Improved methods to estimate days and temperature to fifty percent mortality of winter wheat (Triticum aestivum L.) under low-temperature flooding and ice encasement

Abstract Low-temperature flooding and ice encasement (LTFIE) cause variable survival of winter wheat (Triticum aestivum L.) in Ontario, which limits the adoption of wheat into crop rotations by growers. The development of novel cultivars capable of withstanding LTFIE is a promising avenue for improvement, but the methods used to assess the survival of winter wheat under LTFIE are restricted. This study developed updated methods to determine the survival of wheat cultivars under LTFIE using controlled environments and, to our knowledge, is the first method since the 1980s to use Canadian eastern soft red winter wheat (CESRW) to conduct cold tolerance studies. Chamber-acclimated plants of AC Carberry (spring wheat control), Branson (CESRW), CM614 (CESRW), and Norstar (hardy Canadian western red winter control) cultivars were used to estimate the days (LD50) and temperature (LT50) to reach 50% mortality under ice and without ice treatments. Norstar had the longest LD50 at 33 days, Branson and CM614 had similar LD50 of 18 and 20 days, and AC Carberry did not reach an LD50 as it died early in both treatments. The LT50 of each cultivar was different; Norstar had the lowest LT50 (−13.6 °C day 0 and −13.2 °C day 7), and AC Carberry had the highest LT50 (−6.6 °C day 0 and −2.7 °C day 7). The detailed methods developed in this study were more reliable compared to older methods based on the more accurate reported LD50 and LT50 of the cultivars, therefore, these methods can be used to screen winter cereals for LTFIE in the future.


Introduction
Winter wheat (Triticum aestivum L.) is an important field crop in eastern Canada, with production covering approximately 570 000 ha in 2023 (Statistics Canada 2023).In Ontario, winter wheat is typically planted in a three-crop rotation with soybeans and corn.Inclusion of winter wheat in the rotation provides soil cover in the fall, reduces the need for tillage, provides good weed suppression, and increases corn and soybean yields (Gaudin et al. 2015;OMAFRA 2017;Janovicek et al. 2021).However, the winter-annual growth habit introduces unique environmental stressors that are not encountered by spring-seeded field crops.One such environmental stress is low temperature flooding and ice encasement (LTFIE), which occurs when water pools on the soil surface due to rainfall or snow melt, followed by a rapid reduction in air temperature during the winter or early spring (Andrews 1996).The incidence of LTFIE damage is significantly higher in field depressions and in poorly drained soils (Andrews 1996;Hayhoe and Andrews 1999).Variable survival due to LTFIE has resulted in the limited adoption of winter wheat into rotations in areas where LTFIE are more pronounced.With increasingly variable winter temperatures due to climate change, it is possible that these winterkill events may become more frequent and severe (Bertrand et al. 2003;Dalmannsdottir et al. 2017;Vaitkeviči ūt ė et al. 2022).
Ice encasement restricts gas exchange between the soil and atmosphere (Andrews 1996;Jackson and Ram 2003;Jackson and Colmer 2005).Oxygen consumption by soil microbes and overwintering plants increases the concentration of CO 2 beneath the ice surface, creating an anoxic environment (Pomeroy and Andrews 1978).The switch to anaerobic respiration accelerates the use of plant carbon reserves, which is associated with a rapid reduction in low temperature tolerance (Bertrand et al. 2003).Anoxic conditions also cause a significant reduction in substrates available for respiration and regrowth and lead to an accumulation of toxic compounds, such as ethanol and lactic acid (Andrews 1977).Soil heaving, repeated ice formation beneath the soil surface due to thaw-refreeze cycles, can expose the crown and root tissue to potentially damaging air temperatures (Johnson and Hansen 1974;Russell et al. 1978).Low temperatures cause plant desiccation through cellular dehydration (Gullord et al. 1975) through the formation of ice crystals in plant cells (Guy 1990;Andrews 1996;Han and Bischof 2004;Chew et al. 2012).Flooding can cause plants to be oversaturated, thereby lowering the metabolic rate (Andrews 1996) and causing lower levels of oxygen (O 2 ) availability for the plant (Jackson and Ram 2003;Jackson and Colmer 2005).Damage attributed to LTFIE has limited winter wheat production in many regions of eastern Canada, and conditions favourable for LTFIE are expected to become more frequent in the Great Lakes Basin due to climate change (Bélanger et al. 2006;Bartolai et al. 2015;He et al. 2019).Producers are largely limited to fall production practices to mitigate these risks, such as ensuring adequate surface and subsurface drainage to reduce pooling of water in the field.However, genetic variation for tolerance to LT-FIE has been observed in numerous grass species (McKersie and Hunt 1987) and may allow for the selection of winter wheat cultivars that are better suited to respond to climatic changes, including LTFIE.Unfortunately, assessing tolerance to LTFIE is not common practice in the winter wheat variety trials conducted in eastern Canada.The wide array of environmental conditions that contribute to winterkill in eastern Canada (lethal temperature, frost heaving, cold-induced desiccation, snow mold, LTFIE, and thaw-refreeze cycles) are generally evaluated under the umbrella of "winter survival" indices, despite evidence that suggests low temperature tolerance and tolerance to anoxia are distinct traits (Bélanger et al. 2006).Methods of assessing the various environmental factors that contribute to winterkill are time consuming and labour intensive, and difficult to deploy in large-scale breeding programs.
Methods of assessing tolerance to LTFIE have remained largely unchanged in 30 years and generally involve the determination of lethal temperature tolerance (LT 50 ) and lethal days under ice (LD 50 ).However, a lack of consensus in the methods used to acclimate and assess plants for tolerance to LTFIE have produced varying LT 50 and LD 50 results for common cultivars (Table 1).Despite the varying methods of assessment, significant variation for tolerance to LTFIE has been observed in winter wheat populations (Andrews and Gudleifsson 1983).Recent studies have also confirmed the importance of assessing whole plants, rather than excised crown tissue, as root tissue is sensitive to LTFIE (Table 1).The inclusion of root tissue may provide more realistic results compared to excised crown tissue.Early studies of winter wheat found variability in survival among cold-acclimated cultivars (Andrews and Pomeroy 1975;McKersie et al. 1982).However, there are gaps in our understanding of the tolerance to low temperature and tolerance to LTFIE in winter wheat cultivars registered for production in Ontario.In this study, we present an updated method for assessing tolerance to LTFIE using two representative eastern Canadian winter wheat cultivars.The development of a standardized method to assess tolerance to LTFIE will allow researchers to better characterize the variation of this trait in elite eastern Canadian winter wheat, and assessment of tolerance to LTFIE will also allow producers to select superior cultivars for their production region.The objective of this study was to develop methods to determine the tolerance of cultivars to LTFIE over time and low temperatures.Using the methods, this study aimed to determine the LD 50 and LT 50 of four cultivars with varying observed winter hardiness under different treatments in controlled environments.It was hypothesized that there are genetic differences for the survival of LTFIE among selected winter wheat cultivars and that these genotypic differences will be reflected in the LD 50 and LT 50 of cold-acclimated plants.

