Seed Science Research, page 1 of 8 doi:10.1017/S0960258512000013 q Cambridge University Press 2012

Water absorption and dormancy-breaking requirements of physically dormant seeds of parahyba ( – )

Thaysi Ventura de Souza, Caroline Heinig Voltolini, Marisa Santos and Maria Terezinha Silveira Paulilo* Departamento de Botaˆnica, Universidade Federal de Santa Catarina, Floriano´polis 88040-900, Brazil

(Received 14 July 2011; accepted after revision 18 January 2012)

Abstract seeds have been a source of controversy (Fenner and Thompson, 2005; Finch-Savage and Leubner-Metzger, Physical dormancy refers to seeds that are water 2006). A definition of dormancy that has been impermeable. Within the Fabaceae, the structure proposed recently is that dormancy is an innate seed associated with the breaking of dormancy is usually property determined by genetics that defines the the lens. This study verified the role of the lens in environmental conditions in which the seed is able to physical dormancy of seeds of Schizolobium para- germinate (Finch-Savage and Leubner-Metzger, 2006). hyba, a gap species of Fabaceae from the Atlantic Five classes of seed dormancy are recognized, and one Forest of Brazil. The lens in S. parahyba seeds of them is physical dormancy (Baskin and Baskin, appeared as a subtle depression near the hilum and 2004), which is caused by a seed (or fruit) coat that opposite the micropyle. After treatment of the seeds prevents absorption of water (Morrison et al., 1998; with hot water, the lens detached from the coat. Baskin and Baskin, 2001; Smith et al., 2002). Blocking water from contacting the lens inhibited water Physical dormancy is known to occur in 17 families absorption in hot-water-treated seeds. High constant (308C) and alternating (20/308C) temperatures pro- of angiosperms, including the Fabaceae (Baskin and moted the breaking of physical dormancy and Baskin, 2000; Funes and Venier, 2006), where it occurs in germination in non-scarified seeds. Maximum percen- many species. Water-impermeability of the coat (or in tage of germination occurred earlier for seeds some species the fruit coat) is caused by the presence of incubated at 20/308C than for those incubated at one or more layers of elongated, lignified Malpighian 308C. Seeds with a blocked lens did not germinate at cells that are tightly packed together and impregnated alternating or high temperatures. This study suggests with water-repellant chemicals (Morrison et al., 1998; that alternating temperatures are probably the cause Baskin and Baskin, 2001; Smith et al., 2002; Baskin, of physical dormancy break of seeds of S. parahyba in 2003). Under natural conditions, it has been suggested gaps in the forest. that physical dormancy is not broken by seeds passing through the digestive tracts of an animal or by cracks in the coat caused by animals (Baskin and Baskin, 2001; Keywords: Fabaceae, lens, physical dormancy, water Fenner and Thompson, 2005). One characteristic that uptake suggests this hypothesis is correct is the presence of a specialized anatomical region in physically dormant seeds that develops an opening where water can enter Introduction the seeds (Baskin and Baskin, 2001). Several types of specialized structures (‘water gaps’) have been found in For , it is important that seed germination occurs 12 of the 17 families that have physical dormancy; for in the right place and at the right time, and, for this example, the carpellary micropyle in Anacardiaceae; reason, most species have mechanisms that delay the bixoide chalazal plug in Bixaceae, Cistaceae, germination, such as seed dormancy (Fenner and Cochlospermaceae, Dipterocarpaceae and Sarcolaena- Thompson, 2005). The definitions of dormancy in ceae; the imbibition lid in Cannaceae; the chalazal plug in Malvaceae; the lens and hilar slit in Fabaceae (Baskin et al., 2000) and the micropyle-water gap complex in *Correspondence Geraniaceae (Gama-Arachchige et al., 2011). However, Email: [email protected] in some Fabaceae (subfamilies Caesalpinioideae and 2 T.V. de Souza et al.

