Breno Régis Santos

Efeito da escarificação e luminosidade na germinação in vitro de sementes de cagaiteira (Eugenia dysenterica DC.)

A cagaiteira e uma especie nativa do Cerrado com elevado potencial frutifero. Como suas sementes apresentam elevada variabilidade, sendo ainda recalcitrantes e dormentes, a germinacao in vitro apresenta-se como uma alternativa para a propagacao da especie, alem de permitir a obtencao de explantes juvenis para a micropropagacao. Com presente trabalho, objetivou-se avaliar o efeito da escarificacao e da luminosidade na germinacao in vitro de sementes de cagaiteira. Sementes desprovidas do tegumento e sementes intactas foram inoculadas em meio MS e mantidas na ausencia e presenca de luz. Sementes desprovidas de tegumento germinadas tanto na presenca quanto na ausencia de luz apresentaram 86,25% e 88,25% de germinacao aos 31 e 71 dias da inoculacao, respectivamente. Apos 150 dias de inoculacao, plântulas provenientes de sementes desprovidas de tegumento, germinadas na presenca e ausencia de luminosidade, apresentaram 10% e 12% de anomalias, respectivamente.

GENETIC TRANSFORMATION OF Eucalyptus camaldulensis BY AGROBALISTIC METHOD

Eucalyptus stands in the setting of worldwide forestry due to its adaptability, rapid growth, production of high-quality and low cost of wood pulp fibers. The eucalyptus convetional breeding is impaired mainlly by the long life cycle making the genetic transformation systems an important tool for this purpose. However, this system requires in vitro eficient protocols for plant induction, regeneration and seletion, that allow to obtain transgenic plants from the transformed cell groups. The aim of this work was to evaluate the callus formation and to optimize the leaves and callus genetic transformation protocol by using the Agrobacterium tumefaciens system. Concerning callus formation, two different culture media were evaluated: MS medium supplemented with auxin, cytokinin (M1) and the MS medium with reduced nitrogen concentration and supplemented with auxin, cytokinin coconut water (M2). To establish the leave genetic transformation, those were exposed to agrobiolistics technique (gene gun), to tissue injury, and A. tumesfasciens EHA 105 contening the vetor pCambia 3301 (35S::GUS::NOS), for gene transference and to establish the callus transformation thoses were exposed only to A. tumefasciens. For both experiments, the influence of different infection periods was evaluated. The M2 medium provided the best values for callus sizea and fresh and dry weight. The leaves genetic transformation using the agrobiolistics technique was effective, the gus gene transient expression could be observed. No significant differences were obtained in the infection periods (4, 6 and 8 minutes). The callus genetic transformation with A. tumefaciens also promotend the gus gene transient expression on the callus co-cultiveted for 15 e 30 minutes. The transformed callus was transfered to a regeneration and selection medium and transformed plants were obtained.

Effect of synthetic auxins on in vitro and ex vitro bromeliad rooting

The tissue culture can contribute to the propagation of several economic species, such as the bromeliads. This research aimed at evaluating the auxins type and concentration in the in vitro and ex vitro rhizogenesis of Neoregelia concentrica bromeliad. N. concentrica shoots were induced in a growth medium with 15.0 µM of 6-benzylaminopurine, for 80 days, followed by sub-cultivation in phytoregulator-free medium, for 45 days. In the in vitro rhizogenesis, the shoots grew in a medium supplemented with indole-3-butyric acid (IBA) or naphthalene-acetic acid (NAA), at the concentrations of 0.0 µM, 1.0 µM, 2.0 µM, 3.0 µM and 4.0 µM. In the ex vitro rhizogenesis, the bases of shoots were immersed, for 60 minutes, in IBA or NAA solutions, at the concentrations of 0.0 µM, 5.0 µM, 10.0 µM and 15.0 µM. After immersion, the shoots were planted in plastic trays with vermiculite. At the end of each rhizogenesis method, the phytotechnical parameters analysis was carried out. For the in vitro rhizogenesis, a higher number of roots were observed when the shoots were cultivated in concentrations higher than 1.0 µM of NAA, when compared to the IBA. However, the rooting rate differed only at 30 days after the in vitro growth, with a higher root induction in the shoots grown with NAA. At 60 days, the rooting rate was higher than 90% and statistically similar in all treatments. In the ex vitro rhizogenesis, a better formation of the rooting system was observed when 5.0 µ M of IBA was applied, with higher rooting averages and number of roots.

