Controlled Environment Room Research Articles

A portable system to quantify seedling damage using stress-induced volatile emissions

This paper describes a portable gas analysis system that was used to quantify stress-induced ethanol in black spruce (Piceamariana (Mill.) B.S.R). Operationally fall-lifted black spruce seedlings, packaged in polyethylene-lined kraft bags, were placed in a greenhouse to simulate conditions in the field, where stock may be exposed to elevated temperatures. The maximum temperature in the greenhouse was 36 °C, and the duration of heat treatment exposure ranged from 3 h to 7 days. After exposure to the heat stress event, the ethanol concentration in the bags of seedlings was measured using a portable gas analysis system. A sample of 20 seedlings were then potted and placed in a controlled-environment room for a 14-day growth period. At the conclusion of the growth period the seedlings were removed from the pots and the number of white roots >1 cm were counted. The terminal buds were dissected to determine viability, and the degree of foliage browning was estimated. The experiment was repeated four times. Ethanol concentration was significantly correlated with subsequent root growth potential, foliage viability, and terminal bud viability.

Plant Growth and Soil Processes in Temperate Grassland Communities at Elevated CO 2

Turves of a Mollic Psammaquent soil were used in controlled environment rooms to examine the response of managed temperate pasture communities to 350, 525 or 700 p.p.m. CO2. Yield of herbage (regrowth over 3-week intervals) increased only slightly with higher C02; however, the botan- ical composition was markedly different. At elevated CO2 Paspalum dilatatum (C4) and Lolium perenne (C3) declined as a proportion of harvested yield despite a stimulation of single leaf photosynthesis that was comparable to that found in Trifolium repens, a species that increased in abundance. Changes in species composition were largely a consequence of C02-induced differences in axillary bud activity. Net primary productivity below-ground was stimulated by CO2. Soil C02-C production was greater in elevated CO2 treatments, and was consistent with a greater input of herbage and root mass and/or metabolites and of more readily decom- posable material. Levels of microbial biomass were un- changed, but enchytraeids were more abundant at elevated CO2. Tracking of 14CO2 into the various C pools also indicated a more rapid turnover of C at elevated CO2 but no change in pool sizes. No consistent effects on net mineralization of N were observed.

The Influence of Elevated CO 2 and Simulated Seasonal Changes in Temperature on Tissue Turnover in Pasture Turves Dominated by Perennial Ryegrass (Lolium perenne) and White Clover (Trifolium repens)

1. Tissue turnover, leaf morphology and population dynamics of perennial ryegrass and white clover were studied in pasture turves grown at ambient (350 μmol mol −1 ) or double ambient (700 μmol mol −1 ) CO 2 concentrations for 217 days in controlled environment rooms. The turves were subjected sequentially to three day/night temperature regimes; 10/4°C, 16/10°C and 22/16°C and harvested at 3-week intervals. The photoperiod was 12 hours for all of the temperature treatments with a mean photon flux density of 480 μE m −2 s −1 . 2. Ryegrass leaf extension and leaf death rates did not differ between CO 2 treatments and there was no effect of CO 2 on rates of leaf appearance in white clover. Weight per unit length of ryegrass laminae was unaffected by elevated CO 2 but lamina weight per unit area, lamina area and petiole weight per unit length in white clover showed a small positive response, especially at the two higher temperatures. Rates of growth and senescence per ryegrass tiller were therefore similar between CO 2 treatments, but rates of growth per white clover growing point were increased by 4, 23 and 13% at 10/4°C, 16/10°C and 22/16°C, respectively, at elevated CO 2 . Responses to CO 2 could not be attributed to any consistent change in morphological characteristics in either species and exposure to elevated concentrations of CO 2 did not appear to change the relationship between growth and senescence per meristem. 3. Total grass tiller populations were similar at both CO 2 concentrations, but ryegrass tiller densities more than halved in both CO 2 treatments as the temperature was increased. The fall was most severe at 700 μmol mol −1 and at the end of the experiment ryegrass tiller densities in this treatment were only 47% of those found at 350 μmol mol −1 . There was no consistent effect of CO 2 concentration on clover growing point numbers and they increased from 800 m −2 to over 3000 m −2 in both treatments with maximum densities occurring at 22/16°C. 4. The results imply that, in plant communities dominated by ryegrass and white clover, exposure to elevated CO 2 concentrations will alter the species composition in favour of white clover. Responses in above-ground dry matter yield to elevated CO 2 will be a balance between the positive response shown by white clover and the negative response of perennial ryegrass. Temperature will have a major influence on the magnitude of this response since both the response of white clover to CO 2 and the ratio of white clover growing points to ryegrass tillers are temperature-dependent

