This behaviour could result in losses by predation and displacement from the nest.     

Freezing and desiccation tolerance of imbibed canola seed

Depending on the environmental conditions, imbibed seeds survive subzero temperatures either by supercooling or by tolerating freezing-induced desiccation. We investigated what the predominant survival mechanism is in freezing canola ( Brassica napus cv. Quest) and concluded that it depends on the cooling rate. Seeds cooled at 3°C h⁻¹ or faster supercooled, whereas seeds cooled over a 4-day period to −12°C and then cooled at 3°C h⁻¹ to−40°C did not display low temperature exotherms. Both differential thermal analysis and nuclear magnetic resonance (NMR) spectroscopy confirmed that imbibed canola seeds undergo freezing-induced desiccation at slow cooling rates. The freezing tolerance of imbibed canola seed (LT₅₀) was determined by slowly cooling to −12°C for 48 h, followed with cooling at 3°C h⁻¹ to −40°C, or by holding at a constant −6°C (LD₅₀). For both tests, the loss in freezing tolerance of imbibed seeds was a function of time and temperature of imbibition. Freezing tolerance was rapidly lost after radicle emergence. Seeds imbibed in 100 μ M abscisic acid (ABA), particularly at 2°C, lost freezing tolerance at a slower rate compared with water-imbibed seeds. Seeds imbibed in water either at 23°C for 16 h, or 8°C for 6 days, or 2°C for 6 days were not germinable after storage at −6°C for 10 days. Seeds imbibed in ABA at 23°C for 24 h, or 8°C for 8 days, or 2°C for 15 days were highly germinable after 40 days at a constant −6°C. Desiccation injury induced at a high temperature (60°C), as with injury induced by freezing, was found to be a function of imbibition temperature and time.