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.