Effects of Elevated CO2 and Simulated Seasonal Changes in Temperature on the Species Composition and Growth Rates of Pasture Turves

Large turves from a ryegrass/white clover based pasture were exposed to 350 or 700 μl l-1 CO2 for a period of 217 d in controlled environment rooms. The temperature was increased during the experiment from 10/4 °C day/night to 16/10 °C and finally to 22/16 °C. The turves were cut to a height of 2 cm at intervals and growth rates calculated from the regrowth.Growth rates over the duration of the experiment were 8% higher at elevated CO2; the difference between CO2 treatments being statistically significant only at the highest temperature. Species composition of the turves at 350 μl l-1 CO2 showed seasonal changes similar to those measured in the field. The effect of CO2 was to exaggerate the normal decline of ryegrass at warmer temperatures and increase the proportion of white clover. About 30% of the total growth rate was from other species (notably Bromus hordeaceus L. and Poa trivialis L.) and this fraction was similar between CO2 levels. Root mass was measured at the end of the experiment and was 50% higher at elevated CO2.The modest above-ground response to CO2 was a result of CO2 stimulation occurring only at the higher temperature. Because of the CO2 × temperature interaction, the effect of CO2 in temperate regions will be seasonal. When this is matched with seasonal growth patterns of herbage species, a complex response of pasture communities to CO2 is possible. In our case, white clover was growing most strongly during the period of greatest CO2 stimulation and consequently its growth was enhanced more than that of ryegrass; however, the cooler season growth of ryegrass gives it a temporal niche which is little affected by CO2 and this may be important for ryegrass stability if it is an inherently poor responder to CO2. The results indicate that for temperate species the effects of competition at elevated CO2 cannot be easily determined from experiments conducted at a single temperature.

Freezing Resistance of Progeny from Open-pollinated Pummelo × Trifoliate Orange Citrus Hybrids

Ten-month-old seedlings, grown from seed extracted from 22 individual pummelo [Citrus grandis (L.) Osbeck] × trifoliate orange [Poncirus trifoliata (L.) Raf.] citrus hybrid trees that survived -15C freezes near Monticello, Fla., were cold-acclimated in controlled-environment rooms and freeze-tested at -6.7C for 4 h. Freeze damage to open-pollinated progeny was ranked by the number of uninjured seedlings and percentage of leaves killed and wood dieback. Morphological segregation was not associated with differences in freeze survival, and the dominant trifoliate gene was readily evident. Progeny from one tree, identified as 98-71, are considered the most likely candidates for further study in developing cold-hardy citrus trees.

Sensory Responses To Environmental Tobacco Smoke From Tobacco-Burning And Tobacco-Heating Cigarettes

Sensory ratings, respiratory behavior and eye blink rate were recorded in 11 nonsmokers exposed to a no-smoking (CONTROL) condition or to environmental tobacco smoke (ETS) from one of three kinds of cigarettes: a Kentucky reference (1R4F), an ultra-low tar (ULT) and one that heats tobacco (TEST).For each two-hour session in a controlled-environment room, two smokers “puffed” unlit cigarettes, for the CONTROL condition, or smoked 16 cigarettes to generate ETS (sidestream plus exhaled mainstream smoke). Concentrations of nicotine and respirable suspended particles (RSP) observed with the 1R4F and ULT cigarettes were 10- to 20-fold higher than those typically found in field sampling studies of office and restaurant smoking environments. The ULT and TEST cigarettes resulted in ETS analyte concentrations that were 77-100% and O-56%, respectively, of those observed with 1R4E Sensory ratings (odor, irritation, annoyance, acceptability) with the two tobacco-burning cigarettes were similar and were much greater than those recorded for the TEST or CONTROL conditions which were, in turn, rated similarly. Respiratory parameters (respiratory rate, tidal volume, expiratory time), which generally failed to discriminate the TEST and CONTROL conditions, were altered during exposure to ETS from the 1R4F and, to a lesser extent, the ULT cigarette. Eye blink rate was not significantly altered by ETS from any of the cigarettes.

