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High-throughput phenotyping of wheat root angle and coleoptile length at different temperatures using 3D-printed equipment

BMC Plant Biology volume 25, Article number: 112 (2025) Cite this article

Abstract

Background

Innovation in crop establishment is crucial for wheat productivity in drought-prone climates. Seedling establishment, the first stage of crop productivity, relies heavily on root and coleoptile system architecture for effective soil water and nutrient acquisition, particularly in regions practicing deep planting. Root phenotyping methods that quickly determine coleoptile lengths are vital for breeding studies. Traditionally, direct selection for root and coleoptile traits has been limited by the lack of suitable phenotyping methods, genetic and phenotypic complexity, and poor repeatability in sampling. In this study, we innovated a method utilizing 3D printing technology to measure root angle and coleoptile length in wheat seedlings. We evaluated seedlings from eight different wheat genotypes across varying temperatures and validated our findings through image processing techniques.

Results

The analysis of variance in root architecture revealed significant differences among genotypes for root angle. Temperature treatments also significantly influenced shoot length, number of roots and total root length. The Tosunbey genotypes exhibited the highest root angle and the lowest root angle was observed in Altindane genotypes. Additionally, we observed that increasing the temperature led to an increase in seedling root length. Similarly, the coleoptile architecture analysis showed significant differences among genotypes in coleoptile length, leaf length, number of roots, and total root length. Temperature treatments and deep sowing applications significantly affected these traits as well. The Tosunbey and Müfitbey genotypes exhibited the longest coleoptile length, whereas the Nevzatbey genotype showed the shortest.

Conclusion

Selecting for a narrow root angle and a high number of seminal roots can result in deeper, more branched root systems. Furthermore, developing wheat genotypes with longer coleoptiles can enhance plant production and early vigor, particularly with deep sowing. Our method, using the eqiupments producing by 3D printing technology enables high-throughput phenotyping of wheat roots and coleoptiles, offering new insights into root and coleoptile system regulation at different temperature conditions. This method can be seamlessly integrated into breeding programs to enhance drought tolerance, rapidly phenotyping populations for root and coleoptile characteristics.

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Introduction

Wheat, a vital global cereal crop primarily grown in temperate zones, is a staple food worldwide [1]. However, wheat faces significant challenges such as climatic factors (precipitation, temperature, drought), land degradation, soil conditions, nitrogen use, and biotic stresses [2,3,4]. Drought, particularly, poses the most severe abiotic stress, threatening global crop production, with potential losses of up to 12% by the century's end in many regions [5].

Roots serve crucial functions beyond anchoring plants in soil [6]. They facilitate nutrient and water uptake, form symbiotic relationships with beneficial microbes, and serve as storage organs. The heterogeneous nature of soils, influenced by environmental factors, poses challenges for studying root systems [7]. This variability in soil conditions necessitates a nuanced understanding of root system architecture (RSA), which defines the morphological and structural organization of roots [8]. RSA plays a pivotal role in plant productivity by enabling efficient access to essential soil resources [9]. In cereals, such as wheat, the radicle emerges first, protected by the coleorhiza, followed by the coleoptile, which rapidly grows to establish the seedling's unique RSA [10, 11]. This early root system development significantly influences seedling establishment and subsequent productivity.

Root architecture traits are critical for crop yield but are often inadequately understood. Assessing root architecture in later plant growth stages is challenging due to the complex, intertwined nature of roots, hindering the differentiation of individual components. Furthermore, studies on roots differ significantly depending on whether they are conducted in isolation or within plant communities, impacting the reliability and applicability of the findings [12]. Various methods have been developed for root phenotyping under controlled and field conditions to study genetic variations beneficial for breeding and agronomy [13, 14]. In controlled environments, such as clear pots, agar, germination paper, rhizotrons, hydroponics, and aeroponics, root phenotyping at seedling stages confines root systems to two-dimensional spaces [15]. In field settings, root phenotyping of unconstrained whole root systems involves harvesting and measuring, often utilizing methods like planted baskets or shovelomics [16, 17]. High-throughput three-dimensional (3D) field methods, such as those by York and Lynch [18], allow rapid assessment of root tip growth and other quantitative traits critical for RSA, advancing breeding efforts. Advanced imaging techniques like X-ray computed tomography (CT), magnetic resonance imaging (MRI), and neutron tomography provide detailed, time-resolved data on root and shoot growth dynamics in natural soils [19,20,21,22]. Despite their capabilities, these methods are generally low throughput due to time and cost constraints, typically assessing fewer plants per study [23]. Recently, Pflugfelder et al. [24] introduced a non-invasive, automated approach using MRI to quantify RSA phenotypes in soil, including neighboring plants, in 4D at plant establishment. These studies underscore the critical role of genotype-specific root characteristics in stress tolerance and yield, highlighting the urgency of rapid and accurate root trait determination for breeding and agronomic advancements. A significant challenge in studying root properties lies in reconciling results between laboratory and field conditions. Variations observed, for instance, between root studies on agar plates versus those in sandy soil, illustrate this complexity [25]. While laboratory root studies are generally less labor-intensive and not constrained by seasons compared to field studies, they offer a controlled and homogeneous environment conducive to more precise and repeatable measurements of genotype root traits [26]. Nevertheless, strong correlations found between laboratory and field data suggest that integrating soil-based phenotyping across field, laboratory, and greenhouse environments can be a strategic and beneficial approach for breeding programs [27]. Recent advances, such as the germination paper technique and transparent pots, facilitate early seedling stage phenotyping shortly after germination [28,29,30,31]. The transparent pot method, particularly noted for its innovative approach, has gained attention. In agricultural research, the ability to efficiently screen numerous genotypes for target traits in broad-scale studies remains crucial. Root phenomics relies on high-throughput pipelines to measure root properties across hundreds of genotypes, demanding methods that are rapid, cost-effective, and practical [32]. Despite advancements, widespread utilization of root genetic information in breeding and selection programs continues to face challenges due to the difficulty in efficiently and accurately phenotyping root traits, particularly in soil [33]. Current wheat root phenotyping studies predominantly focus on the very early seedling stage, employing limited platforms for whole root system measurement, which necessitates substantial labor and space for phenotyping large genotype sets [34].

