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Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND 58105
* Corresponding author (dmeyer{at}ndsuext.nodak.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Abbreviations: LT50, temperature that kills 50% of seedlings
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Tolerance to freezing temperature varies with multiple factors, and it is difficult to determine how much freezing damage is the result of temperature alone. A crop species may have tolerance varying with different growth stage, duration of freezing temperature, soil moisture, acclimation/declamation cycles, and other associated factors. Frost may cause damage at any age of the plant. For example, yield reductions from frost occurred from 0 to R7 growth stages for two early maturing soybean cultivars in Canada (Saliba et al., 1982). Hume and Jackson (1981) evaluated 30 soybean genotypes of different sources and maturity groups at -2, -2.5, and -3°C at the cotyledon, unifoliolate, and first trifoliolate leaf stages. Prefreezing growth temperatures in the greenhouse were 15/9, 20/14, and 25/19°C (day/night). They found that the greatest soybean mortality occurred at the unifoliolate stage at -3°C when growth temperature in the greenhouse was 25/19°C compared with the lower growth temperatures. With exceptions, temperature drop in the spring does not occur abruptly in the northern Great Plains but over a 2- or 3-d period. The seedlings were not hardened in Hume and Jackson's (1981) experiments, temperature was dropped sequentially from prefreezing to freezing over a 9.5-h period. But dropping the temperature from 9, 14, or 19°C at night to a freezing temperature within 9.5-h generally does not happen in the northern Great Plains. A weather front could drop the air temperature abruptly, but rarely does a freezing temperature occur the first night, which allows some seedling acclimation prior to freezing.
In another report, Hicks (1978) indicated that soybean was more tolerant to freezing temperature at the unifoliolate than at the third-trifoliolate leaf stage. However, plants from both stages could resume growth within 2 h after freezing at -3.8°C.
Seedling age is important for tolerance to low temperature. Calder et al. (1965) reported that legume seedlings were more susceptible to frost injury in the vegetative stage (up to 48 d) compared with later stages. However, Peltier and Tisdal (1932) and Tisdal and Pieters (1934) reported that 2-wk and older seedlings were more tolerant to freezing than younger seedlings. None of these reports showed uniform survival rate of the seedlings at a particular temperature.
Many previous experiments (Megee, 1935; Steinmetz, 1926; Jung and Smith, 1961; Calder et al., 1966) have evaluated factors related to overwintering of forage legumes. Very few have evaluated spring-seeded seedling tolerance to freezing temperature. Arakeri and Schmid (1949) grew alfalfa, sweetclover, red clover, alsike clover, and white clover in the greenhouse at 20°C. Seedlings of different growth stages were hardened at 4°C for 15 d and then again at 10°C for 2 d in the growth chamber. Seedlings were held in the freezing chamber at -10°C for 8 h before transferring them to the greenhouse for 2 wk to obtain survival counts. Across the legume species, prefreezing growth stages represented by 1, 2, and 3 wk in the greenhouse (hypocotyl arch through 3- to 4-leaf stages) were more tolerant to freezing temperature than were older seedlings. However, survival percentage was more by the fourth week for alfalfa, and after the fifth week for alsike, red, and white clovers. Sweetclover seedlings younger than 9 wk did not survive the freeze. Their data suggested that species and seedling age both influenced frost tolerance. However, the long hardening period used did not reflect typical northern Great Plain conditions where hardening period during the spring rarely exceeds 3 d, and rarely does the duration of minimum temperature last for more than 1 to 4 h.
Calder et al. (1965) hardened alfalfa seedlings at 2°C for 48 h. They found that unhardened alfalfa seedlings were killed at -4.5°C temperature and did not regrow within two weeks. However, Tisdal and Pieters (1934) reported that about 90% of unhardened alfalfa and red clover seedlings survived at -4.1°C and no seedling death occurred with the hardened seedlings. In these experiments, they found that tolerance of seedlings to freezing temperature increased with hardening.