Mimosoideae) the lens is absent (Gunn, 1984, 1991) and 1981; Matheus and Lopes, 2007), the seed coats were after treating some legume seeds to break physical made impermeable in four ways: (1) extrahilar region dormancy, cracks develop in the extrahilar region blocked with paraffin; (2) hilar region blocked with or in the hilum that permit entrance of water into the paraffin; (3) hilum blocked with Super Bonderw glue seeds (Hu et al., 2008, 2009). (Henkel, Jundiai, Brazil); and (4) lens blocked with Several artificial techniques are used to break physical Super Bonderw glue. A control group was of non- dormancy in seeds, including mechanical, thermal and dormant, non-blocked seeds. Twenty seeds were chemical scarification, enzymes, dry storage, percussion, utilized for each treatment. Seeds were placed in low temperatures, radiation and high atmospheric transparent plastic boxes of 11 £ 11 £ 3.5 cm on two pressures (Baskin and Baskin, 2001). Studies on seeds layers of filter paper (Whatman No. 1, Whatman with physical dormancy have contributed greatly to International Ltd, Maidstone, England) with 10 ml of our understanding of water gaps, the effects of various distilled water. The boxes were stored at 208C with a factors (e.g. drying, heating, low temperatures and photoperiod of 12 h/12 h. Incubated seeds were alternating temperatures) in breaking physical dor- counted at intervals of 2 or 3 d for 19 d, during which mancy under natural conditions, and the rate and path time germination was observed. of water entrance into seeds that have become permeable (Baskin and Baskin, 2001). Under natural conditions, it is Analysis of seed coat features known that temperature is an important environmental factor for breaking physical dormancy in seeds (Baskin The hilar regions of five intact and five thermally and Baskin, 2001). Va´zquez-Yanez and Orozco-Segovia scarified seeds were fixed in 2.5% glutaraldehyde (1982) verified that the highly fluctuating temperature in a 0.1 M sodium phosphate buffer at pH 7.2 and that occurs in gaps, but not in forest understorey, breaks dehydrated in a graded ethanol series. Sections of physical dormancy in gap forest species. 40 mm thickness were cut using a sliding microtome. (Fabaceae–Caesalpinioideae) Histochemical tests were made utilizing Sudan IV for is a pioneer woody species from the Atlantic Forest of suberin, cutin, oils and waxes; acid phloroglucinol Brazil that occurs mostly in gaps and along forest and iron chloride for lignin (Costa, 1982); and toluidine borders, with physically dormant seeds and anemo- blue for polychromatic reactions to lignin and cellulose choric seed dispersal (Carvalho, 2003). The imperme- (O’Brien et al., 1965). Images were taken with a digital able seed coat of this species can be broken artificially camera connected to an optical microscope (Leica MPS by boiling water or mechanical scarification (Caˆndido 30 DMLS). For scanning electron microscopy (SEM) et al., 1981; Freire et al., 2007; Matheus and Lopes, 2007). analyses, the dehydrated pieces of five intact and five The aim of this work was to study the seeds of scarified seeds were immersed in hexamethyldesila- S. parahyba with the objectives of: (1) locating the water sane (HMDS) for 30 min, as a substitute for critical gap in the seeds; (2) describing the anatomical point drying (Bozzola and Russell, 1991) and then structure of the water gap; and (3) testing the effect mounted on aluminium stubs and blocked with a of alternating temperatures on breaking the physical gold layer (40 nm thick). The pieces were viewed using dormancy of the seeds. a Jeol JSM 6390 LV scanning electron microscope. To verify the presence of callose in the seeds, sections of non-fixed samples of the hilar and extrahilar Materials and methods regions of five intact seeds were immersed in 0.05% aniline blue with a 0.1 M potassium phosphate buffer Seed collection at pH 8.3 (Ruzin, 1951). As a control, some sections were immersed only in the potassium phosphate buffer. The Seeds of S. parahyba, which remain enclosed in the sections were observed using an Olympus BX41 similar-shaped papery envelope of endocarp resembling microscope, with a mercury vapour lamp (HBO 100) a wing, were collected from the ground soon after wind and a blue epifluorescence filter (UMWU2), at 330– dispersal, during spring, in a section of Atlantic Forest 385 nm excitation and 420 nm emission wavelengths. located in the municipality of Florianopolis, Santa Images were taken with a Q-imaging digital camera 0 00 0 00 Catarina, Brazil (27835 36 S, 48835 60 W). The endocarp (3.3 mpixel QColor3C) and the software Q-captures was removed, and the seeds were stored in plastic bottles Pro 5.1 (Q Images, Surrey, British Columbia, Canada). at room temperature until they were used. Effect of alternating temperatures on germination Location of the water entrance region and dormancy break