Good establishment is crucial for rapid leaf area development in wheat crops, as poor establishment results in fewer and later-emerging plants, reduced leaf area, and fewer tillers. Additionally, poorly established crops face increased soil moisture loss through evaporation and heightened competition from weeds, leading to fewer spikes and diminished grain yield. The coleoptile plays a pivotal role in ensuring good establishment by shielding the emerging first leaf. Its length and interaction with soil physical properties determine a cultivar's ability to emerge from deeper soil layers [35]. Coleoptile length (CL) is a critical agronomic trait in cereal crops due to its direct impact on the optimal depth for seed sowing, thereby enhancing seedling establishment, especially in drought-prone areas with limited topsoil moisture retention [36]. In cereal crops, sowing depth is largely determined by the maximum potential of coleoptile length, influencing the practice of timely sowing without delay [37]. The coleoptile, a protective sheath covering the primary leaf of germinating monocotyledonous seedlings, safeguards emerging shoots as they penetrate through the soil surface [38]. In addition to scientific efforts focused on enhancing root characteristics for drought tolerance, cultivation techniques such as deep planting are crucial for mitigating drought effects, particularly in arid regions. Deep planting facilitates efficient water use from deeper soil layers and supports robust root system development, contingent upon sufficient coleoptile length for seedlings to emerge successfully [39]. Understanding the genetic basis of coleoptile length and seedling emergence requires further investigation, considering both genetic backgrounds and environmental factors such as soil texture, seed-zone moisture, temperature, light availability, and crop residue [40]. Planting depth decisions hinge primarily on genotype-specific coleoptile length, as seeds planted deeper than the coleoptile's length cannot emerge effectively, thereby compromising plant density and yield potential [41]. Phenotyping studies on coleoptile length typically employ methods such as pot cultures, germination paper assays, or field measurements to assess this crucial trait [42].

Temperature is one of the three primary environmental factors influencing plant phenology and physiology, alongside solar radiation and soil moisture [43]. It plays a critical role not only in agronomic yield but also in various aspects of plant productivity, including root and shoot growth, nutrient and water uptake, and key physiological processes such as photosynthesis, respiration, and transpiration [44]. Studies exploring the relationship between root and shoot traits have often been limited to a small number of cultivars and conducted primarily at early growth stages under fixed temperature conditions. While many seeds germinate optimally under controlled laboratory settings, they frequently fail to emerge successfully in field environments [45]. Temperatures above a plant's optimal range have been shown to negatively affect physiological functions, including root activity, flowering, fertilization, seed set, and overall yield. Conversely, low temperatures delay seed germination [46], reduce growth rates, and impair plant vigor [47]. Both heat and cold stress generally inhibit root elongation. However, under field conditions, these stresses rarely produce identical root architectures. Temperature extremes are typically preceded by warmer or cooler periods that permit some root growth. Consequently, heat-stressed roots are more likely to exhibit an elongated structure, whereas cold-stressed roots tend to develop a more compact form [48]. In drought-prone agricultural regions, topsoil moisture is often insufficient for seed germination, necessitating deeper sowing to access adequate moisture [43] and cooler temperatures [46]. In such water-limited environments, varieties with longer coleoptiles are preferred. For example, winter wheat cultivated in the low-water regions of the Pacific Northwest, United States, is typically sown at depths of 10 to 20 cm [43]. However, it is noteworthy that coleoptile length significantly decreases at germination temperatures exceeding 15°C [48]. Understanding these interactions between temperature, root and shoot development, and germination dynamics is essential for optimizing crop management strategies in diverse agro-environmental conditions.

Genetic improvements in grain production are pivotal goals for plant breeding programs, which can be enhanced by understanding root and coleoptile traits that contribute to overall plant performance. The main challenge lies in developing non-destructive phenotyping systems that accurately capture RSA and coleoptile system architecture (CSA). These systems need to facilitate continuous monitoring of root and coleoptile development under varying growth conditions. Furthermore, there is a critical need for high-throughput phenotyping systems to efficiently evaluate numerous genotypes as part of breeding programs, independent of seasonal variations. Conducting phenotyping studies regardless of the growing season is crucial as the architecture of root and coleoptile systems significantly affects the timing and efficiency of soil water extraction. However, progress in selecting these architectural traits in breeding programs has been impeded by the absence of suitable phenotyping methods [31].

