Progress in tree biotechnology initially trailed behind agricultural crops due to their long life cycle, difficult tissue culture and regeneration protocols, and their abundance in natural forests. However, rapid global deforestation rates, together with an increased world demand for pulp, paper and timber products, have prompted scientific and commercial focus to improve genetic timber stocks. South Africa, a tree-poor country (where indigenous forests are protected), has relied almost solely on exotic plantations to meet its demand for timber. A pioneer study investigating the feasibility of using direct (biolistic) and indirect (Agrobacterium-mediated) methods for gene transfer was undertaken in Pinus patula Schiede et Deppe, a Mexican softwood and a forerunner for saw timber, pulpwood and paper in the South African forest industries. The aim of the transformation methods was to impart herbicide resistance to the trees. This was achieved via the introduction of a bar-GUS pAHC25 cassette under the control of the ubiquitin promoter. To provide target material for transformation, two in vitro micropropagation pathways were used: somatic embryogenesis and organogenesis. Both embryonal suspensor masses (ESM) and somatic embryos at various stages of development were initially used as target explants for the biolistic study using an established in vitro protocol. A stepwise selection was implemented in order to allow transformed (particularly bombarded) cultures the opportunity to regenerate under selection pressure using MSG3 maintenance medium supplemented with BASTA® herbicide at 1 mg l ¯¹ followed by 3 mg l¯¹ active ingredient at the next subculture. Biolistic transgene delivery was more efficient when sorbitol was included in the pre-bombardment medium enabling use of higher vacuum and shooting pressures, without lowering the regeneration potential of ESM significantly. Bombarded material from two genotypes (Lines 2 and 3) was regenerated to produce mature somatic embryos using an optimized regeneration regimen. The indirect study with Agrobacterium tumefaciens (LBA4404), transformed with the pAHC25 vector via triparental mating or heat shock, used a variety of target tissues including: mature somatic embryos, ESM and mature zygotic embryos (MZE's) - a novel in vitro system for P. patula. The Agrobacterium-mediated method resulted in optimized decontamination conditions using a combination of liquid MSG3 (or sterile dH₂O for mature embryos) supplemented with 500 mg l ¯¹ cefotaxime, with rotation, and sterile 65 mm Whatman No. 3 filter paper stacks, which avoided excess filtering and stress to transformation material. Further efforts to aid regeneration during the indirect study included L-proline post-transformation, though no mature somatic embryos were regenerated at the conclusion of the Agrobacterium-mediated study. Recovery of transformed ESM in both studies was best during the active growth phase 4-6 d after subculture. Regeneration with good somatic embryo potential was an exigent aspect in both transformation studies. Expression of positive histochemical GUS activity in all transformed material was confirmed by polymerase chain reaction (PCR) analysis indicating that Pinus patula tissue was amenable to transformation. A new bar PCR regime was implemented in P. patula. In the biolistic study, a higher transformation efficiency of bar amplicons (53%) than GUS amplicons (45%) was observed, reflecting their non-linked status on the pAHC25 transformation vector. This is the first report of biolistic transformation of P. patula that will allow for the production of transgenic ESM. The production of transgenic P. patula holds great promise for commercial development in the South African forestry industry. The application of transgenic trees in the timber industry is numerous but the aims most relevant to P. patula include wood modification and disease resistance to pathogens like pitch canker fungus. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

This research was an attempt to use a dicot transformation vector to transform a monocot. The initial purpose of this thesis was to transform orchids with the sense and antisense coat protein genes from the Odontoglossum ringspot virus (ORSV) in an effort to mitigate viral symptoms in transgenic plants using the transformation vector, Agrobacterium tumefaciens. However, it soon became apparent that much time would be needed to develop a transformation protocol. The transformation vectors used included the Agrobacterium tumefaciens disarmed strain LBA4404 with the binary plasmid pB1121, the disarmed strain At699 with the binary plasmid pCNL65, and the wild-type strain Chry5. The marker gene on the binary plasmids of both disarmed strains was p-glucuronidase (GUS).Several transformation protocols were used in an effort to determine if this transformation system would work on orchids. Transformation was not