Analytical methods to evaluate seed dormancy in legumes
Legumes are known to have multiple health benefits. The seed coat in particular contains a number of bioactive compounds which possess antioxidant activity. Of great interest is the relationship between the chemical composition of the seed coat and its role in seed dormancy. Modern technological approaches are currently used to further identify these compounds and determine their role in seed development; these include NRIS FTIR, mass spectroscopy, electron microscopy and chromatography. The following review describes the constituents of the legume seed coat and their respective roles in dormancy and other stages of seed development. The review also highlights technologies used to analyze these components and concludes with a discussion of the implication of these findings for crop improvement.
Key Words: legume, seed coat, dormancy, mass spectroscopy, Fourier transform infrared spectroscopy.
Legumes/leguminous plants include a wide diversity of families of flowering plants. There have been approximately 20,000 species of the legume family recorded to date. The most commonly known legumes are peanuts (Arachis hypogea), Medicago (Medicago trancatula), broad bean (Vicia faba), soybean (Glycine max), pea (Pisum sativum), scarlet runner bean (Phaseolus coccineus), and lotus (Lotus japonicus). Legumes also contain a highly diverse number of bioactive compounds such as phenolics, tannins and flavonoids that have been identified as being beneficial to human health. These compounds have the ability to reduce chronic conditions such as cardiovascular disease and even some cancers. Many of these compounds play a role in defending the seed against pathogens or abiotic stresses such as water permeability, or have defined role in seed development, dormancy and germination.
New developments in plant experimental biology and analytical chemistry have greatly assisted in the advancement of legume science, specifically with respect to seed metabolism and its link to human nutrition. Many of the techniques currently utilized include genomics, transgenic plants, histological methods and biochemistry. Legumes are among the most well studied for examining seed dormancy. Since seed dormancy, growth and development are in general regulated at the metabolic level, the concentrations of proteins, sugars and bioactive compounds play large roles in influencing plant growth and nutritional content (Jayasuriya et al., 2010, 2012).
Measurement of the changing concentrations of these metabolites during development within different seed tissue types will help to gain fresh insight as to the role of these molecules in growth and development (Borisjak et al., 2014).
Analytic technologies can help to further identify the role of specific genes in metabolic pathways involved in dormancy, growth and development. To date, these approaches have shown that seed development and metabolism is a highly complex process. The end result of such studies could lead to the manipulation of seed nutritional content, as well as better knowledge of improved dormancy and germination conditions (Verdier et al., 2013, Lopez-Pedrouso et al., 2014).
Seed and Seed Coat composition
Seed morphology at early and late stages of development are illustrated in Figure 1. Seeds are composed of an embryo, endosperm and seed coat. At early stages of development, the seed coat is composed of ovule integuments, a hilum (where the seed was connected to the maternal plant), an outer integument containing palisade or macrosclereid cells, and an outer cuticle (Figure 2). A subepidermal layer of cells becomes differentiated into osteosclereids and contains air-filled intercellular spaces. The most innermost part of the seed coat contains parenchyma cells, and the innermost layers of these come in contact with the endosperm (Smykal et al., 2014). Growth of all seed components is regulated by transcription factors, with the integuments undergoing differentiation to become highly complex structures that protect the embryo. At first, the embryo and endosperm undergo development within the seed coat. The endosperm occupies the majority of space until the seed approaches maturation. At this point, the embryo expands and the endosperm degenerates. Signals for maturation are derived from the maternal plant (Smykal et al., 2014).
Different layers of the seed coat have different functions, including transport of metabolites, synthesis of defense-related compounds and establishment of dormancy and protection. For example, the endothelium is the site of proanthocyanidin synthesis (Smykal et al., 2014). As the seed matures, the seed coat outer layers thicken. Secondary metabolites such as tannins are synthesized in the inner tegument layer of the seed coat. Compounds such as lignins, which offer structural support, as well as peroxidases and chitinases, which play roles in defense against pathogens, also predominate in seed coats. The seed coat is also a source of phytohormones which are involved in seed development. Finally, the seed coat plays an important role in regulating seed dormancy and germination by regulating water uptake.
