This study focused on induction of somatic embryogenesis of Magnolia spp. and cultivars utilizing leaf and seed (immature and mature) tissues with attempted micropropagation experiments. In a preliminary experiment, direct embryo regeneration was successful in a single leaf tissue of M. ‘Yellow Bird’. After various micropropagation experiments, microshoot proliferation rates decreased. As a result of minimal leaf material, mature seeds were utilized but had contamination issues. Subsequent experiments utilized immature seeds. M. ‘Leonard Messel’ and M. stellata had significantly greater embryo regeneration rates and M. ‘Rosea’, M. stellata, and M. kobus had greater callus induction rates. Woody Plant Medium had significantly greater rates of embryo regeneration as compared to Yellow Poplar medium. Further experimental measures including various collection times of immature seeds are necessary for an efficient regeneration protocol to support potential research utilizing floral-inducing genes to induce rapid breeding cycles for selection of magnolias with diverse floral characteristics.

Effects of the in ovo injection of Inovocox EM1 vaccine (EM1 vaccine) suspended in commercial diluent on developing broiler embryos were investigated in 3 trials. Effects of the EM1vaccine administered by in ovo injection on broiler embryogenesis and posthatch performance was determined by evaluating site of injection (SOI), embryo staging (ES), hatchability, and chick quality parameters. Oocyst output, microscopic lesion scores, and grow-out performance were further examined through day 35 posthatch. In these studies, it was shown that oocyst output began at day 3 posthatch (6 days post-injection), and peaked at day 7 posthatch (10 days post-injection). The EM1 vaccine had no effects on hatchability, various and chick quality parameters that were examined in the study. Similarly, grow-out performance through day 35 posthatch was not affected by the EM1 vaccine. SOI and ES provided information on the accuracy of in ovo vaccine delivery to the embryos, and were found to be significantly influenced by embryo age. In conclusion, in ovo injection of the EM1 vaccine has no detrimental effect on broiler embryogenesis, hatching chick quality, or the performance characteristics of Ross × Ross 708 broilers.

Date palms (Phoenix dactylifera L.) and papayas (carica papaya L.) are two commercially important plantation crops. Their economic potential in South Africa and worldwide is increasing. However, due to disease, pests and socio-economic reasons, planting material is in short supply. Micropropagation provides a method for rapidly propagating selected superior cultivars for commercial and environmental interests. A satisfactory process for the regeneration of elite cultivars should result in individuals phenotypically and genetically identical to the explant from which they were derived. However, due to somaclonal variation generated during in vitro culture, the true-to-typeness is questionable. For this reason a southern African survey for off-types on date palms produced using somatic embryogenesis was conducted. Plant growth variations such as leaf variegation, seedless fruit, broad leaves, compact growth habit and parthenocarpic fruit were recorded and possible explanations for each phenomenon given. Factors influencing the date palm initiation process such as decontaminating agents, plant growth regulators, explant type and nurse cultures were investigated. A double decontamination process with 2.6% and 1.3% sodium hypochlorite was most effective at reducing contamination. Alternative plant growth regulators, TIBA and NAA were ineffective as a substitute to 2,4-D for somatic embryogenesis. The size of the explant and "nurse cultures" played an important role in explant growth and initiating callogenesis. A "nurse culture" reduced the time in culture significantly. The problem areas in the three commercial tissue culture techniques used for date palms were outlined. In the second part of the study, factors influencing initiation, multiplication and rooting of papaya were determined. Presoaking with antibiotic, Rifampicin, and various fungicides had a positive effect on decontaminating papaya explants, while Bronocide™ had little effect. Various methods and materials were used to optimize papaya multiplication and rooting in vitro. The growth and multiplication of papaya was optimal at 50 g l ¯¹ sucrose. Gelling agent, Gelrite, increased multiplication rates significantly but had a negative effect on overall growth causing