achieved even though a number of experimental conditions were varied. These included using two different types of orchid tissue, callus and protocorms; using two different species of orchids, Cattleya Chocolate Drop x Cattleytonia Kieth Roth and Cymbidium maudidum; varying the time the plant tissue was exposed to the bacteria from 1 hour to 96 hours; performing experiments with and without the wound signal molecule acetosyringone; and exposing the tissue to the virulent strains of A. tumefaciens mentioned previously.This research also developed GUS assay conditions necessary to decrease the number of false positives due to bacterial contamination. These conditions included chloramphenicol in the GUS assay buffer. / Department of Biology

Vibrio parahaemolyticus is an opportunistic human pathogen that occurs naturally in a non-pathogenic form in coastal estuarine and marine environments worldwide. Following the acquisition of virulence-associated genes, V. parahaemolyticus has emerged as a significant pathogen causing seafood-borne illnesses. The mechanisms and conditions that promote the emergence of disease causing V. parahaemolyticus strains are not well understood. In addition, V. parahaemolyticus clinical strains isolated from disease-associated samples and environmental strains from sediment, water, and marine organisms have been identified with considerable diversity; however, the evolutionary relationships of disease-causing strains and environmental strains are not known. In the following research, the evolutionary relationships of V. parahaemolyticus clinical and environmental strains are examined. In addition, the contribution of genetic elements and molecular mechanisms such as deficiency of DNA repair to the evolution of V. parahaemolyticus clinical and environmental strains is shown. Molecular analysis of the evolutionary relationships of V. parahaemolyticus clinical and environmental strains demonstrated separate lineages of pathogenic and non-pathogenic strains with the exception of several environmental strains that may represent a reservoir of disease-causing strains in the environment. Sequence characterization of plasmids isolated from diverse environmental Vibrios indicated a role of plasmids in strain evolution by horizontal transfer of housekeeping genes. In addition, analysis of plasmids from V. parahaemolyticus clinical and environmental strains indicated the existence of a plasmid family distributed among V. parahaemolyticus, V. campbellii, and V. harveyi environmental strains. Sequence characterization of a plasmid of this family from a V. parahaemolyticus environmental strain indicated the contribution of these plasmids to the emergence of the clonal pandemic strains. Investigation of the role of molecular mechanisms to the evolution of V. parahaemolyticus strains showed that inactivation of the DNA repair pathway methyl-directed mismatch repair (MMR) increased the accumulation of spontaneous mutations leading to increased nucleotide diversity in select genes. The research findings in the following chapters demonstrate a considerable contribution of genetic elements and molecular mechanisms to the evolution of genetic and phenotypic diversity.

Our objective was to address current cell source limitations in engineering pancreatic â-cells for the treatment of type 1 diabetes by investigating retroviral genetic modification of murine embryonic stem cells (mESC) with a murine stem cell virus (MSCV) encoding proendocrine transcription factor Neurogenin 3 (Ngn3). We found that expression of Ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mESCs. To overcome this obstacle and enhance the efficiency of retroviral gene transfer to mESCs in general, we employed a virus-polymer complexation method to deliver more transgenes to mESCs. Despite increased transgene delivery and integration in mESCs, transgene expression did not increase. Results suggest mESCs may be restricted in several steps of retrovirus transduction. We then investigated which steps of the virus lifecycle restrict efficient transduction of mESCs by using a recombinant MMuLV-derived retrovirus and a recombinant HIV-1-derived lentivirus to compare three major steps in the transduction of mESCs and NIH 3T3 cells - virus binding, virus integration, and transgene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 fibroblasts. We also detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels in NIH 3T3 cells, suggesting the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels, suggesting lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. We then investigated whether depletion of linker histone 1 in mESCs would alleviate silencing of retrovirus transgenes and improve gene transfer by transducing histone H1c, H1d, H1e triple null mESCs with different recombinant vectors. We found this did not improve viral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine.

