Induction of somatic embryogenesis in immature seeds of guavatree cv. Paluma

The biotechnological techniques may help solve many problems of guava culture, such as the high perishability of fruits. Somatic embryogenesis can generate highly multiplicative cell cultures and with high regenerative potential, serving as basis for genetic transformation. The aim of this work was to obtain somatic embryogenesis of guava (Psidium guajava L.) cv. Paluma. Immature seeds were used, and they were inoculated in MS environment containing 400 mg L-1 of L-glutamine, 100 mg L-1 myo-inositol, 60 g L-1 sucrose, 100 mg L-1 ascorbic acid and supplemented with different types and concentrations of growth regulators. Embryogenic callus appeared after 37 days of culture in an environment containing 1.0 mg L-1 2.4-D + 2.0 mg L-1 2-ip, in 7% of the explants. After 65 days of culture, the treatment containing 0.5 mg L-1 CPA showed 20% of explants with direct embryos, while the treatment with 1 mg L-1 had 14% of explants with direct embryos and 7% of explants with embryogenic callus. In 66.6% of embryos regenerated with 0.5 mg L¹ CPA there was the formation of secondary embryos. The use of IASP and BAP, aiming embryogenesis proliferation, led to an increase in the cellular proliferation, but calli apparently lost their embryogenic potential.

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.

Somatic embryogenesis from zygotic embryos of Euterpe oleracea Mart.

The aim of this work was to study the morphogenetic responses of zygotic embryos of açai palm (Euterpe oleracea Mart.) submitted to several conditions of in vitro culture. Several research experiments were conducted, in laboratory, using vegetable material collected from açai palm plants at Embrapa Amazon Oriental, Belém-PA, Brazil. It was possible to verify the expression of a direct, repetitive and no-synchronized model of somatic embryogenesis in mature zygotic embryos cultivated in primary MS medium supplemented with 2,4-D (339.36 muM) and transferred to a secondary MS medium in the presence of NAA (0.537 muM) and 2iP (12.30 muM). The conversion of somatic embryos into seedlings was reached after 210 days with the transfer of the cultures to a third medium with sucrose and mineral salts concentrations reduced to a half, without growth regulators.

Somatic embryogenesis and the effect of particle bombardment on banana Maçã regeneration

A plant regeneration method with cell suspension cultures of banana, and the effect of biobalistic on regeneration potential are described in this report. Somatic embryos of banana were obtained from indirect embryogenesis of male inflorescence of banana cultivar Maçã (AAB group). Part of the calluses formed (40%) showed embryogenic characteristics (nonfriable, compact and yellow color). The cell suspension, originated from embryogenic calluses, contained clusters of small tightly packed cells with dense cytoplasms, relatively large nuclei and very dense nucleoli. After four months of culture, somatic embryos started to regenerate. The maximum number of regenerated plants was observed between 45 and 60 days after embryo formation.In the first experiment, 401 plants were regenerated from approximately 10 mL of packed cells. In the second experiment, 399 plants were regenerated from a cell suspension six months older than that of the first experiment. Cell transformation using particle bombardment with three different plasmid constructions, containing the uid-A gene, resulted in a strong GUS expression five days after bombardment; however, plant regeneration from bombarded cells was much lower than nonbombarded ones.

Somatic embryogenesis and plant regeneration in elite clones of Theobroma cacao

The objective of this work was to evaluated a procedure for somatic embryogenesis and regeneration of cacao (Theobroma cacao L.) elite clones. Petal explants from cacao clones TSH 565 and TSH 1188 were cultured on PCG and SCG-2 media, for calli growth. Somatic embryos were formed on the surface of embryogenic calli after transfer to embryo development (ED) medium. Clone TSH 565 showed a higher embryogenic potential than TSH 1188. The best combination of carbon source for embryo induction in ED medium was genotype-specific. Embryogenic callus formations increased in micropore tape-sealed Petri dishes, irrespective of cacao genotype. Mature somatic embryos were successfully converted into plantlets.