The threat of spring frost delays planting of many species. Delayed planting of dry bean may cause greater economic losses than does frost through the reduction of yield and quality of seed (Blaylock, 1995). Delayed planting may also cause crop failure because of early fall frost. Although the risk of fall frost may be reduced by planting early in May, the risk of a killing spring frost is increased (Halvorson et al., 1995). Sims et al. (1989) reported that dry bean should be seeded as early as machinery can safely be used in the field in Montana. Each day of delay in seeding from 1 June to 7 June resulted in 29 kg ha-1 decrease in yield across four cultivars of pinto bean. They also suggested that even for warm-season beans, seeding should not be delayed beyond the first week of June for the crop to mature before the first killing frost in fall.
The limited studies evaluating freezing temperature tolerance of seedling legumes were conducted under hardening and temperature duration conditions usually atypical of that prevailing in the northern Great Plains during the spring. Therefore, our objectives were to determine freezing temperature tolerance of 10 legume species at four seedling ages simulating conditions more often prevailing in the field and to determine the 50% killing temperature (LT50) for each legume species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Legume species were seeded in pots and placed in the greenhouse. Pot size was 100 by 100 by 90 mm. Rooting medium included a 1:1 Fargo clay soil (fine, montmorillonitic, frigid Vertic Haplaquoll) and Sunshine (a greenhouse growth medium, Sun Gro Horticulture Canada Ltd., Bellevue, WA) mixture. Forage legumes (alfalfa, red clover, sweetclover, alsike clover, white clover, and sainfoin) were seeded from 10 to 20 mm deep and grain legumes (pinto bean, navy bean, soybean, and field pea) from 20 to 30 mm deep. Ten to 12 seeds for forage legumes and three to four seeds for grain legumes were seeded and subsequently thinned to six and two plants per pot, respectively. The greenhouse was maintained at 25/19°C day/night temperature. Sodium vapor lights were used to supplement light intensity from about 2 m above the plant canopy during the day (about 14 h). Average light intensity was 1392 µmol m-2 s-1.
Plants at 1, 2, 3, or 4 wk of seedling age were transferred to a controlled environmental chamber maintained at 3.5 to 4°C for 3 d for hardening. Then, plants were put in a growth chamber at 15/5°C day/night temperature for 2 d after hardening. From the growth chamber, the plants were placed in a programmable freezing chamber at 3°C, and the temperature decreased 1°C h-1 until the desired minimum temperature was reached. Duration of minimum temperature was 1 h. The temperature was increased from the minimum temperature at 1°C h-1 until 3°C was reached. After freezing, the plants were transferred to the growth chamber for 1 d before they were transferred to the greenhouse (environmental conditions same as before). Unfrozen controls for each species were maintained in the growth chamber while freezing occurred. The plants were evaluated for 2 wk following freezing.
Number of dead plants, plant height, and above-ground biomass were determined right after the plants were transferred to the greenhouse. Regrowth potential and leaf chlorosis (bleaching injuries) were determined on survived plants 10 to 14 d after freezing. Chlorosis was determined by visual examination of the yellowing of leaf area for frozen plants compared to unfrozen control. Dry biomass weight on survived plants was determined by drying samples at 60 to 65°C for 72 h in a force-air oven. Initiation of new shoot (leaf) and shoot size during a 2-wk period in the greenhouse was considered as the criteria for regrowth potential. Regrowth potential was visually assessed on a 0 to 4 scale, where 4 was considered equal to the nonfrozen control plants and 0 had no regrowth. Percentage of plant height, dry biomass, leaf chlorosis, and regrowth potential were calculated on the basis of nonfrozen plants.