Of the seed coat, the endothelium, or innermost layer, is the metabolically active layer and is the site of synthesis of prostaglandins (PA). During early stages of seed development, the seed coat becomes a temporary storage site of carbohydrates and proteins. During the filling period, an expansion of the branched parenchyma takes place; this is followed by a complete separation between the seed coat and the embryonic tissue. The outermost seed walls also change to become more callose-rich (β-1,3 glucans). The vacuoles of these thick walled outermost cells are high in tannin content, underscoring their importance in seed coat hardening. Tannins are polymeric flavonoids that act as secondary metabolites for a variety of other functions. During seed coat development, tannins are first present around the hilum, indicating their role in defense against fungal pathogens. At least twenty genes involved in flavonoid metabolism have been characterized and are believed to play a role in seed dormancy. Tannins, on the other hand, can complex with minerals and proteins in the gastrointestinal tract and reduce the nutritional availability of nutrients when seeds are used as a food source (Dahl et al., 2012).
The variety of tannins differs among different legumes. For example, lentils produce catechin and gallocatechin units in their seed coats, while common beans produce predominantly catechin (Diaz et al., 2010). Other compounds found in abundance in seed coats are lignins, which provide mechanical strength and water impermeability. Peroxidases and catchol oxidases, abundant in seed coats, promote the polymerization of these soluble units into insoluble polymers. These compounds also play a role in defense against pathogens and a variety of stresses, both biotic and abiotic. In addition to this, there is much interest in the nutritional and medicinal properties of these compounds (Smykal et al., 2014).
Other compounds found in the seed coat include various pigments, which provide colour due to the presence of specific bioactive compounds, such as anthocyanins and carotenes (Chon, 2013). For example, a number of chalcone synthases, expressed in the seed coat, are involved in flavonoid biosynthesis to produce compounds that act as UV protectants and insect repellants, among a variety of other functions (Smykal et al., 2014). Chitinases and peroxidases, also involved in plant defense, are produced in the seed coat during the later stages of seed development. Suberin is a compound that prevents water permeability of the seed. In wild peas, catechol oxidase activity is linked to seed dormancy. The presence of a number of other phenolic compounds are also associated with increased seedhardness. Hardness of wild soybean (Glycene soja) is correlated to the presence of certain major phenolics namely epicatechin, cyanidin 3-O-glucoside, and delphinidin 3-O-glucoside. The concentration of epicatechin, for example, can vary throughout different conditions including humidity and gas present during seed storage. Epicatechin has been shown to play a functional role in affecting hardness of seeded wild soybean (Zhou et al., 2010). It has been demonstrated that the presence of phenolic acids affects the pre-harvest sprouting of wheat, rye, and triticale. Other phenolic compounds, including chlorogenic and caffeic acids, are found in many seed coats and can not only inhibit the germination process, but can leach out into the soil and prevent neighbouring seeds from germinating (Mandal et al., 2010). In this way, the constituents of the seed coat can influence dormancy and germination
Figure. Early and Late Stage of seed development. Top; Arabidopsis, bottom; soybean.
Source: National Science Foundation http://seedgenenetwork.net/arabidopsis
FIGURE 1. The arrangement of Fabaceae seed coat share rather common structural features. (A) Transversal section of the seed coat of wild Pisum sativum subsp. elatius (left), with a schematic drawing (right); epidermal cells differentiate into macrosclereids, which are characterized by a cuticle-covered surface. The outer parts of the macrosclereids (sclereid caps) are frequently separated by a region of the cell wall (light line) with specific features, resulting in different optical and staining properties. The central part of the testa differentiates into osteosclereids with a specific shape caused by thickened secondary cell wall. The innermost layers of parechymatous cells frequently die during differentiation and only disintegrated fragments are left. (B) Generalized scheme of the seed coat morphology commonly found in Fabaceae seeds showing the most important structural features, including the hilum, lens differentiated on the raphe and micropylar pore. (C) Transversal section of a Pisum sativum seed coat in the area of the hilum. The macrosclereids of the hilar scar are covered with counter palisade tissue with a central fissure above the tracheid bar, which is surrounded by star-shaped parenchyma interconnected to intercellular spaces of a layer of osteosclereids. Source: Front. Plant Sci., 17 July 2014 | doi: 10.3389/fpls.2014.00351
Legume cotyledons are principally composed of fiber and starches with arabinose and glucose being the most abundant sugars, respectively. Cotyledons exhibit show a vast reduction in the number of compounds which possess antioxidant activity, such as phenolic compounds and flavonol structures when compared with components found within the seed coat (Duenas et al., 2006). For example, while the seed coat is very rich in catechins, and procyanidins, the cotyledon contains mainly hydroxybenzoic and hydroxycinnamic acids in low concentrations. In addition to this, esters of trans-p-coumaric acid have been identified in the cotyledon, while the stilbene trans-resveratrol-5-glucoside has been detected in the seed coat. In general, legume embryos undergo differentiation into a series of highly specialized storage organs, which can be easily identified via histochemistry. Differentiation of cotyledons occurs gradually, from the inner adaxial region and out to the outer layers resulting in a heterogenous population of cells that differ in physiological age with each other. Eventually, cell expansion stops in the centre initially and then gradually in the more outer layers as the cotyledon reaches maturity. The patterns of starch accumulation and mitotic activity are spatially distinct. The next section discusses the cotyledon composition in more detail during seed development and dormancy.
