The Role of the Internal Structure of Fabaceae Seeds in the Processes of Dormancy and Germination

Abstract

The germination processes of Fabaceae seeds are well studied based on physiological parameters. However, in many cases, especially in wild seeds, there is a predominance of dormancy processes that must be reversed to finally produce germination, generally applying scarification processes. In the anatomical studies of seeds, a certain conformation of the structure of the cover is appreciated, with a predominance of sclerenchymatic tissues and waxy covers that are the cause of the difficulty of the entry of water to produce the imbibition of the seed. Mechanical or chemical scarifications are usually recommended to produce effective scarification. The characterization of the anatomical details of the seed coat allows us to predict the appropriate scarification technique with which optimal seed germination can be obtained.

1. Introduction

The seed is the main reproductive organ of the spermatophytes. In nature, the seed is a basic food source for many animals and can be stored alive for long periods, thus ensuring the preservation of valuable plant species and varieties [1]. The seed plays a fundamental role in the dispersal, renewal, persistence of plant populations, forest regeneration, and ecological succession. For this reason, it is considered to be one of the main resources for the agricultural and silvicultural management of plant populations, for reforestation and the conservation of plant germplasm.

Knowledge about the germination of native species is considered the initial step in ecological restoration processes [2]. This knowledge is not only related to germination requirements, but also includes storage techniques to ensure its availability in the medium and long term [3]. Although there are few studies on the propagation of native Andean species [4], the importance of the ecological requirements of seeds in relation to ecological restoration programs is currently recognized [5]. Among the physiological processes that control germination is dormancy, which allows the seeds to remain for long periods of time in the soil until the environmental conditions are favorable for the establishment of seedlings [6]. Several types of dormancy have been described, including physical dormancy, which is determined by the characteristics of the seed (or fruit) cover and prevents the absorption of water by the embryo [7]. This type of dormancy is considered one of the most evolved in angiosperms and one of the families that presents them in a notorious way is the Fabaceae, which has the so-called hard seeds in which the characteristics of the thickening of the seed coat that prevent the adequate hydration of the embryo and therefore the triggering of the germination process; [8, 9]. The identification of the anatomical components of physical dormancy in seeds and the methods for the seed to get out of this state are key to understanding the germination and dispersal process of species that present this type of dormancy [9, 10]. This paper reports the effects of mechanical scarification in the germination of three promising Fabaceae seeds from the Peruvian Andean zone in relation to the structural anatomical characteristics of the seed coat.

2. Taxonomics aspects

The Fabaceae is one of the largest and most important families of angiosperms and is considered monophyletic in both morphological and molecular analyses [11]. Geographically, the Fabaceae are trees, shrubs, and herbs distributed in the South American Neotropics. In Peru around 145 genera and 1000 species have been registered, the endemic species occupy mainly the so-called Mesoandean regions, such as the humid and dry puna and the humid montane forests, between 1100 and 4800 meters of altitude [12]. The genus with the largest number of endemic species is Lupinus, in contrast to the genus Astragalus with few endemic species. Many of the Fabaceae species are promising phytostabilizing species, and there is a priority need to carry out detailed taxonomic studies and a greater collection of specimens of these genera [12]. Astragalus garbancillo Cav. (Figure 1A, D), It is a species that forms dense shrubby perennial tufts that in very cold places are small-sized plants, frequently glabrous and erect or sometimes decumbent stems. The fruit is an oblong legume, sometimes compressed and puberulent with 3–4 seeds [13]. Lupinus ballianus C.P. Sm. (Figure 1B and E), is a shrub approximately 1.50 meters tall, with branches and petioles with an attenuated pubescence [13]. Lupinus condensiflorusC.P.Sm. (Figure 1C and F), is a shrubby species 60 to 110 cm long, with sericeous-adpressed stems and branches. Fruits are 2.9 cm x 2.9 cm x 0.8 cm broad, hairy, with 3–5 seeds, and rostrate [14].

Figure 1.

Evaluated species and their seeds. A, D Astragalus garbancillo. B, E Lupinus ballianus. C, F Lupinus condensiflorus.

