Eliane Romanato Santarém

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.

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.

Efeito do estresse hídrico na germinação e crescimento inicial de três espécies de leguminosas

Seeds of Senna macranthera, S. multijuga e Mimosa bimucronata were submitted to different osmotic potentials using PEG 6000 (0; -0.15; -0.49 and -1.03MPa). The objective of this study was to evaluate the effect of osmotic stress on germinability and initial growth of seedlings. Water stress of -0.15 and -0.49MPa increased the time required for germination in 12, 24 and 48 hours for M.bimucronata, S. multijuga and S. macranthera, respectively. All species showed a reduced germinability in the Ψ = -1.03MPa. Such results suggest a limit of tolerance between the osmotic potentials of -0.49 and -1.03MPa. Senna macranthera seedlings were more sensitive to osmotic stress, reducing shoot and radicle elongation when levels of external water stress were increased. Senna multijuga and M. bimucronata increased the radicle lenght at Ψ = -0.15MPa. Reduction of fresh weight was observed for all levels of osmotic stress in all species.

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.

Herbicide activity of natural compounds from Baccharis spp Herbicidal activity of natural compounds from Baccharis spp. on the germination and seedlings growth of Lactuca sativa and Bidens pilosa

We evaluated the effects of aqueous and ethanolic extracts from 3-Baccharis species (B. dentata, B. uncinella and B. anomala) on the germination and seedlings growth of Lactuca sativa (model species) and Bidens pilosa (weed), and by chemical analysis, the phenolic compounds were determined in extracts. Aqueous and ethanolic extracts of B. dentata, B. uncinella and B. anomala were tested at 2.5, 5, 7.5 and 10% concentrations. Allelopathic effects of three Baccharis species were variable. The B. pilosa was more sensitive to the extracts than L. sativa and germination was inhibited with ethanolic extracts of B. anomala and B. uncinella from 5 to 10%. Aqueous extract of B. dentata and B. uncinella at 10% concentration reduced the germination of B. pilosa by 80%. The ethanolic extracts of B. uncinella (2.5 to 10%) and B. anomala (5 to 10%) caused 100% mortality of seedlings. Total phenolic compounds were more abundant in aqueous extracts. Amongst the phenolics, catechin was most abundant (1.61 to 6.16 mg g DM) in aqueous and ethanolic extracts of Baccharis species tested. This study showed that Baccharis uncinella may be used as an alternative bioherbicide to control the weeds in agroecosystems.

Environmental and health risk assessments of genetically modified eucalypts in Brazil

Motivated by the Brazilian Ministry of Agriculture, Farming and Supply (MAPA), we created in 2009 the “Collaborating Center in Agriculture Defense Relative to the Biosafety of Genetically Modified Eucalypts” (Project “CDA Eucalyptus”) in order to collect information and conduct research to assess the biosafety of GM eucalypts in the Brazilian context. The Normative Resolution Nr. 5 of the National Biosafety Technical Commission (CTNBio) is the official document presenting all information needed to propose the commercial release of GMOs in Brazil. Based on this document and along with the personnel of the Suzano Paper & Cellulose Co., we conducted a series of experiments with GM and non-GM eucalypts planted in a test field in the state of Sao Paulo to start collecting the necessary information. Two independent groups of transgenic plants, harboring two different transgene constructions along with non-GM control plants are being assayed. The genetic traits, the identity or names of the transgenes as well as the identity of each tree individual will not be revealed due to intellectual property request still pending. Each group of plants was represented by four independent events in triplicates (2 groups x 4 events x 3 clonal trees + 3 non-GM clonal trees), therefore totaling 27 individuals under analysis. Samples were identified by random numbers and all assays were conducted in a simple-blind or a double-blind fashion. Tests concluded until now included (i) the detection of transgene regulatory sequences in purified DNA samples by conventional PCR and RT-qPCR, confirming the expected sampling conducted; (ii) extraction, chemical characterization and analysis of the antifungal effects of essential (volatile) oils extracted from leaves; (iii) pollen germination in vitro; (iv) flower morphology; (v) seed production; (vi) initial seedling development; (vii) leaf allelopathy; (viii) measurements of total phenolic compounds in leaves and roots; and (ix) effects of leaf extracts on the viability of human colon cells. All results obtained from experiments (ii) to (ix) revealed no statistical differences between GM- and non-GM-derived samples. A second round of experiments will be conducted to confirm these results. Proteomic and transcriptomic profiling of GM and non-GM trees are under analysis, as well as a series of experiments that include the chemical, nutritional and biological analysis of honey samples derived from bee hives located in fields of GM versus non-GM plants; and bee (Apis mellifera) population dynamics.