Дисертації з теми "VITRO PROPAGATION"

The development of a successful protocol for micropropagating seagrass provides a valuable tool for seagrass-restoration programs and a facility to study their biology (especially their physiology). This work reports on some of the culture requirements of some seagrasses that are commonly found in Western Australia: Posidonia coriacea, P. sinuosa, P. australis and Halophila ovalis. The protocol developed for H. ovalis allows very rapid multiplication and sustainable growth of cultures while the protocol developed for Posidonia requires further development. The culture of Posidonia cariacea proved to be problematic however experimental media that provided insights into its culture conditions. The carbohydrate source was the most important medium component as it affected the development of roots and leaves. The presence of sucrose in the culture media enhanced leaf growth (especially glucose) but decreased the proportion of white roots. More fresh weight, roots, leaves and the proportion of white roots were observed in Posidonia when they were grown in glucose-based media than in mannitol-based media. When mannitol was present in the media, the proportion of white roots was high, which could be attributed to its osmotic effects. Similar responses to sucrose, glucose and mannitol were also observed for P. australis and P. sinuosa. Halophila ovalis was able to grow rapidly on most experimental media. Growth was enhanced by the presence of sucrose in the media and was essential for rapid and sustained growth. Other media components altered the growth of this species, in particular levels of nitrogen (most importantly NH4) influenced root growth and morphology. When H. ovalis is grown in media in moderate or high levels of NH4, root length was significantly reduced and root hair was limited. When NH4 was omitted from the medium, roots were significantly longer and root hairs were prolific. Posidonia coriacea and Halophila ovalis have different growth strategies under natural conditions. H. ovalis is an early succession species that grows rapidly and responds to increased nutrients. P. coriacea is slower growing, colonises later and is Jess responsive to environmental changes than H. ovalis. While the growth responses observed for P. coriacea were significant (in some cases), the differences between means were considerably smaller when compared with H. ovalis. This may be due to the different growth strategies of these species or a lack of fundamental requirement in the conditions under which P. coriacea was grown. Much of what is reported in this thesis for Posidonia will need repeating if the reasons for these differences are identified in the future. In summary, in this thesis I have demonstrated that in vitro propagation of these seagrass species is possible, It is necessary for species-specific protocols to be developed which take into consideration the growth strategies employed by each species. This is particularly significant as many researchers attempt to draw comparisons between species and protocols. The protocols developed in this research increase the knowledge of the biology of these seagrasses and can be incorporated into transplantation protocols in the future.

Field performance of micropropagated (MP) and conventionally propagated (CP) red raspberry (Rubus idaeus L. cv. Comet and Festival) was examined under hedgerow and stool cane management systems for 3 seasons (1989 to 1991). All MP plants established well compared to 58% survival rates 45 days after planting and 92% survival rates after replanting for CP plants. The MP plants were more vigorous compared with the CP plants for the duration of this study as indicated by more and taller canes. MP 'Festival' in 1990 yielded 2.2 MT$ cdot$ha$ sp{-1}$, almost half the yield of established commercial plantings in Quebec, while yields from CP 'Festival' and MP and CP 'Comet' were negligible. The MP 'Festival' crop (8.42 MT$ cdot$ha$ sp{-1}$) also outyielded CP 'Festival' (6.8 MT$ cdot$ha$ sp{-1}$) and both MP (5.72 MT$ cdot$ha$ sp{-1}$) and CP (4.91 MT$ cdot$ha$ sp{-1}$) 'Comet' in the second fruiting year. Propagation method had no effects on winter hardiness, photosynthetic capacity nor leaf and stem morphology of either cultivar. The results indicated that MP plants were superior to CP plants for both nursery propagation and fruit production due to their more consistent establishment and increased vigor. Red raspberry plantlets were successfully hardened in vitro on low-sucrose or sucrose-free media through CO$ sb{2}$ enrichment (1500 ppm) and relative humidity reduction (90%) using a forced ventilation system in specially constructed plexiglass chambers. Enriched CO$ sb{2}$ significantly increased general vigor, root formation, root growth, plantlet growth and plantlet photosynthetic capacities. Sucrose in the medium promoted plantlet growth but depressed photosynthesis. In vitro relative humidity at 90% decreased stomatal apertures and improved plantlet ex vitro performance but did not affect the CO$ sb{2}$ uptake rates of cultured plantlets or ex vitro transplants. The maximum CO$ sb{2}$ uptake rates of plantlet leaves were about 52-69% that of greenhouse control pla

Des protocoles couvrant l'ensemble des étapes de la micropropagation depuis le conditionnement des pieds-mères, et l'introduction in vitro des explants primaires jusqu'à l'enracinement et le sevrage des plantules, ont été mis au point, en attachant une attention toute particulière à l'obtention d'un clonage efficace à long terme à partir de matériel juvénile et adulte. A cette occasion, des variations du mode de croissance des pousses développées in vitro correspondant à une disparition progressive de la croissance rythmique endogène caractéristique de l'espèce, ont été observées. L'étude des conditions permettant leur apparition a montré le rôle primordial de la BA, en présence de BA, l'importance de l'alimentation azotée en tant que facteur de contrôle du modèle de croissance. La réalisation de bilans azotes (n total, n insoluble, n soluble, n minéral, acides aminés et amides libres) au sein des vitroplants, a permis d'associer l'apparition des différents modes de croissance décrits à une accumulation d' n soluble (no#3#, nh#4#+, asparagine, arginine, proline) dans les tiges feuillées, indépendamment des effets induits par l'introduction in vitro elle-même. Ces dosages ont également montré l'importance du cal basal qui assure l'interface entre le milieu de culture et la tige feuillée. Diverses hypothèses permettant d'expliquer le rôle de l'azote dans les mécanismes de contrôle de la croissance rythmique du chêne, ont été proposées

Iris sanguinea is a perennial flowering plant that is typically cultivated through seeds or bulbs. However, due to limitations in conventional propagation, an alternate regeneration system using seeds was developed. The protocol included optimization of sterilization, stratification and scarification methods as iris seeds exhibit physiological dormancy. In addition to chlorine-based disinfection, alkaline or heat treatment was used to break seed dormancy and reduce contamination. When seeds were soaked in water at 80 °C overnight, and sterilized with 75% EtOH for 30 s and 4% NaOCl solution for 20 minutes, contamination was reduced to 10% and a 73.3% germination was achieved. The germinated seedlings with 2-3 leaves and radicle were used as explants to induce adventitious buds. The optimal MS medium with 0.5 mg L−1 6-benzylaminopurine, 0.2 mg L−1 NAA, and 1.0 mg L−1 kinetin resulted in 93.3% shoot induction and a proliferation coefficient of 5.30. Medium with 0.5 mg L−1 NAA achieved 96.4% rooting of the adventitious shoots. The survival rate was more than 90% after 30 days growth in the cultivated matrix. In conclusion, a successful regeneration system for propagation of I. sanguinea was developed using seeds, which could be utilized for large-scale propagation of irises of ecological and horticultural importance.

Thesis (M.S.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed July 2, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 30-34).

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticultural Sciences in the Faculty of Applied Sciences at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY Supervisor: Dr L Kambizi Co-supervisor: Dr NP Makunga Cape Town December 2013
Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a member of the Rutaceae family, and indigenous to the fynbos botanical biome of the Western Cape of South Africa. It is commonly known as buchu. Extracts as well as powdered leaves have traditionally been used for the treatment of various ailments. The increase in the international demand for A. betulina for health as well as food and beverage benefits, have raised concerns over exploitation of wild populations and the lack of horticultural information necessitates this study to evaluate the propagation of this economical important species. The main objective of this study was to establish a simple and highly productive micropropagation protocol for A. betulina through experimenting with nodal explants. Testing of the effect of various treatments (physical scarification, chemical scarification, GA, stratification, smoke and combinations thereof) on the in vitro germination of A. betulina seeds was done to elucidate the factors which control seed germination. The study revealed that the physical scarification and smoke-induced germination had a significant effect on germination percentages. In terms of germination rate, the radical generally started to appear after approximately 10 days in the physical scarification with smoke treatment. Initial decontamination methods with the exposure of various concentrations of NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather than fungal or bacterial contamination. Interestingly, contamination rates of explants were influenced by the stage of maturity of the explant material. This plant material was used to test different strengths of regeneration media, to ensure that the explants receive ample nutrients. Results made exhibited that ½ MS was the best strength for growing A. betulina nodal explants. Compared comparison between in vitro derived explants and ex vitro collected explants showed that the ex vitro derived explants had significant results, but the explants lost vigour soon after the initial exponential growth leading to the explants dying off. Furthermore, ex vitro decontaminated plant material was not economically viable to continue with. Seedlings derived from germinated seeds appeared to be the preferred method of propagation as this spent the least time in culture and produced a stable plant with an established root system, which is essential during the hardening off process after in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was applied to ½ MS media. For both shoot length and multiple shoot production, a combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant results. Interestingly, a higher phytohormone concentration of NAA is necessary to develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it resulted in multiple adventitious roots, but fewer calli was observed in this treatment. Micropropagation becomes valuable as little attention between subcultures is needed; making it less labour intensive compared to conventional nursery propagation systems where weeding watering and spraying of plants are labour intensive. In the traditional world of medicine, more so in Southern Africa, extracts are prepared by adding boiling water to the plant material; however commercial ethanol is used as an extractant. Establishment of the essential oil quality of the in vitro cultures post exposure to various treatments was done. Analysis of essential oils from A. betulina resulted in the identification of twenty one compounds. The results showed qualitative as well as quantitative differences amongst the samples used in the study. The highest relative concentration of limonene was observed in the callus of nodal explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this treatment making it ideal for limonene production. This suggests that liquid culture with the same treatment may produce more calli making it ideal for the production of limonene.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The Symplocos uniflora species is a native tree that reaches up to 10m tall, belonging to the Symplocaceae family. It is popularly known as pau-de-canga, maria-mole-do-banhado and sete-sangria. Virtually, there is no information in the literature on reproduction, growth and development of this species, whose studies of forms of propagation is essential. Thus, the present study intended to examine the sexual and vegetative propagation of the species S. uniflora through protocols for seed germination and micropropagation. Fruit and vegetative material of adult plants from the Botanical Garden of UFSM were collected. The fruits without pulp, containing the seeds were used to evaluate the effect of temperature, storage time, light, collection time, the application of chemical and mechanical scarification, besides the use of gibberellic acid on seed germination of S. uniflora. Plants grown in a greenhouse were used as donor explants (nodal and apical segment) in order to obtain the direct organogenesis. In the study, MS medium and different combinations of auxin naphthaleneacetic acid (NAA) and cytokinin 6-benzylaminopurine (BAP) were used. The presence of light is essential for the germination of Symplocos uniflora, being classified as positive photoblastic preferred. The use of pre-germination treatment with concentrated sulfuric acid is favorable for overcoming seed dormancy. Of the substrates analyzed, the filter paper showed the best germination percentages and speed of germination. Plantmax® substrate offered the highest length of air shoots. In micropropagation, the average percentage of air shoots was 29% with no occurrence of root formation.
Symplocos uniflora (Symplocaceae) é uma árvore que atinge até 10 m de altura, nativa e conhecida popularmente como pau-de-canga, maria-mole-do-banhado e sete-sangria. O trabalho visou estudar a propagação sexuada e vegetativa da espécie, através de protocolos para a germinação de sementes e micropropagação. Endocarpos concrescidos com as sementes (diásporos) foram utilizados para estudos da germinação das sementes avaliando temperatura de armazenamento (10 e 25ºC), temperaturas de incubação (15, 20, 25 e 30ºC), regimes de luz (fotoperíodo de 16 horas e escuro contínuo), época de coleta dos frutos (janeiro e março de 2013), escarificação química (ácido sulfúrico concentrado por 10 minutos), escarificação mecânica (desponte do endocarpo) e ácido giberélico (GA3), 1,5 m L-1. Plantas cultivadas em casa de vegetação, com 18 meses de idade, foram utilizadas como doadoras de explantes (segmento nodal e apical), visando à obtenção da organogênese direta. No estudo foi utilizado o meio MS e diferentes combinações (tratamentos) de ácido naftalenoacético (ANA) e 6-benzilaminopurina (BAP), em mg L-1: 0,0; 0,1; 0,2 e 0,4 e 0,0; 1,0; 2,0 e 4,0, respectivamente. A temperatura de 25ºC promoveu a maior porcentagem de germinação das sementes (24%). Frutos maduros apresentaram as melhores porcentagens de germinação (30%) e índice de velocidade de germinação (1,19). A presença de luz favoreceu à germinação das sementes, sendo classificadas como fotoblásticas positivas preferenciais. O tratamento pré-germinativo com ácido sulfúrico concentrado por 10 minutos promoveu a superação da dormência das sementes. Na germinação in vitro o uso de GA3 no meio de cultura propiciou os melhores índices de velocidade de germinação (0,18), não influenciando na porcentagem da mesma. No cultivo in vitro, a maior porcentagem de explantes com brotações aéreas (29%) ocorreu na combinação de 0,2 mg L-1 de ANA e 2,0 mg L-1 de BAP, sem ocorrência de formação de raízes. Os explantes utilizados e brotações obtidas in vitro apresentaram taxas de oxidação variando de 10 a 70%.