In this study, we aimed to develop a method like the transparent pot method, which is regarded as one of the most efficient and straightforward approaches in phenotyping, by utilizing two distinct pieces of equipment designed using a 3D printer. This method eliminates the need for computer-aided imaging processes and root photography, which are commonly used but are also labor-intensive and costly for measuring root angles. Instead, the angles and numbers of roots can be determined simultaneously directly on the equipment. The root and coleoptile devices developed in this study not only reduce peat usage but also mitigate the risk of roots being uncovered by peats. The compact and portable design of the equipment allows for phenotyping under a range of climate conditions, facilitating the analysis of many genotypes. Given their suitable dimensions, the devices are adaptable to various temperature and climate conditions (e.g., in plant growth chambers), enabling measurements that more accurately reflect natural conditions, particularly in winter-sown plants. Furthermore, these devices integrate existing laboratory and field phenotyping methods, combining the practical advantages of laboratory-based approaches with the physiological relevance of field-based techniques, thereby leading to significant labor and time savings. The primary objective of this research was to develop cost-effective, high-throughput phenotyping methods that facilitate the selection of desirable root and coleoptile architectural traits. While there were no restrictions on genotypic characteristics, the focus was on identifying phenotypic traits that align well with field conditions. Advances in various fields, including the use of 3D printing technology, have enabled cost-effective solutions in agricultural research. This study utilized two 3D-printed pieces of equipment, designed and patented [49, 50], for root and coleoptile phenotyping. These tools allowed for practical, simultaneous, accurate, and rapid measurements of root traits, shoot length, and coleoptile length across eight wheat genotypes, all conducted without the need for image processing technology. Additionally, the tools were evaluated under three different temperatures to assess their performance in varied environmental conditions.

Materials and methods

The study was conducted using a panel of eight wheat genotypes (Table 1). In the selection of plant materials, emphasis was placed on incorporating drought-tolerant varieties commonly grown in Türkiye, alongside genotypes with contrasting traits, to enable the comprehensive assessment of phenotypic variations in relevant characteristics. The phenotyping process for these wheat genotypes was divided into three stages; 1) setting up the experiment on the platform; 2) acquisition of RSA and CSA without images; and 3) analysis of acquired data.

Table 1 The common name of eight different genotype assessed for its RSA and CSA
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Root angle measuring equipment

In this study, two distinct pieces of equipment were designed for measuring seminal root angles and coleoptile lengths. The equipment used to measure seminal root angles was designed using ThinkerCAD software. This product consists of two main parts: a base and a cover with channels divided according to the angle values where seminal root angles are determined. Four different wheat genotypes were germinated simultaneously within this equipment to measure their seminal root angles. The area designated for seed placement has a depth of 0.9 cm, with space intentionally left below for root growth facilitation. At the bottom of this seed placement area, there are channels set at 10-degree angles. A central guide channel, designed longer than the others, is positioned at zero degrees. Each adjacent channel deviates by 10 degrees to the right and left of this central guide, collectively spanning 180 degrees (Fig. 1A). To ensure optimal conditions for seed germination and root angle measurement, a container made of plexiglass was fabricated to house the plates, preventing light exposure during the planting process (Fig. 1B, C). The component highlighted in orange in Fig. 1 was 3D printed using carbon fiber filament, chosen for its robustness and durability in experimental settings. This setup was integral to accurately determining seminal root angles across different wheat genotypes, providing essential data for understanding root development and its implications for plant performance under varying conditions. This research utilized the equipment was patented in Turkish patent and trademark institution [50] (Fig. 2).

Fig. 1
figure 1

Root angle measuring equipment; A) open view of root angle measuring equipment, B) closed view of root angle measuring equipment and C) open and closed images of the root angle measurement equipment from different angles

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Fig. 2
figure 2

Comparison of Images with and without image processing in Bayraktar genotype: A) image without image processing, B) image with image processing

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Coleoptile lenght measuring equipment

The equipment designed to determine coleoptile length is depicted in Fig. 3. It comprises 10 channels, each incrementally spaced by 1 cm, ranging from 4 cm for the shortest coleoptile length to 13 cm for the longest. The width of the seed planting sections is carefully configured to accommodate easy planting of wheat seeds. At the bottom of each channel, small holes are incorporated to facilitate root growth, while ensuring the seeds remain securely in place. These holes are designed to be narrow enough to prevent seeds from slipping through. The seeds to be sown are sieve sized, ranging between 2.5–2.8 mm. A 0.4 cm thick lid covers the plate, creating a dark environment conducive to seed germination (Fig. 3A, B, C). This research utilized the equipment was patented in Turkish patent and trademark institution [51] (Fig. 4).

Fig. 3
figure 3

Coleoptile lenght measuring equipment; A) internal view of the equipment where coleoptile length will be determined, B) the equipment on which the coleoptile length will be determined must have a lid. C) open and closed view of the equipment from different angles to determine the coleoptile length

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Fig. 4
figure 4

Comparison of conventional and the newly designed apparatus in Müfitbey genotype; A) conventional calculating, B) calculating with new equipment

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Determination of root of genotypes using rooting equipment

The experiment involved filling plates with approximately 60 g of Klasmann TS1 peat before planting eight wheat seeds per plate, with four replicates per genotype. The amount of peat was kept equal so that the roots were subjected to the same pressure in each replicate. The seeds were planted flat with embryos positioned at the bottom of the plates (Fig. 5). The experimental design followed a factorial completely randomized design (CRD) method comprising two factors: temperature (10 °C, 15 °C, and 20 °C) and genotype (Altindane, Bayraktar, Ekiz, Gerek 79, Karahan, Müfitbey, Nevzatbey and Tosunbey). Following irrigation, the plates were shielded from light exposure within a container. Germination occurred under controlled climatic conditions in a Daihan, ThermoStable GC-459 model plant growth cabinet, maintaining 70% humidity, 16 h of light, and 8 h of darkness. After the emergence of shoots reaching 3–4 cm in length, typically 7–10 days after planting, the plates were removed from the container. Careful washing with a peat spray irrigation head revealed the roots for clear photographic documentation. Root angles of the genotypes were determined by counting the channels entered by the first two seminal roots. Previous studies indicate that these first two seminal roots are primary determinants of root angle, as they are typically the longest among the root system [35]. To validate these results, measurements were also conducted using image processing software to confirm the root angles of the same seeds. Comparing the root angle results obtained from our designed plates with those from image processing demonstrated the effectiveness and reliability of our plate method. Measurements were taken on different days at three different temperatures. All measurements were made for all varieties at 10°C on the 14th day, at 15°C on the 10th day, and at 20°C on the 7th day. Additionally, the average number of roots, root length, and shoot length of each genotype were measured using a ruler. For all temperature treatments, seeds of each variety (4 replicates—40 seeds) were germinated and measured.