Cultivar selection
The cultivars used in this study were 'AC Carberry', 'Branson', 'CM614', and 'Norstar'.Branson and CM614 are commercial cultivars in eastern Canada, AC Carberry is a Canada western hard red spring wheat (DePauw et al. 2011) and Norstar, first registered in 1977(CFIA 2015a), is an established winter hardy check in the literature first developed for western Canada (Fowler et al. 1999;Fowler 2012).Branson, CM614 are soft red winter wheats registered for production in Ontario (CFIA 2015b(CFIA , 2023)).This is the first study to our knowledge that addresses LTFIE in Canadian eastern soft red winter wheat cultivars since the 1980s (McKersie and Hunt 1987).

Growth room
Wheat seeds were seeded in 5 cm depth, 8 × 12 cell inserts (T.O.Plastics Inc., Clearwater, MN, USA) filled with a 80:20 (v:v) sand-peat mixture (Hutcheson Sand & Mixes, Huntsville, ON, Canada).The cells were filled leaving 1 cm of space from the top of the cell to allow ice to pool in each insert.A 1 cm layer of Turface MVP substrate (Turface Athletics Co., Buffalo, IL, USA) was placed at the bottom of each cell to prevent the sand-peat mixture from eroding.A 12 cell insert (containing 12 plants of a single genotype) constituted an experimental unit.