La culture du manioc est l'une des plus importantes en Thaïlande. L'évolution de la demande en amidon, en aliments pour animaux et en biocarburant nécessitera une augmentation annuelle de 4 à 6 millions de tonnes de racines. Une augmentation de la production peut être obtenue par la diffusion de clones sélectionnés. Celle-ci est limitée par la méthode de propagation traditionnelle. La propagation en masse par embryogenèse somatique (ES) est une méthode prometteuse pour accélérer la diffusion des clones chez les fermiers. Ce travail avait pour but de mettre en place trois procédés d' ES (embryogenèse primaire, secondaire et indirecte) pour cinq clones thaïlandais : R5, R7, R9, R72, R90. Le clone R9 a été sélectionné récemment pour la production d'éthanol. Des embryons primaires ont été obtenus pour deux clones (R9 et R90). Des cultures d'ES secondaires (cycliques) ont été établies en milieux solide et liquide pour le clone R9. . . . .
Cassava is one of the most important economic crops in Thailand. The increase demand for starch, animal feed and carburant ethanol will require an addition 4-6 million tons of fresh roots every year. A short term increase in production can be obtained from the dissemination of new clones with higher productivities. However, the diffusion rate of selected clones is limited by the traditional vegetative propagation method of this crop. Mass propagation by somatic embryogenesis (SE) can be a very promising technique to faster their diffusion to the farmers. This work aimed to establish different SE processes (primary, secondary and indirect SE) for five cassava clones selected by the Rayong Field Crops Research Center : R5, R7, R9, R72, R90. R9 has recently been selected for its suitability to produce ethanol. Primary somatic embryos were obtained from foliar explants for two clones (R9 and R90). Secondary SE cultures, also called cyclic, were established on solid and liquid media for the clone R9. . .

Orientadores: Clarice Weis Arns, Fernando Rosado Spilki
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O Metapneumovirus aviário (aMPV) pertence à família Paramyxoviridae, subfamília Pneumovirinae, gênero Metapneumovirus. O vírus, relatado pela primeira vez no Brasil em 1995, é o agente etiológico da Rinotraqueíte em perus (TRT) e está associado também à Síndrome da Cabeça Inchada (SHS) em frangos e poedeiras comerciais. O presente estudo foi dividido em três partes. Na primeira foi avaliada a suscetibilidades de oito sistemas celulares para a propagação de amostras virais do aMPV subtipos A e B. As células chicken embryo related (CER), Vero e baby hamster kidney cells (BHK-21) demonstraram ser as mais apropriadas para a multiplicação de ambos os subtipos. Além disso, cultivo de anel de traquéia (TOC) e cultivo primário de embrião de galinha (CEF) foram permissíveis à infecção por aMPV após terem sido isolados e propagados em CER. As curvas da cinética viral foram realizadas nas três linhagens celulares e ambos os subtipos tiveram títulos mais altos em CER durante as primeiras 30 horas após a infecção. Não foram observadas diferenças significativas entre os títulos obtidos em células CER e Vero, demonstrando que as células CER são tão adequadas à propagação do aMPV quanto as células Vero. A segunda parte do trabalho consistiu em analisar a virulência de uma amostra de aMPV subtipo B após sofrer passagens seriadas em células CER. Cinco variantes provenientes das passagens em CER foram inoculadas em galinhas e a excreção viral foi analisada. Os resultados obtidos com as amostras de traquéia demonstram que a virulência do aMPV diminui gradualmente enquanto o título viral aumenta com o número de passagens. Em contrapartida, nas amostras de seio nasal foi observado aumento da carga viral, demonstrando que não houve diminuição no fitness viral para este órgão. As seqüências do gene G das amostras utilizadas para desafio foram obtidas, porém este gene parece não ser afetado pela propagação em células CER. Na terceira e última parte deste estudo, foi avaliada a proteção viral conferida por uma vacina comercial contra isolados brasileiros do aMPV subtipos A e B em frangos de corte. Para tanto, uma amostra de cada subtipo foi avaliada quanto à sua virulência. O isolado do subtipo B foi detectado em um período mais longo e em maiores quantidades quando comparado ao subtipo A. Os resultados da analise da proteção demonstram que algumas aves imunizadas receberam proteção viral completa contra o vírus virulento heterólogo. Porém, a mesma vacina forneceu proteção viral parcial quando as aves foram desafiadas com o vírus virulento homologo ao vacinal
Abstract: Avian metapneumovirus (aMPV) belongs to the Paramyxoviridae family, Pneumovirinae subfamily, within the genus Metapneumovirus. The virus, first described in Brazil in 1995, is responsible for an acute rhinotracheitis in turkeys (TRT) and is associated with swollen head syndrome in broiler chickens and layer hens. The present study is divided in three parts. In the first part, eight cell culture systems were evaluate for the propagation of aMPV subtypes A and B. The chicken embryo related (CER) cells, Vero and baby hamster kidney cells (BHK-21) cells were shown to be the most appropriate for propagation of both subtypes of aMPV. In addition, propagation of aMPV in chicken embryo fibroblasts (CEF) and tracheal organ culture (TOC) remained efficient after the primary isolation and several passages of viruses in the CER cell line. The growth curves were created using CER, Vero and BHK-21 cell lines. Compared with growth, both yielded higher titres in CER cells during the first 30 hours after infection, but no significant difference was observed in the results obtained from CER and Vero cells. This data show that CER cell are adequate for aMPV propagation, giving similar results to Vero cells. The second part of this study was conducted to analyze the virulence of an aMPV subtype B strain after serial passage in CER cells. To accomplish this, chickens were infected with 5 different variants derived from serial passage and the amount of viral shedding was determined. The results of tracheal samples showed that the virulence decreases gradually with passage, while in vitro viral titre increases. However, an increase in viral shedding was observed in sinusal samples, demonstrating no decrease in fitness for this organ. The G gene sequences of challenge samples were analyzed, however this gene appears to not be affected when aMPV is propagated in CER cells. Finally, the last part of this study aimed to examine a commercially available vaccine in broiler chickens in terms of it ability to provide virological protection against aMPV subtypes A and B. To accomplish this, the virulence of each virulent strain was analyzed. The results demonstrate that the subtype B virulent strain could be observed longer and in larger quantities compared to subtype A. A complete heterologous virological protection was provided by the subtype B vaccine; however, a lack of complete homologous virological protection was observed when chickens were challenged with the homologous subtype B strain
Doutorado
Microbiologia
Doutor em Genetica e Biologia Molecular

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2018.
Drosanthemum micans and Drosanthemum hallii are endangered succulent shrubs of horticultural and medicinal value. They are restricted to the Succulent Karroo, which is one of the world’s biodiversity hotspots. The species risk extinction from illegal over-harvesting for water-wise gardens, erosion by occasional flush floods from ephemeral rivers, competition from alien invasive species, overgrazing and clearing of land for agriculture and human settlement. Although seeds and cuttings may be used in propagating these species, they often require seasonal collection and planting and cuttings struggle to establish, hence the need for in-vitro propagation as an alternative solution. Thus, the main objective of the study was to develop a method for rapid in-vitro shoot and root multiplication and acclimatization of D. micans and D. hallii. To initiate shoot formation, disinfected leaf and stem nodal explants were cultured in Murashige and Skoog (1962) media supplemented with different rates (0, 10, 20 or 30μM) of 2-isopentyladenine, 6-Benzyladenine and kinetin for D. hallii or 2-isopentyladenine, 6-Benzyladenine and Thiadiazuron for D. micans. Shoots from explants were rooted in varying rates (0, 10, 20 or 30μM) of IAA for root initiation. Three media, which were used in previous studies, were tested for acclimatization of rooted explants in i) vermiculite, ii) sand (50%): vermiculite (50%) or iii) sand (75%): perlite (25%). For quantitative evaluation of plant stress, chlorophyll fluorescence index (Fv/Fm) was measured as a proxy for plant stressf stress. It emerged that stem nodal explants of D. hallii tend to produce multiple shoots whilst leaf explants tended to produce callus when cultured in full-strength Murashige and Skoog (1962). Shoot multiplication was optimal in both D. hallii and D. micans at 10 μM of kinetin. Root formation in both D. hallii and D. micans only occurred when shoots were transferred to a full-strength Murashige and Skoog (1962) media without any phytohormones added. The intensity of tissue browning increased at higher levels of cytokinins, suggesting an interaction of plant growth regulators with exudates from explants. Different acclimatization media tested showed no significant differences in the level of stress (Fv/Fm). It is recommended that Murashige and Skoog (1962) media with10 μM kinetin be used for shoot development and multiplication, followed by transfer of the shoots to fresh full-strength Murashige and Skoog (1962) media without hormones for root development. Acclimatization of the rooted explants was possible in one of the following media: i) vermiculite, ii) sand (50%): vermiculite (50%) or iii) sand (75%): perlite (25%) and in a misted greenhouse (ca. 60% RH), with gradual weekly reductions in humidity by 10% over 2 weeks.

