Crop Science 43:96-100 (2003)
© 2003 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Cold Tolerance in Oilseed Rape over Varying Acclimation Durations
C. L. Rife*,a and
H. Zeinalib
a Dep. of Agronomy, 2004 Throckmorton Plant Science Center, Kansas State Univ., Manhattan, KS 66506-5501
b Agronomy Dep., College of Agriculture, Tehran Univ., Karaj, Tehran, Iran
* Corresponding author (crife{at}oznet.ksu.edu)
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ABSTRACT
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Winter rapeseed (Brassica napus L.) promises to be an important crop in the southern Great Plains. To increase the crop's consistency in the region, cultivars with improved winter hardiness and management recommendations that utilize all of a genotype's winter hardiness need to be developed. Better understanding of the variability in cold tolerance over the winter season need to be determined to help achieve these goals. The objectives of this study were to evaluate variability in cold tolerance over prolonged acclimation durations in oilseed rape by means of laboratory freezing tolerance procedures, and determine changes in freezing tolerance brought about by high temperature treatments. A warming period to simulate possible deacclimation events was included both before and after vernalization saturation had taken place. Maximum cold tolerance was achieved by 3 d of acclimation in the spring cultivar and between 6 and 9 d in the winter cultivars. A 7-d warming period reduced cold tolerance in all lines tested, but it did not reduce cold tolerance to the level of unacclimated plants. All tested lines were able to recover cold tolerance to the same level as before the deacclimation treatment after reexposure to 5°C conditions for 7 d. Cold tolerance did tend to decrease with prolonged acclimation durations. This information can help in the planning of strategies to develop rapeseed lines better adapted to the winter conditions of the southern Great Plains.
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INTRODUCTION
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OILSEED RAPE has become a major crop in North America. North American cropland dedicated to rapeseed production has increased from 2 632 000 ha in 1986 to 5 573 000 ha in 2000 (Canola Connection, 2001; NASS, 2001). Nearly all of this production is due to the cultivation of spring annuals grown in the Canadian Prairie Provinces and the northern Great Plains of the USA. Expansion of spring annuals production farther south is limited by high temperature stress during the flowering and seed development stages (Raymer et al., 1990). Winter rapeseed has been successfully grown in the Pacific Northwest, southern Great Plains, Midwest, and southeast regions of the USA. When hardy cultivars are successfully established, they almost always survive the winters in USDA Plant Hardiness Zones 6, 7, and 8. The hardiest cultivars will routinely survive winters in the southern parts of zone 5 but survival is not as consistent as farther south (Rife et al., 2001). However, winter hardiness and freezing tolerance are still concerns for improving production consistency in many regions of the country.
Low temperature is one of the primary stress factors that limit growth and productivity of winter annual plants. Winter plants have evolved survival mechanisms that are temperature regulated to cope with low temperature stress. The vernalization response and low temperature acclimation are among the most important of these winter survival mechanisms. Vernalization is defined as acceleration of the ability to flower by a chilling treatment (Chouard, 1960). Plants acclimate to survive metabolic lesions because of intracellular ice formation, as well as to survive the dehydrative effects of frost (Kacperska, 1984). Fowler et al. (1996) found that after the vernalization requirement was met in wheat (Triticum aestivum L.) and rye (Secale cereale L.), cold acclimation declined. Laroche et al. (1992) did not observe this reduction in cold acclimation in rapeseed but they estimated cell survival on excised leaves to determine freezing tolerance and not crown meristem survival.
The relationship between vernalization requirements and cold tolerance is not clear. Andersson and Olsson (1961) indicated that selecting plants with high winter hardiness levels and long vernalization requirements are closely linked. However, in environments such as Japan's, many rapeseed cultivars have evolved long vernalization requirements but possess little tolerance to low temperatures (Sovero, 1993). Markowski and Rapacz (1994) compared vernalization requirements and frost resistance of winter rape lines derived from doubled haploid and found little relationship between these traits. Rapacz and Markowski (1999) found a significant correlation between vernalization requirement and both frost resistance and field survival when looking at older, high erucic acid cultivars. This correlation was not present in the 00 rapeseed cultivars they studied and was probably due to the recent influx of genetic material from spring genotypes. In cereals, most research findings indicate that although genotypes with longer vernalization requirements are generally more frost resistant, it is possible to find genotypes that require shorter vernalization period but exhibit high levels of cold tolerance (Brule-Babel and Fowler, 1988; Doll et al., 1989). Long vernalization requirements will delay a plant from entering the reproductive growth phase, a cold sensitive plant growth stage (Fowler et al., 1996). Since both mechanisms are advantageous for survival under winter conditions, it is probable that they evolved together. However, it does not appear that they are always controlled by the same genetic mechanisms or are closely linked.
