ABSTRACT Arbuscular mycorrhizal fungi (AMF) and dark septate fungi (DSF) are symbionts that are associated with the roots of plants, including epiphytic lycophytes and ferns. Paris-type mycorrhiza and glomoid structures are the most common forms of colonization in these plants. This work aimed to evaluate the occurrence of these symbionts in the roots of epiphytic lycophytes and ferns as well as the diversity of AMF spores recovered from substrate associated with the roots of eleven species. Roots of Asplenium gastonis, Campyloneurum aglaolepis, C. nitidum, Niphidium crassifolium, Pecluma pectinatiformis, Phlegmariurus mandiocanus, Pleopeltis hirsutissima, P. pleopeltifolia andSelaginella microphyllahad hyphae and vesicles typical of AMF colonization, but not arbuscules.Campyloneurum nitidum, Pecluma pectinatiformis, Phlegmariurus mandiocanus, Pleopeltis pleopeltifolia and Selaginella microphyllahad melanized hyphae and microsclerotia typical of DSF. All species colonized by DSF were also colonized by AMF. Seventeen spore morphotypes of AMF were identified, of which six were acaulosporoid and eleven glomoid. Glomus aff. formosanum andAcaulospora aff. lacunosa were the most abundant and frequent species. Epiphytic lycophytes and ferns host concurrently AMF and DSF but colonization is scanty in their roots. For the first time, acaulosporoid spores and intraradical vesicles are reported for this group of plants.

The development of micropropagation techniques in orchid has many advantages such as the reproduction of elite, rare, sterile and/or disease-free genotypes. The aim of this study was to evaluate the use of organic supplements (coconut water and banana pulp) in the in vitro development of seedlings of Laeliocattleya (Hadrolaelia purpurata x Cattleya intermedia), comparing their response to plant growth regulators: auxins and cytokinins. For this, the following four experiments were carried out: 1. Organic supplements compared to different concentrations ofkinetin (N6-furfuryl aminopurine; KIN) and naphthaleneacetic acid (NAA), 2. Organic supplements compared to different concentrations of 3-indolebutyric acid (IBA) and N6-benzyladenine (6BA), 3. Organic supplements compared to different concentrations of IBA and KIN, and 4. Organic supplements compared with different concentrations ofANA and 6BA. Coconut water was statistically superior to the others (P <0.01) for the variable number of leaves, in all experiments performed with 7.3 leaves per seedling cultivated in vitro. While banana pulp showed the best results in the number of roots (P <0.01) for all experiments, with an average value of 7.4 roots. The addition of banana pulp and/or coconut water is suitable for the production of orchid hybrids, which present a positive effect on the development of seedlings. Its low cost is also a plus in relation to the use ofplant growth regulators.

El desarrollo de técnicas de micropropagación en orquídeas ofrece múltiples ventajas como la reproducción de genotipos superiores, raros, estériles o libres de enfermedades. El objetivo del presente trabajo fue evaluar el uso de suplementos orgánicos (agua de coco y pulpa de banana) en el crecimiento y desarrollo in vitro de plántulas del híbrido intergenérico Laeliocattleya (Hadrolaelia purpurata x Cattleya intermedia), comparando su respuesta con fitorreguladores de auxinas y citoquininas. Para ello se realizaron los siguientes cuatro experimentos: 1. Suplementos orgánicos comparados con diferentes concentraciones de kinetina (N6-furfuril amino purina; KIN) y ácido naftalenacético (ANA), 2. Suplementos orgánicos comparados con diferentes concentraciones de ácido 3-indolbutírico (IBA) y N6-benzyladenine (6BA), 3. Suplementos orgánicos comparados con diferentes concentraciones de IBA y KIN, y 4. Suplementos orgánicos comparados con diferentes concentraciones de ANA y 6BA. El suplemento orgánico de agua de coco fue estadísticamente superior al resto (P<0,01), para la variable número de hojas, en todos los experimentos realizados, con 7,3 hojas por plántula cultivada in vitro. En tanto que la adición de pulpa de banana mostró los mejores resultados en el número de raíces (P<0,01), para todos los experimentos en estudio, con un valor medio de 7,4 raíces. Se concluye que la adición de pulpa de banana o de agua de coco es conveniente para la producción de orquídeas híbridas por presentar un efecto positivo en el desarrollo de las plántulas, y por su costo reducido en relación con el uso de reguladores de crecimiento.

No presente estudo foram adicionados ao meio de cultura Knudson-C (KC), suplementos específicos (micronutriente, peptona, carvão ativado) e complexos (água de coco e polpa de banana) para estimular a propagação in vitro das espécies de orquídeas Hadrolaelia purpurata (=Laelia purpurata) e Encyclia randii. O número maior de plântulas de H. purpurata foi observado no meio KC contendo 90 g L-1 de polpa de banana com iluminação contínua (24h). O efeito positivo da adição de polpa de banana foi descrito para a formação de plântulas e folhas de E. randii. O número de plântulas de E. randii mantidas com iluminação contínua também foi alto em meio contendo água de coco. Um efeito positivo da adição de peptona foi descrito somente na formação de plântulas de E. randii mantidas sob iluminação contínua. O maior número de plântulas de H. purpurata e folhas de E. randii ocorreu com iluminação contínua. O sucesso na germinação assimbiótica de sementes com três anos de idade, das duas espécies de orquídeas nativas do Brasil, de regiões diferentes, com custo reduzido, é uma técnica oportuna para ser usada em viveiros comerciais, e para a conservação ex-situ destas espécies nativas e exploradas de orquídeas.

In the current study, complex (coconut water and ripe-banana pulp) and specific (micronutrients, peptone and activated charcoal) supplements were added to Knudson-C medium (KC) medium to stimulate the in vitro propagation of Hadrolaelia purpurata (=Laelia purpurata) and Encyclia randii orchid species. The highest number of H. purpurata seedlings occurred in KC medium containing 90 g L-1 banana pulp maintained with 24-hour illumination. Positive effects of the banana pulp addition in KC medium were reported for the formation of seedlings and leaves from E. randii seeds. The seedling number from seeds of E. randii maintained with continuous illumination was also high in KC medium containing coconut water. Positive effects due to the addition of peptone were reported only in the case of seedling formation from E. randii seeds maintained under continuous illumination. The highest number of H. purpurata seedlings and leaves of E. randii occurred in cultures maintained in continuous illumination. The successful asymbiotic germination of the three-year-old seeds of two Brazilian native species from different regions at reduced costs offers an opportunity for the use of the techniques in commercial nurseries and for ex situ conservation of the native and explored orchid species.

Nenhum estudo prévio tem investigado o uso de grafite como substituto do carvão ativado em orquídeas. No presente trabalho, concentrações diferentes de carvão ativado ou de grafite foram adicionadas ao meio KC para escurecer o meio de cultura e estimular a propagação in vitro de Cattleya bicolor e de um duplo híbrido de orquídea ('BLC Pastoral Innocence'). As plântulas foram inoculadas em meio KC sem reguladores de crescimento; os efeitos de concentrações de carvão ativado (0,0; 1,5; 3,0; 4,5; 6,0 and 7,5 g L-1) e de grafite (0,0; 1,5; 3,0; 4,5; 6,0 and 7,5 g L-1) foram testados separadamente. Foram inoculadas quinze plântulas em cada frasco e os experimentos foram conduzidos sob esquema de planejamento fatorial com dois fatores: meios de cultura e luminosidade; o meio de cultura com onze níveis (diferentes concentrações de grafite ou de carvão ativado) e luminosidade com dois níveis (fotoperíodo de 14h e iluminação contínua). Para cada combinação do nível fatorial foram feitas três repetições. Após seis meses de cultivo, foram registrados os números médios de brotos e raízes induzidos em cada concentração de carvão ativado e de grafite. O maior número de brotos nas plântulas de C. bicolor foi observado no meio contendo 6,0 e 7,5 g L-1 de grafite, e o maior número de raízes foi verificado no meio contendo 6,0 g L-1 de carvão ativado. No duplo híbrido o maior número de brotos e raízes foi observado no meio contendo 4,5 g L-1 de carvão ativado. O grafite não pode ser recomendado para substituir o carvão ativado.

No previous studies have investigated the use of graphite instead of activated charcoal in orchids. In this work, different concentrations of activated charcoal or graphite were added to KC medium to darken the culture medium and stimulate the in vitro propagation of Cattleya bicolor and of a double hybrid orchid ('BLC Pastoral Innocence'). The seedlings were inoculated on growth regulator-free KC medium; the effects of activated charcoal (0.0, 1.5, 3.0, 4.5, 6.0, and 7.5 g L-1) and graphite (0.0, 1.5, 3.0, 4.5, 6.0, and 7.5 g L-1) concentrations were tested separately. Each flask contained fifteen seedlings, and the experiments had a randomised, two-way factorial design. The two tested variables were culture medium and illumination levels; eleven culture levels (different concentrations of graphite or activate charcoal) and two illumination levels (14-hr photoperiod or continuous illumination) were evaluated. Three replicates of each combination were utilised. After six months of culture, the average numbers of induced buds and roots per seedling were recorded for each concentration of activated charcoal or graphite. Whereas the largest number of buds in C. bicolor seedlings occurred on media containing 6.0 or 7.5 g L-1 graphite, the largest number of roots occurred on media containing 6.0 g L-1 activated charcoal. In the hybrid 'BLC Pastoral Innocence', the largest number of buds and roots was reported in medium with 4.5 g L-1 activated charcoal. When using graphite in place of activated charcoal, we obtained disparate the results in root formation that suggest that graphite is not a recommended substitute for activated charcoal.