Germination control by a hard seed coat: insights from a tropical legume

Seeds can have a blockage to prevent germination, especially for species living in a seasonal climate region, which affects germination timing and the plant life cycle¹. The blockage (i.e. seed dormancy) presents distinct structural and physiological features allowing the classification of dormancy into different classes^2,3. The current classification system for seed dormancy includes five classes inside two subdivisions (exogenous and endogenous) with subclasses and levels for most kinds of germination blockage^2,3. The diversity and complexity of these dormancy classes were updated recently⁴. However, seed (or dispersal unit) features may hamper seed dormancy classification, as in the case of the stony endocarp in palm diaspores and seeds with a hard and thick coat. Additionally, other seed features, such as the presence of aril, may affect seed germination, impacting an accurate classification.

Physiological dormancy (PD) is the most common kind of dormancy in all habitats all over the world; however, in the tropical zone, there is a high percentage of species having seeds with physical dormancy (PY), notably in more seasonal habitats³. In tropical deciduous forests and savannas, the recurrence of PD and PY is similar³. However, the presence of seed dormancy decreases considerably in aseasonal environments (e.g., in lower latitudes as in tropical rainforests) since this germination blockage is not an adaptive trait in habitats with a longer growing season^1,3. The other kinds of dormancy, such as morphological (MD), morphophysiological (MPD) and combinational (i.e., PD + PY), are not prevalent in any habitat worldwide³. In Brazilian Cerrado, dormancy in seeds is highly frequent, with PY occurring in several species^5,6,7. Leguminous species are widespread in the Cerrado vegetation, and PY is also frequently associated with this plant family^1,8,9.

Copaifera is a pantropical genus of plants comprising 33 species, wherein 27 species are distributed all over Brazil^10,11,12. From this genus, C. langsdorffii Desf. is a widespread tree species living in tropical forests, and it can colonize dissonant environments such as the Amazon rainforest and Cerrado (Brazilian savanna)¹². C. langsdorffi produces dark brown/black seeds with a bright yellowish orange aril covering the hard seed coat (Fig. 1A, B). Most of the seed comprises a large embryo with aligned seed structures (lens, hilum and micropyle) in the seed coat (Fig. 1C). These seeds are desiccation tolerant (orthodox)^13,14 and in some reports have slow germination (i.e., ≥ 30 d to 50% germination)¹⁵, but there is conflicting information in the literature about the presence or absence of dormancy.

Some authors have reported dormancy in C. langsdorffii seeds or the need for pre-germinative treatments to achieve higher germination^{16,17,18,19,20,21,22,23,24}. Additionally, reports in the literature describe C. langsdorffii seeds as possibly having more than one kind of dormancy. PY, PD + PY, ‘chemical dormancy’ or even the generalistic classification “occasional dormancy” have been reported for this species^22,23,25. In contrast, other authors described these seeds as non-dormant^7,26,27,28. The information that the seed aril interferes in C. langsdorffii germination is also found in the literature. According to Carvalho²⁰ and Souza et al.²⁷, aril removal is required for germination since this pulpy part of seeds has inhibitory substances avoiding germination. Non-dormancy (ND) is prevalent in tropical rainforests, reducing the proportion of ND in tropical savannas³; however, the investigated species inhabits both environments. Plant species can also produce seeds with different levels of dormancy or vary the proportion of dormant and non-dormant seeds to spread germination over time (‘bet-hedging strategy’, see Gremer & Venable²⁹; Pausas et al.³⁰). If this occurs for C. langsdorffii, variation in the proportion of dormant seeds could be related to the inconsistencies in dormancy classification for this species. Thus, C. langsdorffii could serve as a model for seed dormancy studies, particularly on the ecological strategy behind the role of the hard seed coat regulating germination.

