Histological changes in banana explants, cv. Nanicão (Musa spp., Group AAA), submitted to different auxins for induction of somatic embryogenesis

The present work analyzes the behavior of banana explants, cv. Nanicão (Musa spp. Group AAA) regarding somatic embryogenesis induction treatments with several auxins. Longitudinal segments of shoot meristematic apices of micropropagated banana plantlets cultivated and rooted in vitro were introduced in culture medium containing dicamba, picloram, 2,4-D or NAA in different concentrations. Explant samples were collected at 0, 7 and 10 days and prepared for light microscopy. Histological sections were used for comparison of the histological changes occurring after induction treatment with different auxins. Embryogenic response was observed only in treatments with picloram or dicamba, with distinct embryogenic regions observed at 14 and 21 days in culture, respectively. Histological sections of embryogenic regions of the explant at 26 days in culture revealed the formation of meristematic regions, structures with multiple root meristems, and somatic embryos at early globular stages. Embryo-like structures morphologically similar to Musa balbisiana zygotic embryos were sectioned and showed a lack of apical meristems and absence of procambial differentiation. These results indicate the induction of non-functional somatic embryos and the need for more studies on developmental aspects and maturation treatments for optimization of the process.

Ovule ontogeny of Tabebuia pulcherrima Sandwith (Bignoniaceae): megasporogenesis and integument development

The ovule of Tabebuia pulcherrima is anatropous, unitegmic and tenuinucellate. The nucellus shows a trizonate structural organization. The integument is initiated by periclinal divisions in the dermal layer (zone I), around the base of the archesporium. Subsequently, cells derived from the subdermal layer (zone II) start to push the dermal cells, so that they shift toward the micropylar region. The archesporial cell differentiates directly into the megaspore mother cell, undergoes meiosis, and originates a linear tetrad of megaspores. The mature embryo sac mother cell is elongated, possess a conspicuous central nucleus, and a characteristic bipolar vacuome with fibrous-granulated content. The inner cell layers of the integument differentiate into an amyloplast-rich endothelium. Patterns of callose deposition in the tetrad and selection of the functional megaspore, as well as the taxonomic value of some characters are discussed.

Effects of iron-ore particles on propagule release, growth and photosynthetic performance of Sargassum vulgare C. Agardh (Phaeophyta, Fucales)

The effect of iron-ore particles on the propagule release and growth of Sargassum vulgare C. Agardh was tested under treatments with different concentrations of iron-ore particles: 0.1, 1.0, 10.0 g.L-1 and a solution of 10.0 g.L-1 of filtered iron-ore. Filtered seawater was used as control. Photosynthesis vs. irradiance (P-I) curves were calculated for S. vulgare in the presence of iron-ore and in seawater. There was no significant difference in the number of propagules released by the receptacles or in the percentage of zygote formation among the treatments. The released propagules acted like aggregation centers for the particles, those more heavily coated with iron (10.0 g.L-1) exhibiting the highest sinking velocity (32.6 ± 9.8 mm.s-1). No difference in the percentage of embryo survival was detected during the first week in culture. After four weeks the embryos grew in all treatments. Maximum frond development (5.3 ± 0.8 mm) was observed in treatment of seawater enriched with Provasoli's medium (PES) while initial filoids did not develop in three treatments without PES and with iron-ore (0.1 g.L-1, 1.0 g.L-1 and 10.0 g.L-1). The values for Pmax, alpha and respiration showed no significant differences between the P-I curves. The calculated value for I K was 106.26 µmol.m-2.s-1 to the control curve and 981.49 µmol.m-2.s-1 to the iron-ore curve. The results indicate that the iron-ore particles in high concentration reduce the growth of S. vulgare as they recovered the embryos, juveniles and young plants. In contrast, the presence of the particles did not affect the release of gametes, percentage of zygote formation or the percentage of embryo survival.

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.