Patterns of Survival and Extinction of Nematodes in Isolated Soil

The effects of local extinction on the structure of a nematode community were examined by monitoring soil nematode populations in cores that were isolated from surrounding soil. The cores were intact hlocks of deciduous forest soil and litter of three different sizes (75, 250, and 920 cm 3 ), maintained in a controlled-environment room, and sampled at 2-month intervals for 16 months. The number of nematode types per sample declined over time, from an initial average of 16 to less than 5 per 100 individuals. Types that were morphologically specialized for feeding on resources that were abundant in the cores persisted. Extinctions were associated with deficiencies of resources and with low initial population size

Growth and flowering of Gypsophila paniculata L. ‘Bristol Fairy’ and ‘Bridal Veil’ in relation to temperature and photosynthetic photon flux

The growth and flowering responses of Gypsophila paniculata L. ‘Bristol Fairy’ and ‘Bridal Veil’ to temperature and photosynthetic photon flux (PPF) were determined by growing plants in controlled environment rooms under all the following combinations of PPF (210, 450 and 710 μmol s −1 m −2) and temperature (12, 20 and 28°C). ‘Bristol Fairy’ showed higher relative growth rate and root: plant ratio, but lower leaf area ratio than ‘Bridal Veil’ over the first 30 days of treatments. Temperature and PPF responses of net photosynthesis were similar in the two cultivars and were characterized by a temperature optimum which shifted from 20°C at 450 μmol s −1 m −2 to between 12 and 20°C at 710 μmol s −1 m −2 Stem elongation (bolting) commenced first at 28°C and 710 μmol s −1 m −2 and was delayed progressively at 20°C and 12°C, and at lower PPF. Plant height at harvest was inversely related to temperature, but increased with increasing PPF. Generally, bolting and flower bud formation occurred earlier in ‘Bridal Veil’ than in ‘Bristol Fairy’; flower buds developed to anthesis more rapidly in ‘Bridal Veil’ at 12°C, but not at 20°C or 28°C. Flower yield was inversely related to temperature and was higher in ‘Bridal Veil’ than in ‘Bristol Fairy’ under all except 28°C growth conditions. Flower yield decreased with PPF in both cultivars, but ‘Bristol Fairy’ displayed more blindness and flower bud abortion at 210 μmol s −1 m −2. The ratio of floret number to inflorescence fresh weight (a measure of inflorescence density) was generally higher in ‘Bridal Veil’ than in ‘Bristol Fairy’. ‘Bridal Veil’ shows considerable promise for commercial production, particularly under lower temperature and PPF conditions.

Selection for increased nodule number in common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) plants were grown for 21–28 days in plastic container-modified Leonard jar assemblies and placed in a controlled-environment room. The nodules on each plant were removed, counted; selected plants were repotted, grown and intercrossed to produce progenies for the next cycle of recurrent selection. Among the ten parent lines, Puebla 152 and WBR 22–34 produced the most nodules and Rio Tibagi and Negro Argel the fewest, when averaged over five experiments. An analysis of number of nodules on F1 plants resulting from crosses made in a partial diallel design among the ten parents revealed highly significants variation for general combining ability (GCA) but not for specific combining ability (SCA). After three cycles of recurrent selection for nodule number per plant, the mean nodule number was 211% of the mean for the 10 parents control. Total nodule weight on selected plants also increased, but individual nodule weight decreased. Nineteen C1 and 18 C2 lines resulting from the individual plants selected for greater nodule number, along with the ten parents and two non-nodulating soybean lines included as non-fixing check plants were grown in a single experiment in a low-N field. C2 lines on average accumulated significantly more N per plant than either the parents or C1 lines, providing evidence for increased N2 fixation measured by the N difference method. These data show that more nodules, possibly resulting from greater susceptibility to nodulation, are an important, heritable component of symbiosis and that selection for increased nodule number resulted in lines capable of fixing more atmospheric N2.

