Alfalfa (Medicago sativa subsp. sativa L.), as the world's most important leguminous forage, plays an indispensable role in modern livestock farming and ecological systems due to its high yield, superior quality, and excellent adaptability. However, China's alfalfa industry has long faced a "bottleneck" in its seed industry, primarily characterized by the insufficient innovation capacity in germplasm resources and a low supply rate of elite proprietary varieties. The root of this challenge lies not only in the relatively late start and underdeveloped technical framework of alfalfa breeding in China, but also in the complex genetic characters obstacles inherent to alfalfa itself—namely, its autotetraploid nature with high heterozygosity and a self-incompatibility mechanism. Therefore, a systematic review of alfalfa's breeding history, current research status, and a forward-looking perspective on biotechnological breeding is of significant referential value for accelerating genetic improvement in China. The history of alfalfa domestication and spread was extensive. Originating from the Transcaucasus region, it was introduced to China via the "Silk Road" and subsequently spread worldwide. The vast enrichment of its genetic diversity benefited historically from the introduction of germplasm from different geographical origins and inter/intraspecific hybridization, particularly through genetic introgression from the stress-tolerant subspecies, M. sativa subsp. falcata. This laid a solid genetic foundation for breeding varieties adapted to diverse ecological niches. Developed countries, such as the United States, have established a comprehensive system for alfalfa breeding that covered germplasm collection, evaluation, and new variety development, with a history spanning over a century. Nevertheless, even in the United States, yield improvement plateaued after 1990, revealing the potential limitations of conventional breeding strategies. Although China has made considerable progress in alfalfa breeding, successfully developing adapted varieties, such as the "Zhongmu" and "Gannong" series, the overall effort still faced significant challenges. Firstly, the number of registered varieties is relatively less (128 as of 2024, far fewer than the 1 738 in the U.S.), which severely limits the ability to select optimal varieties for China's diverse ecological environments. Secondly, conventional breeding strategies themselves have constrained the effective utilization of heterosis. Historically, breeders often employed a strategy of population mixing and recurrent selection with varieties from different geographical origins. However, this approach aggregated favorable genes to some extent, and it also led to a homogenization of the genetic backgrounds among different breeding populations, thereby diminishing the potential for generating strong heterosis through subsequent hybridization. Consequently, breaking through the limitations of traditional methods by introducing efficient modern biotechnologies has become an inevitable choice for contemporary alfalfa breeding. In recent years, modern biotechnology centered on genomics has presented unprecedented opportunities to overcome the challenges in alfalfa breeding. China has achieved notable success in foundational genomics research, completing tasks ranging from genetic analysis of germplasm resources and genetic mapping to the key molecular markers and the development of elite germplasm. These genomic resources have made it possible to dissect the genetic regulatory networks of complex traits, such as yield, quality, and stress resistance at the molecular level. Looking ahead, alfalfa breeding in China should adopt a strategy that integrates conventional breeding with modern biotechnology, transitioning from source innovation to the "Breeding 4.0" era of precision design. The integration of modern biotechnologies—including genomics, marker-assisted selection, and gene editing—to conduct precise and efficient molecular design breeding is the core pathway to resolving the "bottleneck" in China's alfalfa seed industry and achieving fast development.
【Background】Soil salinization is one of the major ecological challenges threatening global agricultural production, severely restricting crop growth, yield formation, and quality improvement. Alfalfa (Medicago sativa), as an important perennial legume forage, is particularly constrained by salt-alkali stress, while the epigenetic regulatory mechanisms underlying its response remain largely unknown. DNA methylation, as a key epigenetic modification, plays an essential role in plant adaptation to abiotic stresses. 【Objective】This study aimed to systematically identify DNA methylation-related gene families in alfalfa, characterize their expression patterns under salt-alkali stress, and further explore the role of DNA methylation in salt-alkali tolerance by applying the DNA methylation inhibitor 5-azacytidine (5-AzaC), thereby providing the theoretical insights for the genetic improvement of salt-alkali-tolerant alfalfa. 【Method】Based on the reference genome of alfalfa, DNA methyltransferase and demethylase genes were identified genome-wide, and their functions were inferred through phylogenetic analysis and conserved domain annotation. RT-qPCR was employed to analyze the expression patterns of these genes under salt-alkali stress. Using the cultivar Gannong No. 3 as plant material, a hydroponic salt-alkali stress system was established. Different concentrations of 5-AzaC were applied as pretreatments, and the optimal concentration was selected for subsequent assays. Plant growth, physiological, and photosynthetic parameters were then measured to evaluate the regulatory role of 5-AzaC in alfalfa salt-alkali tolerance. 【Result】A total of 13 DNA methyltransferase genes and 4 DNA demethylase genes were identified in alfalfa, all of which were localized to the nucleus with complete conserved domains. Expression analysis revealed that MsCMT4, MsCMT6, MsCMT8, and MsDML2 were significantly upregulated under salt-alkali stress, indicating the involvement of both methylation and demethylation processes in stress responses. Physiological analyses showed that 100 μmol·L-1 5-AzaC significantly alleviated growth inhibition caused by salt-alkali stress, so plant height, fresh weight, and dry weight increased by 12.62%, 23.50%, and 18.67%, respectively, compared with the control. Regarding chlorophyll metabolism, 5-AzaC suppressed the expression of chlorophyll degradation-related genes (PAO, CAO, NYC), thereby delaying pigment degradation. Photosynthetic analysis indicated that 5-AzaC treatment markedly increased net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), as well as the quantum efficiency of photosystem II (YII) and photochemical quenching (qP), suggesting enhanced stability and efficiency of the photosynthetic system. In terms of osmotic adjustment, 5-AzaC promoted soluble sugar accumulation (+37.21%) but had no significant effect on soluble protein. Reactive oxygen species (ROS) measurements showed that 5-AzaC reduced H2O2 and O2-·levels by 22.8% and 35.8%, respectively, while superoxide dismutase (SOD) and catalase (CAT) activities increased by 13.58% and 21.82%, respectively, indicating that 5-AzaC enhanced antioxidant capacity and alleviated oxidative damage. 【Conclusion】This study systematically characterized DNA methylation-related gene families in alfalfa and their responses to salt-alkali stress, revealing the pivotal role of DNA methylation in shaping salt-alkali tolerance. Exogenous application of 5-AzaC improved alfalfa tolerance by maintaining photosystem stability, enhancing photosynthetic efficiency, promoting osmolyte accumulation, and strengthening ROS scavenging capacity. These findings provided the new experimental evidence for understanding the epigenetic mechanisms of forage adaptation to abiotic stress and offer theoretical guidance for the improvement and utilization of salt-alkali-tolerant alfalfa germplasm.
【Objective】Salt stress can severely damage plant cells, inhibit plant growth and development, and consequently lead to a substantial reduction in yield. Kunitz trypsin inhibitor (KTI), a representative type of serine protease inhibitors, is primarily involved in regulating physiological processes in plants, such as growth and development, pest and disease resistance, and responses to abiotic stress. Exploring and analyzing the molecular mechanisms underlying the regulation of salt stress by the alfalfa KTI would contribute to the provision of novel candidate genes for molecular breeding of salt-tolerant alfalfa. 【Method】In this study, a salt stress induced KTI was cloned from Medicago sativa ‘Zhongmu No. 4’, which was named MsKTI3. Through bioinformatics methods, the structural characteristics of the MsKTI3 and its encoded protein were deeply analyzed. Sequence alignment and evolutionary analysis were carried out with homologous genes of other species. The real-time fluorescence quantitative PCR (RT-qPCR) method was used to analyze the expression patterns of the MsKTI3 in different tissues and under different stress conditions. With the aid of the tobacco transient expression system, the subcellular localization of the MsKTI3 protein was analyzed. An MsKTI3 overexpression vector was constructed, and the MsKTI3 overexpressing lines of Arabidopsis thaliana were successfully obtained by the Agrobacterium tumefaciens-mediated method. Meanwhile, the Medicago sativa plants with MsKTI3 overexpression in roots were obtained by the Agrobacterium rhizogenes-mediated method. Phenotypic analysis and physiological index determination of the related lines were carried out under salt stress conditions. 【Result】Bioinformatics analysis indicated that the coding sequence (CDS) of the MsKTI3 gene was 627 bp, encoding 208 amino acids. The relative molecular weight was 23 220.81 Da, and the theoretical isoelectric point was 8.57. Phylogenetic tree analysis revealed that the amino acid sequence of MsKTI3 shared a high homology with that of MtKTI3 of Medicago truncatula, reaching 97%. RT-qPCR was employed to analyze the expression pattern of the MsKTI3. The results demonstrated that the expression level of the MsKTI3 was the highest in roots. Moreover, during the initial stage of NaCl (200 mmol·L-1) and ABA (150 μmol·L-1) stress, the expression level generally exhibited an up-regulation trend. Subcellular localization results showed that the MsKTI3 protein was located in the plasma membrane. Twelve overexpressing Arabidopsis lines were generated via the Agrobacterium-mediated method. Under salt-stress conditions, the germination rate of the Arabidopsis lines overexpressing the MsKTI3 was higher than that of the wild type, and the damage degree of the overexpressing seedlings was lower than that of the wild type. The relative electrolyte leakage (IEL) and malondialdehyde (MDA) content of the overexpressing plants were significantly lower than those of the wild type (P<0.05), while the chlorophyll (Chl) content and catalase (CAT) activity were significantly higher than those of the wild type (P<0.05). MsKTI3 was transferred into the roots of alfalfa using Agrobacterium rhizogenes. Phenotypic analysis indicated that overexpression of MsKTI3 in roots enhanced the salt tolerance of alfalfa. Additionally, the CAT activity of the overexpressing plant roots was higher than that of the control plants. 【Conclusion】The MsKTI3 gene played a positive regulatory role in responding to salt stress, and overexpression of MsKTI3 could improve the salt tolerance of plant.
【Objective】Soil salinization and alkalization are significant limiting factors for the growth and yield of alfalfa (Medicago sativa L.). To elucidate the molecular mechanisms underlying the response of alfalfa seedlings to salt or alkali stress, this study employed proteomic analysis to identify key differentially expressed proteins and their associated metabolic pathways under saline-alkali stress, so as to provide a theoretical foundation for elucidating the mechanisms of saline-alkali tolerance. 【Method】This study used Zhongmu No. 4 alfalfa as the experimental material. Seeds were subjected to salt stress (30 mmol·L-1 NaCl and 30 mmol·L-1 Na2SO4) and alkali stress (10 mmol·L-1 Na2CO3 and 10 mmol·L-1 NaHCO3). TMT labeling combined with liquid chromatography- mass spectrometry (LC-MS/MS) was employed to analyze the proteomic changes in alfalfa seedlings after stress treatments. 【Result】In total, 6 829 proteins were identified, including 489 differentially expressed proteins (DEPs) (274 up-regulated and 215 down-regulated) under alkali stress and 376 DEPs (218 up-regulated and 158 down-regulated) under salt stress. GO annotation analysis revealed that the DEPs were primarily associated with biological processes, such as cellular metabolism, organic metabolism, and stress response, as well as cellular components including intracellular organelles, cytoplasm, and membranes. KEGG pathway enrichment analysis indicated that under alkali stress, DEPs were significantly enriched in pathways, such as photosynthesis, phenylpropanoid biosynthesis, and isoflavonoid biosynthesis, while under salt stress, they were mainly enriched in photosynthesis, isoflavonoid biosynthesis, and glutathione metabolism. Further analysis of the enriched proteins demonstrated that alfalfa seedlings enhanced their resistance to salt-alkali stress by up-regulating key proteins in the isoflavonoid biosynthesis pathway (HI4OMT and CYP81E). The upregulation of key enzymes such as HI4OMT and CYP81E significantly enhanced the accumulation of isoflavonoids, which facilitated reactive oxygen species scavenging and maintained cellular homeostasis in plants. Furthermore, under alkaline stress, key enzymes (PAL, CAD, and COMT) in the phenylpropanoid biosynthesis pathway were markedly upregulated, promoting the synthesis of lignin and flavonoids. This process strengthened cell wall integrity and antioxidant capacity, enabling plants to adapt to high-pH environments and cope with alkaline stress. Under salt stress, alfalfa seedlings upregulated critical enzymes (PRDX6, GPX, and GST) in the glutathione metabolism pathway, maintaining redox homeostasis and enhancing ROS scavenging, thereby improving salt stress tolerance. 【Conclusion】In summary, this proteomic study elucidated key proteins and metabolic pathways involved in the response of alfalfa seedlings to salt-alkali stress, providing a theoretical foundation for understanding the molecular mechanisms of salt-alkali tolerance in alfalfa. Simultaneously, this study provided potential candidate proteins and metabolic pathways for breeding salt-alkali tolerant alfalfa. Subsequent research could further validate the functions of these key proteins and employ advanced biotechnological breeding approaches to develop new alfalfa varieties with enhanced salt-alkali tolerance, thereby addressing the challenges posed by soil salinization-alkalization to alfalfa production.
【Background】Drought stress is one of the major abiotic factors limiting global agricultural productivity. Systematic elucidation of transcriptional regulatory mechanisms during drought and rehydration processes is crucial for improving crop drought tolerance and advancing molecular breeding strategies. Medicago ruthenica Sojak cv. Zhilixing, a perennial leguminous forage species, exhibits strong ecological adaptability and drought resistance.【Objective】This study aimed to identify key regulatory modules and core functional genes involved in drought and rehydration responses through transcriptome analysis and co-expression network construction, thereby revealing the underlying molecular mechanisms. 【Method】Four treatment stages were established, including normal irrigation (Group A), mid-term drought stress (Group B), late-stage drought stress (Group C), and post-rehydration (Group D), to simulate the transcriptional response of M. ruthenica under progressive drought and recovery conditions. High-throughput RNA sequencing was performed to obtain gene expression profiles, followed by weighted gene co-expression network analysis (WGCNA) to construct expression modules. Principal component analysis (PCA) and KEGG pathway enrichment were conducted to assess expression variation and functional clustering. Modules and hub genes closely associated with drought tolerance were identified, and six representative genes from the MEmagenta and MEdarkgreen modules were selected for qRT-PCR validation to assess data consistency and biological relevance. 【Result】PCA revealed clear separation of samples across PC1 and PC2 dimensions, indicating stage-specific effects of drought and rehydration treatments on gene expression patterns. Differential expression analysis showed the greatest number of up- and down-regulated genes during mid-stage drought (Group B), suggesting rapid activation of stress- responsive mechanisms. In the late-stage drought (Group C), fewer differentially expressed genes were observed, while pathways related to fatty acid degradation and carbohydrate metabolism were significantly enriched, suggesting a shift toward homeostatic regulation. During rehydration (Group D), most gene expression levels gradually recovered, although some signaling and defense pathways remained active, indicating ongoing adaptive modulation.WGCNA identified four modules significantly correlated with specific treatments (|r| > 0.6). The MEdarkgreen module (r = 0.93), highly expressed in Group C, was enriched in MAPK signaling, endoplasmic reticulum (ER) stress response, lipid metabolism, and flavonoid biosynthesis. Hub genes BZIP17 and IRE1B were implicated in ER stress signaling, protein folding regulation, and transcriptional reprogramming, indicating their key roles in prolonged drought adaptation. The MEmagenta module (r = 0.82), highly expressed under normal conditions (Group A), was enriched in ABA and JA signaling pathways, as well as flavonoid metabolism. Its core genes, ABF4 and MYC2, are involved in stomatal regulation and secondary metabolic control, functioning as crucial regulators during the early drought response. Additional genes such as NAC072 and CLPD contribute to chloroplast protein stability and reactive oxygen species scavenging, supporting their multifaceted roles in stress mitigation. KEGG enrichment results were highly consistent with module functions, confirming the reliability of functional annotations. qRT-PCR analysis showed that all six selected genes were significantly upregulated during mid and late drought stages (Groups B and C) and downregulated upon rehydration (Group D), mirroring RNA-seq expression patterns and validating the biological relevance of the identified modules and genes. 【Conclusion】This study reveals dynamic transcriptional regulation patterns in M. ruthenica under drought stress and rehydration. Two key modules, MEmagenta and MEdarkgreen, were identified as strongly associated with drought adaptation, encompassing hub genes such as ABF4, MYC2, BZIP17, and IRE1B. These genes play central roles in signal transduction, metabolic adjustment, and stress response, representing core components of the molecular mechanisms underlying drought adaptation in M. ruthenica. The findings provide a theoretical foundation and candidate targets for elucidating drought-responsive pathways and advancing molecular breeding of drought-resilient forage crops.
【Objective】This study aimed to explore the function of MsPT5, a phosphate transporter in alfalfa, so as to provide the theoretical basis and genetic resources for analyzing the response of alfalfa to low phosphate stress and cultivating phosphate nutrition efficient alfalfa. 【Method】Alfalfa was subjected to both full phosphate and low phosphate stress treatments, and root systems were selected for transcriptome sequencing. Weighted gene correlation network analysis was used to screen for the key gene MsPT5 in response to low phosphate stress in alfalfa; the experiment was carried out to clone the CDS sequence of MsPT5 gene, and the transmembrane domain of MsPT5 protein was analyzed using DNAMAN and TMHMM online websites; Super1300: GFP plant expression vector was used as the backbone, and the overexpression vector Super1300:MsPT5-GFP was constructed through enzyme digestion and homologous recombination; the recombinant vector Super1300:MsPT5-GFP was transformed into Agrobacterium GV3101 using the freeze-thaw method, and Agrobacterium was injected into tobacco for subcellular localization observation; Super1300:MsPT5-GFP was transferred into Arabidopsis through Agrobacterium mediated inflorescence infection, and homozygous strains were screened for inorganic phosphate content determination, biomass statistics, and arsenate phenotype detection; the tissue culture method was employed to obtain MsPT5 transgenic alfalfa, and the positive plants were screened for biomass statistics, phosphate content, and protein content determination. 【Result】MsPT5 was a member of the PHT1 family, with 12 transmembrane domains located on the cytoplasmic membrane; the physiological indicators of Arabidopsis thaliana showed that the inorganic phosphate content of MsPT5 overexpressing strains was 23.02 and 22.30 nmol·mg-1, respectively, while the inorganic phosphate content of the wild-type control was 19.97 nmol·mg-1. The biomass of MsPT5 overexpressing strains were 0.047 g/plant and 0.054 g/plant, respectively, while the biomass of the wild-type control was 0.026 g/plant. Overexpression of MsPT5 could increase the inorganic phosphate content and biomass of Arabidopsis; the results of arsenate phenotype detection showed that the MsPT5 overexpression material exhibited a significant arsenic toxicity phenotype; the physiological indicators of alfalfa showed that the biomass of MsPT5 transgenic alfalfa was 19.39 g/plant, 18.62 g/plant, and 16.65 g/plant, respectively, while the biomass of the wild-type control was 15.14 g/plant. The phosphate content of MsPT5 transgenic alfalfa was 0.37%, 0.39%, and 0.38%, respectively, while the wild-type control had a phosphate content of 0.30%. The protein content of MsPT5 transgenic alfalfa was 21.37%, 21.54%, and 19.91%, respectively, while the protein content of the wild-type control was 18.04%. The biomass of MsPT5 transgenic alfalfa was significantly increased, while the phosphate and protein content were significantly higher than the control. 【Conclusion】 MsPT5 was an important gene in alfalfa's response to low phosphate stress. Overexpression of MsPT5 could increase plant phosphate absorption capacity and phosphate content, and improve alfalfa yield and quality. Therefore, MsPT5 was important for cultivating high-yield and high-quality alfalfa.
