Dormancy-breaking requirements of Sophora tomentosa and Erythrina speciosa (Fabaceae) seeds Requisitos para romper la latencia en semillas de Sophora tomentosa y Erythrina speciosa (Fabaceae)
Carolina Maria Luzia Delgado1*, Alexandre Souza de Paula1, Marisa Santos1 & Maria Terezinha Silveira Paulilo1
Abstract
The physical dormancy of seeds has been poorly studied in species from tropical forests, such as the Atlantic Forest. This study aimed to examine the effect of moderate alternating temperatures on breaking the physical dormancy of seeds, the morphoanatomy and histochemistry of seed coats, and to locate the structure/region responsible for water entrance into the seed, after breaking the ]]>
Sophora tomentosa and Erythrina speciosa. To assess temperature effect, seeds were ]]>
S. tomentosa or E. speciosa. These results may indicate that these seeds require larger fluctuation of temperature than the applied or/and longer period of exposition to the temperature fluctuation. Blocking experiments water inlet combined with SEM analysis of the structures of seed coat for both species showed that besides the lens, the hilum and micropyle are ]]>
E. speciosa the immersion of scarified seeds into an aniline aqueous solution showed that the solution first entered the seed through the hilum. Both species showed seed morphological and anatomical features for seed coats of the subfamily Faboideae. Lignin and callose were found around all palisade layers and the water ]]>
Key words: physical dormancy, seeds, tropical woody Fabaceae, water gaps.
Resumen
La latencia física de las semillas ha sido poco estudiada en las especies de los bosques tropicales, como el bosque atlántico. Este estudio tuvo como objetivo examinar el efecto de las temperaturas moderadas alternantes en romper la latencia física de las ]]>
Sophora tomentosa y Eythrina speciosa, dos especies ]]>
S. tomentosa o E. speciosa. Estos resultados pueden indicar que estas ]]>
E. speciosa la inmersión de semillas escarificadas en una solución acuosa de anilina mostró que la solución entró por primera vez a la semilla a través del hilio. Ambas especies mostraron características morfológicas y ]]>
Palabras clave: latencia física, semillas, Fabaceae tropicales. Mature seeds of many plant species, particularly those in the Fabaceae, do not germinate readily under favorable environmental conditions because they are impermeable to water and/or gases (Argel, & Paton, 1999; Bewley, & Black, 1994). This type of impermeability is referred to by Baskin and Baskin (2001) as physical dormancy and is caused by a layer of palisade cells in the seed coat, which have thick, lignified secondary walls (macrosclereids) containing hydrophobic substances ]]>
In physically dormant seeds there are specialized anatomical structures in the coat that develop an opening where water can enter (Gama-Arachchige, Baskin, Geneve, & Baskin, 2013). Under natural conditions, it is also known that temperature is an important environmental factor that influences the breaking of physically dormant seeds (Kondo, & ]]>
The family Fabaceae is the third largest family of angiosperms and the second most economically important family (Judd,Campbel, Kellongg, Steens, & Donogue, 2009). Several species of this group play a vital role in global biogeochemistry because they have nodules with ]]>
This study utilized two species of Fabaceae (subfamily Faboideae), Sophora tomentosa L. and Erythrina speciosa Andr., which have physically dormant seeds. Sophora tomentosa occurs in restinga (Brito et al., 2010), which is a sunny and windy environment near the sea that has water and nutrient shortages (Bresolin, 1979). This species has autochorous and hydrochorous seed dispersal (Bechara, 2003), and produces mature fruits throughout the year but mostly in the summer (Dec-Feb) when there is more rain (Brito ]]>
Erythrina speciosa is a neotropical tree distributed throughout the Southern and Southeast regions of Brazil (Lorenzi, 2002). It is typically found in fluvial forest and coastal moist plains, as well as flood-prone habitats, and is always in open and secondary formations (Klein, 1969; Medina et al., ]]>
This work focused on the following aspects: 1) the structure and chemical composition of the seed coats, 2) the structure of the region where water enters the seeds, and 3) the effect of moderate alternating temperatures on breaking the physical dormancy of the seeds.