Acclimation chamber
At the three-leaf, one-tiller stage (Z21), plants were transferred to a PGR 900 cold acclimation chamber (Constant Controls Inc., Aurora, ON, Canada).The ambient temperature was held at a constant 3.5 • C with 10/14 h (light/dark) photoperiod, light intensity of 135 μmol m −2 s −1 at canopy level, and a relative humidity of 75%-80% for a period of 4 weeks to achieve consistent acclimatization (Fowler et al. 1996;Limin and Fowler 2006;Fowler 2008).Previous studies have reported that winter wheat cultivars had the greatest temperature tolerance to low temperatures following a 4 week acclimation period (Fowler et al. 1996;Limin and Fowler 2006).Plants in the acclimation chamber were watered every 4 days with chilled water until the soil was saturated at least 12 h before entering the Model PGW36 cold chamber (Z1) (Controlled Environments Ltd., Winnipeg, MB, Canada).This was done to ensure the soil would freeze and to limit the dripping of water from the base.

Cold chamber Z1 and recovery refrigeration
Following the 4-week acclimation period, the trays were transferred to cold chamber Z1.The conditions in the chamber were set to a 10/14 h (light/dark) photoperiod, a light intensity of 125 μmol m −2 s −1 at canopy level, and a relative humidity of 72%.The plants were placed in Z1 at approximately 3 • C and dropped 0.5 • C every 30 min until it reached −4 • C, where it was maintained until the end of the study.Following a 24-h period at −4 • C when the soil froze, ice water was applied to half of each tray to ensure formation of ice on the top layer.Ice water was applied using a manual pump spot-sprayer filled with cold water and ice chips.The formation of ice typically took 8-12 h after application for most trays with a final ice thickness of approximately 1 cm.Two trays were removed from Z1 at time points 0, 4, 8, 12, 16, 20, 25, and 32 days under ice encasement and placed in a refrigerator held at a constant 4 • C (Model #DUF140E1WDD, Danby Appliances, Guelph, ON, Canada).The two trays per time point were held in the refrigerator for 2 days to allow the ice to fully thaw before transferring to the greenhouse.At each time point, the two trays were replaced with two blank trays that contained the same soil medium (80:20 sand-peat mixture) to limit airflow differences within Z1.Every 4 days, the trays within Z1 were re-randomized to account for environmental differences within the chamber.

Greenhouse
Once the plants were fully thawed in the refrigerator, they were moved to a greenhouse where they were allowed to regrow for 3 weeks at approximately 20 • C with a 16/8 h (light/dark) photoperiod.Following the 3-week period, survival was determined by visually assessing any regrowth in the above soil tissue of individual plants for each cultivar.Scores of 0 (dead/no growth) and 1 (alive/growth) were given for each individual plant.

Experimental design and statistical analysis for LD 50
The LD 50 experiment was set up as a randomized split-split plot design with three factors (time point, treatment, and cultivar).The time point was the main plot factor, the treatment was the split-plot factor, and the cultivar was the split-splitplot factor.There were eight levels for the time point (0, 4, 8, 12, 16, 20, 25, and 32 days held at −4 • C), two levels for treatment (ice and without ice), and four levels for cultivar (AC Carberry, Branson, CM614, and Norstar).Within each time point, there were two replicates that were represented by two trays, for a total of 16 trays.Each tray included four cultivars per treatment and two treatments.Each cultivar per treatment was the experimental unit, and there were 128 experimental units over two replicates.Each experimental unit had twelve samples (plants per cultivar) that were scored for survival.
The distribution was non-normal (non-Gaussian) due to the binary scoring system utilized to assess survival.LD 50 values were derived in SAS 9.4 (SAS Institute 2013) using PROC PRO-BIT by log-transformation of survival data and days under ice using the base 10 log.Means separation was performed using LSMEANS within the PROC PROBIT procedure and the treatment by cultivar interactions were SLICED (SAS Institute 2013).A threshold of α = 0.05 was used to establish statistical significance.To further validate the findings and determine differences, 95% fiducial limits were used for each LD 50 curve derived from PROC PROBIT and depicted using the ggplot2 package in R version 4.2.2 (R Core Team 2022).