Seed dormancy classification proposed by Baskin and Baskin² and recently updated⁴ makes the comprehension of distinct kinds of seed germination blockage clearer; however, some researchers still do not follow this guide for an accurate dormancy classification. Additionally, some difficulties are found, particularly for seeds (or dispersal units) with a hard coat, or pivotal tests are forgotten, such as the imbibition tests for identifying seed permeability. Permeability tests determine if the coat confers a blockage to the water entrance (i.e., PY) or only a mechanical barrier to radicle protrusion (in this case, conferring PD), making the dormancy classification more assertive³¹. Thus, we carried out this work aiming to understand the ecological strategy to regulate germination in a widespread tropical species (C. langsdorffii), contributing to the efforts for the precise understanding of the hard coats on germination control. It may also help clarify whether the term “hardseededness” is appropriate to describe physical dormancy or water-impermeable seed coats.

Seed collection

C. langsdorffii seeds were collected in 2020 (2020 C, August-September) during the natural dispersal period in Brazil (Iraí de Minas city, Minas Gerais State – 18º59’ 23” S, 47º28’33” W). The seed aril, when still present, was manually removed. The seeds were kept in plastic boxes and then stored in plastic bags under laboratory conditions until the beginning of the experiments (right after collection). A second collection was carried out in 2021 (2021 C) (at the same local) to obtain arillate seeds (seed + aril). The investigations using the 2021 seedlot started right after collection, aiming to investigate the influence of seed aril on germination.

Morphological characterization of seeds

For the external morphology study, seeds of C. langsdorffii were removed from the fruits and photographed using a stereomicroscope (Zeiss Stemi 2000 C, Carl Zeiss Microscopy, Jena, Germany) equipped with a digital camera (Taida TD-HU708A, Shenzhen Sanqiang Taida Optical Instrument, Shenzhen, China). For scanning electron microscopy investigation, de-arillated seeds were mounted directly onto aluminium stubs, coated with gold using a sputter coater (Leica EM SCD050, Leica Microsystems, Wetzlar, Germany), examined under a scanning electron microscope (Zeiss EVO MA 100, Zeiss, Jena, Germany) and the images were digitally recorded. For light microscopy study, de-arillated seeds were imbibed, fixed in FAA 50³², dehydrated in an ascending ethanolic series to ethanol absolute, and embedded in historesin (Historesin, Leica Microsystems, Heidelberg, Germany) following the manufacturer’s instructions. The material was sectioned using a rotary microtome (Leica RM 2235, Leica Biosystems, Nussloch, Germany) into slices approximately 6 μm thick, stained with 0.05% toluidine blue³³, modified with acetate buffer (pH 4.7), and mounted in a synthetic mount media (Entellan, Merck, Darmstadt, Germany). The slides were examined and photographed using a microscope (Olympus BX51, Olympus, Southall, UK) with a digital camera (Olympus DP70, Olympus, Southall, UK), and images were digitally recorded. The images were organized into plates using image editing software (Photoshop, Adobe, Redwood City, USA) and some images backgrounds were replaced.

Seed dormancy and germination in C. langsdorffii

The following experiments aimed to investigate the presence/absence of seed dormancy in the species, whether seed aril influences dormancy and germination, and whether seed size affects seed coat permeability (i.e., PY). Germination tests using intact (without aril) and scarified (using sandpaper, on the opposite side to the hilum) seeds were carried out for both seed collections. An additional treatment using intact arillate seeds was carried out for 2021 C. The effects of thermal treatments on seed germination were also investigated (for 2020 C) using immersion in water at 100 °C for 15 s and 80 °C (initial temperature) for 15 minutes^8,34. The seeds were then kept in Gerbox© on moistened germination paper using distilled water and incubated at 25 °C and constant light. Germinated seeds were scored at 3-d intervals over 30 days, and the criterion for germination was the protrusion of the radicle. The number of imbibed, intact (without imbibition) and dead seeds were also evaluated. Imbibition leads to a noticeable change in seed color and size, making imbibed seeds easy to detect. The seeds were considered dead when the tissues began to liquefy and/or were surrounded by fungi. These seeds were cut to verify if the embryo was firm and white or deteriorated.

Seed coat permeability was also investigated through imbibition tests. Seeds (2020 C) were separated into (1) intact and (2) scarified seeds. For 2021 C, seeds were then separated into three groups: (1) intact seeds without aril, (2) scarified seeds without aril, and (3) intact seeds + aril. Additionally, 2020 C seeds were separated into two groups: (1) large or (2) small seeds. These two groups were selected based on the seed weight using a precision scale (0,0001 g). The weight of large (heavy) seeds was ≥ 0.7 g and for small (light) seeds was ≤ 0.2 g (based on a previous characterization of the seedlot using 200 seeds). Thirty seeds for each group, for both seed collections, were individually weighed and kept in germination conditions at 25 °C under constant light. Seeds were blotted dry before each weighing, which occurred during 240 h. Intact seeds absorbing water indicate the absence of PY.

Investigation of water entrance in the seeds

To investigate water entrance through the seed coat, a dye-tracking experiment [based on Jayasuriya et al.³⁵, Gama-Arachchige et al.^36,37, Rodrigues-Junior et al.⁸ was carried out using methylene blue 0.1% [modified from Johansen³². Seeds were immersed in the solution for 12, 24 and 48 h, blotted dry and sectioned longitudinally to observe the presence of dye and its route in the seed tissues. Seeds were analysed under a stereomicroscope (Zeiss Stemi 2000-C), and pictures were taken with a digital camera (Taida TD-HU708A).

In a second investigation, seed structures were sealed to determine if the water penetrated the seed in a specific region [based on Jayasuriya et al.³⁵, Turner et al.³⁸, Rodrigues-Junior et al.³⁴. Five treatments were selected for this experiment, using super glue (ethyl cyanoacrylate) to block the following seed structures: (1) lens, (2) micropyle, (3) hilum + micropyle, (4) hilar region (lens + hilum + micropyle), and (5) control (non-blocked seeds). The seeds were kept in lab conditions for 48 h to allow the superglue to dry. Twenty-five seeds of each treatment were individually weighed and then kept in germination conditions at 25 °C. Seed weight was measured again after 1, 2, 4, 6, 8, 10, and 15 days. Variation of seed weight was evaluated individually during all the experimental period.

Effect of drying and storage on seed germination

As drying can induce seed dormancy, the purpose of this experiment was to investigate if seed drying can induce the acquisition of dormancy in C. langsdorffii. Seeds (2020 C) were kept in a closed plastic box (32 × 19 × 9.5 cm, 5 L) containing dry silica gel (826 g) to reach approximately 5% of relative humidity (RH). Temperature and RH inside the drying box were measured continuously during the experiment using a datalogger (AKSO AK174). Seed samples remained in the drying box on Petri dishes (six samples of 45 seeds) for 1, 2, 4, 8, 12 and 16 days. Non-dried seeds were the control in this test. After each sample removal, 20 seeds (four replicates of five seeds) were used for water content determination³⁹ and 25 for imbibition test. For imbibition test, dried seeds were individually weighed and kept in germination conditions for five days before another weighing to investigate if seed drying can induce dormancy (i.e., physical dormancy). For the seeds dried for 8, 12, and 16 days, an additional weighing was carried out after 10 days in germination conditions to evaluate the seed weight.

Additionally, intact seeds without aril (2020 C) were stored in laboratory conditions (25 ± 3 °C) for 1, 1.5, 2 and 3.5 years. After storage, the seeds were kept in germination conditions at 25 °C under constant light. Four replicates of 25 seeds were used for each test, and the germination was evaluated at 3-d intervals for 30 days. The results were compared to non-stored (fresh) seeds (control) to investigate seed viability and storage tolerance, as well as a possible induction of seed dormancy during storage.

Influence of Aril and scarification on seedling emergence

Seeds (2021 C) were separated into three groups: (1) arillate seeds, (2) seeds without aril, and (3) scarified seeds without aril. The seeds were then buried at a 2 cm-depth in plastic pots containing soil from Cerrado and kept in a covered (60% shade cloth cover) greenhouse with an automated watering system. A clear plastic cover above the experiment was used to avoid seed removal by the rain. The emergence evaluation occurred every week for 60 weeks, and all pots were verified at the end of the experiment to check seed mortality.

Statistical analyses

The experimental design for all essays was completely randomized, except for the seedling emergence test, which was designed in randomized blocks. Germination, imbibition and seedling emergence data were analyzed with a generalized linear models (GLMs) (negative binomial), and the means were compared using Tukey’s test using software R⁴⁰. To analyze the data of blocking experiment, a regression analysis was performed, and the fit of the model evaluated using the coefficient of determination (R²) (P ≤ 0.05) Sigmaplot^® software was used to design the graphs (Systat, San José, CA, USA).

Morphological characterization of seeds

The seeds of C. langsdorffii are ellipsoid, with a rigid, dark brown, slightly glossy seed coat (Fig. 1A, B). They are partially covered by an orange aril (a hilar-originated outgrowth) (Fig. 1A, B). The micropyle is punctiform and sometimes covered by remnants of the aril (Fig. 1C). The hilum is linear with remnants of the funiculus and aril (Fig. 1C). The lens is inconspicuous, showing a slight elevation at the base of the raphe (Fig. 1C). The seed coat exhibits fracture lines (Fig. 1D, E) and tiny pores (Fig. 1E, F). The seed coat consists of an exotesta with juxtaposed palisade cells covered by a thin mucilaginous layer (Fig. 1G). The mesotesta comprises three distinct regions. The outermost layer consists of hourglass-shaped cells (Fig. 1G). The median and inner layers are composed of crushed cells (Fig. 1G). The median layer has conspicuous intercellular spaces and slightly thickened walls (Fig. 1G), while the inner layer consists of cells with thinner walls and cytoplasm containing phenolic compounds (Fig. 1G). The remnants of crushed cells can be observed between the seed coat and the embryo, resulting from embryo growth (Fig. 1G).

Seed dormancy and germination in C. langsdorffii

For 2020 C, intact and scarified seeds had the highest germination percentages (P < 0.001; CV = 0.8), 71 and 67%, respectively. Seeds subjected to 80 °C for 15 min also had high germination (Fig. 2A). However, those seeds subjected to 100 °C for 15 s had a strong decrease in germination, reducing to 7% (Fig. 2A). Intact and scarified 2021 C seeds had the average germination of 43 and 9%, respectively, whereas arillate seeds had 23% of germination (Fig. 2B). The percentage of dead seeds increased significantly for the seeds that were subjected to mechanical scarification. All parameters evaluated for the seeds, germinated (P < 0.001; CV = 1.9), imbibed (P < 0.001; CV = 7.1) and dead seeds (P < 0.001; CV = 0.2) differed statistically among the treatments (Fig. 2B).

Scarified seeds of both 2020 C and 2021 C had a noticeable increase in seed weight right after 3 h of imbibition, exceeding 100% weight increase at the end of 240 h in germination conditions (Fig. 3A, B). In contrast, intact seeds had a slower imbibition, with a weight increase only starting after 48 h in germination conditions (Fig. 3A, B). Despite this initial slow imbibition, intact seeds increased seed weight substantially after 240 h, with 83 and 63% of weight increase for 2020 C (P = 0.002, CV = 0.2) and 2021 C (P < 0.001, CV = 1.0) seeds, respectively (Fig. 3A, B). However, the increase in weight for intact seeds was statistically lower than scarified seeds after 240 h of imbibition.

The presence of aril does not prevent water uptake, but it affects the imbibition (Fig. 3B). A high increase in seed weight at the first hours of imbibition occurred, followed by a constant decrease due to aril degradation. Regarding seed size, large seeds had an average seed weight of 0.76 g, whereas small seeds had 0.29 g. Despite having a similar pattern of weight increase, there was a difference in relation to water absorption after 240 h of imbibition between the seed sizes (P = 0.002, CV = 0.3); small seeds had a 95% weight increase while large seeds had 87% (Fig. 3C).

To identify if weight increase occurred in every single intact seed (i.e., not only in the average seed weight), the individual pattern of imbibition for each seed was analysed. Some intact seeds (2020 C and 2021 C) exceeded 100% weight increase after 240 h of imbibition, while most seeds exceeded 50% weight increase. However, few seeds had a little weight increase after 240 h of imbibition. For 2020 C, one seed (from those 25) had only a 2% weight increase, whereas for 2021 C, seven seeds did not surpass a 3% weight increase (Supplementary Data Fig. S1A, B).

Investigation of water entrance in the seeds

Figure 4 details a longitudinally sectioned C. langsdorffii seed after 48 h of immersion in the methylene blue. The dye penetrated the seed exclusively through the hilar region (Fig. 4A, B). At this time, the embryo was not stained, only the seed coat in the hilar region—particularly under the hilum and micropyle (Fig. 4B).

The blockage of seed structures did not prevent water absorption by the seed. However, water uptake rates had little difference among treatments (Fig. 5). Seeds with lens or micropyle sealed had higher weight increase than the other seed structures. Seeds had 117% and 114% weight increase when the micropyle or lens were blocked, respectively. However, control seeds (non-blocked seeds) had a 107% increase in weight. Seeds with the hilar region or hilum + micropyle blocked had a lesser increase in weight (Fig. 5).

Effect of drying and storage on seed germination

The conditions in the drying box were 4.9 ± 1.8% RH and 23 ± 0.5 °C during the evaluation period. The water content for non-dried seeds was 11.4% and dropped down until day 12 (7.9%), and then stabilized until 16 d of drying (Fig. 6A). The increase in weight after 5 d of imbibition following distinct drying periods is shown in Fig. 6A. Non-dried seeds had 49% of weight increase after 5 d imbibition. The increase in weight reduced along with the extension of drying, attained 28% of weight increase after 16 d of drying (Fig. 6A); however, there was no statistical difference amongst drying periods (P = 0.078, CV = 3.1). Additionally, the increase in weight after 10 d of imbibition has no statistical difference following 8, 12 and 16 d of drying, with an increase of 40, 48 and 55% in seed weight, respectively (P = 0.421, CV = 1.0) (Fig. 6B).

Freshly harvested seeds and those stored for 1, 1.5, 2 and 3.5 years had the first seeds germinated between the sixth and ninth days, but the germination of stored seeds reduced drastically (Fig. 7). For non-stored seeds, 71% of germination was attained, whereas 57, 42, 18 and 5% of seeds germinated for 1, 1.5, 2 and 3.5 year stored seeds (P < 0.01, CV = 1.1) (Fig. 7A). The decrease in germination percentage was due to the increase in seed mortality in the germination tests. For non-stored seeds, 16% of seed death was attained, increasing to 36, 40, 75 and 81% after seed storage for 1, 1.5, 2 and 3.5 y, respectively (P < 0.001, CV = 1.3) (Fig. 7B).

Influence of Aril and scarification on seedling emergence

Seedling emergence started in the second week of evaluation for all treatments. Intact (without aril) and scarified seeds had higher emergence in the first weeks compared to the arillate seeds (Fig. 8). In the subsequent weeks, intact seeds still had a higher emergence percentage, reaching 25% after eight weeks, and then seedling emergence has stabilized. There was no difference between scarified and arillate seeds regarding seedling emergence, with higher seedling emergence for intact seeds (P = 0.001, CV = 1.0). For scarified seeds, the emergence stabilized in the fourth week, reaching 15%, whereas for arillate seeds the emergence continued until the tenth week, but also reached only 15% of seedling emergence (Fig. 8). At the end of 60 weeks, there were no intact or imbibed seeds in the pots, only remains of the seed coat.

Seed dormancy is recurrent in leguminous trees, and plant species living in seasonal tropical areas have a significant probability of producing dormant seeds^1,3. Amongst the five seed dormancy classes in Baskin’s classification^2,3, PY is frequently reported for trees, especially for leguminous trees^41,42, and this kind of dormancy is commonly reported for species in seasonal tropical environments, such as the Cerrado^5,6,43. Seeds of C. langsdorffii are quite hard and have slow imbibition (seed weight may not vary after 48 h in germination conditions, or even longer). All information described above seems to lead to a possible presence of dormancy in the seeds. However, our results have confirmed the absence of seed dormancy, exogenous or endogenous, for the studied species. These seeds have a fully developed embryo at the time of dispersal (lacking MD or MPD), and their seed coat is not water-impermeable but regulates imbibition (i.e., does not exhibit PY). High germination in 30 d of evaluation also excludes the low growth potential of the embryo or a possible mechanical restriction to germination (lacking PD).

