Orientador: Angelo Luiz Cortelazzo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T04:47:50Z (GMT). No. of bitstreams: 1 Leitao_CarlosAndreEspolador_D.pdf: 13217650 bytes, checksum: a26e4baf6ed272da154648cc5de96142 (MD5) Previous issue date: 2007 / Resumo: Rodriguezia venusta é uma orquídea com potencial ornamental nativa do Brasil e do Peru. Pertence à subtribo Oncidiinae que se encontra na subfamília Epidendroideae que. é, de longe, a maior subfamília de Orchidaceae. Apesar desta família ser amplamente estudada quanto à morfologia e taxonomia, são raros os trabalhos de anatomia, principalmente em se tratando de estruturas secretoras, como se levou a cabo no presente trabalho. Amostras de botões florais, brácteas e folhas em expansão foram processadas para a microscopia e analisadas. Na face abaxial da bráctea que subtende cada flor, em especial na região de sua inserção, encontram-se tricomas que secretam material que reage positivamente a testes para detecção de carboidratos e mucilagem. Como, provavelmente, esta secreção atua como lubrificante durante o crescimento do botão floral sobre a bráctea, concluiu se que a denominação "coléteres" é a mais adequada neste caso. Em se tratando das flores, os tecidos estigmático e transmissor são constituídos por tipos celulares semelhantes, que se caracterizam pela presença de células alongadas que se mantêm unidas por suas paredes terminais, formando assim uma estrutura reticulada. Nos espaços intercelulares ocorre copiosa secreção predominantemente mucilaginosa. Na superfície do estigma e uma porção apical do canal delimitado pelo tecido transmissor, ocorre uma cuticula. R. venusta possui também nectários extraflorais nas folhas em expansão e na bráctea que subtende os botões, além de um nectário floral que se insere na base da face abaxial do labelo. Os nectários extraflorais são anatomicamente semelhantes entre si enquanto o nectário floral é estruturalmente mais complexo e revestido por uma epiderme constituída por tricomas nectaríferos / Abstract: Rodriguezia venusta is an orchid with ornamental potential, native to Brazil and Peru. This specie belongs to subtribe Oncidiinae that is in Epidendroideae, the notorioas biggest Orchidaceae's subfamily. Although the wide morphology and taxonomy studies on this family, the works concerning anatomy are rare, specially focusing the secretory structures, in order that is the objective of the present work. Floral buds, bracts and expansion leaves samples were carried out to microscopy analysis. ln the abaxial side of the bract subtending each flower, especially at the portion of they insertion, there are material secreting trichomes that react positive to carbohydrates and mucilage histochemical tests. Due the probable lubricant action of this secretion during the floral bud growth passing aver the bract, it is expected that "colleters" is the most convenient term to designate these structures. ln the flowers, the stigmatic and transmitting tissues have similar cellular types characterized by the elongated shape. The cellular unions only at the terminal walls give a net shape to the structure. ln the intercellular spaces copious secretion occurs constituted predominantly of mucilage. There is a cuticle on the stigma surface and at the canal delimited by transmitting tissue in it apical region. ln addition, R. venusta has extrafloral nectaries in the growing leaves and in the floral bud subtending bract, and also a floral nectary inserted at the lip base. The extrafloral nectaries are anatomically similar, whereas floral nectary is more complex structurally, being covered by nectariferous trichomes / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Made available in DSpace on 2016-02-26T12:59:12Z (GMT). No. of bitstreams: 1 JackelineSchultzSoares.pdf: 419520 bytes, checksum: 681b65601587b028c9aa1b2b88798f13 (MD5) Previous issue date: 2010-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Orchidaceae differs of most botanical families by its seeds did not have enough reservations for germination. The present work aimed: 1. to study the influence of plant growth regulators BAP (6-benzylaminopurine) e GA3 (gibberellic acid), at concentrations of 0.0; 2.0 and 5.0 mg L-1, as pre-germinative treatment in the process of initial development of Dendrobium nobile Lindl seedlings; 2. to determine the volume of coconut water (0; 50; 100; 150 and 200 mL L-1), that, added to culture media, promoted better germination and in vitro cultivation of D. nobile. Seeds of D. nobile that were produced by manual self-pollinating were used as material of study. After