Materials and Methods
Collection of seeds: Seeds of Sophora tomentosa were collected at Daniela’s beach in July, ]]>
Erythrina speciosa in a fragment of Atlantic Forest in November, 2009. Both areas are located in the municipality of Florianópolis, Santa Catarina, Brazil (27º35’36’’ S - 48º35’60’’ W). An approximate quantity of one thousand seeds were collected from numerous plants and used in the study. ]]>
Germination of seeds: Intact seeds were sterilized by immersing them in 5% sodium hypochlorite for five minutes and then washed three times in distilled water. The seeds were placed in transparent plastic boxes on two sheets of filter paper (Whatman No.1, Whatman International Ltd. Maidstone, England) moistened with distilled water. Intact seeds were incubated at 15°C, 20°C, 25°C, 30°C and 35°C and a 12h/12h alternating temperature ]]>
S. tomentosa and E. ]]>
scarified in water at 98°C for 5min and 15min, respectively, were used in the experiments.
Identification of the site where water enters the seeds: Hot water scarified seeds had parts ]]>
Sophora tomentosa seed coats were blocked in the following regions: a) micropyle plus hilum and lens, b) micropyle plus hilum, and c) lens. Erythrina speciosa seed coats were blocked in the a) micropyle plus hilum plus ]]>
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Seed coat features: The hilar region of five intact seeds of both species were cut with a scalpel and fixed in 2.5% glutaraldehyde, in a 0.1M sodium phosphate buffer at pH 7.2, and dehydrated in a graded ethanol series. Sections that were 40µm thick were cut using a sliding microtome. Histochemical tests were then made utilizing Sudan IV (2g/ ethanol 95% 100mL/gliceryne 5mL) for suberin, cutin, oils and waxes; acid ]]>
S. tomentosa and E. speciosa after 5min and 15min, respectively, in water at 98ºC) were dried according Horridge and Tamm (1969) with a CO2 critical point dryer (Leica EM CDP 300). The dried samples were adhered to aluminum stubs, with double-sided carbon tape, and coated with 20nm of gold using a Leica MS SCD500 sputter coater. Samples were observed and documented using a Jeol (model XL30 JSM 6390 LV) scanning ]]>
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Dye tracking of water movement in the hilar pad: Forty seeds of each species (20 hot water scarified and 20 non-scarified) were immersed in an aqueous solution of 1% aniline blue (modified from Jayasuriya, Baskin, & Baskin, 2007). After 15min, 30min and 60min, five seeds of each treatment of each species were transversely and longitudinally sectioned through the hilum area. The sections (40µm thick) were made with a sliding ]]>
A completely randomized design was used for the germination experiments for data analysis. Arcsine-transformed germination data were analyzed using one-way ANOVA with the software Bioestat (Ayres, Ayres, Ayres, & Santos, 2005). Tukey’s test was performed to compare treatments after ANOVA. ]]>
Results
Seed germination: For intact seeds of Sophora tomentosa
and Erythrina speciosa incubated at 30ºC, the germination was around 13% and 2%, respectively, while 100% of the scarified seeds of both species germinated. For intact seeds of both species there was no germination at the lowest temperature tested (15°C) with the exception of 1.66% in one experiment using
S. tomentosa (Table 1). At the upper limit temperature (35ºC) S. tomentosa seeds germinated but those of E. speciosa
did not. The alternating temperatures studied did not promote germination of the species in relation to the constant temperature.
Identification of the site where water enters the seeds: Treatments utilizing glue to block the hilar region of scarified seeds showed, for both species, that the lens, micropyle and hilum are involved in water absorption (Fig. 1). In non-blocked seeds of S. tomentosa (Fig. 1a) treated with hot water, almost 100% absorbed water after 10 days, but when the hilar region was blocked only 10% of the seeds absorbed water. When the hilum-micropyle complex or when the lens was blocked, almost 90% of seeds absorbed water, showing that all of these structures need to be blocked for there to be a large reduction in water intake. In heat-treated and non-blocked seeds of E. speciosa (Fig. 1b) about 90% had imbibed water after 12 days, but when the hilar region was blocked only 10% of the seeds absorbed water. In seeds where the hilum, lens, and micropyle were blocked separately, 35%, 50% and around 90%, respectively, of the seeds imbibed water, which indicates that the hilum and lens were only partly effective in inhibiting water uptake and the micropyle was completely ineffective. However, during the first four days of incubation, blocking the micropyle greatly reduced water uptake compared to seeds without a blocked micropyle.