plants to become vitrified. The addition of activated charcoal reduced vitrification but also reduced multiplication rate. Activated charcoal greatly improved overall growth of papaya and reduced leaf senescence. No vitrification was observed in multiplying papaya cultures where agar and Gelrite combinations were used, but multiplication rate was reduced compared to cultures grown on Gelrite alone. callus removal from the bases of papaya at subculturing reduced multiplication rate and influenced elongation, growth and leaf senescence. Lower concentrations magar and Gelrite improved rooting percentages, but did not provide good support. Damaged roots and lower rooting percentages were observed on plantlets treated with IBA for four weeks compared to those exposed for only two days. A one hour pulse with a higher concentration (5 mg l ¯¹) of IBA greatly improved rooting percentage and further eliminated a second subculture onto an IBA-free medium after two days. Good, strong roots with root hairs were produced on vermiculite medium containing equal volumes of DS salts and vitamins. Modified lids with cotton-wool plugs also reduced leaf abscission. In vitro grafting using stericrepe proved impractical, while grafting in vitro unrooted papaya plants onto ex vitro seedlings was more successful, using wedge and slant grafts. Grafts sealed with pegs and Parafilm™ were less effective. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

Orientador: Marcelo Carnier Dornelas / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T19:57:11Z (GMT). No. of bitstreams: 1 Cazoto_JulianaLacorte_D.pdf: 5127995 bytes, checksum: 832b6cadc45544f0ef0e0353cad06755 (MD5) Previous issue date: 2012 / Resumo: Durante o desenvolvimento vegetal, o meristema apical caulinar e o meristema apical radicular são determinados durante o processo de embriogênese, enquanto que outros órgãos como folhas, flores e frutos são produzidos durante o desenvolvimento pós-embrionário. Durante o desenvolvimento vegetal, a expressão de diferentes fatores de transcrição em um determinado local e tempo culminam com a ativação de domínios celulares responsáveis pelo estabelecimento e manutenção dos meristemas. Foi hipotetizado que os mesmos genes que são ativados durante o desenvolvimento embrionário zigótico são também ativados durante a embriogênese somática. Portanto, o padrão de expressão de genes preferencialmente expressos durante o desenvolvimento embrionário em espécies modelo nesses dois processos foi analisado e comparado. Para este propósito, o processo de embriogênese zigótica e somática em Passiflora edulis foram caracterizados anatomicamente. Posteriormente, genes preferencialmente expressos durante a embriogênese somática e zigótica foram identificados e caracterizados. Foram selecionados genes das famílias YABBY, WOX e Aux/IAA. Estes genes foram encontrados por meio de análises de bioinformática no banco de dados de etiquetas de sequências expressas (ESTs) de Passiflora, PASSIOMA. As sequências putativas de aminoácidos codificadas pelos genes estudados, juntamente com seus possíveis homólogos em Arabidopsis, foram alinhadas e analisadas. O padrão de expressão desses genes foi estudado por RT-PCR, qPCR e hibridização in situ durante diferentes estágios do desenvolvimento embrionário somático e zigótico em P. edulis. A caracterização morfo-anatômica do embrião zigótico nessa espécie resultou na elaboração de um cronograma que identifica, a partir do dia da polinização, os diferentes estágios de desenvolvimento do mesmo. Além disso, os dados de expressão gênica sugerem que os mecanismos moleculares responsáveis pelos processos de embriogênese zigótica e somática apresentaram padrão de expressão similar no início do desenvolvimento embrionário vegetal em P. edulis / Abstract: During plant development, the shoot apical meristem (SAM) and the root apical meristem (RAM) are established during the embryo formation process, while the other organs as leaves, flowers and fruits are produced during the post-zygotic development. During plant embryogenesis, the expression of different transcription factors in a given time and place culminates with the activation of cellular domains responsible for the establishment and maintenance of the meristems. We hypothesized that the same genes that are activated during zygotic embryo development are also activated during somatic embryogenesis. Therefore we analyzed and compared the expression patterns of genes preferably expressed during embryo development in these two processes. For this purpose, we