Annals of Botany 1–10, 2007 doi:10.1093/aob/mcm070, available online at www.aob.oxfordjournals.org Morphology and Anatomy of Physical Dormancy in Ipomoea lacunosa: Identification of the Water Gap in Seeds of Convolvulaceae (Solanales) K. M. G. GEHAN JAYASURIYA1 ,JERRYM.BASKIN1 , ROBERT L. GENEVE2 and CAROL C. BASKIN1,3,* 1Department of Biology, University of Kentucky, Lexington, KY 40506, USA, 2Department of Horticulture, University of Kentucky, Lexington, KY 40546, USA and 3Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA Received: 15 January 2007 Returned for revision: 13 February 2007 Accepted: 26 February 2007 † Background and Aims Convolvulaceae is the most advanced plant family (asterid clade) that produces seeds with physical dormancy (water-impermeable seed coat). There are several different opinions about the nature of the specialized structure (‘water gap’) in the seed coat through which water initially enters seeds of Convolvulaceae, but none of them has been documented clearly. The primary aim of the study was to identify the water gap in seeds of Ipomoea lacunosa (Convolvulaceae) and to describe its morphology, anatomy and function. † Methods Light microscopy, scanning electron microscopy, tissue-sectioning, dye-tracking and blocking experi- ments were used to describe the morphology, anatomy and function of the water gap in seeds of I. lacunosa. † Key Results Dormancy-breaking treatments caused slits to form around the two bulges on the seed coat adjacent to the hilum, and dye entered the seed only via the disrupted bulges. Bulge anatomy differs from that of the rest of the seed coat. Sclereid cells of the bulges are more compacted and elongated than those in the hilum pad and in the rest of the seed coat away from the bulges. † Conclusions The transition area between elongated and square-shaped sclereid cells is the place where the water gap opens. Morphology/anatomy of the water gap in Convolvulaceae differs from that of taxa in the other 11 angio- sperm plant families that produce seeds with physical dormancy for which it has been described. Key words: Convolvulaceae, Ipomoea, seed coat anatomy, seed dormancy, seed germination, palisade layer, physical dormancy, water gap, water-impermeable seed coat. INTRODUCTION seeds of these 12 families, and they differ in origin, mor- phology and anatomy (Baskin et al., 2000). Physical dormancy (PY) in seeds is caused by a water Ipomoea lacunosa belongs to Ipomoeeae, the most impermeable seed (or fruit) coat (Baskin and Baskin, advanced tribe in Convolvulaceae (Stefanovic et al., 1998, 2004), and it is known to occur in only 16 families 2003). This species is a summer annual vine native to of angiosperms (J. M. Baskin et al., 2000, 2006): one eastern USA (Abel and Austin, 1981; Gleason and monocot family (Cannaceae) and 15 eudicot families. Cronquist, 1991). Ipomoea lacunosa is resistant to glypho- Within the eudicots, 14 of the families are in the rosids, sate (Koger et al., 2004; Koger and Reddy, 2005) and thus and only one (Convolvulaceae) is in the highly evolutionary difficult to control in crop fields. Therefore, it is a common advanced asterid clade (Baskin and Baskin, 2000; noxious weed in agricultural crops, especially in corn J. M. Baskin et al., 2006). The phylogenetically isolated (Anonymous, 1995), cotton, soybeans (Anonymous, 2000) position of the Convolvulaceae and the fact that it is the and rice (Anonymous, 2001). most advanced family (APG, 2003) with PY (Baskin Seeds of I. lacunosa buried in soil can remain viable et al., 2000) make it an important family to study to for at least 39 years (Toole and Brown, 1946). Gomes advance our understanding of the evolutionary relationships et al. (1978), Crowley and Buchanan (1980) and Oliveira of PY. and Norsworthy (2006) tested the effects of various Impermeability of the seed (or fruit) coat to water deve- environmental factors on germination of I. lacunosa lops during maturation drying of the seed (Van Staden seeds, but only Gomes et al. (1978) tested the effect of et al., 1989; Baskin and Baskin, 1998) or fruit (Li et al., scarification on germination. They found that scarified 1999). A palisade layer(s) in the seed (or fruit) coat is seeds generally germinated to significantly higher percen- (are) responsible for the impermeability. The seed tages across a range of temperatures than non-scarified becomes permeable to water when an opening is formed ones. This suggests, but does not prove, that seeds of via a specialized anatomical structure (‘water gap’) in the I. lacunosa are water impermeable. They did not compare palisade layer(s), allowing water to enter the seed (Baskin water uptake (imbibition) in scarified versus non-scarified et al., 2000). A water gap has been described in 12 of the seeds, and scarification can break dormancy in seeds with 16 families known to have PY (Baskin et al., 2000). shallow physiological dormancy (see Baskin and Baskin, Further, several different kinds of water gaps occur in 2004) as well as in those with PY (see C. C. Baskin et al., 2006). * For correspondence. E-mail: [email protected] # The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Page 2 of 10 Jayasuriya et al. — Seed Water Gap Anatomy in Convolvulaceae There are several claims of identification of the route of from the Petri dish, blotted dry with filter paper, weighed to water entry into seeds of Convolvulaceae. However, none of the nearest 0.0001 g and returned to the wet filter paper in these studies clearly documented a water gap. Koller and the dish. Imbibition experiments were carried out under Cohen (1959) reported that a plug-like structure near the ambient room conditions. Imbibition curves [increase in micropyle opens during the dormancy break, thereby allow- seed mass (fresh weight basis) over time] of scarified and of ing water to enter the seeds of Convolvulus lanatus, non-scarified seeds were constructed and compared to deter- C. negevensis and C. secundus. However, they did not mine if scarification was required for seeds to imbibe (PY) describe the structure in detail, nor did they clearly or not (physiological dormancy or no dormancy). present evidence that led them to this conclusion. Callihan (1933) described the hilum slit as the route of Germination water entry into ethanol-treated seeds of Convolvulus arven- sis, which he observed to open at high RH. However, this Germination tests of manually scarified and of non- was just a speculation, since he came to this conclusion scarified (control) seeds were conducted in both light/dark after observing photomicrographs of the seeds and did not (14/10 h) (approx. 40 mmol m –2 s–1, 400–700 nm, cool document water uptake by the seeds. white fluorescent light) and constant darkness at (12/12 h) Hutchison and Ashton (1979) used the blocking method daily temperature regimes of 35/20, 30/15, 25/15, 20/10 (lanolin–petroleum jelly) to investigate where water entered and 15/6 8C. Darkness was provided by wrapping Petri seeds of Cuscuta campestris that had been scarified with dishes in aluminium foil. Three replicates of 25 seeds concentrated H2SO4. They concluded that the acid-scarified were used for each treatment. Seeds were incubated on seeds took up water uniformly over the entire seed coat. moist sand in 5.5-cm-diameter Petri dishes, and the Lyshede (1984) studied scanning electron micrographs of number of seeds germinated in light was counted every 2 seeds of Cuscuta campestris made permeable by slight d for 14 d. Dark-incubated seeds were checked for germina- abrasion with sand paper and of those of C. pedicellata, tion after 14 d. Petri dishes were allocated in a completely which germinated without treatment, and she came to the randomized design on incubator shelves. Germination per- same conclusion as that of Hutchison and Ashton. centages of non-treated and of manually scarified seeds Lyshede suggested that papillae present on the seed coat were compared to determine if seeds have PY. are involved in water uptake. Thus, there is no clear description of the route of water entry into seeds of Breaking dormancy in intact seeds Convolvulaceae. The purpose of the present study was 3-fold: (1) to document whether seeds of I. lacunosa have The following treatments were applied to intact (not PY, which has been shown to occur in I. purpurea mechanically scarified) seeds in an attempt to break PY: (Brechu-Franco et al., 2000), I. hederacea (Thullen and seeds dipped in boiling water for 2, 4 and 6 s; seeds dry Kelly, 1983), I. pandurata (Horak and Wax, 1991), heated at 90 8C for 1, 5 and 10 min; seeds heated for 1, I. crassicaulis [syn. I. carnea Jacquin ssp. fistulosa 2, 3 and 6 h at 35 8C on wet sand; and seeds heated for 1, (Martius ex Choisy) D.F. Austin] (Misra, 1963), 2, 3 and 6 h at 35 8C on dry sand. Treated seeds were I. coccinea (Hardcastle, 1978), I. pes-caprae (Martinez tested for germination in the 25/15 8C light/dark regime et al., 2002) and various other Ipomoea species described above. Three replicates of 25 seeds were used (Ogunwenmo, 2006); (2) to document the presence (or for each treatment. Germination percentages in each treat- not) of a water gap in seeds of this species; and (3) to ment were compared with those of control (non-scarified describe the water gap morphologically and anatomically. seeds incubated at 25/15 8C) to determine the effectiveness of the dormancy-breaking treatment. MATERIALS AND METHODS Morphological changes during imbibition Seed collection Seeds made non-dormant by treating them for 3 h at Seeds were collected from numerous plants of I.