Studying cold tolerance in the field is difficult. Field sites often exhibit either complete survival or complete winterkill. Because of this variability, laboratory procedures to measure freezing tolerance have been developed by a number of investigators. These include plant tissue water content (Brule-Babel and Fowler, 1988), ion leakage from plant cells after a freezing stress (Teutonico et al., 1993), and changes in luminescence (Brzostowicz and Barcikowska, 1987). Meristem regrowth after plants are subjected to freezing temperatures is also commonly used to estimate cold tolerance (Fowler et al., 1981; Andrews and Morrison, 1992). Laboratory freezing tolerance procedures have allowed investigators to gather information on cold tolerance that would otherwise be unobtainable in the field.
The objectives of this study were to (i) evaluate variability in cold tolerance over prolonged acclimation durations in oilseed rape and (ii) determine changes in freezing tolerance brought about by high temperature treatments.
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MATERIALS AND METHODS
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Three oilseed rape cultivars with varying degrees of vernalization requirements and cold tolerance were used in all experiments. ‘A112’ was developed for the winter annual cropping system of the Southeast USA and was released in 1990. A112 has little vernalization requirement and poor winter hardiness. ‘Ceres’ is a winter rapeseed developed in Germany and released around 1986. Ceres is moderately winter hardy and requires less than 6 wk at 5°C to vernalize. ‘Plainsman’ is a winter rapeseed developed in Kansas and released in 1998. Plainsman requires less than 8 wk at 5°C to vernalize. When evaluated at 60 environments in the Great Plains, Plainsman averaged 74% winter survival compared to 64% for Ceres (Rife et al., 2001).
Plants were seeded and grown in plastic flats (206 by 521 mm) containing 48 cells (eight six packs). Each cell measured 25 by 50 mm. Cells were separated by an air space of approximately 10 mm. This allowed cold air to act upon all seedlings in the same manner during the freezing treatment. Flats were over planted and thinned to one plant per cell to maintain equal seedling size and an even stand. Each six pack was an experimental unit, and the plant in each cell represented an observational unit. The soil medium was approximately a 6:1:1 soil (Wamego Silt Loam soil [Fine, Mixed, Mesic Typic Argiudoll]): peat: pearlite ratio by weight. The medium was amended with both macro- and micronutrients at the rates (g nutrients kg-1 medium) of 2.88 N, 3.49 P, 2.48 K, 4.62 S, 1.32 Ca, 16.05 x 10-4 B, 80.25 x 10-4 Cu, 1929 x 10-4 Fe, 40.70 x 10-4 Mn, 8.03 x 10-4 Mo, and 160.9 x 10-4 Zn. After establishment, seedlings were fertilized weekly with a 20-9-17 water-soluble fertilizer (Peters, Allentown, PA).
Four weeks after planting, the seedlings were moved from the greenhouse to a growth chamber (Conviron, Asheville, NC) and hardened. At this stage, the seedlings were equivalent to the Sylvester-Bradley and Makepeace (1984) growth stage 1.05 and had about five true leaves exposed. The growth chamber conditions were 12-h (300 µmol m-2 s-1 PAR) day length and a 5°C temperature. After the appropriate hardening duration, plants were placed randomly in a low-temperature incubator for the freezing treatment. Initial temperature in the incubator (Percival, Boone, IA) was 1°C. The temperature was subsequently lowered to -1°C over a period of 2 h, and held at -1°C for 12 h, to ensure that nucleation occurred evenly. The temperature then was reduced 1°C per hour. During this treatment, plants were removed from the incubators at the appropriate temperatures (-6, -8, -10, or -12°C) and moved back to the growth chamber. Twenty-four hours after the last treatment was removed from the incubator, all plants were returned to the greenhouse. After 3 wk, the plants were analyzed for root and shoot regrowth. Plant survival was defined as plants that developed and maintained photosynthetically active leaf tissue and developed and maintained active roots.
The three cultivars were evaluated at different acclimation durations (0, 3, 6, 9, 12, 20, 30, 40, 60, and 80 d) by the Cold Tolerance Determination procedure described above. Experimental design was a three factor, three replicate factorial with acclimation duration and freeze temperatures (-6, -8, -10, or -12°C) split on the three cultivars in a completely randomized design. Because of the lack of acclimation, temperatures of -2, -4, -6, and -8°C were used to evaluate the seedlings at the 0 d duration to calculate the LT50s.