Fig. 5
figure 5

Root architectural; wheat seedlings phenotyped for seminal root traits in a high-throughput system using with 3D printer: A) photograph of the seed of the Altindane genotype germinated after 6 days in covered plate, B,C) preparing the root for washing by removing the cover of the plate, D) determining the angles of the washed roots (50°) and analyzing with image processing to confirm the root angle determined on the plate, E) container in which the root plates are placed and F) plants germinated in containers in the growth chamber

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Determination of coleoptile length of genotypes using coleoptile equipment

Before planting the eight wheat seeds, each plate was filled with approximately 120 g of Klasmann TS1 peat, and ten seeds from each genotype were carefully placed into designated channels (Fig. 6). The seeds were positioned horizontally with their embryos facing upwards on the plate side. The sieve analysis value for the seeds ranged between 2.5–2.8 mm, chosen to accommodate variations in seed size across different wheat varieties, ensuring that even smaller-seeded types could remain securely within the channels without the risk of falling out as coleoptiles emerged. Following irrigation, the plates were placed in a container to shield them from light exposure, like the setup for the root angle equipment. Germination proceeded under control conditions in a Daihan, ThermoStable GC-459 model plant growth cabinet, maintaining temperatures of 10 °C, 15 °C, and 20 °C, with 70% humidity, 16 h of light, and 8 h of darkness. Coleoptile length, influenced by soil temperature, typically decreases by 50% as temperature increases [52]. Hence, 15°C was identified as the optimal temperature for accurate coleoptile length measurements [53]. Seeds were monitored daily at consistent intervals, and the emergence date of the coleoptile was recorded once its tip appeared. The length of the longest channel through which each genotype's coleoptile emerged was recorded as its coleoptile length. The experiment followed a factorial experiment based on completely randomized design (CRD) involving three factors: temperature (10 °C, 15 °C, and 20 °C), genotypes (Karahan, Müfitbey, Bayraktar, Gerek 79, Altındane, Nevzatbey, Ekiz, and Tosunbey), and deep sowing depths (ranging from 4 to 13 cm in 1 cm increments), with four replicates per treatment combination.

Fig. 6
figure 6

Coleoptile architectural: A) identification of seeds whose coleoptiles come out of the soil by removing the cover of plate 2; B) preparing the root for washing by removing the cover of the plate, C) container in which the coleoptile plates are placed and germination of Nevzatbey genotype seeds, and D) plants germinated in containers in the growth chamber

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Additionally, apart from coleoptile length, the average root length, root number, and shoot length of each seed within these plates were also measured using a ruler. For all temperature treatments, seeds of each variety (4 replicates—40 seeds) were germinated and measured.

Statistical analysis and evaluation of data

Before proceeding with the analysis of variance, the normal distribution of the residuals was checked using the Kolmogorov–Smirnov test in SPSS. Statistical analysis of all data related to root and coleoptile architecture was conducted using a factorial expriment based on completely randomized design (CRD) with four replications. Differences among the means were compared using Duncan's multiple range test at 5% probability level.

Results

Root architecture system (RAS) measurement

The primary purpose of this equipment was to determine the root angles of the various plant varieties. In this study, we took inspiration from the clear pot experiments of Richard et al. [31] We grew roots along the lines where the root plates were placed. At the time of measuring for the seminal root angle (i.e., five days after planting), the first pair of seminal roots on each side had elongated.

The analysis of variance revealed significant differences among the genotypes for root angle, shoot length, number of roots, and total root length, while significant differences among temperature treatments were observed only for shoot length and total root length (Table 2). Mean comparisons among genotypes indicated that Tosunbey exhibited the highest mean root angle (114.17 degrees) and shoot length (14.39 cm), while Nevzatbey had the highest number of roots (3.91). In contrast, Altindane showed the lowest root angle (58.33 degrees) and shoot length (9.17 cm), Gerek 79 had the lowest number of roots (3.16), and Karahan had the lowest total root length (28.83 cm) (Fig. 7A, B and Table S 1).

Table 2 Analysis of variance of effects different genotype, and temperate rooting levels on root angle (degree; °), shoot length (cm), number of roots (number), and total root length (cm) of wheat seedling
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Analysis of variance also indicated significant two-way interactions (genotype × temperature, G × T) for root angle, shoot length and number of roots (Table 2). The highest root angle (132.50 degree) was observed in Nevzatbey at 20°C temperature, and the highest shoot length (20.55 cm), the highest number of roots (4.75 number) was found in Tosunbey at 20°C, Ekiz at 20°C temperature. In contrast, the lowest root angle (50.00 degree) and shoot length (5.12 cm), the lowest number of roots (3.00 number) were both observed in Altindane at 10°C temperature, Bayraktar at 20°C temperature and Altindane at 20°C temperature, Bayraktar at 20°C temperature, Gerek at 10°C temperature, Karahan at 15°C temperature, Mufitbey at 15°C and 20°C temperature (Fig. 7C and Table S 1).

