Propagação in vitro e aclimatização de Lippia rotundifolia, uma espécie endêmica dos Campos Rupestres Brasileiros

The 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.A 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

Spore germination, early development and some notes on the effects of in vitro culture medium on Frullania ericoides (Nees) Mont. (Frullaniaceae, Marchantiophyta)

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.

Avaliação do metabolismo oxidativo em Pitcairnia encholirioides L. B. Sm. (Bromeliaceae) in vitro e ex vitro e sob desidratação

Bromeliaceae is essentially a neotropical family, whose representatives are widely used for landscaping. One major source of supply for the consumer market is the extraction, which coupled with the destruction of the natural environment, has led to irreparable losses in biodiversity of the family, especially in the Atlantic Forest, where about 40% of bromeliad species are under some category of threat. Pitcairnia encholirioides L. B. Sm. is a critically endangered species. In 2004, a unique population of this species was found, with about 900 individuals in a very degraded rocky outcrop, subject to fire and trampling by animals, located in Santa Maria Madalena, RJ. The use of micropropagation can guarantee high multiplication rates, providing necessary material for the consumer market, effectively obviating the extraction of plants in their places of origin. In addition, installation and in vitro conservation of germplasm banks have special importance, ensuring the survival of rare species and/or endemic and providing micropropagated plants for reintroduction initiatives. Following the procedures of in vitro multiplication and rooting, the final stage of micropropagation is the ex vitro acclimatization, a critical period for the plants due to water loss, which causes water stress and, consequently, oxidative stress, which can cause metabolic problems and high losses. In this study, Pitcairnia encholirioides was grown for 150 days in vitro in a culture medium supplemented with two growth regulators (GA3 or NAA), and two concentrations of sucrose (15 or 30 g L-1) in two types of sealing of test tubes (hermetic seal with lids and PVC film and seal with lids that allowed gas exchange), totaling eight treatments, after which the plants were transferred to ex vitro conditions in a greenhouse, staying for more than 180 days. Two of these treatments were selected for analysis of dehydration when their plants were subjected to 30, 42 or 54 days without irrigation, apart from control irrigated periodically, being subsequently rehydrated for 90 days. After periods of in vitro cultivation and maintenance of the plants in the greenhouse, they were analyzed for proline content, total protein, activities of antioxidant metabolism enzymes SOD, CAT, POD and PPO, besides the content of photosynthetic pigments. Apart from these parameters, after dehydration and rehydration, levels of carbohydrates were also assessed. In general, the in vitro culture in medium containing the lowest concentration of sucrose and hermetically sealed tubes proved to be harmful, which was evidenced by lower levels of proline, increased 4 activity of antioxidant enzymes and lower accumulation of photosynthetic pigments. After the acclimatization period, no differences were found between treatments for most parameters, or data varied so as to not allow a generalization about the residual effects of sucrose, the type of cover and of the growth regulators used in the in vitro cultivation. During the period in which the plants were subjected to water stress, higher accumulation of proline and total soluble proteins occurred, and also increased levels of photosynthetic pigments in tissues from plants grown in vitro in medium containing 30 g L-1 sucrose and GA3. There was also, in general, sharper decrease in enzyme activity in medium with the lowest concentration of sucrose and NAA, as well as fluctuations in pigment. Relations between pigments suffered slight reduction in both treatments, and total soluble carbohydrates, sucrose, reducing sugars and starch increased with increasing stress. After rehydration, all plants showed high resilience, presenting for all analyzes values very similar to those of control plants not subjected to dehydration. The results indicate that the in vitro culture in medium supplemented with 30 g L-1 sucrose and tubes that allow gas exchange are the most appropriate. Moreover, the concentration of sucrose added to the culture medium also influences the development of plants after acclimatization, with best results found in plants grown in media supplemented with higher amount of this carbohydrate. It is believed that plants grown under these conditions would have higher capacity of resistance to transplanting and could survive for prolonged periods of water stress, which are often subjected in the natural environment.Bromeliaceae é uma família essencialmente neotropical, cujos representantes são muito utilizados para