A series of experiments were established to determine the effect of a warming period followed by a subsequent reacclimation period on the cold tolerance of rapeseed plants both before and after meeting the vernalization requirement. Seedlings were established and subjected to the freezing procedure as outlined in the Cold Tolerance Determination.
Experiment 1 consisted of four acclimation treatments. Treatment 1 was acclimated for 21 d at 5°C. Treatment 2 was acclimated for 21 d at 5°C and then 7 d at 15°C. Treatment 3 was acclimated for 21 d at 5°C, 7 d at 15°C, and then 7 d at 5°C. Treatment 4 was acclimated for 21 d at 5°C, 7 d at 15°C, and then 14 d at 5°C. At the end of each acclimation period, that treatment was subjected to the freezing procedure. This deacclimation–reacclimation cycle represents a warming period prior to vernalization saturation for Ceres and Plainsman.
Experiment 2 consisted of four acclimation treatments. Treatment 1 was acclimated for 63 d at 5°C. Treatment 2 was acclimated for 63 d at 5°C and then 7 d at 15°C. Treatment 3 was acclimated for 63 d at 5°C, 7 d at 15°C, and then 7 d at 5°C. Treatment 4 was acclimated for 63 d at 5°C, 7 d at 15°C, and then 14 d at 5°C. At the end of each acclimation period, that treatment was subjected to the freezing procedure. This deacclimation–reacclimation cycle represents a warming period after vernalization saturation for all cultivars.
Experimental designs were three factor, three replicate factorials with acclimation treatments and freeze temperatures split on the three cultivars in completely randomized designs. Analyses of variance and mean comparisons were conducted to determine the significance of treatment differences in all three experiments. SAS Institute, Inc. (1989) and MSTAT-C (1988) were used for statistical computations. The temperature at which 50% of the plants were killed (LT50) was calculated using a simple linear regression model (Pomeroy and Fowler, 1973; Fowler et al., 1981). These procedures were used for all experiments.
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RESULTS AND DISCUSSION
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Plants exposed to freezing temperature well above those of the LT50 suffered some tissue damage to the untrimmed leaves. New leaf and root growth was rapid and substantial before the termination of the experiment 3 wk after the freezing treatment. Plants exposed to temperatures substantially colder than the LT50 usually suffered complete desiccation within the 3 wk. Most of the differential survival occurred at temperatures near the LT50. In most cases, survival or death was obvious. Some plants did maintain active plant tissue but failed to produce new root or leaf growth within 3 wk. These plants were counted as dead.
Several studies have cited problems associated with plants remaining in soil during the freezing treatment (Andrews and Morrison, 1992). These problems were based upon the use of a large flat containing multiple seedlings. Plants around the edges of the flat were exposed to conditions more adverse than those in the center and consistent results were difficult to obtain. These problems were not observed with the six-cell packs used in this study. The small soil volume was solidly frozen before the point where the plants were exposed to lethal temperatures. The temperature of the soil had a small lag (about 0.5°C warmer) when compared with the air temperature of the chamber. This lag was consistent in all cells measured and affected all plants similarly. Results were consistent among experiments and no obvious discrepancies were observed.
Significant differences were found among all treatments, as well as significant interactions among factors. Across all acclimation durations and freezing treatments, A112 had the least cold tolerance (32.2% survival, -8.15 LT50) followed by Ceres (52.1% survival, -9.56 LT50) and Plainsman (56.6% survival, -10.13 LT50). A112 was different from Ceres and Plainsman (P > 0.01) and differences between Ceres and Plainsman were significant (P > 0.10). Across all cultivars and acclimation durations, mean survival for the freezing treatment temperatures of -6, -8, -10, and -12°C were 90, 64, 27, and 7%, respectively (P > 0.01). Differences in acclimation durations were also observed (P > 0.01). However, most of this was due to differences in the first few acclimation treatments.
The greatest increase in cold tolerance for all three cultivars occurred after only 3 d of acclimation (Fig. 1). A112 reached its maximum cold tolerance by this time. Ceres and Plainsman continued to increase in cold tolerance, reaching levels that were not significantly different from the maximum sustained LT50 (the LT50 level maintained between 20 and 60 d) by Days 6 and 9, respectively. The two biannual cultivars exhibited a strong peak in cold tolerance at Day 12. However, only Plainsman's response was significantly different from its maximum sustained LT50. All three cultivars tended to show a reduction in cold tolerance between Day 20 and 80. Again, only Plainsman's response was significant. These responses contributed to the cultivar by acclimation duration interaction (P > 0.01).