six months of sowing and keeping in germination and growth chamber with controlled temperature and photoperiod (12 hours and 23 ºC ±), seedlings were removed from pots and evaluated about number, percentage of germination, fresh mass, height, diameter and number of psedobulbs, number of leaves, number of roots and length of the greatest root. Completely randomized experimental design was used. Every variables and vegetal attributes were submitted to variance analysis and when they were significative, to regression analysis. D. nobile seeds germinated more in the absence of plant growth regulators and treatments with BAP or GA3 for imbibitions of seeds influenced statistically in vitro development of D. nobile. The addition of 200 mL L-1 of coconut water to culture media promoted the best results for plant heights, number or roots, number of pseudobulbs and fresh mass of D. nobile. / Orchidaceae diferencia-se da maioria das famílias botânicas por suas sementes não possuírem reservas suficientes para a germinação. O presente trabalho objetivou: 1- estudar a influência dos reguladores hormonais BAP (6-benzilaminopurina) e GA3 (ácido giberélico), nas concentrações de 0,0; 1,0; 2,0 e 5,0 mg L-1, como tratamento pré-germinativo no processo de germinação e desenvolvimento inicial de plântulas de Dendrobium nobile Lindl; 2- determinar o volume de água de coco (0; 50; 100; 150 e 200 mL L-1 ), que, acrescido ao meio de cultura proporcionou melhor germinação e cultivo in vitro de D. nobile. Foram utilizadas como material de estudo sementes de D. nobile produzidas mediante autopolinização manual Após seis meses do semeio e permanência em câmara de germinação e crescimento com temperatura e fotoperíodo controlados (12 horas e 23 ºC ± 2) as plântulas foram retiradas dos frascos e avaliadas quanto ao número, porcentagem de germinação, massa fresca, altura, diâmetro e número de pseudobulbos, número de folhas, número de raízes e comprimento da maior raiz. Foi utilizado o delineamento experimental inteiramente casualizado. Todas as variáveis e atributos vegetais foram submetidos à análise de variância e quando significativos à regressão. As sementes de D. nobile germinaram mais na ausência de reguladores vegetais, e os tratamentos com BAP ou GA na embebição das sementes influenciaram 3estatisticamente o desenvolvimento in vitro de D. nobile. A adição de 200 mL L-1 de água de coco ao meio de cultura propiciou os melhores resultados para altura de plantas, número de raízes, número de pseudobulbos e massa fresca de D. nobile.

Made available in DSpace on 2016-02-26T12:59:35Z (GMT). No. of bitstreams: 1 JackelineSchultzSoares.pdf: 419520 bytes, checksum: 681b65601587b028c9aa1b2b88798f13 (MD5) Previous issue date: 2010-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Orchidaceae differs of most botanical families by its seeds did not have enough reservations for germination. The present work aimed: 1. to study the influence of plant growth regulators BAP (6-benzylaminopurine) e GA3 (gibberellic acid), at concentrations of 0.0; 2.0 and 5.0 mg L-1, as pre-germinative treatment in the process of initial development of Dendrobium nobile Lindl seedlings; 2. to determine the volume of coconut water (0; 50; 100; 150 and 200 mL L-1), that, added to culture media, promoted better germination and in vitro cultivation of D. nobile. Seeds of D. nobile that were produced by manual self-pollinating were used as material of study. After six months of sowing and keeping in germination and growth chamber with controlled temperature and photoperiod (12 hours and 23 ºC ±), seedlings were removed from pots and evaluated about number, percentage of germination, fresh mass, height, diameter and number of psedobulbs, number of leaves, number of roots and length of the greatest root. Completely randomized experimental design was used. Every variables and vegetal attributes were submitted to variance analysis and when they were significative, to regression analysis. D. nobile seeds germinated more in the absence of plant growth regulators and treatments with BAP or GA3 for imbibitions of seeds influenced statistically in vitro development of D. nobile. The addition of 200 mL L-1 of coconut water to culture media promoted the best results for plant heights, number or roots, number of pseudobulbs and fresh mass of D. nobile. / Orchidaceae diferencia-se da maioria das famílias botânicas por suas sementes não possuírem reservas suficientes para a germinação. O presente trabalho objetivou: 1- estudar a influência dos reguladores hormonais BAP (6-benzilaminopurina) e GA3 (ácido giberélico), nas