Wet heat treatments are known to be efficient in breaking dormancy in seeds^44,45,46. Additionally, similar treatments whose C. langsdorffii seeds were subjected has been reported for other species as effective in breaking PY^2,8,34,35,37. Mechanical scarification is one of the most effective treatments to release PY in seeds because it results in removal of a part of the water-impermeable seed coat that prevents germination^2,3. However, all treatments described did not increase germination for C. langsdorffii seeds. Thus, these results corroborate our statement that the investigated species does not have dormant seeds, particularly PY. However, a fraction of seeds still do not germinate within 30 days of germination tests (Fig. 2B). As shown in Supplementary Data Fig. S1A and B, certain seeds exhibited restricted water uptake, displaying only a slight increase in weight even after several days under germination conditions. These seeds likely require an extended period to complete germination.

Imbibition test is fundamental for classifying seed dormancy – without this test we cannot investigate seed impermeability accurately³¹. Imbibition tests must be conducted carefully since seed components such as aril or mucilage can absorb water, leading to a misinterpretation of the test⁹. For C. langsdorffii, intact, arillate or scarified seeds absorbed water during the imbibition test, thus excluding PY. These distinct conditions only affected the water absorption rate (see Fig. 3). The aril speeds up this process, and intact seeds still absorb water, but at a slower rate. Seed scarification also hastens imbibition, indicating that the seed coat controls water uptake; however, this fast imbibition does not mean higher germination percentages (see Figs. 2 and 3). Additionally, there was no acquisition of dormancy following seed drying – PY is only acquired with a reduction of seed water content⁴⁷ – but only delayed water uptake with the prolongation of drying time (Fig. 6A, B).

Distinct types of diaspore tissues impose mechanical resistance and/or impermeability to water in seeds, postponing germination^41,48. Hard (or stony) coat as those presented by the palm dispersal unit (seeds + endocarp) provides a strong mechanical resistance to embryo elongation but does not prevent water entrance into the seeds, only controlling imbibition rate^49,50,51,52. On the other hand, those of Rhus (Anacardiaceae) and Nelumbo (Nelumbonaceae) species act as a water-impermeable barrier to prevent the start of germination^37,53,54. Regarding the germination blockage by water-impermeability coverings (i.e., PY), fruit coat (e.g., pericarp or endocarp) can prevent water absorption by the seeds, as in the plant families Anacardiaceae, Lauraceae and Nelumbonaceae, or (in most cases) the seed coat, as the case of the water-impermeable palisade layer recurrently described for legume plants^{8,34,37,41,55}. Some seeds also have both physical and physiological components of dormancy (PY + PD, combinational dormancy), and in this case, even after breaking PY by scarification, the permeable seeds still do not germinate until releasing PD³. However, the seed coat in C. langsdorffii does not exert enough resistance to hinder germination or prevent water uptake. The juxtaposed palisade layer only limits water entry through most of the seed coat (Fig. 1G), while the hilar region acts as the main permeable zone in the seed, influencing imbibition dynamics (Fig. 4).

Rubio-de-Casas et al.¹ stated that nondormant seeds evolved in climates with long growing seasons or lineages with larger seeds. C. langsdorffii is a species widespread in the tropics, including the Amazon Forest, an environment with a long suitable condition for seedling growth. Additionally, this species produces large seeds. However, even lacking dormancy, the slow germination (due to the limited water uptake by the hard seed coat) may enhance the establishment success for the species inhabiting contrasting conditions, as the highly seasonal region of Cerrado in Brazil (also known as the Brazilian savanna). Slow water absorption prevents germination out of the growing season caused by a short suitable condition due to occasional rainfall. Then, this species did not stagger germination over time by producing seeds with different levels of dormancy (since they are non-dormant), but rather by controlling water flux into the seeds (see Supplementary Data Fig. S1). Regarding persistence in the soil, the high predation rate could be a serious problem for a large seed with a relatively slow germination. However, the physical defensive trait conferred by the thick/hard seed coat in C. langsdorffii associated with the slow imbibition (avoiding the release of olfactory cues) may reduce predation risk, potentially contributing to seed survival. Thus, seeds of the leguminous C. langsdorffii exhibit physical traits that contribute to protection against predation while remaining non-dormant [see Dalling et al.⁵⁶ for seed defence]. This permeable seed is protected from predation but ready to germinate when the conditions for seedling establishment are suitable.