characterized anatomically both somatic and zygotic embryogenesis in Passiflora edulis (passion fruit). Later, we identified and characterized genes preferably expressed during somatic and zygotic embryogenesis in this species. Among the identified genes are members from YABBY, WOX and Aux/IAA families. These genes were selected by bioinformatics analysis from the PASSIOMA database of Passiflora reproductive expressed sequence tags (ESTs). The putative protein sequences encoded by the studied genes, together with possible horologes in Arabidopsis, were analyzed by sequence alignment. The expression patterns of these genes were studied by RT-PCR, qPCR and in situ hybridization during different developmental stages of P. edulis somatic and zygotic embryogenesis. The morpho-anatomical characterization of zygotic embryos in this specie resulted in a schedule elaboration that identifies, from the first day of pollination, the different developmental stages of the embryo. Moreover, the gene expression data suggest that the molecular mechanisms responsible for the processes of zygotic and somatic embryogenesis presented similar expression pattern during the beginning of the plant embryo development in P. edulis / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal

MYB30 est un facteur de transcription d’Arabidopsis thaliana connu pour son rôle de régulateur positif des défenses via la production d’acides gras à très longue chaîne (VLCFA). La cuticule, une couche hydrophobe protectrice qui recouvre les parties aériennes de la plante, est composée majoritairement de molécules lipidiques dérivées des VLCFA. Dans cette thèse, nous avons montré que le gène MYB30 est exprimé dans les cellules épidermiques de l’embryon, à une étape du développement qui coïncide avec la mise en place de la cuticule embryonnaire. Nos travaux révèlent que lors de l’embryogenèse, MYB30 contrôle l’expression de gènes de biosynthèse de la cuticule et la production de composés cuticulaires. En accord avec cela, nous avons montré que MYB30 est requis pour la formation d’une cuticule embryonnaire fonctionnelle. De plus, l’expression de MYB30 est dépendante de GASSHO1 et GASSHO2 (GSO1/2), deux récepteurs de type Receptor Like Kinase (RLK) impliqués dans une voie de signalisation spécifique à la graine, nécessaire pour la formation de la cuticule chez l’embryon. Nos données indiquent également que MYB30 régule négativement l’expression de GSO2, et que MYB30 pourrait agir en aval des deux RLK pour la formation de la cuticule embryonnaire. D’autre part, nous avons montré que ces deux RLK jouent également un rôle positif dans l’activation des réponses immunitaires de la plante adulte. Enfin, notre étude indique que MYB30 est aussi impliqué dans l’élongation cellulaire chez les plantules étiolées probablement via la régulation de la production de composés pariétaux. En conclusion, ces travaux identifient de nouveaux rôles pour MYB30, et le placent à l’interface entre les réponses de défense et le développement de la plante. / MYB30 is an Arabidopsis thaliana transcription factor known for its role of positive regulator of defense responses through the production of very long chain fatty acids (VLCFAs). The cuticle, a hydrophobic and protective layer that covers the aerial parts of the plant, is mainly composed of VLCFA-derived lipid molecules. In this thesis, we have shown that the MYB30 gene is expressed in the epidermal cells of the embryo, at a developmental stage that corresponds to the establishment of the embryonic cuticle. Our work reveals that during embryogenesis, MYB30 controls the expression of cuticle biosynthetic genes and the production of cuticular compounds. In agreement, we have shown that MYB30 is required for the formation of a functional embryonic cuticle. In addition, MYB30 expression is dependent on GASSHO1 and GASSHO2 (GSO1/2), two Receptor Like Kinases (RLKs) involved a seed-specific signaling pathway, necessary for the cuticle formation in the embryo. Our data also indicates that MYB30 negatively regulates GSO2 expression and that MYB30 may act downstream of the two RLKs in embryonic cuticle formation. Moreover, we have shown that these two RLKs also play a positive role in the activation of immune responses in adult plants. Finally, our study indicates that MYB30 is additionally involved in cell elongation in etiolated seedlings, likely by regulating the production of cell wall components. In conclusion, this work identifies new roles of MYB30, placing it at a crossroads between defense responses and plant development.

