Seed Anatomy and Water Uptake in Relation to Seed Dormancy in Opuntia tomentosa (Cactaceae, Opuntioideae) A. OROZCO-SEGOVIA1,*, J. MARQUEZ-GUZMAN2 �� �� , M. E. SANCHEZ-CORONADO1 �� , A. GAMBOA DE BUEN1 , J. M. BASKIN3 and C. C. BASKIN3,4 1Instituto de Ecolog��a, �� Universidad Nacional Autonoma �� de Mexico, �� Apartado Postal 70-275, Ciudad Universitaria, 04510 Mexico, �� D. F., Mexico, �� 2Departamento de Biolog��a, �� Facultad de Ciencias, Universidad Nacional Autonoma �� de Mexico,�� Ciudad Universitaria, 04510 Mexico, �� D. F., Mexico, �� 3Department of Biology, University of Kentucky, Lexington, KY 40506-0225, USA and 4Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546-0312, USA Received: 23 October 2006 Returned for revision: 15 November 2006 Accepted: 8 December 2006 ��� Background and Aims There is considerable confusion in the literature concerning impermeability of seeds with ���hard��� seed coats, because the ability to take up (imbibe) water has not been tested in most of them. Seeds of Opuntia tomentosa were reported recently to have a water-impermeable seed coat sensu lato (i.e. physical dormancy), in combination with physiological dormancy. However, physical dormancy is not known to occur in Cactaceae. Therefore, the aim of this study was to determine if seeds of O. tomentosa are water-permeable or water-imperme- able, i.e. if they have physical dormancy. ��� Methods The micromorphology of the seed coat and associated structures were characterized by SEM and light microscopy. Permeability of the seed-covering layers was assessed by an increase in mass of seeds on a wet substrate and by dye-tracking and uptake of tritiated water by intact versus scarified seeds. ��� Key Results A germination valve and a water channel are formed in the hilum���micropyle region during dehy- dration and ageing in seeds of O. tomentosa. The funicular envelope undoubtedly plays a role in germination of Opuntia seeds via restriction of water uptake and mechanical resistance to expansion of the embryo. However, seeds do not exhibit any of three features characteristic of those with physical dormancy. Thus, they do not have a water-impermeable layer(s) of palisade cells (macrosclereids) or a water gap sensu stricto and they imbibe water without the seed coat being disrupted. ��� Conclusions Although dormancy in seeds of this species can be broken by scarification, they have physiological dormancy only. Further, based on information in the literature, it is concluded that it is unlikely that any species of Opuntia has physical dormancy. This is the first integrative study of the anatomy, dynamics of water uptake and dormancy in seeds of Cactaceae subfamily Opuntioideae. Key words: Mexico �� Valley, Opuntia tomentosa, physical dormancy, physiological dormancy, seed anatomy, seed germination, water uptake by seeds. INTRODUCTION Seeds with a water-impermeable seed or fruit coat have physical dormancy (PY sensu Baskin and Baskin, 2004), and such seeds are known to occur in 16 families of angios- perms but no gymnosperms (Baskin et al., 2000, 2006 Baskin and Baskin, 2003). In all cases, PY is associated with a water-impermeable layer(s) of palisade cells (macro- sclereids) (Baskin et al., 2000). However, considerable con- fusion occurs in the literature concerning seeds with ���hard��� seed coats. These seeds have a seed (or fruit) coat that is hard when pinched with one���s fingers, but they may or may not be water-permeable, depending on the species. The only way to determine if seeds with hard (to-the-touch) coats are water-permeable is to conduct imbibition studies. If seeds fail to imbibe water, they have PY. To add to the confusion, mechanical or acid scarification of water- permeable, hard (to-the-touch) seeds may promote germi- nation. Low growth potential (or ���push power���) of the embryo is the major reason why imbibed seeds with fully developed embryos fail to germinate these seeds have physiological dormancy (PD). If the coat of such seeds is scarified, mechanical restriction is decreased, and the embryo is able to expand (germinate). However, when physiological dormancy is broken in intact seeds, the embryo gains sufficient growth potential to overcome the restraint of the seed coat (Baskin and Baskin, 2004). Seeds of Opuntia species have hard (to-the-touch) seed covers, and pressures of 440.83���44.35 daN may be required to break them (Aguilar, 2003). The hardness of the Opuntia seed covers is related to the presence of a funicular envelope, which completely encloses the seed (Archibald, 1939). Further, physiological dormancy is present in seeds of many cacti (Baskin and Baskin, 1998 C. C. Baskin and J. M. Baskin, unpubl. res.), including those of Opuntia spp., e.g. O. macrorhiza (Timmons, 1942), O. compressa (Baskin and Baskin, 1977), O. tomentosa (Olvera-Carrillo et al., 2003), O. rastrera (Mandujano et al., 1997, 2005) and O. stricta (Reinhardt et al., 1999). Exposing seeds of O. macrorhiza (Timmons, 1942) and of O. compressa (Baskin and Baskin, 1977) to outdoor winter temperatures in Kansas and Kentucky, respectively, was effective in breaking dormancy. Various dormancy-breaking treatments, * For correspondence. E-mail aorozco@miranda.ecologia.unam.mx # The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org Annals of Botany 1���12, 2007 doi:10.1093/aob/mcm001, available online at www.aob.oxfordjournals.org

including scarification with sulfuric acid (Archibald, 1939 Wiggins and Focht, 1967 Baskin and Baskin, 1977 Potter et al., 1984 Olvera-Carrillo et al., 2003 Mandujano et al., 2005), mechanical scarification (Johnson, 1918 Archibald, 1939 Hamilton, 1970a, b Pilcher, 1970 Baskin and Baskin, 1977 Pendley, 2001 Mandujano et al., 2005), after- ripening (Mandujano et al., 1997, 2005 Reinhardt et al., 1999 Pendley, 2001), cold stratification (Timmons, 1942 Baskin and Baskin, 1977 Olvera-Carrillo et al., 2003), leach- ing (Wiggins and Focht, 1967 Pilcher, 1970 Hamilton, 1970a Pendley, 2001), passage through the digestive tract of animals (Timmons, 1942 Potter et al., 1984 Mandujano et al., 1997 Gimeno and Vila, 2002), soaking in hot water (Reinhardt et al., 1999), and some combinations of these treatments have been reported to enhance germination of Opuntia seeds. However, these treatments do not always promote germination and, in some cases, treated seeds have germinated to lower percentages than those in the control (e.g. Wiggins and Focht, 1967 Baskin and Baskin, 1977 Mandujano et al., 2005). Gibberellic acid (GA3) alone seems to be only moderately effective in promoting germina- tion of seeds of Opuntia spp. However, it induces a high per- centage of germination in combination with other treatments such as rinsing/washing (Pendley, 2001) or chemical or mechanical scarification (Sanchez-Venegas, �� 1997). In a recent paper, Olvera-Carrillo et al. (2003) reported PY in seeds of Opuntia tomentosa Salm-Dyck (Cactaceae), but this class of dormancy is not (otherwise) known to occur in the Cactaceae (Baskin et al., 2000 Baskin and Baskin, 2003). Further, seeds of O. tomentosa required gibberellin and acid scarification to germinate, but scarification time for full germination varied from 5 to 90 min, depending on seed batch (Olvera-Carrillo et al., 2003). However, after 15���18 months of dry storage, seeds only germinated following scarification, and GA3 (1000 and 2000 ppm) reduced germination. Although Olvera-Carrillo et al. (2003) showed that scarification significantly increased germination of O. tomentosa seeds, they did not compare water uptake (imbibition) of scarified versus non-scarified seeds. Further, it was observed that non-scarified seeds of O. tomentosa did not germinate, and they did not appear to swell when placed on a wet substrate. Thus, the purpose of the present study was to address questions concerning water uptake in the hard (to-the-touch) seeds of O. tomentosa. In particular, the anatomy of the seed cover layers was investigated in relation to imbibition and germination, with the primary objectives being to determine if the seed cover layers are water-impermeable or water-permeable, i.e. whether or not seeds have PY, and the pathway of water entry into the seed. MATERIALS AND METHODS Seed collection Opuntia tomentosa (cactus pear) occurs in the Central Plateau and the south-west of Mexico (Guzman et al., 2003), where it grows in open xerophilous shrubland vegetation developed on a lava field (Rzedowski, 1994 Scheinvar, 1982). Ripe fruits were collected from August to November in 1998, 1999, 2000, 2003 and 2005 from more than 12 adult plants growing at the El Pedregal de San Angel Reserve (Mexico, City, 198190N, 998110W, 2240 m a.s.l.). After dormancy break, seeds from these collections had high viability (77 + 11.4 %, A. Orozco- Segovia et al., unpubl. res.). Mean annual rainfall at the reserve is 803 mm, 93 % of which occurs from May to October, and the mean annual temperature is 15.5 8C (Barradas et al., 1999). Precipitation during the collection years is shown in Fig. 1. Seeds were removed from fruits and stored dry in paper bags at room temperature (23���25 8C, 20���50 % relative humidity) until used in the study some treatments were limited by seed availability. Seed micromorphology and anatomy The primary purpose of this part of the study was to characterize the micromorphology of the seed coat and the funicular envelope in relation to water uptake. Longitudinal cuts in seeds collected in 1998 and in 2003 were made in 2004 with a tissue chopper (Sorvall, TC-2 Smith & Farquhar, Newtown, CT, USA). The samples were mounted on aluminium stubs using carbon double tape and coated with approx. 200 nm of gold with a sputter coater (Desk II Denton Vacuum Inc., Norristown, NJ, USA). Finally, seeds were observed with a scanning electron microscope (JSM-5310LV Electron Optics Div., Medford, MA, USA). In 2004, non-scarified seeds and seeds scarified for 45 or 90 min (1998 and 2003 collections) in concentrated sulfuric acid (see below) were immersed for 12, 24, 48, 72, 96, 120 and 144 h in methylene blue (1 % in water Sigma-Aldrich Qu��mica �� S. A. de C. V., Mexico) �� or for 96 h in neutral red stain (1 % in water, Sigma-Aldrich Qu��mica �� S. A. de C. V., Mexico). �� Then, the seeds were cut longitudinally and observed with a photomicroscope (Olympus Provis AX-70 Olympus, Tokyo, Japan). Additionally, longitudinal cuts in seed covers (seed coat and funicular envelope), stained with red oil ���O��� to determine if lipids were present, and funicular cells, disassociated in HNO3, were examined under the photomicroscope. To identify tannins, longitudinal cuts Precipitation (mm) 0 100 200 300 400 1998 1999 2000 2003 2005 Month J F M A M J J A S O N D FIG. 1. Amount and monthly distribution of precipitation during the seed collection years. Orozco-Segovia et al. ��� Seed Water Uptake in Opuntia tomentosa Page 2 of 12