Soybean is an important oilseed crop in the US Midsouth, where an average air temperature of 30 °C is considered ideal for germination and seedling emergence [1
]. However, soybean planting dates vary from early March to late May depending upon the type of production system followed, namely, early soybean production system (ESPS) and conventional soybean production system (CSPS) [2
]. The CSPS involves May and later plantings of soybean varieties belonging to maturity group (MG) V-VIII, which allows rapid seed germination and emergence [4
]. Whereas ESPS involves planting early-maturing varieties, MG III and IV, from late-March to early-April [5
]. Soybean acres and yields are consistently increased in the US Midsouth since the shift from CSPS to ESPS, which provides benefits of early season rainfall, avoids reproduction stage from mid-summer drought and high temperatures, prevents late-season insect-attack, and potential early harvest [1
]. However, farmers may risk the exposure of early-growth (seed germination and seedling emergence) of soybean to chilling injury under ESPS, leading to uneven and poor stand establishment [5
]. Thus, planting too early under EPSP and too late under CSPS could expose soybean seedling growth to both low- and high-temperatures, respectively, in the US Midsouth.
During the early germination process of soybean, low temperatures can significantly reduce the rate of imbibition, the ability of embryo tissue to expand, and mitochondrial respiration [6
]. Further, susceptibility to chilling injury increases with decreasing initial moisture content in the embryo [6
]. The rate of hypocotyl elongation significantly decreases with decreasing temperature below 30 °C [8
]. Interestingly, after effects of low temperatures during the seedling stage can substantially extend the vegetative growth rate, and increase number of axillary branches, the rate of dry weight per plant and pod setting [9
]. Whereas, the effects of high temperatures are mostly studied and considered damaging on the reproductive growth and yield potential in soybean, especially under the CSPS system [5
]. Many argue that the success of the ESPS system was due to continuously increasing global air temperatures over the years [10
] and they emphasize the importance of determining heat/cold tolerance among the available soybean cultivars during early-growth stages. Also, southernmost states of the US with higher spring temperatures are deprived of the ESPS [12
Currently, numerous soybean varieties are available that are recommended for a given region that may differ in their tolerance to low and high temperatures [1
]. Therefore variety selection along with other planting decisions (i.e., planting date, seed rate, and row spacing) is a key to profitable soybean production in a specific environment [4
]. Temperature and photoperiod predominantly affect morphological and physiological growth and development of soybean plant among other environmental variables [15
]. While the phenological response to temperature can primarily determine soybean variety selection for cultivation in a given geographical location during early growth-stages with little interaction of photoperiod [15
], however, photoperiod modifies the response to temperature with changing geographical locations and therefore serves as a basis for classifying the cultivars by maturity group [17
]. Studies in the past have determined genotypic variability in phenological responses to temperatures for the traits such as germination, plant height, node number, net photosynthesis, leaf area, and fruit number per plant by either varying the planting date in the field [2
] or utilizing controlled-environment facilities [10
]. However, photoperiod can become a confounding factor when using planting date as a variable to determine the cultivar response to temperature [16
]. Therefore, soybean cultivars tolerance to low or high temperatures within or across MGs at constant photoperiods can be best achieved by utilizing controlled-environment facilities.
Also, root architecture is increasingly studied in US Midsouth crops such as rice, corn, and cotton in identifying responses involved in stress tolerance during seedling growth [22
], however, little is known about the soybean root system under stressful conditions [3
]. Early assessments of whole-root systems without breaking off the finer parts was nearly impractical in the past [29
]. For this reason, previous studies mostly screened cultivars for abiotic stress tolerance based on above-growth traits, like height, leaf area, and node numbers [18
]. However, the introduction of root phenotype systems, like hydroponics, gels, wax-petroleum layers, and WinRHIZO root scanner, have offered plant and soil scientists to evaluate root system architecture traits with minimal destruction [23
]. Recent studies have successfully exploited the above technologies to define the relationship between temperature stress tolerance and root traits, including root length, diameter, thickness, surface area, and lateral root numbers [27
]. Further, differences in correlation between root and shoot traits to different abiotic stresses were also found during the seedling stage [27
]. Therefore, combined analysis of above- and belowground growth and developmental traits are important in identifying cultivars for abiotic stress tolerance.
