ABSTRACT In bryophytes, establishment can occur by a sexual or asexual process, but the production of spores enables colonization of a wider range of habitats and substrates than can asexual propagules. Successful germination is critical for establishment in a new environment. This paper addresses germination and sporeling development in Frullania ericoides, a leafy liverwort species. Fresh spores were inoculated in vitro in different culture strengths of Knop’s nutrient solution (one-fourth strength, half strength, full strength, one and a half strength and double strength), in order to evaluate the effects of this solution on spore germination and on the development of external protonema. On the first assessment, spore germination was observed at all the concentrations. Germination was endosporic, with cell division and proliferation, resulting in a globular protonema, within the spore wall. Beginning at the fourth week, the development of tightly concave primordial leaves was observed in all but the double-strength medium. Throughout the period of study, the treatments with lower concentrations exhibited external protonema with greater lengths. The double-strength treatment was statistically different from other treatments in at least two parameters. The results of this study demonstrate the potential of in vitro culture techniques for bryophyte spore studies and germplasm preservation.

RESUMOA importância na medicina popular, assim como as ameaças em seu ambiente, tornam necessária a realização de estudos envolvendo a propagação em larga escala de plantas do gênero Lippia. Embora a propagação in vitro seja amplamente disseminada para plantas medicinais, nenhum artigo foi publicado ainda com L. rotundifolia. O presente estudo teve como objetivo estabelecer um protocolo eficiente para a micropropagação dessa espécie. Segmentos nodais, retirados de plantas coletadas na Cadeia do Espinhaço, foram desinfetados antes da inoculação em meio MS suplementado com PVPP (1 g L-1), sacarose (3%) e ágar (0,7%). Os tubos de ensaio foram mantidos em sala de crescimento sob condições controladas. O tratamento de desinfestação e a adição de PVPP aos meios de cultura resultaram na redução da infecção microbiológica assim como nas taxas de oxidação fenólica, proporcionando mais de 90% de culturas assépticas e viáveis . Na fase de multiplicação foram testadas diferentes combinações de BAP e ANA adicionadas ao meio MS. O tratamento que resultou em maiores taxas de multiplicação foi de 0,33 µM de BAP. Os efeitos do ANA foram avaliados no enraizamento in vitro. A 0,44 µM de ANA, o enraizamento foi 70% superior ao observado no controle. A aclimatização foi realizada em bandejas com substrato, revestidas com plástico translúcido e mantidas à sombra. As plantas foram transferidas para casa de vegetação após 15 dias e transplantadas para canteiros após 30 dias. As plantas aclimatizadas floresceram após um ano da transferência para condições de campo, demonstrando que os procedimentos de cultivo in vitro não afetaram o desenvolvimento vegetativo e reprodutivo das plantas, o que confirma o potencial da micropropagação para redução dos riscos de extinção de L. rotundifolia.

ABSTRACTThe importance in folk medicine, combined to threats in their environment, becomes necessary to carry out studies involving large-scale propagation of Lippia genus. Although the tissue culture propagation is widely disseminated for medicinal plants, for L. rotundifolia any article was published yet. The present study aimed to establish an efficient protocol for micropropagation of L. rotundifolia. Nodal segments, taken from plants collected in the Espinhaço Range, were disinfected, and cultures were initiated on MS medium with PVPP (1 g L- 1), sucrose (3%) and agar (0.7%). The culture were maintained in a growth room at controlled conditions. Disinfestation procedures and the supply of PVPP on culture media resulted in both reduced contamination and phenol oxidation rates, with more than 90% of viable cultures. In the multiplication phase were tried different BAP and NAA combinations supplied to the MS medium. The treatment that resulted in highest multiplication rates was 0.33 µM BAP. The effects of NAA were evaluated for in vitrorooting. At 0.44 µM, rooting was 70% higher than that observed in the control. The acclimatization was held in trays with substrate, coated with translucent plastic and kept under shade. The plantlets were transferred to the greenhouse after 15 days and transplanted to plant beds after 30 days. The acclimatized plantlets bloomed one year after the transference to field conditions, showing that the in vitro culture did not affect the vegetative and reproductive development, which confirms the potential of micropropagation to reduce the extinction risk of L. rotundifolia.