After artificially breaking dormancy of the seeds by Seeds were immersed in 5% sodium hypochlorite for placing them in water at 988C for 1 min (Caˆndido et al., 5 min and then washed three times in distilled water. Physical dormancy in seeds of Schizolobium 3

For some of the seeds, the region with the lens was with the blocked lens was only 1.0%. However, 50% of covered using Super Bondw glue, which made the the scarified seeds with only the hilum blocked seeds impermeable. Then the seeds were placed in germinated (Fig. 1). The germination levels at the last transparent plastic boxes on a 5 cm autoclaved layer of day of incubation were similar for seeds blocked in the sand moistened with distilled water. The boxes were lens and in the hilar regions, but significantly different stored at 208C, 308C and a 12 h/12 h alternating for scarified seeds and scarified seeds blocked in the temperature regime of 20/308C with a photoperiod of extrahilar region and hilum (P # 0.05). 12 h. Four boxes, each with 20 blocked seeds and another four, each with 20 non-blocked seeds, were Analysis of seed coat features used for each treatment. Germinated seeds were counted at intervals of 2 or 3 d for 29 d. To verify the In S. parahyba, the hilar region is near the wide end of effect of the temperature on the breaking of dormancy the seeds and consists of the hilum, micropyle and of the seeds, three boxes with 20 seeds (of known mass) lens, with the hilum positioned between the micropyle were stored at 208C, 308C, and at a 12h/12h alternating and lens (Fig. 2a, b). temperature of 20/308C with a daily photoperiod of The seed coat consists of one layer of thick walled, 12 h. Every day the mass of the seeds was measured tightly packed, columnar palisade cells (macroscler- until the beginning of germination. The mass of each eids or Malpighian cells) and sclerenchymatous tissue; seed, after and before the incubation period, was used osteosclereids (‘hourglass cells’) are not present to calculate the amount of absorbed water. (Fig. 2c–f). The seed coat is covered by a thin cuticle. In front view, the macrosclereid cells have a hexagonal Data analysis shape (Fig. 2d). The palisade layer is thinner in the lens region than in the rest of the coat (Fig. 2e). It is possible A completely randomized design was used in all to see a light line crossing the macrosclereids in the experiments. Arcsine-transformed germination data palisade layer (Fig. 2e, f). Below the sclerenchymatous were analysed using one-way ANOVA with the tissue and above the endosperm is the tegmen, formed software Statistica (Statsoft, 2001). Tukey’s tests were by a layer of crushed cells with thin walls between two performed to compare treatments. cuticle layers, which reacted positively to Sudan IV (not shown). The macrosclereids and the subjacent sclerenchymatous tissue reacted negatively for lignin Results when exposed to iron chloride, phloruglucinol and toluidine blue. However, the macrosclereids reacted Location of the water entrance positively for cellulose when exposed to toluidine blue. The cuticle reacted positively to Sudan IV. The upper portion of the macrosclereids, mainly After 19 d of incubation, germination of scarified seeds the light line, showed aniline blue-induced fluore- exposed to boiling water, as well as the germination of scence, indicating the presence of callose (Fig. 3a). scarified seeds with the blocked extrahilar region, was In non-scarified seeds, the lens is a slight depression at about 80% (Fig. 1). Germination of scarified seeds with the side of the hilum and opposite the micropyle the blocked hilar region (i.e. the hilum plus lens) and (Fig. 3b). In thermally scarified seeds, the hilum and micropyle do not show alterations, but in the lens 100 Control Extrahilar Hilar Hilum Lens 90 region a crack forms between the macrosclereids, 80 70 exposing the underlying tissue (Fig. 3c). 60 50 40 30 Effect of alternating temperatures on dormancy Germination (%) 20 break and germination 10 0 012345678910111213141516171819 Seeds incubated at alternating temperatures of Days of incubation 20/308C and 308C absorbed about 10 g of water during 3 d of incubation, while those at 208C did not absorb Figure 1. Germination curves for seeds of Schizolobium water (Fig. 4). The amount of absorbed water on the parahyba that were thermally scarified (control) and with third day of incubation was similar for seeds incubated extrahilar, hilum and lens regions blocked after scarification. Germination at day 19 of incubation was not significantly at 20/308C and 308C (about 10 g) but significantly different for seeds with blocked lens and hilar regions, but different for seeds at 208C(P # 0.05). significantly different for scarified seeds and scarified seeds Seeds incubated at alternating temperatures of with blocked extrahilar region and hilum (Tukey’s test, 20/308C reached the maximum percentage of germi- P # 0.05). Bars indicate standard deviation. nation (about 80%) after 13 d of incubation, while those 4 T.V. de Souza et al.