Fig. 7
figure 7

A Response of different genotypes to seedling growth in wheat; 1) root angle, 2) shoot length, 3) number of roots and 4) total root length. B Response of different temperatures to seedling growth in wheat: 1) shoot length, and 2) total root length. C Response of genotype and temperature interaction in wheat seedlings for: 1) root angle, 2) shoot length, and 3) number of roots

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Considering the effect of different temperatures on root angle, our study identified Altindane as the best-performing genotype, while Tosunbey was found to be the least favorable in this regard.

Throughput and repeatability of root phenotyping with equipment validation using image processing

Several free image processing programs are available for determining plant root angles, with ImageJ, RootNav, and SmartRoot being among the most notable. These programs calculate root angles with user assistance. However, effective use of these software programs requires a certain level of expertise and experience. Additionally, user-induced measurement errors can occur while using these image processing programs. For instance, Fig. 3 shows a measurement example using ImageJ. In this program, the user must select three points to determine the root angle, and the position of each point directly affects the calculated angle. Consequently, incorrect positioning of these points can lead to measurement errors [42, 53].

The root angle determination apparatus we designed allows for faster, low-cost, and highly reliable measurements, eliminating the need for image processing programs and minimizing human errors. Figure 3 demonstrates a comparison of the results obtained using the designed apparatus and those obtained through image processing. Both measurement techniques yielded similar results for the Bayraktar genotype. The designed apparatus measured root angles of 110°, while the image processing results were 109° and 106°.

Coleoptile architecture system (CAS) measurement

Our primary aim is to measure coleoptile length across all varieties. The secondary goal is to measure the number of roots, shoot lengths, and root lengths. For coleoptile measurements, we waited for all seeds to germinate and emerge at the soil surface. This period varied according to temperature and variety. We identified which seeds had their coleoptiles reach the soil surface and which remained underground.

The analysis of variance revealed significant differences among genotype, temperature and deep sowing applications for coleoptile length, leaf length, number of roots, and total root length (Table 3). The comparison of mean values among genotypes revealed that the highest coleoptile length (8.37 cm) was found in the Mufitbey genotype, the highest leaf length (11.42 cm) in the Altindane genotype, the highest number of roots (4.26) in the Tosunbey genotype, and the highest total root length (40.28 cm) in the Nevzatbey genotype. In contrast, the lowest values in coleoptile length (7.04 cm) were observed in Altindane genotype, the lowest leaf length (7.61 cm) in the Bayraktar genotype, the lowest number of roots (3.13 number) in the Altindane and Gerek 79 genotypes, the lowest total root length (20.32 cm) in the Karahan genotypes (Fig. 8A and Table S2). Regarding temperature treatments, the highest mean coleoptile length (7.83 cm) was observed at 10 °C, the longest leaf length (12.71 cm) at 20 °C, the greatest number of roots (3.76) at 15 °C, and the longest total root length (34.81 cm) at 20 °C while the lowest means were recorded at 20 °C with 7.06 cm for coleoptile length, 5.81 cm for leaf length at 10 °C, 3.63 cm for at 10 °C and 34.81 cm for total root length, respectively (Fig. 8B and Table S2). When considering the deep sowing applications, the highest coleoptile length (9.12 cm) was found at 13 cm deep sowing, the highest leaf length (9.92 cm) at 6 cm deep sowing, and the highest total root length (32.91 cm) at 7 cm deep sowing. Conversely, the lowest values were observed at a sowing depth of 4 cm, with a coleoptile length of 4.44 cm and a leaf length of 7.94 cm and the shortest total root length (25.65 cm) were observed at a sowing depth of 12 cm (Fig. 8C and Table S2).

Table 3 Analysis of variance of effects of different genotypes, and temperate rooting levels on coleoptile length (cm), leaf length (cm), number of roots (number), and total root length (cm) of wheat seedling
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Fig. 8
figure 8

A Response of different genotypes in seedling growth of wheat: 1) coleoptile length, 2) leaf length, 3) number of roots, and 4) total root length. B Response of different temperatures in seedling growth of wheat: 1) coleoptile length, 2) leaf length, 3) number of roots, and 4) total root length. C Response of different deep sowing conditions in seedling growth of wheat: 1) coleoptile length, 2) leaf length, and 3) total root length. D Response of genotype and temperature interaction in seedling growth of wheat: 1) coleoptile length, 2) leaf length, 3) number of roots, and 4) total root length. E Response of genotype and deep sowing interaction in seedling growth of wheat: 1) coleoptile length, 2) leaf length, and 3) total root length. F Response of temperature and deep sowing interaction in seedling growth of wheat: 1) coleoptile length, and 2) leaf length. G Response of genotype, temperature, and deep sowing interaction in seedling growth of wheat: 1) coleoptile length, and 2) leaf length

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The analysis of variance revealed significant two-way interactions between genotype and temperature (G × T) for coleoptile length, leaf length, number of roots, and total root length. Additionally, the interaction between genotype and deep sowing (G × D) showed significant differences in leaf length. Furthermore, significant differences were found in coleoptile length and leaf length due to the interaction between temperature and deep sowing (T × D) (Table 3).