- Sucrose and Starch Profiles
Besides the morphological level, cotyledon development can be examined at the gene expression level. In the past, this has been accomplished using in situ hybridization of mRNAs which encode storage proteins, as well as by examining the spatial distribution of different metabolites including sucrose and hexose within cotyledonous tissues . Determining how these metabolites are expressed during cotyledon maturation is integral to understanding seed metabolism. Initially, sugars and other metabolites such as hexose and glucose were examined using enzymatic or chromatographic analysis. To undertake this required a mixture of cell types by homogenization, thus altering or even destroying the original state of the metabolite. New, noninvasive technologies have thus emerged as the method of choice for examining plant metabolite distribution in vivo. The use of bioluminescence technologies, for example, allows a quantitative measurement of the metabolite distribution in tissue sections at high resolution by coupling the reaction of a particular metabolite of interest to glucose‐6‐phosphate oxidation, which in turn is linked to a luciferase light reaction enzyme assay The bioluminescence intensity is proportional to the concentration of the metabolite in a given tissue, and distribution can be visualized by imaging using microscopy as well as a photon counting processor.
Using this process, glucose concentrations have been mapped to be at higher concentrations in non‐differentiated parts of the early cotyledon, while mature, fully differentiated regions contain low concentrations of glucose (Mason et al, 2014). This indicates that glucose distribution is related to the stage of cell development in cotyledons. These results were shown to correlate with sucrose synthase mRNA transcript levels, further supporting the utility of this method.
Analysis of sugar content in the seed coat determined that the concentration of hexose is increased, and this special sugar status appears to initiate cell division. This is due to the presence of invertase, located within the seed coats. The end result of high hexose levels is an increase in cell number within the embryo and a delay in seed maturity. This is the case for large‐seeded genotypes such as V. faba An increase in glucose levels is also associated with mitotic activity. Mutant plants such as the growth‐deficient pea embryo E2748, illustrate that these sugar profiles are regulated by the seed coat.
It is important to note that during development, oxygen profiles change dramatically within the embryonic cotyledon. The oxygen content within the seed coat dramatically decreases, thus providing an hypoxic environment for legume embryo development. The O2 concentration in embryonic tissue were lowest the earliest stages of development. ATP concentrations were also measured, and while low in early cotyledons increased during development, starting from the abaxial region and migrating toward the interior (Verdier et al., 2013).
An increase of sucrose during embryogenesis is correlated with increased transcript levels of enzymes involved in its synthesis, such as sucrose synthase and ADP‐Glc pyrophosphorylase. Hexose levels decrease over development with a concomitant increase in starch accumulation levels.
Seeds of legumes/leguminous plants are indispensable in our daily life. These types of seeds are rich in proteins, oils and carbohydrates. Cultivation of legumes needs to be implemented in places where the climate is favored for germination and growth of leguminous crops. In general, a legume seed has a hard seed coat which is originated from its maternal tissue of the ovule integuments. A seed coat provides both protection and nourishment for the embryo sac and development of embryo. Seed coats are substantively demonstrated having biological effects on the seeds, such as their development in size and shape. Moreover, the colors of seed coats vary as a result of heredity (Yang et al., 2010); pigmentation of the seed coat generally occurs due to the presence of proanthocyanidins and other bioactive compounds such as anthocyanins and carotenes. Hardseedness, which is predicted by such compounds as well as the thickness of the seed coat, thus plays a significant role with respect to the maintainance or breakage of seed dormancy. In addition to their association with seed dormancy, many of these phenolic compounds also possess antioxidative characteristics, which are advantageous to human health and nutrition. The property of antioxidation of seeds is reportedly subjected to change depending on the organic compounds used for the treatment of legume seeds (Nithiyanantham, 2012).
