The co-occurrence of keywords in literature offers a gateway to understanding the intrinsic connections, research hotspots, and predictive development trends in an academic field. Table 2 shows the top 20 most frequently occurring keywords in the field of alfalfa drought tolerance research. Notably, “drought stress” and “medicago-sativa” are predominant, followed by “plant-growth” and “yield”. This pattern indicates the significant impact of drought on both the growth and yield of alfalfa, highlighting the importance of understanding the drought-plant growth-yield relationship as a foundational research area in this field. The presence of “gene-expression” as a key keyword reflects the focus on genetic regulation as a means for alfalfa to combat drought stress. The inclusion of “arabidopsis” suggests its use as a model organism for gene function, genetics research, and related areas, pointing towards transgenic research in alfalfa as a current research hotspot. Based on these keywords, the current research in alfalfa drought tolerance encompasses (1) Enhancing plant growth and crop yield, and (2) Transgenic research, physiological and biochemical response mechanisms, and nutrient management as key research hotspots.

Keyword clustering analysis elucidates the relationships between keywords, revealing research hotspots. The visualization of this analysis in Figure 6 identifies 12 core clusters in alfalfa drought resistance research: (1) Red cluster focuses on alfalfa yield, plant physiological responses to drought, and water resource management. (2) Green cluster delves into alfalfa growth and forage quality studies. (3) Blue cluster explores alfalfa seedling drought resistance using polyethylene glycol, weed control, and regeneration management techniques. (4) Yellow cluster investigates stress signal transduction related protein genes involving Ca²⁺ as a second messenger molecule. (5) Purple cluster covers inter-root microbial research. (6) Light blue cluster relates to alfalfa in animal husbandry production and irrigation management. (7) Orange cluster deals with symbiotic nitrogen fixation, crossbreeding, and related transcription factors. (8) Brown cluster examines antioxidant defense mechanisms during water stress. (9) Aqua red cluster focuses on genetic diversity analysis in agronomic traits. (10) Light brown cluster investigates osmotic regulation and related gene regulation during water deprivation. (11) Light green cluster examines drought-resistant morphological characteristics and environmental interactions. (12) Gray cluster studies photosynthesis and leaf morphology adaptations to drought stress.

A comprehensive analysis of the results in Table 2 and Figure 6 underscores two main research aspects: alfalfa’s response mechanisms to drought and drought-resistant technology research. These findings align with the conclusions derived from the analysis of the top 30 most influential papers, indicating a consistent focus within the field.

Figure 7 elucidates the categorization of research themes in the context of alfalfa drought resistance. The lower right quadrant, termed ‘Basic Themes,’ encompasses physiological and biochemical responses. These themes are pivotal to the field yet underdeveloped, signaling a need for further investigation. Contrarily, the upper right quadrant, ‘Motor Themes,’ comprises well-established and significant keywords, corroborating the findings presented in Table 2 . ‘Niche Themes’ occupy the upper left quadrant, featuring keywords such as “drought stress,” “gene-expression,” “arabidopsis,” “abscisic-acid,” and “oxidative stress.” Although their significance and developmental extent are marginally lower than those in the ‘Motor Themes,’ their importance remains substantial. The lower left quadrant represents ‘Emerging or Declining Themes,’ with keywords like “down-regulation” and “transgenic alfalfa.” The inclusion of Figure 8 in the analysis aids in further delineating these research frontiers.

The ‘Trend Topics’ section facilitates a visual analysis and projection of research focal points. A notable concentration of research activity, represented by the largest nodes, occurred between 2017 and 2019, particularly in areas such as “drought stress” and “gene-expression.” This trend highlights the significant attention devoted to these topics. Furthermore, the emergence of keywords like “regulator,” “bacteria,” and “gene-expression” up until 2022 suggests their potential as persistent research interests. Likewise, the appearance of terms such as “microbial biomass,” “genome-wide identification,” and “encodes” up to 2023 indicates their likelihood as future research directions. Cross-referencing Figures 7 and 8 reveals that the themes in the lower left quadrant of Figure 7 are gaining traction as emerging research areas. In conclusion, the current research frontiers in alfalfa drought resistance encompass gene expression studies, investigations into related microorganisms, and transgenic alfalfa research.

Under drought conditions, alfalfa undergoes significant morphological adaptations to sustain growth and development, particularly in its leaves and root system. Firstly, leaves, being the primary interface with the external environment, are highly responsive to environmental changes. Drought stress alters leaf morphology in alfalfa, affecting metrics such as leaf area ratio (LAR), specific leaf area (SLA), and leaf weight ratio (LWR). SLA significantly decreases with decreasing moisture content (Gu and Wan, 2021). These changes in SLA or LAR can be instrumental in identifying drought-resistant alfalfa varieties (Erice et al., 2010). Secondly, the root system, a vital component of the soil-plant-atmosphere continuum, exhibits a positive correlation with drought tolerance. It plays a crucial role in adapting to drought-stressed environments. Under drought stress, as soil moisture diminishes, there is an increase in both the volume and length of alfalfa’s root system, and a concurrent decrease in root diameter. This results in an expanded root-soil contact area, enhancing the plant’s capacity to absorb and utilize water from deeper soil layers (Zhu et al., 2008). Recent studies have demonstrated that larger leaves and robust roots during drought contribute to the accumulation of higher biomass yield (Prince et al., 2022). Moreover, the root-crown ratio in alfalfa is particularly sensitive to drought stress. An increase in this ratio under water stress conditions is indicative of a reduction in water consumption and an enhancement in water uptake (Erice et al., 2010), serving as a significant morphological index for assessing alfalfa’s drought resistance. The root-crown ratio stress index further reflects the drought resistance performance of alfalfa (Ma et al., 1993; Han et al., 2006).

In response to water deficits, alfalfa employs a range of physiological and biochemical strategies to mitigate drought stress. These responses can be categorized into five primary aspects.

In recent years, owing to the extensive application of molecular biology technology (Huang et al., 2022; Zhao et al., 2022a; Zhao et al., 2022b), research on alfalfa drought resistance has progressed to the molecular level, such as identification of relevant stress genes, transcriptomics, metabolomics, and proteomics (Zhang et al., 2014; Li et al., 2021). The primary focus of this molecular investigation is the study of functional genes (proteins) associated with drought resistance in alfalfa. Transcription factors have emerged as key players in the response of alfalfa to drought stress (Zhao et al., 2022c). MicroRNA156 (miR156), for instance, regulates the Squamosa promoter binding protein (SPL) gene family, which acts as transcription factors that subsequently modulate the expression of downstream genes, thus regulating various plant growth and developmental networks (Cardon et al., 1999). Arshad and colleagues (Arshad et al., 2017) conducted experiments to explore the role of miR156d in alfalfa’s response to drought stress. Their findings revealed that, in comparison to the wild-type control (WT), alfalfa genotypes overexpressing miR156 (referred to as miR1560E) exhibited significantly higher drought tolerance. The miR1560E genotype not only displayed increased survival rates and reduced water loss but also maintained higher stomatal conductance, enhanced accumulation of compatible solutes (e.g., proline), and elevated levels of abscisic acid (ABA) and antioxidants during drought stress, when compared to the WT. Similarly, alfalfa plants with reduced expression of miR156-targeted SPL13 demonstrated decreased water loss, enhanced stomatal conductance, elevated chlorophyll content, and improved photosynthetic assimilation. These results suggest that miR156 enhances drought tolerance in alfalfa, partially by suppressing the expression of SPL13. Expression analysis of transcription factor genes related to the endoplasmic reticulum stress signaling pathway in alfalfa confirmed that DTT treatment reduced the functionality of bZIP60 and bZIP28 genes involved in the ER stress pathway. The most critical time for plants to tolerate drought (PEG) is the 8th hour after treatment, during which bZIP60 plays a more active role than bZIP28 in the stress pathway; the leaf tissues were more affected than the root tissues (Oğuz et al., 2022).

Transcriptomics reflect the transcriptional expression and regulation of genes in different genotypes across specific cells, tissues, or organisms under various adversities and developmental stages, thereby identifying candidate genes and revealing interactions among different gene regulatory pathways (Mutz et al., 2013; Palovaara et al., 2013). Feyissa et al. further demonstrated that the miR156/SPL13 module alleviates drought stress in alfalfa through tissue-dependent regulatory molecules and physiological processes (Feyissa et al., 2020). Feyissa et al. further demonstrated that the miR156/SPL13 module alleviates drought stress in alfalfa through tissue-dependent regulatory molecules and physiological processes (Wan et al., 2021). Feyissa et al. further demonstrated that the miR156/SPL13 module alleviates drought stress in alfalfa through tissue-dependent regulatory molecules and physiological processes (Fang et al., 2023). Moreover, the sensitivity of alfalfa seeds to ABA is heritable, and screening for ABA during seed germination can help select alfalfa lines with better drought tolerance (Xu et al., 2024).

Metabolomics studies aim to identify specific traits associated with stress tolerance by examining the molecular phenotypes of plants under abiotic stress. Feyissa and colleagues (Feyissa et al., 2019) further elucidated the regulation of drought stress in alfalfa by coordinating gene expression with metabolites and physiological strategies involving miR156/SPL13 and WD40–1/DFR. Modest levels of miR156 overexpression led to the suppression of SPL13 and an increase in WD40–1, thereby fine-tuning DFR expression to enhance anthocyanin biosynthesis. This, in conjunction with the accumulation of other stress relief metabolites and physiological responses, contributed to improved drought tolerance. Dominant classes of differential metabolites include amino acids, organic acids, sugars, and alkaloids, such as 6-gingerol, salicylic acid (SA), indole-3-acetic acid (IAA), gibberellin A4 (GA4), abscisic acid (ABA), trans cinnamic acid, sucrose, L-phenylalanine, L-tyrosine, succinic acid, and nicotinic acid, essential for drought stress tolerance in alfalfa (Wang et al., 2024).

Proteomics studies have increasingly reported on the response of alfalfa to drought stress. A total of drought stress-responsive proteins that play a role in a variety of cellular functions in the alfalfa root system were identified in the study by Rahman and colleagues (Rahman et al., 2016). These functions include energy metabolism, signaling pathways, antioxidant mechanisms, stress defense mechanisms, transcriptional and translational processes, regulation of reactive oxygen species (ROS), abscisic acid (ABA) biosynthesis, calcium signaling and storage processes. Ma et al. investigated the proteomic changes during the germination stage of Zhongmu NO.3 seeds under 200 mol·L^-1 NaCl and 180 g·L^-1 PEG stress, identifying 17 differentially abundant proteins (DAPs) mainly involved in defense responses, energy metabolism, protein synthesis and degradation, oxidative stress, and carbohydrate metabolism. Osmotic stimulation, used as a pretreatment, accelerates germination and improves the uniformity of seedling growth (Ma et al., 2017).

Currently, conventional techniques continue to dominate the breeding of alfalfa varieties, with methods such as selective breeding and crossbreeding being the prevalent approaches. Simultaneously, molecular techniques have been progressively integrated into alfalfa breeding strategies. Selection breeding comprises various methods, including single selection, mixed selection, modified mixed selection, group selection, and rotational selection. For instance, Li et al (Li et al., 2011)successfully bred the drought- and cold-resistant Longmu 808 alfalfa by employing single-plant selection within the original population of Longmu 803 alfalfa, a process involving the selection of the best specimens while eliminating the least suitable.

Hybrid breeding, on the other hand, involves crossing individuals from different populations and genotypes to generate pure varieties through selection among their hybrid progeny. Notable Chinese registered varieties, such as Caoyuan No.1, Caoyuan No.2, and Gannong No.1, were bred through crosses between the tetraploid subsp. sativa and the diploid subsp. falcata (Shi et al., 2017). However, achieving a stable and mature variety through hybrid breeding requires multiple generations of backcrossing and faces challenges similar to standard crossbreeding.

An alternative approach is breeding based on male sterile lines, which offers a comprehensive method to produce genuine hybrids but is hampered by the difficulty of generating and maintaining stable male sterile lines. The CAU group employed a reverse genetic strategy, leveraging genome editing tools, to create genetically male sterile alfalfa and corresponding maintenance lines (Ye et al., 2022). Following ten generations of crossings between male sterile lines and maintenance lines, transgene-free male sterile lines were obtained, which can serve as parent materials for hybrid seed production.

Overcoming the challenge of cultivating purebred parents in alfalfa breeding is complicated due to its partial self-incompatibility and inbreeding suppression, whose molecular basis remains unclear. Molecular breeding, a contemporary approach, facilitates the rapid, stable, and directed creation of new varieties or species at the molecular level (Cai and Wang, 2003). As alfalfa genomics continues to advance, molecular marker technology can pinpoint drought-resistant genes or genes closely associated with drought resistance in alfalfa. This enables the swift identification of drought-tolerant varieties, shortening the breeding cycle and, in theory, reducing costs (Charles and Yu, 2018). However, its practical implementation hinges on the accuracy of marker data and thorough validation, particularly considering alfalfa’s tetraploid nature and complex genetic characteristics.

In tandem with the progress in genetic engineering, transgenic technology has emerged as a novel avenue for enhancing alfalfa’s drought resistance and improving variety traits. Zhang and colleagues (Zhang et al., 2005) reported the successful activation of alfalfa’s waxy production and the conferment of drought tolerance through the introduction of the WXP1 gene from the model legume Tribulus terrestris. This resulted in a notable increase in leaf wax accumulation and enhanced drought tolerance in transgenic alfalfa.

Additionally, Nie et al. (Nie et al., 2017) observed increased proline accumulation in alfalfa through the overexpression of the AmDHN gene, a change that potentially induces the expression of genes related to the proline synthesis pathway, ultimately bolstering drought tolerance. Fehér-Juhász and colleagues (Fehér-Juhász et al., 2013) demonstrated that ectopic expression of the GsWRKY20 gene led to increased osmoregulatory substance accumulation, improved water-holding capacity, decreased membrane permeability, reduced MDA content, and enhanced drought tolerance in transgenic alfalfa. Mckersie and colleagues (McKersie et al., 2000) transplanted the Mn-SOD gene from blue snowy tobacco into alfalfa using Agrobacterium, with field experiments confirming the significant enhancement of cold and drought resistance in transgenic alfalfa plants.

These advancements in breeding and biotechnological approaches hold promise for mitigating the impact of drought on alfalfa production, offering potential solutions for sustaining alfalfa cultivation in the face of global climate challenges.

Cultivation techniques aimed at enhancing water stress resistance in alfalfa encompass several strategies. The first strategy involves water conservation and drought resistance, which includes techniques such as seed triggering (Li et al., 2011; Antoniou et al., 2020), the utilization of irrigation systems (Annicchiarico et al., 2012; Ismail and Almarshadi, 2013; Huang et al., 2018), and adjustments in the timing of product harvest (Liu et al., 2018). Furthermore, the traditional application of organic fertilizers and other materials like sludge serves to protect alfalfa nodules from oxidative stress (Antolín et al., 2010). The application of plant growth-regulating substances, such as melatonin (Cardon et al., 1999), and the utilization of beneficial bacterial strains, including cytokinin-engineered bacteria (Xu et al., 2012), have been shown to improve drought resistance in alfalfa.

Cultivation of alfalfa is not limited to field management, and the establishment of a suitable alfalfa standardized growth and development patterns and designing ideal cultivation practices for different ecological zones is imperative. Alfalfa can be cultivated either as a pure stand or in rotation or intercropping systems, and a rational model necessitates a balanced relationship with companion crops. This presents both advantages and challenges (Buddhaboon et al., 2011; Grieder et al., 2021; Peng et al., 2022; Xu et al., 2022; Wang et al., 2023).

To enhance drought resistance, the cultivation process incorporates selecting appropriate alfalfa varieties and applying effective nutrient management, weed control, and pest management. These elements are pivotal in achieving optimal harvests and are crucial for sustaining alfalfa production under drought conditions (Putnam, 2021; Undersander, 2021; Graham et al., 2022; Wang et al., 2022; Yin et al., 2022). Additionally, mechanization of the entire production process is essential for alfalfa cultivation (Putnam, 2021). This requires alfalfa plants with an upright growth habit and robust resistance to lodging.