Plant embryogenesis is an intriguing developmental process that is controlled by many genes. AGAMOUS Like 15 (AGL15) is a MADS-domain transcriptional regulator that accumulates preferentially during this stage. However, at the onset of this work it was unknown which genes are regulated by AGL15 or how AGL15 is regulated. This dissertation is part of the ongoing effort to understand the biological roles of AGL15. To decipher how AGL15 functions during plant development, a chromatin immunoprecipitation (ChIP) approach was adapted to obtain DNA fragments that are directly bound by AGL15 in vivo. Putative AGL15 targets were isolated, and binding and regulation was confirmed for one such target gene, ABF3. In addition, microarray experiments were performed to globally assess genes that are differentially expressed between wild type and agl15 young seeds. Among them, a gene, At5g23405, encoding an HMGB domain protein was identified and its response to AGL15 was confirmed. Preliminary results suggest that the loss-of-function of At5g23405 might have an effect on somatic embryogenesis, consistent with AGL15 repression of the expression of this gene. Lastly, to address the question about how the regulator is regulated, the cis elements controlling the expression of AGL15 must be identified. Deletion analysis of the AGL15 promoter indicated the presence of putative positive and negative cis elements contributing to the expression of AGL15. Further analysis suggested that AGL15 regulates the expression of its own gene and this regulation may partially be explained by the direct binding of the protein to the AGL15 promoter. The data presented in this dissertation demonstrate that ChIP can be used to identify previously unsuspected targets of AGL15. Based on ChIP, a ChIP-chip technique is being developed in the lab to allow a more global analysis of in vivo binding sites. The identification of target genes and cis elements in AGL15 promoter is a step towards characterization of the biological roles of AGL15.

Recent studies of hoplonenemertean planuliform larvae have clarified their development and provided insight into larval evolution within the phylum. However, an assessment of viviparous development using modern techniques is lacking. To help facilitate a comprehensive comparative evaluation of developmental diversity within hoplonemerteans, we have conducted a confocal laser scanning microscopy investigation of the development in Prosorhochmus americanus, one of the few viviparous hoplonemertean species. Phalloidin staining provides evidence of a modified transitory larval epidermis, and reveals that the foregut, midgut, proboscis, central nervous system, and body wall musculature form early in development, consistent with observations for planktonic and encapsulated hoplonemertean larvae. However, invaginations characteristic of these larvae were not observed. Acetylated tubulin labeling and light microscopy shows that embryos are uniformly ciliated, and some specimens possess a caudal ciliary cirrus and/or apical tuft which are characteristic of planktonic larvae. These are interpreted as vestigial structures in the non-swimming P. americanus embryos. The findings provide additional evidence that hoplonemerteans exhibit a form of metamorphosis in their life history and thus exhibit indirect development. However, a comparative assessment of larval features in P. americanus suggests an evolutionary trend towards direct development in this species.

Influence de la micro-propagation par embryogenèse somatique sur la variation somaclonale chez le caféier (Coffea arabica): évaluation des changements au niveau phénotypique, cytologique, génétique et épigénique. La variation somaclonale (VS) est une préoccupation majeure de tous les systèmes de micropropagation. Elle est décrite comme un changement phénotypique présent chez les vitroplants et pourrait être générée par une large gamme de mécanismes génétique et épigénétiques. Des hybrides de Coffea arabica hautement productifs sont distribuées sous forme clonale par embryogenèse somatique (ES) en Meso-Amérique. L’objectif de ce travail chez le caféier est d’évaluer la conformité génétique des plants multipliés par ES et de comprendre les mécanismes impliqués dans les SV. Nous avons évalué les variations dans les plantes régénérées au niveau phénotypique, cytologique, génétique (mutations/AFLPs, transposition génétique/S-SAP) et épigénétique (méthylation/MSAP) en utilisant deux approches complémentaires. Tout d’abord, nous avons étudié chez deux hybrides des conditions de cultures industrielles supposées peu mutagènes i.e. une courte période de prolifération (6 mois) et faible apporte en auxine (0-1.4 µM 2,4-D). Deux systèmes de prolifération i.e. l’embryogenèse secondaire et les suspensions embryogènes seront comparés, le dernier étant plus productif et économique. Les