Germination of seeds relates to a process involving transformation from a dormant state into a stage of development. There are several conditions essential for the germination of seeds. It is vital for the agricultural industry to incorporate the most effective crop growing conditions to optimize yield. Some cultivars of seeds require only moisture for germination, while some others need extra treatment, e.g. scarification of seed coats or dormancy periods in order to maximize the germination rate. Structure and permeability of seed coats affect the germination of seeds. Seeds selected for agricultural use generally require moisture only; however, many legume crops require seed treatment prior to germination. Dormancy levels is a key element for efficient production of certain crops. Much effort has been placed on the study of seed dormancy to improve agricultural productivity (Moreir and Pausas, 2012, Gama-Arachchige et al., 2012). Crop production will be decreased if the dormancy levels of seeds are too high, while yield loss due to pre-harvest sprouting occurs if the seed dormancy levels are too low. Dormancy levels can be determined chemically by measuring the amount of phaseolin present in the seed, for example, or physically by identifying the types of water-gap regions created using microscopy (Gama-Arachchige et al., 2013, López-Pedrouso et al., 2014).
Dormancy determines population dynamics as it is a mechanism to optimize germination time in response to environmental changes. Often seeds are dispersed at various degrees of dormancy to ensure that some will survive and germinate in the ideal environment, thus reducing the possibility of extinction. The actual state of dormancy cannot be directly measured, and stored seeds frequently change in their phase of dormancy (Thompson and Ooi, 2013). This heterogeneity in dormancy pattern can also be the result of other factors such as the nutritional status and age of the maternal plant. Both abscisic acid and gibberellins metabolism are required to maintain physiological dormancy Physical dormancy is linked to the hardness and water impermeability of the legume seed coat. Dormancy can be overcome and germination can proceed in the presence of various environmental factors, chiefly, moisture and temperature increases, and in certain cases, fire. The seed coat plays an important role in dormancy by regulating water intake. Many of the compounds mentioned earlier, such as the proanthocyanidins and carotenes, for example, are believed to play a significant role in this control. The rate of germination in legumes has increased extensively through domestication; this took place by selective breeding for thinner seed coats. As a result, domesticated legumes respond to water and break dormancy much more easily; they also often more easily by cooking and are easier to eat (Abbo et al., 2014).Thus, selective breeding for thinner seed coats has enabled domestic legumes to germinate more frequently and serve as a better nutritional source tan their wild counterparts (Campos-Vega et al., 2010, Bouchenak and Lamri-Senhadji, 2013).
Legumes are ideal examples for the study of seed development, and many relevant mechanisms have been elucidated using them as model systems (Smykal et al., 2014, Thomas and Dugham, 2014). A majority of these mechanisms studied using legumes as models are directly related to the advancement of science. Originally, legume biology was used to explain the molecular, biochemical, and physiological activities related to seed development. Later, in the 1990s, legume model systems were used to explain the ultrastructure and histochemistry of seed development. State-of-the-art, biological and biochemical tools including q-RT-PCR, EST sequencing, microarrays, and in-situ hybridization have been used to study the seed development of legumes. In a recent study, seedhardness has been estimated by the entire transcriptome analyses of seeds (Verdier et al., 2013). Other methods, including microscopy, near-infrared spectroscopy, chromatography and mass spectrometry have been used to examine the components of legume seed during dormancy. The following section highlights analytical methods used to characterize the components of legume seeds. A list of some of the techniques used to identify and characterize legume seed bioactive compounds is presented in Table 1.
Initially, the compositional analysis of seeds and its relationship to dormancy has been measured using simplistic methods such as determining seed weight and size relative to the stage of development. Microscopic examination of individual whole seeds or sections of stained seeds have also been used as a means to identify differences in components and their relationship with dormancy (Schulmerich et al. 2012). More sophisticated technologies were later introduced as they became available.