A transformação genética pode contribuir substancialmente para o melhoramento genético do feijão, permitindo a introdução de genes que contribuam para o aumento da produtividade e estabilidade da produção. A metodologia de transformação genética de feijão (Phaseolus vulgaris), ora disponível (biobalística em embriões) apresenta baixa eficiência, o que dificulta o seu uso em pesquisas envolvendo a transferência de genes e não permite seu uso de forma ampla em programas de melhoramento genético da cultura. Um método efetivo e reprodutível de regeneração de plantas, a partir de células ou tecidos, é essencial em estudos de genética e melhoramento envolvendo a transferência de genes pela engenharia genética. Os métodos de transformação somente terão sucesso se tivermos previamente estabelecido um protocolo eficiente de regeneração de plantas a partir de tecidos potencialmente transformáveis. O objetivo principal deste trabalho é o desenvolvimento de um protocolo de transformação genética de feijão via Agrobacterium. O primeiro passo no desenvolvimento deste protocolo foi o estabelecimento de um sistema eficiente de regeneração de plantas a partir de calos. O passo seguinte foi o estabelecimento de metodologia da transformação de calos via Agrobacterium. / The genetic transformation can contribute substantially with the bean (Phaseolus vulgaris L.) breeding, allowing the introduction of genes to improve productivity and its stability. The transformation methodology of bean, now available (biolistic in embryos), is not efficient, which prevents its use in bean breeding programs. A reproducible and effective method of plant regeneration, from cells or tissues is essential in genetics studies and plant breeding, involving the genetic engineering. The transformation methods will only work if we can previously establish an efficient plant regeneration protocol from tissues with potential for transformation. The aim of this work was to develop an efficient transformation protocol of bean via Agrobacterium. The first step was to establish an efficient system of plant regeneration from callus, followed by the establishment of a transformation methodology via Agrobacterium.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_dissertacao_juliana_oliveira_de_carvalho.pdf: 19466 bytes, checksum: 575be4d4f71c27a4d8e3c7e3d7bacb3a (MD5) Previous issue date: 2018-07-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O Rio Grande do Sul é considerado o maior produtor de arroz irrigado do Brasil. Contudo, devido às condições climáticas da região, o potencial produtivo da lavoura de arroz ainda é limitado pela incidência de doenças fúngicas. A utilização de cultivares resistentes ou tolerantes é uma forma alternativa sustentável para reduzir as perdas na produção orizícola. A transformação genética, aliada a cultura de tecidos, vem auxiliando os programas de melhoramento vegetal na geração de plantas resistentes e tolerantes a estresses bióticos e abióticos, contribuindo para a sustentabilidade da orizicultura. Lectinas são proteínas que se ligam a carboidrados e estão envolvidas em diferentes processos biológicos, inclusive na defesa de plantas contra diversos tipos de patógenos. Desta forma, o objetivo do presente trabalho foi introduzir o gene da lectina bvl de Bauhinia veriegata em diferentes explantes da cultivar IRGA 426 a fim de se obter plantas transformadas geneticamente com o gene bvl. Para a obtenção das plantas transformadas foram usados calos organogênicos, mesocótilos e a técnica de transformação de botões florais. No experimento para a obtenção de calos via organogênese indireta, o 2,4-D na concentração de 2,0 mg L-1 foi efetiva, induzindo uma média de 47,3 calos, sendo 89% calos organogênicos aptos para à regeneração. Na regeneração dos calos, observou-se a formação média de 12,73 brotações no meio com 0,5 mg L-1 de ANA e 2,5 mg L-1 de BAP. Já na regeneração do mesocótilo, o incremento de BAP no meio de cultivo reduziu diretamente o comprimento das brotações primárias, no entanto esse efeito foi compensado pelo aumento da multiplicação dos explantes, principalmente com 5 mg L-1 de BAP, onde observou-se uma média de 21,16 brotações. Após o processo de infecção com o vetor pH7WGD2::bvl e seleção com antibiótico, os calos da cv. IRGA 426 apresentaram hiperhidricidade e não foram regenerados. Dos mesocótilos que passaram pela transformação, 20,66% sobreviveram ao meio de seleção. A análise de PCR revelou uma eficiência de transformação de 6,35% em relação ao total de brotos e, a partir do Western Blot, confirmou-se que 3 plantas estavam expressando a lectina BVL. No método de transformação imersão floral, em todas as sementes putativamente transformadas o gene gfp estava ativo, sendo observada a expressão transiente. De acordo com os resultados obtidos, os protocolos de regeneração dos explantes foram eficientes. Análises moleculares das plantas transformadas deverão ser realizadas para determinar o número de cópias do