3. Methodology

3.1 Method of collecting fruits of species of Astragalus and Lupinus

The fruits of the seeds of the evaluated species were collected in different localities of the Peruvian high Andean region (Table 1). Aspects of the reproductive phenology of the species were previously defined, through a previous visit to the natural populations of these species, taking into account that the beginning of flowering, fruiting, fruit ripening, and seed dispersal; they are different if we compare it with the fruits of non-domesticated plants [15]. The ripe fruits were manually detached from the floral clusters and then stored in Kraft paper bags. Once the collection was finished, the fruits were wrapped with a paper towel to reduce humidity [16]. The criteria established to determine the maturity of the fruits were the change in coloration and the reduction in moisture content. It was observed that in most of the fruits and seeds of the high Andean plant species, maturation ends in the month of July, while the dispersal of the seeds begins in the month of August and ends in the month of October, this physiological process coincides with the dry season that prevails in the Andean region of the country.

Localities	District	Province	Region	Altitude (m)	Coordinates UTM
Santa Rosa	Aquia	Bolognesi	Ancash	3, 798	265,166, 8,894,157
Cerro bendita	Lachaqui	Canta	Lima	3, 780	322,491, 8,722,856
Caruya- Rio blanco	Chicla	Huarochiri	Lima	3, 889	363,325, 8,701,373

Table 1.

Places of origin, altitude and geographic coordinates (UTM) of fruits, and seeds of Andean species of Astragalus and Lupinus evaluated.

3.2 Determination of seed moisture content

Seed moisture was determined based on dry weight (dw) by weighing them in Petri dishes on an analytical balance. For each species, five replicates and 20 seeds per Petri dish were weighed in the case of Lupinus ballianus and L. condensiflorus and 100 seeds in the case of Astragalus garbancillo, because the seeds are small in the latter. For the determination of the dry weight, the seeds were dried in an oven at 105°C for 4 hours. Seed moisture content was expressed in terms of the weight of water contained in seed as a percentage of the total weight of the seed before drying, known as wet weight based on fresh weight (fw) [17, 18] and was calculated using the following equation [19]:

$$\text{Moisture Content}\left(\%\text{dw}\right)=\left(\text{fresh weight}\right)–\left(\text{dry}\text{weight}\right)/\left(\text{dry}\text{weight}\right)]\text{x}100$$E1

3.3 Seed pre-treatment by mechanical scarification

A part of the evaluated seeds was subjected to a mechanical scarification treatment to ensure their hydration and ensure the appropriate conditions to achieve optimal germination. The outer coverings of the seeds were scraped using fine sandpaper, taking care not to deeply damage the testa. The scarified seeds were disinfected in a 30% sodium hypochlorite solution for 20 minutes, rinsed several times in distilled water and then sown in Petri dishes conditioned with filter paper and sterile water, hermetically covered and incubated in a growth chamber at 21°C during the day, 15°C at night, with a photoperiod of 12 hours of light and 12 hours of darkness and with a relative humidity of 80% during the day and 90% at night. A germinated seed was considered when it presented the emergence of a radicle of at least 0.5 mm long. Finally, on the tenth day, the calculation of the accumulated percentage of germinated and non-germinated seeds was made according to the species and treatment evaluated.

3.4 Experimental design and statistical treatment

The experiment was carried out under laboratory conditions using a completely randomized experimental design. They were evaluated under two light conditions (with and without light) and with two scarification treatments (with mechanical scarification, and without scarification). For each species, four treatments were evaluated, with five repetitions for each treatment (Number of Petri dishes). For the germination test, 20 pre-treated seeds of each species were added to each Petri dish. The distribution of the experimental units (Petri dishes with seeds) within the growth chamber was carried out at random. In the statistical analysis, it was carried out according to the experimental design, and the variance analysis (ANOVA) was carried out, and the multiple comparison test of means by Tukey (α = 0.01), using the Infostat program version 2016e.

3.5 Observation of the internal structure of the seed

Representative seeds of the evaluated species were cut transversally by freehand, rinsed in 50% sodium hypochlorite, washed, stained with 1% toluidine blue, and mounted in diluted glycerine for observation at 100 and 400 magnifications in light microscopy [20]. For the observation of the seeds in scanning electron microscopy, the material was treated following the steps of fixation with Carnovsky, post fixation with 1% osmium tetroxide, dehydration with a battery of alcohols, drying of the samples at a critical point with CO₂. Mounting of the samples with double-sided adhesive tape, conductive tape, and gold plating on an ion coating. [21]. Observations were made between 90 and 5000 magnifications in the INSPECT S50 Scanning Electron Microscope (FEI, Hillsboro, Oregon), from the equipment laboratory of the Faculty of Biological Sciences of the National University of San Marcos.