Os objetivos deste trabalho foram estabelecer protocolos eficientes para a multiplicação e enraizamento in vitro, bem como para a aclimatização ex vitro de Aegiphila verticillata, uma espécie arbórea típica dos campos rupestres brasileiros. Culturas assépticas foram estabelecidas a partir de sementes, e dois experimentos de multiplicação foram realizados. No primeiro experimento, foram utilizados 6-benzilaminopurina (BAP – 0; 2,5; 5; e 7,5 μM) + ácido α-naftaleno acético (ANA – 0; 0,2; 0,4; e 0,6 μM) e, no segundo, sulfato de adenina, cinetina ou thidiazuron (0; 5; 7,5; 10; e 12,5 μM). Após 90 dias, foram avaliados o número e a qualidade das brotações. Houve mais de 90% de germinação das sementes e reduzida taxa de contaminação (2%). Na etapa de multiplicação, o meio que promoveu o melhor desenvolvimento qualitativo e quantitativo das culturas foi o suplementado com 7,5 μM de BAP + 0,4 μM de ANA. No experimento de enraizamento, foram utilizados ANA, ácido indol acético (AIA) ou ácido indol butírico (AIB) (0; 0,1; 0,2; 0,3; e 0,4 μM). Após 90 dias, foram avaliados o número de raízes e a qualidade do sistema radicular. Nessa análise, não foram encontradas diferenças entre o controle e os demais tratamentos. Plântulas enraizadas foram aclimatizadas inicialmente por 30 dias em bandejas de isopor, período após o qual foram transferidas para vasos, em casa de vegetação. Apenas 3% das plantas submetidas à aclimatização inicial morreram, e 70% daquelas transferidas para a casa de vegetação sobreviveram e apresentaram desenvolvimento normal. Os resultados encontrados neste trabalho são os primeiros relatos envolvendo a propagação in vitro e a aclimatização ex vitro de Aegiphila verticillata e possibilitam o fornecimento de um suprimento contínuo dessa espécie medicinal nativa, ameaçada de extinção devido às atividades antrópicas.

The objective of this work was to establish an efficient protocol for in vitro multiplication and rooting, as well as ex vitroacclimatization of Aegiphila verticillata, a woody species found in Brazilian rocky fields. Aseptic cultures were established by seeds and two multiplication analyses were performed. In the first, we employed 6-benzylaminopurine (BAP – 0, 2.5, 5 and 7.5 μM) + α-naphthalene acetic acid (NAA – 0, 0.2, 0.4 and 0.6 μM) and, in the second, were studied adenine sulfate, kinetin and thidiazuron (0, 5, 7.5, 10 and 12.5 μM). After 90 days, we assessed the quantitative and qualitative shoot propagation. There were more than 90% seed germination and low contamination (2%). In multiplication phase, the culture medium that promoted the best quantitative and qualitative culture development was supplemented with 7.5 μM BAP + 0.4 μM NAA. In the rooting assay, were used NAA, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (0, 0.1, 0.2, 0.3 or 0.4 μM). After 90 days, the root number and rooting quality were evaluated. In this analysis, differences were not found between the control and the other treatments. Rooted plantlets were acclimatized in styrofoam trays for 30 days, after which they were transferred to pots in the greenhouse. Only 3% of the plants subjected to initial acclimatization died and 70% of the plants transferred to the field conditions survived and showed normal development. The results founded in this work are the first involving in vitro propagation and ex vitroacclimatization of Aegiphila verticillata and provide a continuous supply of this medicinal native species, endangered due anthropogenic activities.

This study aimed to establish and propagate in vitro plants of Bouchea fluminensis, a medicinal species known in Brazil as gervão-falso ("false verbena"), evaluating the influences of different growth regulators on in vitro multiplication and rooting stages, as well as examining ex vitro acclimatization of rooted plants. Explants were established on Murashige and Skoog medium at half strength of salts and vitamins without growth regulators. For multiplication, the explants were subjected to combinations of 6-benzyladenine (BA; 0, 2.5, 5.0 and 7.5 µM) and α-naphthalene-acetic acid (NAA; 0, 0.2, 0.4 and 0.6 µM). The medium found to induce the greatest number of shoot was that containing 5 µM of BA (NAA-free). For rooting, we evaluated three auxins (NAA, indole-3-acetic acid and indole-3-butyric acid; 0.1, 0.2, 0.3 and 0.4 µM), as well as a control. No differences were observed between the control and the other treatments. The auxin-free medium was deemed the most suitable, because it ensures the lowest cost in the micropropagation procedures. We obtained 100% survival of the acclimatized seedlings, and the plants showed normal vegetative and reproductive development, suggesting that the micropropagation did not alter the biological cycle of this species. The results show the importance and potential of micropropagation for biodiversity conservation of Bouchea fluminensis.