concentrações de 0,0; 1,0; 2,0 e 5,0 mg L-1, como tratamento pré-germinativo no processo de germinação e desenvolvimento inicial de plântulas de Dendrobium nobile Lindl; 2- determinar o volume de água de coco (0; 50; 100; 150 e 200 mL L-1 ), que, acrescido ao meio de cultura proporcionou melhor germinação e cultivo in vitro de D. nobile. Foram utilizadas como material de estudo sementes de D. nobile produzidas mediante autopolinização manual Após seis meses do semeio e permanência em câmara de germinação e crescimento com temperatura e fotoperíodo controlados (12 horas e 23 ºC ± 2) as plântulas foram retiradas dos frascos e avaliadas quanto ao número, porcentagem de germinação, massa fresca, altura, diâmetro e número de pseudobulbos, número de folhas, número de raízes e comprimento da maior raiz. Foi utilizado o delineamento experimental inteiramente casualizado. Todas as variáveis e atributos vegetais foram submetidos à análise de variância e quando significativos à regressão. As sementes de D. nobile germinaram mais na ausência de reguladores vegetais, e os tratamentos com BAP ou GA na embebição das sementes influenciaram 3estatisticamente o desenvolvimento in vitro de D. nobile. A adição de 200 mL L-1 de água de coco ao meio de cultura propiciou os melhores resultados para altura de plantas, número de raízes, número de pseudobulbos e massa fresca de D. nobile.

O ambiente epifítico é considerado um dos habitats mais desafiadores para as plantas, pois a disponibilidade hídrica e nutricional pode ser bastante escassa. Além disso, as plantas que colonizam este ambiente estão mais expostas aos fatores ambientais, dentre eles a luminosidade se mostra bastante importante, uma vez que esta atua tanto como fonte de energia na fotossíntese quanto como sinal ambiental em repostas fotomorgênicas. Sabe-se, por exemplo, que a luz exerce forte influência sobre a morfogênese radicular de plantas em geral, porém impacta ainda mais o desenvolvimento de orquídeas epífitas, dada a frequente exposição de suas raízes aéreas à incidência luminosa. Tendo em vista que a auxina ácido indolil-3-acético (AIA), o ácido abscísico (ABA) e o etileno são moduladores cruciais no controle da arquitetura radicular na maioria das plantas, sendo também mediadores chave em várias respostas fotomorfogênicas, este estudo propôs-se a investigar o possível envolvimento destes hormônios durante diferentes respostas morfo-fisiológicas desencadeadas pela exposição à luz do sistema radicular de plantas de C. fimbriatum. A ausência de incidência luminosa sobre às raízes resultou em maiores taxas de crescimento e volume radicular, porém, com menor acúmulo de biomassa em relação às raízes expostas à luz. O incremento na biomassa em raízes expostas à luz esteve correlacionado ao espessamento da parede celular na região cortical, o qual ocorreu em resposta especificamente à luz azul. Em termos gerais, a exposição das raízes à luz induziu o aumento nos níveis de AIA e ABA, enquanto que os teores de ACC foram superiores em raízes protegidas da incidência luminosa. Estes resultados sugerem que a luz pode modular o desenvolvimento radicular de C. fimbriatum através de um fino controle hormonal que depende de ajustes coordenados dos níveis de AIA ABA e ACC. Também foi investigado o potencial envolvimento das auxinas e do ABA durante a remobilização de carboidratos entre pseudobulbos e folhas de plantas que tiveram seus sistemas radiculares expostos à (ou protegidos da) luz. Os resultados revelaram que a manutenção das raízes sob condições de escuro levou ao aumento dos teores de AIA e de todas as fontes de carbono estudadas (especialmente de glicose e frutose) nos pseudobulbos, enquanto que as raízes cobertas apresentaram apenas um leve aumento no conteúdo de AIA. O tratamento concomitante das raízes com a condição de escuro e a aplicação de um inibidor do transporte polar de auxina causou uma diminuição abrupta nos teores de AIA em todos os órgãos analisados e a elevação do conteúdo de ABA no sistema radicular. De maneira interessante, essa última condição experimental induziu um conspícuo acúmulo de carboidratos nos pseudobulbos, principalmente de sacarose. Assim, os dados deste trabalho reforçam a importante participação do AIA e ABA como possíveis mediadores da sinalização desencadeada pela luz incidente no sistema radicular de C. fimbriatum, cujas respostas induzidas regulam não somente a morfogênese de tecidos radiculares, mas também influenciam na regulação da partição de carbono no sistema caulinar por meio de um provável mecanismo de sinalização à longa distância / The epiphytic