(a) (b) mi hi le

mi hi le

µ 0.5 µm 400 m

(c) (d)

mc

20 µm 5 µm

(e) (f)

le mc mc

sc sc 100 µm 200 µm

Figure 2. Optical micrographs (OM) and scanning electron micrographs (SEM) of the seed coat of Schizolobium parahyba: (a) front view of the hilar region (SEM); (b) longitudinal section of the hilar region (OM); (c) extrahilar region of seed coat, showing macrosclereids (SEM); (d) front view of macrosclereids (SEM); (e) longitudinal section of peripheral tissues of hilar region showing palisade layer, subjacent sclerenchymatous tissue and shorter macrosclereids in the region of the lens; arrow points to the light line (OM); (f) longitudinal section of lens region showing palisade layer and sclerenchymatous tissue; arrow points to the light line (OM). le, lens; hi, hilum; mc, macrosclereids; mi, micropyle; sc, sclerenchymatous tissue. incubated at a constant temperature of 308C reached entrance into seeds after physical dormancy is broken only 7% of germination in the same period. The (Hyde, 1954; Zeng et al., 2005; Hu et al., 2008), as well as percentages of germination at 20/308C and 308C at the cracks in the cuticle of the seed coat (Morrison et al., last day of incubation were not significantly different, 1998; Hu et al., 2009). Water entrance in the region of the but both were significantly different from germination lens has been reported for legume seeds by several at 208C(P # 0.05) which was less than 2% (Fig. 5). authors (Dell, 1980; Hanna, 1984; Van Staden et al., 1989; Seeds with blocked lens did not germinate. Serrato-Valenti et al., 1995; Morrison et al., 1998; Baskin et al., 2000; Burrows et al., 2009; Hu et al., 2009). The seeds of S. parahyba lack a conspicuous lens, as Discussion observed by Gunn (1991) for the subfamily Caesal- pinioideae, and it is distinguished on the seed coat as a Location of the water entrance region subtle depression close to the hilum and opposite the micropyle. Our experiments in which the hilar and In physically dormant seeds, dormancy break involves extrahilar regions of thermally scarified seeds were disrupting an impermeable seed (fruit) coat, thereby blocked, and also the SEM images of the lens region creating an opening for water to enter (Baskin and after breaking seed dormancy with boiling water, Baskin, 2001). However, the initial site where water showed that the lens is the only region involved in the enters after physical dormancy is broken varies in the absorption of water in seeds of S. parahyba. In the study Fabaceae (Hu et al., 2008; Valtuen˜a et al., 2008). The where dormancy was broken by alternating tempera- hilum and micropyle have been reported to allow water ture, the seeds that had blocked lenses did not Physical dormancy in seeds of Schizolobium 5

18 (a) 16 20°C 30°C 20/30°C 14 12 10 8 6 Imbibition (g) 4 2 0 0123 Days of incubation * Figure 4. Imbibition curves (water absorbed in grams) for intact seeds of Schizolobium parahyba incubated at 208C, 308C 100 µm and 20/308C. Imbibition at the third day of incubation was similar for seeds incubated at 308C and 20/308C, but (b) significantly different for seeds at 208C (Tukey’s test, P # 0.05). Bars indicate standard deviation. did not affect the location where the water initially entered, which was always through the lens. Unfortu- nately, there are no data on this subject in the literature mi hi le about the genus Schizolobium.

Anatomical structure of the seed coat

Our study showed that the seed coat of S. parahiba is 200 µm composed of a coat and tegmen. The coat originates from the outer integument of the ovule and the tegmen from (c) the inner integument (Corner, 1951). It is considered an exotestal seed because the main mechanical layer of the coat lies in the outer epidermis of the outer integument (Corner, 1951). As described for other species of Fabaceae mi hi le (Corner, 1951), the coat of S. parahyba consists of a layer of palisade cells with thick walls, that are packed tightly together, a light line and sclerenchymatous tissue. However, the layer of osteosclereid cells (also called ‘hourglass cells’) that usually lies below the palisade layer is not present. Smith et al. (2002) reported that ‘hourglass cells’ are not universally present in Fabaceae. 500 µm The light line lies just beneath the cuticle, as in Glycine max (Harris, 1983; Ma et al., 2004), but in Figure 3. Morpho-anatomical aspects of the seeds of S. parahyba, this line crosses the palisade layer in the Schizolobium parahyba. (a) Longitudinal section of the tegument middle third of the macrosclereids, as in other species stained with aniline blue. Arrow indicates the light line with high fluorescence and upper portion of the macrosclereı´ds. 100 20°C 30°C 20°C/30°C 90 Asterisk indicates the sclerenchymatous tissue which did not 80 show fluorescence. (b) Front view of hilar region of a seed (not 70 60 thermally scarified). (c) Thermally scarified seed, showing 50 cracks (arrow) between the macrosclereids and subjacent tissue 40 30 Germination (%) of the lens. le, lens; hi, hilum; mi, micropyle. 20 10 0 germinate, indicating that the lens is broken by 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829 alternating temperature. However, Hu et al. (2009) Days of incubation obtained results that indicated that the primary site of Figure 5. Germination curves for intact seeds of Schizolobium water entry, after the breaking of physical dormancy, parahyba incubated at 208C, 308C and 20/308C. Germination can vary for Vigna oblongifolia and that it depended on on day 29 of incubation was not significantly different for the treatment (boiling water or sulphuric acid). For seeds at 308C and 20/308C, but significantly different for Sesbania sesban, however, Hu et al. (2009) found that the seeds at 208C (Tukey’s test, P # 0.05). Bars indicate standard treatment method used to break physical dormancy deviation. 6 T.V. de Souza et al. of Fabaceae (Serrato-Valenti et al., 1995; Leython and seeds were exposed to alternating temperatures of Ja´uregui, 2008). The origin of the light line has been 20/308C and a constant temperature of 308C. Germina- discussed by many authors, and Kelly et al. (1992) tion of the seeds also occurred at these temperature suggested that it is an optical phenomenon generated regimes. These data are consistent with previous by the juxtaposition of the inner cellulose of the studies, which suggest that the two factors that palisade cells and the outer suberized caps. For Pisum influence the breaking of physical dormancy of seeds sativum, Harris (1983) noted that the light line becomes are a high constant temperature and fluctuating discernable with a light microscope, at the junction of temperature (Bewley and Black, 1994; Argel and the cellulosic tips of the macrosclereids and the line of Paton, 1999; Baskin and Baskin, 2001). The alternating the subcuticular layer, and may represent the suberin temperature that breaks the physical dormancy of a caps. Martens et al. (1995) indicated that the light line seed depends on the amplitude of the fluctuation in Trifolium repens is caused by an alteration of cellulose (Quinlivan, 1966). Seeds of Trifolium subterraneum microfibrillar orientation in palisade cell walls. Ma et al. soften in response to temperatures that fluctuate (2004) reported that in G. max the light line is not between 308C and 608C each