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Frost tolerance and hardening capacity during the germination and early developmental stages of four white spruce (Picea glauca) provenances C. Coursolle, F.J. Bigras, and H.A. Margolis

Abstract: Frost tolerance during the germination stages of four white spruce (Picea glauca (Moench) Voss) provenances (between 45°37′ and 50°17′N) was studied at four different developmental stages (imbibed seed, radicle, cotyledon, and young seedling), and their hardening capacity was determined for the latter three stages. Hardening capacity was examined by submitting radicle-stage germinants to two temperature–photoperiod treatments (20:15°C – 16-h photoperiod or 5:5°C – 8 h) for 14 days and by submitting cotyledon and young seedling stage germinants to four treatments (20:15°C – 16 h; 20:15°C – 8 h; 5:5°C – 16 h; 5:5°C – 8 h). Frost tolerance was determined immediately after these treatments. Latitude of origin showed no clear pattern with respect to either frost tolerance or hardening capacity at any of the developmental stages. Imbibed seeds had the greatest degree of frost tolerance. With the exception of the most northern provenance, radicle-stage germinants did not respond to a 5:5°C day:night temperature and 8-h photoperiod hardening treatment. A low-temperature treatment of 5:5°C increased the frost tolerance of cotyledon and young seedling stage germinants, while their response to a shortened photoperiod (8 h) was quite variable. However, an 8-h photoperiod did enhance the effect of the low-temperature treatment at the young-seedling stage. Thus, the timing of germination in the field appears to be an important factor in the ability of germinants to tolerate freezing stress. Key words: germination stage, frost tolerance, hardening capacity, photoperiod, temperature. Résumé : La tolérance au gel de quatre provenances d’épinette blanche (Picea glauca (Moench) Voss) (situées entre 45°37′ et 50°17′N) a été étudiée à quatre stades de germination (graine imbibée, radicule, cotylédon et jeune semis) et la capacité d’endurcissement des trois derniers stades a été déterminée. Afin d’étudier la capacité d’endurcissement, les germinants au stade radicule ont été soumis pendant 14 jours à deux traitements de température–photopériode (20:15°C – 16 h, 5:5°C – 8 h) et les germinants aux stades cotylédon et jeune semis à quatre traitements (20:15°C – 16 h, 20:15°C – 8 h, 5:5°C – 16 h, 5:5° – 8 h) de même durée. La tolérance au gel a été mesurée à la fin des traitements. La latitude d’origine n’a pas eu un effet évident sur la tolérance ou la capacité d’endurcissement. Les graines imbibées sont les plus tolérantes au gel. À l’exception de la provenance la plus nordique, les germinants au stade radicule n’ont pas réagi au traitement d’endurcissement de 5:5°C et de 8 h de photopériode. Le traitement de basse température (5:5°C) a augmenté la tolérance au gel des germinants aux stades cotylédon et jeune semis, mais la réponse au traitement de courte photopériode (8 h) était variable. Cependant, une photopériode de 8 h augmente l’effet du traitement de basse température pour les germinants au stade jeune semis. Donc, la date de germination sur le terrain semble être un facteur important pour la capacité des germinants de tolérer un stress de gel. Mots clés : stade de germination, tolérance au gel, capacité d’endurcissement, photopériode, température.

Introduction Successful seedling establishment and early growth is one of the most important phases in the life cycle of a plant, since it determines the persistence of a species in a given habitat (Sakai and Larcher 1987). Therefore, it is important to understand how the early developmental (germination) stages of plants react and adjust to environmental stresses that can affect their

Received June 5, 1997. F.J. Bigras.1 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, Sainte-Foy, QC G1V 4C7, Canada. C. Coursolle and H.A. Margolis. Centre de recherche en biologie forestière, Université Laval, Sainte-Foy, QC G1K 7P4, Canada. 1

Author to whom all correspondence should be addressed. e-mail: [email protected]