The antioxidant defense system response was evaluated in two rice cultivars (Oryza sativa L.), Fernandes (CNA-1158) and Maravilha (CNA-6843-1), treated with toxic levels of aluminum. After exposure to aluminum we determined plant growth, H2O2 and O2•- contents, lipid peroxidation, antioxidant enzymes activities and ascorbate and dehydroascorbate contents. Al predominantly accumulated in roots of both cultivars but it reduced root and shoot growth only in the Maravilha cultivar. Treatment with aluminum resulted in a reduction of 84 and 60% in the levels of H2O2 in the roots of the cultivars Maravilha and Fernandes, respectively, and of 26% in the levels of O2•- only in the roots of Fernandes cultivar. Increased lipid peroxidation was observed only in the roots of the Maravilha cultivar. In general, the antioxidant enzyme activities were higher in roots and increased in the presence of aluminum, especially in the Fernandes cultivar. The levels of ascorbate were higher in leaves and increased with aluminum treatment, while dehydroascorbate decreased in roots of both cultivars after aluminum treatment. However, the ascorbate/dehydroascorbate ratio increased in the roots of both cultivars after treatment with aluminum. Ascorbate, dehydroascorbate and ascorbate/dehydroascorbate levels found here point to an efficient regeneration of ascorbate, essential for the homeostasis of cellular metabolites involved in reactive oxygen species removal by rice plants treated with aluminum. Therefore, the higher tolerance of Fernandes to aluminum relative to Maravilha cultivar may be the result of better growth of the root system and shoots, higher antioxidant enzyme activities and a best use/regeneration of ascorbate.

Este trabalho teve por objetivo determinar a influência do Al sobre a composição de ácidos graxos em raízes de duas cultivares de sorgo [Sorghum bicolor (L.) Moench] com tolerância diferencial ao Al. Após exposição das plantas ao Al, raízes foram coletadas, os lipídios extraídos e determinada sua composição em ácidos graxos. O palmítico e, especialmente, o linoléico foram os principais ácidos graxos identificados nos extratos lipídicos de raízes das duas cultivares, independentemente da presença do Al. Nas plantas tratadas com Al, os teores dos ácidos esteárico e oléico aumentaram apenas na cultivar tolerante, mas os teores do ácido linolênico reduziram nas duas cultivares. Essas alterações na composição de ácidos graxos, o índice de ligações duplas para ácidos graxos contendo 18 carbonos (ILD C18) e as relações ácidos graxos saturados/ácidos graxos insaturados (AGI/AGS) e ácido linoléico/ácido palmítico (18:2/16:0) sugerem que ocorre redução na fluidez dos lipídios da cultivar tolerante, porém aumento na sensível. A redução na fluidez dos lipídios na cultivar tolerante, comparativamente à da sensível sugere ter a cultivar tolerante maior capacidade de reter íons e metabólitos essenciais e, ou limitar a entrada do Al em suas raízes.

The objective of this work was to determine Al effect on fatty acid composition of roots from two sorghum (Sorghum bicolor (L.) Moench) cultivars with differential tolerance to aluminum. After exposure to Al, roots were collected, lipids extracted and the fatty acid composition determined. Palmitic and, especially, linoleic acid were the major fatty acids identified in the root lipid extracts of both cultivars, regardless of the presence of aluminum. Stearic and oleic acids contents increase only in the tolerant cultivar, while linolenic acid decreased in both cultivars after Al treatment. These changes in fatty acid composition, as well as in the C18-fatty acid double bound index (DBI C18) and in the ratios of unsaturated fatty acid/saturated fatty acid (UFA/SFA) and linoleic acid/palmitic acid (18:2/16:0) indicated of a reduction of the lipid fluidity of the tolerant cultivar, but an increase in the sensitive one. The observed reduction in lipid fluidity in the tolerant relative to the sensitive cultivar suggests of a better capacity of the tolerant cultivar to retain essential ions and metabolites and/or to limit the entry of toxic Al ions into roots.