Seed coat features: In the seed coats of S. tomentosa and E. speciosa the hilum, lens and micropyle are in line, the lens and micropyle are on opposite sides of the hilum, and the middle of the hilum has a groove (Fig. 2a, Fig. 2b, Fig. 2c, Fig. 2d). In S. tomentosa
the hilum covers the micropyle. The seed coat structure of the two species is very similar and consists of the extra-hilar region with one layer of columnar palisade cells, one layer of osteosclereids, spongy tissue and crushed cells. The columnar palisade cells are covered by a thick cuticle. In the hilar region there are two layers of palisade cells, the counterpalisade layer and, underneath, the palisade layer. A light line can be seen at the top of the palisade layer in both the hilar and extra-hilar regions. Tracheid bars are visible in the spongy tissue. Histochemical experiments showed ]]>
S. tomentosa (Fig. 2a) and E. speciosaseeds (Fig. 2c) revealed that in all non-treated seeds the tissues of this region remained intact with no disruptions. However, S. tomentosa seeds ]]>
Fig 2b). In scarified seeds of E. speciosa the lens region ruptured and the hilum grove was more extended compared to the control (Fig. 2d). Dye treatments used to visualize the water movement in the hilar ]]>
S. tomentosa; however, in seeds of E. speciosa the dye penetrated the lens and moved toward the vascular bundle (Fig. 2e). ]]>
Discussion
Studies of species from tropical forests have shown that alternating temperatures of 15ºC and 20ºC (minimum) and 30ºC and 40ºC (maximum) effectively broke the ]]>
S. tomentosa or E. speciosa. Temperature fluctuation in Brazilian dunes can be over 25ºC (Franco et ]]>
The lens has been reported as being the site of initial water intake in physically dormant seeds of Fabaceae species (Baskin, Baskin, & Li, 2000; Morrison, McClay, Porter and Rish, 1998; Serrato-Valenti, Vries, & Cornara, 1995; Souza et al., 2012). However, in seeds of S. tomentosa, blocking only the lens or the micropyle-hilum complex does not prevent the entry of water into a scarified seed, which indicates that both structures are equally related to the entry of water. This is further supported because there is a significant inhibition of the entry of water into the scarified seed only when these two structures are blocked. The SEM analysis of E. speciosa showed that in scarified ]]>
Sophora alopecuroides evidence of water intake through the hilum after sulphuric acid scarification of seeds (which showed a wider hilum fissure than non-scarified seeds) and in seeds buried in the field. However, they observed that depending on ]]>
S. tomentosa and E. speciosa in the present work, it was interesting to notice that when only the micropyle ]]>
For both species studied, the major features of the seed coat were similar, despite the differences in humidity and sunlight that occur where they grow. In fact, as discussed by Souza and Marcos-Filho (2001), the morphological features of the Fabaceae seed coat are relatively insensitive to environmental conditions and contain distinct taxonomic features. Both ]]>
S. tomentosa the micropyle is covered by the hilum as observed by Hu et al. (2008) in
S. alopecuroides. Lignin and callose, found around all palisade layer(s), are reported to be impermeable to water and would be responsible for the impediment of water absorption by the physically dormant seeds of Fabaceae (Baskin et al., 2000; Ma,Cholewa, Mohamed , Peterson, & Gijzen, 2004; Varela, & Albornoz 2013) suggest the permeability of Anadenanthera colubrina var. cebil seeds to water is because of the absence of lignin in the palisade layer. Lignin is also ecologically important because it protects the seed against predation (Souza and Marcos Filho, 2001). According to (Dalling Davis, Schutte, & ]]>
S. tomentosa because the fruits of this species are indehiscent and remain on the plant for several months, which makes them and the seeds within them ]]>
E. speciosa, a species found in wet areas with autochorous seed dispersal, the seeds can fall to the wet ground and stay there for a long time. In this case, the presence of lignin is very important because it prevents microbial access. Strong seed coat impermeability also protects against deterioration as seeds age (Brancalion, Novembre, Rodrigues, & Marcos Filho, 2010).
Acknowledgments
This study received financial support from Coordenação de Aperfeiçoamento do Ensino Superior (CAPES), Brazil. ]]>
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1. Departamento de Botânica, Universidade Federal de Santa Catarina, Florianópolis-SC, Brasil; delgado_carol@yahoo.com.br, alexandredepaula_07@hotmail.com, marisa.santos@ufsc.br, paulilo@ccb.ufsc.br
Received 18-III-2014. Corrected 08-IX-2014. Accepted 10-X-2014.