day over a period of merely an optical phenomenon caused by chemical several weeks or months, which are similar to modifications, but is a real structure formed where the fluctuations that occur on open soils in Mediterranean secondary walls are tightly appressed to one another. and tropical climates (Hagon, 1971; Quinlivan, 1971; Baskin and Baskin (2001) suggested that the light line Taylor, 1981). Germination of species from tropical is due to differences in refraction of light by the top coastal dunes increased when temperature fluctu- and bottom portions of the macrosclereids, which ations were greater than 208C and lasted for more than differ in chemical composition. In S. parahyba it was 45 d (Moreno-Casasola et al., 1994). In Thermopsis possible that the light line originates at the junction of lupinoides (Fabaceae), which grows on dunes in Japan, the upper portion of the macrosclereids with callose alternating temperatures of 258C/358C promoted and the inner portions without callose. breaking of physical dormancy (Kondo and Takahashi, The seed coat of Fabaceae contains several sub- 2004). In water-soaked seeds of Ipomoea lacunosa stances, including polysaccharides, lignin, proteins, (Convolvulaceae), dormancy was broken by an phenolic compounds, pigments, waxes, fats and alternating temperature of 35/208C or a constant resinous matter, that protect the embryo or create a temperature of 358C (Jayasuriya et al., 2008). The barrier to water (Bewley and Black, 1994). In S. parahyba, regimes of temperatures tested for S. parahyba occur in the cell wall of the macrosclereids is composed of gaps in the Atlantic rainforest, the natural environ- cellulose, as indicated by histochemical tests, but ment where this species grows. Thus, we suggest that suberin and lignin, which have been found in the seed temperature is probably the factor involved in break- coats of legumes (Kelly et al., 1992), were not present. In ing the physical dormancy of the seeds of this species another species of subfamily Caesalpinioideae, Cassia in natural habitats, as reported for Heliocarpus donnell- cathartica, Souza (1981) also found macrosclereids that smithii, a gap tree species from Mexico and Costa Rica only had walls made of cellulose. The presence of (Va´zquez-Yanes and Orozco-Segovia, 1982). callose in the upper portion of the macrosclereid cells, and especially in the light line, in S. parahyba has also been observed in other Fabaceae species (Serrato- Acknowledgements Valenti et al., 1993; Ma et al., 2004), and its function is associated with the impermeability of the coat to water (Bhalla and Slattery, 1984; Serrato-Valenti et al., 1993). This study received financial support from Coordena- The present study showed that in the lens region c¸a˜o de Aperfeic¸oamento do Ensino Superior (CAPES), the macrosclereids were shorter than in the rest of the Brazil. tegument. This has been observed in other legume species (Serrato-Valenti et al., 1995; Baskin et al., 2000), References and it was suggested that this site is physically the weakest part of the seed coat and thus more easily Argel, P.J. and Paton, C.J. (1999) Overcoming legume broken by treatments (Serrato-Valenti et al., 1995; hardheadedness. pp. 247–265 in Loch, D.S.; Ferguson, J.E. Baskin et al., 2000; Hu et al., 2009). (Eds) Forage seed production: tropical and sub-tropical species. Wallingford, CAB International. Baskin, C.C. (2003) Breaking physical dormancy in seed – Effect of alternating temperatures on dormancy focusing on the lens. New Phytologist 158, 227–238. Baskin,C.C.andBaskin,J.M.(2001) Seeds: Ecology, break and germination biogeography and evolution of dormancy and germination. London, Academic Press. Laboratory experiments showed that the physical Baskin, J.M. and Baskin, C.C. (2000) Evolutionary consider- dormancy of S. parahyba seeds was broken when the ation of claims of physical dormancy-break by microbial Physical dormancy in seeds of Schizolobium 7

action and abrasion by soil particles. Seed Science Research Hanna, P.J. (1984) Anatomical features of the seed coat of 10, 409–413. Acacia kempeana (Mueller) which relate to increased Baskin,J.M.andBaskin,C.C.(2004) A classifi- germination rate induced by heat treatment. New cation system for seed dormancy. Seed Science Research Phytologist 96, 23–29. 14, 1–16. Harris, W.M. (1983) On the development of macrosclereids Baskin, J.M., Baskin, C.C. and Li, X. (2000) , in seed coats of Pisum sativum L. American Journal of anatomy and evolution of physical dormancy in seeds. Botany 70, 1528–1535. Species Biology 15, 139–152. Hyde, E.O.C. (1954) The function of the hilum in Bewley, J.D. and Black, M. (1994) Seeds: Physiology of some Papilionaceae in relation to the ripening of the development and germination (2nd edition). New York, seed and the permeability of the coat. Annals of Botany 18, Plenum Press. 241–256. Bhalla, P.L. and Slattery, H.D. (1984) Callose deposits make Hu, X.W., Wang, Y.R., Wu, Y.P., Nan, Z.B. and Baskin, C.C. clover seeds impermeable to water. Annals of Botany 53, (2008) Role of the lens in physical dormancy in seeds of 125–128. Sophora alopecuroides L. (Fabaceae) from north-west China. Bozzola, J.J. and Russell, L.D. (1991) Electron microscopy. Australian Journal of Agricultural Research 59, 491–497. Principles and techniques for biologists. Boston, Jones and Hu, X.W., Wang, Y.R., Wu, Y.P.and Baskin, C.C. (2009) Role of Bartlett. the lens in controlling the water uptake in seeds of two Burrows, G.E., Virgona, J.M. and Heady, R.D. (2009) Effect Fabaceae (Papilionoideae) species treated with sulphuric of boiling water, seed coat structure and provenance on acid and hot water. Seed Science Research 19, 73–80. the germination of Acacia melanoxylon seeds. Australian Jayasuriya, K.M., Baskin, J.M., Geneve, R.L., Baskin, C.C. Journal of Botany 57, 139–147. and Chien, C.T. (2008) Physical dormancy in seeds of the Caˆndido, J.F., Conde´, A.R., Silva, R.F., Maria, J. and holoparasitic angiosperm Cuscuta australis (Convolvula- Ledo, A.A.M. (1981) Estudo da causa da dormeˆncia em ceae, Cuscuteae): dormancy-breaking requirements, sementes de guarapuvu (Schizolobium parahybum (Vell.) anatomy of the water gap and sensitivity cycling. Annals Blake) e me´todos para sua quebra. Revista A´ rvore 5, of Botany 102, 39–48. 224–232. Kelly, K.M., Van Staden, J. and Bell, W.E. (1992) Seed coat Carvalho, P.E.R. (2003) Espe´cies arbo´reas brasileiras. Brası´lia, structure and dormancy. Plant Growth Regulation 11,201–209. EMBRAPA. Kondo, T. and Takahashi, K. (2004) Breaking of physical Corner, E.J.H. (1951) The leguminous seed. Phytomorphology dormancy and germination ecology for seeds of 1, 117–150. Thermopsis lupinoides. Journal of the Japanese Society of Costa, A.F. (1982) Farmacognosia. Lisboa, Fundac¸a˜o Calouste Revegetation Technology 30, 163–168. Gulbenkian. Leython, L. and Ja´uregui, D. (2008) Morfologı´a de la semilla Dell, B. (1980) Structure and function of the strophiolar y anatomı´a de la cubierta seminal de cinco especies de plug in seeds of Albizia lophanta. American Journal of Calliandra (Leguminosae-Mimosoideae) de Venezuela. Botany 67, 556–563. Revista Biologia Tropical 56, 1075–1086. Fenner, M. and Thompson, K. (2005) The ecology of seeds. Ma, F., Cholewa, E., Mohamed, T., Peterson, C.A. and Jzen, Cambridge, Cambridge University Press. M.G. (2004) Cracks in the palisade cuticle of soybean Finch-Savage, W.E. and Leubner-Metzger, G. (2006) seed coats correlate with their permeability to water. Seed dormancy and the control of germination. New Annals of Botany 94, 213–228. Phytologist 171, 501–523. Martens, H., Jakobsen, H.B. and Lyshede, O.B. (1995) Freire, J.M., Coffler, R., Gonc¸alves, M.P.M., Santos, A.L.F. Development of the strophiole in seeds of white clover and Pin˜ a-Rodrigues, F.C.M. (2007) Germinac¸a˜oe (Trifolium repens L.). Seed Science Research 5, 171–176. dormeˆncia de sementes entre e dentro de populac¸o˜es Matheus, M.T. and Lopes, J.C. (2007) Termoterapia em de guapuruvu (Schizolobium parahyba (vell.) Blake) semente de Guarapuvu´ (Schizolobium parahyba (Vell.) oriundas dos municı´pios de Paraty e Miguel Pereira-R.J. Blake). Revista Brasileira de Biocieˆncias 5, 330–332. Revista Brasileira de Biocieˆncias 5, 168–170. Moreno-Casasola, P., Grime, J.P. and Martinez, L. (1994) Funes, G. and Venier, P. (2006) Dormancy and germination A comparative study of the effects of fluctuations in in three Acacia (Fabaceae) species from central Argen- temperature and moisture supply on hard coat tina. Seed Science Research 16, 77–82. dormancy in seeds of coastal tropical legumes in Mexico. Gama-Arachchige, N.S., Baskin, J.M., Geneve, R.L. and Journal of Tropical Ecology 10, 67–86. Baskin, C.C. (2011) Acquisition of physical dormancy Morrison, D.A., McClay, K., Porter, C. and Rish, S. (1998) and ontogeny of the micropyle–water-gap complex in The role of the lens in controlling heat-induced break- developing seeds of Geranium carolinianum (Geraniaceae). down of coat-imposed dormancy in native Australian Annals of Botany 108, 51–64. legumes. Annals of Botany 82, 35–40. Gunn, C.R. (1984) Fruits and seeds of genera in subfamily O’Brien, T.P., Feder, N. and McCully, M.E. (1965) Poly- Mimosoideae (Fabaceae). United States Department of chromatic staining of plant cell walls by toluidine blue. Agriculture Technical Bulletin 1681, 1–194. Protoplasma 59, 368–373. Gunn, C.R. (1991) Fruits and seeds of genera in subfamily Quinlivan, B.J. (1966) The relationship between temperature Caesalpinioideae (Fabaceae). United States Department of fluctuations and softening of hard seeds of some legume Agriculture Technical Bulletin 1755, 1–408. species. Australian Journal Agricultural Research 17, Hagon, M.W. (1971) The action of temperature fluctuations 625–631. on hard seeds of subterranean clover. Australian Journal of Quinlivan, B.J. (1971) Seed coat impermeability in legumes. Experimental Agriculture and Animal Husbandry 11, Journal of the Australian Institute of Agricultural Science 37, 440–443. 283–295. 8 T.V. de Souza et al.

Ruzin, S.E. (1951) Plant microtechniques and microscopy. Taylor, G.B. (1981) Effect of constant temperature treatments New York, Oxford University Press. followed by alternating temperatures on the softening of Serrato-Valenti, G., Cornara, L., Ferrando, M. and hard seeds of Trifolium subterraneum L. Australian Journal Modenesi, P. (1993) Structural and histochemical of Plant Physiology 8, 547–558. features of Stylosanthes scabra (Leguminosae; Papilio- Valtuen˜ a, F.J., Ortega-Olivencia, A. and Rodriguez-Rian˜ o, noideae) seed coat as related to water entry. Canadian T. (2008) Germination and seed bank biology in some Journal of Botany 71, 834–840. Iberian populations of Anagyris foetida L. (Leguminosae). Serrato-Valenti, G., De Vries, M. and Cornara, L. (1995) Plant Systematics and Evolution 275, 231–243. The hilar region of Leucaena leucocephala Lam. (De Wit) Van Staden, J., Manning, J.C. and Kelly, K.M. (1989) seeds: structure, histochemistry and the role of the lens Legumes seeds – the structure:function equation. in germination. Annals of Botany 75, 569–574. pp. 417–450 in Stirton, C.H.; Zarucchi, J.L. (Eds) Advances Smith, M.T., Wang,B.S.P.andMsanga,H.P.(2002) in legume biology, monographs on systematic botany. Dormancy and germination. pp. 149–176 in Vozzo, J.A. St. Louis, Missouri Botanical Garden. (Ed.) Tropical tree seed manual. Agriculture Handbook 721. Vazquez-Yanes, C. and Orozco-Segovia, A. (1982) Seed Washington DC, USDA Forest Service. germination of a tropical rain forest pioneer tree Souza, L.A. (1981) Estrutura do tegumento das sementes (Heliocarpus donnel-smithii) in response to diurnal fluctu- de Cassia cathartica Mart. (Leguminosae). Cieˆncia e Cultura ation of temperature. Physiologia Plantarum 56, 295–298. 34, 71–74. Zeng, L., Cocks, P.S., Kailis, S.G. and Kuo, J. (2005) The role Statsoft (2001) Statistica (data analysis software system). of fractures and lipids in the seeds coat in the loss of Version 6. Available at http://www.Statsoft.com hardseededness of six Mediterranean legume species. (accessed 31 January 2012). Journal of Agricultural Science 143, 43–55.