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growth. One important stress factor is the capability of a seedling to adapt to and tolerate freezing temperatures that can occur almost anytime in northern forests (Christersson and von Fircks 1988). Considerable attention has been paid to the frost tolerance and hardening ability of forest tree seedlings during their first years of growth, yet little is known about these factors during the stages of germination and early development. Information available from the few existing studies of forest tree species as well as from studies on agricultural crops indicate that frost tolerance is generally greatest in intact seeds, decreases abruptly once the seeds have germinated, and eventually increases with chronological or physiological age (Arakeri and Schmid 1949; Andrews 1960; Dantuma and Andrews 1960; Cary 1975; Gray and Steckel 1983; Cremer and Mucha 1985; Sakai and Larcher 1987). Previous studies have shown that the hardening ability, as well as the degree of hardening, of plants seems to be affected by chronological age (Brown and Bixby 1976; Cloutier et al. 1990). According to Timmis and Worrall (1975), 3- (1 cm © 1998 NRC Canada


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long epicotyl) and 7-week-old (2 cm long epicotyl) seedlings of Pseudotsuga menziesii (Mirb.) Franco reacted to a lowtemperature – short-photoperiod treatment by increasing their frost tolerance, and the degree of response increased with age. However, 1-week-old seedlings (no epicotyl, cotyledons only) were unable to increase their frost tolerance. Chen and Li (1978) also reported similar results for Cornus stolonifera Michx. seedlings, with the greatest frost tolerance occurring under a combination of low temperature and short photoperiod. As well, 4-month-old seedlings showed a greater response to low temperatures compared with 2-month-old seedlings. However, these studies involved frost tolerance measurements after treatments of at least 7 days duration. This may cause difficulty in determining whether hardening capacity was due to the treatment applied or to aging alone, since plants may have passed through more than one developmental stage during the treatment period. This problem could be alleviated by using a control treatment, which would indicate if any natural hardening due to aging had occurred over the course of the experimental period. Furthermore, frost tolerance of older seedlings has also been reported to vary with latitude of origin of the seedlings (Toivonen et al. 1991; Simpson 1994; Coursolle et al. 1997). Therefore, it would be interesting to determine if this is the case for germinants going through their early developmental stages. The objectives of the current study were to evaluate the frost-tolerance levels of four provenances of white spruce seedlings during their initial stages of growth (imbibed seeds, radicles, cotyledons, and young seedlings) and to evaluate the hardening capacity of germinants at the radicle, cotyledon, and young-seedling stages.

Table 1. Latitude, longitude, and altitude of provenances used.

Materials and methods

Experiment 4: young-seedling stage Stratified seeds were sown in peat pots and placed in growth chambers as in experiment 3 and allowed to grow to the young-seedling stage (epicotyl 1–3 mm in length), which occurred 59 days after sowing. Watering and fertilization (starting 44 days after sowing) were carried out regularly. The same treatments as in experiment 3 were then applied to the young seedlings. A second lot was also sown 14 days after the first, as in the cotelydon experiment, so as to obtain an age control at the end of the 14-day treatment period. No fertilization was given during the treatments. All young seedlings were subjected to freezing tests immediately after the 14-day treatment period. Developmental stage was evaluated as for cotyledons (Table 2).

Plant material Four white spruce (Picea glauca (Moench) Voss) provenances originating between 45°37′ and 50°17′N in Quebec were used (Table 1). All seeds used for the study were first stratified in the dark in a cold room at 2°C for 21 days inside closed Petri dishes on moist filter paper. Growth conditions and treatments Treatments, growth stages, and age of seedlings used for each experiment are shown in Table 2. Experiment 1: imbibed-seed stage Stratified seeds were placed on three layers of wet filter paper in Petri dishes and allowed to imbibe water for 48 h at room temperature in the dark. Imbibed seeds were then subjected to freezing tests. Experiment 2: radicle stage Stratified seeds were allowed to germinate and grow on wet filter paper in parafilm-sealed Petri dishes placed in growth chambers with 20:15°C day:night temperatures and 16-h photoperiods at 200 µmol⋅m–2⋅s–1 (160 W cool white fluorescents) until germinants had reached the radicle stage (i.e., radicle 2–5 mm in length, mean 3.7 mm), about 8 days after seeds were placed in Petri dishes. Germinants were then placed in growth chambers programmed to give one of two treatments over a 14-day period: (i) 20:15°C – 16-h photoperiod and (ii) 5:5°C – 8-h photoperiod. A second lot of stratified seeds was started 14 days after the first lot and allowed to germinate and grow (under 20:15°C – 16-h photoperiod conditions) to the radicle stage. This produced a control (age control) that had just reached

Provenance

Latitude (N)

Longitude (W)

Altitude (m)