La coltura in vitro, applicata alla propagazione e miglioramento genetico della biodiversità vegetale, può rappresentare uno strumento efficace per affrontare i problemi attuali come i cambiamenti climatici, le nuove esigenze dei consumatori e indirettamente lo sviluppo delle aree rurali. Inoltre, può assumere un ruolo strategico nel miglioramento genetico e nella propagazione delle cultivar al fine di ottenere genotipi resistenti, con frutti migliori dal punto di vista organolettico e piante capaci di adattarsi ai cambiamenti climatici. Il miglioramento genetico attraverso i metodi convenzionale è limitato da molti fattori infatti, gli alberi da frutto sono caratterizzati da un lungo periodo di giovanilità, elevata eterozigosi e molto spesso incompatibilità sessuale. La tecniche in vitro della coltura di antere e/o microspore isolate, per ottenere piante aploidi o doppi aploidi, riescono a superare alcuni limiti del miglioramento genetico tradizionale e fornire in un solo passaggio linee completamente omozigoti interessanti per studi di genomica, mutazioni, mappatura e trasformazione genetica. La tecnologia in vitro oltre ad essere applicata al miglioramento genetico, risulta un metodo complementare ai metodi convenzionali di propagazione agamica, in quanto, il materiale vegetale risulta protetto da eventuali attacchi di patogeni, il processo è indipendente dalle condizioni stagionali, permette uniformità genetica ed elevati tassi di moltiplicazione in uno spazio ridotto. La coltura in vitro è resa possibile dal fenomeno della totipotenza; cioè la capacità che hanno le cellule, organi o tessuti di de-differenziarsi e acquisire capacità meristematiche dando origine ad organi, tessuti o a un individuo intero, anche diverso da quello di partenza. Questa ricerca ha avuto come obiettivo l’induzione dell’embrio enesi ametica in vari genotipi di agrumi, tramite coltura in vitro di antere. Inoltre, si è studiata la messa a punto di un efficiente protocollo di conservazione e propagazione, attraverso la tecnologia del seme sintetico, rispettivamente in Fico, Luppolo e Cappero. Gli esperimenti sono stati condotti nel 2017, 2018 e 2019 presso l’Universit de li Studi di Palermo (UNIPA) e l’Instituto Valenciano de Investi aciones A rarias (IVIA). Per quanto riguarda la coltura di antere di agrumi è stata ottenuta embriogenesi somatica nella varietà Moro (2x), portando alla rigenerazione di embrioni che dopo le analisi della ploidia e molecolari sono risultati tetraploidi eterozigoti; nei genotipi Marisol (4x), Clemenules (4x), Moro Los Valles (4x), Sanguinelli (4x) e Sanguinelli (2), è stata osservata divisione simmetrica del nucleo, strutture multinucleate e la formazione di embrioni che in se uito all’analisi citofluorimetrica sono risultati per la maggior parte diploidi. L’analisi molecolare, effettuata con i marcatori SSR invece, ha rilevato risultati singolari, infatti, nei genotipi Marisol, Clemenules e Sanguinelli (2x e 4x), anche se sono state osservate microspore multinucleate e la maggior parte dei rigenerati erano diploidi, dall’analisi sono risultati tutti eterozi oti, mentre in Moro Los Valles, imarcatori hanno rilevato che dei diploidi rigenerati tre erano omozigoti, due omozigoti mutati (variabilità gametoclonale) e due eterozigoti mutati. La tecnologia del seme sintetico è stata applicata alla varietà Houmairi di Ficus carica L., in particolare, è stato valutato l’effetto di due Regolatori di crescita (PGR): 6-benzilaminopurine (BAP) e meta-Topolina (mT), aggiunti all’endosperma artificiale, e della conservazione a 4° C per 30 iorni delle microtalee incapsulate. La più alta percentuale di semi vitali e che hanno portato ad una maggiore ripresa vegetativa si è avuta nella tesi che non ha previsto conservazione a freddo prima della semina e quando l’endosperma artificiale veniva addizionato con il BAP. In maniera diversa, la percentuale maggiore di conversione si è avuta, sia per i semi conservati che non quando l’endosperma veniva addizionato con mT. La propagazione in vitro, è stata applicata anche, alla varietà Cascade di Humulus lupulus L. con l’obiettivo di realizzare un protocollo efficiente di micropropagazione. Partendo dal prelievo in campo del materiale vegetale, si è proceduto alla sterilizzazione, stabilizzazione della coltura asettica nonché alla moltiplicazione, radicazione e acclimatazione alle condizioni ex vitro. Per la moltiplicazione sono stati testati 3 PGR: BAP, mT e Thidiazuron (TDZ) in diverse combinazioni, mentre per la radicazioni 2 PGR auxinici: Acido indol-3-acetico (IAA) e Acido indol-3-butirrico (IBA). L’interazione TDZ/BAP ha mostrato il più alto tasso di germogliamento anche se con un maggiore sviluppo di callo tale da considerare il mezzo con 2mg Lˉ¹ di TDZ il migliore nonostante dia un germogliamento inferiore così come per la presenza di callo. Per quanto riguarda la maggiore percentuale di radicazione si è avuta nel mezzo contenente 2 mg Lˉ¹ di IBA anche se differiva per pochi punti percentuali dal mezzo che conteneva 1 mg Lˉ¹ di IAA. Infine, è stata valutata l’influenza del rivestimento di alginato di calcio e di tre diversi regolatori di crescita (PGR): 6-benzilaminopurina (BAP), meta-topolina (mT) e zeatina (ZEA), sulla vitalità, la ricrescita e la conversione dei propaguli. di due genotipi siciliani di Capparis spinosa (L.). Le microtalee di cappero sono state sezionate e collocate in diversi endospermi artificiali a base di sali e vitamine Murashige e Skoog, arricchiti di mT o ZEA o BAP. I semi sintetici ottenuti sono stati seminati su un mezzo MS addizionato con 0,4 mg / Lˉ¹ di acido acetico naftalenico e 0,7 mg / Lˉ¹ di acido gibellerico. Dopo 60 giorni, sono stati rilevati i seguenti parametri: vitalità, ricrescita, numero e lunghezza dei germogli e delle radici, conversione. I risultati confermano che l'incapsulamento non ha influito negativamente sulla vitalità, che ha mostrato la percentuale più alta con BAP in Tracino e con ZEA in Scauri. Risultati simili sono stati ottenuti nella ricrescita, con differenze statisticamente significative tra i tre PGR testati: Tracino ha mostrato la migliore ricrescita in capsule arricchite con BAP, Scauri con ZEA. Inoltre, la conversione dei semi sintetici è stata fortemente influenzata dalla PGR ed era più elevata nell'endosperma artificiale addizionato con BAP in Tracino e ZEA in Scauri.
In vitro culture, applied to the propagation and genetic improvement of plant biodiversity, can be an effective tool to face current problems such as climate change, and new consumer needs. Moreover, it can take on a strategic role in genetic improvement and propagation of cultivars in order to obtain genotypes resistant to biotic and abiotic stresses, with fruits improved from an organoleptic point of view and plants able to adapt to climate change. Genetic improvement through conventional methods is limited by many factors. Fruit trees are characterized by a long period of juvenitlity, high heterozygosity and, very often, by sexual incompatibility. The in vitro techniques of the cultivation of isolated anthers and/or microspores, to obtain haploid or double haploid plants, are able to overcome some limits of traditional genetic improvement and provide in a single step completely homozygous lines of interest for genomic studies, mutations, mapping and genetic transformation. In addition, in vitro technology is a complementary method or can even support conventional methods of agamic propagation, since the plant material is protected from possible attacks by pathogens, their production is independent of seasonal conditions, this tecquique allows genetic uniformity and high multiplication rates, in a small space. In vitro culture is made possible by the phenomenon of totipotency, that is, the ability of cells, organs or tissues to de-differentiate and acquire meristematic skills, giving rise to organs, tissues or to an entire individual, even different from the original one. This research had as its objective the induction of gametic embryogenesis in various citrus genotypes through in vitro culture of anthers, and the development of an efficient protocol for Fig, Hop and Caper through synthetic seed technology and micropropagation, respectively. The experiments were conducted in 2017, 2018 and 2019 at the University of Palermo (UNIPA) and the Valencian Institute of Agricultural Research (IVIA). As far as the cultivation of citrus anthers is concerned, somatic embryogenesis was obtained in the Moro variety (2x), leading to the regeneration of embryos which after ploidy and molecular analyzes were heterozygous tetraploids; and Marisol (4x), Clemenules (4x), Moro Los Valles (4x), Sanguinelli (4x) and Sanguinelli (2) genotypes, symmetric division of the nucleus, multinucleated structures and formation of embryos that following the analysis was observed cytofluorimetric results for the most part were diploid, molecular analysis, carried out with SSR markers instead, found singular results, in fact, in the Marisol, Clemenules and Sanguinelli genotypes (2x and 4x), even if multinucleated microspores and the most regenerated were diploid, from the analysis they were all heterozygous, while in Moro Los Valles, the markers found that three regenerated diploids were homozygous, two mutated homozygotes (gametoclonal variability) and two mutated heterozygotes.In this research, synthetic seed technology has been applied to the Houmairi variety of Ficus carica L., in particular, the effect of two “Plant rowth Re ulators” (P R): 6-benzylaminopurine (BAP) and meta-Topolina (mT), added to the artificial endosperm, and of the preservation at 4 ° C for 30 days of the encapsulated microcuttings. The highest percentage of viable seeds that led to a greater vegetative recovery occurred in the thesis that it did not foresee cold storage before sowing and when the artificial endosperm was added with BAP. In a different way, the highest percentage of conversion occurred, both for the stored seeds and not when the endosperm was added with mT. In vitro propagation was applied to the Cascade, variety of Humulus lupulus L. with the aim of achieving an efficient micropropagation protocol. Starting from the collection in the field of the plant material, sterilization, stabilization of the aseptic culture as well as the multiplication, rooting and acclimatization to ex vitro conditions were carried out. For the multiplication 3 PGR were tested: BAP, mT and Thidiazuron (TDZ) in different combinations, while for the rooting 2 PGR auxinic: indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). The TDZ / BAP interaction showed the highest germination rate even though with a greater callus development such as to consider the medium with 2 mg lˉ¹ of TDZ the best despite giving a lower budding as well as for the presence of callus. As regards the greater percentage of rooting, it occurred in the medium containing 2mg lˉ¹ of IBA without significant difference from the medium that contained 1 mg L of IAA. Finally, the influence of the calcium alginate coating the cuttings along with the three different growth regulators (PGR) were evaluated: 6-benzylaminopurine (BAP), meta-topolina (mT) and zeatin (ZEA), on vitality, regrowth and the conversion of the propagules of two Sicilian genotypes of Capparis spinosa (L.) (Tracino and Scauri, from Pantelleria Island). Caper microcutting have been sectioned and placed in different artificial endosperms based on Murashige and Skoog, enriched with mT or ZEA or BAP. The synthetic seeds obtained were seeded on an MS medium added with 0.4 mg / L of naphthalene acetic acid and 0.7 mg / L of gibelleric acid. After 60 days, the following parameters were detected: vitality, regrowth, number and length of shoots and roots, conversion. The results confirm that encapsulation did not negatively affect vitality, which showed the highest percentage with BAP in Tracino and with ZEA in Scauri. Similar results were obtained in regrowth, with statistically significant differences between the three PGRs tested: Tracino showed the best regrowth in capsules enriched with BAP, Scauri with ZEA. Furthermore, synseed conversion was strongly influenced by PGR and was higher in artificial endosperm added with BAP in Tracino and ZEA in Scauri genotype.

Tissue-cultured shoots and plantlets usually have leaves with non-functional, open stomata and little epicuticular and cuticular wax, resulting in excess evapotranspiration after transplantation. Various strategies were evaluated to decrease ex vitro acclimatization difficulties for 'Silvan' blackberry, including transplanting unrooted shoots, increasing the medium agar concentration from 6 to 9 or 12 g/l and diluting the basal medium. Increased medium agar concentrations and medium dilution did not improve survival or growth. Stomatal function resumed sooner in new leaves of plantlets than shoots. High relative humidity ($>$95%) and low light intensity (90 $ mu$mol s$ sp{-1}$ m$ sp{-2}$) negatively affected stomatal closure both on acclimatizing transplants and greenhouse-grown plants. Guard cells developed on leaves in vitro were physiologically active but had apparent anatomical abnormalities that inhibited closure. A rapid clearing and staining method was developed for examination of foliar morphology using intact in vitro blackberry (Rubus sp. 'Silvan') and strawberry (Fragaria x ananassa Duch. 'Totem') plantlets and sections of greenhouse-grown 'Silvan' and 'Totem' leaves. This method involved three steps: (1) removing the chlorophyll by autoclaving in 80% ethanol; (2) dissolution of the protoplasm using 5% NaOH at 80$ sp circ$C; (3) post-alkali treatment with 75% bleach (4.5% NaClO) at room temperature for tissue-cultured plantlets and at 55$ sp circ$C for greenhouse-grown leaves. Aqueous safranin (10 mg/l) was used for staining.

A família Orchidaceae apresenta imensa diversidade taxonômica, bioquímica, fisiológica e genética. Por essa razão não existe um modelo único que possibilite o sucesso na propagação in vitro para todas as suas espécies e variedades. Assim, esse trabalho teve como objetivo estudar aspectos envolvidos com a propagação, o desenvolvimento inicial in vitro e a aclimatização de Brassavola martiana Lindl., espécie ocorrente no estado do Tocantins. Testou-se os efeitos dos meios de cultura de Knudson C [KC], de Vacin e Went [VW] e de Murashige e Skoog nas concentrações de 100 e 50% de seus macronutrientes [MS e ½MS] na germinação e desenvolvimento inicial in vitro. Analisou-se também a influência da idade das sementes no processo germinativo. Foram avaliados os efeitos de diferentes concentrações de sacarose (0%, 1%, 2%, 3%, 4%, 5% e 6%) bem como do ácido naftalenoacético (ANA) e da benziladenina (BA) em combinações de 0; 0,57; e 2,28 μM na multiplicação e desenvolvimento de plantas, em experimentos separados. Verificou-se também a possibilidade de se multiplicar a espécie por meio de segmentos caulinares estiolados. Por fim, foi desenvolvido um protocolo para aclimatização de B. martiana utilizando-se substratos comerciais. O meio ½MS foi o mais promissor para a germinação de B. martiana, enquanto que o KC foi o melhor para o desenvolvimento dos protocormos e formação de novas plantas. Para a obtenção de uma germinabilidade superior a 60% recomenda-se utilizar sementes com pelo menos 7 meses de idade. A adição de 3% de sacarose ao meio de cultura favoreceu a multiplicação e desenvolvimento in vitro. O uso de concentrações iguais de ANA e BA (0,57 μM) favoreceu o desenvolvimento tanto da parte aérea quanto das raízes. A utilização de segmentos caulinares estiolados foi eficaz para a propagação de B. martiana. Para a aclimatização, as plantas de B. martiana devem inicialmente serem transferidas para recipientes comunitários contendo o substrato Bioplant e após oito semanas transplantadas para vasos individuais contendo o substrato Ouro Negro e cultivados durante 90 dias. A porcentagem de sobrevivência das plantas foi considerada satisfatória.
The Orchidaceae family presents immense taxonomic, biochemical, physiological and genetic diversity. For this reason, there is no single model that allows the success of in vitro propagation for all species and varieties. Thus, the objective of this work was to study aspects involved in the propagation, initial in vitro development and acclimatization of Brassavola martiana Lindl., A species occurring in the state of Tocantins. The effects of the cultivation media of Knudson C [KC], Vacin and Went [VW] and Murashige and Skoog at the concentrations of 100 and 50% of their macronutrients [MS and ½MS] on initial in vitro germination and development. The influence of seed age on the germination process was also analyzed. The effects of different concentrations of sucrose (0%, 1%, 2%, 3%, 4%, 5% and 6%) as well as naphthaleneacetic acid (ANA) and benzyladenine (BA) were evaluated in combinations of 0; 0.57; and 2.28 μM in plant multiplication and development, in separate experiments. It was also verified the possibility of multiplying the species by means of stretched cauline segments. Finally, a protocol for the acclimatization of B. martiana was developed using commercial substrates. The ½MS medium was the most promising for the germination of B. martiana, while the KC was the best for the development of protocormos and formation of new plants. To obtain germinability higher than 60%, it is recommended to use seeds at least 7 months old. The addition of 3% sucrose to the culture medium favored multiplication and development in vitro. The use of equal concentrations of ANA and BA (0.57 μM) favored the development of both shoot and roots. The use of styrofoam segments was effective for the propagation of B. martiana. For acclimatization, B. martiana plants should initially be transferred to community vessels containing the Bioplant substrate and after eight weeks transplanted into individual pots containing the Ouro Negro substrate and cultured for 90 days. The percentage of plant survival was considered satisfactory.