Heavy metals availability and soil fertility after land application of sewage sludge on dystroferric Red Latosol

Sewage sludge is the solid residue obtained from urban sewage treatment plants. It is possible to use the sludge in a sustainable way as fertilizer and as soil conditioner due to its high levels of organic matter and nutrients. Besides pathogens and volatile organic compounds, the residue may also contain heavy metals which may accumulate and contaminate crops and the food chain. The aim of this study was evaluates the changes in the fertility of dystrophic Red Latosol and in the availability of heavy metals following application of sewage sludge. It was assessed whether organic matter supplied to the soil as large amounts of sewage sludge would decrease availability of heavy metals in the soil due to of insoluble compounds formation. From this, an experiment was carried out in polyethylene pots using lettuce plant for test. Sewage sludge were applied to the soil in concentrations equivalent to 60, 120 and 180 t ha-1, and a control without sludge, in four replicates, in a completely randomized design. The results show that sewage sludge led to an increase of organic matter contents, of the cation exchange capacity (CEC) and of nutrients found in the soil. It also improved plant growth up to a concentration of 120 t ha-1. Availability of heavy metals, however, was reduced in sludge concentrations starting with 120 t ha-1.

Growth curve, biochemical profile and phytochemical analyses in calli obtained from the procambium segments of Bacupari

ABSTRACT Garcinia brasiliensis , popularly known as Bacupari, is native to the Amazon and commonly used in folk medicine for its therapeutic properties. This plant is rich in bioactive compounds like benzophenones. However, there are no works about the in vitro establishment and achievement of secondary metabolites in this plant. Thus, the aim of this work was to determine the growth curve and to perform the biochemical and phytochemical analyses in calli obtained from the procambium segments of Bacupari. The growth curve of calli followed a sigmoidal pattern, with four distinct phases (lag, exponential, linear, deceleration). Total soluble sugars were higher on the inoculation day and the reducing sugars on the 20 th day. Amino acids increased from the 60 th day up to the stabilization on the 120 th day. The protein content varied, but it seemed to be related to the amino acids metabolism. The phytochemical screening showed the presence of phenolic and flavonoid compounds in the calli and the HPLC analysis allowed the identification of Fukugetin, Guttiferone A and 7-epiclusianone. Key words: Bacupari, benzophenones, calli

Métodos para a superação da dormência fisiológica de Caryocar brasiliense Camb.

O pequizeiro e uma frutifera nativa dos cerrados brasileiros com grande potencial economico. Neste trabalho, objetivouse avaliar o efeito de substâncias potencialmente estimuladoras da germinacao. As sementes foram extraidas dos carocos e colocadas para germinar em rolos de papel embebidos com as seguintes solucoes: agua destilada (Controle); 2mmol L-1 KNO3 (Nitrato); 2mmol L-1 Etephon (ET); 1mmol L-1 GA3 (GA); 1mmol L-1 GA3 + 2mmol L-1 Etephon (GA + ET). Os resultados de percentagem de germinacao e o tempo medio para germinacao foram, respectivamente, de 54,0% e 9,3 dias no tratamento GA; 47,3% e 11,0 dias no tratamento GA + ET; 32,0% e 12,2 dias no tratamento Controle; 30,7% e 13,1 dias no tratamento Etephon e 20,1% e 13,1 dias no tratamento Nitrato. No tratamento GA + ET houve queda da taxa de germinacao ate os nove dias da semeadura, com relacao ao tratamento GA, o que indica uma possivel inibicao da germinacao de sementes de pequizeiros pela presenca de etileno.