fins paisagísticos. Uma das principais fontes de abastecimento do mercado consumidor de bromélias é o extrativismo o que, associado à destruição do ambiente natural, tem levado a perdas irreparáveis na biodiversidade da família, especialmente na Mata Atlântica, onde cerca de 40% das espécies encontram-se sob alguma categoria de ameaça. Pitcairnia encholirioides L. B. Sm. é uma espécie “criticamente em perigo” de extinção, sendo conhecida somente uma população dessa espécie, com cerca de 900 indivíduos, encontrada em 2004 em um afloramento rochoso muito degradado e sujeito ao fogo e ao pisoteio de animais, no município de Santa Maria Madalena, RJ. A utilização das técnicas de micropropagação pode reverter os riscos de extinção garantindo taxas elevadas de multiplicação, fornecendo material necessário ao mercado consumidor, evitando, dessa forma, o extrativismo das plantas nos seus locais de origem. A instalação e a conservação in vitro de bancos de germoplasma possuem especial importância, garantindo a sobrevivência de espécies raras e/ou endêmicas e fornecendo plantas para iniciativas de reintrodução. A etapa final da micropropagação é a aclimatização ex vitro, realizada após os procedimentos de multiplicação e enraizamento in vitro. A aclimatização é um período crítico para as plantas devido à perda de água, o que causa estresse hídrico e, como consequência, estresse oxidativo, podendo provocar problemas metabólicos e perdas elevadas. No presente trabalho, Pitcairnia encholirioides foi cultivada in vitro por 150 dias em meio de cultura adicionado de dois reguladores de crescimento (GA3 ou ANA), além de duas concentrações de sacarose (15 ou 30 g L-1) e sob dois tipos de vedação dos tubos de ensaio (vedação hermética com tampas e filme de PVC ou vedação com tampas que permitiam trocas gasosas), totalizando 8 tratamentos, período após o qual as plantas foram transferidas para condições ex vitro, em casa de vegetação, permanecendo por mais 180 dias. Dois desses tratamentos foram selecionados para as análises de desidratação, quando suas plantas foram submetidas a 30, 42 ou 54 dias sem irrigação, além do controle irrigado periodicamente, sendo posteriormente reidratadas durante 90 dias. Após os períodos de cultivo in vitro e permanência das plantas em casa de vegetação, foram realizadas análises dos conteúdos de prolina, proteínas totais, atividades das enzimas do metabolismo antioxidativo SOD, CAT, POD e PPO, além 2 dos teores de pigmentos fotossintetizantes. Além desses parâmetros, após desidratação e reidratação foram também avaliados os teores de carboidratos. De maneira geral, o cultivo in vitro em meio adicionado da menor concentração de sacarose e em tubos vedados hermeticamente se mostrou prejudicial, o que foi evidenciado pelo menor acúmulo de prolina, aumentos nas atividades das enzimas antioxidativas e menor acúmulo de pigmentos fotossintéticos. Após o período de aclimatização, não foram encontradas diferenças entre os tratamentos para a maioria dos parâmetros analisados, ou os dados oscilaram de maneira a não permitir uma generalização quanto aos efeitos residuais da sacarose, do tipo de tampa e dos reguladores de crescimento utilizados durante o cultivo in vitro. No período em que as plantas foram submetidas à desidratação progressiva, houve maior acúmulo de prolina e proteínas solúveis totais, além de aumento nos teores de pigmentos fotossintéticos nos tecidos provenientes de plantas cultivadas in vitro em meio contendo 30 g L-1 de sacarose e GA3. Houve também, em geral, queda mais acentuada nas atividades das enzimas em meio suplementado com a menor concentração de sacarose e ANA, além de oscilações nos teores de pigmentos. As relações entre os pigmentos sofreram leve redução em ambos os tratamentos, e os teores de carboidratos solúveis totais, sacarose, açúcares redutores e amido aumentaram com o prolongamento do estresse. Após a reidratação, todas as plantas demonstraram elevada capacidade de recuperação, apresentando em todas as análises realizadas valores muito similares aos das plantas do controle, não submetidas à desidratação. Os resultados obtidos no trabalho permitem concluir que o cultivo in vitro em meio adicionado de 30 g L-1 de sacarose e em tubos de ensaio que permitem trocas gasosas é o mais apropriado para plantas de P. encholirioides. Ademais, a concentração de sacarose adicionada ao meio de cultura também influencia o desenvolvimento das plantas após aclimatização, sendo encontrados melhores resultados em plantas cultivadas nos meios de cultura suplementados com a maior concentração desse carboidrato. Acredita-se que as plantas cultivadas nessas condições apresentariam maior capacidade de sobrevivência ao transplantio e maior resistência a períodos prolongados de estresse hídrico, aos quais, frequentemente, estão submetidas no ambiente natural.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Desempenho ecofisiológico de Syzygium jambos (L.) Alston, espécie exótica invasora cultivada sob diferentes substratos e intensidades luminosas