The overall objective of this study was to quantify the temperature effects on root and shoot growth of 64 soybean cultivars during the early-growth stage using the sunlit controlled-environment facility. The specific objective was to classify the soybean cultivars for their degree of tolerance to low- and high-temperatures.
2. Materials and Methods
This experiment was conducted in Soil-Plant-Atmosphere-Research (SPAR) units, a sunlit controlled-environmental facility located at the Environmental Plant Physiology Laboratory, Mississippi State University, MS, USA during the 2016 growing season [32
]. The experiment consisted of a collection of 64 soybean cultivars from maturity groups (MG) III, IV, and V (Table 1
) that are most commonly grown in the US Midsouth and were evaluated under three different day/night temperature treatments (TTs) namely, low temperature (LT; 20/12 °C), optimum temperature (OT; 30/22 °C), and high temperature (HT; 40/32 °C). The experiment was organized in completely randomized design with two factorial arrangements (64 cultivars × 3 TTs) replicated three times spatially using nine different SPAR units such that three replications of each treatment combination (cultivar and TT) were represented by three SPAR units. Treated seeds of sixty-four soybean cultivars were sown in 576 polyvinyl chloride (PVC) plastic pots (10 cm diameter and 45.5 cm tall), each filled with sandy soil and 250 g of gravel at the bottom. The pots were placed in the SPAR units at the time of sowing. Immediately after sowing, TTs were imposed and continued until harvesting, 20 days after sowing (DAS). Initially, four seeds were seeded in each pot at a depth of 2 cm and then thinned to 1 plant after emergence. Plants were irrigated three times per day through an automated, computer-controlled drip system with full-strength Hoagland’s nutrient solution at 0700, 1200 and 1700 h. All SPAR units were maintained at 400 ppm CO2
throughout the experiment.
Physiological parameters such as chlorophyll content were measured using chlorophyll estimates measured and presented as Soil-Plant-Analysis-Development (SPAD) units (SPAD-502, Minolta Camera Co. Ltd., Osaka, Japan) and canopy temperature using an infrared thermometer (MI-230, Apogee Instruments, Inc., Logan, UT, USA) were measured on the day before the harvest between 10:00 to 12:00 a.m. Shoot parameters such as plant height (PH), mainstem node number (NN), and leaf area (LA) using leaf area meter (Li-3100, Li-COR Inc., Lincoln, Nebraska, USA) were measured at the time of harvest. Root parameters such as cumulative root length (CRL), root surface area (RSA), root diameter (RD), lateral root numbers (i.e., numbers of root tips (RT), forks (RF), crossings (RC)), and root volume (RV) were measured and analyzed using the Win-RHIZO optical scanner according to the methods described previously [27
]. After that, plant-component dry weights, stems, leaves, and roots, were obtained by oven-drying at 80 °C, and root/shoot ratio was calculated accordingly.
2.2. Cumulative Stress Response Indices
Cumulative stress response indices for LT (CLTRI) and HT (CHTRI) were calculated to classify soybean cultivars based on their degree of tolerance to LT and HT, respectively. Koti et al. [33
] defined cumulative stress response index (CSRI) as the sum of relative individual component responses under each treatment. Accordingly, individual stress response indices for LT (ILTRI) and HT (IHTRI) for each cultivar were obtained by dividing the value of parameter obtained at LT or HT by the value of the same parameter obtained at OT. The calculations were done for all measured parameters. Then, CLTRI and CHTRI were calculated for each cultivar by summing ILTRI and IHTRI, respectively. Finally, soybean cultivars were classified as sensitive, moderately sensitive, moderately tolerant, and highly tolerant to LT or HT based on CLTRI or CHTRI values, and an increment of one standard deviation, respectively, as described by Koti et al. [33
2.3. Data Analysis
Considering all SPAR chambers have the same growth conditions, except temperature, the assignment of temperature treatments to a given SPAR unit was randomized and cultivars were completely randomized within each unit, therefore, the study was treated as a completely randomized design for statistical analysis purposes. Proc ANOVA analysis procedure (ANOVA) was performed on the replicated values of the measured parameters using PROC GLM procedure in SAS (SAS Institute, Inc., Cary, NC, USA) to determine the effect of cultivar, TT, MG, and their interaction. Post ANOVA means comparison was made using least significant difference (LSD = 0.05). Pearson’s correlation coefficients for pairs of shoot, root, and physiological traits were calculated at α level of 5%. Sigma plot 13.0 (Systat Software, Inc., San Jose, CA, USA) was used to generate graphs and correlations using best-fitted regression functions.
2.4. Principal Component Analysis (PCA)
The principal component analysis was performed to identify the parameters that best describe either low or high-temperature tolerance to response variables and to classify cultivars into different temperature tolerant groups. The analysis was conducted with the PRINCOMP procedure of SAS (SAS Institute, Inc., Cary, NC, USA) and the results were summarized in biplots using SigmaPlot 13 (Systat Software, Inc., San Jose, CA, USA), which are the plots of the mean principal component scores (PC scores) for the treatments of first two principal components. PCA was performed on the correlation matrix of 64 soybean cultivars and 16 response variables comprising plant height (PH), mainstem nodes number (NN), leaf area (LA), stem weight (SW), leaf weight (LW), root weight (RW), total weight (TW), root length (RL), root surface area (RSA), root average diameter (RAD), root volume (RV), canopy temperature (CT), root tips (RNT), root forks (RNF), root crossings (RC), and root-shoot ratio (RS). The superimposed biplot was developed by plotting eigenvectors for the 16 responses as solid circles and cultivars as open stars projecting from the origin into various positions. The values of eigenvectors and PC scores were used to classify soybean cultivars into LT and HT tolerant groups.
The identification of LT and HT tolerance in soybeans is vital for effective management and production under ESPS and CSPS in US Midsouth. Further, information on cultivar-specific tolerance to a degree of temperatures can be exploited in breeding programs to develop tolerant genotypes that are highly suited for cold or hot environments. Most of the studies in the past have utilized planting date as a criterion to evaluate cultivar’s ability to grow under a given production system [2
]. However, several confounding weather factors co-vary during the growing season that limits the results of such studies to validate cultivar’s tolerance to low or high-temperature tolerance [16
]. The present study is distinct in that it utilizes controlled conditions to identify cultivar-specific tolerance to LT or HT during early-growth, keeping other environments constant. Secondly, this study characterized both shoots as well as root growth and development to determine the temperature tolerance in the soybean cultivars. The present study evaluated soybean cultivars belonging to MG III, IV, and V, which are recommended ideal for the US Midsouth environments based on previous literature describing the interactive effects of agronomic practices, environments, and MG [2
]. The present study showed vigorous seedling growth in cultivars belonging MG IV and V when compared to MG III, which supports recent studies that favored MG IV and V to utilize under ESPS in the US Midsouth [2
Among TTs, LT caused more severe reductions in the shoot, root, and physiological parameters of soybean seedlings than HT. This was expected because, in general, soybean is regarded a warm season crop [9
], and considered sensitive to chilling that may occur within a certain range of temperatures during most of the stages of life cycle [7
]. The highest damage from chilling injury was observed during germination and seedling emergence of soybean, which showed the severity of damage increase linearly with decreasing temperatures, and finally leading to the death of seedlings [6
]. Also, chilling injury during seedling growth of soybean has been identified as a major constraint in the success of ESPS in the US Midsouth [5
]. Cool and wet conditions developed from early season rainfall may hinder germination of April-planted soybeans under ESPS [4
]. According to Wuebker et al. [26
], seeds flooded for one day after the start of imbibition showed 18% decrease in germination at 15 °C than at 25 °C. Similar to the present study, the findings on early-season planting (April–May) of other crops grown in US Midsouth such as cotton and corn reported LT as most damaging for seedling growth among various abiotic stress factors [27
]. The lesser degree of damage from imposed levels of HT further suggests that like most species, soybean also have a higher temperature optimum for vegetative development than reproductive development [11
]. Higher mean values for chlorophyll content as well as canopy temperatures under HT effects than LT further strengths the arguments mentioned above. SPAD values and canopy temperatures are important parameters to evaluate plant photosynthetic efficiency and acclimation [37
]. The higher chlorophyll content attributed to higher photosynthetic rate might have positively contributed to greater plant component dry weights observed under HT treatments in this study.