Athrixia phylicoides (bush tea) is one of many plants from the Asteraceae family used as a traditional herbal medicine. With very few cultivated plants, natural growing plants currently serve as the main resource for plant material. The plant is not yet commercialised and its medicinal value is known and used only by a few people. With the long term aim at commercial scale propagation, this study consists of three parts. Firstly we developed a protocol for in vitro propagation of A. phylicoides. Secondly, the ultrastructure and morphology of leaves were studied microscopically and thirdly, comparisons were made between in vitro and ex vitro grown plants. Nodal segments of greenhouse plants were used to establish cultures. Better growth and less wilting was recorded on explants surface sterilised with NaOCl compared to Ca(OCl)2 after establishment. The addition of growth regulators IBA (indole-3-butyric acid) and BAP (6-benzylaminopurine) to the culture medium did not seem to affect the growth response of explants during the multiplication phase. Hyperhydricity was a problem throughout our trials. The development of hyperhydricity symptoms seems to be related to seasonal changes in the stock plant material used to initiate cultures, rather than the composition of growth medium or growth room temperatures. The occurrences of hyperhydricity symptoms were inconsistent and unpredictable. A rooting medium with added BAP and decreased sucrose levels resulted in a higher rooting percentage compared to the control medium, free from BAP and with a higher sucrose concentration, which yielded no rooting. However, in another experiment, in vitro rooting occurred spontaneously after subdividing and transfer of microshoots to fresh control medium. The addition of GA3 to the establishment medium (but not to the subsequently used multiplication and rooting media) yielded a slightly higher percentage of rooting. However, cultures initially established on GA3 medium yielded fewer roots per explant and roots were shorter than those of explants established on hormone free medium. The medicinal properties of plants are often linked to the production of essential oils. We hypothesised that the medicinal value of A. phylicoides can be linked to the production of the aromatic essential oils released by leaves. A microscopic study of leaves provided some preliminary insight of the mechanisms involved in the production of medicinally active products. Electron- and light microscopic examination of leaves were used to identify and study structures that are apparently involved in the production and secretion of essential oils. Two types of trichomes were identifyed – nonglandular and glandular trichomes. These glandular trichomes are multicellular with a subcuticular storage space and are present only on the adaxial surface of leaves. In the case of medicinal plants, it is essential that the medicinal properties of the plant are not altered by the method of propagation. This was our motivation for comparing the morphology and ultrastructure of leaves of plants that were grown in their natural environment to that of plants grown in vitro. Leaf surfaces of in vitro grown plants were smaller and the number of glandular trichomes per surface area was less on in vitro grown plants. There were no noticeable changes in the morphology of glandular trichomes.
Dissertation (MSc(Agric))--University of Pretoria, 2009.
Plant Production and Soil Science
unrestricted

Introduzione: la patologia di Alzheimer (AD) è la causa più comune di demenza ed è caratterizzata da depositi extracellulari di -amiloide e da aggregati intracellulari chiamati grovigli neurofibrillari (NFTs) formati dalla proteina tau iperfosforilata. Poiché è stato dimostrato che il declino cognitivo dei pazienti di AD correla con la diffusione degli aggregati di tau più che con la presenza di -amiloide, è fondamentale capire i meccanismi molecolari che causano la propagazione di tau. Studi hanno dimostrato come la diffusione temporale e spaziale degli NFTs sia costante nei vari pazienti e che sia dovuta alla propagazione attraverso il network assonale tra neuroni connessi a livello sinaptico. Ad oggi infatti, alcuni degli “early phase Clinical Trials” e degli studi si stanno concentrando sulla propagazione di tau al fine di poter prevenire la sua internalizzazione da parte delle cellule riceventi, ancora sane, sia con uso di molecole che con anticorpi. Ci sono stati diversi studi per riprodurre in vitro un modello di network neuronale al fine di valutare in maniera appropriata delle terapie mirate prima che esse venissero testate in vivo, e la maggior parte di questi si basano sull’uso di microfluidica. Tuttavia, in questi modelli spesso sono stati utilizzati degli approcci poco fisiologici come l’over-espressione o la mutazione di tau o l’utilizzo di tags fluorescenti che possono compromettere l’affidabilità di questi sistemi. Per questo motivo è necessario lo sviluppo di una nuova metodologia che possa migliorare questo approccio e che potrà in fine essere utilizzata per un efficiente ed affidabile screening di nuove terapie per la malattia di Alzheimer. Scopo del lavoro: il principale obiettivo di questa ricerca è stato quello di stabilire una nuova piattaforma neuronale umanizzata di microfluidica, che potesse modellare e che permettesse lo studio, dell’aggregazione e propagazione di tau in modo sia qualitativo che quantitativo. I dispositivi di microfluidica rappresentano un’alternativa miniaturizzata per ricapitolare la propagazione di aggregati proteici come quelli di tau; infatti, essi permettono la coltura di popolazioni neuronali sinapticamente connesse ma fisicamente isolate che possono essere indotte allo sviluppo di aggregati e, conseguentemente, alla loro propagazione. Questo modello potrebbe essere ideale per testare gli effetti di potenziali terapie contro tau che vadano a contrastare la propagazione trans neuronale. Materiali & Metodi: al fine di sviluppare un modello di propagazione nei dispositivi di microfluidica, è stato in primo luogo validato un protocollo per stimolare l’aggregazione di tau endogena in neuroni corticali di ratto (RCN) utilizzando come induttore del materiale derivato da cervelli umani di pazienti AD (hAD seed). Successivamente, usando le stesse condizioni, sono stati sviluppati diversi protocolli di microfluidica per RCN che hanno mostrato non solo aggregazione ma anche propagazione di tau utilizzando tecniche quali High Content Imaging (HCI) ed un software di immagine che è stato sviluppato internamente. Infine, è stato sviluppato una versione umanizzata e miniaturizzata di questo protocollo al fine di avere una piattaforma fisiologicamente rilevante per poter testare terapie contro tau umana. Risultati: in questo progetto è stato sviluppato un modello cellulare umanizzato che permette lo studio della propagazione endogena di tau, da neurone a neurone, usando dispositivi di microfluidica. Dopo una prima fase di ottimizzazione, è stato dimostrato che utilizzando hAD seed è possibile indurre in neuroni corticali piastrati in dispositivi di microfluidica, l’aggregazione della proteina tau endogena e la sua propagazione in maniera trans-neuronale in modo quantificabile. Inoltre, questo modello è stato validato statisticamente ed è stato successivamente convertito in uno miniaturizzato per aumentare il “throughput” della piattaforma passando così da 6 a 16 unità di microfluidica contenute in una piastra. Infine, questo metodo è stato umanizzato al fine di studiare l’aggregazione e la propagazione della forma umana di tau usando una cultura primaria di neuroni murini che esprimono solo la proteina tau umana. E’ stato dimostrato che Anle 138b, una molecola conosciuta in letteratura per le sue capacità antiaggreganti, può bloccare l’aggregazione e il trasferimento trans-neuronale di tau, suggerendo che questo nuovo sistema possa essere usato per valutare i meccanismi di propagazione di tau e trovare nuove terapie. Conclusioni: In questo lavoro è stato sviluppato con successo un metodo di microfluidica quantitativo e riproducibile che può modellare le taupatie sporadiche umane come la malattia di Alzheimer. Usando sia RCN che neuroni corticali hTau trattati ed indotti con hAD seed, è stata osservata e quantificata la formazione e la propagazione di aggregati endogeni di tau ed è stato testato l’effetto di una molecola inibitoria su questi due meccanismi. È stato inoltre dimostrato come questi modelli possano essere anche utilizzati per condurre studi esplorativi come quelli volti al monitoraggio dell’attività e della connettività neuronale e gli studi sul ruolo dei diversi tipi cellulari presenti in cultura, nell’aggregazione e propagazione di tau. Soprattutto, è stato dimostrato come questo modello possa essere utilizzato come piattaforma di screening per testare potenziali inibitori della propagazione di tau murina ed umana.
Background: Alzheimer’s disease (AD) is the most common cause of dementia, characterized by the presence of extracellular -amyloid plaques and intracellular neurofibrillary tangles (NFTs) composed of aggregated and hyperphosphorylated tau. Since it has been shown that tau aggregates correlate with cognitive decline much better than -amyloid formations, it is important to understand how tau can spread in the brain. Moreover, the spatiotemporal spread of tau observed during clinical manifestation suggests that it propagates along the axonal network between synaptically connected neurons. On these bases, some early-phase Clinical Trials are aiming to target tau during transcellular spreading in order to prevent its internalization by recipient neurons, using both compounds and antibodies. For the experimental evaluation of candidates before in vivo studies, there have been many attempts to replicate this network in vitro, most of which used microfluidic approaches; however, these experiments have often utilized parameters that may reduce the physiological relevance of the assay, such as by overexpressing tau, using fluorescent tags, or by introducing MAPT mutations. New methods that can improve these assays are required to help the screening of efficient and effective treatments. Aim of the work: The main purpose of this research project has been to establish a humanized, in vitro neuronal microfluidic platform to recapitulate and study tau aggregation and propagation in a qualitative and quantitative way. Microfluidic devices represent a miniaturized alternative tool to recapitulate tau spreading conditions, by enabling the culture of synaptically connected, but environmentally isolated, neuronal populations that can be seeded, thereby inducing endogenous tau aggregation and subsequent propagation. This model system could be ideal for testing the effect of potential tau therapeutics that modulate transneuronal tau propagation. Material & Methods: Before developing the microfluidic propagation assay, a rat cortical neuron (RCN) aggregation assay that uses seeding-competent material from human AD brains (hAD seed) to induce endogenous aggregation was validated. Subsequently, the same conditions were used to develop a RCN microfluidic assay that can show endogenous tau aggregation, and consequent propagation, using High Content Imaging (HCI) and a proprietary interactive computer program for image quantification. Finally, using a transgenic mouse line that expresses human MAPT, a humanized and miniaturized version of the assay has been developed in order to have a physiologically relevant, medium-throughput platform to test tau therapies. Results: After a phase of optimization, it has been shown that hAD seed induces endogenous rodent tau aggregation and transneuronal propagation in a quantifiable manner in a microfluidic culture model. Moreover, this assay was statistically validated and further converted to a medium-throughput format allowing the user to handle 16 two-chamber devices simultaneously in the footprint of a standard 96 well plate. Furthermore, this assay was humanized in order to study hTau aggregation and propagation using primary neurons from a mouse model that expresses human tau only. It has been proved that Anle 138b, a literature small molecule that has been previously shown to impair protein aggregation, can block the transneuronal transfer of tau aggregates, suggesting that this novel system can be used to evaluate mechanisms of tau spreading and to find therapeutic interventions. Moreover, preliminary experiments have shown that the aggregation of endogenous tau induces not only an increase of neuronal excitability but also an activation of astrocytes which might also have a role in tau pathology. Conclusions: This work has been successfully developed a robust and quantitative microfluidic assay that can model an isolated mechanism of tau propagation. Using both RCNs and hTau mouse cortical neurons seeded with hAD seed, it was possible to quantify the formation and propagation of endogenous tau inclusions and demonstrate that a putative inhibitor of tau propagation is active in this assay. It was also shown that these models can be further employed for exploratory studies, such as monitoring the functional activity and connectivity of the neuronal cultures, as well as investigating the role of different cell types in tau aggregation and propagation. Most importantly, this manuscript exhibits the latent potential of microfluidic assays as screening platforms for the preclinical evaluation of tau propagation inhibitors.