transgene no gDNA e o papel da lectina BVL na fisiologia vegetal e defesa da planta de arroz. / Rio Grande do Sul is the largest producer of irrigated rice in Brazil. However, due to the region's climate, the productive potential of crop farms is still limited by fungal diseases. The use of tolerant or resistant cultivars is a sustainable alternative to reduce loss in rice production. Genetic transformation, coupled with tissue culture, has been assisting vegetable breeding in the production of crops resistant and tolerant to abiotic/biotic stress, contributing to the sustainability of rice culture. Lectins are proteins that bind to carbohydrates and are involved in several biological processes, including defense against diseases in plants. The main goal of this study was the insertion of the Bauhinia variegata BVL Lectin gene in different explants of cultivar IRGA 426 to obtain transformed plants with the BVL gene. To obtain the transformed plants, organogenic callus, mesocotyls and flower buds transformation technique were used. In the experiment for obtaining callus through indirect organogenisis, 2,4-D at 2,0 mg L-1 was effective, inducing the formation of an average of 47,3 calluses, of which 89% of embriogenic callus were apt for regeneration. In the regeneration step, an average of 12,73 sprouts in medium with 0,5 mg L-1 of ANA and 2,5 mg L-1 of BAP was observed. In the mesocotyls regeneration, the BAP increment in the growing medium directly reduced the length of primary shoots; however, this effect was compensated by the increse of explant multiplication, mainly with 5 mg L-1 of BAP, where an average of 21,16 sprouts were observed. After infection with the pH7WGD2::bvl vector and antibiotic selection, the callus of cv. IRGA 426 showed hyperhydricity and were not regenerated. Of the mesocotyls that went through transformation, 20,66% survived the selection medium. PCR analysis revealed a transformation efficiency of 6,35% in all sprouts and, through Western Blot, 3 plants were confirmed to express the BVL lectin. In the floral immersion transformation method, in all transformed seeds the GFP gene was active, with transient expression being observed. Based on the results obtained, the explant regeneration protocols were efficient. Molecular analysis of the transformed plants should be made to determine the number of copies of the transgene in gDNA and the role of BVL lectin in the plant physiology and its role in the plant's defense.

Plantas transgênicas constituem interessantes alternativas para a produção de compostos com elevado valor agregado como polímeros industriais, proteínas farmacológicas e lipídios nutracêuticos. Como candidata à biofábrica, a cana-de-açúcar (Saccharum spp.) apresenta características agronômicas favoráveis, além de versatilidade de matéria-prima, que é empregada em fins tradicionais, como produção de álcool e açúcar, e até mesmo para geração de energia ou como forragem para alimentação de gado. Em nutrição animal, uma molécula bastante estudada é o ácido linoleico conjugado (CLA), que tem atividades anticarcinogênica, antidiabética, antiaterosclerose e moduladora do sistema imune e metabolismo de lipídios. O isômero t10,c12- CLA tem sido particularmente promissor na agropecuária por conta de inibição da síntese de gordura do leite e na melhoria do desempenho reprodutivo de vacas. Com o objetivo de avaliar a cana-de-açúcar como sistema biotecnológico para produção de CLA, o gene da isomerase do ácido linoleico de Propionibacterium acnes (pai) foi isolado e clonado, previamente caracterizado em Escherichia coli e, em seguida, utilizado na biolística de calos embriogênicos para regeneração de plantas transgênicas. No sistema procariótico, foi induzida a expressão de pai e a proteína heteróloga foi verificada como uma banda de 50 kDa, porém não houve alteração evidente do perfil de ácidos graxos da bactéria. Na cotransformação de cana-de-açúcar, o vetor pHA9, que contém o gene de seleção da neomicina fosfotransferase (nptII), foi bombardeado com uma das duas construções desenvolvidas com o promotor da poliubiquitina 1 de milho (pUbi1) dirigindo a expressão de pai adicionado ou não de sequência sinal da proteína de reparo de DNA recA para direcionamento para cloroplasto. Das cultivares CTC2 e IACSP9303046, foram obtidas eficiências de transformações de 2,2% e 1,7-7,1%, respectivamente. Um total de 156 plântulas foram regeneradas após regime de seleção com geneticina. Em seguida, 115 plântulas transgênicas foram identificadas por PCR para nptII e, entre essas, 29 e 48 foram PCRpositivas para pai e rec-pai, respectivamente. De 50 plantas aclimatizadas, 12 foram analisadas por Southern blot para nptII e 4 para pai, confirmando-se a transformação genética. Para comprovar a expressão de mRNA de pai, 27 plantas foram averiguadas por RT-PCR e RT-qPCR e indicaram produção de transcritos estudados. A análise de expressão das proteínas de folha por Western blot em 21 plantas selecionadas não produziu