Experimental design and statistical analysis for LT 50
The LT 50 experiment was set up as a randomized split-splitsplit plot design with four factors (time point, treatment, cultivar, and temperature).The time point factor was the main plot, treatment was the split plot, cultivar was the splitsplit plot, and temperature was the split-split-split plot.There were two levels for time point (day 0 and day 7 held under ice at −4 • C), and within each time point there were two treatment levels (ice and no ice), four cultivar levels (AC Carberry, Branson, CM614, and Norstar), and six temperature levels (−4, −7, −10, −13, −16, −19 • C).Each time point had one replicate per temperature, represented by one tray.Each tray included four cultivars and two treatments.Each cultivar per treatment was the experimental unit, resulting in 288 experimental units over three replicates.Each experimental unit had twelve plants observed for survival.
The same environments and growth conditions described above were used for the LT 50 experiments; however, some modifications were made.In the LT 50 experiments, one tray represented one temperature point per time point, and there were two time points with six temperature points each, for a total of 12 trays.Six trays were removed at each time point (0 and 7 days under ice encasement) from the cold chamber Z1.The cold chamber Z1 ramped down to −4 • C and was maintained at this temperature.The six trays were placed in a chest freezer (Model #40-21B; Scientemp Corp. Adrian, MI, USA) set to a program that began at −4 • C and decreased by 3 • C every hour (−4, −7, −10, −13, −16, −19 • C) and held at each temperature point for an additional hour until a temperature of −19 • C was reached.At each temperature point, one tray was removed from the freezer and placed in a 4 • C refrigerator to thaw for 2 days.The six trays at the first time point were replaced with six blank trays that contained the same soil medium to limit airflow differences within Z1.Every 4 days, the trays within Z1 were re-randomized to account for environmental differences.Temperatures in the chest freezer were recorded every 10 min using two Watchdog Data Logger B-Series Temp 2K temperature loggers (Model #B100; Spec-Fig.1. Lethal days to reach 50% mortality (LD 50 ) for each winter wheat cultivar with both ice and no ice treatments pooled.Norstar is displayed in blue, CM614 in grey, and Branson in orange.The dotted lines and shaded areas surrounding each curve represent the 95% fiducial limits.Fiducial limits for Norstar were capped at 100 days, which is why it ends abruptly.Lines with days labelled beside them were to show the days at which 50% mortality was reached for each cultivar.The horizontal line at 50% was the reference line for the 50% mortality point.trum Technologies Inc. Aurora, IL, USA).One of the temperature loggers was used to record the air temperature and the other logger was used to measure the soil temperature at the crown depth (approximately 2.5 cm).
The statistical limits for this experiment were the same as for the LD 50 experiment.

Results
Lethal days to reach 50% mortality (LD 50 ) AC Carberry was excluded from the results, as it did not reach an LD 50 due to early death at 4 days in −4 • C under ice and no ice treatments.The treatments of ice and no ice were not statistically different from each other, and there was no significant treatment by cultivar interaction (Supplmentary Table S1).Among the other cultivars with both ice and no ice treatments pooled, Norstar had the longest LD 50 (33 days), while Branson (18 days), and CM614 (20 days) were not significantly different from each other (Fig. 1).The observed differences were based on the 95% confidence limits around the LD 50 for each cultivar and were used to characterize the survival of wheat under LTFIE, as it shows how the length of time that different cultivars can tolerate freezing temperatures and ice encasement.These results also help validate the methodology used to estimate the LD 50 since the winter hardy control (Norstar) had the longest LD 50 .
Table 2. Lethal temperature ( • C) to reach 50% mortality (LT 50 ) for day 0 and day 7 at −4 • C, followed by a gradual decrease in temperature by all four wheat cultivars interaction for three replicates for the ice and without ice treatments combined.Lethal temperature to reach 50% mortality (LT 50 ) Norstar had the lowest LT 50 at day 0 (−13.6 • C) compared to the other cultivars (Table 2).Branson and CM614 were not statistically different from each other but had lower LT 50 (−11.9• C and −12.7 • C) at day 0 compared to AC Carberry (−6.6 • C), which had the highest LT 50 (Table 2).However, on day 7, Norstar and CM614 were not statistically different from each other and had the lowest LT 50 (−13.2• C and −12.6 • C, respectively) (Table 2).On day 7, Branson and CM614 were Table 3. Lethal temperature ( • C) to reach 50% mortality (LT 50 ) for the ice and without ice treatments by all four wheat cultivars interaction for three replicates for day 0 and day 7 combined.2).Norstar, Branson, and CM614 appeared to show no differences in LT 50 when comparing day 0 and day 7 (Table 2), while AC Carberry had a lower LT 50 at day 0 (−6.6 • C) compared to day 7 (−2.7 • C) (Table 2).Under the ice treatment, Norstar and CM614 were not statistically different from each other and had the lowest LT 50 (−13.6• C and −12.7 • C, respectively) compared to the other cultivars (Table 3).Branson had a statistically higher LT 50 than Norstar under ice (−12.4 • C), but statistically lower than AC Carberry (−4.4 • C).A similar trend was observed under the no ice treatment, with the average LT 50 similar to the ice treatments, but not statistically different between Norstar and CM614 (Table 3).However, Branson had a statistically lower LT 50 under ice (−12.4 • C) than it did under no ice (−11.2 • C) (Table 3).
The observed differences in LT 50 help to further characterize wheat survival of wheat under LTFIE, as they demonstrate that prolonged periods under ice and exposure to freezing temperatures over time impact the survivability of different cultivars.These results also help to validate the methodology developed to estimate LT 50 as the winter hardy control (Norstar) had the lowest LT 50 , while the nonhardy control (AC Carberry) had the highest LT 50 .