analyses moléculaires AFLP et MSAP sur 145 somaplants montrent que les polymorphismes génétique et épigénétique entre plantes mères et somaplants sont extrêmement réduits, i.e. dans l’intervalle 0-0,003% et 0,07-0,18% respectivement, sans différence significative entre les systèmes de prolifération. Pour les deux hybrides testés, des observations phénotypiques massives en pépinière et au champ ont révélé de très faibles niveaux de VS (0,9% pour 800.000 plantes). Des analyses cytologiques ont mis en évidence des nombres de chromosomes anormaux (41-43, 45) chez la plupart des variants et des nombres normaux (44) chez les plants ayant un phénotype normal. Des conditions expérimentales a priori mutagènes ont également été appliquées en utilisant des périodes de prolifération prolongées (4, 12 et 27 mois) chez trois lignées embryogènes indépendantes de la variété Caturra en présence de concentrations élevées en régulateurs de croissance (4.5 μM 2,4-D, 17.8 μM 6-BA), afin de comprendre les mécanismes liés vieillissement des cultures interviennent sur les VS. L’étude des 180 somaplants régénérés a montré que le temps de prolifération affecte fortement la fréquence de VS et d’une manière hautement similaire pour les 3 lignées embryogènes. Aucun variant n’a été trouvé après 4 mois de prolifération alors que 30% et 94% de variants phénotypiques ont été caractérisés chez les plants issus de cultures de 12 et 27 mois, respectivement. Quels que soient l’âge de culture et la lignée embryogène, aucun polymorphisme n’a été trouvé chez les 124 somaplants et un très faible nombre des changements des méthylation avec les MSAP (0,049-0,087%). Cependant, de façon similaire aux somaplants produits en conditions industrielles, les variants phénotypiques montrent systématiquement des nombres de chromosomes anormaux (41-43) et les plants normaux le nombre de chromosomes attendu. Ce travail montre que l’ES s’appuyant sur des suspensions embryogènes peut garantir une propagation conforme des variétés sélectionnées de C. arabica. Il démontre également l’importance de l’âge des cultures sur l’apparition de VS et donc le caractère non aléatoire du phénomène. Les changements génétiques et épigénétiques sont particulièrement limités durant l’ES. Le principal changement chez la plupart des variants est l’aneuploidie, ce qui montre que les aberrations mitotiques jouent un rôle majeur dans les VS chez le caféier. / Influence of micropropagation through somatic embryogenesis on somaclonal variation in coffee (Coffea arabica): assessment of variations at the phenotypical, cytological, genetic and epigenetic level Somaclonal variation (SV) is a major concern in all micropropagation systems. It is described as the phenotypic variation displayed in in vitro-derived regenerants and it is believed to be originated from a large array of genetic and epigenetic mechanisms. Highly productive Coffea arabica hybrids are clonally disseminated in Meso-American region through somatic embryogenesis (SE). The objective of the present work in coffee is to evaluate the trueness-to-type of SE and to understand the mechanisms involved in SV. We assessed the variations in the propagated plants at the phenotypic, cytogenetic, genetic (mutations/AFLP, genetic transposition/S-SAP) and epigenetic (methylation/MSAP) level by using two complementary approaches. First, with 2 hybrids we studied industrial culture conditions expected to be weakly mutagenic thanks to the combined use of short term proliferation period (6 months) and low auxin supply (0-1.4 µM 2,4-D). Two proliferation systems i.e. secondary embryogenesis and embryogenic suspensions were compared, the latter being more productive and economic. AFLP and MSAP molecular analyses on 145 somatic seedlings showed that genetic and epigenetic polymorphisms between mother plants and emblings were extremely low, i.e. ranges of 0–0.003% and 0.07–0.18% respectively, with no significant difference between the proliferation systems. For the two hybrids tested, massive phenotypic observations in nursery and field plots showed very low levels of SV (0.9% from 800,000 plants). Cytological analysis showed abnormal chromosome numbers (41-43, 45) in most of coffee somaclonal variants and normal numbers (44) in phenotypically normal plants. Stressful experimental conditions were also applied by using extended proliferation periods (4, 12 and 27 months) for three independent embryogenic lines established for the Caturra var. in presence of high growth regulator concentrations (4.5 μM 2,4-D, 17.8 μM 6-BA) to understand the mechanisms of culture ageing on SV. The proliferation time strongly affected the SV frequency among the 180 regenerated plants and in a highly similar way with the three embryogenic lines. No variant was found after 4 months proliferation although 30% and 94% phenotypic variants were observed in plants derived from 