Seed Science Research http://journals.cambridge.org/SSR Additional services for Seed Science Research: Email alerts: Click here Subscriptions: Click here Commercial reprints: Click here Terms of use : Click here Breaking physical dormancy of Cassia leptophylla and Senna macranthera (Fabaceae: Caesalpinioideae) seeds: water absorption and alternating temperatures Alexandre Souza de Paula, Carolina Maria Luzia Delgado, Maria Terezinha Silveira Paulilo and Marisa Santos Seed Science Research / Volume 22 / Issue 04 / December 2012, pp 259 ­ 267 DOI: 10.1017/S096025851200013X, Published online: 26 July 2012 Link to this article: http://journals.cambridge.org/abstract_S096025851200013X How to cite this article: Alexandre Souza de Paula, Carolina Maria Luzia Delgado, Maria Terezinha Silveira Paulilo and Marisa Santos (2012). Breaking physical dormancy of Cassia leptophylla and Senna macranthera (Fabaceae: Caesalpinioideae) seeds: water absorption and alternating temperatures. Seed Science Research, 22, pp 259­267 doi:10.1017/S096025851200013X Request Permissions : Click here Downloaded from http://journals.cambridge.org/SSR, IP address: 150.162.33.156 on 11 Jun 2013 Seed Science Research (2012) 22, 259–267 doi:10.1017/S096025851200013X q Cambridge University Press 2012 Breaking physical dormancy of Cassia leptophylla and Senna macranthera (Fabaceae: Caesalpinioideae) seeds: water absorption and alternating temperatures Alexandre Souza de Paula, Carolina Maria Luzia Delgado, Maria Terezinha Silveira Paulilo and Marisa Santos* Departamento de Botaˆnica, Universidade Federal de Santa Catarina, Floriano´polis 88040-900, Brazil (Received 4 December 2011; accepted after revision 15 June 2012; first published online 26 July 2012) Abstract several types of specialized structures (‘water gaps’) have been found in 12 of the 17 families. The family This study analysed the anatomical structure of the Fabaceae has a large number of species with physically seed coats, identified the location of water uptake and dormant seeds (Villers, 1972; Morrison et al., 1998) and evaluated the effects of alternating temperatures and three types of water gaps are recognized: the lens, hilar heat treatment on the breaking of physical dormancy slit (Baskin et al., 2000) and micropyle (Hu et al., 2008, of two species of Fabaceae (Caesalpinioideae), 2009). Events in nature, such as high and alternating Cassia leptophylla and Senna macranthera,fromthe temperatures, are known to break physical dormancy Atlantic forest of Brazil. The seed coats of both species effectively in some species, allowing water to enter consisted of a cuticle (extra-hilar region) or remaining through a gap (Fenner and Thompson, 2005). Previous funicle region (hilar region), subcuticular layer, pali- studies of species from several environments have sade layer with lignin, osteosclereids, sclerified parenchyma and white cells. The palisade layer was shown that alternating temperatures between 15 and 8 formed by elongated macrosclereids with a light line of 35 C break physical dormancy of species that grow on callose. In thermally scarified seeds of C. leptophylla, dunes in Japan (Kondo and Takahashi, 2004), of the water entered through the micropylar canal, and in agricultural weed Ipomoea lacunosa (Jayasuriya et al., S. macranthera the water entered through the lens. 2007) and in tree species that grow in gaps in a rain Alternating temperatures that ranged from 15 to 308C forest in Mexico (Va´zquez-Yanes and Orozco-Segovia, did not break physical dormancy of either species; 1982), in non-climax tree species from the evergreen however, exposure to 508C broke seed hardcoated- Atlantic forest (Souza et al., 2012) and a semi- ness, allowing the entrance of water in both species. deciduous forest in Brazil (Abdo and Paula, 2006). When exposed to alternating temperatures of 74/158C and 60/158C, the highest temperatures broke physical Keywords: Fabaceae, hardcoatedness, lens, physical dormancy of seeds of species from an arid region of dormancy, water intake Australia (Quinlivan, 1966). Among the Fabaceae, studies of physical dormancy of seeds have mainly focused on species within the Introduction subfamily Faboideae because this group contains important agricultural legumes (Baskin and Baskin, Physical dormancy is determined by the imperme- 2001). There are fewer studies on the subfamily ability of seed coats to water, which is caused by the Caesalpinoideae and studies on effects of alternating presence of one or more layers of Malpighian cells that temperatures to break physical dormancy are rare in are tightly packed together and impregnated with seeds of tree species from Brazilian forest ecosystems, water-repellant substances, such as lignin, callose and such as the Atlantic rain forest, one of the most threatened wax (Baskin, 2003; Smith et al., 2003). Seeds of species ecosystems in Latin America (Myers et al., 2000). with physical dormancy are known in 17 families of Knowledge about the ecology of seed germination angiosperms (Gama-Arachchige et al., 2010) and of species from the Atlantic rain forest is important for conservation