USO DE BIORREATORES DE IMERSÃO CONTINUA, TEMPORÁRIA E MEIO DE CULTURA SEMISSÓLIDO NA PRODUÇÃO DE CLONES DE Eucalyptus camaldulensis

The plant micro-propagation in bioreactor systems is regarded as one way to reduce cost by automation and production scheduling. This research was carried out in order to obtain an efficient procedure for clone production of Eucalyptus camaldulensis on different types of bioreactor including continuous and temporary immersion bioreactor. To do so, the apical meristems (1 mm) and the apical meristems with adjacent tissue (2,5 mm) were used as initial explants. These tissues were cultured, for 60 days, in semisolid culture medium supplemented with 1 mg L-1 indole acetic acid (IAA) and 0.32 mg L-1 benzylaminopurine (BA). After 60 days, the meristems with adjacent tissue were transferred to a continuous immersion bioreactor and maintained in dark or light conditions. In order to verify the effect of the explant source on bioreactor multiplication, the explants subcultured from meristems multiplied in semisolid culture medium and the meristems multiplied in continuous immersion bioreactor were tested and maintained in dark conditions. After establishing this parameters, the multiplication experiments were carried out in continuous and temporary immersion and the multiplied explants were then rooted in MS medium supplemented with 0, 2, 4, 8 and 20 mg L-1 indole butyric acid (IBA) and kept in the dark or under controlled lighting conditions. After that, the rooting the plants were acclimatized in mist chamber. The meristem with adjacent tissue favored a greater number of buds/explants. The continuous immersion bioreactor in the dark provided higher shoots number and multiplication rate. The rooting was better on culture medium without auxin and kept in the dark for 15 days or the culture medium supplemented with auxin and maintained under light with 100% plantlet rooting. The Eucalyptus camaldulensis acclimatization was efficient, with high survival rate (76%). It was possible to establish the procedure for bioreactor micro-propagation of Eucalyptus camaldulensis large-scale clones.A micropropagacao em sistemas de biorreatores e considerada como uma forma de reduzir os custos de producao por meio do escalonamento de automatizacao do processo. O objetivo desse trabalho foi desenvolver um protocolo eficiente de producao de mudas de Eucalyptus camaldulensis em diferentes tipos de sistema, incluindo biorreator de imersao continua e temporaria. Para isso, meristemas apicais (1 mm) e meristemas apicais com tecido adjacente (2,5 mm) foram usados como explantes iniciais. Esses tecidos foram cultivados, por 60 dias, em meio de cultura suplementado com 1 mg L-1 de acido indolacetico (AIA) e 0.32 mg L-1 de benzilaminopurina (BAP). Apos 60 dias, os meristemas com tecidos adjacentes foram transferidos para biorreatores de imersao continua ou temporaria e mantidos no escuro ou sob condicoes controladas de luminosidade. Para verificar o efeito da fonte de explante na multiplicacao em biorreator foram testados explantes subcultivados de meristemas multiplicados em meio de cultura semissolido e meristemas multiplicados em biorreator de imersao continua e mantidos no escuro. Despois de estabelecer esses parâmetros, os experimentos de multiplicacao foram realizados em biorreatores de imersao continua e temporaria. Os explantes multiplicados foram enraizados em meio de cultura MS suplementado com 0, 2, 4, 8 e 20 mg L-1 de acido indolbutirico (AIB) e mantidos no escuro ou sob condicoes controladas de luminosidade. Depois do enraizamento as plantas foram aclimatizadas em câmara de nebulizacao. Os meristemas com tecidos adjacentes favoreceram um maior numero de gemas/explantes. O biorreator de imersao continua e mantido no escuro promoveu maior numero de brotacoes e maior taxa de multiplicacao e o melhor enraizamento ocorreram no meio de cultura isento de auxina, mantido no escuro por 15 dias ou o meio de cultura suplementado com auxina, mantido na luz apresentando 100% de enraizamento. A aclimatizacao do Eucalyptus camaldulensis foi eficiente com taxa de sobrevivencia de 76%. Portanto, foi possivel desenvolver um metodo eficiente de micropropagacao em biorreator para a producao de mudas Eucalyptus camaldulensis em larga escala.