12 and 27 month-old cultures, respectively. Regardless the culture age and the embryogenic line, no polymorphisms were found in the 124 plants analyzed and very limited methylation changes with MSAP markers (0.049-0.087%). However, similarly to plants derived from industrial conditions, phenotypic variants systematically showed abnormal chromosome numbers and normal plants systematically showed normal numbers. This work showed that SE based on embryogenic suspensions is reliable for true-to-type propagation of selected C. arabica varieties. It also demonstrated the importance of culture age on SV and hence the non random nature of this phenomenon. The genetic and epigenetic alterations are particularly limited during SE. The main change in most of phenotypic variants was aneuploidy showing that mitotic aberrations play a major role in SV in coffee.

Araucaria angustifolia é uma conífera nativa do Brasil de importância econômica, que, após intenso desmatamento, possui sua distribuição limitada a aproximadamente 3% da área original. Atualmente está incluída na lista de espécies ameaçadas de extinção, na categoria de perigo crítico de acordo com a IUCN (International Union for Conservation of Nature). A embriogênese somática é um processo em que, através da técnica de cultivo in vitro, células isoladas ou um pequeno grupo de células somáticas dão origem a embriões. Este é um sistema com potencial de aplicação em conservação, reflorestamento, propagação em larga escala, e melhoramento vegetal em espécies arbóreas recalcitrantes e em risco de extinção. Um dos principais fatores limitantes, nos sistemas de embriogênese somática em arbóreas, é a baixa freqüência de maturação e conversão de embriões somáticos em plântulas. Durante a fase de maturação, os embriões somáticos acumulam substâncias de reserva, reduzem a atividade metabólica e adquirem tolerância à desidratação. A suplementação do meio de cultura com determinados reguladores de crescimento e agentes osmóticos, os quais permitem a progressão do desenvolvimento normal dos embriões somáticos, promove um estresse hídrico. Esta situação mimetiza aquela que ocorre durante a embriogênese zigótica quando do desenvolvimento da semente. O óxido nítrico (NO) e espécies reativas de oxigênio (ROS) são moléculas que atuam nas respostas das plantas aos vários estresses ambientais e estão envolvidas em processos de desenvolvimento como embriogênese e germinação de sementes, gravitropismo, formação de raízes. Este trabalho teve como objetivo avaliar o efeito de promotores de maturação, ABA e os agentes osmóticos, PEG e maltose, no conteúdo de NO e ROS em culturas embriogênicas de A. angustifolia. Foi observado que os agentes promotores da maturação reduzem a síntese endógena de NO e ROS. Essa redução é dose dependente. Foram pesquisadas duas linhagens de células com diferentes capacidades de maturação. Para as culturas embriogênicas com competência de maturação esses promotores induzem a maturação e nas culturas que não maturam, promovem a morte celular programada. / Araucaria angustifolia is a native conifer of economic importance in Brazil. Due its massive deforestation, this species is now limited to only 3% of the original area. A. angustifolia is currently on the list of endangered species in the category of critically endangered according to IUCN (International Union for Conservation of Nature). Somatic embryogenesis is a process that, through the technique of in vitro culture, isolated cells or a small group of somatic cells gives rise to embryos. This is a system with potential application in conservation, reforestation, large-scale propagation and breeding of tree species . A limiting factor in the systems of somatic embryogenesis in trees is the low frequency of embryo maturation and conversion into plantlets. During the maturation, somatic embryos accumulate storage substances, reduce metabolic activity and acquire tolerance to dehydration. The water stress is made by supplementation of culture medium with growth regulators and osmotic agents, which allow the normal development of somatic embryos. This situation mimics that occurs during development of the seed. Nitric oxide (NO) and reactive oxygen species (ROS) are molecules that act in plant responses to various environmental stresses and are involved in developmental processes such as embryogenesis and seed germination, gravitropism, root formation. This study aimed to evaluate the effect of promoters of maturation, ABA and osmotic agents, PEG and maltose in the generation of NO and ROS in embryogenic cultures of A. angustifolia. It was observed that the promoters of maturation reduces the endogenous synthesis of NO and ROS. We investigated two cell lines with different maturation capacities. For responsive cell line these promoters induce maturation and for blocked cell line, promote programmed cell death.