Examining the mean values for G × T interactions, the highest coleoptile length (8.72 cm) was observed in the interaction between the Tosunbey genotype and the 10 °C temperature treatment, whereas the lowest (6.04 cm) was found in the interaction between the Nevzatbey genotype and the 10 °C temperature treatment. The highest leaf length (14.26 cm) occurred in the interaction between the Gerek 79 genotype and the 20 °C temperature treatment, while the lowest (3.09 cm) was recorded in the interaction between the Bayraktar genotype and the 10 °C temperature treatment. The highest number of roots (4.70 number) was observed in the interaction between the Karahan genotype and the 10 °C temperature treatment. The highest total root length was found in the interaction between the Nevzatbey genotype and the 20 °C temperature treatment. Conversely, the lowest coleoptile length (6.04 cm) was found in the interaction between the Nevzatbey genotype and the 10 °C temperature treatment. The lowest leaf length (3.19 cm) was found in the interaction between the Bayraktar genotype and the 10 °C temperature treatment. The lowest number of roots (3.00 number) was observed in the interaction between the Bayraktar genotype and the 10 °C temperature treatment. The lowest total root length (18.23 cm) was found in the interaction between the Karahan genotype and the 20 °C temperature treatment (Fig. 8D and Table S2). In terms of G × D interactions, the highest coleoptile length (11.12 cm) was observed in the interaction between the Müfitbey genotype and the 13 cm deep sowing treatment. The longest leaf length (15.08 cm) was observed in the interaction between the Altindane genotype and the 5 cm deep sowing treatment. The greatest total root length (45.67 cm) was observed in the interaction between the Nevzatbey genotype and the 5 cm deep sowing treatment. Conversely, the lowest coleoptile length (4.11 cm) was found in the interaction between the Ekiz genotype and the 4 cm deep sowing treatment. The shortest leaf length (4.48 cm) was found in the interaction between the Tosunbey genotype and the 12 cm deep sowing treatment. The shortest total root length (15.17 cm) was found in the interaction between the Karahan genotype and the 10 cm deep sowing treatment (Fig. 8E and Table S2). For T × D interactions, the highest coleoptile length (10.29 cm) was determined in the interaction between 10 °C temperature and 13 cm deep sowing treatment, the highest coleoptile length (14.95 cm) was determined in the interaction between 20 °C temperature and 6 cm deep sowing treatment, whereas the lowest coleoptile length (4.38 cm) was found in the interaction between 20 °C temperature and 4 cm deep sowing treatment and the lowest leaf length (5.17 cm) was found in the interaction between 10 °C temperature and 12 cm deep sowing treatment (Fig. 8F and Table S2).

The analysis of variance revealed significant three-way interactions between genotype and temperature (G × T × D) for coleoptile length and leaf length (Table 3). Examining the mean values for G × T × D interactions, the highest coleoptile length (12.10 cm) was observed in the interaction between the Bayraktar genotype at 10 °C temperature with 12 cm deep sowing treatment, whereas the lowest (3.88 cm) was found in the interaction between the Ekiz genotype at 10 °C temperature with 4 cm deep sowing treatment. The highest leaf length (22.00 cm) occurred in the interaction between Altindane genotype at 20 °C temperature and 5 cm deep sowing treatment, while the lowest (2.28 cm) was observed in the interactions between Ekiz genotype at 15 °C temperature and 10 cm deep sowing treatment (Fig. 8G and Table S2).

Considering the different temperatures and deep sowing in terms of coleoptile length, our study determined that Ekiz was the best-performing genotype, while Nevzatbey exhibited the lowest coleoptile length.

Throughput and repeatability of coleoptile phenotyping with equipment validation using conventional methods

Although the manual measurement of coleoptile length is widely employed in agricultural research, it presents several disadvantages, including labor intensity, time consumption, susceptibility to human error, and subjective evaluations. Ensuring uniform germination and maintaining consistent environmental conditions are critical for the accuracy and reliability of these measurements [36]. Consequently, in our study, plants were cultivated in a climate chamber to address these challenges. When evaluating thousands of genotypes in modern breeding programs, manual methods have proven inadequate, underscoring the necessity for high-throughput phenotyping technologies [51]. Moreover, physical damage during manual handling can adversely affect both the accuracy of measurements and the subsequent growth and development of plants [54]. To mitigate these issues, we developed a coleoptile measurement apparatus that eliminates human errors, enables faster and easier analysis, and does not require any measuring instruments. This apparatus facilitates measurements without the need for image processing programs or experienced personnel. Figure 6 illustrates the conventional measurement method, while Fig. 6. depicts our new measurement technique. An additional advantage of this new technique is the simultaneous observation of all coleoptile lengths. Our results demonstrated that under 10°C, the coleoptile lengths of the Müfitbey cultivar, measured using both the conventional and the newly designed apparatus, yielded similar outcomes.

Discussion

Seedling establishment marks the crucial initial phase of crop growth and productivity, where plant roots and coleoptiles play essential roles in functions vital for plant performance, including water and nutrient uptake [55]. Water availability remains a significant limiting factor for wheat production in rain-fed agricultural systems globally. Among the least explored areas in drought tolerance research are root characteristics and architecture, particularly the challenges associated with studying subsoil structures. Investigating roots under field conditions presents substantial challenges, requiring specialized equipment and skilled labor. However, advancements in root research under controlled settings, such as laboratories or greenhouses, have been notable due to evolving technologies and methodologies. Despite their limited scale, linking data on root characteristics with genotypic traits enhances the effectiveness of studies in this field.

The advent of innovative approaches in root research has expanded the scope of this field. RSA plays a critical role in determining the timing and efficiency of soil water extraction yet breeding programs have been impeded by the lack of effective phenotyping methods for root architectural traits. This study aimed to develop cost-effective, high-throughput phenotyping methods to facilitate the selection of desirable root architectural traits. Several methods have been developed and utilized for phenotyping root characteristics, including soil sampling [56], thermography [57], image processing [58], X-ray computed tomography [59], as well as field techniques like mini-rhizotrons [60] and non-soil methods [61], which can be conducted in laboratory settings. However, many of these approaches are characterized by low throughput. Laboratory-based methods may also struggle to replicate field-like conditions [62]. Researchers encounter numerous challenges with each root phenotyping method. These challenges include high costs associated with infrastructure and the need for staff proficient in coding and computer technology. Additionally, there is a demand for robust, high-throughput laboratory screening methods that can effectively predict field performance.

Many existing root phenotyping methods face limitations in efficiently and economically assessing many genotypes. To address these challenges, our study focuses on developing 3D-printed equipment designed to simplify the measurement of root characteristics. This equipment aims to offer a more accessible and cost-effective solution, reducing reliance on complex image processing techniques and specialized expertise. Our goal is to streamline the phenotyping process, making root studies more straightforward and contributing to more efficient breeding programs through rapid, cost-effective screening of numerous samples. Our equipment is akin to the transparent pot method, known for its efficiency in root phenotyping. However, the transparent pot method has drawbacks, such as roots being obscured by peat in some genotypes, which hinders accurate photographing for image processing. On average, only 4.9 roots out of six seeds planted to determine root angle could be photographed, necessitating repeated measurements for certain genotypes with lower averages. By minimizing peat use, our equipment aims to prevent root obscuration issues, ensuring more consistent and reliable measurements. Furthermore, our equipment features a portable and user-friendly design, facilitating phenotyping across diverse climatic conditions and with a wide range of genotypes. This innovation combines the advantages of both laboratory and field phenotyping methods, enhancing the efficiency and effectiveness of root phenotyping in supporting breeding programs and agricultural research.

In this study, we used a panel of eight wheat genotypes grown at different seedling temperatures to evaluate a method for measuring seminal root angle and number using our custom-made equipment. The analysis of variance in root architecture revealed significant differences among genotypes, while temperature treatments significantly influenced shoot length and total root length. Our results showed that the Tosunbey genotype exhibited the highest root angle and shoot length. Genotype and temperature interaction was shown that the highest root angle was observed in Nevzatbey at 20°C whereas the lowest seen in Altindane genotype at at 10°C treatment. It appears that root angles show a negative relationship with temperature, suggesting that genotypes with consistently low root angles across varying temperatures could be preferred for different planting conditions. The Altindane genotype, which maintains a stable root angle regardless of temperature changes, emerges as the most ideal in this regard. These results highlight the variability among genotypes in their responses to different seedling growth temperatures, particularly in root distribution traits like length, angle, and number. Such insights are crucial for selecting genotypes that can adapt effectively to diverse environmental conditions, enhancing agricultural productivity and resilience. The first roots to emerge during wheat germination are seminal roots, which play crucial roles in root system architecture, particularly seminal root angle and number [31, 63]. These traits are pivotal for adapting to different types of drought conditions at various growth stages. A narrow root angle and higher root number facilitate efficient nitrogen uptake and improved water access, particularly beneficial under terminal drought conditions. Conversely, a shallow root system with a wider angle enhances phosphorus absorption from the topsoil and better exploration of superficial soil layers to capture in-season rainfall [64]. The selection of wheat genotypes with optimal root traits, such as deep root systems with narrow angles in arid regions or shallow root systems with wider angles in irrigated areas, is critical for maximizing productivity. Drought-resistant genotypes typically exhibit higher dry matter content and greater root dimensions, particularly in the upper segments of the root system (0–15 cm soil depth), where root growth angles typically range from 0–30° or 30–60° [65]. The variability in seminal root angles among wheat genotypes underscores the importance of selecting those with narrower angles, facilitating deeper soil penetration for enhanced water uptake and prolonged drought tolerance [26]. This approach contrasts with genotypes suited to wetter conditions, where wider root angles optimize energy use by minimizing the need for deep root exploration [66]. Understanding and harnessing this genotypic variability in root traits are crucial for mapping quantitative trait loci (QTLs) and identifying molecular markers that contribute to developing wheat varieties with tailored root architectures for optimal adaptation to drought and other environmental stresses [67].

Recent efforts have focused on identifying constraints to crop growth and yield, with increasing emphasis on breeding wheat cultivars with root systems that possess desirable traits for efficient resource utilization [68]. Wheat seedlings typically exhibit compact root systems with deeper masses characterized by narrow angles and higher numbers of seminal roots [69]. Root system architecture is therefore a critical target for improving seedling establishment in wheat [70]. Compared to aboveground traits, studying root characteristics has posed challenges for plant breeders [71], primarily due to the lack of efficient and reliable root phenotyping methods and limited understanding of the genetic regulation of root development [72]. Different phenotyping systems may serve complementary roles in addressing these challenges. Traditional root phenotyping techniques in field environments face difficulties due to genetic and phenotypic complexity, as well as limited repeatability in root sampling [13]. To measure root system architecture and early vigor effectively, traditional root phenotyping techniques are increasingly being replaced by high-throughput methods like our system. Laboratory-based methods bridge the gap between genotypes and phenotypes, enabling exploration of below-ground components and early vigor to enhance crop yield. In contrast, field-based methods for root studies are labor-intensive and often require plot destruction for sample collection [27]. This integrated approach ensures physiological relevance while maintaining controlled conditions necessary for precise measurements and reproducibility in root phenotyping studies. Our developed equipment for measuring coleoptile lengths in wheat genotypes streamlines the assessment process, ensuring quick and easy measurements while maintaining the health of experimental materials. It adapts to various temperature conditions, providing consistent and reliable measurements across environments. This versatility is crucial for identifying wheat genotypes with early and robust vigor, essential for maximizing yield potential and resilience. Integrated with root phenotyping methods, our equipment enhances efficiency and accuracy in breeding studies focused on improving wheat performance under diverse conditions. Technology advancements like 3D printing have facilitated personalized and sustainable manufacturing across sectors [73]. Our equipment, designed for controlled environments, ensures precise measurements of coleoptile lengths under ideal conditions, essential for effective breeding selection. Compared to traditional methods, our equipment offers practical advantages such as minimal soil or peat usage, simultaneous measurement of multiple seeds, and portability for experiments in different climates. By integrating these tools, our study aims to revolutionize root and coleoptile phenotyping, offering practical, cost-effective solutions for identifying genotypes with desirable traits and supporting the development of resilient crop varieties.