Growth Curve and Development of the Internal Calli Structure of Eucalyptus camaldulensis Dehn

The objective of this work was to elucidate the growth curve of Eucalyptus camaldulensis Dehn. calli analyzing their anatomical modifications. A sigmoid aspect of the growth curve of the calli fresh matter was observed, with five different phases (lag, exponential, linear, deceleration and decline). In the lag phase, the highest growth percentage 87%, was observed, which reduced during the evaluation period to 17% in the linear phase. As for the anatomical analyses, cellular multiplications was observed during the lag and exponential phases and increase in cell size during the linear phase, promoting the calli volume growth and the establishment of the globular conformation.

CRESCIMENTO E ASPECTOS SINTOMATOLÓGICOS NA ACLIMATIZAÇÃO DE IPÊ-ROXO

This work evaluated the influence of seal type and substrate type on pre-acclimatization, and symptomatological aspects relating to acclimatization of ipe-roxo. For the pre-acclimatization, ipe-roxo plants obtained through an in vitro zygotic embryo culture were aseptically inoculated in test tubes with different types of seal (cotton plug, plastic cap + parafilm and plastic lid) and different substrates (agar, vermiculite and Plantmax ® ) supplemented with a WPM culture medium with 1 gL -1 of activated charcoal, 10 mgL -1 of citric acid and 30 gL -1 of sucrose added. For the acclimatization, in vitro ipe-roxo plants were transplanted into 56-cm o C and 1 atm for 20 minutes. Once transplanted, the plants were irrigated with different concentrations of the MS medium (25%, 50%, 75%, 100% and 150%), using distilled water as control. After 30 days, the effects of seal type and substrate type on pre-acclimatization were examined. Cotton can be recommended where Plantmax ® or vermiculite is used as substrate. In the acclimatization it was noted that where plants were irrigated with distilled water and concentrations < 50% they showed symptoms of nutritional deficiency. Similar symptoms were observed when using MS medium at a concentration equal to or greater than 100%. The 75% MS concentration was found to be the most effective at maintaining the nutritional vigor of ipe-roxo seedlings in the process of acclimatization .

Proteic and phenolics compounds contents in Bacupari callus cultured with glutamine and nitrogen sources

In this study was evaluated the influence of glutamine supplementation on the endogenous content of amino acids, proteins, total phenolics, flavonoids and proanthocyanidins in Bacupari callus. The explants were inoculated in MS medium, MS with half concentration of the nitrogen salts (MS½) and nitrogen-free MS, supplemented with glutamine (5, 10, 30 and 60mM) named as Gln5, Gln10, Gln30 and Gln60. Amino acids and proteins were analyzed after 20, 80 and 140 days and the secondary metabolites on the 140th day. There was no difference in the amino acids on the 20th day. On the 80th day the treatments MS and MS½ presented the lowest levels. On the 140th day MS and MS½ presented the lowest amino acid concentration and Gln10 the highest. Concerning proteins, there was difference only on the 140th day, being the highest concentrations observed in Gln5, and the lowest in MS½ treatment. Total phenolics content was higher in the treatment Gln60 and lowest in MS. Treatments Gln5, Gln10, Gln30 and MS½ were statistically equal. For flavonoids, the highest values occurred in the treatments Gln30, Gln60 and MS½ and the lowest in Gln5, Gln10 and MS. Similarly, for the proanthocyanidins the highest concentrations were observed in treatment Gln60 and the lowest in Gln5 and MS. In conclusion, the treatment with 60mM of glutamine favors the protein accumulation and production of secondary metabolites in Bacupari callus.