environment is considered one of the most challenging for plants, due to frequent scarcity of water and nutrients. Furthermore, the plants that colonized this biotope are usually more exposed to the environmental cues. Light is considered one of the most important signals controlling plant development because it can act as both an energy source for photosynthesis and an environmental signal for photomorphogenic responses. Besides, light can influence the root morphology of most plants, with even stronger impacts expected in aerial roots of epiphytic orchids due their frequent exposition to direct light. Since indole-3-acetic acid (IAA), abscisic acid (ABA) and ethylene are crucial hormonal signals modulating the root architecture in most plant species, and key mediators during numerous photomorphogenic responses, this study investigated the potential involvement of these hormones in different morpho-physiological responses regulated by either the darkness treatment or the light exposure of Catasetum fimbriatum root system. The absence of light incidence on the roots resulted in higher root volume and growth rate, but lower dry mass accumulation than the light-exposed ones. The higher accumulation of biomass in the light-exposed roots was closed correlated with a more intense cell wall thickening in the root cortex, which appeared to be specifically induced by the blue light. In general, root exposure to light induced increasing levels of ABA and AIA, while the ACC content was higher in roots protected from light. This suggests that light might modulate C. fimbriatum root development through a fine-tuned hormonal mediation, which depends on coordinated adjustments of IAA, ABA and ACC levels. This study also investigated the potential involvement of auxin and ABA during the (re)mobilization of carbohydrates in pseudobulbs and leaves of plants that had their root systems either exposed to (or protected from) light. The results revealed that covering the roots increased in pseudobulbs the levels of AIA and all carbon sources studied (specially glucose and fructose), while the covered roots showed slightly higher levels of AIA. The concomitant treatment with NPA and root covering caused a sharp decrease of AIA levels in all organs and an ABA increase in the root system. Interestingly, this last condition induced a conspicuous carbohydrate accumulation in pseudobulbs, with sucrose as the predominant form. The data obtained in this study reinforce the remarkable participation of IAA and ABA as possible mediators of the signaling cascades triggered by the light incidence on C. fimbriatum root system, which was capable of inducing photomorphogenic responses not only in root tissues, but was also able to influence the carbon portioning in the shoot system by a potential long-distance signaling mechanism

Made available in DSpace on 2016-01-26T18:56:32Z (GMT). No. of bitstreams: 1 Jessica Fontes Fileti.pdf: 657960 bytes, checksum: d27058552310d001b7d5b1306d3396dd (MD5) Previous issue date: 2015-08-04 / Orchid seeds are the smallest and rich in lipids, which turn them in an ideal model to deterioration studies as large populations can be worked in small samples. Controlled deterioration (CD) is a procedure that uses a control in the seed water content during ageing process. The objective of this work was to verify the controlled deterioration effect over orchid seeds. This work was done by the first time with five tropical orchid species: Cattleya brevicaulis, C. tenebrosa, C. amethystoglossa, C. tigrina and Encyclia granitica submitted to three temperatures 41, 43 and 45ºC during the periods of 0, 1, 3, 6, 9, 15, 24 and 48 hours. The variables Total germination, Germination Velocity Index (GVI), Tetrazolium test, MDA content, SOD and PRX activities were evaluated. Results were more expressive after 15 horas of exposition to temperatures of 43 and 45ºC. Total germination, GVI and Tetrazolium test exhibited decreases with the increase in the controlled deterioration. Cattleya tigrina and C. tenebrosa showed expressive increase of MDA content after 48h of controlled deterioration, the other species (C. amethystoglossa, C. brevicaulis and Encyclia granitica) did not. Enzyme activities (SOD and PRX) showed decreases in all temperatures. Total germination, GVI and Tetrazolium test were more efficient to detect variations in the aged seeds. / Sementes de orquídeas são diminutas e constituídas em grande parte por lipídeos, o que as torna um modelo ideal para estudos de deterioração, uma vez que grandes populações podem ser trabalhadas em pequenas amostras. A