As técnicas de cultura in vitro têm contribuído para a optimização de protocolos de propagação do género Juglans. Neste trabalho foi testada a técnica de microenxertia de J. regia em híbrido ‘Paradox’. Foi efectuado o enraizamento ex vitro em simultâneo com a enxertia. Foi avaliada a influência da presença de folhas no enxerto e no porta-enxerto na taxa de sucesso da técnica, assim como o despiste de Cherry leaf roll virus. Verificou-se que a presença de folhas foi um requisito obrigatório para o sucesso da enxertia e do enraizamento. A enxertia apresentou uma taxa média de sucesso de 84,6 % e a taxa de aclimatização aos 60 dias foi 86,4 %. Detectaram-se amostras suspeitas de serem positivas para a presença de Cherry leaf roll virus. A técnica de microenxertia apresentada mostrou-se viável tecnicamente e eventualmente com condições de competir com as técnicas tradicionais de enxertia em viveiro; ABSTRACT: Trials about walnut (Juglans regia L.) micrografting. In vitro culture techniques have contributed to the optimization of propagation protocols of the genus Juglans. Here the micrografting technique was tested with J. regia grafted in a 'Paradox' hybrid. Rooting ex vitro was performed simultaneously with grafting. The influence of the presence of leaves on the graft and rootstock on the success rate of the technique was evaluated, as well as the screening of Cherry leaf roll virus. It was found that the presence of leaves, in the graft or in the rootstock, or in both, was a mandatory requirement for the grafting success. The grafting success presented a final global average of 84.6% and the acclimatization rate at 60 days was 86.4%. Samples putatively positive for the presence of Cherry leaf roll virus were detected. The micrografting technique here presented seems to be technically feasible and maybe able to compete with the traditional techniques of nursery grafting.

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In this work, the propagation in vitro of Anthurium andraeanum cv. Eidibel through somatic embryogenesis was investigated, evaluating the induction of the embryogenic cultures (Experiment I); pre-maturation of the somatic embryos (Experiment II); and maturation of the somatic embryos with subsequent regeneration of the plants (Experiment III). In Experiment I, the subdivision of plants of anturio established in vitro was used, to obtain the explant sources. Analyzed in DIC with factorial 5 x 5 x 5, with five explant types (whole leaves were cut across the midrib of leaves; petioles; stem segments with one bud; and root segment without the apex radicular; each explant was cut into pieces of about 1.0 cm); five auxins (Picloram, ANA, AIB, 2.4-D and Picloram) in five concentrations (0.0; 2.5; 5.0; 7.5 and 10.0 μM) and five additional witness, in other words, each explant source added to the treatment without auxin. The repetition was composed by five Petri dishes (90 x 15 mm), containing 25 mL of Pierik medium, and each unit experimental nine explant/placa. Cultures were maintained in growth room, at 25 ± 2 ºC, in the darkness. After 60 days of cultivation, it was evaluated the presence of embryogenic cultures, shoots, roots and mass of the produced callus. The production of the first callus was observed in petioles and stem explants, and its production was in average containing 7.5 μM ANA and 10.0 μM Picloram. In the concentrations of 10.0 μM, there was no different statistics among the treatments with the auxins ANA, 2.4-D and Picloram, being superior to the others. For the production of shoots, the explant with the largest average was the stem segments, in average without the auxins. The proliferation of roots was observed in stem segments and roots explants, mainly in average with AIB. Histochemistry analysis were determined, through the double stained with acetocarmine and Evan's blue and lugol test. However, it was observed in histological cuts that the callus produced in Pierik medium containing 10.0 μM of ANA presented well developed embryos, with protoderm and procambium, with polarization signs. For the proliferation of the embryogenic cultures, the subdivision of the selected callus was accomplished, and inoculated in Petri dishes with Pierik medium containing 10.0 μM of ANA, in five successive subcultures, with 60 days each. In the experiment II, the callus was inoculated, produced in the proliferation of the embryogenic cultures, with about 90 mg weight, in Erlenmeyers of 125 mL, containing 25 mL of average liquid , Pierik and AA2, with different concentrations of 2.4-D (0.00; 4.52; 9.05 μM) and kinetin, (0.00; 0.47; 2.32 μM), analyzed in DIC with factorial 2 x 3 x 3, maintained at growth room at 25 ± 2 ºC, in the darkness, staying under orbital agitation of 100 rpm. After 45 days of cultivation, it was evaluated a mass of the embryogenic callus; production of somatic embryos; production of secondary somatic embryos; oxidation percentage; coloration, texture of the embryogenic callus and development of the somatic embryos produced. The production of somatic embryos was better in Pierik medium with 0.47 μM of kinetin, being observed a smaller production of secondary somatic embryos, smaller oxidation percentage, with friable texture of the callus, being one of the treatments with larger development of the embryos, proven for the histological cuts, in which it was observed embryos in the globular stadium, with polarization signs, even mature embryos, with the presence of primary leaf and meristematic zone. In the Experiment III, the callus produced were inoculated in Erlenmeyers containing 25 mL of liquid and semi-solid medium, Pierik and AA2, suplemented with the kinetin concentrations (0.0; 1.16; 2.32; 4.64 μM), analyzed in DIC with factorial 2 x 2 x 4. Cultures were maintained under a 16 hours of light, photoperiod at 36 μmol m-2 s-1 provided by cool white fluorescent lamps at 25 ± 2 ºC. The liquid medium staying under orbital agitation of 100 rpm. After 45 days of cultivation, it was evaluated the number of callus with mature embryos; percentage of conversion of plants; oxidation percentage; and number of embryos with complete maturation. Pierik medium containing 2.32 μM of kinetin is recommended, in which, it was much better to the number of embryogenic callus with mature embryos, number of embryos with complete maturation and for the best conversion in plants. The plants were acclimatized ex vitro in the bench of the laboratory and transferred, in the end of two months to a greenhouse. The present study evidenced that the induction and proliferation of embryogenic cultures starting from stem segmentswere dependent on the type and concentration of the auxins. For the pre-maturation and maturation of the somatic embryos, it is necessary the retreat or the reduction of the auxin in the medium, adding ideal concentrations of cytokinins, and obtaining high conversion of the somatic embryos in plants, in the final process.
O presente trabalho teve como objetivo estabelecer a propagação in vitro de Anthurium andraeanum cv. Eidibel via embriogênese somática, avaliando a indução das culturas embriogênicas (Experimento I); pré-maturação dos embriões somáticos (Experimento II); e maturação dos embriões somáticos e posterior regeneração das plantas (Experimento III). No experimento I, adotou-se a subdivisão de plantas de antúrio estabelecidas in vitro, para a obtenção das fontes de explante. Utilizou-se o delineamento inteiramente casualizado (DIC), em disposição fatorial 53, representados por cinco tipos de explantes (folhas inteiras e seccionadas ao meio; pecíolos; segmentos nodais com uma gema; e segmento de raiz sem o ápice radicular; cada explante com ~ 1,0 cm); cinco auxinas (AIA, ANA, AIB, 2,4-D e Picloram) em cinco concentrações (0,0; 2,5; 5,0; 7,5 e 10 μM) e cinco testemunhas adicionais, ou seja, cada fonte de explante adicionada ao tratamento sem auxina. A repetição foi composta por cinco placas de Petri (90 x 15 mm), contendo 25 mL de meio Pierik, e cada unidade experimental nove explantes/placa, mantidas em sala de crescimento, a 25 ± 2 ºC, no escuro. Após 60 dias de cultivo, avaliou-se a presença de calos embriogênicos, brotos, raízes e massa freca dos calos produzidos. A produção dos primeiros calos foi observada em explantes de pecíolo e segmento nodal, e a sua produção ocorreu, principalmente, nos meios acrescidos de ANA e Picloram, nas concentrações de 7,5 e 10,0 μM, respectivamente. Em 10,0 μM, não houve diferença estatística entre os tratamentos com as auxinas ANA, 2,4-D e Picloram, sendo superiores aos demais. Para a produção de brotos, o explante com a maior média foi o segmento nodal, em meio sem a adição de auxina. Observaram-se a proliferação de raízes em explantes de segmento nodal e radicular, principalmente em meio suplementado com AIB. A partir de análise histoquímica, determinaram-se, por meio da dupla coloração com carmim acético e azul de Evans e teste de lugol, características embriogênicas dos calos. No entanto, observou-se em cortes histológicos que os calos produzidos em meio Pierik acrescido de 10,0 μM de ANA apresentaram embriões bem desenvolvidos, com presença de procâmbio e protoderme, demostrando sinais de polarização. Para a proliferação das culturas embriogênicas, foi adotada a subdivisão dos calos selecionados, e inoculados em placas de Petri com meio de cultura Pierik acrescido de 10,0 μM de ANA, em cinco subcultivos sucessivos, com 60 dias cada. No experimento II, foram inoculados os calos produzidos na fase de proliferação, com cerca de 90 mg, em Erlenmeyers de 125 mL, contendo 25 mL de meio de cultura líquido, Pierik e AA2,com diferentes concentrações de 2,4-D (0,00; 4,52; 9,05 μM) e cinetina, (0,00; 0,47; 2,32 μM), analisados em DIC com fatorial 2 x 3 x 3, mantidos em sala de crescimento a 25 ± 2 ºC, no escuro, permanecendo sob agitação orbital de 100 rpm. Avaliadas aos 45 dias de cultivo, quanto a: massa dos calos embriogênicos; produção de embriões somáticos; produção de embriogênese somática secundária; porcentagem de oxidação; coloração, textura dos calos embriogênicos e desenvolvimento dos embriões somáticos produzidos. A produção de embriões somáticos foi superior no meio Pierik acrescido de 0,47 μM de cinetina, observando-se a menor produção de embriogênese somática secundaria, menor porcentagem de oxidação, com textura friável dos calos, sendo um dos tratamentos com maior desenvolvimento dos embriões, comprovado pelos cortes histológicos, no qual observaram embriões no estádio globular, com sinais de polarização, até embriões maturados, com a presença de folha primária e zona meristemática. No Experimento III, os calos produzidos na fase de pré-maturação foram inoculados, em Erlenmeyers contendo 25 mL de meio líquido e semi-sólido, Pierik e AA2, suplementados com as concentrações de cinetina (0,0; 1,16; 2,32; 4,64 μM), formando um DIC com fatorial 2 x 2 x 4, mantidos em sala de crescimento, a 25 ± 2 ºC, sob fotoperíodo de 16 horas, irradiância luminosa de 36 μmol m-2 s-1. Os meios líquidos permaneceram sob agitação orbital de 100 rpm. Avaliou-se aos 45 dias de cultivo quanto ao número de calos com embriões maturados; porcentagem de conversão em plantas; porcentagem de oxidação; e número de embriões com maturação completa. Recomenda-se o meio Pierik suplementado com 2,32 μM de cinetina, no qual, foi superior para o número de calos embriogênicos com embriões maturados, número de embriões com maturação completa e principalmente pela melhor conversão em plantas. As plantas produzidas foram aclimatizadas ex vitro na bancada do laboratório e transferidas, no final de dois meses, para casa de vegetação. O presente estudo evidenciou que a indução e proliferação de calos embriogênicos a partir de segmentos nodais de antúrio foi dependente do tipo e da concentração das auxinas. Para as fases de pré-maturação e maturação dos embriões somáticos, é necessária a retirada ou a redução da auxina no meio de cultura, adicionando concentrações ideais de citocinina para cada fase, e assim, obtendo máxima conversão dos embriões somáticos em plantas, no final do processo.

O setor de produção de coco é atingido por vários problemas que afetam sua produtividade especialmente o uso difundido de plantas sem seleção devido a características intrínsecas da cultura que dificultam a seleção de cultivares com características superiores. O uso eficiente dos recursos genéticos do coco tem encontrado dificuldades na coleta e troca de germoplasma. A cultura de tecidos tem por finalidade viabilizar a troca segura de germoplasma e facilitar o desenvolvimento de variedades produtivas em um programa de melhoramento genético, porém a regeneração de plantas através de técnicas in vitro tem apresentado dificuldades adicionais no estabelecimento de protocolos viáveis. O presente trabalho teve por objetivo estabelecer um protocolo viável para a regeneração de embriões zigóticos para ser utilizado em programas de melhoramento genético e para o transporte seguro de germoplasma, testando diferentes meios básicos de cultura, presença de reguladores vegetais, concentração de sacarose e fontes de aminoácidos. A regeneração de embriões foi mais efetiva em meio de cultura Y3 (Eeuwens, 1976) com concentração de Fe2SO4 41,7 mg.L -1 e Na2EDTA 55,8 mg.L -1 na presença de glicina e ausência de mio-inositol, contendo carvão ativado. A presença de reguladores vegetais e a caseína hidrolisada foram limitantes inclusive inibindo a germinação. A presença de altas concentrações de sacarose (60 g/L) mostrou melhores resultados.
The sector of coconut production is reached by several problems that affect his productivity especially the spread use of plants without selection due to intrinsic characteristics of the culture that difficult the selections for cultivars whit superiors characteristics. The efficient use of the genetic resources of the coconut has been having difficulties in collecting and germplasm exchange. The tissue culture has for purpose to make possible the safety change of germplasm and to facilitate the development of productive varieties in a program of genetic improvement; however the regeneration of plants through in vitro techniques has been presenting additional difficulties in the establishment of viable protocols. The present work had for objective to establish a viable protocol for the regeneration of coconut zigotic embryos to be used in programs of genetic improvement and for the safe movement of germplasm, testing different basic media of tissue culture, presence of growth regulators, sucrose concentration and sources of amino acids. The regeneration of embryos was more effective in Y3 (Eeuwens, 1976) media with concentration of Fe2SO4 41,7 mg.L -1 and Na2EDTA 55,8 mg.L -1 whit presence of glycine and absence of meso-inositol, containing activated charcoal. The growth regulators and hydrolysate casein presence even inhibited germination. The presence of high sucrose concentrations (60 g/L) showed better results.

A novel technique was developed to immobilize plant cells. The cells are deposited on a surface of man-made fibrous material which provides for strong binding of the plant tissue biomass growing in the submerged culture. It was shown that the plant cells need to be fully viable for the attachment process to occur.
The scale-up of this technique to laboratory size specifically designed bioreactors was performed successfully. The cell immobilizing matrix was formed into a vertical spirally wound configuration to provide for a high immobilizing area-to-volume ratio (0.8-1.2 cm$ sp{-1}$). A modified airlift (riser-to-downcomer area ratio of 0.03 and vessel height-to-diameter (H/D ratio of 3) and a low H/D ($ sim$1.5) mechanically stirred vessel delivered the optimum bioreactor performance characterized by low foaming of the broth and highly efficient plant cell attachment and retention ($ geq$96%).
The growth of Catharantus roseus plant cells was investigated in these bioreactors. This process was found not to be mass transfer limited above minimal mild mixing and aeration levels ensuring sufficient supply of nutrients, especially oxygen (k$ sb{ rm L}$a $ sim$ 10-15 h$ sp{-1}$) to the immobilized biomass.
The gentle surface immobilization technique developed in this work did not hinder the biosynthesis potential of the SIPC. In fact, it appeared to induce a partial secretion of some valuable compounds into the culture medium. The mildness, easiness, efficiency, mass transfer characteristics, scale-up potential and biomass loading capacity (11-13 g d.w./L) of the surface immobilization technique make it superior to all other immobilization techniques used to culture plant cells. In addition, its bioreactor overall biomass concentration compares favourably to suspended plant cell concentrations attainable in bioreactors (15-20 g d.w./L).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Mate (Ilex paraguariensis) is an important species in Southern Brazil for its economical and cultural values. The present work had the objectives of analyzing the propagation of mate by cuttings, disinfection and in vitro establishment of nodal segments from adult plants and micropropagation of plants originated from immature embryos. Mate cutting were submitted to three (0, 4000 and 8000 mg L-1) IBA doses and two lengths (3 and 10 cm) of cutting. The cuttings were evaluated at 135 days for survival, rooting and formation of roots, leaves and shoots. In the disinfection experiments, nodal segments of one-year-old shoots were immersed in alcohol solution of 70% for two and four min., in 2% of NaOCl for 15, 25 and 35 min. and inoculated in ¼ of MS medium. The percentage of contaminated, oxidized and healthy explants were evaluated at 15 days. In micropropagation, mate seedlings from immature embryos were submitted to different culture mediums (MS, ¼ MS and WPM) for 30 days and evaluated for number of leaves and length of seedlings. For the multiplication, different doses (0; 0,01; 0,1; 1,0 and 2,0 mg L-1) of BAP were tested in ¼ MS culture medium. At 50 days, they were evaluated for the number of shoots, leaves and internodes, shoots length and height. Different doses (0; 0,5; 1,0 and 1,5 mg L-1) of IBA were evaluated for rooting and kept explants either for 15 or 30 days in the medium. In vitro plants of mate were acclimatized in different (vermiculite, sand or coconut shells) substrates, with controlled temperature and light environmental conditions. The highest percentage of rooted cuttings, in both evaluated length, were with 4000 mg L-1 of IBA. Cuttings with 3 cm length produced the highest percentage of rooted cuttings. Nodal segments of one-year-old shoots have the highest survival, with the disinfection with alcohol 70% for four minutes and NaOCl 2% for 25 min. The ¼ of MS salts medium can be used as basal medium for in vitro culture of mate plants. The basal medium with 2 mg L-1 of BAP increases the number of adventitious shoots and promotes shoot proliferation after some sub cultivations, enabling in vitro plant maintenance as microstump, as shoot supply. In vitro rooting can be achieved in one cycle of 30 days of cultivation, with the addition of 3 mg L-1 of IBA to the medium with ¼ of MS salts. In vitro plants of mate can be acclimatized in coconut shells or sand in controlled temperature and light conditions.
A erva-mate (Ilex paraguariensis) é uma espécie de grande importância no sul do Brasil, pelo valor econômico e cultural que apresenta. O presente trabalho teve por objetivos analisar a propagação de erva-mate por estaquia, o estabelecimento e desinfestação in vitro de segmentos nodais de plantas adultas e a micropropagação de plantas oriundas de embriões imaturos. Para a estaquia, testaram-se três doses de AIB (0, 4000 e 8000 mg L-1) e dois comprimentos de estacas (3 e 10 cm). As estacas foram avaliadas quanto à sobrevivência, o enraizamento, o número de raízes, de folhas e de brotos e o comprimento de raízes e de brotos, aos 135 dias. No experimento de desinfestação, segmentos nodais de brotações de ano de erva-mate foram imersos em etanol 70%, por 2 e 4 min. e, depois, em NaOCl 2%, por 15, 25 e 35 min e, então inoculados em meio com ¼ do MS. Aos 15 dias avaliaram-se a percentagem de explantes contaminados, oxidados e sadios. Na micropropagação, plântulas de erva-mate, obtidas de embriões imaturos, foram submetidas a diferentes meios de cultura (MS, ¼ MS e WPM) por 50 dias, com avaliação do incremento obtido para as variáveis número de folhas e comprimento de plântulas. Para a multiplicação dessas plantas, foram testadas a adição de diferentes doses (0; 0,01; 0,1; 1,0 e 2,0 mg L-1) de BAP ao meio de cultura com ¼ do MS. Foram avaliados o número de brotos, folhas e entrenós, o comprimento dos brotos e a altura das plantas, aos 50 dias de cultivo. No enraizamento de brotos de erva-mate foram utilizadas doses (0; 0,5; 1,0; 1,5; 3,0 e 6,0 mg L-1) de AIB, por 15 e 30 dias. As avaliações constaram da percentagem de enraizamento e de calo obtidas, do número e comprimento de raízes, do número de folhas e altura das plantas, aos 30 dias de cultivo. Plantas de erva-mate, produzidas in vitro, foram aclimatizadas em diferentes substratos (vermiculita média, areia e casca de coco) com temperatura e luminosidade controladas. A maior percentagem de estacas enraizadas, em ambos os comprimentos, foi com a dose de 4000 mg L-1 de AIB, sendo que as estacas de 3 cm obtiveram uma maior percentagem de enraizamento. Segmentos nodais de brotações de ano de erva-mate apresentam a maior sobrevivência com a desinfestação com álcool 70% por 4 min. e em NaOCl 2% por 25 min. O meio com ¼ dos sais do MS pode ser utilizado como o meio de cultura base para o cultivo in vitro de plantas de erva-mate. A adição de 2 mg L-1 de BAP ao meio de cultura base aumenta o número de brotos adventícios em erva-mate, além de formar tufos após alguns subcultivos, possibilitando a manutenção de plantas como microcepas, para o fornecimento de brotos. O enraizamento in vitro de erva-mate pode ser realizado em apenas uma fase de 30 dias, com a adição de até 3 mg L-1 de AIB ao meio com ¼ dos sais do MS. Plantas de erva-mate, produzidas in vitro, podem ser aclimatizadas nos substratos casca de coco ou areia em ambiente com temperatura e luminosidade controladas.

The present work is a study of two techniques for multiplication and conservation of Fevillea trilobata L., and an evaluation of popular knowledge of the related plant. For the multiplication study, techniques were used for propagation ´in vitro´and cutting. For the evaluation of popular knowledge of gindiroba was applied a semi-structured questionnaire to sellers who sell seeds and / or natural products in the Municipal Market Albano Franco and in fair-free in Mosqueiro Beach, both located in Aracaju / SE, beyond some sellers from the municipality of Carmópolis / SE. Plant-matrices of gindiroba, sources of explants for micropropagation experiments, were grown in the mini-garden of the Department of Biology / UFS, from seeds provided by EMBRAPA-CPATC. ´In vitro´propagation, the culture medium used was Murashige & Skoog (MS), supplemented with vitamins of Morel and Wetmore, 3% sucrose, 0.01% casein and myo-inositol, and 0.6% agar plus the BAP (0, 1, 2, 4 and 6 mgL-1), pH adjusted to 5.8, temperature 27 º ± 1 º C, photoperiod of 12 hours and irradiance of 45μmol.m-2.s-1 with cool white light. The cutting experiment was conducted in the greenhouse of the Department of Biology / UFS. For this technique had been developed two experiments. The first one with medium plant cuttings in juvenile phase and the second with cuttings of plants in reproductive phase. The treatments were formed by the combination type of cutting (stem and stem + leaf) with concentration of AIB (0, 1 and 2mg L-1) in substratum sand + clay soil (2:1). The results were submitted to analysis of variance, where significant interactions were analyzed by the Tukey test at 5% level of probability. The data were analyzed by the software GraphPad Prism, version 4. According to the results of the in vitro propagation, callus formation was higher in seedlings treated with 1 mg/L-1 BAP. For the variable of sprouting of leaves, the treatment 2 showed a statistically significant difference. The cutting experiment 1, cutting + leaf without immersion in AIB showed higher formation of new roots and leaves. For cuttings in reproductive stage, cutting + leaf immersed in 1 mg L-1 of AIB was more effective in the formation of roots and leaves. Cuttings in juvenile and reproductive phase of treatments 4, 5 and 6 had presented dehydration, although some of them have given roots and small leaves. Stakes younger and without immersion in AIB, were most suitable for the technique of cutting. According to the results of the interviews, gindiroba is used as a medicinal plant in Sergipe. However the users are unaware of the biotechnological potential of this species.
O presente trabalho é um estudo sobre duas técnicas para multiplicação e conservação da Fevillea trilobata L., bem como uma avaliação sobre o conhecimento popular da referida planta. Para o estudo de multiplicação foram utilizadas as técnicas de propagação in vitro e estaquia. Para a avaliação do conhecimento popular da gindiroba foi aplicado um questionário semi-estruturado a vendedores que comercializam sementes e/ou produtos naturais no Mercado Municipal Albano Franco e da feira-livre da praia do Mosqueiro, ambos situados em Aracaju/SE, além de alguns proprietários do município de Carmópolis/SE. Plantas-matrizes de gindiroba, fontes de explantes para experimentos de micropropagação, foram cultivadas no do mini-horto do Departamento de Biologia/UFS, oriundas de sementes fornecidas pela EMBRAPA-CPATC. Na propagação in vitro o meio de cultura utilizado foi o Murashige & Skoog (MS), suplementado com vitaminas de Morel e Wetmore, sacarose a 3%, caseína e mio-inositol a 0,01%, ágar a 0,6%, acrescido de BAP a (0; 1; 2; 4 e 6 mgL-1 ), pH ajustado em 5,8, temperatura de 27º±1º C, fotoperíodo de 12 horas e irradiância de 45μmol.m-2.s-1 com luz branca fria. O experimento de estaquia foi conduzido na estufa do Departamento de Biologia/UFS. Para essa técnica foram desenvolvidos dois experimentos, sendo o primeiro com estacas medianas de planta em fase juvenil e o segundo com estacas de plantas em fase reprodutiva. Os tratamentos utilizados foram formados pela combinação tipo de estaca (caule e caule+folha) com concentração de AIB(0; 1 e 2mgL-1), em substrato areia+solo argiloso (2:1). Os resultados obtidos foram submetidos à análise de variância, onde as interações significativas foram analisadas através do teste de Tukey em nível de 5% de probabilidade. Os dados foram analisados como auxilio do software GraphPad Prism, versão 4. De acordo com os resultados obtidos com a propagação in vitro, houve maior formação de calos em plântulas tratadas com 1 mgL-1 de BAP e para a variável brotamento de folhas, o tratamento 2 apresentou diferença significativa estatisticamente. O experimento 1 de estaquia, estaca+folha sem imersão em AIB apresentou maior formação de raízes e folhas novas. Já para estacas em fase reprodutiva, estaca+folha imerso em 1mg L-1 de AIB foi mais efetivo na formação de raízes e folhas. Estacas em fase juvenil e reprodutiva dos tratamentos 4, 5 e 6 apresentaram desidratação, embora algumas delas tenham emitido raízes e pequenas folhas. Estacas mais jovem e sem imersão em AIB, mostraram-se mais adequadas para a técnica de estaquia. De acordo com os resultados das entrevistas, a gindiroba é utilizada em Sergipe como planta medicinal. Entretanto os usuários desconhecem o potencial biotecnológico dessa espécie.

Thesis (MTech (Horticulture)--Cape Peninsula University of Technology, 2017.
A study was conducted to investigate the effects of various media composition and wounding treating on the in vitro propagation of Argyroderma subalbum and A. testiculare explants derived from mature plants, antioxidants and plant growth regulators (PGR) concentrations. One experiment consisted of 3 medium types including Murashige and Skoog (MS) medium strength, vitamin supplement. Fifteen replicates were used for each treatment. The shoots were then sub-cultured to ten replicate regenerated medium consisting of varying levels and combination of indole-3-acetic acid (IAA) and 10 μM 6-Benzyladenine (BA) supplements. In another experiment consisted of varying levels of auxins with MS medium strength, activated charcoal (AC) and vitamin supplements ten replicates were used for each treatment. Results indicated the positive role of cytokinins types’ 6-Benzyladenine (BA), 2-isopentyladenine (2iP) and Kinetin in inducing callus formation from wounded explants. The highest rate of friable callus formation of wounded explants was observed in media containing vitamin supplementation with BA at 10 μM. Callus formation significantly increased with the addition of vitamins at 10 μM on BA, 2iP and kinetin. With regards to the effects of various media composition and wounding explants on in vitro growth and regeneration of A. subalbum and A. testiculare, significant results were achieved with BA, 2iP and kinetin concentrations on explants discoloration and callus formation. The antioxidant treatment, AC did not reduce explants discoloration, but the induction of the callus was developed furthermore, results showed that IAA with BA concentrations without addition of AC there was significantly difference on both species but A. subalbum dominated with browning intensity (Chapter 3). Only sub-culturing of the explants succeeded in preventing explants discoloration and subsequently increased the number of shoots. The interaction between Indole-3-acetic acid (IAA) concentrations combined with BA resulted in the most effective technique in reducing explants discoloration at the media contact point. This study provides an insight into the contributing factor and methods of overcoming the major problem of phenolic oxidation and promoting the in vitro growth and regeneration of A. subalbum and A. testiculare.

Par son mode de propagation clonale(greffage), le pommier est un modèle intéressant pour les études épigénétiques. On observe en effet, une variabilité phénotypique au sein d’une même variété,suggérant une contribution de mécanismes épigénétiques. Au début de cette thèse les mécanismes de transmission de marqueurs épigénétiques, tel que la méthylation de l’ADN,étaient encore peu étudié chez les plantes pérennes.Pour mieux comprendre comment les marques épigénétiques sont transmises et contribuent à des traits phénotypiques, nous avons défini deux axes de recherche principaux: (i) En utilisant la technologieCRISPR-Cas9 nous avons créé des lignées de pommier afin d’obtenir une diminution systématique de la méthylation de l’ADN. (ii) Afin d'évaluer la transmission des marques épigénétiques lors de la reproduction sexuée et asexuée, nous avons comparé les épigénomes d’arbres adultes, de greffons et de semis obtenus par autofécondation sur une lignée modèle double-haploïde. Les analyses à l’échelle de génomes complets ont montré une absence de modification globale au niveau de la méthylation de l’ADN entre un arbre adulte, un greffon et un semis de pommier.Cependant nous avons détecté des régions spécifiques présentant une différence de méthylation de l’ADN (DMRs). Fait intéressant, nous avons détecté plus de DMRs entre le semis et le greffon ou l'arbre qu’entre l'arbre et le greffon, ce qui suggère que l'arbre et le greffon sont plus proche au niveau épigénétique. Nos résultats de phénotypage,d’analyses transcriptomique et de méthylome confirment la différence entre arbre adulte et semis.Mais également que les greffons sont à l’interface entre juvénilité et maturité
Thanks to its clonal propagation (grafting), apple tree is an interesting model for epigenetic studies. Indeed, phenotypic variability can be observed within the same variety, suggesting a potential contribution of epigenetic mechanisms. At the beginning of this thesis, the mechanisms of transmission of epigenetic marks, such as DNA methylation, were still little studied in perennial plants.To better understand how epigenetic marks are transmitted and contribute to phenotypic traits, we have defined two main axes of research: (i) UsingCRISPR-Cas9 technology we have created apple mutant lines in order to obtain a systematic decrease in DNA methylation. (ii) In order to assess the transmission of epigenetic marks during sexual and asexual reproduction, we compared the epigenomes of adult trees, grafts and seedlings obtained by self-fertilization on a model doubled-haploid line. Analyses at the whole epigenome level indicated that there is no overall change in DNA methylation between an adult tree, a grafted tree and an apple seedling. However, we were able to detect specific regions that are differentially methylated (DMRs). Interestingly, more DMRs were detected between seedlings and grafted tree or adult tree than between the tree and grafted tree, suggesting that the tree and grafted tree are closer epigenetically. Our phenotypic, transcriptomic and methylome analysis results confirm the difference between adult trees and seedlings. They also confirm that grafted tree is at the interface between juvenility and maturity

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Although the louro-pardo (Cordia trichotoma Vell.) is native forest specie with high timber potential, there are still scarce studies that approach the production of seedlings of this species by vegetative propagation. The objective of this work was to evaluate the vegetative propagation of the louro-pardo by the techniques of cuttings and micropropagation. It was tested the presence or absence of 8000 mg L-1 of indolbutiric acid (IBA) and two types of cuttings (basal and apical). At 40 days, the cuttings were evaluated for survival, rooting, the presence of callus and shoots, the number and length of shoots and the number of leaves. For the establishment of aseptic seedlings, seeds of louro-pardo were treated with 2% or 5% of sodium hypochlorite, for 0, 5, 10, 15 or 20 min. The seeds were inoculated in WPM basic culture medium. At 30 days, the percentages of disinfection, fungal contamination and / or bacteria and the average time of germination were evaluated. For multiplication, apical segments were inoculated in basic medium, plus 0; 0,25; 0,50 or 0,75 mg L-1 of 6-benzilaminopurin (BAP). At 45 days were evaluated the length, number of leaves and internodes of the shoots, the percentage of callus and the survival of the explants. In the multiplication, using microstumps kept in vitro, was tested whether or not the addition of 1,5 g L-1 of activated charcoal to the basic medium on the production of shoots. At 30 and 60 days, concerning to the first and second cuts of microstumps, were evaluated the number and length of shoots, number of leaves and internodes of the shoots, and the number of microcutting. Rooting in vitro microcuttings were maintained in basic medium plus 1,5 g L-1 of activated charcoal and IBA (1,5 or 2,0 mg L-1). At 45 days were evaluated the presence of roots and callus, the percentage of survival and sprouting. In cutting, were observed the formation of shoots on the cuttings, but they are not rooted. The cutting type and dose of IBA did not influence the rooting and survival. In micropropagation was found that the sterilization of seeds with 5% of sodium hypochlorite for 5 min. allowed the in vitro establishment of aseptic seedlings. The induction of shoots in the explants was not influenced by different doses of BAP. The presence of activated charcoal in the culture medium favored the formation and growth of shoots in microstumps. The use of 1,5 or 2,0 mg L-1 IBA did not promote the rooting of microshoots of louro-pardo.
Apesar de o louro-pardo (Cordia trichotoma Vell.) ser uma espécie florestal nativa com elevado potencial madeireiro, ainda são escassos os estudos que abordam a produção de mudas dessa espécie pela propagação vegetativa. O objetivo deste trabalho foi avaliar a propagação do louro-pardo pelas técnicas de estaquia e micropropagação. Para a estaquia foi testada a presença ou ausência de 8000 mg L-1de ácido indolbutírico (AIB) e dois tipos de estacas (basais e apicais). Aos 40 dias, as estacas foram avaliadas quanto à sobrevivência, o enraizamento, a presença de calos e de brotos, o número e o comprimento de brotos e o número de folhas. Para o estabelecimento de plântulas assépticas, sementes de louro-pardo foram tratadas com 2% ou 5% de hipoclorito de sódio, por 0, 5, 10, 15 ou 20 min. As sementes foram inoculadas em meio de cultura base WPM. Aos 30 dias foram avaliadas as porcentagens de desinfestação, de contaminação por fungos e/ou bactérias e o tempo médio de germinação. Para a multiplicação, ápices caulinares foram inoculados em meio de cultura base, acrescido de 0; 0,25; 0,50 ou 0,75 mg L-1 de 6-benzilaminopurina (BAP). Aos 45 dias avaliou-se o comprimento, o número de folhas e de entrenós dos brotos, as porcentagens de calo e de sobrevivência dos explantes. Na multiplicação, utilizando microcepas mantidas in vitro, testou-se a adição ou não de 1,5 g L-1 de carvão ativado ao meio de cultura base na produção de brotos. Aos 30 e 60 dias, referentes ao primeiro e ao segundo corte das microcepas, foram avaliados o número e o comprimento dos brotos, o número de folhas e de entrenós dos brotos, e o número de microestacas. No enraizamento in vitro, microestacas foram mantidas em meio de cultura base, acrescido de 1,5 g L-1 de carvão ativado e AIB (1,5 ou 2,0 mg L-1). Aos 45 dias foram avaliadas a presença de raízes e calos, a porcentagem de sobrevivência e de brotação. Na estaquia, foi observada a formação de brotos nas estacas, contudo estas não enraizaram. O tipo de estaca e a dose de AIB utilizada não influenciaram no enraizamento ou na sobrevivência. Na micropropagação foi verificado que a assepsia das sementes com 5% de hipoclorito de sódio, por 5 min., possibilitou o estabelecimento in vitro de plântulas assépticas. A indução de brotos nos explantes não foi influenciada pelas diferentes doses de BAP. A presença de carvão ativado no meio de cultura base favoreceu a formação e o crescimento dos brotos nas microcepas. A utilização de 1,5 ou 2,0 mg L-1 de AIB não promoveu o enraizamento adventício das microestacas de louro-pardo.

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Blueberry cultivation is recent in Brazil, and the area expansion of this production is limited by the lack of quality seedlings. The in vitro propagation make possible, in little time, to produce of mass form, high quality plants. In this work, the first objective was to study some involved factors in the establishment, multiplication and rooting of blueberry rabbiteye group, seeking yield increase of the seedlings production through micropropagation. The second objective was to study some involved factors seedlings development of the Aliceblue, Climax, O Neal and Georgiagem cultivars, seeking make available to producers seedlings sufficiently developed and adapted to orchard implantation. The work was developed in 2 stages. The first stage was constituted for 4 trials micropropagation related, and consisted respectively in the in vitro establishment, multiplication of the material previously in vitro established and rooting sprout obtained in the multiplication stage. Was verified that zeatin is necessary for in vitro establishment Powderblue and Florida cultivars, be able to utilize the smaller concentration (9 μM). Cultivars Woodard, Bluebelle and Bluegem explants presented the most bud number. The most rooting percentage was obtained with 6 µM of IBA added in culture media. The basal stakes presented the most root quantity. The 6 µM concentration of growth regulator ANA provide the most root length. The second stage of this work was constituted for 2 trials vegetative establishment related. In the first trial were utilized 2 blueberry cultivars of rabbiteye group and 4 substrates tips. In the second trial were utilized 2 cultivars blueberry group and 4 substrates tips. It recommended 70% sawdust + 20% coconut fiber + 10% bovine manure mixture to the most growth aerial part, the most mass of dry matter aerial and root, and the most sprout number. For the rabbiteye group, Climax cultivar presented the most vegetative development, parallel O´Neal cultivar highbush group.
O cultivo do mirtilo no Brasil é recente, e a expansão da área de produção é limitada pela disponibilidade de mudas de qualidade. A propagação in vitro possibilita em curto espaço de tempo, produzir de forma massal, plantas sadias com alta qualidade. Neste trabalho, o primeiro objetivo foi estudar alguns dos fatores envolvidos no estabelecimento, na multiplicação e no enraizamento de cultivares de mirtilo do grupo rabbiteye , visando o aumento do rendimento na produção de mudas através da micropropagação. O segundo objetivo foi estudar alguns dos fatores relacionados ao desenvolvimento de mudas das cultivares Aliceblue, Climax, O Neal e Georgiagem, visando disponibilizar ao produtor uma muda suficientemente desenvolvida e adaptada para a implantação de pomares. O trabalho foi realizado em duas etapas. A primeira etapa foi constituída por 4 experimentos relacionados a micropropagação e consistiram respectivamente em estabelecimento, multiplicação do material já estabelecido in vitro e enraizamento de brotações obtidas na fase de multiplicação. Foi verificado que zeatina é necessária para o estabelecimento in vitro das cultivares Powderblue e Florida, podendo ser utilizada a menor concentração (9 μM). Explantes das cultivares Woodard, Bluebelle e Bluegem na posição vertical do explante apresentaram maior número de gemas. A maior porcentagem de enraizamento na cv. Climax foi obtida com a adição de 6 µM de AIB no meio de cultura. As estacas basais apresentaram maior quantidade de raiz. O fitorregulador ANA a 6 µM, proporciona maior comprimento de raiz. A segunda etapa deste trabalho constituiu-se de dois experimentos relacionados ao desenvolvimento vegetativo. No primeiro foram utilizadas 2 cultivares de mirtilo do grupo rabbiteye e 4 tipos de substratos. No segundo experimento foram utilizadas 2 cultivares de mirtilo do grupo highbush e 4 tipos de substratos. Recomenda-se a mistura 70% serragem + 20% fibra de coco + 10% esterco bovino para maior crescimento da parte aérea, maior massa da matéria seca da parte aérea e do sistema radicular e maior número de brotações. Para o grupo rabbiteye a cultivar Climax foi a que apresentou maior desenvolvimento vegetativo, paralelamente à cultivar O´Neal para o grupo highbush'.

Primary afferent sensory neurons can be incredibly long single cellular structures, often traversing distances of over one metre in the human. Cutaneous sensory stimuli are transduced in the periphery by specialised end-organs or free nerve endings which enable the coding of the stimulus into electrical action potentials that propagate towards the central nervous system. Despite significant advances in our knowledge of sensory neuron physiology and ion channel expression, many commonly used techniques fail to accurately model the primary afferent neuron in its entirety. In vitro experiments often focus on the cell somata and neglect the fundamental processes of peripheral stimulus transduction and action potential propagation. Despite this, these experiments are frequently used as a model for cellular investigations of the receptive terminals. Crucially, somal responses may not represent the functional expression of ion channels in the axon and end terminals. The aim of this thesis was to develop a system using compartmentalised culture chambers and ratiometric calcium imaging to directly and accurately compare the sensitivity and functional protein expression of isolated neuronal regions in vitro. Using this preparation I demonstrate that the nerve terminals of cultured DRG neurons can be depolarised to induce action potential propagation, which has both a TTX-resistant and TTX-sensitive component. Furthermore, I show that there is a differential regulation of proton sensitivity between the sensory terminals and somata in cultured sensory neurons. I also go on to show that capsaicin sensitivity is highly dependent on embryonic dissection age. This novel approach enables a comprehensive method to study the excitability characteristics and regional sensitivity differences of cultured sensory neurons on a single cell level. Examination of the sensory terminals is crucial to further understand the properties and diversity of DRG sensory neurons.

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Citronella of Java (Cymbopogon winterianus Jowitt) is an aromatical plant with large usage in the manufacture of mosquito repellents because it contains essential oil rich in citronelal. The application of tissue culture on a large scale in medicinal plants production has increased significantly, since the conventional methods of propagation limit the potential use of some plants. Due that, one citronella of Java protocol was established with the objective of getting an alternative technique for the propagation of that culture. Four experiments ware conducted to evaluate the asepsis time, the explant type, the effect of vegetal regulators on the "in vitro" propagation of citronella of Java, as well as the micro-propagated seedling acclimatization. The gotten results had disclosed that the use of sodium hypochlorite (0.5%) during 30 minutes promoted a satisfactory asepsis index. The shoot tips explants type showed greater potential for the ?in vitro? development. It was verified that the use of benzilaminapurine (BAP) cytokynin is indispensable to succeed in the citronella of Java "in vitro" establishment, being able to be used in the concentration of 2.0 mg L-1. However, the use of indolbutyric acid (IBA) in the different tested concentrations did not present any results that justify its use, neither in the citronella of Java multiplication nor in the root establishment. The root establishment was evidenced when micro-propagated seedlings had been inoculated only in MS culture medium. It was verified that the check (without vegetal regulator) had a direct organogenesis, while such event was not verified in the other treatments. In the micro-propagated seedling acclimatization it was verified that the use of the floating method, it was essential for the seedlings survival, resulting in high survival index
A citronela de Java (Cymbopogon winterianus Jowitt) é uma planta aromática muito utilizada na fabricação de repelentes contra mosquitos por conter óleo essencial rico em citronelal. A utilização da cultura de tecidos, como forma de propagação de plantas medicinais tem aumentado significativamente, pois os métodos convencionais de propagação limitam o potencial de uso de algumas plantas. Sendo assim, um protocolo regenerativo para a citronela de Java foi estabelecido com o objetivo de se obter uma técnica alternativa para a propagação dessa cultura. Para isso foram realizados quatro experimentos envolvendo a avaliação do tempo de assepsia, do tipo de explante, do efeito de reguladores vegetais na propagação in vitro da citronela de Java, bem como a aclimatação das plântulas micropropagadas. Os resultados obtidos revelaram que a utilização do hipoclorito de sódio (0,5%) por 30 minutos promoveu índice satisfatório de assepsia. Os explantes com maior potencial ao desenvolvimento in vitro foram os ápices caulinares. Verificou-se que o emprego da citocinina benzilaminopurina (BAP) é indispensável para o sucesso do estabelecimento in vitro da citronela de Java, podendo ser utilizado na concentração de 2,0 mg L-1. No entanto, a utilização do ácido indolbutírico (IBA) nas diferentes concentrações testadas não apresentou resultados que justifique seu emprego, tanto na multiplicação quanto no enraizamento das plântulas de citronela de Java. O enraizamento foi evidenciado quando as plântulas micropropagadas foram inoculadas somente em meio de cultura MS. Foi constatado que na testemunha (sem regulador vegetal) houve uma organogênese direta, não sendo verificada nos demais tratamentos. Na aclimatação das plântulas micropropagadas verificou-se que a utilização do método floating , foi fundamental para a sobrevivência das plântulas, tendo sido observado alto índice de sobrevivência

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nus. To obtain peach plants that produce fruits of high quality and to obtain high productivity, it is necessary a modern and tecnified nursery plant production. The objective for this study was to establish an appropriated and low cost methodology for production of nursery Prunus plants. The experiments were carried out at the Faculdade de Agronomia Eliseu Maciel Universidade Federal de Pelotas during the period from 2003 to 2005. For the in vitro establishment phase were used explants of peach rootstocks cvs. Mr. S. 2/5, Sírio, Tsukuba, Nemaguard, Nemared and Flordaguard. The variables evaluated on the establishment phase were: type of explant; culture medium solidifiers; light quality; bud location; type of culture medium; and different BAP concentrations in the media culture. Regarding to the in vitro multiplication phase, the peach rootstocks cultivars tested were: Mr. S. 2/5; Sírio; and Tsukuba, which were evaluated about the effects of the following variables: light quality; BAP concentrations; type of explant; and type of culture medium. Regarding to the in vitro rooting phase, the peach rootstock tested was the cv. Mr. S. 2/5, which was evaluated about the following variables: light quality; vermiculite as a substitute of agar in culture media; and sucrose and AIB concentrations in culture medium. In the phase of in vitro establishment it was observed that: in general the best explant was nodal segment; the best culture medium solidifier was agar; the culture medium solidifier phytagel caused explants vitrification; and the nº 088 green filter for light promoted shoot morfogenesis on rootstock cv. Mr. S. 2/5. On the in vitro multiplication phase it was observed that: light quality had no effect on sprouting; addition of BAP 1,2 mg L-1 to the culture media had no effect on adventitious shooting in the Tsukuba rootstocks; the sprouts from shoot tip explants had more growth; and the MS culture medium gave the best results. On the in vitro rooting phase it was observed that: sucrose was essential for in vitro rooting; vermiculite substituted for agar in medium without affecting in vitro rooting; and the different light filters did not increase the.
Na cultura do pessegueiro, a implantação de um sistema moderno e tecnificado de produção de mudas é determinante para a obtenção de frutos de qualidade e uma alta produtividade. Desta forma, a qualidade genética e sanitária do material propagativo é imprescindível para alcançar o sucesso no empreendimento agrícola. Com o objetivo de adequar uma metodologia de propagação e de redução dos custos para a produção de mudas, foram desenvolvidos uma série de experimentos com porta-enxertos de Prunus spp. nas fases de estabelecimento, multiplicação e enraizamento in vitro. Os experimentos foram realizados na Faculdade de Agronomia Eliseu Maciel durante o período de 2003 a 2005. Para a fase de estabelecimento foram utilizados os porta-enxertos cvs. Mr. S. 2/5, Sírio, Tsukuba, Nemaguard, Nemared e Flordaguard. Nesta fase foram testados os tipos de explantes, solidificantes do meio de cultura, qualidade da luz através da utilização de filtros, efeito da localização da gema no ramo, tipos de meio de cultura e diferentes concentrações de BAP. Na segunda fase, a de multiplicação, para os porta-enxertos cvs. Mr. S. 2/5, Sírio e Tsukuba, avaliou-se a qualidade daxvii luz, diferentes concentrações de BAP, tipos de explantes e meios de cultura. Na terceira, a de enraizamento in vitro, avaliou-se a qualidade da luz, a vermiculita como substituto do ágar no meio de cultura, diferentes concentrações de sacarose e AIB no enraizamento do porta-enxerto de Prunus cv. Mr. S. 2/5. Na fase de estabelecimento observou-se que o melhor tipo de explante é o segmento nodal e o ágar foi o melhor solidificante, pois, o phytagel causou vitrificação. Em relação à qualidade da luz, o filtro verde nº 088 favoreceu a morfogênese das brotações do porta-enxerto cv. Mr. S. 2/5. A melhor região no ramo para a coleta dos explantes pode variar de acordo com o porta-enxerto. Na fase de multiplicação verificou-se que a qualidade da luz não influenciou a formação das brotações. Não houve formação de brotações adventícias no porta-enxerto cv . Tsukuba cultivado no meio acrescido de até 1,2 mg L-1 de BAP. Dos explantes testados na cv . Tsukuba, aquele contendo o ápice proporcionou o maior crescimento dos explantes. O meio de cultura MS foi o que apresentou os melhores resultados na multiplicação. Para a fase de enraizamento verificou-se que a adição de sacarose no meio de cultura é imprescindível e que a vermiculita pode substituir o ágar no meio. Os filtros utilizados para modificar as condições de luz não contribuíram para aumentar o potencial de enraizamento.