One of the most well established technique for determining seed constituents is near-infrared spectroscopy (NIRS). The ease of sample preparation makes NIRS suitable for evaluating a large number of samples. Plans et al. (2012) used NIRS to analyze seed coats of 90 widely variable bean samples, using either intact beans or their ground seed coats. The authors found that while the whole bean samples produced rough screening data, the ground seed coat samples were capable of quality screening that would be adaptable for breeding in bean research. NIR spectra were found to be distinct for different colors of seeds, indicating that NIR could be used for identifying multiple seed traits in single bean seeds without the need of sample preparation (Hacisalihoglu et al., 2010). Esteve Agelet et al. (2012) examined the performance of four near-infrared spectrophotometers and their associated spectral collection methods using soybean as their model. The performance of all instruments differed from each other with no single one giving the best overall performances. Transmission Raman spectroscopy (TRS) in particular is the one of the best technologies to constituents of legumes such as soybeans. Schulmerich et al. (2012) used TRS to measure the constituents of soybean and were able to predict the oil and protein content without destroying the bean.
Seed composition has also been examined using a variety of separation techniques. As mentioned previously, a large variety of phenolic compounds, including phenolic acids epicatechin, catechin, proanthocyanidins and other flavonoids have been identified in the skins of legumes (Ma et al., 2014). High-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS(n)) enabled the separation and identification of these and other phenolic constituents, based on molecular ions and MS(n) fragmentation patterns acquired by ESI-MS in the negative-ion mode. Quantification was performed based on peak areas of the UV (free phenolic compounds) or fluorescence signals from the HPLC chromatograms and calibration curves of commercial standards.
Using ultra performance liquid chromatography (UPLC) methodology, chromatographic separation of 12 soy intrinsic isoflavone forms was achieved in soybean by Fiechter et al (2010). Derived native isoflavone profiles implied a certain variability, comprising conjugated forms, especially glycosides, as the predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated the more bioavailable free aglycones as prevailing compounds
Other separation techniques have been used to examine seed composition. For example, Tang et al., (2013),used an HPLC-based technique to identify in the medicinal plant seed of Strychnos nux-vomica L. chlorogenic acid (a phenolic acid) and loganin (an iridoid glycoside) whose bioactive activities correlate with therapeutic effects. An optimal ultrasonic extraction procedure was carried out, then applied to determine the components of the seed coat, seed leaf, endosperm and whole seed.
Over the past few years, mass spectrometry imaging (MSI) is increasingly used in the studies of plant sciences (techniques, like Hucisalihaglu et al., 2010). In particular, matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry imaging using the power of high resolution time of flight (ToF) mass spectrometry offers additional information regarding the spatial distribution of specific molecules within biological tissues (Grassl et al., 2011).
Several studies use MS techniques analyze plant tissues. For example, certain MS-based proteomics techniques are utilized in the studies of rice (Agrawal et al., 2009). Rice, one of the major food crops, has been studied as a model cereal under either normal or stress conditions to examine the function of individual proteins (Agrawal et al., 2009). However, factors including levels of expression, post-translational modification profiles and interactions with other proteins cannot be examined adequately based on sequence knowledge. In addition to establishing proteomes during growth and development of plants, these techniques can be further used to study the effects due to the changes of environmental factors, e.g. disease, and genetic relationships. Similarly, proteomics study proteins which participate in photosynthesis, bioflavonoid biosynthesis, and the production of bioactive compounds , e.g. alkannins, shikonins, and those found in traditional Chinese medicines (TCMs) (Lu et al., 2013). Additionally, MS techniques are also used in the studying varieties of plant science, e.g. fruit ripening, plant pathogen interactions, and legume symbiosis (Chan et al., 2013; Yang et al., 2013; Ahmad et al. 2014; Lee et al., 2013). A rather special capability of MS based proteomics is to identify and characterize allergens, which is beneficial for numerous clinical manifestations ranging from mild to life-threatening systemic conditions, e.g. to prevent foods from generation of allergens (Nakamura et al., 2013; Harrer et al., 2010). Thus, mass spectral identification provides good assistance in enhanced data collection in many aspects of plant cell biology.