Interestingly, the present study found varied response of shoot and root parameters to the effects of TTs. The shoot growth was more adversely affected under LT but showed rapid increases under HT effects, when compared to OT. This supports previous reports of rapid germination and emergence on late-season planting (May or later) of soybean under CSPS [2
]. Little is known on the effects on abiotic on the root system of soybeans compared to other major crops such as corn, rice, and cotton of US Midsouth during seedling growth [27
]. Root hydraulic conductivity is considered most sensitive to low temperatures irrespective of soil moisture status [39
]. The low temperatures can induce assimilate partitioning regarding higher RAD, and lower CRL and RSA to maintain root hydraulic conductivity in plants [3
]. Higher mean RAD observed under LT effects in this study was in agreement with Singh et al. [27
] and Wijewardana et al. [28
] that found significantly greater mean RAD in cotton and corn seedlings under LT effects, respectively. Moreover, in agreeing with previous findings, RAD was negatively correlated with all the other shoot and root parameters (Table S1
). Similar to RAD, RS also exhibited a negative correlation (Table S1
), however, all the correlations were significantly different (p
< 0.001). Further, increased lateral root numbers (RNF, RNC, and RNT) under HT corroborate the findings of Khaled et al. [3
] that showed mean lateral root numbers in soybeans were significantly increased (12.7%) in CSPS (June planting) compared to ESPS (April planting). Further, increased lateral root number may have positively contributed to increased root biomass (canopy temperatures) observed under HT effects in this study.
According to PCA, RL, RV, TW, and LA were identified as the traits that best described the temperature tolerance in soybean. Similar to the CLTRI and CHTRI procedure, PCA also identified 4714LL and GT476CR2 as cold tolerant, S47K5 and CZ5225LL as cold sensitive, 45A46 and CZ5242LL as heat-tolerant, and S48RS53 as heat sensitive. Therefore, the findings from PCA were in reasonable agreement with the CLTRI and CHTRI methods where all traits were used in the analysis and the classification of soybean cultivars for low- and high-temperature tolerance. Both positive and negative response in the shoot and root parameters under HT effects supports a positive correlation obtained between CLTRI and CHTRI calculated for a shoot or root parameters separately. The cultivars are showing a reduction in a shoot or root parameters under high temperatures ascribed to their low tolerance to imposed levels of HT or vice versa. A strong and positive correlation between CHTRI and CLTRI indicates that temperature treatments operate likewise on seedling growth and development. For instance, cultivars like 5115LL and JTN-5110 were found sensitive to both LT and HT, while cultivars like 5N393R2 and 45A46 showed tolerance to both HT and LT. The identified tolerance among the tested cultivars based on CHTRI and CLTRI will help farmers in selecting cultivars suited best for a specific region as well as a production system followed, with an aim to maximize benefits regarding temperature tolerance.