Morphology and anatomy of the fruit and seed in development of Chorisia speciosa A. St.-Hil. - Bombacaceae

The structure of the fruit and seed in development of Chorisia speciosa are described with the main purpose of clarifying the origin and nature of the hairs that cover the seeds and aiding future taxonomical and ecological studies of the group. The fruit is an ellipsoid loculicide capsule and presents the exocarp formed by 7-10 cells layers, with very thick walls and evident simple pits. A great number of mucilage secretory cavities and ramified vascular bundles, accompanied by fibers, occur in the parenchymatic mesocarp. The endocarp derives from the ventral epidermis of the ovary wall, whose cells undergo a gradual elongation, become lignified, and constitute the trichomes which cover the mature seeds. The fruit aperture occurs by means of a suture evident in the ovarian wall in the middle region of the carpel leaf. Anatropous and bitegmic ovules, provided by a hypostase, give rise to campilotropous and bitegmic seeds. The testa is uniseriate, the exotegmen is completely formed by macrosclereids, and mucilage secretory cavities occur in the mesotegmen. The endotegmen, which is differentiated in the endothelium, is crushed in the mature seed. The plicate embryo, which occupies practically the entire seminal cavity, is found between endosperm layers, both being rich in lipids.

Changes in polyamines content associated with zygotic embryogenesis in the Brazilian pine, Araucaria angustifolia (Bert.) O. Ktze.

Changes in the polyamine content were analyzed in different embryo developmental stages and tissues during seed development in the conifer Araucaria angustifolia (Bert.) O. Ktze. Free polyamine contents varied according to the tissue and stage of embryo development, the highest levels occurring in the embryonic axis at the early stages, when putrescine and spermidine were most abundant. The levels of spermidine were higher from the stage where cotyledons arise, whereas putrescine decreases. The putrescine/spermine+spermidine ratio was higher during the initial phases of seed development, corresponding to cell multiplication and elongation, with a decrease in the final stages, corresponding to stabilization of the dry matter content.

In vitro somatic embryogenesis and adventitious root initiation have a common origin in eggplant (Solanum melongena L.)

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

Female gametophyte development in Adesmia latifolia (Spreng.) Vog. (Leguminosae - Papilionoideae)

The female gametophyte has a monosporic origin and a Polygonum type development. The female gametophyte growth consumes a large part of the neighboring nucellar cells and, in the micropylar region, part of the nucellar epidermis and internal integument. The mature gametophyte is composed of only four cells due to the ephemeral characteristic of the antipodals. The synergids are pear-shaped cells with the formation of filiform apparatus. Their nuclei lie in the micropylar region and large vacuoles in the chalazal region, while the egg cell presents an opposite polarization. The central cell accumulates starch grains and the two polar nuclei approach the egg. Occasional development of two gametophytes was recorded. The closest to the micropyle develops fully, while the chalazal one remained in the tetranucleated stage. The embryological characters of A. latifolia are compared with those of other taxa within the Leguminosae, and the reproductive importance of multiple gametophyte formation for this species was discussed.

Anomalous somatic embryos in Acca sellowiana (O. Berg) Burret (Myrtaceae)

Somatic embryogenesis represents a valuable tool for the studies on the basic aspects of plant embryo development. Today this process is used as a potencial technique for large-scale plant micropropagation although, so far, it has been applied to only a small number of species. However, when somatic embryos are malformed they are considered economically useless. In Acca sellowiana (O. Berg) Burret, an important fruit-producing crop, large amounts of anomalous somatic embryos (76.3%) were found just after 40 days of culture of explants in a 2,4-D containing medium. Among the anomalous forms found in the cotiledonary stage, 12.2% consisted of fused embryos, 40.4% displayed fused cotyledons, 13.0% presented supernumerary cotyledons, and 10.7% showed absence or poorly developed cotyledons, including those without the shoot apical meristem. Histological analyses indicated that the altered embryos were formed either directly from cotyledons, hypocotyl and radicle of the zygotic embryos used as explants, or indirectly from calli formed from these tissue parts. It is suggested that the formation of anomalous somatic embryos, as well as a low frequency of conversion into emblings reflect physiological and/or genetic disturbances triggered by the presence of 2,4-D in the medium. In vitro experimental alternative approaches are discussed in order to lessen the occurrence of malformed somatic embryos.

Multiple dormancy and maternal effect on Miconia ferruginata (Melastomataceae) seed germination, Serra de Caldas Novas, Goiás, Brazil

Species with mostly asexual reproduction are interesting subjects for germination studies since variation would be more easily linked to environmental factors. Miconia ferruginata DC. is an apomictic treelet in Brazilian cerrado areas on rocky outcrops. Germination of seeds collected from individuals occurring in the Serra de Caldas Novas State Park, Goiás, was studied in three experiments under controlled conditions. Germination characteristics differed among individuals and were correlated with altitude and soil Al content. Seeds from plants growing at lower altitudes, with lower soil aluminium content, presented malformed seeds with absence of embryo which rendered lower, but better synchronized germination. The nested analysis showed that from the total variance, 78.14% for germinability, 54.56% for uncertainty of the germination process, and 68.30% for the quantity of seeds without embryo was attributed to the altitudinal effect. Individuals nested within altitude contributed up to 16.93% for the total variance. It means that there is low variability among individuals of the same altitude and high variability among individuals from different points of the slope, making clear that for the studied population the environmental effect is stronger than the genetic component to determine the seed quality. The testa of the seeds provides a mechanical dormancy which seems to be associated also with phenolic compounds, which help to disperse germination through time. Photoblastism was also registered for seeds of this species.

Morphology and anatomy of the developing fruit of Maclura tinctoria, Moraceae

(Morphology and anatomy of the developing fruit of Maclura tinctoria, Moraceae). Maclura tinctoria (L.) D. Don ex Steudel was selected for the present study due of its economic and medicinal importance. The purpose of this investigation is to present a detailed description of the fruit development, specially by: (a) defining the fruit type presented by the species, and (b) characterizing the seed type of the species based upon the presence or not of mechanical tissue on the seed-coat. The fruit originates from the subglobose female inflorescence which consists of small unipistillate flowers with superior ovary, unilocular and uniovular apical placentation. The mature fruit is multiple, constituted of small drupes. The ovule is ana-campylotropous, suspended, bitegmic and crassinucellate. The mature seed is flattened, slightly ovated, cream colored, with unspecialized membrane coat with thin-walled cells more or less crushed. The seed has parenchymatic endosperm with lipophilic content. The embryo is straight, with two cotyledons of the same size. Ontogenetic studies reveal that the fruits are infrutescences. The fleshy edible part is derived from the perigone and inflorescence axis. The drupes consist of a single pyrene of macrosclereids.

Viability and vigor of jamun (Syzygium cumini) seeds

Jamun (Syzygium cumini L. Skeels) (Black plum, Damson plum) fruits weigh between 2-5 g at maturity. Fresh seeds represented 20-80% of the total fruit weight; the seed coat and cotyledons contributed 6% and 94% to the total seed weight respectively, while the weight of the embryonic axis was insignificant. Only the embryonic axis stained with Tetrazolium, not the cotyledons. The seeds are polyembryonic with up to four embryos, of which at most three germinate. Decoated seeds germinated faster than coated seeds under nursery conditions, with high significant germination percentages, dry matter production rates and vigor indices. The lack of staining of the cotyledon by tetrazolium was probably due to the presence of an impermeable layer. Decoating seeds for faster germination is recommended.

Egg size, yolk mass extrusion and hatching behavior in two cryptic speciesof Anastrepha fraterculus (Wiedemann) (Diptera, Tephritidae)

Variations in egg length were observed for two populations of cryptic species of Anastrepha fraterculus (Wiedemann). The eggs of type I flies were smaller than those of type II individuals. For both types, in regard to yolk mass extrusion, four classes of embryos were detected. Class 1: embryos that extrude masses at both extremities; class 2: embryos in which extrusion occurs only at the anterior pole; class 3: embryos that eliminate mass only at the posterior pole, and class 4: embryos that do not extrude any mass. Embryo class frequencies were similar for populations belonging to the same type, but different between types. Individual females may produce eggs from different embryo classes, but for any given female the pattern remains constant during a long period of oviposition. Variation in size of the extruded masses was similar for both populations. Individual females produced embryos with a small range of mass diameters, and different females produced masses of different mean size. However, individual mass size remained constant during oviposition. The results suggest the existence of genetic components involved in the control of this unusual process. Larvae of both types presented, just before eclosion, similar unusual behaviors: they ingest the anterior extruded mass, rotate 180°, absorb the posterior mass and eclose near the posterior pole. Data show that cryptic A. fraterculus type I and type II differs in regard to egg size as well as to the phenomenon of yolk mass extrusion

Inhibition of carbachol-induced inositol phosphate accumulation in the embryonic retina promoted by kainate and veratridine

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 µM carbachol was used, the estimated IC50 value for kainate was 0.2 µM and the maximal inhibition of ~50% was obtained with 1 µM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 µM veratridine, but not 50 mM KCl, inhibited ~65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 ± 38.0 to 2044.5 ± 299.9 cpm/mg protein, retinal response decreased to 861.6 ± 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 µg/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 µg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 µM acetylcholine alone and cumulative concentrations of 1 µM to 10 mM were unaffected. At venom concentrations higher than 50 µg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 µg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

Distribution of versican and hyaluronan in the mouse uterus during decidualization

Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-µm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.