Intact Leaves Exhibit a Climacteric-Like Rise in Ethylene Production before Abscission

The rate of ethylene production by intact, attached leaves of cotton plants (Gossypium hirsutum L.) during aging and senescence was studied using a continuous flow system that allowed air around enclosed leaves to be scrubbed to collect and assay ethylene. Senescence of lower leaves began around 150 d after planting in a controlled environment room. A progressive decline in the ethylene production rate was observed when comparing the 3rd, 6th, and 10th leaves from the base with each other. Ethylene production rates of individual leaves also declined over a 50-d period. However, as leaves began to appear chlorotic, a peak of ethylene production occurred that lasted for about 4 d followed by abscission. This peak involved a 3-fold or greater increase in the rate of ethylene production. The data indicate that intact leaves experience a climacteric-like surge in ethylene production after visible symptoms of senescence appear. This "ethylene climacteric" is apparently the signal that initiates hydrolysis of cell walls in the abscission zone.

Regulation of the photosynthetic capacity of primary bean leaves by the red:far‐red ratio and photosynthetic photon flux density of incident light

The main objective of the present work was to examine the effects of the red:far‐red ratio (R:FR) prevailing during leaf development on the photosynthetic capacity of mature leaves. Plants of Phaseolus vulgaris L. cv. Balin de Albenga were grown from time of emergence in a controlled environment room, 25 ± 3°C, 12‐h photoperiod, with different light treatments:a) high photosynthetic photon flux density (PPFD) = 800 μmol m−1 s−1+ high R:FR= 1.3;b) low PPFD= 300 μmol m−2 s−1+ high R:FR= 1.3; c) high PPFD=800 μmol m−2 s−1+ low R:FR= 0.7; d) low PPFD= 300 μmol m−2s−1+ low R:FR=0.7. With an R:FR ratio of 1.3, a decrease in irradiance during leaf growth reduced photosynthesis when measured at moderate to high PPFD; but when measured at low PPFD, leaves expanded under low irradiance actually had photosynthesis rates higher than those of leaves grown in high irradiance. A low R:FR ratio during development reduced the photosynthetic capacity of the leaves. In leaves expanded under R:FR = 0.7 and high irradiance photosynthesis was reduced by 42 to 89%, depending on the PPFD at which measurements were made, whereas for leaves developed at R:FR = 0.7 and low irradiance photosynthesis decreased by 21 to 24%, compared to leaves under R:FR = 1.3 and similar irradiance. The reduced photosynthetic capacity under R:FR = 0.7 and high irradiance. In natural environments, leaves may experience low R:FR conditions temporarily during their development, and this may affect their future photosynthetic capacity in full sunlight.

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.

Effects of environmental variables and cropping on plant water status of custard apple{Annona cherimolaXAnnona squamosa)

Several studies were conducted to develop reliable methods of measuring and sampling custard apple trees for determination of plant water status. Compared with laboratory dewpoint hygrometer, in-situ psychrometer and relative water content, the pressure chamber (because of its portability and reliability of measurement) was the most suitable technique for measuring plant water status. Custard apple leaves were shown to have a low modulus of elasticity (<8.5 MPa) and leaves were shown to adjust osmotically diurnally (0.14 MPa) and after several slowly developing water stress cycles (0.29 MPa). The level of osmotic adjustment is low compared with other crop species. In both controlled environment room and field studies, air temperature was shown to be the most important environmental variable to influence plant water status. Bearing trees exhibited higher stress levels than non-bearing ones and flowers and fruitlets exhibited more stress than leaves suggesting that reproductive organs may be more appropriat...

