Evolutionary reversal of physical dormancy to nondormancy: evidence from comparative seed morphoanatomy of Argyreia species (Convolvulaceae).

Argyreia is the most recently evolved genus in the Convolvulaceae, and available information suggests that most species in this family produce seeds with physical dormancy (PY). Our aim was to understand the evolution of seed dormancy in this family via an investigation of dormancy, storage behaviour, morphology and anatomy of seeds of five Argyreia species from Sri Lanka. Imbibition, germination and dye tracking of fresh intact and manually scarified seeds were studied. Scanning electron micrographs and hand sections of the hilar area and the seed coat away from the hilar area were compared. Scarified and intact seeds of A. kleiniana, A. hirsuta and A. zeylanica imbibed water and germinated to a high percentage, but only scarified seeds of A. nervosa and A. osyrensis did so. Thus, seeds of the three former species are non-dormant (ND), while those of the latter two have physical dormancy (PY); this result was confirmed by dye-tracking experiments. Since >90% of A. kleiniana, A. hirsuta and A. zeylanica seeds survived desiccation to 10% moisture content (MC) and >90% of A. nervosa and A. osyrensis seeds with a dispersal MC of ~12% were viable, seeds of the five species were desiccation-tolerant. A. nervosa and A. osyrensis have a wide geographical distribution and PY, while A. kleiniana, A. hirsuta and A. zeylanica have a restricted distribution and ND. Although seeds of A. kleiniana are ND, their seed coat anatomy is similar to that of A. osyrensis with PY. These observations suggest that the ND of A. kleiniana, A. hirsuta and A. zeylanica seeds is the result of an evolutionary reversal from PY and that ND may be an adaptation of these species to the environmental conditions of their wet aseasonal habitats.

Dormancy, germination, and associated seed ecological traits of 25 Fabaceae species from northern India.

Fabaceae produce seeds with water-impermeable seed coats, i.e., physical dormancy (PY). We hypothesized that the proportion of PY seeds will increase with the dryness of the habitat, and some key seed ecological traits will be strongly associated with different levels of PY. Fresh seed of 25 Fabaceae species collected in northern India were used for imbibition and germination experiments to determine the proportion of seeds with PY and of nondormant (ND) seeds compared to their Sri Lankan congeners. Seed coat:seed mass ratio (SCR), 1000 seed weight, seed shape index (SSI), embryo type and median germination time of ND seeds were determined. Four imbibition and germination patterns were identified among seeds of the studied species. Seeds collected from Indian populations had a higher proportion of PY seeds than those of Sri Lankan populations. We identified a type of embryo called 'spatulate axile' that had not been identified before among the studied species. Species with ND seeds had a lower SCR and a higher SSI than those with PY. Our hypothesis was confirmed since populations from drier habitats in India produce a higher proportion of PY seeds than those from Sri Lanka. A low SCR ensures minimal resistance to germinating seeds, while seeds with a high SSI have a lower tendency to incorporate into the soil seed bank. Thus, these seed traits aid the fast germination of ND seeds, often dispersed just before the rainy season.

Seed dormancy of Cardiospermum halicacabum (Sapindaceae) from three precipitation zones in Sri Lanka.

This study investigated seed germination of Cardiospermum halicacabum, a medicinally important invasive species. We compared mass, moisture content (MC), dormancy and dormancy-breaking treatments and imbibition and germination of scarified and non-scarified seeds of C. halicacabum from a low-elevation dry zone (DZ), low-elevation wet zone (WZ1) and mid-elevation wet zone (WZ2) in Sri Lanka to test the hypothesis that the percentage of seeds with water-impermeable seed coats (physical dormancy, PY) decreases with increased precipitation. Seed mass was higher in WZ2 than in DZ and WZ1, while seed MC did not vary among the zones. All scarified DZ, WZ1 and WZ2 and non-scarified DZ and WZ1 seeds imbibed water, but only a few non-scarified WZ2 seeds did so. When DZ and WZ1 seeds were desiccated, MC and percentage imbibition decreased, showing that these seeds have the ability to develop PY. GA3 promoted germination of embryos excised from fresh DZ and WZ1 seeds and of scarified WZ2 seeds. At maturity, seeds from DZ and WZ1 had only physiological dormancy (PD), while those from WZ2 had combinational dormancy (PY+PD). Thus, our hypothesis was not supported. Since a high percentage of excised embryos developed into normal seedlings; this is a low-cost method to produce C. halicacabum plants for medicinal and ornamental purposes.