resultados conclusivos quanto à detecção de PAI. Essas mesmas foram analisadas por Cromatografia Gasosa, mas não foi detectado acúmulo de CLA. Embora seja possível a introdução e expressão do gene pai em cana-de-açúcar, é necessário avaliar em maior detalhe os fatores que possam afetar positivamente a produção de CLA, como tipo de tecido, compartimentos subcelulares para direcionamento proteico ou coexpressão de fosfolipases. / Transgenic plants are attractive alternatives for the production of high value compounds as industrial polymers, pharmacological proteins and nutraceutical lipids. As a candidate for biofactory, sugarcane presents favorable agronomic characteristics and versatility of raw material which is used for traditional purposes such as ethanol and sugar production and even for power generation or as forage feeding of cattle. In animal nutrition, a widely studied molecule is the conjugated linoleic acid (CLA), which has anticarcinogenic, antidiabetic, antiatherosclerosis and immune system and metabolism of lipids modulator activities. The isomer t10,c12-CLA has been particularly promising in agriculture because of inhibition of milk fat synthesis and improvement of the reproductive performance of cows. Aiming to evaluate the sugarcane as a system for biotechnological production of CLA, the linoleic acid isomerase gene from Propionibacterium acnes (pai) was isolated and cloned, previously characterized in Escherichia coli and then used for biolistic of embryogenic calli for regenerating transgenic plants. In the prokaryotic system, the expression of pai was induced and the heterologous protein was verified as a band of 50 kDa, but there was no obvious change in fatty acid profile of the bacterium. In cotransformation of sugarcane, pHA9 vector containing the selection gene neomycin phosphotransferase (nptII) was bombarded with one of the two constructions developed with the promoter of maize polyubiquitin 1 (pUbi1) driving the expression of pai, either added or not with the signal sequence of DNA repair protein recA for targeting to chloroplast. Cultivars CTC2 and IACSP93-3046 were obtained with transformation efficiencies of 2.2% and 1.7 a 7.1%, respectively. A total of 156 plantlets were regenerated after selection with geneticin regimen. After that, 115 transgenic plantlets were identified by PCR for nptII and among then, 29 and 48 were PCR-positive for pai and rec-pai, respectively. Of 50 acclimatized plants, 12 were analyzed by Southern blot for nptII and 4 for pai, confirming the genetic transformation. In order to prove the expression of pai mRNA, 27 plants were verified by RT-PCR and RT-qPCR and indicated the production of the studied transcripts. Expression analysis of leaf proteins by Western blot in 21 plants selected produced no conclusive results regarding the detection of PAI. Those were analyzed by gas chromatography and no accumulation of CLA was detected. Although it was possible to introduce and express the pai gene in sugarcane, it is necessary to evaluate in more detail the factors that may positively affect the production of CLA, as tissue type, subcellular compartments for protein targeting or coexpression of phospholipases.

Atualmente a cultura da cana de açúcar tem ganhado destaque no cenário mundial devido a seu potencial uso na produção de bioenergia o qual poderia ser beneficiado desenvolvendo-se cultivares com aumento da produtividade de biomassa por unidade de área, o que é por sua vez, determinada pela arquitetura da planta. A brotação lateral é um dos principais fatores que regulam a arquitetura dos vegetais. Recentemente, esta fase do desenvolvimento tem sido estudada intensivamente em plantas consideradas modelo, elucidando em parte as vias genéticas, ambientais e hormonais que regulam este processo. Dentro desta vias, microRNAs, uma classe de pequenos RNAs não codantes que modula pós-transcricionalmente a expressão de genes endógenos, parecem ser importantes reguladores. Em cana de açúcar, a brotação lateral é importante para a arquitetura dos ramos laterais, germinação de gemas e perfilhamento. Entretanto, devido a sua complexidade genética e ausência de mutantes defectivos na brotação lateral, estudos nesta área ao nível molecular são limitados. Neste contexto, este trabalho teve por objetivos estudar em cana de açúcar a via microRNA156/fatores de transcrição do tipo SQUAMOSA promoter-binding-protein (SPL), a qual é associada à regulação do perfilhamento, bem como caracterizar molecularmente o processo de emergência de gemas axilares. Ferramentas computacionais usando o banco publico de ESTs TIGR gene índex permitiram identificar e classificar no genoma de cana de açúcar, diferentes genes associados ao processo de brotação lateral e resposta hormonal. Entres estes, seis genes SPL regulados pelo miR156 foram identificados, sendo um deles (SsSPL1) homólogo a SPLs envolvidas diretamente na regulação do perfilhamento. A expressão da SsSPL1 foi monitorada em diferentes