Syzygium jambos (L.) Alston is a species of the Myrtaceae family, popularly known as “rose-apple” and originated in tropical Asia. It has great phenotypic plasticity to contrasting environmental conditions and high invasiveness, being able to adapt to both open areas as closed canopy. Its individuals form a dense crown, preventing colonization of native plants and the establishment of more light dependent species. It is therefore of great ecological importance. Despite being an invasive species in many tropical oceanic islands and continental areas, works aimed at understanding the ecophysiological mechanisms underlying this capacity are scarce. Thus, this study aimed to describe the morphological, biochemical and metabolic alterations in S. jambos plants grown under different light conditions and fertilization systems, and verify the feasibility of using the SPAD-502 chlorophyll meter in indirect determination of the levels of leaf photosynthetic pigments. For that, seeds were collected from one individual established in the Institute of Biological Sciences of UFJF and placed to germinate, during which the mobilization of cotyledon carbohydrates was assessed. The seeds were planted in plastic cups, and after raising to about 11 cm they were transplanted into pots of 25 L, filled with substrate formed by land and sand (control), substrate added with bovine manure and a third with the addition of chemical fertilizer. The plants were transferred to different light environments, called low radiation (BR), intermediate radiation (RI) and high radiation (AR), where they stood for about 10 months. The results of this study showed significant adaptive responses of S. jambos plants to different light environments, such as lower development of the aerial part, higher allocation of biomass in the roots, shorter internodes and the presence of leaves in a more vertical position in plants at full sun. In turn, plants under low radiation showed leaves with larger areas than plants under higher light intensities, in addition to significantly higher levels of photosynthetic pigments. There was a significant positive correlation between the levels of photosynthetic pigments measured spectrophotometrically and the SPAD indices, obtained with the help of chlorophyll meter, validating its use in indirect quantification of these levels in S. jambos plants. Regarding the antioxidative metabolism, there were different strategies to combat the formation of reactive oxygen species. Overall, the study highlights the increased activity of antioxidant enzymes in shadier environments, as well as increased production of carotenoids in the leaves developed in full sun. The photosynthetic and fluorescence parameters showed significant responses to changes in light condition. Regardless of the ambient light, plants of S. jambos showed growth and development while remaining viable. The present data allow us to conclude that the morphological, physiological and biochemical attributes presented by the invasive species Syzygium jambos in contrasting light conditions are of great importance for their acclimation and permanence in different natural environments, contributing to its invasiveness.Syzygium jambos (L.) Alston é uma espécie da família Myrtaceae, conhecida popularmente como “jambo-rosa” e originária da Ásia Tropical. Apresenta grande plasticidade fenotípica a condições ambientais contrastantes e uma elevada capacidade invasora, sendo capaz de adaptar-se tanto a áreas abertas quanto de dossel fechado. Seus indivíduos formam uma copa densa, impedindo a colonização de plantas nativas e o estabelecimento de espécies mais dependentes de luz. É, por isso, de grande importância ecológica. Apesar de ser uma espécie invasora de diversas ilhas oceânicas e áreas continentais tropicais, trabalhos que visem entender os mecanismos ecofisiológicos subjacentes a essa capacidade são inexistentes. Dessa forma, objetivou-se descrever as alterações morfológicas, bioquímicas e metabólicas de plantas de S. jambos cultivadas em diferentes condições de luminosidade e fertilização, além de verificar a viabilidade da utilização do clorofilômetro portátil SPAD-502 na determinação indireta dos teores de pigmentos fotossintéticos foliares. Para tal, sementes foram coletadas de um indivíduo estabelecido no Instituto de Ciências Biológicas da UFJF e germinadas, período durante o qual a mobilização de carboidratos cotiledonares foi avaliada. As sementes foram plantadas em copos descartáveis, e após o crescimento até cerca de 11 cm foram transplantadas para vasos de 25 L, preenchidos com substrato formado por terra e areia (controle), substrato adicionado de esterco bovino e um terceiro com adição de adubo químico. As plantas foram transferidas para diferentes ambientes luminosos, denominados de baixa radiação (BR), radiação intermediária (RI) e alta radiação (AR), permanecendo por cerca de 11 meses. Os resultados do presente trabalho demonstraram importantes respostas adaptativas das plantas de S. jambos aos diferentes ambientes luminosos, como, por exemplo, menor desenvolvimento da parte aérea, maior alocação de biomassa nas raízes, menor comprimento de internódios e a presença de folhas em posição mais vertical em plantas a pleno sol. Por sua vez, as plantas sob baixas radiações apresentaram folhas com áreas maiores, além de teores de pigmentos fotossintéticos significativamente maiores, que as plantas sob maiores intensidades luminosas. Houve correlação positiva significativa entre os teores de pigmentos fotossintéticos aferidos espectrofotometricamente e os índices SPAD obtidos com auxílio do clorofilômetro portátil, validando sua utilização na quantificação indireta desses teores em plantas de S. jambos. Com relação ao metabolismo antioxidativo, foram evidenciadas diferentes estratégias de combate à geração de espécies reativas de oxigênio. De forma geral, ressalta-se a maior atividade de enzimas antioxidativas nos ambientes mais sombreados, bem como a maior produção de carotenoides nas folhas desenvolvidas a pleno sol. Os parâmetros fotossintéticos e de fluorescência evidenciaram importantes respostas à variação na luminosidade. Independentemente do ambiente luminoso, as plantas de S. jambos apresentaram crescimento e desenvolvimento, permanecendo viáveis. Os dados do presente trabalho nos permitem concluir que os atributos morfológicos, fisiológicos e bioquímicos apresentados pela espécie invasora Syzygium jambos em condições contrastantes de luminosidade são de grande importância na sua aclimatação e permanência em ambientes naturais diversos, contribuindo para a sua capacidade invasora.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerai

In vitro propagation and acclimatization of Lippia rotundifolia, an endemic species of Brazilian Campos Rupestres

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

Spore germination, early development and some notes on the effects of in vitro culture medium on Frullania ericoides (Nees) Mont. (Frullaniaceae, Marchantiophyta)

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

MICROPROPAGATION AND ACCLIMATIZATION OF Aegiphila verticillata Vell.: AN ENDANGERED WOODY SPECIES

The objective of this work was to establish an efficient protocol for in vitro multiplication and rooting, as well as ex vitro acclimatization 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 vitro acclimatization of Aegiphila verticillata and provide a continuous supply of this medicinal native species, endangered due anthropogenic activities

An efficient system for in vitro propagation of Bouchea fluminensis (Vell.) Mold. (Verbenaceae)

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