O presente trabalho teve como objetivo caracterizar o desenvolvimento da semente de Araucaria angustifolia através da proteômica comparativa, buscando compreender as alterações fisiológicas e metabólicas que ocorrem durante esse processo. Inicialmente, foram avaliados três diferentes metodologias de extração de proteínas. A metodologia composta por solução de extração contendo 7 M de uréia, 2 M de tiouréia, 1% de ditiotreitol, 2% de Triton-100, 1 mM de fluoreto de fenilmetilsulfonil e 5 µM de pepstatina, seguido de precipitação em 20% de ácido tricloroacético apresentou géis de maior resolução e reprodutibilidade, tendo sido escolhida como metodologia de extração protéica para o estudo das alterações no proteoma da semente de A. angustifolia. Uma dificuldade associada ao estudo do proteoma de espécies não sequenciadas é a baixa representatividade nos bancos de dados protéicos, resultando em identificações baseadas em homologia. Estratégias proteômicas baseadas em fracionamento em gel resultam em grandes contaminações por fragmentos de queratina. Sendo assim, foi desenvolvido um programa de remoção de espectros de baixa qualidade para utilização em proteômica baseada em homologia. As análises mostraram que o programa reduz o tempo de busca, melhora a qualidade dos alinhamentos e não resulta em perda de identificações positivas. Finalmente, utilizando as metodologias descritas, foram estudadas as alterações no proteoma durante o desenvolvimento da semente de A. angustifolia. Noventa e seis proteínas foram identificadas e agrupadas de acordo com sua função biológica e padrão de detecção. Os resultados obtidos permitiram o estabelecimento de marcadores protéicos no início e final do desenvolvimento embrionário. A análise das proteínas abundantes no início da embriogênese indica um maior controle no metabolismo oxidativo em relação aos estádios finais. Contrariamente, o final da embriogênese é caracterizado por um alto metabolismo de assimilação de carbono e acúmulo de proteínas de reserva. As implicações dos resultados obtidos no controle e melhoramento de sistemas de embriogênese somática na espécie também foram discutidas. / The aim of the present work was to characterize the seed development of Araucaria angustifolia through proteomics in order to understand the physiological and biochemical changes during this process. For that, initially, three different protein extraction methods were evaluated. The extraction based on protein solubilization in 7 M urea, 2 M thiourea, 1% dithiothreitol, 2% Triton-100, 1 mM phenylmethylsulphonyl fluoride, 5 µM pepstatin, followed by 20% trichloroacetic acid precipitation showed the highest gel resolution and reprodutivity and, thus, was chosen to be used in the analysis of the proteome of A. angustifolia seeds. One aspect that hampers the proteome study of unsequenced species is the low protein representativity in databases. So, protein identification is usually carried out through homology. Strategies based on 2-DE result in high keratin contamination. In the present work a spectra filtering software was developed and evaluated for use in homology driven proteomics. The software reduced the time of search, improved alignment quality and did not result in lost of positive identifications. Finally, using the described strategies, the changes in the proteome of A. angustifolia seeds were studied. Ninety six proteins were identified and classified according to their biological functions and expression profiles during seed development. The identified proteins may be used as protein markers of early and late embryogenesis. Proteins involved in the control of oxidative metabolism were highly expressed during the early stages of seed development; while, carbon metabolism and storage proteins were highly expressed in late stages. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed.