Root Phenotyping: A Contribution to Understanding Drought Stress Resilience in Grain Legumes

Abstract

Global climate change predictions point to an increase in the frequency of droughts and floods, which are a huge challenge to food production. During crop evolution, different mechanisms for drought resilience have emerged, and studies suggest that roots can be an important key in understanding these mechanisms. However, knowledge is still scarce, being fundamental to its exploitation. Plant-based protein, especially grain legume crops, will be crucial in meeting the demand for affordable and healthy food due to their high protein content. In addition, grain legumes have the unique ability for biological nitrogen fixation (BNF) through symbiosis with bacteria, which contributes to sustainable agriculture. The exploitation of root phenotyping techniques in grain legumes is an important step toward understanding their drought resilience mechanisms and selecting more resilient genotypes. Different methodologies are available for root phenotyping, including the paper pouch approach, rhizotrons and the semi-hydroponic system. Additionally, different imaging techniques have been employed to assess root traits. This review provides an overview of the root system architecture (RSA) of grain legumes, its role in drought stress resilience and the phenotyping approaches useful for the identification of accessions resilient to water stress. Consequently, this knowledge will be important in mitigating the effects of climate change and improving grain legume production.

1. Introduction

Legumes, belonging to the Fabaceae family, are among the most widely cultivated crops worldwide. They can be divided into forage legumes and grain legumes (pulses), serving as important sources of food, fodder, oil and fiber [1,2]. Grain legumes, considered poor man’s meat, have an important place in human nutrition, especially in the dietary patterns of low-income people from developing countries [3]. They are mainly appreciated for their high protein content and as a source of slow-release carbohydrates [3,4]. They also contain several beneficial compounds to health, such as folate, lignans, saponins, antioxidants, dietary fiber and resistant starch, offering potential protection against some cancers, diabetes and obesity [5]. In addition, one of the most important attributes of grain legumes is their unique capacity for biological nitrogen fixation (BNF) through bacterial symbiosis, playing an important role in natural and agricultural ecosystems [4,6]. These attributes are useful in sustainable farming through crop rotations that allow increasing soil fertility and reduce requirements for commercial nitrogen fertilizers, and consequently, they are a valuable strategy to mitigate climate change [7]. The use of grain legumes to increase crop productivity is a promising strategy for addressing low-yielding crops and improving sustainable agriculture [8].

Nowadays, the scientific community faces huge challenges related to climate change and population growth, and agricultural productivity is fundamental to reducing agriculture’s environmental impact. Agriculture, especially crop production and food security, is considered one of the most affected sectors by climate change. Its impact on agricultural production is mainly attributed to the increase in air temperature (including during the night period) and drought periods [9,10].

Drought is one of the most important abiotic stresses that negatively affect plant production [11]. This stress essentially affects the physiological and biochemical processes of crops, causing growth reduction and a decrease in chlorophyll contents [11,12]. Plant responses to drought depend on numerous factors, including their growth stage and genetic potential, as well as the duration and severity of stress, but in all cases, the final yield is significantly decreased. Understanding plants’ natural drought tolerance mechanisms is a key step for the selection of cultivars and breeding programs [13]; however, the available information is still limited, and most studies are focused on the aerial part of plants [12].

Most of the water content is found inside cells, while the remaining is primarily located in the cell walls. A water deficit occurs when the rate of water loss through transpiration from the leaf surface exceeds the rate of water absorption by the roots. Root system architecture (RSA) is an underexplored trait probably due to the dense nature of soils, which makes phenotyping roots in situ more challenging than that of the aerial part of the plant [14]. RSA has been reported as an important trait to better understand crops’ ecophysiology and abiotic stress tolerance.

This review of the State of the Art intends to provide a comprehensive overview of the current advancements in grain legume roots research and their role in drought stress resilience. At the same time, different approaches for root phenotyping are presented for the identification of grain legume drought-resilient genotypes that, in the future, can be useful to integrate into breeding programs.

2. Drought Stress Responses in Grain Legumes

Drought stress is a huge threat and is the most unpredictable constraint, often occurring alongside other stresses, such as heat, nutrient deficiencies and salinity [12]. This stress is considered one of the most prevalent environmental factors, leading to a significant amount of agricultural crop losses [15]. Drought stress can be related to a huge variety of factors, namely, irregular and limited rainfall, high evapotranspiration, soil water retention and soil field capacity. This abiotic stress significantly negatively influences crop production and yield [16]. Drought induces devasting effects on plants, essentially affecting their morphology and physiological, biochemical and molecular processes. This results in growth reduction, decreased chlorophyll contents, reduced ascorbic acid and increased proline accumulation and hydrogen peroxide production [11,12] (Figure 1).

Plants exhibit various self-protection responses against drought, which can be classified into three types: drought avoidance, drought escape and drought tolerance. In brief, drought avoidance involves morphological and physiological adaptations (such as reducing the number of stomata, increasing root growth and rolling leaves); drought escape refers to the use of strategies like early flowering, increased nitrogen levels, and rapid growth; and drought tolerance is related to the accumulation of osmolytes (e.g., proline and glycine), as well as the production of antimicrobial compounds [15,18]. These responses depend on numerous factors, including the growth stage of the plant and its genetic potential and the duration and severity of stress (Figure 1). However, in all cases, the final yield is significantly reduced [12].

Plants’ first response to drought stress is to halt growth. The reduction in water flow caused by hydric stress also limits the transport of nitrogen fixation products and suppresses nitrogenase activity. To survive water stress, plants reduce transpiration and mobilize metabolites for the synthesis of protective compounds necessary for osmotic adjustment. Grain legumes frequently produce antioxidants and molecules that help to maintain the balance of osmotic potential in cells as a response to drought stress [16].

During the evolution process, plants have developed sophisticated resistance mechanisms to cope with abiotic stresses, including stress perception, epigenetic modification and the regulation of transcription and translation [19]. These mechanisms help to regulate key morphological and physiological traits, such as root and leaf structures and water accumulation [20]. The scientific community recognizes that these processes are controlled by a set of genetically programmed and regulated mechanisms (Figure 1); however, they remain highly complex and not yet fully understood, especially in grain legumes [17].

3. Roots and Their Role in Drought Stress Resilience

Understanding the natural drought resilience mechanisms of plants is a key step for the selection of the most interesting cultivars that will be useful in the development of breeding programs for this stress. However, these efforts have been mainly focused on aboveground traits. Root traits are underexplored, probably due to the dense nature of soils, which makes phenotyping roots in situ more challenging compared to the aboveground part of plants [14]. Nonetheless, roots are crucial for better understanding crops’ ecophysiology and abiotic stress resilience. This organ anchors plants in soil, acquires water and nutrients and promotes interactions with the soil microbiome and other plants [21,22]. The roots are the first plant organs to sense water stress and play a key role in transmitting this signal to other parts of the plant [15]. Changes in water availability and soil moisture directly influence root morphology, anatomy and, consequently, molecular adaptations [23].

3.1. Morphological and Physiological Changes in Roots Under Drought Stress

Adapting root system architecture (RSA) to environmental events can improve advantageous agronomic traits, such as yield, drought resistance and tolerance to nutrient deficiencies [24,25]. RSA refers to the spatial distribution of the root system in a specific soil matrix, allowing the evaluation of multiple traits, namely, root biomass, root length, root diameter, root angle and root volume [26]. Wang et al. [26] assert that RSA depends on the plant species and soil composition, resulting in an interplay between genetic and environmental influences. Legume root systems commonly adhere to the structure of tap roots, consisting of primary roots and lateral roots. Moreover, legumes have the ability to form nitrogen-fixing nodules through symbiosis with rhizobia [26,27].

Generally, and according to differences in spatial morphology, crop root systems can be classified into tap roots and fibrous root systems [28]. Grain legume crops have different root systems, and it is difficult to indicate a specific root system for this group of plants. Predominantly, their root system is characterized by tap root systems, comprising a primary root, lateral roots, root hairs and occasionally adventitious roots [28,29]. The common bean (Phaseolus vulgaris L.) and cowpea (Vigna unguiculata Walp. L.) have epigean germination, which is differentiated by embryonic root systems that include both the primary and basal roots [30]. On the other hand, the root system of chickpea (Cicer arietinum L.) is characterized by a dominant root with primary lateral roots and dense or sparsely dispersed secondary branches. Chickpea exhibits variations in rooting and branching patterns, with some genotypes showing distinctive traits, such as long lateral roots, deeper roots, sparse and short branches and thin roots [31]. Reports indicate that the root system of soybean (Glycine max L.) also differs amongst its varieties [32]. Their main roots can be described as well-developed (tap root), poorly developed or branched (which is longer).

Root systems play a significant role in developing drought-resilient crops because roots are the primary organ to sense the first indications of a decline in water potential and, consequently, drought [15]. In recent years, some efforts have been undertaken to evaluate plant root responses to water deficits, revealing that drought directly affects root growth and development, decreases water and nutrient uptake, limits root system penetrability, and impacts soil microbial populations and activity [21,33].

Plants have developed several strategies as a response to drought stress, including roots that have evolved to be highly responsive and adaptable to their environment (Figure 2).

In response to drought stress, plants develop a more robust root system by increasing root length and biomass density—traits that help crop survival and achieve better yields under drought conditions [24]. Roots are highly responsive and adaptable to their environment, with their morphology, development and physiology having a strong genotype influence [34].

Drought affects soybean roots in various ways depending on the severity of the stress. Some effects are the increase in root volume, length and total surface area during the seedling stage [25]. In medicago (Medicago sativa L.), drought stress reduced stem and root development, potentially due to cell wall roughening. As previously mentioned, drought stress impacts plant growth differently across various plant developmental stages, and medicago is particularly vulnerable during the plantlet, flowering and seedling stages [35].

In some grain legumes, such as common bean, chickpea and cowpea, one of the primary drought tolerance mechanisms is the development of long, deep roots capable of penetrating the lower layers of the soil. This adaptation allows them to survive for long periods of water stress by accessing water reserves available in the deeper layers [36,37,38].

One of the most critical characteristics of the root system is its size, which is essential for acquiring water from deeper soil layers. Adjustments in root and stem allometry are necessary to compensate for water scarcity. To survive drought, plants develop specific root system traits, such as deeper roots with larger diameters to absorb water uptake and penetrate compacted soils. Additionally, they produce a greater number of long lateral roots and root hairs that improve water absorption [34].

Root morphology, including length, diameter and surface area, plays a crucial role in determining a crop’s ability to obtain nutrients and water [24,25]. Root diameter and tissue density influence the overall length and surface area of root systems in relation to the biomass allocated to them. According to Zhang et al. [28] and Ye et al. [32], grain legumes benefit from a deep and proliferative root system due to its positive impact on water and nutrient absorption, which helps their adaptive capacity under drought stress.

The morphology and anatomical characteristics of roots exhibit flexibility under stress conditions. Grain legumes respond by increasing the number of fibrous roots, decreasing the diameter of lateral roots and altering root biomass, alongside developing deeper and more proliferative rooting systems under stress conditions (Figure 2). The number and size of root cells, as well as the size and density of xylem vessels, significantly influence water flow, resulting in variations in the roots’ hydraulic conductivity. This adaptation functions as a defensive mechanism employed by plants during drought conditions [39]. The amount of water absorbed by the roots is also affected by the distribution and availability of water in the soil. Hydraulic conductivity decreases in water-stressed (extremely dry) soils, which affects water redistribution within the soil. Furthermore, the volume of soil from which individual root segments can extract water is reduced under such conditions [40]. Changes in root growth and allometry play an important role in enhancing hydraulic conductivity and improving plant productivity under stress. According to the “balanced growth” hypothesis, some plants respond to drought by increasing root growth while reducing shoot development. This adaptive growth pattern improves plant hydraulic status during moderate to severe drought stress by increasing the root surface area, promoting the development of new root tips and increasing the plant’s overall water uptake capacity [41].

Some root physiological responses include the regulation of phytohormones, the accumulation of osmolytes (such as proline) and the activation of reactive oxygen species (ROS) scavengers. Meanwhile, biochemical responses are mainly linked to enzymes involved in signaling pathways (reviewed in [15,33]). The maintenance of primary root functions relies on a set of specific molecular regulations, which require further investigation.

Adjusting to intermittent stress involves a priming response initiated by prior exposure to a mild stress factor, which enhances resilience to more severe stress conditions. This type of defensive priming is commonly observed in many plants as a reaction to both biotic and abiotic stressors [42].

Plants are influenced physiologically and morphologically by stress; however, some processes, such as nitrogen fixation and cell elongation, are more affected than others, such as photosynthesis, which is relatively less vulnerable. The timing of water stress during the plant life cycle significantly impacts production and yield components, including total biomass, vegetative biomass, number of seeds, seed weight and water content [43]. Drought escape is one of plants’ essential adaptation mechanisms, characterized by rapid growth and development to complete the life cycle before drought conditions appear. Grain legumes, including soybean, common bean, cowpea, lentil, chickpea, and pea, have the ability to shorten their life span to avoid drought by maintaining a high-water potential, improving water absorption and reducing water loss (reviewed by Khatun et al. [44]). To improve water-use efficiency (WUE), grain legumes, such as common bean and cowpea, develop deeper root systems [12,45]. However, if the drought occurs at an earlier stage, plants employ osmolyte synthesis and a high WUE as a more advanced drought tolerance strategy [12].

3.2. Biochemical and Molecular Changes in Roots Under Drought Stress

In response to drought stress, plants synthesize specific plant hormones to regulate drought resistance mechanisms. One key signaling pathway involved in drought response is abscisic acid (ABA) signaling. It is produced in large quantities at the root tips and transported through the xylem to the leaves, where it reduces stomatal conductance, thereby decreasing the rate of transpiration as drought intensifies. Additionally, ABA mediates root elongation, enabling plants to access water in deeper soil layers [44,46]. ABA also increases water absorption and transport, promotes the production of superoxide radicals and H₂O₂ and improves the activity of antioxidant enzymes. The overexpression of ABA-induced genes serves as a crucial signal for drought tolerance. Another essential hormone is jasmonic acid, which, in coordination with other hormones, contributes to drought survival. Jasmonic acid plays an important role in root growth, fruit ripening, tendril coiling and viable pollen production [44,47].

Drought adaptation traits include root length density and penetration; however, water extraction is influenced not only by these traits but also by other factors that change water movement within the roots. Root hydraulic conductivity, which determines the amount of water that roots can absorb from the soil, varies significantly among species, genotypes and root types. Anatomical features and membrane transporters, such as aquaporins, play a crucial role in water transport through xylem vessels during drought stress, directly affecting hydraulic conductivity [39]. Water moves through the xylem via three pathways—the apoplastic, symplastic and transcellular—supported by aquaporins. Under drought stress, the aquaporin and symplastic pathways become more active [39]. Aquaporins facilitate the movement of water across cell membranes, contributing to homeostasis. The genes encoding aquaporin proteins, located in the cell membrane, play an important role in root hydraulic conductivity and other physiological functions [48]. Some members of the aquaporin family have been identified based on their subcellular localization and sequence. Plant aquaporins are classified into five major groups: NIPs (NOD26-type intrinsic proteins), PIPs (plasma membrane intrinsic proteins), SIPs (small basic intrinsic proteins), TIPs (tonoplast intrinsic proteins) and XIPs (uncharacterized intrinsic proteins) [49,50]. When activated by drought stress, these aquaporins help elongate primary roots and reduce membrane damage. For instance, aquaporins like PvXIP1;2 have been shown to play an important role in the drought stress response of common bean varieties, with significant differences in gene expression between drought-resistant and drought-susceptible genotypes [51]. Similarly, Tayade, et al. [50] demonstrated that specific aquaporin genes, such as PIP-type genes (VaPIP1-6, VaPIP1-7, VaPIP2-1 and VaPIP2-2), TIP-type genes (VaTIP3-1 and VaTIP3-2) and XIP-type genes (VaXIP1-1 and VaXIP2-1) play a crucial role in maintaining cellular water and osmotic homeostasis in the adzuki bean (Vigna angularis L.). These findings provide an opportunity to deepen our understanding of drought resilience mechanisms.

Regulatory genes encode proteins involved in stress response regulation, including stress receptors, protein kinases, components of protein degradation machinery, alternative splicing factors and transcription factors (TFs). These proteins play a crucial role in controlling signal transduction pathways and modifying the expression and gene products of multiple stress-related genes [52]. Dehydration-responsive element-binding (DREB) proteins are transcription factors (TFs) from the APETALA2/Ethylene Responsive Element Binding Factor (AP2/ERF) family that use an ABA-independent signal transduction pathway [52,53]. The transcription factor DREB2A is one of the most promising genes involved in dehydration tolerance in crop plants and has already been studied in common bean, chickpea and cowpea [54,55,56]. Also, small heat shock proteins (sHSPs) are a diverse protein family in plants and are primarily induced by heat stress [57]. Several studies have shown that sHSPs are highly expressed during heat stress, as well as in other environmental conditions such as oxidative stress, drought, cold, salt and heavy metals [58,59]. These proteins have an important role in protecting plants from stress-induced damage and facilitating damage repair [59]. Under extremely dry conditions, the Hsp17.7 gene has been shown to be upregulated in drought-tolerant cultivars in cowpea [56]. These genes can be used as indicators of the drought tolerance of grain legumes and other crops.

In recent years, some studies have explored the molecular basis of drought tolerance via segregation mapping and quantitative trait loci (QTL) analysis in major crops, including soybean, common bean, pea, chickpea and cowpea. Several QTLs associated with root system architecture have been identified in the main legume crops (reviewed by Ye et al. [32] and Khatun et al. [44]). This approach is also an important step in screening drought-resilient genotypes, which is essential for the development of multidisciplinary studies.

4. Different Techniques for Root Phenotyping

Plants require a robust root system to counteract abiotic stresses and adapt to environmental changes. However, the study of root development and growth is extremely challenging since root systems are dynamic and involve phenotypic traits that vary in expression across different genotypes [60].

Genetic traits are increasingly being studied as a strategy to improve plant resilience to abiotic stresses. Multiple genes influence RSA by regulating gravitropic and radial growth in response to drought, allowing plants to develop either shallow or deep roots depending on the conditions. In addition to genotyping quantitative traits, phenotyping has emerged as a significant challenge in improving crop tolerance to abiotic stresses [34,61]. Advances in both genotyping and phenotyping are crucial for the development of stress-resilient crop varieties.

Research on root system features has advanced alongside technological progress. Studying root systems remains challenging due to their complex three-dimensional structure and branching within the soil. Some of the methods used for studying RSA are destructive, highlighting the need for innovative technologies to study RSA more efficiently and the development of non-destructive approaches to root phenotyping [26].

Root phenotyping holds equal importance to shoot phenotyping because roots are essential for water and nutrient absorption, and their efficiency is closely related to the architecture and function of the root system [34]. As shown in

Table 1. Different root phenotyping technologies applied to several legume species.

Root Phenotyping Techniques		Software	Species	References
2D approach	Soil	WinRhizo™ Pro 2019 software	Adzuki bean (Vigna angularis L.)	[62]
	Perspex sheets	WinRhizo™ Pro 2019 software	Mung bean (Vigna radiata L.)	[63]
	Paper pouch	SmartShooter software version 3.0	Mung bean (Vigna radiata L.)	[64]
	Agar medium	GrowScreen-Root platform	Pea (Pisum sativum L.)	[65]
		ImageJ software version 1.53	Mung bean (Vigna radiata L.)	[66]
	Semi-hydroponic system	WinRhizo Pro software (version 2009)	Lupin (Lupinus angustifolius L.)	[67]
		WinRhizo Pro software (version 2009)	Soybean (Glycine max L.)	[68]
	Rhizobox	WinRhizo Pro software (version 2009)	Soybean (Glycine max L.)	[69]
	Rhizotron	GrowScreen-Root platform	Faba bean (Vicia faba L.)	[70]
3D approach	Computed X-ray tomography (CTX)	RootForce approach	Common bean (Phaseolus vulgaris L.)	[71]
	Magnetic resonance imaging (MRI)	GrowScreen-Root platform	Common bean (Phaseolus vulgaris L.)	[72,73]
	Positron emission tomography	MeVisLab version 2.8.2	Pea (Pisum sativum L.)	[74]
	Root imaging with a photogrammetric camera	ImageJ version 1.52p	Chickpea (Cicer arietinum L.)	[75]
	Soil	Digital Imaging of Root Traits (DIRT) platform (https://quantitative-plant.org/software/dirt, accessed on 19 March 2025)	Common bean (Phaseolus vulgaris L.) Cowpea (Vigna unguiculata L. Walp.)	[30]

, numerous research projects on several grain legume crops have been conducted over the years using different imaging approaches, technologies and software for root phenotyping.

Traditionally, root measurements have been performed by projecting the root system onto a transparent surface. Consecutive markings are made to track root growth, allowing researchers to calculate root growth rates over specific time periods. This method allows long-term monitoring of root development without causing disturbance, but the root development kinetics obtained using this approach lack precise temporal resolution [76].

In the past, root-length images were manually analyzed using a ruler. Recent studies have explored innovative approaches to studying the growth of roots, including soil-free methods, such as hydroponics, semi-hydroponics and gel-based systems. These improvements facilitate the assessment of root images, and software tools have recently been employed to analyze quantitative traits that characterize root system structure [26,77].

Recent technological improvements have made it easier to visualize the distribution of roots in the soil. Currently, two approaches are available for root phenotyping evaluation: two-dimensional (2D) and three-dimensional (3D) approaches. However, these approaches need to be complemented by additional techniques and technologies to improve data and imaging acquisition [26,61].

Depending on the target root trait, numerous image analysis methods are available [26]. Two-dimensional root phenotyping approaches are commonly used to study root development in soil-based and soil-free environments, including hydroponic, rhizotrons, rhizoboxes, agar gel and paper pouch systems (reviewed by Wang et al. [26]).

Paper pouch systems are frequently employed for crops such as maize (Zea mays L.) and mung bean (Vigna radiata L.), offering a simple and cost-effective method for studying root growth [26,64,78]. In this system, roots grow vertically on germination paper and are covered by a polyethylene sheet to simulate a light-free soil environment. Root images are then captured and quantified using available software, such as RootNav (version 2.0), WinRhizo Pro (version 2009), GiA Roots software, SmartShooter (version 3.0) or GrowScreen-Root platform [26,69,70,77,79].

Another approach involves the use of an agar medium, which is non-destructive and transparent, allowing easy observation of root growth. This approach has been commonly used to study the arabidopsis root morphology [66]. In pea (Pisum sativum L.), this approach has been applied to monitor root phenotypes from seedlings to mature plants [65]. Several advances have refined the agar-based systems, such as plates, polypropylene containers or transparent pots, and GrowScreen-Agar, to make it simpler to identify the quantitative traits of the RSA [26], and this methodology is frequently used for legume roots [26,66].

Rhizotrons or rhizoboxes are non-destructive systems developed to monitor roots on transparent surfaces, allowing researchers to study root growth directly in soil and, at the same time, soil and rhizosphere activities ex situ. It is a non-invasive technology that is frequently utilized as an alternative to field testing [69]. The system consists of a compartment filled with soil for plant growth with a transparent glass panel to observe the roots and has been applied to various species, including soybean [69], fava beans (Vicia faba L.) [70] and chickpeas (Cicer arietinum L.) [31]. These two-dimensional methodologies use complementary software to analyze root images to obtain more automatic growth parameters. Several imaging systems have been developed to capture and analyze root images from rhizotron systems, including EZ-Rhizo, Smart Root, WinRhizo and Root Nav software’s [62]. Another interesting phenotyping platform is GrowScreen-Rhizo, which evaluates the root phenotype of faba bean germplasm [70].

Semi-hydroponic systems, on the other hand, allow plants to grow in nutrient solutions instead of soil. This system allows long-term growth, fast development, cost-effective, and non-destructive measurements with minimal interference in root growth [26]. It has been widely applied to legumes like soybean [26,69], narrow-leafed lupin (Lupinus angustifolius L.) [67] and chickpea [31]. The main advantage of this system is the capacity to stimulate diverse growth conditions. However, the artificial growth conditions may not accurately replicate real soil environments, particularly under water stress. In some cases, it is necessary to simulate drought using substances like polyethylene glycol (PEG) and mannitol to induce osmotic stress in plants and mimic drought conditions [26,80].

The 2D techniques have been successfully used for high-throughput root phenotyping, but some parameters (namely, root arrangement, root volume, surface area occupied by the root and thickness) cannot be determined using these techniques [81]. To overcome this limitation, researchers have implemented and developed 3D phenotyping approaches, which have been widely applied to legume crops [26]. X-ray computed tomography (CTX) has emerged as a non-invasive and non-destructive technology to visualize root systems and internal structures in 2D and 3D within soil or a similar medium. This method relies on the attenuation of electromagnetic waves [82,83]. Similarly, magnetic resonance imaging (MRI) is a versatile technology that provides insights not only into RSA but also anatomical features and water content in the surrounding soil [84].

The Digital Imaging of Root Traits (DIRT) is a high-throughput computing and collaboration platform for field-based root phenomics that allows the researcher to store images of plant roots [85]. Recently, a DIRT/3D system was created and consists of a 3D root scanner and a software package for reconstructing field-grown roots into a 3D point cloud model, allowing automated, time-efficient, and high-quality imaging directly in the field. Consequently, this technology provides rapid and effective data acquisition [86]. These technologies are essential for the development of root phenotyping, but they are costly and time-consuming to install, with variations in the soil profile potentially affecting results [26]. Unfortunately, these 3D approaches are mainly applied to cereals, while their potential remains unexplored in legumes [26].

Crop improvement through phenotyping traits associated with physiological responses to abiotic stress remains a significant challenge. Traditional phenotyping approaches rely on manual measurements, making data acquisition difficult and time-consuming and unsuitable for analyzing large populations. Precise, high-throughput phenotyping approaches have become essential with the advancements in genomic selection, the study of drought stress-related traits and physiological improvements [87].

High-throughput phenotyping methods have emerged as effective tools for assessing plant growth, biomass and nutritional status. These approaches include visible light imaging, hyperspectral imaging and fluorescence imaging [88]. Joint analysis will bring new insights to research and the identification of drought stress genotypes.

5. Conclusions and Future Directions

Climate change presents a significant threat to the sustainability of crop production, with severe impacts on grain legume yields and a subsequent increase in global food insecurity. Grain legumes play an important role in addressing economic and societal challenges due to their high protein content and ability to fix nitrogen, contributing to a healthy diet and agriculture sustainability. Drought stress, a major abiotic stress, negatively affects plant growth at all stages—from seedling development to reproduction and maturity—and has significant effects on physiological, biochemical, metabolic and molecular mechanisms, leading to reduced plant performance. For these reasons, it is fundamental that researchers explore new strategies to maintain crop production under climate change scenarios.

The exploitation of adaptive traits to these challenges is critical for advanced breeding activities, and roots play a pivotal role in helping plants adapt to water stress. Root system architecture (RSA) remains a scantily explored trait, likely due to challenges due to soil density. In situ root phenotyping can provide valuable insights into crop ecophysiology and resilience to abiotic stress. The adoption of novel technologies and strategies for image acquisition and analysis offers a promising approach to generating valuable data for identifying germplasm with advantageous RSA traits targeted to increase production while mitigating climate change. A major challenge in this field is the identification of the best high-throughput root phenotyping methodology to be used in grain legumes. These methodologies will allow the selection of a promising RSA that is more resilient to drought stress.

In conclusion, the root system plays a crucial role in plant development and growth, directly influencing agricultural production. Integrating root phenotyping data with molecular and genetic technologies is essential for selecting grain legume genotypes with root traits adapted to extreme climate conditions. This approach will improve the resilience of grain legume crops, enabling them to maintain growth and yield under drought and other environmental stresses, thereby contributing to sustainable agriculture.