deterioração controlada é um tratamento que apresenta controle do teor de água das sementes durante o envelhecimento. O objetivo deste trabalho foi avaliar o efeito da deterioração controlada sobre sementes de orquídeas. Este trabalho foi conduzido pela primeira vez em cinco espécies de orquídeas: Cattleya brevicaulis, C. tenebrosa, C. amethystoglossa, C. tigrina e Encyclia granítica submetidas a três temperaturas 41, 43 e 45ºC durante os períodos de 0, 1, 3, 6, 9, 15, 24 e 48 horas. As variáveis de germinação total, índice de velocidade de germinação (IVG), teste de tetrazólio, além dos testes bioquímicos como acúmulo de Malondialdeído (MDA), atividade enzimática de Superóxido Dismutase (SOD) e Peroxidase (PRX), foram avaliadas. Os resultados foram mais expressivos a partir de 15 horas de exposição às temperaturas de 43 e 45ºC. A germinação total, o IVG e o teste de tetrazólio apresentaram decréscimos com o aumento da deterioração controlada para todas as espécies. Cattleya tigrina e C. tenebrosa apresentaram incrementos expressivos de MDA após 48 h de deterioração controlada, ao contrário de C. amethystoglossa, C. brevicaulis e Encyclia granitica. A atividade enzimática das duas enzimas testadas (SOD e PRX) apresentaram decréscimos nas três temperaturas. A germinação total, o IVG e o teste de tetrazólio foram mais eficientes para detectar as variações de viabilidade e vigor nas sementes deterioradas.

Made available in DSpace on 2016-07-18T17:51:13Z (GMT). No. of bitstreams: 1 Jessica Fontes Fileti.pdf: 657960 bytes, checksum: d27058552310d001b7d5b1306d3396dd (MD5) Previous issue date: 2015-08-04 / Orchid seeds are the smallest and rich in lipids, which turn them in an ideal model to deterioration studies as large populations can be worked in small samples. Controlled deterioration (CD) is a procedure that uses a control in the seed water content during ageing process. The objective of this work was to verify the controlled deterioration effect over orchid seeds. This work was done by the first time with five tropical orchid species: Cattleya brevicaulis, C. tenebrosa, C. amethystoglossa, C. tigrina and Encyclia granitica submitted to three temperatures 41, 43 and 45ºC during the periods of 0, 1, 3, 6, 9, 15, 24 and 48 hours. The variables Total germination, Germination Velocity Index (GVI), Tetrazolium test, MDA content, SOD and PRX activities were evaluated. Results were more expressive after 15 horas of exposition to temperatures of 43 and 45ºC. Total germination, GVI and Tetrazolium test exhibited decreases with the increase in the controlled deterioration. Cattleya tigrina and C. tenebrosa showed expressive increase of MDA content after 48h of controlled deterioration, the other species (C. amethystoglossa, C. brevicaulis and Encyclia granitica) did not. Enzyme activities (SOD and PRX) showed decreases in all temperatures. Total germination, GVI and Tetrazolium test were more efficient to detect variations in the aged seeds. / Sementes de orquídeas são diminutas e constituídas em grande parte por lipídeos, o que as torna um modelo ideal para estudos de deterioração, uma vez que grandes populações podem ser trabalhadas em pequenas amostras. A deterioração controlada é um tratamento que apresenta controle do teor de água das sementes durante o envelhecimento. O objetivo deste trabalho foi avaliar o efeito da deterioração controlada sobre sementes de orquídeas. Este trabalho foi conduzido pela primeira vez em cinco espécies de orquídeas: Cattleya brevicaulis, C. tenebrosa, C. amethystoglossa, C. tigrina e Encyclia granítica submetidas a três temperaturas 41, 43 e 45ºC durante os períodos de 0, 1, 3, 6, 9, 15, 24 e 48 horas. As variáveis de germinação total, índice de velocidade de germinação (IVG), teste de tetrazólio, além dos testes bioquímicos como acúmulo de Malondialdeído (MDA), atividade enzimática de Superóxido Dismutase (SOD) e Peroxidase (PRX), foram avaliadas. Os resultados foram mais expressivos a partir de 15 horas de exposição às temperaturas de 43 e 45ºC. A germinação total, o IVG e o teste de tetrazólio apresentaram decréscimos com o aumento da deterioração controlada para todas as espécies. Cattleya tigrina e C. tenebrosa apresentaram incrementos expressivos de MDA após 48 h de deterioração controlada, ao contrário de C. amethystoglossa, C. brevicaulis e Encyclia granitica. A atividade enzimática das duas enzimas testadas (SOD e PRX) apresentaram decréscimos nas três temperaturas. A germinação total, o IVG e o teste de tetrazólio foram mais eficientes para detectar as variações de viabilidade e vigor nas sementes deterioradas.