Patterns of protein and carbohydrate accumulation during somatic embryogenesis of Acca sellowiana

The aim of this work was to quantify the protein, starch and total sugars levels during histodifferentiation and development of somatic embryos of Acca sellowiana Berg. For histological observations, the samples were dehydrated in a battery of ethanol, embedded in historesin and stained with toluidine blue (morphology), coomassie blue (protein bodies) and periodic acid-Schiff (starch). Proteins were extracted using a buffer solution, precipitated using ethanol and quantified using the Bradford reagent. Total sugars were extracted using a methanol-chloroform-water (12:5:3) solution and quantified by a reaction with anthrone at 0.2%. Starch was extracted using a 30% perchloric acid solution and quantified by a reaction with anthrone at 0.2%. During the somatic embryogenesis' in vitro morphogenesis and differentiation processes, the total protein levels decreased and the soluble sugars levels increased during the first 30 days in culture and remained stable until the 120th day. On the other hand, total protein levels increased according to the progression in the developmental stages of the somatic embryos. The levels of total sugars and starch increased in the heart and cotyledonary stages, and decreased in the torpedo and pre-cotyledonary stages. These compounds play a central role in the development of somatic embryos of Acca sellowiana.

Screening of Brazilian soybean genotypes with high potential for somatic embryogenesis and plant regeneration

The aim of this work was to identify Brazilian soybean (Glycine max) genotypes with potential to respond to in vitro culture stimuli for primary somatic embryo induction, secondary embryo proliferation and plant regeneration. Differences among eight tested cultivars were observed at each stage. Two cultivars, IAS-5 and BRSMG 68 Vencedora, were selected for the evaluation of the capacity for embryo differentiation and plant regeneration. These cultivars had high embryo induction frequencies, repetitive embryogenic proliferation, and low precocious embryo germination in the initial experiment. The effect of abscisic acid (ABA) and charcoal addition on plant regeneration was investigated. The addition of ABA to proliferation medium and of ABA and activated charcoal to maturation medium increased embryo differentiation rates, which resulted in a higher number of regenerated plants. The BRSMG 68 Vencedora cultivar was found to have a high potential for embryo induction, embryo proliferation and plant regeneration. The potential of this cultivar for somatic embryogenesis was similar to that observed for cultivar IAS-5, which is currently used for soybean transformation in Brazil. BRSMG 68 Vencedora may be a good alternative genotype for soybean genetic engineering via somatic embryogenesis protocols.

Somatic embryogenesis and in vitro plant regeneration from pejibaye adult plant leaf primordia

The aim of this work was to evaluate a protocol for plant regeneration by means of somatic embryos obtained from isolated adult pejibaye leaf primordia, and to describe histological origin of embryos and morphogenetic response. Explants were cultivated in modified MS medium. Mesophyll parenchymatous cells originated meristemoids (preembryonic complex formation) induced with 7.1 µM BAP in the first two subculture periods. After polarized structures with 12.9 µM NAA and 3.55 µM BAP were formed in the third subculture, somatic embryos developed and regenerated normal plants. The mesophyll parenchymatous cells display high capacity of direct response to the auxin and cytokinin.

JUVENILE-MATURE GENETIC CORRELATIONS IN Pinus taeda CLONES PROPAGATED VIA SOMATIC EMBRYOGENESIS

ABSTRACT This study aimed to estimate the genetic correlation among selection ages (juvenile - adult) and efficiency of early selection for the height, diameter, and volume traits of individuals from Pinus taeda families propagated via somatic embryogenesis. This study was carried out by genetic-statistical analysis, estimation procedure of variance (Reml), and prediction components of breeding values (Blup), using the Selegen-Reml/Blup software. Genetic correlations among juvenile ages and rotation age were performed by applying the linear model developed by Lambeth (1980). In accordance with results of the established model, the early selection can be performed in clones of Pinus taeda with high selection efficiency. Ages from 4 to 6 years old are enough to select Pinus taeda clones propagated via somatic embryogenesis for harvesting at 8 and 12 years old; and 6 to 10 years old are enough to select them for harvesting at 20 years old. On the basis of the genetic correlations estimates from the environments, the clones' selection of Pinus taeda propagated via somatic embryogenesis should be developed specifically for each environment. The clones' selection can be performed considering the diameter due to the high correlation between volume and diameter.

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.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

Reduction of QTc interval dispersion. Potential mechanism of cardiac protection of pyridostigmine bromide

OBJECTIVE: Parasympathetic dysfunction is an independent risk factor in individuals with coronary artery disease, and cholinergic stimulation is a potential therapeutical option. We determined the effects of pyridostigmine bromide, a reversible anticholinesterase agent, on electrocardiographic variables of healthy individuals. METHODS: We carried out a cross-sectional, double blind, randomized, placebo-controlled study. We obtained electrocardiographic tracings in 12 simultaneous leads of 10 healthy young individuals at rest before and after oral administration of 45 mg of pyridostigmine or placebo. RESULTS: Pyridostigmine increased RR intervals (before: 886±27 ms vs after: 1054±37 ms) and decreased QTc dispersion (before: 72±9ms vs after: 45±3ms), without changing other electrocardiographic variables (PR segment, QT interval, QTc, and QT dispersion). CONCLUSION: Bradycardia and the reduction in QTc dispersion induced by pyridostigmine may effectively represent a protective mechanism if these results can be reproduced in individuals with cardiovascular diseases.

Endothelium-dependent relaxation in response to poly-L-arginine in canine coronary arteries: implications about hyperpolarization as a mechanism of vasodilatation

OBJECTIVE: To study the mechanism by which poly-L-arginine mediates endothelium-dependent relaxation. METHODS: Vascular segments with and without endothelium were suspended in organ chambers filled with control solution maintained at 37ºC and bubbled with 95% O2 / 5% CO2. Used drugs: indomethacin, acetycholine, EGTA, glybenclamide, ouabain, poly-L-arginine, methylene blue, N G-nitro-L-arginine, and verapamil and N G-monomethyl-L-arginine. Prostaglandin F2á and potassium chloride were used to contract the vascular rings. RESULTS: Poly-L-arginine (10-11 to 10-7 M) induced concentration-dependent relaxation in coronary artery segments with endothelium. The relaxation to poly-L-arginine was attenuated by ouabain, but was unaffected by glybenclamide. L-NOARG and oxyhemoglobin caused attenuation, but did not abolish this relaxation. Also, the relaxations was unaffected by methylene blue, verapamil, or the presence of a calcium-free bathing medium. The endothelium-dependent to poly-L-arginine relaxation was abolished only in vessels contracted with potassium chloride (40 mM) in the presence of L-NOARG and indomethacin. CONCLUSION: These experiments indicate that poly-L-arginine induces relaxation independent of nitric oxide.

Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission

Infective stages of Leishmania (Leishmania) amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L.) chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of [quot ]metacyclic[quot ] promastigotes in the mouthparts of the vector is discussed.

Anti-schistosomal drugs: observations on the mechanism of drug resistance to hycanthone, and on the involvement of host antibodies in the mode of action of praziquantel

This paper reports recent observations from our laboratory dealing with the anti-schistosome drugs hycanthone (HC) and praziquantel (PZQ). In particular, we discuss a laboratory model of drug resistance to HC in Schistosoma mansoni and show that drug sensitive and resistant lines of the parasite can be differentiated on the basis of restriction fragment length polymorphisms using homologous ribosomal gene probes. In addition, we summarize data demonstrating that effective chemotherapy of S. mansoni infection with PZQ in mice requires the presence of host anti-parasite antibodies. These antibodies bind to PZQ treated worms and may be involved in an antibody-dependent cellular cytotoxicity reactions which result in the clearance of worms from the vasculature.