A randomized complete-block design with four replicates was used. Each freezing chamber set comprised 10 legume species by one freezing temperature by one growth stage. Each set had two separate runs in the freezing chamber. Data were analyzed by analyses of variance and regression by means of SAS (SAS Institute, 1990). Analyses were performed on both actual and arcsine transformed data, but the actual data were used since the transformation did little for the analyses. Run in the freezing chamber was considered a random factor where as temperature, growth stage, and species were considered fixed effects in the analyses. Logistic regression analyses were done to estimate parameters of LT50 prediction equations for each species. The model used was
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= Ln 
= 0sc. Therefore, the final prediction equation of LT50 was
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The equation for predicting percentage seedling death at temperatures other than LT50 was
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Confidence interval (95%) of LT50 was calculated as
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Where variance of LT50 = variance of a(-1/ß)2 + variance of ß(a/ß2)2 + 2covariance of (a,ß)(1/ß)(a/ß2).
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RESULTS |
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All forage legumes had 100% survival for all four ages at -2°C (Table 1). Field pea was resistant to freezing at -2°C up to 3 wk and soybean at 1 wk of age. Killing temperature for pinto and navy beans started at -2°C. Six to 19% of pinto and navy bean plants were killed at -2°C for 1 to 4 wk of age.
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All forage legumes had 100% survival for 1 wk of age at -6°C except red clover (Table 1). At -6°C, sainfoin and white clover had the highest survival rate at 2 wk of age. However, at 3 wk of age, alsike and white clover seedlings survived better than alfalfa, red clover, and grain legumes. Survival rate at 4 wk of age increased numerically compared with 2- and 3-wk-old seedlings for all forage legumes. Among the grain legumes, field pea and soybean had greater tolerance to freezing temperature at 1 wk of age than pinto and navy beans. Moreover, field pea and soybean had little tolerance to freezing temperature at other ages. Pinto and navy bean had little tolerance at -6°C, and almost 100% seedling death occurred at all ages.
All grain legumes were killed at -8°C temperature regardless of seedling ages (Table 1). Alsike clover had greater survival rate (50%) at 1 wk of age than other small-seeded legumes. Alfalfa and alsike clover were similar at 2 and 3 wk of age, but alsike clover had greater survival than other forage legumes at these ages. Seedling survival rate was similar for all forage legumes and sainfoin at 4 wk of age. This indicates that species with greater freezing tolerance than other species at 1 wk did not maintain the differences when they grew older.
The LT50 for alsike clover was the coldest (-7.43°C) of legumes tested while red clover was the warmest (-6.31°C) of the forage legumes (Table 2). The LT50 for soybean and field pea were equal. The LT50 of navy bean was slightly colder than that of pinto bean. Three tolerance groups emerged from the 10 species studied, the forage legumes were the most tolerant, dry edible beans were the least tolerant, and soybean and field pea were intermediate. Equations from Table 2 predicted that substantial seedling loss would occur for forage legumes at temperatures below -6°C with the exception of red clover, which was sensitive to freezing temperature below -5°C. Likewise, significant seedling loss of pinto and navy beans started at -3°C and for soybean and field pea at -4°C.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Variation in frost tolerance among legume groups could be associated with differences in cell structure. Gusta et al. (1983) reported that plant tissue could avoid freezing by allowing ice crystals to grow in the extracellular spaces. To avoid damage to live cells, water must diffuse from within the cell to extracellular areas and form ice crystals quickly enough to maintain a critical freezing point below the ambient temperature. Freezing tolerance is favored in tissues having a high specific surface area (small cells), high permeability to water, and (or) low free-water content, which decreases the amount of water that diffuses out of the cells to achieve equilibrium (Gusta et al., 1983). Cell structural differences among the legume groups probably explains the differences in frost tolerance we observed.
Dry edible beans (pinto and navy) had different frost tolerance at temperatures below their LT50 (-3.25°C). The first-trifoliolate leaf (3-wk) stage of pinto bean and the unifoliolate leaf (2-wk) stage of navy bean were the most susceptible to frost damage when freezing temperature went below the LT50 (Table 1). Hume and Jackson (1981) reported that the unifoliolate leaf stage of soybean was more susceptible than the hypocotyl arch and first-trifoliolate leaf stages, which was similar to navy bean but different from pinto bean in our study.