Seed dormancy of 80 tropical montane forest species in Sri Lanka, the first dormancy profile for a tropical montane forest community.

Community-level seed dormancy studies are important in understanding the dynamics of plant communities and adaptations of species to their habitat. Our aim was to develop a seed dormancy profile for tropical montane forests of Sri Lanka, which are a global biodiversity hotspot, and compare it to the profile made using a world database for this vegetation type. Germination, imbibition and embryo length : seed length ratio of seeds were determined for 80 Sri Lankan montane forest species. Seeds of 31 species were fast-germinating with a median length of germination (MLG) <30 days and the remaining 49 were slow-germinating with MLG >30 days. Embryos of six fast-germinating species grew prior to radicle emergence, indicating morphological dormancy (MD). The other 25 fast-germinating species had non-dormant (ND) seeds. Manually scarified seeds of two species imbibed significantly more water than non-scarified seeds, revealing physical dormancy (PY). Embryos of 20 slow-germinating species grew prior to radicle emergence, confirming morpho-physiological dormancy (MPD). The remaining slow-germinating species had a fully developed embryo and thus physiological dormancy (PD). The percentage of species with ND seeds and with MD, MDP, PD and PY was 31, 7.5, 25, 34 and 2.5, respectively. Species with dormant seeds (70%) dominate the Sri Lankan montane forest community similar to the world database, with 85% dormant seeds. Seed dormancy may be an adaptation that prevents seeds from germinating during the Sri Lankan dry season from December to March when conditions are unfavourable for seedling growth due to low water availability.

Diversity of epicotyl dormancy among tropical montane forest species in Sri Lanka.

Fruiting season of many Sri Lankan tropical montane species is not synchronised and may not occur when conditions are favourable for seedling establishment. We hypothesised that species with different fruiting seasons have different seed dormancy mechanisms to synchronise timing of germination with a favourable season for establishment. Using six species with different fruiting seasons, we tested this hypothesis. Germination and imbibition of intact and manually scarified seeds were studied. Effect of GA3 on germination was examined. Embryo length:seed length (E:S) ratio of freshly matured seeds and of those with a split seed coat was determined. Time taken for radicle and plumule emergence and morphological changes of the embryos were recorded. The radicle emerged from Ardisia missionis, Bheza nitidissima and Gaetnera walkeri seeds within 30 days, whereas it took >30 days in other species. Embryos grew in seeds of B. nitidissima and G. walkeri prior to radicle emergence but not in Microtropis wallichiana, Nothapodytes nimmoniana and Symplocos cochinchinensis. A considerable delay was observed between radicle and plumule emergence in all six species. Warm stratification and/or GA3 promoted germination of all species. All the tested species have epicotyl dormancy. Seeds of B. nitidissima and G. walkeri have non-deep simple morphophysiological epicotyl dormancy, and the other four species have non-deep physiological epicotyl dormancy. Differences in radicle and epicotyl dormancy promote synchronisation of germination to a favourable time for seedling development. Therefore, information on dormancy-breaking and germination requirements of both radicle and epicotyl are needed to determine the kind of dormancy of a particular species.

Physiological epicotyl dormancy and recalcitrant storage behaviour in seeds of two tropical Fabaceae (subfamily Caesalpinioideae) species.

BACKGROUND AND AIMS: Physiological epicotyl dormancy in which the epicotyl elongates inside the seed before the shoot emerges has been reported for only a few tropical rainforest species, all of which are trees that produce recalcitrant seeds. In studies on seeds of Fabaceae in Sri Lanka, we observed a considerable time delay in shoot emergence following root emergence in seeds of the introduced caesalpinioid legumes Brownea coccinea and Cynometra cauliflora. Thus, our aim was to determine if seeds of these two tropical rainforest trees have physiological epicotyl dormancy, and also if they are recalcitrant, i.e. desiccation sensitive. METHODOLOGY: Fresh seeds were (i) dried to various moisture levels, and (ii) stored at -1 and 5 °C to determine loss (or not) of viability and thus type of seed storage behaviour (orthodox, recalcitrant or intermediate). To identify the kind of dormancy, we tested the effect of scarification on imbibition and monitored radicle emergence and epicotyl growth (inside the seed) and emergence. PRINCIPAL RESULTS: FRESH SEEDS OF BOTH SPECIES HAD HIGH MOISTURE CONTENT (MC): 50 % for C. cauliflora and 30 % for B. coccinea. Further, all seeds of C. cauliflora and the majority of those of B. coccinea lost viability when dried to 15 % MC; most seeds of both species also lost viability during storage at -1 or 5 °C. Intact seeds of both species were water permeable, and radicles emerged in a high percentage of them in <30 days. However, shoot emergence lagged behind root emergence by 77 ± 14 days in B. coccinea and by 38 ± 4 days in C. cauliflora. Further, plumule growth inside seeds of C. cauliflora began almost immediately after radicle emergence but not until ∼30-35 days in B. coccinea seeds. CONCLUSIONS: Seeds of both species are recalcitrant and have physiological epicotyl dormancy. The kind of physiological epicotyl dormancy in seeds of C. cauliflora has not been described previously; the formula is C(nd) (root)-[Formula: see text] (epicotyl).

Recalcitrancy and a new kind of epicotyl dormancy in seeds of the understory tropical rainforest tree Humboldtia laurifolia (Fabaceae, Ceasalpinioideae).

We report a new kind of seed dormancy and identify the storage behavior category for an important understory rainforest tree that also is used as an ornamental. While studying seed dormancy of Fabaceae species in Sri Lanka, we observed a considerable delay in emergence of the plumule following radicle emergence in Humboldtia laurifolia. Because epicotyl dormancy has not been reported in Fabaceae, we undertook a detailed morphological study of seed germination in this species. Our aims were to document desiccation tolerance/intolerance and epicotyl dormancy in seeds of H. laurifolia. Drying and low temperature storage were used to evaluate storage behavior of the seeds and imbibition, germination, and seed coat anatomy to categorize seed dormancy in two seed collections. Plumule development before its emergence and effects of light and temperature on plumule emergence were monitored. All seeds that were dried to 15% moisture content or stored at -1°C lost viability. Plumules began to grow 20 ± 5 d from radicle emergence and emerged after 40 ± 3 d. Dark and high illuminance further delayed plumule emergence. Seeds are recalcitrant and have a hitherto unreported kind of epicotyl dormancy, for which we propose the formula .

Phylogeny of seed dormancy in Convolvulaceae, subfamily Convolvuloideae (Solanales).

BACKGROUND AND AIMS: The water gap is an important morphoanatomical structure in seeds with physical dormancy (PY). It is an environmental signal detector for dormancy break and the route of water into the non-dormant seed. The Convolvulaceae, which consists of subfamilies Convolvuloideae (11 tribes) and Humbertoideae (one tribe, monotypic Humberteae), is the only family in the asterid clade known to produce seeds with PY. The primary aim of this study was to compare the morphoanatomical characteristics of the water gap in seeds of species in the 11 tribes of the Convolvuloideae and to use this information, and that on seed dormancy and storage behaviour, to construct a phylogenetic tree of seed dormancy for the subfamily. METHODS: Scanning electron microscopy (SEM) was used to define morphological changes in the hilum area during dormancy break; hand and vibratome sections were taken to describe the anatomy of the water gap, hilum and seed coat; and dye tracking was used to identify the initial route of water entry into the non-dormant seed. Results were compared with a recent cladogram of the family. KEY RESULTS: Species in nine tribes have (a) layer(s) of palisade cells in the seed coat, a water gap and orthodox storage behaviour. Erycibe (Erycibeae) and Maripa (Maripeae) do not have a palisade layer in the seed coat or a water gap, and are recalcitrant. The hilar fissure is the water gap in relatively basal Cuscuteae, and bulges adjacent to the micropyle serve as the water gap in the Convolvuloideae, Dicranostyloideae (except Maripeae) and the Cardiochlamyeae clades. Seeds from the Convolvuloideae have morphologically prominent bulges demarcated by cell shape in the sclereid layer, whereas the Dicranostyloideae and Cardiochlamyeae have non-prominent bulges demarcated by the number of sub-cell layers. The anatomy and morphology of the hilar pad follow the same pattern. CONCLUSIONS: PY in the subfamily Convolvuloideae probably evolved in the aseasonal tropics from an ancestor with recalcitrant non-dormant seeds, and it may have arisen as Convolvulaceae radiated to occupy the seasonal tropics. Combinational dormancy may have developed in seeds of some Cuscuta spp. as this genus moved into temperate habitats.

A proposed mechanism for physical dormancy break in seeds of Ipomoea lacunosa (Convolvulaceae).

BACKGROUND AND AIMS: The water-impermeable seeds of Ipomoea lacunosa undergo sensitivity cycling to dormancy breaking treatment, and slits are formed around bulges adjacent to the micropyle during dormancy break, i.e. the water gap opens. The primary aim of this research was to identify the mechanism of slit formation in seeds of this species. METHODS: Sensitive seeds were incubated at various combinations of relative humidity (RH) and temperature after blocking the hilar area in different places. Increase in seed mass was measured before and after incubation. Scanning electron microscopy (SEM) and staining of insensitive and sensitive seeds were carried out to characterize these states morphologically and anatomically. Water absorption was monitored at 35 and 25 degrees C at 100 % RH. KEY RESULTS: There was a significant relationship between incubation temperature and RH with percentage seed dormancy break. Sensitive seeds absorbed water vapour, but insensitive seeds did not. Different amounts of water were absorbed by seeds with different blocking treatments. There was a significant relationship between dormancy break and the amount of water absorbed during incubation. CONCLUSIONS: Water vapour seals openings that allow it to escape from seeds and causes pressure to develop below the bulge, thereby causing slits to form. A model for the mechanism of formation of slits (physical dormancy break) is proposed.

Physical dormancy in seeds of the holoparasitic angiosperm Cuscuta australis (Convolvulaceae, Cuscuteae): dormancy-breaking requirements, anatomy of the water gap and sensitivity cycling.

BACKGROUND AND AIMS: Dormancy in seeds of Cuscuta (Convolvulaceae, tribe Cuscuteae) is due to a water-impermeable seed coat (physical dormancy). In nondormant seeds of several species of this family, bulges adjacent to the micropyle have been identified as the initial route of water entry into seeds (water gap). However, there are claims that water enters seeds of Cuscuta spp. via the entire seed coat. Although several studies have been done on seed coat anatomy of Cuscuta, none has identified and/or characterized the morphology/anatomy of a water gap. Thus, the primary aim of this research was to identify and describe the morphology and anatomy of the water gap in seeds of Cuscuta australis. It was also determined if sensitivity cycling to dormancy-breaking treatments occurs in seeds of this species. METHODS: Light microscopy, scanning electron microscopy, tissue-sectioning and dye-tracking and blocking experiments were used to investigate the morphology and anatomy of the water gap. Treatments simulating natural conditions were used to break seed dormancy. Storage of seeds at different temperatures was tested for their effect on sensitivity to dormancy-breaking treatment. KEY RESULTS: Dormancy-breaking treatments caused the tightly closed hilar fissure to open. Staining was observed in cells below the hilum area but not in those below the seed coat away from the hilum. Sensitivity to dormancy-breaking treatment was induced by storing seeds dry and reduced by storing them wet. CONCLUSIONS: Whereas bulges adjacent to the micropyle act as the water gap in other species of Convolvulaceae with physical dormancy, the hilar fissure serves this function in Cuscuta. Cuscuta australis can cycle between insensitivity <--> sensitivity to dormancy-breaking treatments.

Cycling of sensitivity to physical dormancy-break in seeds of Ipomoea lacunosa (Convolvulaceae) and ecological significance.

BACKGROUND AND AIMS: Although a claim has been made that dormancy cycling occurs in seeds of Ipomoea lacunosa (Convolvulaceae) with physical dormancy, this would seem to be impossible since the water gap cannot be closed again after it opens (dormancy break). On the other hand, changes in sensitivity (sensitive <--> non-sensitive) to dormancy-breaking factors have been reported in seeds of Fabaceae with physical dormancy. The primary aim of the present study was to determine if sensitivity cycling also occurs in physically dormant seeds of I. lacunosa. METHODS: Treatments simulating conditions in the natural habitat of I. lacunosa were used to break seed dormancy. Storage of seeds at temperatures simulating those in spring, summer, autumn and winter were tested for their effect on sensitivity change. Seeds made non-dormant were stored dry in different temperature regimes to test for dormancy cycling. In addition, seeds collected on different dates (i.e. matured under different climatic conditions) were used to test for maternal effects on sensitivity to dormancy-breaking factors. KEY RESULTS: Sensitivity was induced by storing seeds under wet conditions and reversed by storing them under dry conditions at low (< or = 5 degrees C) or high (> or = 30 degrees C) temperatures, demonstrating that seeds of I. lacunosa can cycle between sensitive and insensitive states. Sensitive seeds required > or = 2 h at 35 degrees C on moist sand for release of dormancy. However, there is no evidence to support dormancy cycling per se. Conceptual models are proposed for sensitivity cycling and germination phenology of I. lacunosa in the field. CONCLUSIONS: Seasonal germination behaviour of physically dormant I. lacunosa seeds can be explained by sensitivity cycling but not by dormancy cycling per se. Convolvulaceae is only the second of 16 families known to contain species with physical dormancy for which sensitivity cycling has been demonstrated.

Seed development in Ipomoea lacunosa (Convolvulaceae), with particular reference to anatomy of the water gap.

BACKGROUND AND AIMS: Disruption of one or both of the bulges (water gap) in the seed coat adjacent to the micropyle is responsible for breaking physical dormancy (PY) in seeds of Ipomoea lacunosa and other taxa of Convolvulaceae. Hitherto, neither ontogeny of these bulges nor onset of PY together with anatomical development and maturation drying of the seed had been studied in this family. The aims of this study were to monitor physiological and anatomical changes that occur during seed development in I. lacunosa, with particular reference to ontogeny of the water gap. METHODS: Developmental anatomy (ontogeny) of seed coat and dry mass, length, moisture content, germinability and onset of seed coat impermeability to water were monitored from pollination to seed maturity. Blocking/drying and dye-tracking experiments were done to identify site of moisture loss during the final stages of seed drying. KEY RESULTS: Physiological maturity of seeds occurred 22 d after pollination (DAP), and 100 % of seeds germinated 24 DAP. Impermeability of the seed coat developed 27-30 DAP, when seed moisture content was 13 %. The hilar fissure was identified as the site of moisture loss during the final stages of seed drying. The entire seed coat developed from the two outermost layers of the integument. A transition zone, i.e. a weak margin where seed coat ruptures during dormancy break, formed between the bulge and hilar ring and seed coat away from the bulge. Sclereid cells in the transition zone were square, whereas they were elongated under the bulge. CONCLUSIONS: Although the bulge and other areas of the seed coat have the same origin, these two cell layers underwent a different series of periclinal and anticlinal divisions during bulge development (beginning a few hours after pollination) than they did during development of the seed coat away from the bulge. Further, the boundary between the square sclereids in the transition zone and the elongated ones of the bulge delineate the edge of the water gap.

Morphology and anatomy of physical dormancy in Ipomoea lacunosa: identification of the water gap in seeds of Convolvulaceae (Solanales).

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 experiments 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 angiosperm plant families that produce seeds with physical dormancy for which it has been described.

Molecular electrostatic potentials: an effective tool for the elucidation of biochemical phenomena.

The electrostatic potential V(r) that is created in the space around a molecule by its nuclei and electrons (treated as static distributions of charge) is a very useful property for analyzing and predicting molecular reactive behavior. It is rigorously defined and can be determined experimentally as well as computationally. The potential has been particularly useful as an indicator of the sites or regions of a molecule to which an approaching electrophile is initially attracted, and it has also been applied successfully to the study of interactions that involve a certain optimum relative orientation of the reactants, such as between a drug and its cellular receptor. A variety of methods for calculating V(r) is available, at different levels of rigor. For large biologically active molecules, multipole expansions and superposition of potentials computed for subunits have been found to be effective. A large number of chemical and biochemical systems and processes have now been studied in terms of electrostatic potentials. Three examples of such applications are surveyed in this paper. These deal with: (a) reactive properties of nucleic acids, including their component bases; (b) biological recognition processes, including drug-receptors and enzyme-substrate interactions; and (c) chemical carcinogenesis, referring specifically to the polycyclic aromatic hydrocarbons and halogenated olefins and their epoxides. For each of these areas, examples of the use of electrostatic potentials in elucidating structure-activity patterns are given.