Comprehensive Assessment of Alfalfa Aluminum Stress Resistance Using Growth and Physiological Trait Analysis

Abstract

Alfalfa (Medicago sativa L.) is an important perennial leguminous forage; however, its high sensitivity to aluminum (Al) stress severely restricts its cultivation in regions with acidic soil. Therefore, this study conducted an integrated assessment of Al stress tolerance by performing systematic evaluations of 11 growth and physiological parameters across 30 alfalfa cultivars under Al stress, and calculated the Al tolerance coefficients based on these parameters. The results revealed that Al stress markedly inhibited root growth and biomass accumulation in alfalfa, thereby triggering increased malondialdehyde (MDA) content in roots across most cultivars, the scope of increase is 0.19–183.07%. Moreover, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased by 7.50–121.44%, 2.50–135.89%, and 3.84–70.01%, respectively. Based on the comprehensive evaluation value (D) obtained via principal component analysis and membership function, the 30 alfalfa cultivars were categorized into four distinct groups: 4 highly Al-tolerant cultivars, 11 moderately high-Al-tolerant cultivars, 9 moderately low-Al-tolerant cultivars, and 6 low-Al-tolerant cultivars. Stepwise linear regression analysis identified root elongation rate, root-to-shoot ratio, root volume, SOD, MDA, CAT, root dry weight, POD, and root length as pivotal indicators for predicting and evaluating Al stress tolerance in alfalfa cultivars. The qRT-PCR analysis showed dynamic changes in ABC transporter gene expression in alfalfa roots over time under aluminum stress. Therefore, this study comprehensively evaluated Al tolerance by systematically investigating the morphophysiological effects of Al stress across 30 alfalfa cultivars using principal component analysis (PCA), membership function, and hierarchical clustering analysis. It provides a practical solution for expanding alfalfa planting in acid soil and improving feed production in acidic environments.

1. Introduction

Aluminum (Al) ranks as the third most abundant element in the Earth’s crust. In natural environments, it predominantly exists in non-toxic forms, such as Al silicates and oxides, within neutral or slightly acidic soils [1]. However, when the soil pH exceeds 6.5, the Al solubility decreases substantially owing to the formation of insoluble Al(OH)₃ precipitates, thereby mitigating aluminum phytotoxicity and microbial toxicity. When soil acidification reduces the pH to levels below 5.0, stable Al compounds gradually dissociate, releasing the biologically toxic hexaaquaaluminum (III) ion, [Al(H₂O)₆]³⁺, and its hydrolysis product, Al³⁺ [2]. These reactive Al ions (Al³⁺) specifically bind to the membrane systems of root tip cells, including the plasma membrane and organelle membranes, as well as the symplast [3]. This binding induces cellular damage through multiple mechanisms, including disruption of the membrane potential, interference with ionic homeostasis, and triggering of oxidative stress. This toxicity manifests as typical Al toxicity symptoms, including inhibition of primary root elongation, blunted and enlarged root tips, short and brittle lateral roots, root cap shedding, and a sharp decline in the number of root hairs [4,5]. These symptoms reduce the efficiency of the root system in terms of water and nutrient uptake, leading to overall plant growth retardation. For instance, Al stress pronouncedly inhibits root elongation and growth in barley (Hordeum vulgare L.) [6], whereas it reduces root biomass, total root length, root surface area, and root volume in Shatian pomelo (Citrus maxima (Burm.) Merr. ‘Shatian Yu’) [7]. Furthermore, Al toxicity induces plasma membrane lipid peroxidation and disrupts calcium ion homeostasis, ultimately diminishing the chlorophyll content and photosynthetic rate [8].

As an abiotic stressor, Al³⁺ induces rapid reactive oxygen species (ROS) generation and accumulation in plant cells, thus causing oxidative stress [9]. Excessive ROS trigger membrane lipid peroxidation, damage cell membrane structures, promote protein degradation, and ultimately induce programmed cell death [10,11]. Suitably, to counteract Al³⁺-induced oxidative stress, plants activate an enzyme-mediated antioxidant defense system to maintain redox homeostasis. This defense network primarily comprises superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) [12,13]. In oxidative stress responses, SOD plays a crucial role by catalyzing the dismutation of superoxide anions (O₂⁻) into oxygen and hydrogen peroxide (H₂O₂), the first step in ROS scavenging. Subsequently, H₂O₂ is further converted to water through the synergistic action of APX, CAT, and POD, thereby reducing oxidative damage and restoring normal cellular functions [14]. Under Al stress, SOD activity in eucalyptus (Eucalyptus) root tips [15], and SOD, CAT, and POD activities in wheat (Triticum aestivum L.) roots [16], are markedly elevated. These findings indicate that plants activate and modulate their intrinsic antioxidant enzyme systems in response to Al-induced oxidative stress. Through this regulatory mechanism, plants effectively mitigate Al-induced oxidative damage and maintain physiological homeostasis.

Alfalfa (Medicago sativa L.), renowned as the “King of Forage Crops”, is a high-quality perennial legume with a long cultivation history and global distribution [17]. China’s rapidly developing livestock industry has witnessed a soaring demand for premium alfalfa products; however, domestic production capacity has struggled to meet this escalating demand [18]. This challenge is exacerbated by the hypersensitivity of alfalfa to acid–Al stress, where even micromolar concentrations of Al³⁺ can induce irreversible root damage [19]. Accordingly, several innovative strategies for expanding alfalfa cultivation in acidic-soil regions have been proposed. As exemplified by genetic engineering approaches to enhance Al tolerance, overexpression of the MsDUF3700 gene effectively improves growth performance under Al stress conditions. Adenosine triphosphate-binding cassette transporters (ABC transporters) are involved in regulating plant growth, development, and tolerance to environmental stresses [20]. Multiple ABC transporters aid aluminum detoxification in crops. The AtALS1 (AtABCB27) protein, functionally similar to rice OsALS1 (OsABCB25), improves root growth under aluminum stress by sequestering aluminum into vacuoles [21]. These improvements include enhanced root architecture and oxidative stress mitigation [22].

Therefore, this study comprehensively evaluated Al tolerance by systematically investigating the morphophysiological effects of Al stress across 30 alfalfa cultivars using principal component analysis (PCA), membership function, and hierarchical clustering analysis. By integrating multidimensional metrics, robust screening criteria for Al tolerance were established, and the gene expression levels of aluminum-tolerant genotypes in root tips were analyzed at different time points under Al stress conditions. These findings provide both an empirical foundation and a superior germplasm resource for breeding Al-tolerant alfalfa cultivars.

2. Results

2.1. Effects of Al Stress on Alfalfa Root Length

Aluminum stress inhibited alfalfa root length and root elongation rate to varying degrees across cultivars. Compared with that under control conditions, root length under Al stress dropped by 1.66–32.47% among the 30 alfalfa cultivars (Figure 1). Notably, the Otana root length decreased significantly, by 5.61 cm (p < 0.05), whereas that of WL168HQ decreased by 0.25 cm. Relative root elongation (RRE) rates were calculated from the control and Al stress treatments to assess the Al-induced inhibition of root growth in 30 alfalfa cultivars. WL168HQ showed the highest root elongation rate (93.8%), thereby indicating lower inhibition of root growth. Otana (19.0%) had the lowest RRE rate, thus indicating the highest degree of root growth inhibition. Under Al stress, the coefficient of variation (CV) values for root length and elongation rate were higher than those under the control treatment (Table S1).

2.2. Analysis of Alfalfa Root Surface Area and Root Volume Under Al Stress

After Al stress treatment, the root surface area and root volume of the 30 alfalfa cultivars were suppressed to varying degrees (Table S2). Under control conditions, the Great cultivar exhibited a markedly higher root surface area (5.74 cm²) than that of the other cultivars, whereas Arora showed a significantly lower root surface area (3.01 cm²; p < 0.05). The Al stress-induced root surface area reduction ranged from 1.10 to 38.67% across all cultivars. Specifically, Algonquin demonstrated a 0.04 cm² decrease (1.10%), and WL440HQ exhibited the most pronounced reduction of 1.28 cm² (38.67%). The CV for the root surface area was 7.73% under control conditions and 9.81% under Al stress (Figure 2).

After Al stress treatment, the root volume inhibition in alfalfa ranged from 1.15 to 35.77%. Among all cultivars, Gannong No. 3 showed the least reduction in root volume (0.0003 cm³), whereas Eclipse exhibited the most substantial volume decrease (0.0093 cm³). The CV for root volume was 9.82% under control conditions and 12.78% under Al stress (Figure 3).

2.3. Analysis of Alfalfa Root Dry Weight (RDW), Shoot Dry Weight, and Root-to-Shoot Ratio Under Al Stress

After Al stress treatment, RDW was significantly reduced in all alfalfa cultivars except WL363HQ, Savoie 5, and Gannong No. 3 (p < 0.05). Under control conditions, RDW exhibited a reduction range of 1.30–3.07 mg·plant⁻¹, whereas Al-stressed plants showed a reduction range of 0.90–2.76 mg·plant⁻¹. After the Al stress treatment, Sersu, Sandy, and Vision exhibited RDW reductions of over 30%, with decreases of 30.77%, 30.42%, and 34.53%, respectively. In contrast, Savoie 5 showed a minimal reduction (8.29%) among all cultivars (Figure 4). Al stress exerted a more pronounced inhibitory effect on RDW than on shoot dry weight. Specifically, shoot dry weight reduction ranged from 3.83% in WL363HQ to 24.63% in the Polar cultivar under Al stress conditions (Figure 5). The root-to-shoot ratio under the control was between 0.157 and 0.425, which was higher than that under Al stress (0.130–0.397). Compared with those under control conditions, the root-to-shoot ratios of Sersu and the nine other cultivars under Al stress decreased significantly (p < 0.05; Table S3). Under Al stress, the CV values of the RDW, shoot dry weight, and root-to-shoot ratio were higher than those under control conditions. (Table S3).

2.4. Effects of Al Stress on Malondialdehyde (MDA) Content in Alfalfa Roots

To evaluate the lipid peroxidation rate, the MDA content in alfalfa roots was measured (Figure 6). Compared with those under the control treatment, the MDA content of WL349HQ under Al stress was the lowest (28.18 nmol·g⁻¹ fresh weight [FW]), and the MDA content of WL377HQ was the highest (85.21 nmol·g⁻¹ FW). Moreover, the Great (183.07%), Vision (149.15%), and Sanditi (130.28%) treatments showed higher increases in MDA content under Al stress than those under control conditions. Conversely, Al stress caused a significant decrease in MDA content in WL363HQ (8.07%), Polar (2.48%), WL168HQ (4.94%), Suda (26.47%), Zhongmu No. 6 (25.36%), and WL349HQ (10.51%) (p < 0.05). The CV value of the Al stress treatment was lower than that of the control treatment. (Table S4).

2.5. Effects of Al Stress on Antioxidant Enzyme Activities in Alfalfa Roots

Compared with those under the control treatment, Al stress significantly increased the SOD, POD, and CAT activities in the roots of most alfalfa cultivars (p < 0.05), and the CV was lower than that under control conditions (Table S5). Under Al stress, SOD activity significantly increased in all cultivars, except McLaren (p < 0.05), with Savoie 5 exhibiting the highest increase (121.44%) (Figure 7).

Under Al stress, POD activity exhibited pronounced cultivar-specific variability. The Sanditi cultivar attained the highest recorded POD activity (4512.17 U·g⁻¹ FW), whereas Gannong No. 3 registered the lowest (1974.78 U·g⁻¹ FW). Moreover, Sanditi demonstrated the most substantial enhancement in POD activity (135.89%), in stark contrast to the WL440HQ, Zhongmu No. 1, and Thunder cultivars, each of which exhibited only marginal increases of less than 5% (Figure 8).

Compared with that under the control conditions, under Al stress, all cultivars except Gannong No. 2, Polar, and Zhongtian No. 1 exhibited a statistically significant increase in CAT activity (p < 0.05). The enhancement ranged from 3.84% for Zhongtian No. 1 to 70.01% for WL440HQ. Conversely, CAT activity in Gannong No. 2 and Polar was inhibited by 3.72% and 3.91%, respectively (Figure 9).

2.6. Al Tolerance Coefficient and Correlation Analysis of Alfalfa

In this study, an Al tolerance coefficient was used to evaluate the differential responses of various alfalfa cultivars to Al stress. The coefficient quantitatively assessed the performance of each cultivar under stress conditions, thereby eliminating discrepancies among the indices and providing a standardized criterion for comparing Al tolerance. The results revealed significant variations in the responses of different alfalfa cultivars to Al stress (

Table 1. Al tolerance coefficient of alfalfa growth and physiological indexes under Al stress.

Cultivars	Root Length	Root Elongation Rate	Root Surface Area	Root Volume	Root DW	Shoot DW	Root/Shoot Ratio	MDA Content	SOD Activity	POD Activity	CAT Activity
Algonquin	0.93 ± 0.035 bcdef	0.75 ± 0.152 defgh	1.00 ± 0.161 a	0.84 ± 0.118 ab	0.89 ± 0.033 abc	0.92 ± 0.028 abcd	0.97 ± 0.018 ab	0.67 ± 0.117 k	1.11 ± 0.036 jkl	1.89 ± 0.132 b	1.39 ± 0.107 cdef
Arora	0.92 ± 0.010 cdef	0.72 ± 0.023 efghi	0.87 ± 0.104 ab	0.82 ± 0.095 ab	0.77 ± 0.035 ghijkl	0.77 ± 0.032 ijk	0.99 ± 0.007 a	1.07 ± 0.011 bcd	1.41 ± 0.091 defghi	1.10 ± 0.133 hij	1.39 ± 0.076 cdef
Eclipse	0.87 ± 0.0410 h	0.54 ± 0.142 kl	0.71 ± 0.073 bc	0.65 ± 0.107 b	0.77 ± 0.068 fghijkl	0.96 ± 0.017 a	0.81 ± 0.086 g	1.00 ± 0.019 defg	1.33 ± 0.124 efghijk	1.15 ± 0.077 fghij	1.22 ± 0.080 ghij
Gannong No. 2	0.95 ± 0.003 abcd	0.86 ± 0.010 abcde	0.96 ± 0.064 ab	0.82 ± 0.100 ab	0.79 ± 0.045 defghijk	0.83 ± 0.036 fghi	0.96 ± 0.033 abc	0.94 ± 0.003 gh	1.18 ± 0.097 ijkl	1.28 ± 0.207 defgh	0.96 ± 0.013 l
Gannong No. 3	0.98 ± 0.007 ab	0.92 ± 0.034 ab	0.98 ± 0.046 a	0.99 ± 0.118 a	0.91 ± 0.006 ab	0.94 ± 0.026 abc	0.96 ± 0.027 abc	0.48 ± 0.02 lm	1.66 ± 0.012 bc	1.11 ± 0.060 ghij	1.53 ± 0.081 bc
Great	0.89 ± 0.016 fgh	0.71 ± 0.033 fghi	0.75 ± 0.099 abc	0.69 ± 0.077 ab	0.87 ± 0.073 abcdef	0.91 ± 0.041 abcde	0.96 ± 0.037 abc	0.35 ± 0.034 n	1.56 ± 0.102 cde	1.37 ± 0.103 de	1.44 ± 0.096 cde
Longdong	0.97 ± 0.041 abc	0.90 ± 0.130 abc	0.93 ± 0.132 ab	0.87 ± 0.236 ab	0.91 ± 0.035 ab	0.91 ± 0.035 abcde	0.99 ± 0.003 a	0.87 ± 0.013 h	1.31 ± 0.097 fghijk	1.18 ± 0.064 fghij	1.15 ± 0.048 jk
McLaren	0.89 ± 0.046 efgh	0.68 ± 0.140 ghij	0.84 ± 0.155 abc	0.81 ± 0.058 ab	0.76 ± 0.040 hijkl	0.88 ± 0.043 defg	0.87 ± 0.045 defg	0.53 ± 0.009 l	1.08 ± 0.041 l	1.14 ± 0.068 fghij	1.32 ± 0.027 efghi
Otana	0.68 ± 0.030 j	0.19 ± 0.083 m	0.89 ± 0.244 ab	0.78 ± 0.226 ab	0.82 ± 0.078 bcdefghi	0.82 ± 0.079 ghij	0.99 ± 0.006 a	0.68 ± 0.013 jk	1.52 ± 0.141 cdefg	1.09 ± 0.020 hij	1.25 ± 0.036 fghij
Overseason	0.92 ± 0.008 defg	0.67 ± 0.050 ghij	0.99 ± 0.200 a	0.90 ± 0.155 ab	0.88 ± 0.005 abcde	0.88 ± 0.012 bcdef	0.99 ± 0.007 a	0.95 ± 0.021 fgh	1.53 ± 0.149 cdef	1.10 ± 0.016 hij	1.33 ± 0.037 efg
Polar	0.97 ± 0.010 abc	0.91 ± 0.026 ab	0.89 ± 0.079 ab	0.73 ± 0.093 ab	0.73 ± 0.046 jklm	0.75 ± 0.022 k	0.97 ± 0.036 ab	1.03 ± 0.009 cdef	1.32 ± 0.207 fghijk	1.19 ± 0.080 fghij	0.96 ± 0.011 l
Salsa	0.90 ± 0.028 efgh	0.71 ± 0.068 fghi	0.97 ± 0.083 a	0.90 ± 0.203 ab	0.77 ± 0.050 ghijkl	0.77 ± 0.047 ijk	0.99 ± 0.007 a	0.75 ± 0.036 ij	1.40 ± 0.077 defghi	1.30 ± 0.142 def	1.32 ± 0.067 efghi
Sanditi	0.95 ± 0.005 abcd	0.82 ± 0.018 abcdef	0.96 ± 0.028 ab	0.95 ± 0.021 ab	0.90 ± 0.049 abc	0.96 ± 0.025 a	0.94 ± 0.028 abcd	0.43 ± 0.054 n	1.33 ± 0.056 efghijk	2.36 ± 0.099 a	1.3 ± 0.067 efghi
Sandy	0.79 ± 0.020 i	0.23 ± 0.034 m	0.97 ± 0.013 a	0.95 ± 0.018 ab	0.69 ± 0.027 klm	0.75 ± 0.060 k	0.92 ± 0.048 abcde	0.65 ± 0.049 k	1.84 ± 0.064 b	1.41 ± 0.145 d	1.22 ± 0.069 ghij
Savoie 5	0.90 ± 0.065 efgh	0.74 ± 0.143 efgh	0.97 ± 0.015 a	0.98 ± 0.050 a	0.92 ± 0.080 a	0.96 ± 0.028 a	0.96 ± 0.057 abc	0.64 ± 0.039 k	2.22 ± 0.088 a	1.25 ± 0.122 defgh	1.13 ± 0.074 jk
Sersu	0.91 ± 0.002 defgh	0.73 ± 0.009 efgh	0.94 ± 0.038 ab	0.95 ± 0.109 ab	0.69 ± 0.024 lm	0.94 ± 0.022 abcd	0.74 ± 0.020 h	0.76 ± 0.024 ij	1.35 ± 0.043 efghi	1.71 ± 0.077 c	1.12 ± 0.026 jk
Suda	0.87 ± 0.038 gh	0.59 ± 0.088 ijkl	0.90 ± 0.109 ab	0.98 ± 0.217 a	0.77 ± 0.030 ghijkl	0.78 ± 0.028 ijk	0.99 ± 0.004 a	1.36 ± 0.006 a	1.40 ± 0.179 defghi	1.38 ± 0.113 de	1.43 ± 0.072 cde
Thunder	0.87 ± 0.025 gh	0.50 ± 0.058 l	0.90 ± 0.262 ab	0.85 ± 0.283 ab	0.80 ± 0.046 cdefghij	0.96 ± 0.015 a	0.84 ± 0.036 fg	0.78 ± 0.008 i	1.33 ± 0.174 efghijk	1.03 ± 0.025 ij	1.14 ± 0.068 jk
Vision	0.97 ± 0.014 abc	0.89 ± 0.053 abc	0.9 ± 0.139 ab	0.83 ± 0.144 ab	0.66 ± 0.109 m	0.77 ± 0.059 ijk	0.86 ± 0.073 efg	0.40 ± 0.021 mn	1.62 ± 0.030 cd	1.22 ± 0.078 efghi	1.17 ± 0.008 ijk
WL168HQ	0.98 ± 0.003 a	0.94 ± 0.011 a	0.99 ± 0.029 a	0.90 ± 0.110 ab	0.81 ± 0.030 bcdefghi	0.91 ± 0.017 abcde	0.89 ± 0.026 bcdef	1.05 ± 0.027 bcde	1.62 ± 0.342 cd	1.27 ± 0.054 defgh	1.25 ± 0.018 fghij
WL298HQ	0.94 ± 0.023 abcde	0.81 ± 0.068 abcdefg	0.99 ± 0.194 a	1.00 ± 0.353 a	0.88 ± 0.040 abcd	0.92 ± 0.021 abcd	0.96 ± 0.026 abc	0.92 ± 0.054 gh	1.67 ± 0.073 bc	1.29 ± 0.066 defg	1.52 ± 0.008 bcd
WL349HQ	0.94 ± 0.015 abcde	0.79 ± 0.048 bcdefgh	0.97 ± 0.168 a	0.96 ± 0.040 ab	0.82 ± 0.035 bcdefghi	0.85 ± 0.020 efgh	0.96 ± 0.024 abc	1.12 ± 0.044 b	1.11 ± 0.042 kl	1.14 ± 0.026 fghij	1.40 ± 0.085 cdef
WL363HQ	0.98 ± 0.007 a	0.92 ± 0.035 ab	0.79 ± 0.077 abc	0.85 ± 0.040 ab	0.86 ± 0.100 abcdefg	0.96 ± 0.016 a	0.89 ± 0.091 bcdef	1.09 ± 0.011 bc	1.42 ± 0.072 defgh	1.76 ± 0.126 bc	1.22 ± 0.089 ghij
WL363HQ	0.92 ± 0.008 cdef	0.65 ± 0.081 hijk	0.88 ± 0.175 ab	0.96 ± 0.306 ab	0.75 ± 0.015 ijklm	0.83 ± 0.032 fghi	0.90 ± 0.053 bcdef	0.46 ± 0.057 lm	1.28 ± 0.077 ghijkl	1.13 ± 0.085 fghij	1.59 ± 0.090 ab
WL440HQ	0.80 ± 0.023 i	0.57 ± 0.042 jkl	0.61 ± 0.037 c	0.73 ± 0.026 ab	0.72 ± 0.036 jklm	0.77 ± 0.031 ijk	0.93 ± 0.007 abcd	0.92 ± 0.019 gh	1.51 ± 0.133 cdefg	1.03 ± 0.065 j	1.71 ± 0.120 a
Zhonglan No.1	0.90 ± 0.007 efgh	0.68 ± 0.009 ghij	0.92 ± 0.126 ab	0.83 ± 0.110 ab	0.87 ± 0.034 abcdef	0.88 ± 0.025 cdefg	0.98 ± 0.010 a	0.64 ± 0.037 k	1.51 ± 0.126 cdefg	1.24 ± 0.058 defgh	1.51 ± 0.087 bcd
Zhongmu No. 1	0.93 ± 0.011 cdef	0.76 ± 0.030 cdefgh	0.96 ± 0.142 ab	0.89 ± 0.113 ab	0.84 ± 0.061 abcdefgh	0.95 ± 0.007 ab	0.89 ± 0.069 cdef	0.70 ± 0.046 ijk	1.22 ± 0.040 hijkl	1.04 ± 0.024 ij	1.18 ± 0.050 hij
Zhongmu No. 3	0.95 ± 0.016 abcd	0.84 ± 0.052 abcdef	0.95 ± 0.129 ab	0.87 ± 0.079 ab	0.78 ± 0.018 efghijkl	0.79 ± 0.015 hijk	0.99 ± 0.008 a	0.99 ± 0.016 efg	1.34 ± 0.112 efghij	1.22 ± 0.060 efghi	1.38 ± 0.184 def
Zhongmu No. 6	0.98 ± 0.011 ab	0.91 ± 0.027 ab	0.87 ± 0.120 ab	0.92 ± 0.251 ab	0.85 ± 0.028 abcdefgh	0.89 ± 0.023 bcde	0.96 ± 0.047 abc	1.34 ± 0.140 a	1.86 ± 0.152 b	1.18 ± 0.147 fghij	1.33 ± 0.105 efgh
Zhongtian No.1	0.96 ± 0.009 abc	0.89 ± 0.021 abcd	0.89 ± 0.094 ab	0.80 ± 0.047 ab	0.76 ± 0.005 hijkl	0.76 ± 0.011 jk	0.99 ± 0.012 a	0.90 ± 0.012 h	1.60 ± 0.062 cd	1.10 ± 0.034 hij	1.04 ± 0.036 kl
DF	29	29	29	29	29	29	29	29	29	29	29
MS	0.013	0.103	0.025	0.025	0.015	0.017	0.012	0.211	0.181	0.253	0.096
p	**	**	ns	ns	**	**	**	**	**	**	**

Note: In the table, the values are presented as means ± standard deviations. Lowercase letters indicate significant differences among the Al tolerance coefficients of different alfalfa cultivars for the same parameter (p < 0.05). DF: degrees of freedom, MS: mean square, **: p < 0.01. ns: no significant difference.

). Under Al stress, a highly significant (p < 0.01) positive correlation was observed between alfalfa root surface area and root volume. Additionally, shoot dry weight (SDW) was significantly (p < 0.01) positively correlated with RDW, and RDW demonstrated a significant (p < 0.05) positive correlation with both root length and root elongation rate. In the antioxidant system, MDA levels were inversely correlated with SOD, POD, and CAT levels (Figure 10). These findings suggest that alfalfa growth is closely linked to its antioxidant defense system. The synergistic action of these mechanisms plays a critical role in enhancing the adaptability of alfalfa to acidic Al-rich environments.

2.7. PCA of Alfalfa Growth Physiological Indices

To streamline the data analysis by reducing the 11 individual Al tolerance coefficient indices to a smaller set of comprehensive indicators, PCA was performed on these metrics. PCA yielded six principal components (PCs) with a total eigenvalue of 9.39, accounting for a cumulative contribution rate of 85.365% (

Table 2. Principal component analysis of Al tolerance coefficients based on growth and physiological indices of 30 alfalfa cultivars under Al stress.

Trait	Component
	1	2	3	4	5	6
Root length	0.271	−0.207	0.249	0.086	−0.001	0.217
Root elongation rate	0.253	−0.196	0.272	0.178	0.022	0.171
Root surface area	0.231	0.17	0.02	−0.451	−0.145	−0.208
Root volume	0.213	0.244	−0.069	−0.355	−0.077	0.464
Root dry weight	0.233	0.186	−0.171	0.386	0.108	−0.364
Shoot dry weight	0.189	−0.162	−0.368	0.215	0.292	−0.02
Root/shoot ratio	0.056	0.411	0.243	0.21	−0.219	−0.447
MDA	0.005	0.019	0.381	0.09	0.146	0.107
SOD	0.028	0.289	−0.042	−0.09	0.727	0.173
POD	0.15	−0.133	−0.244	−0.098	−0.39	−0.132
CAT	−0.038	0.271	−0.138	0.383	−0.369	0.661
Eigenvalue	2.827	1.739	1.694	1.281	0.989	0.86
Contribution rate (%)	25.702	15.813	15.396	11.647	8.99	7.817
Cumulative contribution rate (%)	25.702	41.515	56.911	68.558	77.548	85.365

). This finding implies that approximately 85.365% of the informational content from the original eleven indices is captured by the six new independent composite indicators. Specifically, PC1 was predominantly determined by root length, root elongation rate, root surface area, root volume, and RDW; PC2 was mainly influenced by the root-to-shoot ratio; PC3 was mainly determined by MDA content; PC4 was largely governed by RDW and CAT activity; PC5 was primarily driven by SOD activity; and PC6 was mainly determined by root volume and CAT activity. Correspondingly, PC1 and PC2 can be classified as “root growth-related factors”, PC3 and PC5 as “physiological regulatory factors”, and PC4 and PC6 as factors associated with both root growth and physiological regulation.

2.8. Comprehensive Evaluation of Al Tolerance of Alfalfa Cultivars

Using Equation (4), the membership function values (U) for all comprehensive indices across the 30 alfalfa cultivars were calculated under Al stress conditions. Subsequently, Equation (5) was used to determine the weights (W) of the six comprehensive indices. By integrating the U values of each cultivar and the corresponding W values, Equation (6) was applied to compute the comprehensive evaluation value (D) for assessing Al tolerance, where a higher D value signifies stronger Al tolerance (

Table 3. Comprehensive index value (CI), membership function value U(X), weight (W), comprehensive evaluation value (D value), and ranking of each comprehensive index value of 30 alfalfa cultivars.

Cultivars	CI1	CI2	CI3	CI4	CI5	CI6	U(X1)	U(X2)	U(X3)	U(X4)	U(X5)	U(X6)	D	Rank
Algonquin	0.959	−0.189	−0.910	0.454	−2.021	−1.193	0.828	0.505	0.239	0.666	0.000	0.246	0.499	20
Arora	−0.608	0.460	1.268	0.434	−0.455	0.179	0.452	0.682	0.783	0.662	0.324	0.579	0.580	12
Eclipse	−1.504	−1.916	−0.712	1.210	1.291	−0.142	0.236	0.032	0.288	0.826	0.686	0.501	0.359	30
Gannong No. 2	0.276	−0.898	1.185	−0.606	−0.328	−1.583	0.664	0.311	0.762	0.442	0.351	0.152	0.505	18
Gannong No. 3	1.427	0.985	−0.592	0.628	0.330	1.084	0.940	0.826	0.318	0.703	0.487	0.798	0.713	2
Great	−0.580	−0.168	−1.145	2.035	0.222	−0.762	0.458	0.510	0.180	1.000	0.464	0.351	0.482	22
Longdong	0.988	−0.107	0.535	0.752	0.154	−1.189	0.835	0.527	0.600	0.729	0.450	0.247	0.626	7
McLaren	−0.767	−1.086	−0.578	0.172	−0.760	0.214	0.413	0.259	0.322	0.606	0.261	0.587	0.394	27
Otana	−2.044	1.622	−1.203	−0.393	0.167	−2.208	0.107	1.000	0.165	0.487	0.453	0.000	0.361	29
Overseason	0.469	1.045	0.222	0.163	0.279	−0.506	0.710	0.842	0.522	0.604	0.476	0.413	0.634	6
Polar	−0.374	−1.093	2.134	−0.337	0.019	−1.337	0.508	0.257	1.000	0.499	0.422	0.211	0.512	17
Salsa	−0.152	0.704	0.569	−0.734	−1.014	−0.206	0.561	0.749	0.608	0.415	0.209	0.486	0.540	15
Sanditi	1.677	−0.474	−1.864	−0.126	−1.694	−0.745	1.000	0.427	0.000	0.543	0.068	0.355	0.494	21
Sandy	−1.387	1.374	−0.807	−2.697	0.244	0.003	0.265	0.932	0.264	0.000	0.469	0.536	0.398	26
Savoie 5	1.110	1.365	−0.948	−0.502	2.808	−0.507	0.864	0.930	0.229	0.464	1.000	0.413	0.679	4
Sersu	0.141	−2.034	−1.182	−2.079	0.121	1.361	0.631	0.000	0.170	0.131	0.444	0.866	0.365	28
Suda	−0.376	1.158	0.879	−0.406	−0.944	0.764	0.507	0.873	0.686	0.484	0.223	0.721	0.593	10
Thunder	−0.533	−0.788	−0.985	−0.496	0.958	−0.297	0.470	0.341	0.220	0.465	0.617	0.464	0.415	25
Vision	−0.426	−1.120	0.531	−1.367	0.344	0.980	0.495	0.250	0.599	0.281	0.490	0.773	0.464	24
WL168HQ	0.957	−0.417	0.523	−0.354	0.929	0.734	0.827	0.442	0.597	0.495	0.611	0.713	0.635	5
WL298HQ	1.120	1.182	−0.255	0.228	0.221	1.100	0.866	0.880	0.402	0.618	0.464	0.802	0.703	3
WL349HQ	0.469	0.295	0.822	0.011	−1.175	0.558	0.710	0.637	0.672	0.572	0.175	0.671	0.610	8
WL363HQ	0.826	−1.386	−0.171	1.086	0.389	0.199	0.796	0.177	0.423	0.800	0.499	0.584	0.563	14
WL377HQ	−0.402	0.248	−0.563	−0.209	−1.254	1.915	0.501	0.624	0.325	0.526	0.159	1.000	0.505	19
WL440HQ	−2.490	0.364	0.068	1.930	−0.195	1.689	0.000	0.656	0.483	0.978	0.378	0.945	0.468	23
Zhonglan No.1	0.043	0.849	−0.515	0.919	−0.316	−0.175	0.608	0.789	0.338	0.764	0.353	0.493	0.576	13
Zhongmu No. 1	0.446	−0.656	−0.381	−0.174	0.239	−0.318	0.705	0.377	0.371	0.533	0.468	0.458	0.512	16
Zhongmu No. 3	0.136	0.349	1.221	0.041	−0.912	0.229	0.630	0.652	0.772	0.579	0.230	0.591	0.606	9
Zhongmu No. 6	0.770	0.514	1.101	0.745	1.665	1.009	0.782	0.697	0.741	0.727	0.763	0.780	0.749	1
Zhongtian No.1	−0.173	−0.183	1.753	−0.329	0.686	−0.850	0.556	0.506	0.905	0.500	0.560	0.329	0.581	11
W							0.301	0.185	0.180	0.136	0.105	0.092

). The Zhongmu No. 6, Gannong No. 3, WL298HQ, and Savoie 5 cultivars exhibited higher D values than the other cultivars, thus indicating an enhanced tolerance to Al stress. Conversely, McLaren, Sersu, Otana, and Eclipse displayed lower D values, suggesting reduced Al tolerance under the same conditions.

The D values of the 30 alfalfa cultivars were analyzed using Euclidean distance and hierarchical clustering methods (Figure 11). The results showed that these cultivars could be classified into four distinct categories. The first category, comprising Zhongmu No. 6, Gannong No. 3, WL298HQ, and Savoie 5, exhibited high Al tolerance. The second category included 11 cultivars, including WL168HQ, Suda, Zhonglan No. 1, and Salsa, which displayed moderately high Al tolerance. The third category consisted of nine cultivars, including WL440HQ, Great, Sanditi, and Algonquin, which showed moderately low Al tolerance. The fourth category encompassed six cultivars, namely McLaren, Sersu, Otana, and Eclipse, which exhibited low Al tolerance.

Using stepwise regression analysis, a D value regression equation for alfalfa cultivars under Al stress was developed, with the D value as the dependent variable and the relative ratios of the 16 indicators as independent variables.

D = −1.260 + 0.150X₁ + 0.452X₂ + 0.298X₃ + 0.126X₄ + 0.096X₅ + 0.112X₆ + 0.351X₇ − 0.073X₈ + 0.453X₉ (R² = 0.999).
X₁ (root elongation rate)

where X₂ (root-to-shoot ratio), X₃ (root volume), X₄ (SOD), X₅ (MDA), X₆ (CAT), X₇ (RDW), X₈ (POD), and X₈ (root length) were identified as crucial indicators affecting alfalfa tolerance to Al stress.

2.9. Expression Analysis of MsABC Genes in Response to Al Toxicity

Studies have demonstrated that ABC transporters facilitate aluminum detoxification in plants. In Arabidopsis thaliana, AtALS3 (AtABCI16), which participates in aluminum toxicity tolerance, contains solely the transmembrane domain (TMD), and functions as a plasma membrane-localized ABC transporter [23]. In wheat, TaMDR1 is associated with aluminum toxicity [24]. FeALS1.1 and FeALS1.2 mediate internal aluminum detoxification by sequestering aluminum into vacuoles in buckwheat [25]. The association between the ABC transporter family and Al tolerance in alfalfa has received limited research attention. Therefore, this study screened 10 MsABC genes from the ABC transporter family (Table S4) and analyzed their dynamic expression patterns in root tip tissues through Al stress treatment at different time points. The expression changes of all selected genes in the root system under aluminum (Al) stress at different time points were analyzed by qRT-PCR (Figure 12).

The relative expression levels of these five genes (MS.gene064142, MS.gene064141, MS.gene030165, MS.gene56746, MS.gene26778) exhibited a fluctuating trend with prolonged Al stress: initially decreasing, then increasing, followed by another decrease, and finally rising again. Among them, the expression levels of MS.gene064142 and MS.gene26778 at 48 h were significantly higher than those at 0 h (p < 0.05). In contrast, no significant differences were observed for MS.gene064141, MS.gene030165, and MS.gene56746 at 0, 4, or 48 h. The expression of MS.gene26779 peaked at 8 h, showing significant differences compared to other time points (p < 0.05). MS.gene020649 displayed an initial decrease, followed by an increase in expression, reaching maximal levels at 4 and 8 h, which were significantly higher than those at other time points (p < 0.05). The expression of MS.gene26775 peaked at 4 h and subsequently declined. For MS.gene21237, expression peaked at 4 h, and was significantly higher than at 0 and 24 h (p < 0.05). Finally, the expression of MS.gene68631 reached its maximum at 48 h, with significant differences compared to all other time points (p < 0.05).

3. Discussion

This study evaluated the growth performance of 30 alfalfa cultivars under Al stress by analyzing root system parameters and enzyme activity. The results indicated considerable variations in the growth responses among the different cultivars when subjected to Al stress.

3.1. Effects of Al Stress on Alfalfa Growth

Under acidic soil conditions, excessive Al markedly inhibits plant development [26]. Panda and Matsumoto [27], Passos et al. [28], and Hijbeek et al. [29] attribute Al phytotoxicity primarily to the direct interaction between Al³⁺ ions and root tissue cells, thereby resulting in cell death. The inhibition of root growth is widely recognized as a key indicator of Al toxicity [30]. In this study, 30 alfalfa cultivars exhibited reduced root parameters after Al stress. Other studies have shown that Al stress impairs root growth in plants. For example, in peanut (Arachis hypogaea L.), Al stress markedly reduced the root fresh weight, dry weight, total root length, total root surface area, and root tip number, thereby indicating severe inhibition of root elongation [31]. Furthermore, under 1.0 mM Al stress, Shatian pomelo exhibited reductions of 37.78% in root length, 27.19% in root surface area, 8.70% in root volume, and 13.40% in RDW compared with those under control conditions. Moreover, root biomass demonstrated a declining trend with increasing Al concentrations [7]. In addition, cucumber (Cucumis sativus L.) subjected to 200 μM Al stress exhibited a considerable reduction in RDW [32]. These findings are consistent with the results of this study, thus further confirming the inhibitory effects of Al on plant root growth.

RRE is a key parameter for assessing plant tolerance to Al stress [33] and is widely used to screen for differences in Al tolerance among cultivars within the same species [34,35]. Under 50 μM·L⁻¹ Al stress, the RRE of wheat decreases, with variations among cultivars ranging from 2.06 to 31.42% [36]. Furthermore, in rice (Oryza sativa L.) subjected to the same Al treatment, cultivars with an RRE below 30% were classified as Al-sensitive, whereas those with an RRE above 45% were considered Al-tolerant [37,38]. Moreover, in Al-tolerant sorghum (Sorghum bicolor (L.) Moench) [39], inhibition of root elongation under Al stress was less pronounced than that in Al-sensitive cultivars. In this study, 30 alfalfa cultivars were subjected to Al stress. The results indicated that 14 cultivars exhibited RRE values exceeding 80%, thereby demonstrating high Al tolerance. In contrast, the Otana and Sandy cultivars displayed RRE values of 19.03% and 23.03%, respectively, indicating markedly greater inhibition of root elongation than that of the other cultivars (Table S1). These findings are consistent with previous studies confirming that the inhibition of root elongation is a common phenotypic manifestation of Al toxicity in plants [40].

Al toxicity primarily manifests as the inhibition of root and bud development [41]. In this study, 30 alfalfa cultivars subjected to Al stress exhibited considerable reductions in root and shoot dry weights, as well as in the root-to-shoot ratio (Table S3). A decline in root biomass is correlated with damage to the root cell wall [42] and plasma membrane [2], accompanied by a reduction in the levels of hemicellulose and pectin components [43]. Moreover, the stunted growth of the aboveground tissues is largely attributed to the gradual translocation of Al³⁺ from the roots to the shoots, thereby substantially impairing crop yield and quality [44,45]. For example, under 1 mM Al stress, the leaf area, perimeter, fresh weight, and dry weight of peanut leaves were substantially reduced by 16.94, 15.99, 26.18, and 13.67%, respectively [46]. Under 200 μM Al stress, rice exhibited inhibited germination, and both the root fresh weight and dry weight were markedly decreased [47]. These results are in agreement with the findings of this study, thereby confirming that Al stress exerts a pronounced inhibitory effect on both root and shoot growth, thus compromising overall plant development.

3.2. Effects of Al Stress on Alfalfa Antioxidant Mechanism

Under normal growth conditions, a dynamic equilibrium exists between the production and scavenging of ROS in plants [48]. However, when plants are subjected to high Al concentrations, this balance is disrupted [49], leading to an excessive accumulation of ROS. Surplus ROS interact with lipids and proteins, thereby triggering protein degradation and lipid peroxidation, which ultimately compromises the integrity of the cell membrane [11,50,51]. Lipid peroxidation serves as a critical indicator of membrane damage, and variations in MDA content are widely considered reliable markers of oxidative injury, reflecting cell membrane stability [52].

In peas (Pisum sativum L.) [53], soybean (Glycine max (L.) Merr.) [54], broad bean (Vicia faba L.) [55], rice [56], and Masson pine (Pinus massoniana Lamb.) [57] under Al stress, the MDA content increases with rising Al concentrations, thus reflecting plasma membrane damage [58]. Moreover, the increase in MDA content is considerably more pronounced in Al-sensitive cultivars than in Al-tolerant ones [55]. In this study, the MDA content of alfalfa under Al stress varied among the cultivars. Compared with those under control conditions, 21 cultivars under AL stress showed an increase in MDA content, which is consistent with previous studies. However, nine cultivars had comparable or markedly lower MDA levels than those of the control (Figure 6). In peanut leaves, the MDA content first increases with Al concentration, and then sharply drops to the highest level, likely due to severe damage to the antioxidant defense system [46]. In another study, the Calabash Rouge genotype of tomato (Solanum lycopersicum L.) [59] had a lower MDA content in roots and shoots under pH 4.2 + Al treatment than under the control conditions. This may be because a low Al³⁺ concentration forms a protective layer on the cell membrane, thereby reducing H⁺ interactions with the cell membrane, and thus lowering cell membrane damage [60]. Based on the aforementioned studies, the decrease in MDA content in these nine alfalfa cultivars under 100 µM Al treatment could be due to antioxidant system damage or a protective layer formed by low Al³⁺ concentrations. Thus, when evaluating plant Al tolerance, changes in MDA content should be considered holistically, and judgment should not be based solely on this index.

SOD, POD, and CAT are the key enzymes in plant antioxidant defense systems [61]. Under oxidative stress, SOD catalyzes the conversion of O₂⁻ to H₂O₂, which is then broken down into water by CAT, ultimately reducing ROS accumulation [62]. POD offers further protection by catalyzing H₂O₂ decomposition [63]. Boosting the activity of these antioxidant enzymes is crucial for minimizing Al-induced root damage in alfalfa [64,65]. The antioxidant enzyme activity in plants typically increases under environmental stress [66], thus countering ROS accumulation to boost oxidative stress tolerance [67]. For instance, upon exposure to Al stress, maize (Zea mays L.) leaves exhibited a notable increase in SOD and CAT activity [68]. Similarly, in buckwheat (Fagopyrum esculentum Moench), Al stress led to considerable elevations in APX, POD, CAT, and SOD activities within the roots. Additionally, marked increases in the concentrations of soluble sugars, soluble proteins, proline, and MDA were observed [69], thus suggesting an enhanced antioxidant response to mitigate oxidative damage. In this study, most alfalfa cultivars exhibited considerably higher SOD, POD, and CAT activities under Al stress than under the control conditions, which is consistent with previous findings. However, certain cultivars, such as McLaren, WL440HQ, and Gannong No. 2, exhibited antioxidant enzyme activities that were markedly lower or not significantly different from those of the control group (Table S5). Based on the comprehensive evaluation results of alfalfa, these cultivars were classified as medium-low Al-tolerant and low-Al-tolerant (Figure 11). Al stress typically induces the generation of ROS in plants, thereby activating antioxidant enzymes, such as SOD, POD, and CAT, to scavenge excess ROS. However, when Al stress exceeds the plant tolerance threshold, a substantial decline in antioxidant enzyme activity occurs, resulting in inadequate ROS detoxification and subsequent oxidative damage [70]. Ma et al. observed that Al-sensitive rice cultivars exhibited decreased or unchanged antioxidant enzyme activity (except for CAT) under Al stress [71]. Moreover, Zhang et al. reported that 50 μM Al treatment reduced SOD activity in Al-sensitive broad bean cultivars [72]. These findings align with the results of this study, thereby indicating that these cultivars have low Al stress tolerance, and ultimately validating the experimental outcomes and evaluation system. Thus, enhancing antioxidant defense by boosting SOD, POD, and CAT activity could be an effective strategy for improving Al stress tolerance in alfalfa.

3.3. Gene Expression Pattern Analysis

ABC transporters, one of the largest and most versatile gene families in higher plants, enhance stress tolerance through multifunctional roles, transporting diverse substrates (heavy metals, phytohormones, inorganic acids) [73,74], modulating cellular homeostasis, and regulating stress signaling. In the process of Al detoxification, ABC transporters mediate the efflux of Al ions from the cytoplasm to the extracellular environment, thereby reducing intracellular Al ion concentrations, alleviating Al induced toxicity [75], and safeguarding normal plant growth and development in Al contaminated soil conditions [76]. Previous studies have demonstrated that Al stress induces significant upregulation of ABC genes (GmABCB23, GmABCB25, and GmABCB48) in soybean roots, with the candidate gene exhibiting markedly enhanced expression under Al exposure [77]. In a study by Singh et al. [20], it was demonstrated that upon Al stress at diverse time points (3, 6, 12, and 24 h), the expression levels of two genes, LcABCI14 and LcABCI17, in lentil (Polygala tatarinowii Regel) roots were the highest, suggesting that these genes may be correlated with aluminum tolerance. In this study, the relative expression levels of MS.gene064141, MS.gene030165, MS.gene56746, and MS.gene26775 in roots at all time points (4, 8, 24, and 48 h) were either lower or showed no significant difference compared to at 0 h. In contrast, the other six genes (MS.gene064142, MS.gene26779, MS.gene020649, MS.gene26778, MS.gene21237, and MS.gene68631) exhibited significantly higher expression levels than at 0 h under Al stress at specific time points, suggesting their potential implication in Al tolerance (Figure 8). Therefore, the candidate genes identified in this study could be further investigated through cloning and development of transgenic lines to elucidate their potential roles in enhancing Al tolerance mechanisms in plants.

4. Materials and Methods

4.1. Plant Materials

This study included 30 alfalfa cultivars: 9 from China, 18 from the USA, 2 from France, and 1 from Canada. For more details, see

Table 4. Alfalfa cultivar information.

Number	Cultivar	Source	Number	Cultivar	Source
1	Algonquin	Canada	16	Sersu	USA
2	Arora	USA	17	Suda	USA
3	Eclipse	China	18	Thunder	USA
4	Gannong No. 2	China	19	Vision	USA
5	Gannong No. 3	China	20	WL168HQ	USA
6	Great	USA	21	WL298HQ	USA
7	Longdong	China	22	WL349HQ	USA
8	McLaren	USA	23	WL363HQ	USA
9	Otana	USA	24	WL377HQ	USA
10	Overseason	USA	25	WL440HQ	USA
11	Polar	USA	26	Zhonglan No.1	China
12	Salsa	USA	27	Zhongmu No. 1	China
13	Sanditi	France	28	Zhongmu No. 3	China
14	Sandy	USA	29	Zhongmu No. 6	China
15	Savoie 5	France	30	Zhongtian No.1	China

.

4.2. Experimental Design

Plump seeds were selected and surface-sterilized with 1.0% (v/v) sodium hypochlorite for 5 min. Thereafter, seeds were rinsed with distilled water five times, and germinated in darkness at 25 °C for three days. Uniformly grown seedlings were selected and transplanted into 21-hole seedling trays (54 cm × 28 cm, hole dimensions: 6.5 cm top diameter, 6 cm depth, 3 cm base diameter) filled with a sterilized perlite–vermiculite (1:3, v/v) soil mix and equipped with drainage trays. Each cultivar was subjected to three replicates per treatment for a total of 180 trays. Seedlings were cultured in 1/2 Hoagland nutrient solution (pH 5.8) for 14 days. The seedlings were then transferred to 0.5 mM CaCl₂ 1/2 Hoagland nutrient solution (pH 4.3) without AlCl₃ for hydroponic pre-treatment for 12 h. After pre-treatment, seedlings were transferred to 0.5 mM CaCl₂ 1/2 Hoagland nutrient solution (pH 4.3) with 100 μM AlCl₃ for Al stress treatment for 14 days, whereas the control group was treated with the same Hoagland nutrient solution without AlCl₃. During the entire culture period, the nutrient solution was renewed every two days. Environmental conditions were set to 16 h of daylight and 8 h of night, with the temperature maintained at 25 °C.

4.3. Phenotypic Trait Assessment

4.3.1. Root Trait Indicators

The complete root systems of plants from both the control and Al-stressed groups were collected. An Epson V800 scanner (Perfection V800 photo; Epson, Suwa, Japan) was used to scan the root images at 300 dpi. The root length, surface area, and volume were analyzed using WinRhizo root image analysis software (WinRhizo Tron Pro 2009, Regent Instruments Inc., Quebec, QC, Canada). Three biological replicates were conducted for each cultivar and treatment, with seven plants per replicate. Alfalfa root length was measured before and after the Al stress and control treatments. The root elongation and RRE rates were calculated as follows:

Root Elongation Rate = ((Root Length after Treatment − Root Length before Treatment)/Root Length before
Treatment) × 100%

RRE Rate = (Root Elongation Rate under Al Stress Treatment/Root Elongation Rate under Control
Treatment) × 100%

The CV indicates the degree of dispersion of each trait, and was calculated using the following formula:

$$\mathrm{C}\mathrm{V}=\mathrm{S}/\mathrm{X}$$

(1)

where S is the standard deviation and X is the average.

4.3.2. Dry Weight

After the alfalfa roots were scanned, the plant surface was used to remove moisture and separate the stems from the roots. Both parts were dried at 65 °C for 72 h until a constant weight was reached. Thereafter, the samples were weighed and their dry weights were recorded separately. To ensure data accuracy and reliability, seven plants per cultivar were measured per treatment, with three replicates. The root–shoot ratio was calculated by dividing the RDW by the shoot dry weight.

4.4. Malondialdehyde (MDA) Content Determination

The MDA content in alfalfa roots was measured using an MDA Content Assay Kit (G0109W, Geruisi Bio, Suzhou, China), following the manufacturer’s instructions. Each plant sample (0.1 g) was homogenized in 1 mL of extraction solution and centrifuged at 12,000× g for 10 min at 4 °C. The supernatant (0.2 mL) was then mixed with 0.3 mL of reaction solution. The mixture was then heated in a water bath at 95 °C for 30 min, before being cooled on ice. Subsequently, the mixture was centrifuged at 12,000× g for 10 min at 25 °C. The absorbance of the supernatant was measured at 532 and 600 nm using a microplate reader (Multiskan GO 1510; Thermo Fisher Scientific, Waltham, MA, USA).

4.5. Antioxidant Enzyme Activity Analysis

SOD activity was measured using the SOD Activity Assay Kit-WST-8 (G0101W, Geruisi Bio, Suzhou, China), POD activity was determined using a POD Assay Kit (G0108W; Geruisi Bio, Suzhou, China), and CAT activity was assessed using a CAT Assay Kit (G0106W; Geruisi Bio). The absorbances of SOD, POD, and CAT were measured at 450, 470, and 510 nm, respectively, using a microplate reader (Multiskan GO 1510; Thermo Fisher Scientific, Waltham, MA, USA). One unit of SOD activity was defined as the amount of enzyme required to achieve 50% inhibition in a xanthine oxidase-coupled reaction system under standardized assay conditions. The POD activity unit was the amount of enzyme per gram of sample that caused an increase of 1 in absorbance at 470 nm/min in the reaction system. A CAT activity unit is the amount of enzyme per gram of sample that catalyzes the decomposition of 1 µM H₂O₂ per minute at 25 °C.

4.6. Analysis of Al Tolerance in Alfalfa

The Al tolerance of 30 alfalfa cultivars was evaluated using the membership function method. Subsequently, a comprehensive analysis of the data (D values) and Al tolerance coefficients was performed. Correlation, principal component, cluster, and regression analyses were conducted, and an optimal regression equation was established [78]. The specific calculations were as follows:

Indicators positively correlated with Al tolerance included root length, root elongation rate, root surface area, root volume, RDW, shoot dry weight, root–shoot ratio, SOD activity, POD activity, and CAT activity.

Al tolerance coefficient = index value under Al stress/index value under control treatment

(2)

MDA content is negatively correlated with Al tolerance:

Al tolerance coefficient = index value under control treatment/index value under Al stress

(3)

The Al tolerance coefficient was calculated using Formulas (2) and (3), and a Pearson correlation analysis was performed for different traits.

$$U\left(X_j\right)=(X_j-X_{min})/(X_{max}-X_{min}) j=1,2\dots \dots n$$

(4)

In Formula (4), U(Xj) is the membership function value of the j-th composite index, Xj is the j-th composite index, X_max is its maximum value, and X_min is its minimum value.

$$W_j=P_j/{\sum }_{j=1}^n{P}_j j=1,2\dots \dots n$$

(5)

In Formula (5), W_j represents the weight of the j-th composite index among all composite indices, and P_j represents the contribution rate of the j-th composite index for each cultivar.

$$D={\sum }_{j=1}^n[U(X_j)\times W_j] j=1,2\dots \dots n$$

(6)

The D value in Formula (6) represents the comprehensive evaluation value of the tolerance of various cultivars to Al stress.

4.7. Quantitative Real-Time PCR Analysis

Hydroponic experiments were conducted using an aluminum-tolerant alfalfa genotype (Zhongmu No. 6), with growth conditions consistent with previous protocols. Seventeen-day-old seedlings were exposed to half-strength Hoagland’s nutrient solution containing 100 μM AlCl₃ (pH 4.3) for 0, 4, 8, 24, and 48 h. Root tip samples were collected after treatment, immediately flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent analyses.

Gene-specific primers (Table S6) were designed using Primer Premier 6.0. Total RNA was isolated from root tips using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). cDNA was synthesized from 1 μg total RNA using the cDNA Synthesis Kit (Transgene, Beijing, China). Quantitative real-time PCR (qPCR) was conducted with the SYBR Green Supermix Kit (Transgene, Beijing, China) using a qPCR machine (Bio-Rad, Hercules, CA, USA). The MsActin gene (MsUBQ) was used as the internal reference [79]. Relative gene expression levels were determined using the 2^−ΔΔCt method [80].

4.8. Data Analysis

IBM SPSS Statistics 27.0.0 (Armonk, NY, USA) was used for one-way analysis of variance, and Duncan’s multiple-range test was used to analyze the experimental data and assess intergroup differences. Aluminum tolerance coefficients were used for PCA and membership function calculations for each cultivar. The D value for alfalfa resistance was analyzed using the hierarchical clustering method. Pearson correlation and linear regression analyses were used to determine the relationship between the D value and the Al tolerance coefficient. The “corrplot” package in R 4.4.2 (Vienna, Austria) was used for the correlation heat maps, and “ggplot2” and “ggdendro” were used for the tree clustering diagram [81]. The R packages “tidyverse”, “ggsci”, and “readxl” were employed to generate boxplots.

5. Conclusions

This study comprehensively evaluated the aluminum (Al) stress tolerance of 30 alfalfa cultivars through integrated analyses of growth, physiological traits, and MsABC gene expression. Al stress significantly inhibited root elongation, biomass accumulation, and root architecture, with cultivar-specific variations in tolerance. Notably, Zhongmu No. 6, Gannong No. 3, WL298HQ, and Savoie 5 exhibited the highest Al tolerance, characterized by robust root growth, elevated antioxidant enzyme activities (SOD, POD, CAT), and lower MDA accumulation. Stepwise regression identified the root elongation rate, root-to-shoot ratio, root volume, SOD, MDA, CAT, RDW, POD, and root length as pivotal predictors for Al tolerance. Furthermore, qRT-PCR revealed dynamic expression patterns of six MsABC transporter genes under Al stress, suggesting their potential roles in Al detoxification.

However, this study has limitations. The hydroponic system may not fully replicate field conditions, and the use of a single Al concentration (100 μM) limits extrapolation to varying soil acidity levels. Additionally, the functional validation of candidate MsABC genes and their mechanistic roles in Al tolerance remain to be explored. Future research should prioritize field trials to validate cultivar performance under natural acidic soils, investigate genetic engineering approaches to enhance MsABC expression, and elucidate synergistic interactions between antioxidant systems and Al exclusion mechanisms. These efforts will advance the development of Al-resistant alfalfa cultivars, facilitating sustainable forage production in acidic regions.