I investigated how orchid biology, floral morphology, and diversity of surrounding floral and pollinator communities affected reproductive success and hybridization of Cypripedium candidum and C. parviflorum. Floral dimensions, including pollinator exit routes were smallest in C. candidum, largest in C. parviflorum, with hybrids intermediate and overlapping with both. This pattern was mirrored in the number of insect visitors, fruit set, and seed set. Exit route size seemed to restrict potential pollinators to a subset of visiting insects, which is consistent with reports from other rewardless orchids. Overlap among orchid taxa in morphology, pollinators, flowering phenology, and spatial distribution, may affect the frequency and direction of pollen transfer and hybridization. The composition and abundance of co-flowering rewarding plants seems to be important for maintaining pollinators in orchid populations. Comparisons with orchid fruit set indicated that individual co-flowering species may be facilitators or competitors for pollinator attention, affecting orchid reproductive success.

I investigated how orchid biology, floral morphology, and diversity of surrounding floral and pollinator communities affected reproductive success and hybridization of Cypripedium candidum and C. parviflorum. Floral dimensions, including pollinator exit routes were smallest in C. candidum, largest in C. parviflorum, with hybrids intermediate and overlapping with both. This pattern was mirrored in the number of insect visitors, fruit set, and seed set. Exit route size seemed to restrict potential pollinators to a subset of visiting insects, which is consistent with reports from other rewardless orchids. Overlap among orchid taxa in morphology, pollinators, flowering phenology, and spatial distribution, may affect the frequency and direction of pollen transfer and hybridization. The composition and abundance of co-flowering rewarding plants seems to be important for maintaining pollinators in orchid populations. Comparisons with orchid fruit set indicated that individual co-flowering species may be facilitators or competitors for pollinator attention, affecting orchid reproductive success.

Terrestrial orchids make up one of the most threatened groups of plants in Australia and the genus Arachnorchis is listed as the fourth most threatened. The process of propagation and re-introduction of terrestrial orchid plants to the wild has proven difficult, and so far, nearly impossible for some species. This may be partly because terrestrial orchids form complex relationships with mycorrhizal fungi and in genera like Arachnorchis the dependency on the fungus appears acute. Arachnorchis has long been considered by amateur growers of terrestrial orchids as one of the most difficult groups to propagate and maintain in cultivation. This lack of knowledge on how to grow Arachnorchis species hinders attempts made by conservation authorities to supplement threatened wild populations in order to achieve a more sustainable future for those species. Natural pollination was absent, but artificial pollination achieved 100% capsule production. Individuals were self-fertile, although seed viability was greater for cross-pollinated samples. This study attempted to track the fate of as many Arachnorchis species as possible from germination through to deflasking and re-emergence, and so destructive and potentially destructive measurements at earlier stages were avoided. This thesis examines germination and subsequent growth of up to eight species of Arachnorchis, but concentrated on A. phaeoclavia, A. tentaculata, A. fulva, A. robinsonii and A. venusta. Two of these are common species: A. pha eoclavia and A. tentaculata, and three carried a threatened classification of &quotrare" or " endangered": A. fulva, A. robinsonii and A. venusta. This study monitored the fate of individuals of the endangered A. fulva in the field and showed that large reproductive plants re-emerged and flowered each year, whereas smaller individuals might be absent in one or more years and were less likely to flower. Germination of all species concentrated on using symbiotic culture (using mycorrhizal fungi), since germination is known to be more rapid, resulting in healthier, more robust seedlings than when plants are grown asymbiotically. Tests using A. fulva and A. venusta, two threatened species, showed similar viability to A. tentaculata and A. phaeoclavia, more common species. Germination was maximised by examining the viability of seeds before and after treatment with surface-sterilising solutions required for