4. Results

4.1 Moisture content of Astragalus and Lupinus seeds

Significant statistical differences (p < 0.0093) were obtained in the moisture content of Astragalus garbancillo seeds compared to Lupinus ballianus and L. condensiflorus seeds. The highest moisture content in the seeds was registered in Astragalus garbancillo, while in the Lupinus species the moisture content remained at values below 10% (Table 2).

Species	Seed moisture content (%)
Astragalus garbancillo	11.74 ^a
Lupinus condensiflorus	9.12 ^b
Lupinus ballianus	8.95 ^b
Least significant difference	2.15

Table 2.

Moisture content of the seeds of the evaluated species.

a, b indicates significant differences between treatments (Tukey, α = 0.0093).

4.2 Germination of the seeds of Astragalus and Lupinus species

In the germination of the seeds of the species, significant statistical differences (p < 0.001) were obtained in the analysis of variance (ANOVA) between the evaluated treatments. In the mean separation analysis by Tukey’s test of the number of germinated seedlings, differences were obtained, being the highest values obtained in the number of germinated seeds with the treatment with mechanical scarification in the species of Astragalus garbancillo and Lupinus condensiflorus compared with the lower value obtained by the seeds of Lupinus ballianus. Additionally, the seeds of A. garbancillo presented the least thickness in the seed coat (Table 3).

Scarification factor	Number of germinated seeds
	A. garbancillo	L. condensiflorus	L. ballianus
With mechanical scarification	18.33 ^a	18 ^a	13 ^a
Without mechanical scarification	1.6 ^b	3.8 ^b	1.83 ^b
Least significant difference	0.72	2.1	3.51
Seed coat thickness (μm)	7.95	8.30	9.0

Table 3.

Accumulated number of germinated seeds of the evaluated species in relation to scarification and thickness of the seed coat.

a, b indicates significant differences between treatments (Tukey, α = 0.01).

In the germination of the seeds of the evaluated species, statistically significant differences (p < 0.001) were obtained with the scarification and light factors according to the analysis of variance (ANOVA). The seeds with mechanical scarification of the testa and light treatment were the ones that germinated in the highest quantity (except L. ballianus) (Table 4).

Scarification factor and light	Number of germinated seeds
	A. garbancillo	L. condensiflorus	L. ballianus
With mechanical scarification	19.4 ^a	15.9 ^a	15.4 ^a
Without mechanical scarification	7.1 ^b	2.5 ^b	4.1 ^b
With lighting	13.6 ^a	10.2 ^a	7.6 ^b
In darkness	12.9 ^a	8.2 ^b	11.9 ^a
Least significant difference	1.25	2.19	1.91

Table 4.

Number of germinated seeds accumulated in the species according to the scarification factor and light evaluated in laboratory conditions.

a, b indicates significant differences between treatments (Tukey, α = 0.01).

4.3 Internal anatomical structure of the seed

In the transverse plane, the internal structure of the seminal layer of the seeds of the evaluated species shows a characteristic color for the species (Figures 1A–3A). An epidermal coat composed of a compact uniseriate layer of palisade sclereids (macrosclereid type), with non-uniformly thickened walls, about 10 microns thick, thicker in Lupinus ballianus and thinner in Astragalus garbancillo (Figures 2B, C, 3B, and 4B). At the level of the hilum region, the palisade layer is thickened. On the outer wall of this palisade layer, there is a refringent linear region in the cell walls, thick in L. ballianus, thin in A. garbancillo, and intermediate in L. condensiflorus. Table 3 shows the comparative results of the seminal coat thickness for the three species. The cells of the adjacent sub-epidermal layer differentiate into hourglass-shaped cells called osteosclereids (Figures 2C, 3B, and 4B). The tissue underlying this layer is a type of colorless large elongated cell parenchyma with a tangentially collapsed appearance. Next, a single-stratified inner layer of quadrangular cells containing Aleurone granules. The innermost tissue, with a positive reaction to Lugol, corresponds to the cotyledon with starch-reserving parenchyma (Figures 2D–4D).

Figure 2.

Anatomical characteristics of the Astragalus garbancillo seed. A, internal view of the seed. B, sagittal section of the seed. C, detail of the seed coat. D, detail of the aleurone layer and cotyledon. Em, embryo. Hi hilum. Ln, lens. Co, cotyledon. Sc, seed coat. Cl, clear line. Pl, palisade layer. Os, osteosclereids layer. P, parenchyma. Al, aleurone layer.

Figure 3.

Anatomical characteristics of the seed of Lupinus ballianus. A, internal view of the seed. B, detail of the seed coat. C, detail of the aleurone layer and cotyledon D, cotyledon storage parenchyma. Em, embryo. Hi hilum. Ln, lens. Co, cotyledon. Sc, seed coat. Cl, clear line. Pl, palisade layer. Os, osteosclereids layer. P, parenchyma. En, endosperm. Al, aleurone layer.

Figure 4.

Anatomical characteristics of the seed of Lupinus condensiflorus. A, internal view of the seed. B, detail of the seed coat. C, detail of the aleurone layer and cotyledon D, cotyledon storage parenchyma. Hi hilum. Ln, lens. Co, cotyledon. Sc, seed coat. Cl, clear line. Pl, palisade layer. Os, osteosclereids layer. P, parenchyma. En, endosperm. Al, aleurone layer. Pa, storage parenchyma.

Under the scanning microscopy view, the thin cuticular surface of the testa appears finely rough and uniform in Astragalus garbancillo (Figure 5A), smooth and uniform in Lupinus ballianus (Figure 6A). and irregularly alveolate in L. condensiflorus (Figure 7A). In the region of the hilum, the funicular tissue can be seen, made up of a fine pubescence with a loose appearance, leaving in the central part the fine fissure of the hilar groove, oriented longitudinally in Lupinus species (Figures 6B and 7B) and transversally oriented in A. garbancillo (Figure 5C). thin refringent line of this palisade layer of sclereids can be highlighted on its outer part (Figures 5D, 6D, and 7C). In this hilar region, the palisade cell layer of the seed coat is particularly thickened (Figures 5B, 6C, and 7C). In L. condensiflorus, the endosperm cells are irregularly polygonal in shape (Figure 7D).

Figure 5.

Scanning microscopy images of Astragalus garbancillo seeds. A. Detail of the surface of testa. B. Longitudinal section at the level of the hilum. C. Surface view of the hilar region. D. Cross section of the seed coat.

Figure 6.

Scanning microscopy images of Lupinus ballianus seeds. A. Detail of the surface of testa. B. Detailed view of the hilar region. C. Longitudinal section of the seed. D. Cross section of seed coat.

Figure 7.

Scanning microscopy images of Lupinus condensiflorus seeds. A. Detail of the surface of testa. B. Detailed view of the hilar region. C. Longitudinal section of the seed. D. Detail of the seed storage parenchyma.

5. Discussion

The seeds of Astragalus garbancillo had the smallest diameter of the seed coat, associated with the palisade layer of sclereids (Figure 2C), which would explain the greater effectiveness of mechanical scarification expressed in the greater number of germinated seeds (Table 3). On the contrary, the seeds of Lupinus ballianus presented the thickest seed coat associated with the largest diameter of the palisade layer and the lowest germination percentages, which indicates that mechanical scarification was not effective for this species. A separate case represents the seeds of L. condensiflorus, which presented a thick seed coat and yet had a high percentage of germination. In the seeds of this species, its seminal coat was characterized by presenting a thin refringent line (Figure 4B), if we compare it with the thick refringent line shown by L. ballianus (Figure 3B), hence we associate this characteristic with effective mechanical scarification and its high percentage of germination. In fact, both species, A. garbancillo and L. condensiflorus, presented high germination percentages with the light factor associated with its thin refringent line (Table 4). Indeed, the presence of the thickened line on the outside of the seed coat and its hydrophobic cuticular layer would be determining a certain degree of impermeability of the seed coat to water and oxygen, which can be broken with a simple mechanical scarifying action. Seed dormancy refers to the state by which viable seed does not germinate when provided with favorable conditions for germination, such as adequate moisture, an appropriate temperature regime, normal atmosphere, and, in some cases, light [22]. This form of dormancy found in the evaluated seeds would correspond to the so-called physical dormancy, where the seminal seed coats (or fruit pericarps) are impermeable to water. This type of impermeability is considered to be one of the most evolved types of dormancy [23]. In this type of dormancy, the impermeability of water in the seed is caused by the presence of palisade cells, which constitutes an impermeable layer to water, so it forms a barrier to its entry [24]. For this reason, the effectiveness of scarification is demonstrated, which is a mechanical or chemical method, by which germination is induced through breakage, abrasion, or softening of the seed coat, making it more permeable to the inhibition of humidity [1].