Discussion
Plant survival under ice encasement is difficult to study in the field due to inconsistent weather conditions year-toyear, within-field variation, and the difficulty of applying and maintaining ice treatments in a natural environment.A reliable method for studying ice encasement in controlled environments would facilitate research in this area and breeding for winter survival in winter cereals.Many previous studies did not use whole plants when conducting controlled low temperature tolerance experiments (Gusta et al. 1978;Fowler and Charles 1979;Olien 1984;Limin and Fowler 1988;Fowler et al. 1996;Livingston 1996;Herman et al. 2006), and the studies that did, utilized conditions that were not consistent with those found in the field environment such as light soil me-dia, uneven application of ice treatments, and sudden shifts in temperature (Table 1; Gusta et al. 1978).Previous research utilized methods that are difficult to replicate and create inconsistent results (Table 1).Factors such as cultivar selection, ambient temperature, and adequate time under ice in controlled environments have a significant impact on LTFIE (Table 1).Therefore, to determine survival and characteristics associated with wheat cultivars under LTFIE, this study established improved methods that included details not specified in previous controlled environment studies on winter wheat.These methods include using a sand-peat mixture to create an even soil temperature around the crown region, providing a 1 cm well around the plant to form sufficient ice coverage, and controlling for spatial variation in the treatment chamber via re-randomization.This study also used four unique cultivars to assess the reliability of the improved methods.Norstar, a known winter-hardy cultivar, and AC Carberry, a spring-type cultivar, were used as controls to confirm that the methods were reliable.As expected, Norstar was observed to be the hardiest cultivar, as it had the longest LD 50 and the lowest LT 50 , while AC Carberry was observed to be the least hardy cultivar, reflected in its high LT 50 value and its inability to reach an LD 50 due to high mortality rates.CESRW cultivars CM614 and Branson also exhibited longer-duration LD 50 and lower LT 50 compared to the spring wheat control.Longer LD 50 and lower LT 50 values have been observed previously in winter-hardy winter wheat (Gusta et al. 1978;Fowler and Charles 1979;Andrews and Gudleiffson 1983;Tanino and McKersie 1985;Limin and Fowler 1988;Fowler et al. 1996), therefore, the observed differences amongst cultivars in this study were indicators that the methods were effective.
The experiments supported the main hypothesis that genotypic differences for survival of LTFIE exist in the winter wheat cultivars studied, and that these genetic differences are reflected in the values of LD 50 and LT 50 .CM614 and Branson had similar LD 50 , which were closer to that of Norstar than AC Carberry, suggesting that there are inherent differences for survival under LTFIE in winter-hardy cultivars.In the LT 50 experiment, the four cultivars were significantly different from each other, with AC Carberry having the highest LT 50 , Norstar having the lowest LT 50 , and Branson and CM614 having LT 50 closer to those of Norstar.The differences in cold temperature tolerance demonstrate that the winter wheat cultivars are hardier than the spring wheat cultivar, and that there are discernable differences between winter-hardy cultivars.However, more research is needed to gain a deeper understanding of specific genetic traits within these cultivars that contribute to the variability of survival.
Lethal days to reach 50% mortality (LD 50 ) Norstar is a known winter-hardy cultivar that has been extensively studied (Andrews and Gudleifsson 1983;Fowler et al. 1996;Fowler et al. 1999;Fowler and Limin 2004;Limin and Fowler 2006;Skinner et al. 2018;Willick et al. 2018;Willick et al. 2020); it should therefore exhibit a longer LD 50 compared to the other cultivars.CM614 and Branson exhibited similar lethal days to reach 50% mortality in this study, which was consistent with their observed winter survival in Ontario varietal performance trials (OCCC 2022).AC Carberry reached 100% mortality on day four, which was expected due to its spring growth habit.Results from other studies that conducted prolonged periods under cold and ice also showed variation in survival between cultivars (Andrews and Gudleifsson 1983;Tanino and McKersie 1985).However, these studies used different methods, including fieldgrown plants, excised plants, and different controlled environments.There is limited research on winter wheat survival under prolonged periods of cold temperatures and ice encasement.
The results show differences in survival among cultivars; however, no impact of ice on any of the cultivars studied was observed for the LD 50 studies.In other words, treatments showed a similar decrease in survival over time with or without ice.The similarities of no ice and ice treatments on cultivar survival may be due to the saturation of the soil with water in all treatments before ice formation, as this would have caused soil ice encasement in both treatment groups.Ensuring that the soil in the no ice treatment group was desaturated may have shown more noticeable differences in treatments.Despite observing no treatment differences, the length of time that the plants survived under ice was significantly longer than found in previous controlled environment experiments (Tanino and McKersie 1985), possibly due to the use of whole plants, acclimation periods, and gradually decreasing temperatures.Studies have shown that longer durations under ice resulted in higher mortality rates, supporting the findings that ice encasement damage may be timedependent (Andrews and Gudleifsson 1983;Tanino and McKersie 1985).
Lethal temperature to reach 50% mortality (LT 50 ) Long periods of ice encasement are associated with higher winter wheat mortality (Andrews and Gudleifsson 1983;Tanino and McKersie 1985).The findings of this research show that this may be due to a decrease in cold tolerance and thus LT 50 .Different LT 50 values were observed for all four cultivars.Norstar had the lowest LT 50 , which was consistent with findings from previous studies noting its tolerance of low temperatures (Fowler et al. 1996;Fowler and Limin 2004;Limin and Fowler 2006;Fowler 2008).The differences in LT 50 observed for Norstar and AC Carberry are not surprising as Norstar is a known winter-hardy cultivar and AC Carberry is a spring wheat cultivar.Variation in LT 50 was observed between the two CESRW varieties, although they exhibited similar winter survival ratings in peformance trials (OCCC 2020).Previous research reported lower LT 50 values, which could be attributed to the use of crown cuttings instead of whole plants, the difference in acclimation process and duration, and the difference in controlled environments (Table 1; Gusta et al. 1978).Another key difference in this experiment compared to previous LT 50 experiments was the duration each temperature point was held.This study ramped down temperatures in increments of −3 • C per hour and held plants at each temperature point for an additional hour to ensure that the plants had adequate exposure to each temperature.
AC Carberry was the only cultivar that showed different LT 50 on days 0 and 7, and had a higher LT 50 on day 7, suggesting that prolonged periods under freezing temperatures decrease its survival.This observed trend is likely due to AC Carberry's spring growth habit, making it unsuitable for prolonged periods of freezing temperatures.Ice coverage only affected Branson, resulting in a higher LT 50 compared to no ice treatments.Testing more cultivars would be required to show if this effect is consistent across different cultivars.Norstar, CM614, and AC Carberry did not differ between ice and no ice treatments.One potential explanation for the lack of differences between ice and no ice treatments could be that oxygen was getting in through the base of the trays, allowing the plants to respire (Freyman and Brink 1967).Another potential explanation could be that ice encasement only affects some wheat genotypes.
The findings showed that differences in survival exist among cultivars, and that survival decreased at lower temperatures.However, as only one cultivar showed a response to ice treatments, there is not enough evidence to suggest differences in survival between plants encased in ice and those not encased in ice.Rather, the severity of damage under ice encasement may be genotype-dependent, and further research would need to be conducted with more genotypes under ice encasement to be confident.

Conclusions
This study successfully developed methods that measured the tolerance of wheat cultivars to LTFIE.These methods used controlled environments to measure LD 50 and LT 50 using four different wheat cultivars with known and unknown tolerance to cold temperatures (AC Carberry, Branson, CM614, and Norstar).The results highlighted differences among cultivars with different survival rates under LTFIE using the same methods.Exposure to decreasing cold temperatures over time showed the most noticeable differences in survival among cultivars.

Table 1 .
Comparison of previous literature that conducted cold tolerance tests to show differences in methodology and results.
Means not sharing a common letter are significantly different at α = 0.05 according to Tukey's honestly significant difference test.notstatistically different from each other in terms of LT 50 , but had lower LT 50 than AC Carberry (−2.7 • C), which had the highest LT 50 (Table Note: