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Differential Overwintering Performance and Mechanistic Insights in Alfalfa Varieties under Nitrogen Regulation

Y
Yongxin Lu1
T
Tian Tian1
Y
Yuhan Liu1
H
Haigang Li1,2
Y
Yang Chen1,2,*
1Inner Mongolia Key Laboratory of Soil Quality and Nutrient Resources, College of Resources and Environmental Sciences, Inner Mongolia Agricultural University, Hohhot, 010018, China.
2Key Laboratory of Agricultural Ecological Security and Green Development at Universities of Inner Mongolia Autonomous Region, Hohhot, 010018, China.

  • Submitted09-04-2026|

  • Accepted20-05-2026|

  • First Online 01-06-2026|

  • doi 10.18805/LRF-955

ABSTRACT

Background: Overwintering is a critical limiting factor for alfalfa production in northern regions, directly influencing yield in the following year.

Methods: A two-factor split-plot experiment was conducted with two nitrogen rates, 0 and 60 kg N ha-1 and ten alfalfa varieties. Winter survival rate, subsequent-year yield, physiological and biochemical traits of root crowns and morphological characteristics were systematically analyzed.

Result: Nitrogen application at 60 kg N ha-1 significantly increased winter survival rate by 3.50% to 7.95% across varieties. Variety Chaoxinxing showed the highest survival rate, which was 28.16% higher than that of Caoyuan No.2. Nitrogen application also increased hay yield by 10.57% to 11.63%. Under the same nitrogen rate, Chaoxinxing outperformed other varieties by 52.42% to 140.51%. Physiological analyses revealed that nitrogen enhanced cold resistance: in February, soluble sugars in root crowns increased by 2.55% to 5.89%, soluble proteins by 4.98% to 16.65% and free proline by 11.42% to 12.27%, while malondialdehyde content decreased by 24.06% to 34.8%. Activities of superoxide dismutase, peroxidase and catalase were also elevated. Nitrogen supply improved root crown diameter and burial depth, with Chaoxinxing and Qishi T showing stronger responses than Caoyuan No.2. Path analysis indicated that osmoregulatory substances, antioxidant enzymes and morphological traits were positively correlated with winter survival rate, whereas malondialdehyde content was negatively correlated.In conclusion, this study provides new mechanistic insights into variety-specific overwintering performance under nitrogen regulation. Combining high hardiness varieties such as Chaoxinxing and Qishi T with appropriate nitrogen application synergistically enhances alfalfa cold tolerance through dual pathways: physiological regulation including osmotic adjustment and antioxidant defense and morphological adaptation including root crown enlargement and deeper burial. These findings offer a practical strategy for stable and high yield alfalfa cultivation in cold and arid regions of northern China.

INTRODUCTION

Alfalfa (Medicago sativa L.) is one of the most important legume forage crops worldwide, characterized by high protein content, excellent palatability and strong adaptability, playing a crucial role in animal husbandry and agricultural ecosystems (Wang and Zou, 2019). In recent years, with the structural adjustment of China’s livestock industry and the expansion of herbivorous animal breeding scale, the demand for high-quality alfalfa has continued to increase, necessitating the urgent adoption of technical measures to enhance alfalfa yield and quality. Alfalfa cultivation in China is predominantly concentrated in the Northwest and Northeast regions, accounting for 70% of the total national planting area. However, these regions experience long, severe winters, where extreme weather events such as spring freezes and cold waves constitute critical factors affecting successful alfalfa overwintering, thereby severely compromising the sustainable utilization of alfalfa. Consequently, safe overwintering has become fundamental to achieving stable and high-yield alfalfa production in northern China (Ni and Wang, 2024)
       
Different alfalfa varieties exhibit varying performance in stress resistance (overwintering) due to their distinct genetic backgrounds (Li et al., 2023). Fall dormancy, as a key genetic trait, is closely associated with cold tolerance and yield potential in alfalfa (Jia et al., 2025). For instance, varieties with different fall dormancy levels demonstrate significant differences in osmotic substance accumulation and antioxidant capacity during low-temperature adaptation (Li et al., 2024; Avci et al., 2018). Previous studies have demonstrated that alfalfa cold resistance is closely associated with the accumulation of osmotic adjustment substances such as soluble sugars and proline in the root crown, which regulate intracellular osmotic balance to reduce freezing points and effectively alleviate physical damage to membrane systems caused by intracellular ice crystal formation, thereby improving winter survival rates of alfalfa (Smith, 1964). Additionally, winter-hardy alfalfa varieties exhibit certain advantages in plant morphology (Schwab et al., 1996). Through the synergistic effects of physiological regulation and morphological adaptation, alfalfa can collectively withstand low-temperature stress, ensuring safe overwintering and stable, high-yield production in cold regions of northern China.
       
Nitrogen exerts profound effects on various physiological processes in alfalfa, including photosynthesis, carbon-nitrogen metabolism and stress resistance (Liu et al., 2013). Although alfalfa can acquire atmospheric nitrogen through symbiotic fixation with Rhizobium, its nitrogen fixation capacity is constrained by multiple factors such as cultivar characteristics, soil conditions, climatic environments and management practices (Ma et al., 2024), which cannot satisfy the requirements for high yield and superior quality. Consequently, exogenous nitrogen supply has become an important approach for enhancing alfalfa yield and quality (Liu et al., 2024). Different alfalfa varieties exhibit variations in nitrogen fixation, uptake and utilization, as well as in the expression of stress resistance-related genes and physiological response mechanisms, which may lead to divergent responsive patterns to nitrogen fertilizer (Wang et al., 2021). Moreover, studies have revealed that the performance of alfalfa in adapting to low temperature is closely associated with nitrogen metabolism (Li, 2025).
       
Based on the aforementioned considerations, this study conducted field experiments to systematically analyze the differences in overwintering performance and yield of different alfalfa varieties under varying nitrogen supply levels. Through the analysis of physiological indicators and morphological characteristics during the overwintering period, this study aimed to elucidate the low-temperature adaptation mechanisms underlying the divergent overwintering performance among varieties and to clarify the regulatory effects of nitrogen on winter survival capacity and production potential, thereby providing theoretical basis and technical support for high-yield and high-quality alfalfa production.

MATERIALS AND METHODS

Experimental site description
 
The field experiment was conducted at the Hailiutu Experimental Base in Tumed Left Banner, Hohhot City, during 2023-2024. The experimental site is located on the Tumed Plain (111o23′ 46″ E, 40o31′ 17″ N, altitude 1008 m) and features a temperate continental monsoon climate. The mean annual temperature is 7.3oC, with an average temperature of 17.6oC during the growing season (May-September). The maximum and minimum annual temperatures are 22.6oC and -11.6oC, respectively. The mean annual sunshine duration is 2744.1 h and the average annual precipitation is approximately 400 mm. The soil physicochemical properties prior to sowing were as follows: total nitrogen 0.99 g kg-1, available phosphorus 28.62 mg kg-1, available potassium 239.67 mg kg-1 and pH 8.09.
 
Experimental materials
 
Ten alfalfa cultivars were used in this study (Table 1).

Table 1: Alfalfa cultivars and their sources.


 
Experimental design
 
This experiment employed a two-factor split-plot design. Factor 1 was nitrogen application rate, comprising two levels: 0 kg N ha-1 (N0) and 60 kg N ha-1 (N60). Factor 2 was variety, consisting of ten alfalfa varieties (Table 1). Each treatment was replicated three times. Main plots were separated by 0.5-m intervals and subplots were divided by small ridges. The subplot area was 4 m × 5 m, with random distribution of variety subplots within each main plot. Alfalfa was sown by drilling in rows spaced 27 cm apart, at a seeding rate of 0.2 kg ha-1. Phosphorus and potassium fertilizers were applied at 120 kg P2O5 ha-1 and 120 kg K2O ha-1, respectively. All fertilizers were applied once annually by trench application after regreening and weeding.
 
Measurement indicators and methods
 
The indoor experiments for the determination of physiological and biochemical traits as well as morphological characteristics were conducted at the Inner Mongolia Key Laboratory of Soil Quality and Nutrient Resources.
 
Winter survival rate
 
On November 15, 2023, three random 1-m row sections were selected within each experimental plot and the number of plants in each section was recorded. The number of surviving plants was surveyed after alfalfa regreening in the following spring. Winter survival rate was calculated using the following formula:

Alfalfa yield was determined at the early flowering stage. Samples were collected on June 15, 2024 (first cut), August 10, 2024 (second cut) and September 20, 2024 (third cut), with three harvests conducted annually.
 
Hay yield
 
Alfalfa was harvested at the early flowering stage. In each plot, three random 1 m × 1 m quadrats were harvested with a stubble height of approximately 5 cm. Fresh herbage was weighed immediately, then subjected to heat inactivation in an oven at 105oC for 30 min, followed by drying at a constant temperature of 65oC until constant weight was achieved. Hay yield per hectare was subsequently calculated.
 
Physiological indicators
 
Beginning when plants entered the wintering period during 2023-2024, roots were excavated from the field at 30-day intervals (November 1, December 1, February 1, March 1 and April 1). Three plants were sampled from each plot. After excavation, alfalfa roots were rinsed thoroughly with distilled water, blotted dry with absorbent paper, immediately frozen in liquid nitrogen and stored at -80oC for subsequent determination of root crown morphology and dynamic changes in physiological indicators during the overwintering period.
       
Soluble sugar content was determined by the anthrone colorimetric method; soluble protein content by the Coomassie brilliant blue G-250 staining method; free proline content by the acid ninhydrin method and malondialdehyde (MDA) content by the thiobarbituric acid method. Superoxide dismutase (SOD) activity was assayed by the nitroblue tetrazolium photochemical reduction method; peroxidase (POD) activity by the guaiacol method and catalase (CAT) activity by the ultraviolet absorption method (Zou, 1995).
 
Root crown diameter
 
Measured at the swollen portion of the root crown using vernier calipers.

Root crown burial depth
 
The vertical distance from the soil surface to the upper end of the root crown.
 
Data processing and statistical methods
 
All data were initially organised using Microsoft Office Excel 2019 and two-way analysis of variance (ANOVA) and significance tests were performed using SPSS 27.0. Graphs were produced using Origin Pro 2021.

RESULTS AND DISCUSSION

Winter survival rate and hay yield
 
Nitrogen application at 60 kg N ha-1 significantly increased winter survival rate of all alfalfa varieties by 3.50% to 7.95% (Fig 1). Variety Chaoxinxing showed the highest survival rate, which was 28.16% higher than that of Caoyuan No.2 under N60 treatment. The interaction between nitrogen and variety was not significant.

Fig 1: Overwintering rates of different alfalfa varieties under varying nitrogen application rates.


       
Hay yield decreased with cutting frequency. Nitrogen application also significantly increased hay yield by 10.57% to 11.63% across varieties and cuts (Fig 2). Chaoxinxing consistently produced the highest yield, outperforming Caoyuan No.2 by 52.42% to 140.51% under N60. The interaction between nitrogen fertiliser and variety was significant only at the second cut (August 2024).

Fig 2: Alfalfa hay yield of different varieties under varying nitrogen application rates.


 
Physiological responses to low temperature
 
Osmotic adjustment substances
 
Soluble sugars, soluble proteins and free proline in root crowns all exhibited a unimodal trend during winter, peaking in February when cold stress was most severe (Fig 3-4, Table 2). Nitrogen fertilization significantly increased their accumulation. For soluble sugars, the increase ranged from 2.55% to 5.89% in February. Soluble proteins increased by 4.98% to 16.65% and free proline by 11.42% to 12.27%. High hardiness varieties (Chaoxinxing, Qishi T, Baimu 341) accumulated significantly higher levels of these osmolytes than low hardiness varieties (Aohan, Jinhuanghou, Caoyuan No. 2). For example, under N60, Chaoxinxing had 19.01% higher soluble sugar and 13.58% higher proline than Caoyuan No.2.

Fig 3: Soluble sugar content in different alfalfa varieties under varying nitrogen application rates.



Fig 4: Proline content in different alfalfa varieties under varying nitrogen application rates.



Table 2: Soluble protein content (ìg g-1) in different alfalfa varieties under different nitrogen application rates.


 
Oxidative stress and antioxidant enzymes
 
Malondialdehyde (MDA) content, an indicator of membrane lipid peroxidation, also peaked in February (Fig 5). Nitrogen application reduced MDA by 24.06% to 34.8%. Low hardiness varieties had significantly higher MDA than high hardiness varieties; under N0, MDA in Aohan was 111.20% higher than in Chaoxinxing.

Fig 5: Malondialdehyde content in different alfalfa varieties under varying nitrogen application rates.


       
Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities followed a similar seasonal pattern, reaching maxima in February (Table 3-5). Nitrogen treatment elevated SOD, POD and CAT activities. High hardiness varieties maintained higher enzyme activities throughout winter. For instance, under N60, SOD activity of Chaoxinxing was 21.23% higher than that of Caoyuan No.2 in February. The interaction between nitrogen and variety was occasionally significant for some indicators (e.g., proline in October and November, SOD and POD in February), indicating that high hardiness varieties responded more strongly to nitrogen supply.

Table 3: SOD activity (U g-1FW) in different alfalfa varieties under varying nitrogen application rates.



Table 4: POD activity (U g-1 FW min-1) of different alfalfa varieties under varying nitrogen application rates.



Table 5: CAT activity (U g-1 FW min-1) of different alfalfa varieties under varying nitrogen application rates.


 
Root crown morphology
 
Nitrogen application significantly increased both root crown diameter and burial depth (Fig 6-7). Across varieties, root crown diameter increased by 4.00% to 16.14% under N60 compared to N0. Chaoxinxing and Qishi T had the largest diameters and deepest burial depths, whereas Caoyuan No.2 and Aohan had the smallest values. Burial depth increased by 4.83% to 4.94% under N60 for all varieties.

Fig 6: Root collar diameter (cm) of different alfalfa varieties under different nitrogen application rates.



Fig 7: Depth of alfalfa root collars below the soil surface (cm) for different varieties under varying nitrogen application rates.


 
Path analysis of nitrogen regulation on alfalfa overwintering performance
 
Path analysis (Fig 8) showed that soluble sugars, soluble proteins, proline, SOD, POD, CAT, root crown diameter and burial depth were all positively correlated with winter survival rate. In contrast, MDA was negatively correlated with winter survival rate. Hay yield was also positively correlated with winter survival rate. These results indicate that nitrogen enhances alfalfa overwintering through coordinated regulation of osmotic adjustment, antioxidant defense and root crown morphology.

Fig 8: Analysis of the pathways by which nitrogen regulates the overwintering performance of alfalfa.


 
Performance of alfalfa in adaptation to low-temperature stress
 
Low temperature stress is the primary factor limiting alfalfa overwintering in northern regions. Plants adapt by modulating osmotic substances and antioxidant enzymes (Liu et al., 2015). In this study, physiological indicators differed significantly among varieties and correlated strongly with winter survival rate.
       
Soluble sugars, soluble proteins and free proline are key osmotic regulators. They lower freezing points, maintain homeostasis and stabilise membranes (Yue et al., 2026). All varieties showed a unimodal sugar curve peaking in February. High hardiness varieties (Chaoxinxing, Qishi T, Baimu 341) accumulated significantly more sugars than Aohan and Caoyuan No. 2 (P<0.05), consistent with Liu et al. (2019). Moreover, Chaoxinxing displayed more stable sugar consumption after winter, favouring rapid spring regrowth.
       
Soluble protein and proline trends were similar, both peaking in February. Chaoxinxing, Baimu 341 and Zhongmu No.3 had higher protein levels than Caoyuan No. 2 (P<0.05), indicating more active protective mechanisms. Proline, a cryoprotectant (Wang and Zhuang, 2008), remained high in Chaoxinxing during late winter, while low hardiness varieties showed sharp declines, reducing cellular stability and regrowth
       
Malondialdehyde (MDA) reflects membrane lipid peroxidation (Liu et al., 2024). MDA peaked in February. Aohan, Jinhuanghou and Caoyuan No. 2 had significantly higher MDA than Chaoxinxing and Zhongmu No. 3 (P<0.05), meaning the latter suffered less oxidative damage. SOD, POD and CAT, key ROS scavengers (Luo et al., 2004), also peaked in February. High hardiness varieties exhibited much higher enzyme activities than low hardiness ones, demonstrating superior ROS elimination capacity.
 
Effects of alfalfa varieties on winter hardiness
 
Variety is the intrinsic determinant of overwintering success (Wang et al., 2019). Genetic background leads to differences in cold resistance, physiology and morphology (Wang et al., 2024). In this study, the ten varieties differed significantly in winter survival, yield, physiological traits and root crown morphology (P<0.01). Chaoxinxing, Qishi T and Baimu 341 performed best, while Caoyuan No. 2, Aohan and Jinhuanghou performed worst.
       
Chaoxinxing had 22.68% higher winter survival than Caoyuan No. 2 under N0, consistent with Wang et al. (2015) in eastern Jilin, confirming that variety selection is critical for safe overwintering.
       
High hardiness varieties accumulated more soluble sugars, proteins and proline, showed higher SOD/POD/CAT activities and lower MDA. These traits collectively alleviate oxidative membrane damage and maintain cellular integrity (Wang et al., 2024). Kang et al. (2010) also found that productive varieties possess stronger physiological adaptability.
       
Root crown diameter and burial depth are important morphological traits (Shi et al., 2009). Chaoxinxing, Qishi T and Baimu 341 had larger diameters and deeper burial than Caoyuan No. 2 and Aohan. Larger root crowns store more carbohydrates and nitrogen, while deeper burial places growing points in thermally stable soil layers, avoiding extreme surface cold (Guo and Shi, 2024; Guo, 2024). These findings align with previous research highlighting the critical role of root crown physiological and morphological dynamics in alfalfa overwintering adaptation (Guo and Shi, 2024).
 
Effects of nitrogen on alfalfa overwintering
 
Nitrogen regulates growth, development and stress resistance. Appropriate N supply enhances yield and winter hardiness by optimising physiology and morphology (Zhang et al., 2016). Here, N60 increased winter survival by 3.50-7.95% and hay yield by 10.57-11.63%, with stronger effects on high hardiness varieties.
       
Physiologically, N promotes accumulation of osmotic substances and antioxidant enzymes (Li et al., 2025). Under N60, soluble sugars, proteins and proline were significantly higher than under N0 (P<0.01), especially in February. This agrees with Liu et al. (2013), showing that N supply affects carbon nitrogen balance and cold related substances. N60 also raised SOD, POD and CAT activities and lowered MDA, meaning N alleviates peroxidation by enhancing ROS scavenging. Wan (2023) confirmed that N improves leaf photosynthesis and antioxidant capacity, enhancing cold adaptation.
       
Morphologically, N significantly increased root crown diameter and burial depth (4.83-4.94% for depth). Larger root crowns store more reserves and deeper burial improves avoidance of surface cold (Chen et al., 2025). Interactions between N and variety were significant for some indicators (proline, SOD, POD) at specific times (e.g., February 2024, P<0.05), indicating differential responses. High hardiness varieties like Chaoxinxing and Qishi T responded more strongly to N than low hardiness ones, possibly due to differences in N metabolism efficiency, carbon N allocation and cold related gene expression (Ma et al., 2026).

CONCLUSION

This study systematically analyzed the overwintering performance and underlying physiological and morphological mechanisms of ten alfalfa varieties under different nitrogen application levels. Nitrogen application significantly enhanced winter survival rate by 3.50-7.95% and hay yield by 8.73-30.31% across varieties. Mechanistically, nitrogen promoted accumulation of osmotic adjustment substances (soluble sugars, soluble proteins, free proline) in root crowns, elevated activities of antioxidant enzymes (SOD, POD, CAT) and reduced malondialdehyde content, thereby alleviating low-temperature oxidative damage. Simultaneously, nitrogen increased root crown diameter and burial depth, optimizing overwintering organ morphology. Significant genetic differences existed among varieties: Chaoxinxing, Qishi T and Baimu 341 exhibited superior low-temperature adaptation with high osmolyte accumulation, strong antioxidant capacity, low MDA and robust root crowns; Caoyuan No. 2, Aohan and Jinhuanghou performed poorly. High hardiness varieties responded more positively to nitrogen supply. In conclusion, variety selection is the foundation for efficient alfalfa overwintering and rational nitrogen application further exploits this potential, offering a synergistic strategy for green and high yield alfalfa production in cold and arid regions of northern China.

ACKNOWLEDGEMENT

The present study was supported by the Inner Mongolia Agricultural University Basic Research Project (BR251301) and Key Laboratory of Agricultural Ecological Security and Green Development at Universities of Inner Mongolia Autonomous Region.

Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article.

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    Full Research Article

    Differential Overwintering Performance and Mechanistic Insights in Alfalfa Varieties under Nitrogen Regulation

    Y
    Yongxin Lu1
    T
    Tian Tian1
    Y
    Yuhan Liu1
    H
    Haigang Li1,2
    Y
    Yang Chen1,2,*
    1Inner Mongolia Key Laboratory of Soil Quality and Nutrient Resources, College of Resources and Environmental Sciences, Inner Mongolia Agricultural University, Hohhot, 010018, China.
    2Key Laboratory of Agricultural Ecological Security and Green Development at Universities of Inner Mongolia Autonomous Region, Hohhot, 010018, China.

    • Submitted09-04-2026|

    • Accepted20-05-2026|

    • First Online 01-06-2026|

    • doi 10.18805/LRF-955

    ABSTRACT

    Background: Overwintering is a critical limiting factor for alfalfa production in northern regions, directly influencing yield in the following year.

    Methods: A two-factor split-plot experiment was conducted with two nitrogen rates, 0 and 60 kg N ha-1 and ten alfalfa varieties. Winter survival rate, subsequent-year yield, physiological and biochemical traits of root crowns and morphological characteristics were systematically analyzed.

    Result: Nitrogen application at 60 kg N ha-1 significantly increased winter survival rate by 3.50% to 7.95% across varieties. Variety Chaoxinxing showed the highest survival rate, which was 28.16% higher than that of Caoyuan No.2. Nitrogen application also increased hay yield by 10.57% to 11.63%. Under the same nitrogen rate, Chaoxinxing outperformed other varieties by 52.42% to 140.51%. Physiological analyses revealed that nitrogen enhanced cold resistance: in February, soluble sugars in root crowns increased by 2.55% to 5.89%, soluble proteins by 4.98% to 16.65% and free proline by 11.42% to 12.27%, while malondialdehyde content decreased by 24.06% to 34.8%. Activities of superoxide dismutase, peroxidase and catalase were also elevated. Nitrogen supply improved root crown diameter and burial depth, with Chaoxinxing and Qishi T showing stronger responses than Caoyuan No.2. Path analysis indicated that osmoregulatory substances, antioxidant enzymes and morphological traits were positively correlated with winter survival rate, whereas malondialdehyde content was negatively correlated.In conclusion, this study provides new mechanistic insights into variety-specific overwintering performance under nitrogen regulation. Combining high hardiness varieties such as Chaoxinxing and Qishi T with appropriate nitrogen application synergistically enhances alfalfa cold tolerance through dual pathways: physiological regulation including osmotic adjustment and antioxidant defense and morphological adaptation including root crown enlargement and deeper burial. These findings offer a practical strategy for stable and high yield alfalfa cultivation in cold and arid regions of northern China.

    INTRODUCTION

    Alfalfa (Medicago sativa L.) is one of the most important legume forage crops worldwide, characterized by high protein content, excellent palatability and strong adaptability, playing a crucial role in animal husbandry and agricultural ecosystems (Wang and Zou, 2019). In recent years, with the structural adjustment of China’s livestock industry and the expansion of herbivorous animal breeding scale, the demand for high-quality alfalfa has continued to increase, necessitating the urgent adoption of technical measures to enhance alfalfa yield and quality. Alfalfa cultivation in China is predominantly concentrated in the Northwest and Northeast regions, accounting for 70% of the total national planting area. However, these regions experience long, severe winters, where extreme weather events such as spring freezes and cold waves constitute critical factors affecting successful alfalfa overwintering, thereby severely compromising the sustainable utilization of alfalfa. Consequently, safe overwintering has become fundamental to achieving stable and high-yield alfalfa production in northern China (Ni and Wang, 2024)
           
    Different alfalfa varieties exhibit varying performance in stress resistance (overwintering) due to their distinct genetic backgrounds (Li et al., 2023). Fall dormancy, as a key genetic trait, is closely associated with cold tolerance and yield potential in alfalfa (Jia et al., 2025). For instance, varieties with different fall dormancy levels demonstrate significant differences in osmotic substance accumulation and antioxidant capacity during low-temperature adaptation (Li et al., 2024; Avci et al., 2018). Previous studies have demonstrated that alfalfa cold resistance is closely associated with the accumulation of osmotic adjustment substances such as soluble sugars and proline in the root crown, which regulate intracellular osmotic balance to reduce freezing points and effectively alleviate physical damage to membrane systems caused by intracellular ice crystal formation, thereby improving winter survival rates of alfalfa (Smith, 1964). Additionally, winter-hardy alfalfa varieties exhibit certain advantages in plant morphology (Schwab et al., 1996). Through the synergistic effects of physiological regulation and morphological adaptation, alfalfa can collectively withstand low-temperature stress, ensuring safe overwintering and stable, high-yield production in cold regions of northern China.
           
    Nitrogen exerts profound effects on various physiological processes in alfalfa, including photosynthesis, carbon-nitrogen metabolism and stress resistance (Liu et al., 2013). Although alfalfa can acquire atmospheric nitrogen through symbiotic fixation with Rhizobium, its nitrogen fixation capacity is constrained by multiple factors such as cultivar characteristics, soil conditions, climatic environments and management practices (Ma et al., 2024), which cannot satisfy the requirements for high yield and superior quality. Consequently, exogenous nitrogen supply has become an important approach for enhancing alfalfa yield and quality (Liu et al., 2024). Different alfalfa varieties exhibit variations in nitrogen fixation, uptake and utilization, as well as in the expression of stress resistance-related genes and physiological response mechanisms, which may lead to divergent responsive patterns to nitrogen fertilizer (Wang et al., 2021). Moreover, studies have revealed that the performance of alfalfa in adapting to low temperature is closely associated with nitrogen metabolism (Li, 2025).
           
    Based on the aforementioned considerations, this study conducted field experiments to systematically analyze the differences in overwintering performance and yield of different alfalfa varieties under varying nitrogen supply levels. Through the analysis of physiological indicators and morphological characteristics during the overwintering period, this study aimed to elucidate the low-temperature adaptation mechanisms underlying the divergent overwintering performance among varieties and to clarify the regulatory effects of nitrogen on winter survival capacity and production potential, thereby providing theoretical basis and technical support for high-yield and high-quality alfalfa production.

    MATERIALS AND METHODS

    Experimental site description
     
    The field experiment was conducted at the Hailiutu Experimental Base in Tumed Left Banner, Hohhot City, during 2023-2024. The experimental site is located on the Tumed Plain (111o23′ 46″ E, 40o31′ 17″ N, altitude 1008 m) and features a temperate continental monsoon climate. The mean annual temperature is 7.3oC, with an average temperature of 17.6oC during the growing season (May-September). The maximum and minimum annual temperatures are 22.6oC and -11.6oC, respectively. The mean annual sunshine duration is 2744.1 h and the average annual precipitation is approximately 400 mm. The soil physicochemical properties prior to sowing were as follows: total nitrogen 0.99 g kg-1, available phosphorus 28.62 mg kg-1, available potassium 239.67 mg kg-1 and pH 8.09.
     
    Experimental materials
     
    Ten alfalfa cultivars were used in this study (Table 1).

    Table 1: Alfalfa cultivars and their sources.


     
    Experimental design
     
    This experiment employed a two-factor split-plot design. Factor 1 was nitrogen application rate, comprising two levels: 0 kg N ha-1 (N0) and 60 kg N ha-1 (N60). Factor 2 was variety, consisting of ten alfalfa varieties (Table 1). Each treatment was replicated three times. Main plots were separated by 0.5-m intervals and subplots were divided by small ridges. The subplot area was 4 m × 5 m, with random distribution of variety subplots within each main plot. Alfalfa was sown by drilling in rows spaced 27 cm apart, at a seeding rate of 0.2 kg ha-1. Phosphorus and potassium fertilizers were applied at 120 kg P2O5 ha-1 and 120 kg K2O ha-1, respectively. All fertilizers were applied once annually by trench application after regreening and weeding.
     
    Measurement indicators and methods
     
    The indoor experiments for the determination of physiological and biochemical traits as well as morphological characteristics were conducted at the Inner Mongolia Key Laboratory of Soil Quality and Nutrient Resources.
     
    Winter survival rate
     
    On November 15, 2023, three random 1-m row sections were selected within each experimental plot and the number of plants in each section was recorded. The number of surviving plants was surveyed after alfalfa regreening in the following spring. Winter survival rate was calculated using the following formula:

    Alfalfa yield was determined at the early flowering stage. Samples were collected on June 15, 2024 (first cut), August 10, 2024 (second cut) and September 20, 2024 (third cut), with three harvests conducted annually.
     
    Hay yield
     
    Alfalfa was harvested at the early flowering stage. In each plot, three random 1 m × 1 m quadrats were harvested with a stubble height of approximately 5 cm. Fresh herbage was weighed immediately, then subjected to heat inactivation in an oven at 105oC for 30 min, followed by drying at a constant temperature of 65oC until constant weight was achieved. Hay yield per hectare was subsequently calculated.
     
    Physiological indicators
     
    Beginning when plants entered the wintering period during 2023-2024, roots were excavated from the field at 30-day intervals (November 1, December 1, February 1, March 1 and April 1). Three plants were sampled from each plot. After excavation, alfalfa roots were rinsed thoroughly with distilled water, blotted dry with absorbent paper, immediately frozen in liquid nitrogen and stored at -80oC for subsequent determination of root crown morphology and dynamic changes in physiological indicators during the overwintering period.
           
    Soluble sugar content was determined by the anthrone colorimetric method; soluble protein content by the Coomassie brilliant blue G-250 staining method; free proline content by the acid ninhydrin method and malondialdehyde (MDA) content by the thiobarbituric acid method. Superoxide dismutase (SOD) activity was assayed by the nitroblue tetrazolium photochemical reduction method; peroxidase (POD) activity by the guaiacol method and catalase (CAT) activity by the ultraviolet absorption method (Zou, 1995).
     
    Root crown diameter
     
    Measured at the swollen portion of the root crown using vernier calipers.

    Root crown burial depth
     
    The vertical distance from the soil surface to the upper end of the root crown.
     
    Data processing and statistical methods
     
    All data were initially organised using Microsoft Office Excel 2019 and two-way analysis of variance (ANOVA) and significance tests were performed using SPSS 27.0. Graphs were produced using Origin Pro 2021.

    RESULTS AND DISCUSSION

    Winter survival rate and hay yield
     
    Nitrogen application at 60 kg N ha-1 significantly increased winter survival rate of all alfalfa varieties by 3.50% to 7.95% (Fig 1). Variety Chaoxinxing showed the highest survival rate, which was 28.16% higher than that of Caoyuan No.2 under N60 treatment. The interaction between nitrogen and variety was not significant.

    Fig 1: Overwintering rates of different alfalfa varieties under varying nitrogen application rates.


           
    Hay yield decreased with cutting frequency. Nitrogen application also significantly increased hay yield by 10.57% to 11.63% across varieties and cuts (Fig 2). Chaoxinxing consistently produced the highest yield, outperforming Caoyuan No.2 by 52.42% to 140.51% under N60. The interaction between nitrogen fertiliser and variety was significant only at the second cut (August 2024).

    Fig 2: Alfalfa hay yield of different varieties under varying nitrogen application rates.


     
    Physiological responses to low temperature
     
    Osmotic adjustment substances
     
    Soluble sugars, soluble proteins and free proline in root crowns all exhibited a unimodal trend during winter, peaking in February when cold stress was most severe (Fig 3-4, Table 2). Nitrogen fertilization significantly increased their accumulation. For soluble sugars, the increase ranged from 2.55% to 5.89% in February. Soluble proteins increased by 4.98% to 16.65% and free proline by 11.42% to 12.27%. High hardiness varieties (Chaoxinxing, Qishi T, Baimu 341) accumulated significantly higher levels of these osmolytes than low hardiness varieties (Aohan, Jinhuanghou, Caoyuan No. 2). For example, under N60, Chaoxinxing had 19.01% higher soluble sugar and 13.58% higher proline than Caoyuan No.2.

    Fig 3: Soluble sugar content in different alfalfa varieties under varying nitrogen application rates.



    Fig 4: Proline content in different alfalfa varieties under varying nitrogen application rates.



    Table 2: Soluble protein content (ìg g-1) in different alfalfa varieties under different nitrogen application rates.


     
    Oxidative stress and antioxidant enzymes
     
    Malondialdehyde (MDA) content, an indicator of membrane lipid peroxidation, also peaked in February (Fig 5). Nitrogen application reduced MDA by 24.06% to 34.8%. Low hardiness varieties had significantly higher MDA than high hardiness varieties; under N0, MDA in Aohan was 111.20% higher than in Chaoxinxing.

    Fig 5: Malondialdehyde content in different alfalfa varieties under varying nitrogen application rates.


           
    Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities followed a similar seasonal pattern, reaching maxima in February (Table 3-5). Nitrogen treatment elevated SOD, POD and CAT activities. High hardiness varieties maintained higher enzyme activities throughout winter. For instance, under N60, SOD activity of Chaoxinxing was 21.23% higher than that of Caoyuan No.2 in February. The interaction between nitrogen and variety was occasionally significant for some indicators (e.g., proline in October and November, SOD and POD in February), indicating that high hardiness varieties responded more strongly to nitrogen supply.

    Table 3: SOD activity (U g-1FW) in different alfalfa varieties under varying nitrogen application rates.



    Table 4: POD activity (U g-1 FW min-1) of different alfalfa varieties under varying nitrogen application rates.



    Table 5: CAT activity (U g-1 FW min-1) of different alfalfa varieties under varying nitrogen application rates.


     
    Root crown morphology
     
    Nitrogen application significantly increased both root crown diameter and burial depth (Fig 6-7). Across varieties, root crown diameter increased by 4.00% to 16.14% under N60 compared to N0. Chaoxinxing and Qishi T had the largest diameters and deepest burial depths, whereas Caoyuan No.2 and Aohan had the smallest values. Burial depth increased by 4.83% to 4.94% under N60 for all varieties.

    Fig 6: Root collar diameter (cm) of different alfalfa varieties under different nitrogen application rates.



    Fig 7: Depth of alfalfa root collars below the soil surface (cm) for different varieties under varying nitrogen application rates.


     
    Path analysis of nitrogen regulation on alfalfa overwintering performance
     
    Path analysis (Fig 8) showed that soluble sugars, soluble proteins, proline, SOD, POD, CAT, root crown diameter and burial depth were all positively correlated with winter survival rate. In contrast, MDA was negatively correlated with winter survival rate. Hay yield was also positively correlated with winter survival rate. These results indicate that nitrogen enhances alfalfa overwintering through coordinated regulation of osmotic adjustment, antioxidant defense and root crown morphology.

    Fig 8: Analysis of the pathways by which nitrogen regulates the overwintering performance of alfalfa.


     
    Performance of alfalfa in adaptation to low-temperature stress
     
    Low temperature stress is the primary factor limiting alfalfa overwintering in northern regions. Plants adapt by modulating osmotic substances and antioxidant enzymes (Liu et al., 2015). In this study, physiological indicators differed significantly among varieties and correlated strongly with winter survival rate.
           
    Soluble sugars, soluble proteins and free proline are key osmotic regulators. They lower freezing points, maintain homeostasis and stabilise membranes (Yue et al., 2026). All varieties showed a unimodal sugar curve peaking in February. High hardiness varieties (Chaoxinxing, Qishi T, Baimu 341) accumulated significantly more sugars than Aohan and Caoyuan No. 2 (P<0.05), consistent with Liu et al. (2019). Moreover, Chaoxinxing displayed more stable sugar consumption after winter, favouring rapid spring regrowth.
           
    Soluble protein and proline trends were similar, both peaking in February. Chaoxinxing, Baimu 341 and Zhongmu No.3 had higher protein levels than Caoyuan No. 2 (P<0.05), indicating more active protective mechanisms. Proline, a cryoprotectant (Wang and Zhuang, 2008), remained high in Chaoxinxing during late winter, while low hardiness varieties showed sharp declines, reducing cellular stability and regrowth
           
    Malondialdehyde (MDA) reflects membrane lipid peroxidation (Liu et al., 2024). MDA peaked in February. Aohan, Jinhuanghou and Caoyuan No. 2 had significantly higher MDA than Chaoxinxing and Zhongmu No. 3 (P<0.05), meaning the latter suffered less oxidative damage. SOD, POD and CAT, key ROS scavengers (Luo et al., 2004), also peaked in February. High hardiness varieties exhibited much higher enzyme activities than low hardiness ones, demonstrating superior ROS elimination capacity.
     
    Effects of alfalfa varieties on winter hardiness
     
    Variety is the intrinsic determinant of overwintering success (Wang et al., 2019). Genetic background leads to differences in cold resistance, physiology and morphology (Wang et al., 2024). In this study, the ten varieties differed significantly in winter survival, yield, physiological traits and root crown morphology (P<0.01). Chaoxinxing, Qishi T and Baimu 341 performed best, while Caoyuan No. 2, Aohan and Jinhuanghou performed worst.
           
    Chaoxinxing had 22.68% higher winter survival than Caoyuan No. 2 under N0, consistent with Wang et al. (2015) in eastern Jilin, confirming that variety selection is critical for safe overwintering.
           
    High hardiness varieties accumulated more soluble sugars, proteins and proline, showed higher SOD/POD/CAT activities and lower MDA. These traits collectively alleviate oxidative membrane damage and maintain cellular integrity (Wang et al., 2024). Kang et al. (2010) also found that productive varieties possess stronger physiological adaptability.
           
    Root crown diameter and burial depth are important morphological traits (Shi et al., 2009). Chaoxinxing, Qishi T and Baimu 341 had larger diameters and deeper burial than Caoyuan No. 2 and Aohan. Larger root crowns store more carbohydrates and nitrogen, while deeper burial places growing points in thermally stable soil layers, avoiding extreme surface cold (Guo and Shi, 2024; Guo, 2024). These findings align with previous research highlighting the critical role of root crown physiological and morphological dynamics in alfalfa overwintering adaptation (Guo and Shi, 2024).
     
    Effects of nitrogen on alfalfa overwintering
     
    Nitrogen regulates growth, development and stress resistance. Appropriate N supply enhances yield and winter hardiness by optimising physiology and morphology (Zhang et al., 2016). Here, N60 increased winter survival by 3.50-7.95% and hay yield by 10.57-11.63%, with stronger effects on high hardiness varieties.
           
    Physiologically, N promotes accumulation of osmotic substances and antioxidant enzymes (Li et al., 2025). Under N60, soluble sugars, proteins and proline were significantly higher than under N0 (P<0.01), especially in February. This agrees with Liu et al. (2013), showing that N supply affects carbon nitrogen balance and cold related substances. N60 also raised SOD, POD and CAT activities and lowered MDA, meaning N alleviates peroxidation by enhancing ROS scavenging. Wan (2023) confirmed that N improves leaf photosynthesis and antioxidant capacity, enhancing cold adaptation.
           
    Morphologically, N significantly increased root crown diameter and burial depth (4.83-4.94% for depth). Larger root crowns store more reserves and deeper burial improves avoidance of surface cold (Chen et al., 2025). Interactions between N and variety were significant for some indicators (proline, SOD, POD) at specific times (e.g., February 2024, P<0.05), indicating differential responses. High hardiness varieties like Chaoxinxing and Qishi T responded more strongly to N than low hardiness ones, possibly due to differences in N metabolism efficiency, carbon N allocation and cold related gene expression (Ma et al., 2026).

    CONCLUSION

    This study systematically analyzed the overwintering performance and underlying physiological and morphological mechanisms of ten alfalfa varieties under different nitrogen application levels. Nitrogen application significantly enhanced winter survival rate by 3.50-7.95% and hay yield by 8.73-30.31% across varieties. Mechanistically, nitrogen promoted accumulation of osmotic adjustment substances (soluble sugars, soluble proteins, free proline) in root crowns, elevated activities of antioxidant enzymes (SOD, POD, CAT) and reduced malondialdehyde content, thereby alleviating low-temperature oxidative damage. Simultaneously, nitrogen increased root crown diameter and burial depth, optimizing overwintering organ morphology. Significant genetic differences existed among varieties: Chaoxinxing, Qishi T and Baimu 341 exhibited superior low-temperature adaptation with high osmolyte accumulation, strong antioxidant capacity, low MDA and robust root crowns; Caoyuan No. 2, Aohan and Jinhuanghou performed poorly. High hardiness varieties responded more positively to nitrogen supply. In conclusion, variety selection is the foundation for efficient alfalfa overwintering and rational nitrogen application further exploits this potential, offering a synergistic strategy for green and high yield alfalfa production in cold and arid regions of northern China.

    ACKNOWLEDGEMENT

    The present study was supported by the Inner Mongolia Agricultural University Basic Research Project (BR251301) and Key Laboratory of Agricultural Ecological Security and Green Development at Universities of Inner Mongolia Autonomous Region.

    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article.

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