Extract of Cimicifuga racemosa (L.) Nutt protects ovarian follicle reserve of mice against in vitro deleterious effects of dexamethasone.

The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.

Extract of Cimicifuga racemosa (L.) Nutt protects ovarian follicle reserve of mice against in vitro deleterious effects of dexamethasone

The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.

Immunolocalization of melatonin receptors in bovine ovarian follicles and in vitro effects of melatonin on growth, viability and gene expression in secondary follicles.

This study aims to investigate the (1) expression of melatonin receptors types 1A/B (MTNR1A/B) in bovine ovaries and (2) the in vitro effects of melatonin on secondary follicle development, antrum formation, viability, and expression of messenger ribonucleic acid (mRNA) for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase-1 (GPX1) and peroxiredoxin 6 (PRDX6). The expression of MTNR1A/B in bovine ovarian follicles was demonstrated by immunohistochemistry. To choose the most effective concentration of melatonin on follicular growth and viability, isolated secondary follicles were cultured individually at 38.5°C, with 5% CO2 in air, for 18 d in TCM-199+ alone or supplemented with 10-11, 10-9, 10-7 or 10-5 M melatonin. Then, melatonin receptor antagonist, luzindole, was tested to further evaluate the mechanisms of actions of melatonin, that is, the follicles were cultured in control medium alone or supplemented with 10-7 M melatonin, 10 µM luzindole and both 10-7 M melatonin and 10 µM luzindole. Follicular growth, morphology and antrum formation were evaluated at days 6, 12 and 18. At the end of culture, viability of secondary follicles was analyzed by calcein-AM and ethidium homodimer-1, and the relative levels of mRNA for SOD, CAT, GPX1 and PRDX6 were evaluated by real time polymerase chain reaction. Immunohistochemistry results showed expression of MTNR1A/B in oocyte and granulosa cells of primordial, primary, secondary and antral follicles. Secondary follicles cultured in medium supplemented with melatonin at different concentrations had well preserved follicles after 18 d of culture. Furthermore, follicles cultured in presence of 10-7 M melatonin presented significantly higher diameters than those cultured in other treatments. The presence of melatonin receptor antagonist, luzindole, blocked the effects of melatonin on follicular growth and viability. In addition, follicles cultured in medium containing only melatonin had significantly higher rates of antrum formation. Follicles cultured in medium containing only melatonin had higher relative levels of mRNA for CAT, SOD and PRDX-6 than those cultured with both melatonin and luzindole. Follicles cultured with luzindole only or both melatonin and luzindole had lower relative levels of mRNA for PRDX6 and GPX1 than those cultured control medium. In conclusion, melatonin promotes growth of bovine secondary follicles through its membrane-coupled receptors, while luzindole blocks the effects of melatonin on follicle growth and reduces the expression of antioxidant enzymes in cultured follicles.

Eugenol influences the expression of messenger RNAs for superoxide dismutase and glutathione peroxidase 1 in bovine secondary follicles cultured in vitro.

This study aimed to investigate the effects of eugenol on growth, viability, antrum formation and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, bovine ovaries were collected from a local slaughterhouse and in the laboratory the follicles were isolated from the ovarian cortex. The follicles were then cultured in TCM-199+ alone or supplemented with different concentrations of eugenol (0.5, 5.0 and 50.0 µM). Follicular diameters and antrum formation were evaluated on days 0, 6, 12 and 18. Viability analysis was performed using calcein and ethidium homodimer. Real-time PCR was used to quantify mRNA levels for SOD, CAT, GPX1 and PRDX6 in cultured follicles. Follicular diameters and mRNA levels in follicles cultured in vitro were compared using analysis of variance and Kruskal-Wallis tests, while follicular survival and antrum formation were compared using the chi-squared test (P < 0.05). The results showed that secondary follicles cultured with eugenol maintained similar morphology and viability to follicles cultured in the control group. A progressive increase in follicular diameter was observed between days 0 and 12 for all treatments, except for follicles cultured with 50 µM eugenol. Eugenol (5.0 and 50.0 µM) increased mRNA levels for GPX1 in cultured follicles, but 0.5 µM eugenol reduced mRNA levels for SOD. The addition of eugenol did not influence mRNA expression for CAT and PRDX6. In conclusion, eugenol supplementation reduces mRNA levels for SOD and increases mRNA levels of GPX1 in bovine secondary follicles cultured in vitro.

Effects of dexamethasone on growth, viability and ultrastructure of bovine secondary follicles cultured in vitro.

