Leandro Vieira Astarita

Multiple shoot formation in Hypericum perforatum L.and hypericin production

Hypericum perforatum is a traditional medicinal plant with wound healing and antidepressive properties. Among the secondary compounds of interest is hypericin, a naphtodianthrone that seems to participate in the medicinal effects of this species. The aim of this work was to obtain an efficient micropropagation system of H. perforatum and to compare the hypericin content between in vitro and field-grown plants. Cultures were initiated from nodal segments of mature plants inoculated onto MS medium supplemented with 4.5 µM BA, kinetin, thidiazuron, individually or in combination with 0.05 µM NAA. Organogenic explants were observed on medium with either BA or kinetin alone or in combination of these with NAA. Subculture of organogenic explants onto the proliferation medium containing 4.5 µM BA promoted the organogenic response. The highest average of shoot production (52.6 shoots) was obtained on those explants induced in the presence of BA and NAA. Rooted plantlets were successfully acclimated. Analysis of hypericin contents showed that levels found in callus represented only 0.11 % of what was detected in adult plants, while shoots and leaves from in vitro plants showed similar hypericin levels to those found in the leaves of the field-grown plants, suggesting that the accumulation of this compound is related to leaf differentiation.

Defense mechanisms of Solanum tuberosum L. in response to attack by plant-pathogenic bacteria

The natural resistance of plants to disease is based not only on preformed mechanisms, but also on induced mechanisms. The defense mechanisms present in resistant plants may also be found in susceptible ones. This study attempted to analyze the metabolic alterations in plants of the potato Solanum tuberosum L. cv. Agata that were inoculated with the incompatible plant-pathogenic bacteria X. axonopodis and R. solanacearum, and the compatible bacterium E. carotovora. Levels of total phenolic compounds, including the flavonoid group, and the activities of polyphenol oxidase (PPO) and peroxidase (POX) were evaluated. Bacteria compatibility was evaluated by means of infiltration of tubers. The defense response was evaluated in the leaves of the potato plants. Leaves were inoculated depending on their number and location on the stem. Multiple-leaf inoculation was carried out on basal, intermediate, and apical leaves, and single inoculations on intermediate leaves. Leaves inoculated with X. axonopodis and with R. solanacearum showed hypersensitive responses within 24 hours post-inoculation, whereas leaves inoculated with E. carotovora showed disease symptoms. Therefore, the R. solanacearum isolate used in the experiments did not exhibit virulence to this potato cultivar. Regardless of the bacterial treatments, the basal leaves showed higher PPO and POX activities and lower levels of total phenolic compounds and flavonoids, compared to the apical leaves. However, basal and intermediate leaves inoculated with R. solanacearum and X. axonopodis showed increases in total phenolic compounds and flavonoid levels. In general, multiple-leaf inoculation showed the highest levels of total phenolics and flavonoids, whereas the single inoculations resulted in the highest increase in PPO activity. The POX activity showed no significant difference between single- and multiple-leaf inoculations. Plants inoculated with E. carotovora showed no significant increase in defense mechanisms such as enzyme activity and phenolic compounds. Therefore, resistance or susceptibility in S. tuberosum cv. Agata might depend on leaf age, type of inoculation performed (single or multiple), and the interaction between plant and pathogen.

Plant regeneration through somatic embryogenesis of yacón [smallanthus sonchifolius (poepp. and endl.) H. Robinson]

Smallanthus sonchifolius has tuberous roots containing large amounts of fructo-oligosaccharides and its medicinal use has increased due to the hypoglycemic properties reported for this species. An efficient system for propagation via somatic embryogenesis is reported using petiole segments cultivated on MS medium supplemented with combinations of BA, kinetin and 2,4-D, under light and darkness conditions. Embryogenic callus was formed in most of the treatments; however, somatic embryogenesis was promoted by the presence of light. Clusters of somatic embryos appeared on callus surface after 50 days of culture. The highest number of embryos was produced on 0.45 µM BA and 4.5 µM 2,4-D. Embryogenic calli were maintained on MS medium containing 4.5 µM BA and 0.045 µM 2,4-D. Embryos converted on hormone-free half-strength MS medium with 2 g.L-1 activated charcoal and plantlets were transferred to non-sterile conditions for acclimatization, showing 100% of survival.