Soybean and field pea were moderately tolerant to frost among the legume species studied. There was no difference between the LT50 of soybean and field pea. All growth stages of soybean were tolerant to -3°C temperature, but only the hypocotyl arch (1 wk of age) and up to 2 wk of age of field pea were tolerant to -4°C (Table 1). Welbaum et al. (1997) reported a similar tolerance in field pea. At 1 wk of age, both of these species had a high tolerance to freezing even when the temperature was colder than the LT50. This was not the case for most of the other species evaluated in this study. Substantial seedling death (over 60% as predicted across ages) started at -5°C for both species. Field pea is normally considered a more frost-tolerant species than soybean in the field, partially because the cotyledons remaining below the soil surface, but that difference was not observed under controlled conditions of this experiment. Also, we did not observe regrowth occurring from below the soil surface on plants killed by freezing temperatures. Possibly the 2-wk evaluation period was too short to allow regrowth to occur. Swensen and Murray (1983) reported that spring field pea grown under controlled environmental conditions had 21 and 0% survival at -3 and -6°C, respectively, after 1 wk of hardening. These results corroborate our results where field pea had 25 and 0% survival at -4 and -6°C, respectively, at 4 wk of age.
The LT50 of soybean was -4.5°C in our experiment. However, Hume and Jackson (1981) reported the LT50 was warmer than -3°C for soybean. We subjected the soybean seedlings to hardening conditions prior to freezing, and the minimum temperature duration was shorter than that used by Hume and Jackson, 1981. Prefreezing hardening (Arakeri and Schmid, 1949) and a short duration of freezing temperature might have increased tolerance of soybean and decreased the LT50 in these experiments.
All forage legumes had high levels of frost tolerance (100% survival) at -4°C at all four ages of growth (Table 1). Hypocotyl arch (1 wk) was the most tolerant growth stage of all forage legume species at -6°C (warmer than LT50), but tolerance at the later growth stages was reduced when temperature was colder than the LT50. This indicates that the LT50 for a particular species is a sensitive critical temperature below which substantial seedling death will occur irrespective of growth stage. Calder et al. (1965) reported that legume seedlings were more susceptible to frost when younger than 48 d compared with older plants. However, several researchers (Peltier and Tisdal, 1932; Tisdal and Pieters, 1934; Arakeri and Schmid, 1949) reported that seedlings younger than 3 wk were more tolerant to freezing temperature, which agrees with our results.
The prediction equations in Table 2 were developed to determine the mortality rate of seedlings down to -8°C under the environmental conditions of this study. Variability in factors like prefreezing temperature (Hume and Jackson, 1981), growth stage (Arakeri and Schmid, 1949), duration of temperature, soil type, and soil moisture may affect how robust the equations are when applied to field conditions. Unfortunately, these equations cannot be verified under field conditions since any freezing event will be a single observation.
Plant injury level was an important characteristic indicating frost tolerance in this study. Among the three tolerance groups, dry beans had the least regrowth even when the plants survived the freezing temperature. Leaf injury or chlorosis was the main cause of death for dry beans. However, both stem and leaf injuries were necessary to kill forage legumes and field pea. In general, it took at least 2 d after thawing to determine if injury had occurred. Therefore, assessment of stand loss the day after a freezing temperature may give an erroneous estimate of frost killing of legume seedlings, especially the forage species.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Received for publication May 19, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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This article has been cited by other articles:
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  J. L. De Bruin and P. Pedersen Soybean Seed Yield Response to Planting Date and Seeding Rate in the Upper Midwest Agron. J., May 7, 2008; 100(3): 696 - 703. [Abstract] [Full Text] [PDF]
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 M. Badaruddin and D. W. Meyer Factors Modifying Frost Tolerance of Legume Species Crop Sci., November 1, 2001; 41(6): 1911 - 1916. [Abstract] [Full Text] [PDF]
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and temperatures.