programmes. Therefore, the goal of this work was to study how physical dormancy is *Correspondence broken in two tree species from the Atlantic rain Email: [email protected] forest that belong to the Caesalpinoideae. The taxa 260 A.S. de Paula et al. chosen, Cassia leptophylla and Senna macranthera,are using an Olympus BX41 microscope (Olympus Corp., non-climax tree species that were selected because of Tokyo, Japan), with a mercury vapour lamp (HBO 100) their commercial and ecological importance and and a blue epifluorescence filter (UMWU2), at an because mature seeds of these taxa were available excitation wavelength of 330–385 nm and a 420 nm during the period of the experiment. These species are emission wavelength. Images were taken with a commercially valuable because of their wood, and Q-imaging digital camera (3.3 megapixel QColor 3C) C. leptophylla is also used in the production of honey and and the software Q-captures Pro 5.1 (Q-Imaging, Surrey, S. macranthera in the regeneration of degraded areas British Columbia, Canada). (Carvalho, 2006). This work focused on the following: Some seeds were subjected to the process of cell (1) the structure and chemical composition of the dissociation (Franklin, 1945, modified by Kraus and integument of the seeds; (2) the structure through which Arduin, 1997). Dissociated samples were stained with water enters the seed; and (3) whether the breaking of toluidine blue, mounted on slides with water and the physical dormancy in seeds is due to the high or cover slips, and examined under a light microscope. the alternating temperature applied, similar to con- The surface of the hilar region was examined with a ditions required for other species of tropical rain forests. scanning electron microscope (SEM) to verify the effect of thermal scarification. Seeds were immersed in water for 2minat968C and were then cut in half (the endosperm Materials and methods was removed, because it is rich in oleaginous sub- stances). The sections of the treated and untreated seeds Seed collection were then stored in a glass bottle, with silica gel, to dry for a month. The dried samples were adhered to Fruits of Cassia leptophylla Vogel and Senna macranthera aluminium supports with double-sided carbon tape, var. macranthera (DC. ex Collad.) H.S. Irwin & Barneby and sputter coated (using a Leica EM 500 SCD) were collected from trees growing in Bosque do with 20 nm of gold. The samples were analysed using a Alema˜o, in the city of Curitiba, Parana´, Brazil Jeol XL30 SEM. Five thermally scarified seeds and 0 00 0 00 (25824 22.89 S, 49817 8.77 W) in July 2009. Seeds were five untreated seeds, for each species, were analysed. removed from the fruits and stored in glass bottles, at room temperature, until they were used. The average mass of each seed was 0.17 g for C. leptophylla and Localization of water entrance in the seeds after 0.041 g for S. macranthera. thermal scarification To verify the path of water entry by treated seeds, a dye Structural analysis of the seed was used, as proposed by Jayasuriya et al. (2007). For this, 20 seeds of each species were submitted to thermal For the morphological analysis, seeds were observed scarification in water, for 2 min at 968C. The 20 scarified using a stereoscopic microscope (Leica EZ4D, Leica seeds and 20 non-scarified seeds (control) were then Microsystems, Wetzlar, Germany) and images were immersed in an aqueous solution of 1% aniline blue. taken with a Sony digital camera. For analysis of the seed The scarified and non-scarified seeds were removed coats, seeds were adhered to a wooden block, with Super after intervals of 15 min, 30 min, 1 h, 2 h and 3 h of Bonderw (Henkel ltda., Itapevi, Sa˜o Paulo, Brasil), and imbibition for analysis, four seeds were removed per the hilar and extra-hilar regions were longitudinally and interval for each treatment. For this analysis, the hilar transversely sectioned (40 mmthick)withasliding and extra-hilar regions of the seeds were sectioned microtome (Micron HM400, Micron, Boise, Idaho, USA). longitudinally. The sections were analysed using a light Some sections were prepared with only water microscope (Leica EZ4D and Leica MPS 30 DMLS) and (control) and others were exposed to the following imaged with a Sony digital camera. histochemical reagents: Sudan IV for suberine, cutin, oils and waxes (Costa, 1982); acid phloroglucinol or iron chloride for lignin (Costa, 1982); toluidine blue for Seed germination and breaking of physical polychromatic reactions to lignin and cellulose dormancy by alternating and high temperatures (O’Brien et al., 1965); and ruthenium red for pectic substances (Gerlach, 1984). Samples were examined Intact seeds were sterilized by immersing them in 5% under a light microscope (Leica DMLS MPS 30) and sodium hypochlorite for 5 min, followed by washing images were taken with a Sony digital camera. them three times in distilled water. The seeds were To verify the presence of callose in the seeds, sections then placed in transparent plastic boxes on two were immersed in 0.05% aniline blue with a 0.1 M sheets of filter paper moistened with distilled water.