Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.     

Understanding the threats to biological diversity in southeastern Brazil

Habitat destruction and the over-exploitation of species are perhaps thetwo most important processes threatening biodiversity. Whilst the growing humanpopulation puts considerable pressure on biological resources, a number ofsocial and economic factors tend to augment over-exploitation of theseresources. Here we show that the over-exploitation of the tropical palm treeEuterpe edulis Mart., as a consequence of social problems,has contributed significantly to its extinction in several forest fragments andalso to the disappearance of many wild animal species which used to be found inareas of the Atlantic Rain Forest (Brazil). Some of these species have been lostas a direct result of people hunting the animals when they went harvestingE. edulis while others disappeared as a consequence of thedecrease in food availability or by alterations in the trophic structure.     

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen.To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3 °C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 °C with dimethylsulfoxide (Me₂SO), ethylene glycol (EG), 1–2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30 min. A first evaluation of freezing rates was made by testing four freezing curves: −1, −3, −6 and −10 °C/min. Then, an optimization was made by testing four freezing curves: −2.5, −3.0, −3.5 and −4 °C/min.The selected temperature for short term conservation has been 3 °C, because only this temperature has allowed good sperm motility conservation after 3 h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me₂SO. EG and MetOH to all concentrations and Me₂SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate −3 °C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.     

Detection and release of allergenic proteins in Parietaria judaica pollen grains

Summary. Rapid diffusion of allergenic proteins in isotonic media has been demonstrated for different pollen grains. Upon contact with stigmatic secretion or with the mucosa of sensitive individuals, pollen grains absorb water and release soluble low-molecular-weight proteins, these proteins enter in the secretory pathway in order to arrive at the cell surface. In this study we located allergenic proteins in mature and hydrated-activated pollen grains of Parietaria judaica L. (Urticaceae) and studied the diffusion of these proteins during the first 20 min of the hydration and activation processes. A combination of transmission electron microscopy and immunocytochemical methods was used to locate these proteins in mature pollen and in pollen grains after different periods of hydration and activation processes. Activated proteins reacting with antibodies in human serum from allergic patients were found in the cytoplasm, wall, and exudates from the pollen grains. The allergenic component of these pollen grains changes according to the pollen state; the presence of these proteins in the exine, the cytoplasm, and especially in the intine and in the material exuded from the pollen grains, is significant in the hydrated-activated studied times, whereas this presence is not significant in mature pollen grains. The rapid activation and release of allergenic proteins of P. judaica pollen appears to be the main cause of the allergenic activity of these pollen grains. Correspondence and reprints: Department of Plant Biology, Faculty of Biology, University of León, Campus de Vegazana, 24071 León, Spain.     

Turnover rate of metallothionein and cadmium in Mytilus edulis

The results demonstrate the first attempt to determine metallothionein turnover in the whole soft tissues of mussels Mytilus edulis exposed to cadmium. Half-lives for metallothionein and cadmium are 25 and 300 days, respectively. As metallothionein degrades the released cadmium induces further synthesis of the protein, to which the metal becomes resequestered. The slow metallothionein turnover rates (compared with mammals) and the lack of significant cadmium excretion testify to the relatively stable nature of the cadmium-metallothionein complex in these invertebrates and supports the view of a detoxifying role for metallothionein in the mussels.     

Phytochrome-mediated long-term memory of seeds

The question is how long phytochrome, stored within the cytoplasm of plant diaspores, may stimulate their germination. This question arose from the observation that soil cultivations in darkness for weed control gave inconsistent results. Namely, after a single nighttime or daytime cultivation during spring and summer, differences in weed emergence became hardly detectable after a period of six weeks. However, after nighttime and daytime cultivations in late autumn, emergence differences persisted for up to nine months. To examine whether this differing memory effect is phytochrome-mediated, seeds of Chenopodium album and Stellaria media were sown in pots with wet peat, either in daylight or after sunset. In the latter, seeds were irradiated with far-red light for one day prior to being covered and buried. For more than two years the far-red irradiated seeds produced significantly reduced emergence, indicating that germination and emergence of weeds in the field may be supported by maternal far-red absorbing seed phytochrome B(fr) over several months or even years. This conclusion allows refining of the strategy of lightless tillage.     

Comparative structure of ovules and seeds inRafflesiaceae

The genera of theRafflesiaceae show a marked diversity in the structure of their ovules and seeds. Evolutionary trends are recognizable in ovule orientation and number of integuments. A change from anatropous ovules inApodantheae andMitrastemoideae towards incomplete anatropy inRafflesieae and orthotropy inCytineae occurs, next to a change from bitegmic ovules inApodantheae towards unitegmy with rudimentary outer integuments inRafflesieae andCytineae and full unitegmy inMitrastemoideae.—The differences in ovule structure are clearly reflected in the seeds. The seeds are essentially exotegmic, have very small embryos and an oily endosperm.—Seed structure strongly confirms the existing subfamilial classification and supports additional arguments for the generic status ofApodanthes. It does not support a separate status of the genusBerlinianche. InRafflesiaceae, seed micromorphology is only of limited use at the species level. As far as known seed dispersal is endo- or exozoochorous in all genera.     

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM^®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.     

Dry artificial seeds and desiccation tolerance induction in microspore-derived embryos of broccoli

Desiccation tolerance of broccoli microspore-derived embryos was induced by exogenous application of abscisic acid (ABA). Embryos, which were desiccated to about 10% water content, were estimated for viability after rehydration. Survival was dependent on the ABA concentration and the development stage of embryo, but not on the length of exposure period to ABA or genotype. Cotyledonary stage embryos acquired the highest desiccation tolerance when treated with 1×10^-4M ABA. Under this condition, on average 27–48% of the desiccated embryos could convert into plants. Embryos treated with 1×10^-6M ABA or no ABA or earlier development-staged embryos, such as globular and heart stages, lost viability after desiccation. A one day exposure to ABA had the similar effect on the induction of desiccation tolerance as a 7-day treatment. The dried embryos maintained their ability of plant conversion after three months of storage under room conditions. The plants derived from the desiccated embryos were not different in the morphology or ploidy level from those from non-desiccated ones.Abbreviations ABA abscisic acid - RH relative humidity     

The expression of dehydrin proteins in desiccation-sensitive (recalcitrant) seeds of temperate trees

Proteins that have homology with dehydrins have been identified immunologically in the desiccationsensitive (recalcitrant) seeds of English oak (Quercus robur L.), European chestnut (Castanea sativa L.), horse chestnut (Aesculus hippocastanum L.), sycamore (Acer psuedoplatanus L.) and silver maple (Acer saccharinum L.), and in the desiccation-tolerant seeds of Norway maple (Acer platanoides L.). The mRNA for a late embryogenesis abundant (LEA) protein (dehydrin) was also detected by Northern blotting, using a cDNA clone (D11) from cotton embryos, in the recalcitrant and orthodox seeds. Medium-stringency washing was required to detect this hybridization. InQ. robur the amount of dehydrin protein increased during seed development, andLEA mRNA was induced by limited desiccation and by abscisic acid. Confirmation of the presence of dehydrin mRNA in matureQ. robur andC. sativa seeds was obtained by in-vitro translation of the extracted polyadenylated RNA followed by analysis of the immunoprecipitation products. Thus the presence of dehydrin proteins is not sufficient to confer desiccation tolerance on truly recalcitrant seeds, nor can their presence or absence be used as clear criteria for identification of recalcitrant seeds.     

Post-dispersal predation and scatterhoarding of Dipteryx panamensis (Papilionaceae) seeds by rodents in Panama

In tropical rain forests of Central America, the canopy tree Dipteryx panamensis (Papilionaceae) fruits when overall fruit biomass is low for mammals. Flying and arboreal consumers feed on D. panamensis and drop seeds under the parent or disperse them farther away. Seeds on the ground attract many vertebrate seed-eaters, some of them potential secondary seed dispersers. The fate of seeds artificially distributed to simulate bat dispersal was studied in relation to fruitfall periodicity and the visiting frequency of diurnal rodents at Barro Colorado Island (BCI), Panama. The frequency of visits by agoutis is very high at the beginning of fruitfall, but in the area close (<50 m) to fruiting trees (Dipteryx-rich area) it declines throughout fruiting, whereas it remains unchanged farther (>50 m) away (Dipteryx-poor and Gustavia-rich area). Squirrels were usually observed in the Dipteryx-rich area. Along with intense post-dispersal seed predation by rodents in the Dipteryx-rich area, a significant proportion of seeds were cached by rodents in the Dipteryx-poor area. Post-dispersal seed predation rate was inversely related to hoarding rate. A significantly greater proportion of seeds was cached in March, especially more than 100 m from the nearest fruiting tree. This correlates with the mid-fruiting period, i.e. during the height of D. panamensis fruiting, when rodents seem to be temporarily satiated with the food supply at parent trees. Hoarding remained high toward April, i.e. late in the fruiting season of D. panamensis. Low survival of scatterhoarded seeds suggests that the alternative food supply over the animal's home-ranges in May–June 1990 was too low to promote survival of cached seeds. Seedlings are assumed to establish in the less-used area of the rodents' home-range when overall food supply is sufficient to satiate post-dispersal predators.     

Endosperms and embryos of mature dry castor bean seeds contain active ribosomes

Mature dry endosperms and embryos of castor bean contain ribosomes which are capable of catalysing protein synthesis in vitro. This observation contradicts previous reports that ribosomes are absent from mature dry endosperms. On hydration of the mature seeds the polyribosome content of both embryos and endosperms increases. These polyribosomes are lost on subsequent dehydration but, again, ribosomes are conserved. REFERENCES     

Hoarding of acorns by granivorous mice and its role in the population processes of Pasania edulis (Makino) Makino

In order to evaluate the role of granivorous mice, Apodemus speciosus Temminck and Apodemus argenteus Temminck, in the regeneration of Pasania edulis (Makino) Makino, an evergreen tree, we conducted field studies examining acorn crops, the population dynamics and hoarding behavior of mice and pre- and postdispersal acorn predation in an evergreen broad-leaved forest dominated by P.edulis in Kagoshima, southern Japan. The study was conducted from 1994 to 2000. Apodemus mice selected sound acorns and hoarded them in the soil after transporting them for distances ranging from 5.4 to 19.9m. This behavior appeared to be beneficial to the establishment of seedlings of P.edulis. However, the positive effects were negated by a high rate of recovery of the acorns hoarded by the mice. A life-table analysis of five cohorts of P.edulis indicated that postdispersal predation of acorns by Apodemus mice was a major contributor to annual variation in the number of new seedlings. A considerable number of acorns germinated only in years with a good acorn crop and a low level of resident mice. These results suggest that Apodemus mice are serious acorn predators rather than important dispersers. After germination, acorns were attacked by the acorn borer at a high rate. From these results, we can conclude that acorn production probably makes a smaller contribution to the regeneration of P.edulis than coppice shoot production at the study site.     

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans

Summary. The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (15)--arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.Correspondence and reprints: Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland     

Protection against suspended sand: the function of the branchial membrane in the blue mussel Mytilus edulis

Blue mussels (Mytilus edulis) living in estuaries have to cope with varying concentrations of suspended sand. Sand flowing through the inhalant siphons comes into the infrabranchial chamber. The inhalant siphon can be partially closed by the branchial membrane. As a result the inward flow decreases, and suspended sand sinks and can be eliminated. Experiments with mussels from three ecologically different locations showed about the same response of the branchial membrane on contact with suspended sand. The presence and function of the branchial membrane appears to be an adaptation of mussels to their estuarine environment.     

An improved clearing method for GUS assay inArabidopsis endosperm and seeds

Precise cellular localization of the GUS stain is notoriously difficult inArabidopsis seeds. Here we report an improved protocol for the clearing of seeds after GUS staining. Incubation in ethanol-acetic acid (EtAc) and Hoyer’s medium allows reliable cellular localization of the GUS, even in seeds from late developmental stages. This method also leads to the staining of nucleoli in the endosperm and embryo, facilitating nuclear counts in endosperm development. An erratum to this article is available at .     

Effects of microhabitat on palm seed predation in two forest fragments in southeast Brazil

The establishment of plants depends crucially on where seeds are deposited in the environment. Some authors suggest that in forest understory seed predation is lower than in gaps, and higher than at the forest edge. However, most studies have been carried out in large forest patches and very little is known about the effects of microhabitat conditions on seed predation in forest fragments. We evaluated the effects of three microhabitats (gaps, forest edge, and understory) on seed predation of two palm species (Euterpe edulis and Syagrus romanzoffiana) in two semi-deciduous forest fragments (230 and 2100 ha) in southeast Brazil. Our objective was to test two hypotheses: (1) Low rodent abundance in small fragments as a result of meso-predator action levels leads to lower seed predation in small fragments. (2) Most mammal species in small fragments are generalists with respect to diet and habitat, so that seed predation is similar in different microhabitats (gaps, forest edge and understory) in the small fragment, but not in the larger one. The study community of small fragments is usually composed of generalist species (in diet and habitat aspects), so we expected the same rate of seed predation among microhabitats (gaps, forest edge and understory) in the tested smaller fragment. The experiment was carried out in the dry season (for E. edulis) and in the wet season (for S. romanzoffiana) in 1999. We conclude that post-dispersal seed predation in forest fragments can be directly connected with mammal communities, reflecting their historical and ecological aspects.