tecidos e órgãos, juntamente com o miR156. Os dados sugerem que a SsPL1, é regulada negativamente pelo miR156, sendo esta via também conservada em cana de açúcar. Foram geradas plantas transgênicas da cultivar RB85486 com o gene endógeno SsmiR156a/b o qual codifica um precursor conservado do miR156 e parece estar associado com a evolução da arquitetura em monocotiledôneas. O acúmulo do miR156 foi avaliado em plantas transformadas via RT-qPCR encontrando variabilidade na sua expressão. Bibliotecas de pequenos RNAs foram geradas em gemas dormentes e em desenvolvimento, permitindo identificar membros de 26 famílias de miRNAs. A expressão de quatro deles, de seus genes-alvo e de outros genes selecionados foi monitorada em gemas dormentes e com 2 e 5 dias após o plantio. Interessantemente, o miR159 foi o mais expresso em gemas axilares de cana e parece ser um fator chave na emergência da gema, já que, segundo os resultados obtidos, esse miRNA parece modular a expressão do seu gene alvo SsGAMyB, o qual é um fator de transcrição implicado na ativação de genes de resposta a giberelina. Durante esta fase inicial do desenvolvimento também foi observado alterações na expressão de genes associados com processos de transdução de sinal associados aos fitohormônios auxina e etileno. Os resultados obtidos indicam que a emergência de gemas laterais é um processo dinâmico em que fitohormônios, fatores de transcrição e microRNAs participam conjuntamente para promover o crescimento e 12 desenvolvimento da nova plântula de cana de açúcar. / Sugarcane is an economically important biofuel crop that recently has become a target for improvement of sustainable biomaterial production due to its high biomass productivity and built-in containment features. Therefore, studies aiming to improve the production of biomass per area are among the most important issues in sugarcane production. Plant biomass is defined, at least in part, by its shoot architecture. Although shoot architecture (branching/tillering) is to some extend influenced by environmental factors, it is determined mainly by the plants genetic program. This includes developmental programs that are regulated by a complex network of genetic pathways that integrate endogenous and environmental cues. Several transcription factors as well as microRNAs are likely part of this network. In this study, we started to investigate the roles of the genetic pathway regulated by the microRNA156 and its targets, the transcription factors SQUAMOSA promoter-binding-protein (SPLs) in sugarcane branching/tillering. We identified six members of the SPL family that were further classified into four subfamilies. In both dicots and monocots, these SPLs are key regulators of the plant shoot architecture. We monitored the expression patterns of SsmiR156 e SsSPL1 in distinct sugarcane tissues/organs. Our observations suggest that miR156 regulates posttranscriptionally SsSPL1 mainly in leaf tissues. We generated transgenic sugarcane plants overexpressing the monocot-specific sugarcane miR156 precursor SsMIR156b/c via biolistic method. This precursor is thought to be important for the evolution of grass shoot architecture. Although we observed higher accumulation of mature SsmiR156b/c in leaf tissues of some transgenic plants as compared with tissues from non-transgenic plants, we could not detect any significant changes in their vegetative architecture. Using deep sequencing approaches, we have generated two small RNA libraries from dormant and outgrown sugarcane lateral buds. Preliminary analyses indicate that a select group of small RNAs are expressed in lateral buds, including over 200 repeat-associated small interfering RNAs (rasiRNAs) and 25 conserved microRNAs (miRNAs). Amongst the miRNAs, miR159 was the most sequenced in the two libraries. We evaluated miR159 accumulation pattern in addition to other selected miRNAs via qRT-PCR in dormant and developing buds. The majority of the evaluated miRNAs accumulate differentially during bud development, though with distinct expression patterns. Interestingly, miR159 accumulates at high levels in dormant buds, but scarcely in developing buds. Conversely, the experimentally confirmed miR159 target, a sugarcane GAMyB-like gene (SsGAMyB), is lowly expressed in dormant buds while its transcripts accumulate at higher levels in developing buds. GAMyB-like genes encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) and abscisic acid (ABA) signaling in germinating seeds. Our data suggest miR159 regulates GAMyB-like genes during sugarcane bud outgrowth. Similarly, SsSPL1 is regulated posttranscriptionally by the miR529, though this gene has sites for both miR529 and miR156. Auxin and ethylene-associated regulatory pathways are affected during sugarcane bud development. Taken together,our data indicate that sugarcane bud outgrowth from rhizomes is a complex developmental process involving hormones, transcription factors as well as microRNAs and other regulatory RNAs.

A transformação genética está se tornando uma importante ferramenta, dentro dos programas de melhoramento genético de citros, e uma alternativa para contornar barreiras naturais da espécie, que dificultam o desenvolvimento de novas variedades pelo melhoramento convencional. Entretanto, os protocolos utilizados para transformação genética de citros têm resultado num baixo número de plantas transgênicas. Com o objetivo de estudar fatores que possam influenciar o processo de transformação genética de citros via Agrobacterium tumefaciens analisou-se o efeito do uso de acetoseringona em diferentes etapas do processo, as condições de incubação dos explantes durante e após o período de co-cultivo e o tipo de corte do explante. Foram utilizados segmentos de epicótilo de plântulas germinadas in vitro da variedade de laranja doce ‘Hamlin’ (Citrus sinensis L. Osbeck.) e da variedade citrange ‘Carrizo’ (C. sinensis x Poncirus trifoliata Raf.), inoculados com a estirpe EHA 105 de A. tumefaciens, contendo o plasmídeo p35SGUSINT, com o gene de seleção que codifica a enzima neomicina fosfotransferase II (nptII) e o gene repórter uidA que codifica a enzima b-glucuronidase (gus). O protocolo básico para transformação genética foi com a inoculação dos explantes por 20 minutos, com o período de co-cultivo de 3 dias em me io de cultura suplementado com acetoseringona (100 mM) e transferidos para meio de cultura de seleção, constituído de meio de cultura EME, suplementado com BAP (1mg L -1 ), canamicina (100 mg L -1 ) e cefotaxime (500 mg L -1 ). As avaliações foram realizadas após 5-6 semanas de incubação, determinando-se o número de explantes com gemas adventícias, o número de gemas adventícias gus + e calculando-se a eficiência de transformação genética, definida pela relação entre o número de gemas gus + regeneradas e o número de explantes inoculados. Pelos resultados obtidos pôde-se observar uma maior eficiência de transformação genética para a variedade citrange ‘Carrizo’; e que a eficiência da transformação genética aumentou, principalmente para a variedade de laranja doce 'Hamlin', quando a incubação do material durante o período de co-cultivo foi feita sob temperaturas inferiores a 27 °C. A suplementação do meio de cultura de co-cultivo com acetoseringona depende do pH deste meio de cultura. A utilização de explantes seccionados longitudinalmente não se mostrou favorável devido ao crescimento excessivo da bactéria na superfície do explante. / Genetic transformation is an important biotechnological tool in citrus genetic breeding programs, and an alternative to overcome natural barriers to the development of new varieties, by conventional breeding. However, the protocols used for citrus genetic transformation have resulted in a low number of transgenic plants. Therefore, this research evaluated some factors that might influence citrus genetic transformation process via Agrobacterium tumefaciens, such as: the addition of acetosyringone in different steps of the process, the explant incubation conditions during and after the period of co-cultivation, and the explant type cut. Epicotyl segments from seedlings of 'Hamlin' sweet orange (Citrus sinensis L. Osbeck) and ‘Carrizo’ citrange (C. sinensis L. Osbeck x Poncirus trifoliata Raf.) were inoculated with EHA 105 strain of A. tumefaciens harboring the binary plasmid p35SGUSINT containing the selection gene for neomicyn phosphotransferase II (nptII) and the reporter gene uidA for b-glucuronidase (GUS). The basic protocol for genetic transformation included the explant inoculation for 20 minutes, and 3 days of co-cultivation in the culture medium supplemented with acetosyringone (100 mM). The epicotyl segments were transferred to selection culture medium, EME culture medium supplemented with BAP (1 mg L -1 ), kanamycin (100 mg L -1 ) and cefotaxime (500 mg L -1 ). The evaluations were done after 5-6 weeks of incubation, determining the number of explants with shoots, the number of gus + shoots, and calculating the genetic transformation efficiency, defined by the relation between the number of gus + shoots and the number of inoculated explants. The highest genetic transformation efficiency was observed in 'Carrizo' citrange. An increase of genetic transformation efficiency, mainly for the 'Hamlin' sweet orange occurred when the segments were incubated under temperatures below 27 o C. The supplementation of the co-cultivation culture medium with acetosyringone depends on pH value of itself. The use of explant sectioned did not favor transformation, probably due to the excessive bacterial growth on explant surface.

A cana-de-açúcar é uma das principais culturas agrícolas no cenário econômico e social brasileiro. Na cultura de cana-de-açúcar o estresse hídrico é o principal fator limitante para o aumento de produtividade, sendo responsável por alterações fisiológicas, bioquímicas e moleculares nas plantas, que podem deflagrar perturbações metabólicas que ativam a morte celular programada (MCP). Sabendo-se que o gene BI-1 apresenta o potencial de reduzir os efeitos da MCP desencadeado por estresses bióticos e abióticos em plantas, este trabalho teve como objetivo analisar plantas transgênicas de cana-de-açúcar que expressam o gene BI-1 de Arabidopsis thaliana (AtBI-1) em condições de estresse hídrico. Também, plantas transgênicas e controle foram inoculadas com o fungo Puccinia melanocephala demonstrando que o processo de transformação genética com o gene AtBI-1 alterou as características pré existentes de resistência a ferrugem marrom nas plantas transgênicas. Os estudos de tolerância ao défict hídrico foram realizados em dois experimentos, o experimento 1 com plantas transgênicas e controles de 90 dias e o experimento 2 com plantas de 60 dias. Plantas do experimento 1 foram analisadas quanto características morfológicas como número de estômatos e tricomas, altura e circunferência do colmo e após ficarem 24 dias sem água foram analisadas quanto a taxa fotossintética, comportamento estomático e conteúdo relativo de água nas folhas, enquanto no experimento 2 as plantas foram analisadas quanto aos teores de prolina, atividades das enzimas guaiacol peroxidase (GPOX), ascorbato peroxidase (APX) e catalase (CAT) após as plantas ficarem 17 dias sob déficit hídrico. Estas enzimas estão envolvidas em processos de desativação de elementos ativos de oxigênio. Os resultados demonstraram que as plantas transgênicas expressando o gene AtBI-1 possuem fenótipo de menor altura, e maior taxa fotossintética, maior comportamento estomático e maior conteúdo relativo de água nas folhas, e assim apresentam maior tolerância ao déficit hídrico que plantas controle. Contudo, houve baixo acúmulo de prolina, baixa atividade da GPOX, APX e CAT nas plantas transgênicas durante o estresse hídrico comparada com as plantas controle do mesmo tratamento. Porém foi observado alta atividade constitutiva da catalase nas plantas transgênicas. A atividade da catalase nestas plantas transgênicas sugere a possibilidade da interação entre AtBI-1 e calmudolinas. Futuros estudos podem contribuir para elucidar se a proteína BI-1 é essencial para a ativação das catalases por calmudolinas. / Sugarcane is one of the main agricultural crops in the Brazilian social and economic scenario. Water stress in the culture of sugarcane is the main limiting factor for increasing productivity accounting for physiological, biochemical and molecular plants that can trigger metabolic disturbances activating programmed cell death (MCP). Knowing that the BI-1 gene has the potential to reduce the effects of MCP triggered by biotic and abiotic stresses in plants, this study aimed to analyze transgenic sugarcane that express the BI-1 gene of Arabidopsis thaliana (AtBI-1) under water stress. Also, transgenic and control plants were inoculated with Puccinia melanocephala fungus demonstrating that the genetic transformation process with the AtBI-1 gene altered the pre-existing characteristics of brown rust resistance in transgenic plants. Studies of tolerance to water deficit were performed in two experiments, the experiment 1 was prepared with transgenic and control plants with 90 days and the experiment 2 used plants with 60 days. Plants from experiment 1 were analyzed as for morphological characteristics such as number of stomata and trichomes, height and diameter of stem after plants being under water for 24 days as were analyzed photosynthetic rate, stomatal behavior, relative water content in leaves while in the experiment 2, plants were analyzed for the levels of proline, enzyme activities of guaiacol peroxidase (GPOX), ascorbate peroxidase (APX) and catalase (CAT) under water deficit for 17 days. These enzymes are involved in deactivation of active elements oxygen. The results demonstrated that the transgenic plants expressing the AtBI-1 gene presented the phenotype of lower height, higher index of leaf area, higher photosynthetic rate, higher stomatal behavior and higher relative water content in leaves than control plants increasing tolerance to drought stress. However, there were low levels of proline, low activity of GPOX activity, APX and CAT in transgenic plants during drought stress compared to control plants of the same treatment, but the observed high constitutive activity of catalase in transgenic plants. Catalase activity in these transgenic plants suggests the possibility of interaction between AtBI-1 and calmudolinas. Future studies may contribute to understand whether the BI-1protein is essential for the activation of catalase by calmudolinas.