Robert L. Geneve

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.

Multiple shoot formation from cotyledonary node segments of Eastern redbud.

A procedure for multiple shoot formation from cotyledonary node explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced higher numbers of shoots than with either cytokinin alone. The highest number of shoots (7.8 to 9.8 shoots per explant) was obtained when explants from 4 to 10 day-old seedlings were treated with a combination of 10 or 15 µM BA and 0.5 or 1.0 µM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 5.8 to 7.2 shoots per explant by cutting through the cotyledonary node prior to culture. Histological studies indicated that the shoots were formed from actively dividing cells located at the axillary bud region. Shoots formed roots in half strength woody plant medium (WPM) supplemented with 10 to 200 µM indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse medium.

Identification and characterization of ten new water gaps in seeds and fruits with physical dormancy and classification of water-gap complexes

BACKGROUND AND AIMS Physical dormancy (PY) occurs in seeds or fruits of 18 angiosperm families and is caused by a water-impermeable palisade cell layer(s) in seed or fruit coats. Prior to germination, the seed or fruit coat of species with PY must become permeable in order to imbibe water. Breaking of PY involves formation of a small opening(s) (water gap) in a morpho-anatomically specialized area in seeds or fruits known as the water-gap complex. Twelve different water-gap regions in seven families have previously been characterized. However, the water-gap regions had not been characterized in Cucurbitaceae; clade Cladrastis of Fabaceae; subfamilies Bombacoideae, Brownlowioideae and Bythnerioideae of Malvaceae; Nelumbonaceae; subfamily Sapindoideae of Sapindaceae; Rhamnaceae; or Surianaceae. The primary aims of this study were to identify and describe the water gaps of these taxa and to classify all the known water-gap regions based on their morpho-anatomical features. METHODS Physical dormancy in 15 species was broken by exposing seeds or fruits to wet or dry heat under laboratory conditions. Water-gap regions of fruits and seeds were identified and characterized by use of microtome sectioning, light microscopy, scanning electron microscopy, dye tracking and blocking experiments. KEY RESULTS Ten new water-gap regions were identified in seven different families, and two previously hypothesized regions were confirmed. Water-gap complexes consist of (1) an opening that forms after PY is broken; (2) a specialized structure that occludes the gap; and (3) associated specialized tissues. In some species, more than one opening is involved in the initial imbibition of water. CONCLUSIONS Based on morpho-anatomical features, three basic water-gap complexes (Types-I, -II and -III) were identified in species with PY in 16 families. Depending on the number of openings involved in initial imbibition, the water-gap complexes were sub-divided into simple and compound. The proposed classification system enables understanding of the relationships between the water-gap complexes of taxonomically unrelated species with PY.

Seed germination of ethylene perception mutants of tomato and Arabidopsis

The involvement of ethylene in determining the time to radicle protrusion was investigated in ethyleneinsensitive gain-of-function (GOF) receptor mutants in tomato and Arabidopsis, as well as in single and double loss-of-function (LOF) receptor mutants in Arabidopsis. Because ethylene evolution from seeds is coincident with radicle protrusion, and the ability to convert 1aminocyclopropane-1-carboxylic acid (ACC) to ethylene is diagnostic for seed vigour, it was hypothesized that ethylene-insensitive mutants would require more time to complete germination compared to wild-type seeds. Mutant Never Ripe (Nr) tomato seeds from two genetic backgrounds refuted this hypothesis, while experiments with wild-type seeds, treated with the ethylene action inhibitors, 2,5-norbornadiene or silver thiosulphate, supported it. However, reciprocal crosses between wildtype and Nr demonstrated that ethylene insensitivity during seed development determined subsequent time to complete germination, rather than the ability of the embryo/endosperm to perceive ethylene in the mature seed during germination. Additionally, seed quality, determined by standard vigour tests, was reduced in Nr compared to wild-type seeds, establishing a disconnection between rapid completion of germination and seed vigour. In Arabidopsis, all ethylene-insensitive GOF, and five of six single LOF mutants, required more time to complete 50% radicle protrusion, while double LOF mutants required the same, or less, time to complete germination compared to wild-type seeds. These findings support a role for ethylene perception in determining the length of time Arabidopsis seeds remain in the lag phase prior to radicle protrusion.

The autumn effect: timing of physical dormancy break in seeds of two winter annual species of Geraniaceae by a stepwise process

BACKGROUND AND AIMS The involvement of two steps in the physical dormancy (PY)-breaking process previously has been demonstrated in seeds of Fabaceae and Convolvulaceae. Even though there is a claim for a moisture-controlled stepwise PY-breaking in some species of Geraniaceae, no study has evaluated the role of temperature in the PY-breaking process in this family. The aim of this study was to determine whether a temperature-controlled stepwise PY-breaking process occurs in seeds of the winter annuals Geranium carolinianum and G. dissectum. METHODS Seeds of G. carolinianum and G. dissectum were stored under different temperature regimes to test the effect of storage temperature on PY-break. The role of temperature and moisture regimes in regulating PY-break was investigated by treatments simulating natural conditions. Greenhouse (non-heated) experiments on seed germination and burial experiments (outdoors) were carried out to determine the PY-breaking behaviour in the natural habitat. KEY RESULTS Irrespective of moisture conditions, sensitivity to the PY-breaking step in seeds of G. carolinianum was induced at temperatures ≥20 °C, and exposure to temperatures ≤20 °C made the sensitive seeds permeable. Sensitivity of seeds increased with time. In G. dissectum, PY-break occurred at temperatures ≥20 °C in a single step under constant wet or dry conditions and in two steps under alternate wet-dry conditions if seeds were initially kept wet. CONCLUSIONS Timing of seed germination with the onset of autumn can be explained by PY-breaking processes involving (a) two temperature-dependent steps in G. carolinianum and (b) one or two moisture-dependent step(s) along with the inability to germinate under high temperatures in G. dissectum. Geraniaceae is the third of 18 families with PY in which a two-step PY-breaking process has been demonstrated.

Patterns of adventitious root formation in English ivy

Adventitious root formation by debladed petiole cuttings of English ivy (Hedera helix L.) proceeds via a direct rooting pattern for the easy-to-root juvenile phase, while the difficult-to-root mature phase roots through an indirect rooting pattern. Juvenile petiole cuttings treated with α-naphthaleneacetic acid (NAA, 100 μM) plus the polyamine biosynthesis inhibitor, difluoromethylarginine (DFMA, 1 mM), formed an increased number of roots per cutting initiated by the indirect rooting pattern. The increased root formation and change in rooting pattern were reversed by the addition of putrescine (1 mM). Delaying auxin application to petiole cuttings for 15 days also induced juvenile petioles to root by the indirect pattern. This could be reversed by rewounding the base of the cutting prior to auxin application after day 15. The data support the use of the terms “competent root-forming cells” and “induced competent root-forming cells” to describe the target cells for the initial events of root formation for the direct and indirect rooting patterns, respectively.

The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 μM α-naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 μM 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 μM 2,4-D and acclimated to greenhouse conditions.

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.

Sensitivity Cycling and Mechanism of Physical Dormancy Break in Seeds of Ipomoea hederacea (Convolvulaceae)

Sensitivity cycling to physical dormancy (PY) break in seeds is known to occur in some Fabaceae and Convolvulaceae species. PY in seeds of species of Convolvulaceae and of some other angiosperm plant families can be broken by storing them dry. However, the mechanism of opening the water gap in the seed coat (dormancy break) during dry storage has not been investigated. In research reported here, we determined whether sensitivity cycling occurs in seeds of Ipomoea hederacea (Convolvulaceae) and investigated the effect of dry storage on opening of the water gap. Seeds can cycle between insensitive and sensitive states to dormancy break, and dormancy can be broken in sensitive seeds by storing them dry at high to moderate temperatures. Seeds with the lower part of the hilum blocked lost a minimal amount of water during storage. Percentage water loss was significantly correlated with percentage dormancy break. Desorption of water through the hilar fissure during dry storage reduced the vapor pressure below the bulges (water gap), which caused slits to form around the bulges (opening of water gap). The amount of water desorption was low in insensitive seeds because the hilar fissure remained closed and thus the water gap did not open.

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.