Prevention of Lettuce Tipburn by Supplying Air to Inner Leaves

Growing environments were controlled to prevent or reduce lettuce tipburn, a physiological disorder in inner developing leaves around a growing point caused by calcium deficiency. Lettuce (Lactuna sativa, L.) of butter head type was grown by hydroponics in a controlled-environment room which had heaters, humidifiers, a refrigerator, electric lamps, and a CO2 control apparatus. With air pumps and flexible pipes, room air was blown onto inner developing leaves totally or partially enclosed by the outer leaves. After transplanting, lettuce plants were exposed to various air supply conditions. Air temperature and RH were 24 C and 85%, respectively, in the light period, and 20 C and 85% in the dark. The PPF was 264 mmol m2sl, and light period was 14 hours. The CO2 concentration was 1500 mmol mol1. Temperature of the nutrient solution was constant at 20 C, pH was 5.8-6.2, and EC was 150 mS/m. The effects of various conditions on tipburn development were studied in relation to growth rate. Air supply to inner leaves prevented tipburn completely up to 60 g f.w., though tipburn occurred at approximately 15 g f.w. in the control. All-day air supply was more effective in preventing tipburn than air supply in the light period or the dark period alone. Air supply to inner developing leaves was proved effective in preventing tipburn without sacrificing a rapid growth rate. The reason for the effect was an increase in transpiration from the leaves encouraged water and calcium uptake by the root and increased calcium concentration in the leaves.

Attainment of puberty in domestic gilts reared under long-day or short-day artificial light regimens

The effect of photoperiod on attainment of puberty was examined in four studies using controlled long- or short-day light regimens. Gilts were reared together under natural photoperiod until divided between two controlled environment rooms at a mean age of 106.6 ± 0.52 days on 25 September 1987 (Study 1, n = 32) or 23 March 1988 (Study 2, n = 26). In Study 3 (21 September 1988; n = 35) and Study 4 (21 March 1989; n = 36) the gilts were transferred to the controlled environment rooms at 56.5 ± 0.82 days of age. In all studies the light: dark ratio was initially 12 h light: 12 h dark (12 L: 12 D) in both rooms and it was then either increased or decreased by 10–15 min per week. In Studies 1 and 2 the ratio was increased to 16 L: 8 D or decreased to 8 L: 16 D over 16 weeks. In Studies 3 and 4 it was increased to 14.5 L: 9.5 D or decreased to 9.5 L: 14.5 D over 12 weeks then returned to 12 L: 12 D over a further 12 weeks. Temperature was maintained at 23°C. In Studies 1 and 2 all gilts commenced daily exposure to mature boars at 173.5 ± 0.52 days of age and 82.9 ± 1.63 kg. In Studies 3 and 4 all gilts remained isolated from boars. In Studies 1 and 2 the light regimens during rearing had no significant effect on either the proportion of gilts which reached puberty in response to boars or the interval from first exposure to boars until puberty (overall, long-day 21 29 , 21.9 ± 2.83 days ; short-day 18 29 , 22.1 ± 2.66 days ). However, when gilts remained isolated from boars more of those housed under the short-day regimens reached puberty in both Study 3 ( 10 18 vs. 1 17 , P < 0.01 ) and Study 4 ( 10 19 vs. 0 17 , P < 0.01 ). The mean age at puberty was 209.3 ± 3.13 days in Study 3 and 214 ± 2.61 in Study 4. These data, obtained from gilts housed at constant cool temperatures under controlled light regimens which closely mimicked natural photoperiods, show conclusively that long days are inhibitory to attainment of puberty in the gilt. This finding provides compelling evidence that photoperiod is the major environmental factor responsible for the delay in puberty often seen during the summer under field conditions.

Fruit shape in apple under various controlled environment conditions

In several experiments done in controlled environment rooms under divergent air and soil temperatures and air humidities, fruit shape (expressed as the width height ratio) was determined throughout the season. Contrary to a number of reports on field surveys, fruit shape showed little or no influence of environmental factors. Seed number per fruit was not affected at all. As fruit shape is thought to be related to seed number, it is postulated that in the field climatic factors influence fruit shape via effects on seed formation during the flower and fruitlet stages, and that the environment has no further effect.

Water movement in yellow-cedar seedlings and rooted cuttings: comparison of whole plant and root system pressurization methods

One-year-old yellow-cedar (Chamaecyparis nootkatensis (D. Don) Spach) seedlings and rooted cuttings were taken from cold-storage facilities and grown in aerated water in a controlled environment room for 21 days. Root areas of seedlings and rooted cuttings were initially comparable, but after 21 days seedlings had produced more than twice as much new root area as rooted cuttings. Water flow resistance through the plant-atmosphere continuum decreased in both stock types over the 21-day period. In both seedlings and rooted cuttings, transpirational flux density increased in a curvilinear manner as new root area increased. Root resistance to water movement decreased sharply with increasing new root area up to 20 cm(2) in both seedlings and rooted cuttings. Further increases in new root area did not result in further changes in root resistance of either stocktype. Plant water movement was measured by both the whole plant and the root system pressurization methods with results differing between the two techniques.