Fourier transform infrared (FTIR) spectroscopy is used to distinguish chemical identifications of plants. These chemical identifications are particularly useful to optimize germination or yields, as well as to help assessment of quality of crops. FTIR spectra of peas (Pisum sativum) and oats (Avena sativa) are obtained from the roots of each plants with treatment by moist paper soil and soil compost mixture respectively, to increase intra-species variability of their chemical composition. The roots were analyzed using FTIR spectra to obtain the sample composition, including the concentrations of lipids, proteins, and carbohydrates, and the spectra were grouped according to their similarity using a dendogram cluster analysis. The significant differences found between pea and oat samples demonstrates the effectiveness of this approach (Naumann et al. 2010). Legume seed has also been under analysis using FTIR (Kudre et al., 2014, Kouvoutsakis et al., 2014, Yu, 2013). Furthermore, researchers have used either of the principle components in legume seeds, e.g. proteins, starch, and oils/fats, as markers to study the differences in MALDI data obtained from different species of legume seeds. Globulins are the major storage proteins in legume seeds, i.e. globulins are very abundant in pea seeds. The concentration of globulins varies depending on the species and varieties of peas. Two forms of globulins have been found in pea, namely vicilins and legumins (distinguished by the sedimentation coefficient: 17S and 11S) respectively.. Thus, vicilins and legumins, analyzed qualitatively and quantitatively by MALDI, generally serve as the markers for profiling of pea seeds. Other biomacromolecules such as polysaccharides, have also been analyzed in pea using MALDI (Franc et al., 2013).
Protein profiling to isolate genetically modified (GM) tubers from non GM tubers has been successfully demonstrated using MALDI. Moreover, MALDI has been used to directly study the composition of plant tissues and analyze the seasonal bud dormancy in critical stages of Japanese apricot, with more than 400 proteins being resolved (Zhuang, 2013).
Other techniques including electron microscopy have also been employed for analysis of seeds. For example, transmission electron microscopy is one technology that has been used to unravel the mechanism of aging in soybean seed (Xin et al., 2014). Similarly,Varela and Albornoz, (2013), used scanning electron microscopy to analyze the seed coat of the tropical legume Anadenanthera colubrina var. cebil.
Statistical analysis such as principle component analysis (PCA) have also been used to examine legume seed constituents. For example, Kim et al., (2013), examined the profiles of 25 phenolic compounds identified from mungbean grains and were subjected to data-mining using PCA, and other forms of analysis. Using this metabolic profiling approach, the quality of food can be better assessed. PCA analysis can also be incorporated into a rapid quantitative screening method that can be used to determine toxic contents of specific polyphenols in yam bean (Lautié et al., 2013). PCA analysis was used after ultra high performance liquid chromatorgraphy (UHPLC) to identify beans that were low in toxicity.
A significant number of legumes such as herbaceous and shrubs have highly dormant seeds because their seeds contain hard impermeable seed coats. The main indication of seed dormancy resulting from a hard seed coat is lack of imbibition. Researchers expect that legumes experience at least a 60 percent imbibition rate in irregular rainfall areas and a maximum of 90 percent seed dormancy. Regardless of the type of legumes, it is important for farmers to analyze the levels of seed dormancy for individual plants so that they can identify the most effective approaches to break dormancy.
The increasing interest in the study of dormancy in legumes has led to the discovery and use of various analytical methods in the evaluation of dormancy among legume seeds. Scientists have studied critical areas such as embryo development and sucrose and starch profiles to develop various methods of identifying dormancy. Histochemistry provides the basis for the study of legume embryos. It allows scientists to determine the patterns of embryo development that is useful in predicting levels of seed dormancy in legumes. Other techniques including NRIS and FTIR spectroscopy are currently utilized to analyze seed content without the need for sample preparation. Chromatography techniques including HPLC and UHPLC are used to separate components further. Statistical analysis such as PCA enables large quantities of legumes to be screened for desired qualities.
There has been the emergence of various methods of studying seed development and growth with the aim of understanding the development of seed dormancy in legumes. The most common analytical approaches include mass spectrometry imaging and nuclear magnetic resonance. These methods enable researchers to understand and quantify metabolites such as sucrose and hexose, antioxidants including epicatechin, and flavonoids, and even lager molecules such as proteins, lipids and polysaccharides within seeds. Such information is useful for scientists to determine levels of dormancy and propose methods of breaking the dormancy.
Legumes/leguminous plants experience high levels of seed dormancy because the seeds are encapsidated in a hard coat. Scientists are interested in identifying levels of dormancy for various plants and as a result have developed various analytical methods of evaluating seed dormancy. Mass spectrometry imaging and nuclear magnetic resonance are found to be among the most common analytical methods.
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