The wheat coleoptile, a sheath-like structure, facilitates the emergence of the first leaf from the embryo to the soil surface [74]. Coleoptile length significantly influences wheat seedling establishment, as it determines the optimal depth for seed sowing, crucial for successful growth [36]. Previous studies indicate that coleoptile length is polygenically controlled, with high heritability and strong additive effects [52]. Longer coleoptiles are often associated with taller plants, which may not always be desirable. Our study reveals significant variations among genotypes in coleoptile length, leaf length, number of roots, and total root length. Temperature treatments and deep sowing applications also show significant differences in coleoptile length. Among genotypes, Müfitbey exhibited the longest coleoptiles, while Nevzatbey had the shortest. Notably, coleoptile length was highest at 20°C and with a sowing depth of 11 cm. Two-way interactions between genotype and temperature significantly affect coleoptile length, suggesting a positive relationship between temperature and coleoptile growth. Genotypes with consistent or increasing coleoptile length with rising temperatures are ideal for various planting conditions. Müfitbey emerges as the most favorable genotype for coleoptile length. Selection of longer coleoptiles is crucial for improving emergence, weed suppression, and grain yield in low rainfall regions. It enables deeper sowing to access underground moisture for germination in dry areas. Developing new wheat varieties with longer coleoptiles and shorter heights could revolutionize breeding and production. Variation in coleoptile length underscores its relationship with sowing depth, crucial for stand establishment, early leaf development, and resistance to environmental stresses [54]. Deeper sowing protects seeds from freezing and pest damage, promoting better water utilization and emergence under drought conditions [75]. However, excessive sowing depth beyond coleoptile capacity may hinder establishment and early growth [76].

Conclusion

This study highlights the application of 3D printing technology in agricultural breeding, focusing on root and coleoptile phenotyping under different temperature conditions. The developed equipment includes a device for simultaneous germination of four seeds, enabling easy measurement of root angles and numbers, and another to assess coleoptile lengths, replacing costly imaging methods. This approach streamlines phenotyping, allowing for efficient and portable analysis across different climates. The study also aids in drought tolerance research by identifying genotypes suitable for deep planting and assessing genetic variations in root and coleoptile traits under varying temperatures. Our results showed that genotypic variations in root and coleoptile traits, with Altindane excelling in root angle and Müfitbey in coleoptile length, while Tosunbey and Nevzatbey showed weaker performances. By using 3D printing, this method accelerates breeding processes, enabling the creation of mapped populations for identifying QTLs linked to root and coleoptile architecture. Ultimately, it enhances the selection of resilient genotypes, improving agricultural productivity and stress tolerance.

Data availability

The data that support the fndings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

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Funding

The authors have not disclosed any funding.

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Authors and Affiliations

  1. Department of Bioengineering, Faculty of Engineering, Karamanoğlu Mehmetbey University, Karaman, 70100, Türkiye

    Nevzat Aydin & Mesut Ersin Sönmez

  2. Plant and Animal Production Department, Karamanoğlu Mehmetbey University, Karaman, Turkey

    Tuğba Güleç

  3. Department of Chemistry and Chemical Processing Technologies, Tecnical Science Vocational School, Karamanoğlu Mehmetbey University, Karaman, 70100, Türkiye

    Bedrettin Demir

  4. Department of Plant Production and Genetics, Urmia University, Urmia, Iran

    Hadi Alipour

  5. Department of Field Crops, Faculty of Agriculture, Necmettin Erbakan University, Konya, 42310, Türkiye

    Aras Türkoğlu

Authors
  1. Nevzat Aydin
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  2. Mesut Ersin Sönmez
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  3. Tuğba Güleç
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  4. Bedrettin Demir
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  5. Hadi Alipour
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  6. Aras Türkoğlu
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Contributions

Conceptualization, N.A.; methodology, N.A., M.E.S., T.G., B.D., H.A., and A.T.; software, N.A., B.D., H.A., T.G., A.T., B.D. and M.E.S; validation, N.A. and A.T., formal analysis, N.A., B.D., H.A., T.G., M.E.S. and A.T., investigation, N.A., B.D., H.A., T.G., M.E.S. and A.T.; resources, N.A.; data curation, N.A. H.A. and A.T.; writing—original draft preparation, A.T. and H.A; writing—review and editing, N.A., H.A., and A.T; visualization, N.A. and A.T., supervision, N.A.; project administration, N.A. and A.T.; funding acquisition, H.A. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Hadi Alipour or Aras Türkoğlu.

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Aydin, N., Sönmez, M.E., Güleç, T. et al. High-throughput phenotyping of wheat root angle and coleoptile length at different temperatures using 3D-printed equipment. BMC Plant Biol 25, 112 (2025). https://doi.org/10.1186/s12870-025-06120-w

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  • DOI: https://doi.org/10.1186/s12870-025-06120-w

Keywords

BMC Plant Biology

ISSN: 1471-2229

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