The effects of phosphate supply on growth of plants from the Brasilian Cerrado: experiments with seedlings of the annual weed, Bidens gardneri Baker (Compositeae) and the tree, Qualea grandiflora (Mart.) (Vochysiaceae).

Plants of the cerrado tree species Qualea grandiflora and the annual herb Bidens gardneri were grown from seed in controlled environment rooms at 30/20° C and 12 hour photoperiod. Seedlings were grown in pots or small tubes containing sand and provided with various amounts of mineral solutions based on the formulation of Hoagland and Arnon but with the phosphate content modified in some cases. In a long-term experiment lasting 213 days, plants supplied with full strength Hoagland's solution all died but plants of Qualea given 1/10 strength solution survived, although they grew very slowly. Low relative growth rates (0.008-0.036 d-1) were also a feature of other experiments with Qualea and calculated rates of net assimilation rate gave values of 3-7 mg CO2 dm-2 h-1. Expansion of the photosynthetic surface proceeded slowly and the cotyledons were the main site of photosynthesis for more than 40 days. The low rates of growth occurred despite significant uptake of phosphorus by young plants and in shortterm experiments growth was independent of the amount of phosphate supplied and accumulated. In contrast, the values of R found for plants of Bidens reached 0.24 d-1. Growth of young plants was dependent on the external supply of phosphorus, being reduced when this was low and also when it was very high. Growth of the photosynthetic surface was also much more rapid than for Qualea and also varied with supply of phosphorus. The results are discussed in the context of the occurrence of these species in the Cerrado.

Influence of nitrogen form and concentration on the nitrate reductase activity of winter barley (Hordeum vulgare L cv Igri)

AbstractWinter barley (Hordeum vulgare L cv Igri) plants were grown for 5 weeks in a controlled environment room using a recirculating nutrient solution culture system at a root and shoot temperature of 10°C.When solution NO3‐N concentration was varied between 0 and 64 mg litre−1, shoot endogenous and induced nitrate reductase activity (NRAe and NRAi respectively) increased asymptotically from zero in plants grown with O mg NO3‐N litre−1 to a maximum activity in solutions containing ⩾ 16 mg NO3‐N litre−1. Shoot Nitrogen Response Index (NRI = NRAi/NRAe) showed an opposite trend decreasing from a maximum of 2.6 at 2 mg NO3‐N litre−1 to a minimum approaching unity when ⩾ 32 mg N litre−1 was supplied.In a separate experiment, nitrogen form (NO3‐N, NH4‐N or a 1:1 mixture) had a marked influence on shoot NRA. Enzyme activity was negligible in tissue from plants absorbing NH4‐N, highest in NO3‐fed plants and intermediate in those supplied with both N forms. NRI, however, approached unity in all treatments.The results provide evidence to suggest that NRI may be used as an indicator of the N status of barley plants grown with varied N nutrition.

Survival of Young Cold-hardened `Hamlin' Orange Trees at – 6.7C

Potted greenhouse-grown, l-year-old `Hamlin' orange [Citrus sinensis (L.) Osbeck] trees on 1.5-year-old rough lemon (C. jambhiri Lush.) rootstock were temperature-conditioned for 6 consecutive weeks in a controlled-environment room to test cold-hardening ability. Holding at 15.6 ± 0.6C during 12-hr days [425 μmol·s-1·m-2 photosynthetic photon flux (PPF) at top of trees] and 4.4C during nights resulted in 100% tree survival and no leaf loss “after 4 hr of – 6.7C in a dark freeze test room. Unhardened greenhouse trees were killed to rootstock. Solute efflux (dS·m-1) from unhardened frozen leaves was &gt; 20-fold that from frozen leaves on hardened trees and nonfrozen leaves on unhardened trees. Oxygen uptake was not significantly impaired in frozen hardened leaves. No 02 uptake was evident for frozen unhardened leaves.