1 2 3 4

45°37′ 47°00′ 48°35′ 50°17′

70°58′ 74°37′ 78°21′ 73°50′

419 400 350 400

the radicle stage when the 14-day treatments applied to the first lot were finished. The two different 20:15°C treatments allowed us to verify if any natural hardening due to age had taken place during the 14-day period. Germinants from the two treatments and the age control were subjected to freezing tests immediately after the treatment period. Developmental stage and radicle length were evaluated both immediately before the treatment period and immediately before the freezing test (Table 2). Experiment 3: cotyledon stage Stratified seeds were sown in 5.7-cm2 peat pots (Magic peat pots, Gardener’s Delight, Brantford, Ont., Canada) on a peat moss – vermiculite mixture (3:1, v/v) and covered with 2 mm of silica. Pots were placed in growth chambers (20:15°C – 16-h photoperiod), irrigated regularly, and allowed to germinate and grow to the cotyledon stage. Germinants at the cotyledon stage (cotyledons fully emerged from seed envelope, 21–26 days after sowing) were then subjected for 14 days to one of four treatments: (i) 20:15°C – 16-h photoperiod, (ii) 20:15°C – 8-h photoperiod, (iii) 5:5°C – 16-h photoperiod, (iv) 5:5°C – 8-h photoperiod. A second lot of stratified seeds were sown 14 days after the first sowing and kept under 20:15°C – 16-h conditions so as to provide an age control. All the germinants were subjected to freezing tests immediately after the treatments had ended. Developmental stage was evaluated immediately before the freezing test (Table 2).

Freezing tests Imbibed seeds, radicles, or washed germinants (cotyledon or youngseedling stage) were placed on moistened filter paper in covered Petri dishes. The dishes were then placed in either a modified freezer (Kenmore model 47148, Sears Inc., Toronto, Ont.) with a programmable controller (controller model 512 and programmer model 519, Power Process Controls, Oakton, Ill., U.S.A.), for seeds and radicles, or in a modified, programmable (MIC 6000 control, Partlow Corp., New Hartford, N.Y.) cold room, for cotyledons and young seedlings. Temperatures in the Petri dishes were recorded with copper constantan thermocouples connected to a data logger (model CR21X, Campbell Scientific, Logan, Utah). The initial temperatures in the freezer and cold room were kept at 1.0°C and 2.0°C, respectively, for seeds and later germination stages for at least 2.5 h. The temperature was then lowered at rates of either 3.0°C/h for the imbibed-seed stage or 1.0°C/h for the other stages with 1-h plateaus after each drop in temperature. Ice crystals were added to Petri dishes at 0°C to promote ice nucleation. Petri dishes were removed at each of five (imbibed seeds) or six (other stages) freezing temperatures, which varied de© 1998 NRC Canada

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Table 2. Treatments applied to germinants during each experiment as well as age and developmental stages at time of treatment application and freezing tests.

Experiment (1) Imbibed seeds (2) Radicles

(3) Cotyledons

(4) Young seedlings

a

Treatments — (1) Age controla (2) 20:15°C, 16 hb (3) 5:5°C, 8 h (1) Age control (2) 20:15°C, 16 h (3) 20:15°C, 8 h (4) 5:5°C, 16 h (5) 5:5°C, 8 h (1) Age control (2) 20:15°C, 16 h (3) 20:15°C, 8 h (4) 5:5°C, 16 h (5) 5:5°C, 8 h

Seedling age at beginning of treatment (days)

Developmental stage at beginning of treatment

Seedling age at time of freezing test

— —

— — Radicle Radicle — Cotyledon Cotyledon Cotyledon Cotyledon — Young seedling Young seedling Young seedling Young seedling

48 h 8 days 22 days 22 days 21–26 days 35–40 days 35–40 days 35–40 days 35–40 days 59 days 73 days 73 days 73 days 73 days

8 8 — 21–26 21–26 21–26 21–26 — 59 59 59 59

Developmental stage at time of freezing test Imbibed seed Radicle Early cotyledon Radicle Cotyledon Young seedling Young seedling Cotyledon Cotyledon Young seedling Young seedling Young seedling Young seedling Young seedling

20:15°C day:night temperature and 16-h photoperiod. Day:night temperature and photoperiod are as indicated.

b

pending on developmental stage, at intervals of 6.0°C and 1.0°C, respectively, and placed with the frost controls (removed at 0°C) in a cold room at 2°C. Germinants were allowed to thaw overnight in the cold room at 2°C. The imbibed seeds or radicle-stage germinants were then returned to sealed Petri dishes containing wet filter paper and the cotyledon and young seedling stage germinants were replanted. Subsequently, germinants from all four growth stages were placed in growth chambers at 20:15°C – 16-h photoperiod. Viability tests Viability (percent survival) of seeds and radicle-stage germinants was determined 30 days after freezing tests and that of cotyledon and young-seedling stage germinants approximately 45 days after freezing tests. Imbibed seeds were judged viable if the root had emerged from the seed coat and produced a germinant with a radicle at least 2 mm long. Radicle, cotyledon, and young-seedling stage germinants were judged viable if growth resumed and was still occurring. Frost tolerance was evaluated as the lethal temperature causing 50% germinant death (LT50). LT50 values were calculated with the PROBIT procedure of SAS (SAS Institute Inc. 1989) using survival counts at each test temperature. Experimental design and statistical analysis Experiment 1: imbibed-seed stage The experimental design was a split-plot with four blocks. Freezing temperatures (including frost control), although not included in the statistical model since they were used to calculate the LT50 values, were randomly assigned to main plots and provenances to subplots (one provenance per Petri dish). A total of 3840 seeds (40 seeds × 6 test temperatures × 4 provenances × 4 blocks) were used in this experiment. A weighted analysis of variance was used to evaluate frost tolerance (LT50 values). LT50 values were weighted by the inverse of the width of their confidence interval (Scheffé 1959) so as to take into account the relative accuracy of the LT50 estimate. Orthogonal polynomial contrasts were calculated to evaluate the provenance effect.

Experiment 2: radicle stage The experimental design consisted of a split plot with four blocks (one block per growth chamber). Temperature treatments (20:15°C and 5:5°C) were applied to whole growth chambers (one treatment temperature per chamber). Frost temperature (including frost control) × treatment combinations were randomly assigned to main plots and provenances to subplots (one per Petri dish). A total of 1680 germinants (5 germinants × 7 test temperatures × 3 treatments × 4 provenances × 4 blocks) were used for the experiment. A weighted analysis of variance was performed on the LT50 data. Orthogonal contrasts were constructed to test the main effects and their interaction. Experiments 3 and 4: cotyledons and young seedlings A split-plot design with four replicates was used for both experiments. The design was the same as for the radicle stage except that two more treatments (20:15°C – 8 h, 5:5°C – 16 h) were added. Each temperature treatment (20:15°C and 5:5°C) was applied to a different growth chamber, and each growth chamber was divided in half, one half for the 16-h photoperiod and one half for the 8-h photoperiod. The photoperiod treatment was assigned randomly within each growth chamber. Germinants from the 8-h photoperiod treatments were covered with a 99.5% lightproof shade cloth (Cravo Equipment Ltd., Brantford, Ont.) to prolong the dark period. In all, 2800 germinants (5 germinants × 7 test temperatures × 5 treatments × 4 provenances × 4 blocks) were used. A weighted analysis of variance was performed on LT50 data. Treatment effects and their interactions were tested using the F test.

Results Experiment 1: imbibed-seed stage The frost tolerance of imbibed seeds varied with the latitude of origin of the seeds (Fig. 1). Provenances 2 and 4 had approximately the same LT50 value (approximately –20°C), while provenances 1 and 3 were less tolerant, having less negative values of –17.8°C and –15.0°C, respectively. © 1998 NRC Canada

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Coursolle et al. Fig. 1. Imbibed-seed stage frost tolerance (LT50; mean + SE) of the four white spruce provenances (P1, 45°37′N; P2, 47°00′N; P3, 48°35′N; P4, 50°17′N). Each point represents the mean of four observations. The P > F value is given for the cubic contrast testing the provenance effect.

Experiment 2: radicle stage Germinants submitted to the 20:15°C regime at the radicle stage were no longer at this stage at the end of the 14-day treatment (Table 2). They were, in fact, freeze tested at the cotyledon or hypocotyl stage and showed the highest degree of frost tolerance with LT50 values of between –5.6° and –7.1°C (Fig. 2). With the exception of provenance 4, the age control (seeds just entering the radicle stage when submitted to freezing tests) and the 5:5°C day:night hardening regime produced similar levels of frost tolerance, indicating that radicle-stage germinants did not react to a temperature hardening treatment. Latitudinal effects showed no clear pattern among treatments and the age control.

Experiment 3: cotyledon stage Germinants submitted to the 20:15°C regimes at the cotyledon stage had developed their first needles by the end of the 14-day treatment period when they were frost tested and thus exhibited a greater degree of frost tolerance compared with the age control (germinants just entering the cotyledon stage at time of freezing test; Fig. 3A). Cotyledon-stage germinants submitted to the 5:5°C regimes were still at the cotyledon stage after the 14-day treatments (Table 2). The effect of temperature and photoperiod both varied with the latitude of origin of the germinants (Figs. 3A and 3B). Neither the two-way interaction of temperature and photoperiod (P = 0.7641) nor the three-way interaction with provenance (P = 0.7327) were significant. The low-temperature treatment (5:5°C) increased the frost tolerance of cotyledon-stage germinants compared with the age control (Fig. 3A). Photoperiod effects were variable depending on the latitude of origin of the germinants (Fig. 3B).

Fig. 2. Radicle-stage frost tolerance (LT50; mean + SE) of the white spruce provenance (P1, 45°37′N; P2, 47°00′N; P3, 48°35′N; P4, 50°17′N) age controls (shaded bars) and germinants submitted to the 20:15°C – 16-h (hatched bars) and 5:5°C – 8-h (open bars) regimes for 14 days. Each point represents the mean of four observations. Radicles exposed to the 20:15°C – 16-h regime were at the cotyledon stage when freeze tested. Age control radicles were 14 days younger than the treated radicles and were just entering the radicle stage when freeze tested. The P > F value is given for the contrast testing the interaction between provenances and treatments (including age control).

Experiment 4: young-seedling stage Frost tolerance varied with respect to hardening temperature, photoperiod, and provenance (Figs. 4A and 4B). Under both photoperiods, germinants submitted to the 5:5°C hardening regime at the young-seedling stage had LT50 values 0.5–2.2°C lower than those submitted to the 20:15°C regime. As with cotyledons, the photoperiod treatment resulted in somewhat more variable reactions. However, provenance 1, 2, and 3 germinants grown under an 8-h photoperiod during the treatments exhibited a greater increase in frost tolerance in response to lower growth temperatures compared with those grown under a 16-h photoperiod (Figs. 4A and 4B). The age control was not significantly different (P = 0.2302) from the mean of the four treatments combined. In fact, results showed that age control seedlings had approximately the same level of frost tolerance as those from the 20:15°C – 16-h photoperiod treatment (Fig. 4B), thus indicating that increases in frost tolerance during the treatment period were a result of the hardening treatments and were not due to the chronological age of the seedlings.

Discussion Frost tolerance of developmental stages While statistical comparisons cannot be made because separate experiments were conducted for each developmental stage, it is apparent that imbibed seeds exhibit a higher frost tolerance (LT50 values of between –15 and –20°C) compared © 1998 NRC Canada

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Fig. 3. Cotyledon-stage frost tolerance (LT50; mean + SE) of the white spruce provenance (P1, 45°37′N; P2, 47°00′N; P3, 48°35′N; P4, 50°17′N) age controls (shaded bars) and germinants submitted to (A) 20:15°C (hatched bars) and 5:5°C (open bars) regimes and under (B) 16-h (cross-hatched bars) and 8-h (solid bars) photoperiods for 14 days. Each point is the mean of eight observations. Age control germinants were 14 days younger than the treated germinants and were just entering the cotyledon stage when freeze tested. Cotyledons exposed to the 20:15°C regimes were at the young-seedling stage when freeze tested. The P > F value is given for the F test of interactions between provenances and (A) temperature treatments or (B) photoperiod treatments.

Fig. 4. Young-seedling stage frost tolerance (LT50; mean + SE) of the four white spruce provenances (P1, 45°37′N (open bars); P2, 47°00′N (hatched bars); P3, 48°35′N (shaded bars); and P4, 50°17′N (solid bars)) submitted to 20:15°C and 5:5°C regimes under (A) 8- and (B) 16-h photoperiods for 14 days, as well as the frost tolerance of the age controls for the four provenances. Each point is the mean of four observations. Age control germinants were 14 days younger than treated germinants and were just entering the young-seedling stage when freeze tested. The P > F value is given for the F test of the triple interaction temperature × photoperiod × provenance.

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with the age controls of the later growth stages, and frost tolerance appears to decrease until the cotyledon stage and then increase thereafter (Fig. 5). These results are similar to those reported for Beta vulgaris L. (Cary 1975) and for Abies alba Mill. (Sakai and Larcher 1987). Other authors (Arakeri and Schmid (1949) for various legumes, Gray and Steckel (1983) for Allium cepa L., and Cremer and Mucha (1985) for Pinus radiata D. Don and Eucalyptus species) have also found that the frost tolerance of imbibed seeds was greater than that of germinated seeds. Results from the radicle-stage experiment also confirm that frost tolerance starts increasing once cotyledons have developed (Fig. 2). The age control was less frost tolerant than the 20:15°C treatment, whose germinants had reached the next germination stage (cotyledon) at the time of frost testing. The frost tolerance of the early stages of germination varies with species and developmental or chronological age. The frost tolerance of hardened Eucalyptus delegatensis R.T. Baker seedlings increased from the cotyledon stage up to 2 months of age (Battaglia and Reid 1993). Andrews (1960), Dantuma and Andrews (1960), and Roberts and Grant (1968) found that hardened seedlings of Secale cereale L., Hordeum vulgare L., and Triticum aestivum L. all showed initial increases in frost tolerance (from 0 to 7 days of age for S. cereale and H. vulgare and from 0 to 4 days for T. aestivum) followed by a decrease to about 21 (S. cereale and H. vulgare) or 14 days (T. aestivum) of age and then an increase in tolerance thereafter. On the other hand, Fuller and Eagles (1978) showed that the frost tolerance of unhardened seedlings of Lolium perenne L. did not increase with chronological age but that tolerance of hardened seedlings did. Results indicate that the frost tolerance of the four germination stages generally varied with respect to the provenance of origin of the seeds, but variations were inconsistent from one developmental stage to another. Coursolle et al. (1997) found that the frost tolerance (after one season of growth and during dehardening at the beginning of the second season) of the same Picea glauca provenances as those used in the present study increased with increasing latitude of origin. Toivonen et al. (1991) and Simpson (1994) also reported that the frost tolerance of Pinus sylvestris L. and Picea glauca seedlings increased with increasing latitude. No such pattern was evident at any of the germination stages during this experiment. For example, provenances 2 and 4 showed similar and higher levels of frost tolerance at the imbibed seed stage compared with provenances 1 and 3, while the frost tolerance of unhardened seedlings decreased from provenance 1 (most southerly) to provenance 4 (most northerly) at the young-seedling stage. This may be an indication that latitudinal effects are difficult to detect or not well defined at early developmental stages. Hardening capacity of different developmental stages According to our results, the hardening capacity of white spruce seedlings in response to 14-day hardening treatments varies with developmental stage. Radicles of three of four provenances did not respond to a lower temperature combined with a shorter photoperiod (Fig. 2), while those at the cotyledon and young-seedling stages responded to a low-temperature treatment with an increase in frost tolerance (Figs. 3 and 4) and exhibited various responses to shorter photoperiods. Radicles submitted to a 5:5°C – 8-h regime responded by

127 Fig. 5. LT50 (mean 6 SE) values of imbibed white spruce seeds and of age controls for radicles, cotyledons, and young seedlings. Each point is the mean of 16 observations.

slowing their development (radicles doubled in length but no cotyledons were present), which resulted in approximately the same level of frost tolerance as the age control at this stage. Cloutier et al. (1990) found that Medicago sativa L. seedlings had to be at least 6 days old when submitted to a 28-day hardening treatment (1°C, 8-h photoperiod) for them to react to the treatment. Unfortunately, no mention of the developmental stage at the beginning or end of the treatment was made. On the other hand, cotyledons and young seedlings showed a definite capacity to harden when submitted to lowtemperature treatments for 14 days. With the exception of provenance 1 cotyledons, the frost tolerance of cotyledons and seedlings submitted to 5:5°C regimes was higher compared with age controls (Figs. 3 and 4). Brown and Bixby (1976) reported the same type of reaction for Robinia pseudoacacia L., in which 4-week-old seedlings were unable to react to a hardening treatment while 8-, 12-, and 16-week-old seedlings did, and their degree of frost tolerance increased with chronological age. Chen and Li (1978) also found that 12-week-old Cornus stolonifera seedlings had a greater response to a low-temperature treatment compared with 8-week-old seedlings. Neither of these studies, however, indicates the developmental stage of seedlings at the beginning of the treatment or when they were freeze tested. Finally, Timmis and Worrall (1975) also reported that 7-week-old seedlings (2-cm epicotyl) of Pseudotsuga menziesii reacted to low temperature treatments by increasing their frost tolerance and that 1-week-old seedlings submitted to low-temperature treatments also reacted in the same way, but not until their epicotyl had started elongating. Frost tolerance responses to photoperiod varied with provenance and were inconsistent, i.e., provenances showed different reactions depending on germination stage. Thus, while the results are difficult to interpret, it would seem that photoperiod is less important (generally less than 1°C differences in LT50 values) than temperature for hardening of early © 1998 NRC Canada

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developmental stages. This may indicate that white spruce seedlings at the cotyledon and young-seedling stage can harden in response to unseasonably cold temperatures while still in a growth phase and before critical daylengths have occurred. This could be important to seedling survival in the field, since germination normally takes place in June and can be as late as July (Fowells 1965). However, three of four provenances at the young-seedling stage showed a greater increase in frost tolerance in response to the low-temperature treatment when it was given under an 8-h photoperiod compared with a 16-h one (Fig. 4). Timmis and Worrall (1975) reported a similar result for 7-week-old seedlings of Pseudotsuga menziesii, which had a greater reduction in frost damage when the cold temperature treatment (6°C compared with 11°C) was applied under an 8-h photoperiod compared with 12- and 16-h ones. Chen and Li (1978) also found that 2-month-old seedlings of Cornus stolonifera reacted to both lower temperatures and shorter photoperiods. A combination of both treatments gave the highest frost tolerance, followed by the low-temperature treatment alone and finally the photoperiod treatment alone. Provenance interactions with hardening treatments were significant for the radicle, cotyledon, and young-seedling stages, but the effects of latitude of origin on hardening capacity were again quite variable and inconsistent from one germination stage to the next. This may indicate that each provenance has specific and different photoperiods and (or) temperatures that trigger the onset of hardening, thus reflecting their ability to adapt to the environmental conditions of their different habitats. On the other hand, these results may simply reflect the fact that latitude of origin does not have an important effect on hardening treatments, a possibility that seems to be supported by two studies dealing with the effects of photoperiod treatments that used 10-week-old seedlings of the same Picea glauca provenances as those used in this experiment (Coursolle et al. 1996, 1997) and reported no significant treatment by provenance interactions.

Conclusions Imbibed seeds of white spruce had a greater frost tolerance than germinants at the radicle, cotyledon, or young-seedling stages. White spruce germinants did not have the capacity to harden in response to low-temperature or photoperiodic treatments until they had reached the cotyledon stage and the latitude of origin of the seedlings had no clear or consistent effect on the hardening capacity of germinants during these early developmental stages. Cotyledons and young seedlings increased their frost tolerance in response to a low-temperature treatment (5:5°C) but an 8-h photoperiod enhanced the response to the low-temperature treatment only for young seedlings. Thus, timing of germination in the field is likely to be an important factor in determining the survival of seedlings. Seedlings having the greatest chance of survival would be those whose germination is timed so that they are (i) no further along than the early radicle stage during the spring frost risk period and (or) (ii) those that have reached the later stages of the cotyledon phase or the young seedling phase when the autumn frost risk period starts. Those at the early radicle stage would probably survive frosts of about –5°C (Fig. 5), while those at the cotyledon stage would be able to start hardening in response to decreasing temperatures. Those at the young-

Can. J. Bot. Vol. 76, 1998

seedling stage, however, can harden in response to both lower temperatures and shortened photoperiods.

Acknowledgements The authors thank Yves Dubuc for technical assistance and Michèle Bernier-Cardou, senior statistician, for help with the statistical analysis. We also thank Germain Couture (Ministère des Ressources naturelles du Québec) and the Centre de Semences Forestières de Berthier for supplying seeds. This research was funded by the Ministère des Ressources naturelles du Quebec, Natural Resources Canada, and the Natural Sciences and Engineering Research Council of Canada.

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