Alteração dos metabólitos secundários em plantas de Hypericum perforatum L. (Hypericaceae) submetidas à secagem e ao congelamento

In recent years, interest in Hypericum perforatum has increased due to its antiviral, antidepressive and apoptosis-inducing properties. Plant material preparation after harvesting often includes dehydration or freezing and further storage, and these procedures can lead to variations in the secondary metabolite profile. In this study the effects of drying, freezing and storage at -20 oC on flavonoid and hypericin content in H. perforatum were evaluated. Leaves and stems of H. perforatum were dried at 25, 30, 50 and 70 oC, frozen in liquid nitrogen or frozen and then stored at -20 oC for 10, 20 and 30 days. Flavonoid and hypericin quantification was performed by High Performance Liquid Chromatography (HPLC). Levels of both flavonoids and hypericin were affected by drying temperature. Drying at 25 oC resulted in lower levels of most of the metabolites analyzed while at 50 oC, levels of free and conjugated rutin, conjugated quercetin and quercitrin as well as hypericin content were maintained when compared to fresh samples. Free apigenin and kaempferol did not show any significant variation, regardless of drying temperature. Freezing did not affect the profile of flavonoids, but it led to significant reduction in hypericin content.

Defense responses in plants of Eucalyptus elicited by Streptomyces and challenged with Botrytis cinerea

AbstractMain conclusionElicitation ofE. grandisplants withStreptomycesPM9 reduced the gray-mold disease, through increasing the levels of enzymes directly related to the induction of plant defense responses, and accumulation of specific phenolic compounds. Members of Eucalyptus are economically important woody species, especially as a raw material in many industrial sectors. Species of this genus are susceptible to pathogens such as Botrytis cinerea (gray mold). Biological control of plant diseases using rhizobacteria is one alternative to reduce the use of pesticides and pathogen attack. This study evaluated the metabolic and phenotypic responses of Eucalyptus grandis and E. globulus plants treated with Streptomyces sp. PM9 and challenged with the pathogenic fungus B. cinerea. Metabolic responses were evaluated by assessing the activities of the enzymes polyphenol oxidase and peroxidase as well as the levels of phenolic compounds and flavonoids. The incidence and progression of the fungal disease in PM9-treated plants and challenged with B. cinerea were evaluated. Treatment with Streptomyces sp. PM9 and challenge with B. cinerea led to changes in the activities of polyphenol oxidase and peroxidase as well as in the levels of phenolic compounds in the plants at different time points. Alterations in enzymes of PM9-treated plants were related to early defense responses in E. grandis. Gallic and chlorogenic acids were on average more abundant, although caffeic acid, benzoic acid and catechin were induced at specific time points during the culture period. Treatment with Streptomyces sp. PM9 significantly delayed the establishment of gray mold in E. grandis plants. These results demonstrate the action of Streptomyces sp. PM9 in inducing plant responses against B. cinerea, making this organism a potential candidate for biological control in Eucalyptus.

Growth inhibition of an Araucaria angustifolia (Coniferopsida) fungal seed pathogen, Neofusicoccum parvum, by soil streptomycetes

BackgroundAraucariaceae are important forest trees of the southern hemisphere. Life expectancy of their seedlings can largely be reduced by fungal infections. In this study we have isolated and characterized such a fungus and investigated the potential of Streptomyces Actinobacteria from the respective rhizosphere to act as antagonists.ResultsThe pathogenic fungus from Araucaria angustifolia seeds was identified by morphological markers (pore-associated Woronin-bodies) as belonging to the Pezizomycotina. Molecular data identified the fungus as Neofusicoccum parvum (Botryosphaeriaceae). Co-cultures on agar of this fungus with certain streptomycete isolates from the rhizosphere, and from the surface of Araucaria roots significantly reduced the growth of the fungus. HPLC analysis of the agar yielded streptomycete-specific exudate compounds which were partly identified. There were differences in compounds between single (bacteria, fungus) and dual cultures (bacteria + fungus).ConclusionStreptomycetes from the rhizosphere of Araucariaceae produce exudates which can suppress the development of pathogenic fungi in their seeds.

Nephroprotective and anti-inflammatory effects of aqueous extract of Melissa officinalis L. on acetaminophen-induced and pleurisy-induced lesions in rats

This study assessed the bioactive properties of an aqueous extract of M. officinalis for its anti-inflammatory activity and its protection against hepatic and renal lesions induced by acetaminophen (APAP). Animals pre-treated with the crude extract in pleurisy induced by carrageenan showed a reduction in the amounts of exudate, in the numbers of leukocytes and polymorphonuclear cells. Intragastric administration of the extract for seven days prior to the APAP-induced lesion showed no protective effect on the liver. The treatment with the extract induced an increase of serum aspartate aminotransferase, indicating a rise of toxicity. Contrarily, the same treatment reduced the APAP induced lesion in kidney, with respect to ν-glutamyltransferase. The results suggested that the extract was not hepatoprotective and could lead to an increase in the lesions induced by the APAP. On the other hand, the extract was nephroprotective against the lesions induced by the APAP and showed an anti-inflammatory effect on pleurisy carrageenan-induced.

Long-term maintenance of in vitro cultures affects growth and secondary metabolism of St. John's Wort.

Hypericum perforatum is a traditional medicinal plant with wound healing and antidepressant properties. Efficiency of micropropagation is often related to the long term maintenance of tissues in culture, which may alter the secondary metabolism of plants. The objective of this study was to evaluate growth and secondary metabolism of in vitro shoots of H. perforatum on short and long term maintenance of cultures (30 and 100 days). The effect of BA and NAA supplementation was evaluated during 30 days of culture. Adventitious shoots were cultivated on MS medium supplemented with 4.4mM BA alone or in combination with 0.05mM NAA for 30 days. A hormone-free medium was used as control. Shoots cultivated for 100 days were maintained in presence of 4.4mM BA. Biomass, multiplication of shoots, contents of phenolic compounds, flavonoids and hypericin were evaluated. No difference between BA and BA+NAA was observed on growth, multiplication of shoots and levels of flavonoids at the end of 30 days of culture. Production of phenolic compounds was promoted by addition of BA+NAA to the medium, whereas hypericin was increased by the presence of BA. The time of culture (30 and 100 days) affected all the parameters analyzed, except the levels of flavonoids in the short term experiment.

Eucalyptus transgenic plants: from genetic transformation protocols to biosafety analysis

Eucalyptus is an exotic plant in Brazil, being originally from Australia. There are about 700 Eucalyptus species described and over 3,000 hybrids. It is one of the most planted tree genus in the world. The great economic interest on Eucalyptus trees is due to their fast growth, high productivity, great adaptability to different types of soils and climates, and also to the high versatility of their wood. Eucalyptus timber has applications for many different purposes such as cellulose pulp and paper production, electric poles, energy, charcoal, lumber and furniture. Given the economic importance of Eucalyptus in Brazil, it is of great interest to generate trees with superior characteristics that may result in considerable gains for the sector, particularly with regard to productivity and wood quality. One main goal of the present work is the definition of an efficient protocol for the genetic transformation and regeneration of transgenic Eucalyptus trees. We have so far obtained callus derived from leaves of E.globulus showing high capacity of in vitro regeneration. These calli were transformed with Agrobacteriumtumefaciens LBA4404 harboring the binary plasmid pGfpKan, containing the green fluorescent protein (gfp) gene as reporter. Regenerated plants were transferred to culture pots with MS medium. Leaves derived from each regenerated plant were collected and analyzed using confocal microscopy to investigate the presence of fluorescence, indicating successful transformation. Molecular assays are also being performed to confirm the independence of transformation events via the pattern of transgene integration into plant genomes. The commercial release of GMOs and its derivatives in Brazil is regulated by the National Technical Biosafety Commission (CTNBio) in terms of the Regulatory Resolution No. 5, March 12, 2008. A series of experiments must be performed in order to prove the equivalence between GM and non-GM plants concerning the effects on human and animal health as well as on the environment. We are conducting the evaluation of global gene expression among different lines of transgenic and non-GM Eucalyptus adult plants, already available in test-fields belonging to FuturaGene in Brazil. Leaf and stem samples of GM and controls were collected and stored at -80 °C. Total RNA and proteins from leaves and stems were extracted and quantified. Messenger RNAs are under sequencing in Illumina platforms, according to the mRNA-Seq protocol (Fasteris S.A.) Our idea is to compare transcript profiles among GM and non-GM tree samples, checking for possible pleiotropic effects of the transgenes. Protein profiles of the same individuals will also be analyzed to verify if the presence of the transgene influences the expression of other proteins in Eucalyptus. In order to do so, total proteins were extracted from samples and fractionated by 1D SDS-PAGE, cut off from gels and processed for posterior Mass Spectrometry sequencing. Financial Support: MAPA/CNPq & FuturaGene.

Comparison of genetically engineered (GE) and non-GE Eucalyptus trees using secondary metabolites obtained by steam distillation

Abstract The genus Eucalyptus is composed of hundreds of species with multiple important uses, making it one of the most valuable forest trees around the world. The genetic improvement of Eucalytpus has been studied especially to improve timber production, wood quality and disease resistance. One of the current approaches to improve Eucalyptus tree species is genetic engineering, and it is a common sense that transgenic plants must be submitted to detailed environmental and health risk assessments in order to guarantee their biosafety for future commercial release. This work aimed to characterize and compare the essential oil composition of leaves from genetically engineered (GE) and non-GE Eucalyptus trees of the hybrid E. grandis x E. urophylla employed in cellulose pulp and paper production. Our results allowed us to conclude that no significant differences in the content or chemical composition of leaf essential oils exist between GE and non-GE Eucalyptus trees.