This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150-200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal-Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

Supplementation of culture medium with knockout serum replacement improves the survival of bovine secondary follicles when compared with other protein sources during in vitro culture.

The present study evaluated the effect of knockout serum replacement (KSR), fetal bovine serum (FBS) and bovine serum albumin (BSA) on the viability and growth of bovine secondary follicles cultured in vitro for 12 days. To this end, secondary follicles were isolated (185-202 µm) and cultured in vitro in TCM-199+ medium supplemented with KSR (5% and 10%), FBS (5% and 10%) or BSA (3 mg/ml) at 38.5°C with 5% CO2 in air. Follicular diameters were evaluated on days 0, 4, 8 and 12. After 12 days of culture, follicular survival analysis was performing by using calcein-AM and ethidium homodimer. Before and after culture, follicles were fixed in paraformaldehyde for histological evaluation. Follicular diameter at different days of culture were compared using the Kruskal-Wallis test, while the percentages of viable follicles were analyzed by chi-squared test (P < 0.05). Results showed that follicles cultured in the presence of KSR at both concentrations presented higher follicular survival rates than those cultured in control medium alone or supplemented with FBS or BSA. Conversely, the presence of KSR, BSA or FBS did not increase follicular diameter after 12 days of culture. Histology analysis showed that, among the tested treatments, follicles cultured in the presence of KSR had preserved rounded oocytes, juxtaposed granulosa cells and intact basal membrane. In conclusion, supplementation of culture medium with KSR increases the follicular survival of bovine secondary follicles cultured in vitro.

Effects of epidermal growth factor and progesterone on development, ultrastructure and gene expression of bovine secondary follicles cultured in vitro.

The aims of this study were to investigate the effects of epidermal growth factor (EGF) and progesterone on the development, viability and the gene expression of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (∼0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5 °C, with 5% CO2 in air, for 18 days, in TCM-199+ (n = 63) alone (control medium) or supplemented with 10 ng/mL progesterone (n = 64), 10 ng/mL EGF (n = 61) or both EGF and progesterone (n = 66). The effects of these treatments on growth, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and cyclin B1 were evaluated, significantly different (p < 0.05). The results showed that there was a progressive increase in follicular diameter in all treatments, but only follicles cultured in medium supplemented with EGF had increased significantly in diameter when compared to follicles cultured in the control medium at the end of the culture period, significantly different (p < 0.05). A positive interaction between EGF and progesterone was not observed. In addition, the presence of EGF, progesterone or both in culture medium did not influence the rate of follicle survival and antrum formation. However, the presence of only progesterone in cultured medium increased the expression of mRNAs for GDF9 and cyclin B1 in oocytes. EGF also significantly increased the levels of mRNAs for cMOS and GDF9 when compared to follicles cultured in control medium. Ultrastructural analyzes showed that cultured follicles in all treatments maintained the integrity of granulosa cells. In conclusion, the EGF promotes the development of secondary follicles cultured in vitro for 18 days and increases the expression of cMOS and GDF9, while progesterone alone or in association with EGF have not a positive effect on follicular growth. However, progesterone increases the expression of GDF9 and cyclin B1 in oocytes.

Genetic effects of in vitro germination and plantlet development in chilli pepper.

The objective of this study was to estimate the genetic effects on in vitro germination and development of chili peppers. For this, four genotypes (UFPB-132, -134, -137, and -390) were used as parents. They were crossed in a complete diallel scheme. The parents (4) and hybrid (12) seeds were germinated in glass bottles containing MS media. The following variables were evaluated: seed germination percentage, deformed seedlings percentage, radicle emission percentage, root length, root number, hypocotyl length, hypocotyl width, definitive leaf number, leaf length, and leaf width. The data were submitted to analysis of variance and the means were grouped by the Scott-Knott criteria (P ≤ 0.05). The diallel analysis was performed according to the Griffing method, method I, fixed model. Significant differences for all analysed variables were observed. Additive and non-additive effects were observed influencing the performance of the genotypes in relation to the evaluated variables. The genitor 132 showed the highest general combining ability for almost all evaluated characteristics, except seed germination percentage and deformed seedlings percentage. This study revealed dominance effects are responsible for genotypic variation for almost all evaluated traits. In addition, we found significant reciprocal effects for all studied characters. For the traits to which non-additive variances were important (germination percentage, deformed seedlings percentage, root number, hypocotyl length, hypocotyl width, and definitive leaf number, leaf length, and leaf width), there was an additional opportunity for developing F1 hybrid.

Genetic diversity of arboreal cotton populations of the Brazilian semiarid: a remnant primary gene pool for cotton cultivars.

Mocó cotton belong to the same species as the cultivated species, Gossypium hirsutum, and cultivated forms were mainly landraces but also developed as cultivars, bearing good fiber quality and drought tolerant when cropped as a perennial species. The northeast Brazil crop system based on this cotton type is finished, with a few small area planted in the three main States, where it was previously cultivated (Ceará, Paraíba, and Rio Grande do Norte), but in others, maintenance is accomplished by single dooryard plants. Plants were found in all visited Northeast Brazil municipalities, sometimes in the North of the country, and were collected for ex situ preservation and evaluation. Most of seeds had no fuzz (62.2%) and 94.6% of the genotypes presented spot in flowers. Seventy-one alleles were revealed in 12 loci. The genetic structure of the population evaluated by microsatellite markers shows two main groups, one comprising the Seridó region where landraces were originated and other comprising the state of Ceará, where a specific breeding program was developed. Genotypes collected in North Brazil States as well as those collected in Bahia, Alagoas, and Sergipe grouped with those collected in Ceará. The Mantel correlogram indicates a significant (P < 0.05) correlation between genetic and geographical distances up to 77 km. The ex situ maintenance and agronomical evaluation are the main concerns for mocó, as the use of the agricultural interesting traits, possibly introgressed to other genotypes, is predicted. The in situ preservation is still of interest since there is more diversity there than in the collected plants and some should be continued due to use as medicinal plant.

Characterization and genetic diversity of pepper (Capsicum spp) parents and interspecific hybrids.

Pepper species exhibit broad genetic diversity, which enables their use in breeding programs. The objective of this study was to characterize the diversity between the parents of different species and their interspecific hybrids using morphological and molecular markers. The parents of Capsicum annuum (UFPB-01 and -137), C. baccatum (UFPB-72), and C. chinense (UFPB-128) and their interspecific hybrids (01x128, 72x128, and 137x128) were used for morphological and molecular characterization. Fruit length and seed yield per fruit (SYF) traits showed the highest variability, and three groups were formed based on these data. CVg/CVe ratio values (>1.0) were calculated for leaf length (1.67) and SYF (5.34). The trait that most contributed to divergence was the largest fruit diameter (26.42%), and the trait that least contributed was pericarp thickness (0.33%), which was subject to being discarded. The 17 primers produced 58 polymorphic bands that enabled the estimation of genetic diversity between parents and hybrids, and these results confirmed the results of the morphological data analyses. The principal component analysis results also corroborated the morphological and random-amplified polymorphic DNA data, and three groups that contained the same individuals were identified. These results confirmed reports in the literature regarding the phylogenetic relationships of the species used as parents, which demonstrated that C. annuum was closer to C. chinense as compared to C. baccatum.

Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1ß on primordial follicle activation and survival in vitro.

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1ß on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1ß (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1ß, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1ß (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1ß promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

Embryogenesis in the anthers of different ornamental pepper (Capsicum annuum L.) genotypes.

The aim of this study was to relate flower bud size with microspore developmental stages and the induction of embryos in the anthers of different ornamental pepper (Capsicum annuum L.) genotypes. Flower buds were randomly collected and visually divided into three classes based on both petal and sepal size. The length and diameter of the bud as well as the length of the petal, sepal, and anther were then measured. The microspore stage was also determined for each anther of the bud where it was found. The data were subjected to analysis of variance (P ≤ 0.01), and the means were separated by Tukey's test (P ≤ 0.01). The broad sense heritability, the CVg/CVe relation, and the Pearson correlation between characters were also determined. Anthers from 10 C. annuum genotypes were cultivated in four culture media types for the induction of embryos. The data were transformed by Arcsin (x) and subjected to analysis of variance (P ≤ 0.01), and the means were separated by Tukey's test (P ≤ 0.01). The majority of anthers in the second class had uninucleate microspores. No correlation was observed between bud size and the number of uninucleate microspores. Genotype 9 specimens grown in M2 medium induced the highest number of embryos (16) compared to the other treatments, which indicates a significant interaction effect between culture media and genotypes.

Natural hybridization between Gossypium mustelinum and exotic allotetraploid cotton species.

Cotton has been collected in Brazil for decades for its conservation, evaluation, and the use of its genetic resources. Gossypium mustelinum is an allotetraploid cotton species that only occurs in Brazil, and little is known about its genetic potential for improvement. However, the species is threatened by habitat fragmentation and interspecific hybridization with exotic species of cotton. In this study, we investigated the rate of natural hybridization in two populations of G. mustelinum in Bahia, Brazil, with G. hirsutum and G. barbadense using a set of microsatellite markers.

Genetic divergence of physiological-quality traits of seeds in a population of peppers.

Brazil has a great diversity of Capsicum peppers that can be used in breeding programs. The objective of this study was to evaluate genetic variation in traits related to the physiological quality of seeds of Capsicum annuum L. in a segregating F2 population and its parents. A total of 250 seeds produced by selfing in the F1 generation resulting from crosses between UFPB 77.3 and UFPB 76 were used, with 100 seeds of both parents used as additional controls, totaling 252 genotypes. The seeds were germinated in gerboxes containing substrate blotting paper moistened with distilled water. Germination and the following vigor tests were evaluated: first count, germination velocity index, and root and shoot lengths. Data were subjected to analysis of variance, and means were compared by Scott and Knott's method at 1% probability. Tocher's clustering based on Mahalanobis distance and canonical variable analysis with graphic dispersion of genotypes were performed, and genetic parameters were estimated. All variables were found to be significant by the F test (P ≤ 0.01) and showed high heritability and a CVg/CVe ratio higher than 1.0, indicating genetic differences among genotypes. Parents (genotypes 1 and 2) formed distinct groups in all clustering methods. Genotypes 3, 104, 153, and 232 were found to be the most divergent according to Tocher's clustering method, and this was mainly due to early germination, which was observed on day 14, and would therefore be selected. Understanding the phenotypic variability among these 252 genotypes will serve as a basis for continuing the breeding program within this family.

Diversity and genetic structure among subpopulations of Gossypium mustelinum (Malvaceae).

Gossypium mustelinum is the only cotton species native to Brazil; it is endemic to the semi-arid region of the northeast. The populations are found near perennial and semi-perennial sources of water, such as ponds or pools in intermittent streams. Problems with in situ conservation derive from human interference in its habitat, mainly because of excessive cattle grazing and deforestation. Establishing efficient strategies for in situ conservation requires knowledge of the genetic structure of the populations. We evaluated the structure and genetic variability of populations of G. mustelinum in the Tocó and Capivara Rivers (State of Bahia). Two hundred and eighteen mature G. mustelinum plants were genotyped with SSR markers. The molecular data were used to estimate the allelic frequencies, the heterozygosity, the F statistics, and the genetic distance among the populations and among individuals. We found high genetic diversity among the populations. The FST indexes for each population were also high and strongly correlated with physical distance. The high estimated level of endogamy and the low observed heterozygosity are indicative that the populations reproduce mainly by self-fertilization and crosses between related individuals. Consequently, strategies for in situ preservation should include at least three occurrence sites of G. mustelinum from each population. For ex situ conservation, the collections should include as many sites as possible.

Estabelecimento in vitro e micropropagação de maracujá silvestre (Passiflora foetida L. ) / In vitro establishment and micropropagation of Passiflora foetida L

Entre as espécies do gênero Passiflora, a P. foetida L. apresenta a maior variabilidade genética e tem grande importância medicinal, pois é usada no tratamento de doenças como asma, icterícia, e na forma de emplastros, para as erisipelas e doenças de pele com inflamação. Portanto, são necessários estudos que visem a micropropagação e conservação. As sementes de P. foetida L. apresentam dormência e muitas vezes, levam alguns meses para germinar, produzindo mudas desuniformes e de baixo vigor. Neste sentido, a cultura de tecidos apresenta-se como uma forma alternativa a propagação. Assim, o objetivo do trabalho foi estabelecer e micropropagar P. foetida L., para formação de um banco de germoplasma. Para tanto, sementes foram escarificadas, desinfestadas e inoculadas em meio MS(½) sem reguladores de crescimento e cultivados por 66 dias. Explantes de hipocótilos obtidos de plantas germinadas in vitro, foram cultivados no mesmo meio suplementado com 1,0 mg L-1 de BAP. Na fase de estabelecimento, 45% dos explantes brotaram e formaram gemas axilares. 88,9% dos explantes de hipocótilo induziram brotação e 11,1% produziram calos. Plântulas regeneradas com 1,82 cm de altura, com raízes foram aclimatadas.

Among the species of the genus Passiflora, P. foetida L. presents highest genetic variability and also great medicinal importance. This species is used in the treatment of diseases such as asthma, jaundice, and in the form of poultices for erysipelas and skin diseases with inflammation. Therefore, studies are needed to preserve them. Its seeds present dormancy and often take several months to germinate. The tissue culture is a alternative form to propagate species. The objective was to establish and micropropagation P. foetida L., and create a germplasm bank. Seeds were scarified, disinfected, and inoculated on MS medium (½) without regulators for 66 days. Hypocotyls explants obtained of seedlings in vitro germinated were transferred and placed in the same medium supplemented with BAP (1,0 mg L-1). In the establishment phase of 45% of the explants sprouted and formed axillary buds. 88,9% of hypocotyl explants induced shoots and 11,1% produced callus. Seedling measuring 1,82 cm length and rooted were acclimatized.

Caracterização de frutos e sementes em acessos de maracujá silvestre (Passiflora foetida L. ) / Characterization of fruits and seeds of wild passion fruit (Passiflora foetida L. )

A espécie Passiflora foetida L.é uma trepadeira exótica cujas folhas e frutos possuem propriedades medicinais. No Brasil, a erva é utilizada na forma de extratos ou emplastros para erisipelas e doenças de pele com inflamação. O objetivo deste trabalho foi caracterizar cinco acessos de Passiflora foetida L. baseados em 17 descritores morfológicos O experimento foi conduzido em casa de vegetação em delineamento inteiramente ao acaso, com 15 repetições por genótipo. As variáveis analisadas foram cor da casca do fruto, formato do fruto, comprimento do fruto, diâmetro do fruto, massa fresca do fruto, comprimento do pedicelo, cor da polpa do fruto, espessura da casca, acidez titulável, sólidos solúveis, potencial hidrogeniônico, razão sólidos solúveis/ acidez titulável, comprimento da semente, diâmetro da semente, espessura da semente, massa fresca das sementes e o número de sementes por fruto. Os dados foram submetidos à análise de variância e teste de médias pelo teste Tukey a 5 por cento de probabilidade. Houve diferença significativa pelo teste F a 5 por cento de probabilidade, entre os genótipos para quase todas as características com exceção da espessura da casca, massa fresca do fruto, comprimento da semente e massa fresca das sementes. A característica que apresentou maior variação foi o teor de sólidos solúveis. E entre os genótipos, o acesso 5 foi o que apresentou as maiores médias pelo teste de Tukey a 5 por cento de probabilidade, em todas as características analisadas, podendo ser utilizado em programas de melhoramento da espécie.

The species Passiflora foetida L. é an exotic vine, which leaves and fruit have medicinal properties. In Brazil, the herbis used in the form of extracts or poultices for erysipelas and skin diseases with inflammation. The aim of this study was to characterize five Passiflora foetida L. accessions based on 17 morphological descriptors The experiment was conducted in a greenhouse in a completely randomized design with 15 replicates per genotype. The variables were the fruit skin color, fruit shape, fruit length, fruit diameter, fresh fruit, length of pedicel, flesh color of the fruit, skin thickness, acidity, soluble solids, hydrogen potential ratio soluble solids / acidity, seed length, seed diameter, thickness of seed, fresh seed and seed number per fruit. The data were submitted to analysis of variance and the means were compared by Tukey test at 5 percent of probability. There was a significant difference by F test at 5 percent,of probability among the genotypes for nearly all traits, except the thickness of the shell, fresh fruit mass, seed length and fresh weight of seed. The variable that most variation was the soluble solids. The genotype 5 had the highest means by Tukey test at 5 percent of probability on all characteristics that can be used in breeding programs of the species.

The identification of sandfly species, from an area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA.

The area around Río Blanco, in the Orán department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species.

Molecular characterization of a virus from the family Luteoviridae associated with cotton blue disease.

Cotton blue disease is an aphid-transmitted cotton disease described in Brazil in 1962 as Vein Mosaic "var. Ribeirão Bonito". At present it causes economically important losses in cotton crops if control measures are not implemented. The observed symptoms and mode of transmission have prompted researchers to speculate that cotton blue disease could be attributed to a member of the family Luteoviridae, but there was no molecular evidence supporting this hypothesis. We have amplified part of the genome of a virus associated with this disease using degenerate primers for members of the family Luteoviridae. Sequence analysis of the entire capsid and a partial RdRp revealed a virus probably belonging to the genus Polerovirus. Based on our results we propose that cotton blue disease is associated with a virus with the putative name Cotton leafroll dwarf virus (CLRDV).

Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks.

Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks. The principal bacterial species isolated were Alcaligenes sp., Bacillus spp. (including B. cereus, B. lentus, B. megaterium, B. pumilus, and B. subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P. agglomerans and B. pumilus being the most frequently isolated species. The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp. Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B. pumilus isolates were more diverse than P. agglomerans isolates, although genetic diversity was not related to the host plants. In vitro interaction studies between G. citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G. citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P. agglomerans.