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Fig. 1. Changes in freezing tolerance (LT50) of three oilseed rape cultivars after different acclimation durations. LSD values represent significance at P = 0.05.
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Deacclimation–Reacclimation
Significant differences were observed among cultivars, acclimation treatments, and freezing treatment temperatures. Significant interactions between cultivar x freezing treatment temperatures and acclimation treatments x freezing treatment temperatures for both pre- and postvernalization saturation studies were also present. Following a 7-d deacclimation period, all cultivars suffered a significant reduction in cold tolerance (P > 0.05) except Plainsman in the post-vernalization saturation study (Fig. 2 and Fig. 3). However, this reduction in the LT50 of Plainsman was significant (P > 0.1). The reduction in cold tolerance to an LT50 of approximately -8.0°C was considerably less than the LT50 of -5.5°C of unacclimated plants that would have been expected if all cold tolerance had been lost. In both studies, all lines did recover all, or even increased cold tolerance after a 7-d reacclimation period. This reacclimation was maintained through 14 d in all lines except Ceres in the prevernalization saturation study where its LT50s were different between Day 21 and 42.
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Fig. 2. The effect of a 7-d deacclimation treatment (Day 21 through 28) followed by low temperature reacclimation (after Day 28) on freezing tolerance (LT50) of three oilseed rape cultivars. The deacclimation treatment was prior to vernalization saturation. LSD values represent significance at P = 0.05.
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Fig. 3. The effect of a 7-d deacclimation treatment (Day 63 through 70) followed by low temperature reacclimation (after Day 70) on freezing tolerance (LT50) of three oilseed rape cultivars. The deacclimation treatment was after the vernalization requirement was met. LSD values represent significance at P = 0.05.
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In all three studies, a spike of increased cold tolerance was observed early in the acclimation or reacclimation period. This was followed by a slight reduction in cold tolerance to a level that was maintained over time. This spike was more evident in the biannual genotypes and more pronounced before vernalization saturation had occurred. The range of freezing treatment temperatures did not allow for accurate LT50s to be calculated for Ceres and Plainsman following this response in the prevernalization study (Fig. 2, Day 35), but the LT50s would have been greater than -12°C. Actual survival at -12°C for Ceres and Plainsman was 100 and 80%, respectively. These events probably coincide with an increased metabolic activity brought about by the warmer temperatures. This may help partially explain why plants in the field survive colder temperatures than plants acclimated at a constant cold temperature.
Under field conditions, rapeseed plants often survive cold events in December and January only to be killed by less severe cold events in February and March (personal observations). One theory as to why this occurs is that after vernalization saturation takes place, rapeseed plants do not have the same ability to recover after a warming event as unvernalized plants. This has been documented in winter cereals. Fowler et al. (1996) found reductions in LT50s of 5°C or more for many cultivars between Day 49 and 84 of acclimation at 4°C. This study suggests that this may not be the case in rapeseed. However, the spike of increased cold tolerance was more pronounced in unvernalized seedlings. Under the variable temperature conditions present during Great Plains winters, this phenomenon could have a substantial impact on winter survival before vernalization saturation and selecting genotypes with increased vernalization requirements could have a positive affect on winter survival.
This study investigated a limited number of lines that are diverse in their response to cold acclimation, vernalization, freezing tolerance, and winter hardiness. In general, these three lines responded to the freezing challenges in similar manners. All lines acclimated rapidly, maintained maximum sustained cold hardiness for a period of time, were able to recover cold tolerance following a warming event, and gave evidence of a trend of reduced cold tolerance after 80 d. However, this trend of reduced cold tolerance over time was substantially less than what Fowler et al. (1996) reported in winter cereals. Differences among these cultivars were also observed. Most notably in the maximum cold tolerance levels and the increase in cold tolerance following a warming event. Evidence has been presented that suggests rapeseed plants may be able to withstand cold temperatures under field conditions more effectively prior to vernalization saturation than after the vernalization requirement has been met. Additional research need to be undertaken to determine if developing lines with extremely long vernalization requirements would have a significant impact on winter survival.
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NOTES
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Contribution no. 02-262-J from the Kansas Agric. Exp. Stn.
Received for publication January 14, 2002.
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REFERENCES
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ABSTRACT
INTRODUCTION