Dormancy is considered to have evolved as a strategy to avoid germination in conditions where seedling survival is low [15, 19]. Seed dormancy breaking also depends on a balance between growth inhibitors and growth promoters. Among the numerous plant germination inhibitors, some are located in the fruit wall or in the seed coat. The stimulating effect of the elimination of the seed coat and the covers associated with germination determines that this is considered in itself as one of the inhibitory sources of germination. In this sense, in Fabaceae seeds, it is usual to find that the quality of light does not greatly affect the germination process [25]. Germination and seedling establishment are critical stages in the biological cycle of plants [26]. Seedling emergence is the event most important phenological of a crop’s establishment. It represents the moment in which a seedling becomes independent of the non-renewable seminal reserves and when photosynthetic autotrophism begins. Emergence time often determines whether a plant competes successfully with its neighbors, whether it is consumed by herbivores, infested by diseases, and whether it flowers, reproduces, and matures at the end of its growth stage [27].

The seed coats of some species have characteristics that help germination and seed emergence. The testa of seeds eaten by animals and by humans can resist digestive processes and allow them to pass through the intestinal tract unharmed and thus facilitate seed dispersal. However, the use of corrosive chemical agents such as sulfuric acid may not always be considered the appropriate simile of this biological process [28]. In studies carried out on the emergence of seedlings in other Lupinus species under greenhouse conditions, it is mechanical scarification that determines the highest percentage of seedling emergence obtained with testa scarification, presenting up to 80% efficiency compared to chemical scarification with sulfuric acid [29]. Although the International Rules for Seed Testing (ISTA) recommends using concentrated sulfuric acid, for 2 to 45 minutes depending on the species, to scarify the test, this method is mentioned to be expensive and dangerous and should be followed with caution [19]. Under natural conditions, it has also been suggested that exposure of the seeds to high temperatures will be responsible for the release of dormancy [30].

The humidity factor is one of the conditions for triggering the seed germination processes. It is considered that the thread acts as a hygroscopic valve, by forming a fissure capable of interacting with the moisture content from the outside [7]. It is known that the combination of an impermeable testa with the valvular action of the thread allows reaching a high degree of desiccation in the hard seeds of Fabaceae, thus obtaining a certain percentage of humidity that is not affected by fluctuations in the external humidity content. of the seed [31]. This moisture is kept in equilibrium with the environment outside the seed and is the most important factor in determining the rate at which seeds deteriorate. For this reason, the moisture content within the seed is also an important aspect to consider in the postharvest of the crop. The determination of the moisture content before storing the seeds makes it possible to accurately predict the storage life potential of the accessions [19]. The highest moisture content recorded for Astragalus garbancillo (Table 2) seeds indicates their greatest potential to preserve their germination power for longer periods of time in relation to the dry season in the Andean region, in fact, we associate this feature with the shorter length of the hilar fissure compared to Lupinus species (Figures 5C, 6B, and 7B). Additionally, we know of the influence of factors such as soil depth and its composition on the germination of especially hard cover seeds, such as Fabaceae seeds, and that they can effectively plan the establishment of species in projects of ecological restoration projects [32].

6. Conclusions

It is concluded that seed dormancy of the studied Andean Fabaceae species is related to the structural characteristics of the seed coat, especially the sclereid layer. Under the evaluated conditions, Astragalus garbancillo seeds had the thinnest sclereid layer compared to seeds of species of the genus Lupinus, with a thicker seed coat. Based on the germination percentages obtained, the mechanical scarification carried out on the seeds of A. garbancillo was the most effective method that allows them to come out of dormancy. The seeds of this species with a thin seminal coat also had a higher moisture content, which is why they have a greater potential for conservation in function and are ideal for ecological restoration programs.