Os efeitos do alumínio (Al+3) sobre a morfologia e o acúmulo de compostos fenólicos foram avaliados em duas cultivares de sorgo (Sorghum bicolor (L.) Moench) com tolerância diferencial ao Al+3. As plantas foram mantidas em solução nutritiva durante dez dias, na presença (185 mM) ou na ausência de Al+3. Os ápices radiculares foram coloridos com hematoxilina férrica, sendo a intensidade da coloração observada na presença do Al+3 muito próxima entre as cultivares, o que inviabiliza a aplicação desse teste, isoladamente, para discriminação entre o genótipo sensível e o tolerante ao Al+3. As análises da morfologia externa e interna dos ápices radiculares de plantas tratadas com Al+3 também foram muito similares entre as duas cultivares, não permitindo a utilização dessas características para seleção entre a cultivar sensível e a tolerante. O maior acúmulo de lignina e, principalmente, a menor produção de compostos fenólicos, observados na presença do Al+3 nas raízes das plantas da cultivar tolerante (BR006R), são parâmetros que possibilitam a discriminação das cultivares quanto à tolerância ao Al+3.

The Al+3 effects on the morphology and on the phenolics accumulation were evaluated in two sorghum cultivars exhibiting differential tolerance to Al+3. The plants were kept in nutrient solution, for ten days, in the presence (185 mM) or Al+3 absence. The root apexes were colored with ferric hematoxylin, and because the intensity of color development is very similar among evaluated cultivars, the use of this method as a selection parameter for Al+3-tolerance is not indicated. Analyses of superficial and internal morphology from tissues treated with Al+3 also expressed very similar alterations among the two cultivars, not allowing its use for selection between sensitive and Al+3-tolerant genotype. The higher lignin accumulation and, mainly, the lower phenolics production in roots of the BR006R cultivar in Al+3's presence, demonstrated the existence of differential tolerance among cultivars.

O objetivo deste trabalho foi avaliar os efeitos do flúor em folhas de plantas aquáticas de Salvinia auriculata Aubl., visando fornecer subsídios para a utilização dessa espécie, no monitoramento da poluição ambiental, causada por esse elemento tóxico. As plantas foram cultivadas sob condições controladas, em vasos com solução nutritiva, e submetidas à aplicação de chuva simulada contendo KF, nas concentrações de 0, 13, 26 e 39 mM, pela manhã, durante cinco dias sucessivos. Os resultados evidenciaram a ocorrência de alterações morfológicas, com o desenvolvimento de lesões nos tricomas e na porção adaxial do limbo foliar. As alterações nas atividades das enzimas peroxidase, polifenol oxidase, superóxido dismutase e catalase indicaram a ocorrência de danos oxidativos em resposta ao flúor, embora testes relacionados à peroxidação dos lipídios tenham apresentado resultados negativos. As alterações na concentração de pigmentos também direcionam para a ocorrência de estresse oxidativo, causado pelo flúor, presente na chuva simulada. Como as alterações morfológicas, enzimáticas e na composição de pigmentos, de plantas de S. auriculata, são passíveis de detecção por métodos relativamente simples, elas podem ser empregadas no biomonitoramento da poluição atmosférica, causada por esse elemento altamente reativo.

The objective of this work was to evaluate the effects of fluoride on leaves of aquatic Salvinia auriculata Aubl., to supply subsidies for the use of this species as an environmental pollution bioindicator. The plants were cultivated under controlled conditions in pots containing nutritive solutions, and were submitted to simulated rain containing KF, in concentrations of 0, 13, 26 and 39 mM, in the morning, during five consecutive days. Results evidenced the occurrence of morphological and anatomical damages involving trichome and adaxial surface of the leaf necrosis. Alterations in the activity of peroxidase, polyphenoloxidase, superoxide dismutase, and catalase enzymes indicated the occurrence of oxidative damages in response to fluoride, although lipid peroxidation tests showed negative results. Alterations in pigment concentration also pointed to the occurrence of oxidative stress, caused by fluoride present in the simulated rain. The morphological, anatomical, enzymatic, and pigment composition alterations in S. auriculata can be used in atmospheric pollution biomonitoring, because they are easy to detect through simple methodologies.