aseptic culture. The highest levels of germination, with limited contamination, were achieved using 0.5% available chlorine for 3 minutes. The most effective fungal isolates (&gt65% germination) were obtained from common species like A. phaeoclavia and A. tentaculata, but there was no correlation between germination and time of year or life stage of the orchid. Collar collection was shown to be non-fatal to robust orchid plants, with large reproductive individuals (at the time of collar collection) re-emerging in the next year and producing a flower bud. Collar collection from small, weedy individuals could be fatal to the plant and isolation of an effective fungus was unlikely. Cross-inoculating seeds with fungi isolated from a different orchid species was not recommended, since the symbiosis failed in all experiments, as late as Stage 4 protocorm development. A range of substrates was used to produce strong seedlings capable of surviving the transfer to nursery conditions with minimal loss. More than 81% of seedlings survived deflasking from non-agar substrates, while the best result from agar was 55%. Some substrates reduced the time involved from seed to plants in the field to as little as 4 months, but aftercare became critical. Sucrose promoted tuberisation, but led to tuber deaths during dormancy. Potting mixes were tested in the nursery and a free-draining loam mix based on a mix used by the Australasian Native Orchid Society was the best medium for deflasking of seedlings. Watering during dormancy should be avoided. The choice of propagule for re-introduction was examined and the best survival to re-emergence was obtained by planting out actively growing seedlings in autumn. Identification of cultures using classical morphology grouped cultures as belonging to the form-genera Epulorhiza and Moniliopsis and suggested that most cultures contained more than one fungus. Identification of the most useful fungal cultures was attempted using molecular techniques such as sequencing the ITS region and mitochondrial DNA. One effective culture, CALAPHAER18 SHTX (cultured from a single monilioid cell) was identified as Serendipita vermifera (Oberwinkler) Roberts. All other cultures tested were mixtures of fungi. The use of specific primers designed to amplify a sequence present in the identified isolate (CALAPHAER18 SHTX) showed that nine mixed cultures also contained a fungus most closely related to Serendipita vermifera. Specific primers also showed that Rhizoctonia solani was not present in any of the 10 isolates from Arachnorchis plants. The molecular work showed that, although the sequenced endophytes from Arachnorchis were all most closely related to Serendipita vermifera, three dist inct groups of fungi were present and these associated with separate species of Arachnorchis. Future work with Arachnorchis species will require the isolation of single fungus cultures and further examination of the development of the orchid plant. In particular, the process of tuberisation and growth in vitro on various non-agar substrates should be investigated further.

Orientador: Marlies Sazima / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T13:13:35Z (GMT). No. of bitstreams: 1 Pansarin_LudmilaMickeliunas_D.pdf: 2285256 bytes, checksum: eb5cb89f4592dded2ffad77b40bf5b7e (MD5) Previous issue date: 2011 / Resumo: O gênero Cirrhaea Lindl. (Orchidaceae) inclui sete espécies distribuídas principalmente pela região Sudeste do Brasil. Neste trabalho foram estudadas a fenologia, a biologia floral e reprodutiva das espécies de Cirrhaea, bem como a morfologia e a anatomia de suas estruturas secretoras. Para isso, observações de campo foram realizadas para investigar os polinizadores e os mecanismos de polinização. Flores frescas foram coletadas e fixadas para os estudos morfo-anatômicos e tratamentos de polinização manual foram feitos para verificar o sistema reprodutivo. Também foi determinada a quantidade de sementes potencialmente viáveis obtidas em cada tratamento e analisadas as fragrâncias. Os estudos de filogenia de Cirrhaea e de evolução dos sistemas de polinização de Stanhopeinae foram realizados a partir do seqüenciamento das regiões trnL-F e matK do DNA de cloroplasto e ITS do DNA nuclear. Os dados obtidos para as espécies de Cirrhaea foram acrescidos aos publicados para os demais gêneros de Stanhopeinae para a elaboração de um estudo sobre a evolução dos sistemas de polinização da subtribo. A filogenia das Stanhopeinae foi comparada com uma hipótese filogenética de abelhas da tribo Euglossini (Apidae) a fim de obter informações sobre a evolução dos sitemas de polinização para a subtribo. Cirrhaea, assim como os demais gêneros de Stanhopeinae, oferece fragrância como recurso floral, que é produzida por osmóforos epidermais localizados nas porções medianas dos labelos, em protuberâncias, como no caso de Cirrhaea dependens e C. nasuta, em depressões e fendas, nas espécies C. fuscolutea e C. longiracemosa e apenas na face interna dos lobos laterais do labelo nas demais espécies. A composição química das fragrâncias é distinta entre as espécies estudadas. Variação na composição da fragrância em populações de uma mesma espécie também pode ocorrer. A fragrância produzida nos osmóforos é fundamental para a atração dos polinizadores, além de ser usada como recurso pelos machos de Euglossini que visitam as flores. Diferentes espécies podem compartilhar um mesmo polinizador ou apresentar polinizadores distintos, refutando a hipótese de que as relações entre orquídeas e euglossines são espécie-específicas. O mecanismo de polinização é do tipo "queda" e semelhante ao previamente descrito para Cirrhaea dependens. As análises filogenéticas e anatômicas indicam que as espécies de Cirrhaea podem ser divididas em três clados: C. dependens e C. nasuta, C. fuscolutea e C. longiracemosa, e C. loddigesii e C. seidelii, demonstando que a morfologia reflete a filogenia do gênero. O grupo irmão de Cirrhaea é Gongora e ambos são polinizados por espécies de Euglossa, Eulaema e Eufriesea. Quando as filogenias de Stanhopeinae e Euglossini são comparadas, verifica-se que as plantas e os polinizadores aparentemente evoluíram de forma independente, uma vez que não há relação correspondente entre os dois grupos e nem especificidade planta-polinizador. / Abstract: The genus Cirrhaea Lindl. (Orchidaceae) comprises seven species distributed mainly in Southeastern Brazil. We have studied the phenology, the floral biology and the reproduction of species of Cirrhaea, as well as their morphology and anatomy of secretory structures. Field observations were conducted to investigate the pollinators and the mechanisms of pollination. Furthermore, fresh flowers were collected and fixed for the studies of morpho-anatomy and hand pollinations were performed in order to verify the reproductive system. The amount of potentially viable seeds obtained in each treatment was also determined, and floral fragrances were analyzed. The phylogenetic hypothesis of Cirrhaea and the study of evolution of pollination systems of Stanhopeinae were developed based on sequencing of the regions trnL-F and matK (cpDNA) and ITS (nrDNA). The data obtained here for Cirrhaea species were added to those previously published for other Stanhopeinae genera in order to prepare the study about the evolution of pollination systems of the subtribe. The study about the phylogeny of Stanhopeinae was compared with a phylogenetic hypothesis of euglossine bees (Apidae) in order to reconstruct the evolution of pollination systems of the subtribe. The genus Cirrhaea, as other Stanhopeinae, produces floral fragrances as resources, which are produced by epidermal osmophores located on the lip midlobe, in protuberances, as in the case of Cirrhaea dependens and C. nasuta, in depressions and slits in C. fuscolutea and C. longiracemosa, and only in the inner surface of lateral lobes in the other species. The chemical composition of the fragrances is distinct among studied species. Variation in fragrance composition among populations of a singular species also occurs. The fragrance produced in the osmophores is fundamental to attract pollinators, besides serving as a resource for Euglossini males visiting the flowers. Different species of Cirrhaea can share the same pollinator or present distinct pollinators, refuting the hypothesis that the relationships between orchids and euglossine are species-specific. The pollination mechanism of all species is similar to that already described for Cirrhaea dependens. The results of the phylogenetic and anatomical studies indicate that species of Cirrhaea can be divided into three clades: C. dependens and C. nasuta, C. fuscolutea and C. longiracemosa, and C. loddigesii and C. seidelii, demonstrating that morphology recovers the phylogeny of the genus. Cirrhaea is sister to Gongora and both genera are pollinated by species of Euglossa, Eulaema and Eufriesea. When the phylogenies of Stanhopeinae and Euglossini are compared, we could conclude that plants and pollinators have evolved independently, since there is neither linear relationship between both groups and nor specificity between plant and pollinator. / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal