A família Bromeliaceae apresenta importância ecológica e econômica, desta forma, o desenvolvimento de protocolos para a micropropagação de espécies dessa família, faz-se necessário, a fim de suprir sua demanda comercial e mesmo ecológica. A escolha do meio de cultura e do explante utilizado durante a micropropagação são fundamentais para um protocolo eficaz. Nesse contexto, o objetivo deste estudo foi avaliar as diferenças quantitativas e qualitativas no desenvolvimento de explantes de Neoregelia sp em meios de cultura e monitorar as vias morfogênicas dos propágulos obtidos em explantes foliares. Para tanto, brotos de microcepas e explantes foliares procedentes de um micro jardim clonal, foram transferidos para os meios de cultura de multiplicação MS, ½ MS e WPM, todos suplementados com 0,050 mg.L-1 ANA e 0,50 mg.L-1 de BAP, onde foram mantidos por 120 dias e submetidos a diversas análises morfofisiológicas. Paralelamente, explantes foliares foram mantidos em meio de cultura MS de multiplicação para o monitoramento das vias morfogênicas durante os processos regenerativos. Para os experimentos com brotos de microcepas verificou-se que o meio de cultura MS proporcionou a melhor taxa de multiplicação, maior crescimento dos brotos, obtendo os valores mais elevados de peso de matéria fresca e seca, além disso, apresentaram maior acúmulo de nitrogênio total e proteico. No entanto, os meios de cultura ½ MS e WPM promoveram uma taxa de multiplicação semelhante a do MS, mas com brotos menores e menos vigorosos, porém, mais homogêneos, com isso, na dependência do objetivo do cultivo in vitro, não deve ser desconsiderada a possibilidade de utilização dos meios de cultura ½ MS e WPM. Os explantes foliares não se desenvolveram bem no meio de cultura WPM, não havendo diferença entre os meios MS e ½ MS, visto que ambos apresentaram resultados satisfatórios. As análises histológicas e histoquímicas identificaram células parenquimáticas, que atuam como células-tronco, manifestando capacidade morfogênica para toti ou pluripotência, dando origem respectivamente a embriões somáticos e gemas adventícias, em resposta aos estímulos in vitro. / The Bromeliaceae family has an ecological and economic importance, therefore, the protocols development for micropropagation of species of this family becomes necessary in order to meet its business and even its ecological demand. The choice of culture medium and the explant used during micropropagation are essential for an effective protocol. Thus, the aim of this study was to evaluate the quantitative and qualitative differences in the explants development of Neoregelia sp in the culture media and monitor the morphogenetic pathways of obtained propagules from leaf explants. Consequently, shoots and leaf explants coming from microcloning garden were transferred to the MS, ½ MS and WPM multiplication culture media, all supplemented with 0.050 mg.L-1 NAA and 0.50 mg.L-1 BAP, where they were held for 120 days and submitted to morphological and physiological analysis. Therefore, leaf explants were kept on MS-medium multiplication for monitoring morphogenetic pathways during the regenerative processes. Furthermore, MS medium showed the best multiplication rate for the sprouts of the microstumps, increased growth of shoots, obtaining the highest values of fresh and dry matter weight, and also showed higher accumulation of total nitrogen and protein. However, the ½ MS and WPM culture media promoted a similar multiplication rate to the MS medium, with the development of the smaller and less vigorous shoots, but with greater homogeneity. This way, depending on the purpose of in vitro culture, their use in the micropropagation for this species should not be disregarded. The leaf explants are not well developed in WPM medium, and don\'t had significant difference between the MS and ½ MS culture medium, as both showed satisfactory results. The histological and histochemical analysis identified the presence of the parenchymatic cells, which act as stem cells, expressing morphogenic ability for toti or pluripotency, leading respectively to somatic embryogenesis or adventitious organogenesis in response to in vitro stimuli.

O crescente aumento no uso da micropropagação de teca como forma de produção de clones com qualidades genotípicas e fenotípicas selecionadas a partir de árvores de elite, determinou a importância desse método, pois origina plantações com maior qualidade e uniformidade, agregando maior valor ao preço da madeira no mercado. O uso de sementes para a obtenção de mudas é uma técnica menos onerosa, porém resulta em plantas com tamanhos desiguais e não há um padrão na qualidade da madeira, essa técnica depende também da época de produção de sementes e, portanto é restrita a um período do ano. A micropropagação permite a clonagem em larga escala das árvores de elite em tempo e espaço reduzidos, podendo ser realizada em qualquer época do ano, além disso, permite a formação de mudas totalmente livres de pragas e patógenos. Faz-se necessário, maiores estudos com meio de cultura para Tectona grandis, pois os materiais relacionados a esse assunto são escassos. Para que a técnica do cultivo in vitro da teca seja incrementada, objetivou-se nesse experimento, otimizar o crescimento dos explantes de três clones diferentes, testando a eficiência de 6 meios de cultura com diferentes formulações nutricionais e constatar qual deles apresenta a melhor resposta para cada clone. O estudo contou com 6 tratamentos (MS, Básico, M1, M2, M3 e M4), durante oito épocas de avaliação (0, 7, 14, 21, 28, 35, 42 e 49 dias), para três clones de Tectona grandis (61, 62 e 68), com três repetições por tratamento/clone, utilizando o delineamento experimental inteiramente casualizado. A avaliação do crescimento foi feita por meio do peso de matéria fresca (PMF), matéria seca (PMS) e a taxa de crescimento relativo (TCR) proporcionado pelos meios. O PMF foi usado para obtenção do PMS e cálculo da TCR. Baseado nos valores do PMS obtidos, para o clone 61, constatou-se a formulação do meio Básico (PMS = 0,38 g), como a mais eficiente. Para o clone 62, o meio mais responsivo foi M4 (PMS = 0,47 g) e no clone 68, destacou-se o meio M3 (PMS = 0,71 g). Quanto a TCR, não foi encontrada diferença estatística significativa para nenhum dos 6 meios de cultura levando-se em conta os três clones. / The increasing use of teak micropropagation as a way of producing clones with genotypic and phenotypic qualities selected from elite trees, established the importance of this method, it leads to higher crop quality and consistency, adding more value to the price of wood business. The use of seeds to obtain seedlings has a less cost, but it results in plants with unequal sizes and wood quality without a pattern. This technique also depends on the time of seed production and therefore is restricted to a year period. Micropropagation allows cloning elite trees in large-scale and reduced time and space. It can be performed at any time of year, in addition, allows the formation of plants totally free of pests and pathogens. It is necessary more studies with culture medium for Tectona grandis, because the materials related to this subject are scarce. To increase the technique of teak in vitro cultivation, this experiment aimed to optimize the growth and development of explants from three different clones, testing the effectiveness of six culture media with different nutritional formulations and find which one offers the best answer to each clone. The study included six treatments (MS, Basic, M1, M2, M3 and M4) for eight periods (0, 7, 14, 21, 28, 35, 42 and 49 days) for three clones of Tectona grandis (61, 62 and 68) with three replicates per treatment/clone, in a randomized experimental design. The growth assessment was performed by the fresh matter weight (FMW), dry matter weight (DMW) and relative growth rate (RGR) provided by the media. The FMW was used to obtain the DMW and to calculate RGR. Based on DMW values obtained for clone 61, was found that the formulation of Basic medium (DMW = 0.38 g) was the most efficient. For clone 62, the most responsive medium was M4 (DMW = 0.47 g) and to clone 68, M3 (DMW = 0.71 g) was the highlighted medium. As the RGR, it was found no statistically significant difference for any of the six culture media taking into account the three clones.

Made available in DSpace on 2017-07-25T19:29:59Z (GMT). No. of bitstreams: 1 francielicolussi.pdf: 2001927 bytes, checksum: 373492674f45bb0ed7d467ad407070d5 (MD5) Previous issue date: 2006-09-01 / The general objective of this research was the evaluation of in vitro culture of Euphorbia heterophylla L. one of the principal weeds that effects yield of important economic crops such as soybean and corn. In addition, some assays considered the evaluation of the species’ potential biochemical value to the pharmaceutical industry. In vitro germination of E. heterophylla seed was tested on cotton batting as substrate, full strength and ½ MS basal medium. Results show a greater percent germination and better growth indices of plant development on the ½ MS medium. Explants tested for organogenesis included hypocotyls, cotyledon and root explants which were plated on media containing the following plant growth regulators (phytohormones): 0.5 mg/L 2ip, 1mg/L Kin + 0.1 mg/L IAA, 0.5 mg/L 2ip + 0.1 mg/L IAA, 1mg/L 2ip + 0.1mg IAA, 1.5 mg.L-1 2iP + 0.1 mg.L-1 AIA added to ½ MS media. Explants were evaluated for the presence of buds, the number of buds/ explant and the number of plantlets that were rooted and re-established. On the average, hypocotyls explants responded with two buds/ explant and 50% of these developed by direct organogenesis on media containing 0.5 mg/L of 2ip. No regeneration was observed on cotyledon or root explants. Root cultures developed in all treatments with or without auxin. These results will assist in future physiological tests such as evaluating the herbicide resistance of this species and also in evaluating possible use in the pharmaceutical industry. With this objective in mind we tested the sensitivity of root cultures in vitro. Roots were placed in ½ MS media containing 0,1,2,4 and 8 times the manufacturer’s recommended dose for the products Scepter® and Flex®. The active ingredients were imazaquin and fomesafen respectively, and they are inhibitors of ALS synthesis and PROTOX. The results obtained showed evidence of suppression of the normal growth of these roots as compared to the control treatment. Therefore, this suggests that an in vitro test of this type can be used to measure resistance this species to herbicides. / O objetivo geral deste trabalho foi estudar o cultivo in vitro de Euphorbia heterophylla. L, que é uma das principais plantas daninhas que afeta a produtividade das grandes culturas como soja e milho. Essa espécie também vem sendo estudado pelos aspectos fitoquímicos para indústria farmacêutica, para isto foram propostos alguns ensaios. Para avaliar a germinação in vitro de sementes de E. heterophylla foram testados substratos de algodão, meio MS e MS/2. Os resultados mostraram maior porcentagem de germinação e melhores índices de desenvolvimento das plantas em meio MS/2. Para o teste de organogênese foram utilizados explantes hipocotiledonares, cotiledonares e radiculares com as seguintes tratamentos: 0,5 mg.L-1 2iP, 1mg.L-1 CIN + 0,1 mg.L-1 AIA, 0,5 mg.L-1 2iP + 0,1 mg.L-1 AIA, 1 mg.L-1 2iP + 0,1 mg.L-1 AIA , 1,5 mg.L-1 2iP + 0,1 mg.L-1 AIA adicionado ao meio MS/2. Foram avaliados o número de explantes com gema, número de gemas por explante, número de plântulas enraizadas e aclimatadas. Obteve-se em média duas gemas por explante hipocotiledonar, em 50% destes, a partir de organogênese direta com o uso de 0,5 mg.L-1 de 2iP. Não houve regeneração a partir dos explantes cotiledonares e radiculares. A cultura racinar desenvolveu-se em todos os tratamentos contendo ou não auxina. Com o objetivo de avaliar a resistência desta planta a herbicidas, testamos a sensibilidade de culturas de raízes in vitro. As raízes foram repicadas em meio MS/2 com as seguintes concentrações: 0; 1; 2; 4 e 8 vezes a dose recomendada pelo fabricante dos herbicidas com ingrediente ativo imazaquin o fomesafen, inibidores da síntese de ALS e PROTOX. Com nomes comerciais de Scepter® e Flex®, respectivamente. Os resultados obtidos evidenciaram um retardo no crescimento normal destas raízes, quando comparada a testemunha.

A transformação genética vem sendo, cada vez mais, incorporada em programas de melhoramento genético de diversas espécies. Esta técnica permite a incorporação de gene(s) exógeno(s) no genoma das plantas, modificando características específicas. Assim, apresenta-se como uma importante ferramenta de auxílio ao melhoramento convencional de citros, que possui uma série de limitações impostas pela sua biologia reprodutiva. Entretanto, a transformação genética requer o estabelecimento prévio de sistemas de regeneração de plantas in vitro como requisito essencial para sua execução. Portanto, o objetivo deste trabalho foi estabelecer as condições de cultivo in vitro para organogênese e transformação genética de laranja 'Hamlin', laranja 'Pera', laranja 'Valência', laranja 'Natal' (Citrus sinenis L. Osbeck) e limão 'Cravo' (Citrus limonia L. Osbeck), realizando-se a caracterização anatômica do processo. Buscou-se, inicialmente, otimizar o processo de organogênese e regeneração de plantas in vitro, a partir de segmentos de epicótilo. Explantes foram introduzidos em meio de cultura MT suplementado com BAP, nas concentrações 0,0; 0,5; 1,0; 1,5; 2,0; 2,5; 3,0; 3,5; 4,0 ou 4,5 mg.L -1 . Avaliaram-se o percentual de explantes responsivos e o número de brotações maiores ou iguais a 1,0 cm por explante responsivo. As brotações obtidas foram transferidas para meios de enraizamento, que se constituíram do MT + 1,0 mg.L -1 de NAA, MT + 1,0 mg.L -1 de IBA e MT na ausência de auxina. A concentração de BAP que melhor favoreceu a indução da organogênese foi 1,0 mg.L -1 para as laranjas doces e 0,5-2,5 mg.L -1 para o limão 'Cravo'. O meio de enraizamento com melhor resposta foi o MT + 1,0 mg.L -1 de IBA, para todos os cultivares estudados. Procedeu-se análise anatômica da organogênese in vitro, nas condições ótimas obtidas no trabalho anterior. As gemas adventícias tiveram origem endógena, formando-se a partir de zonas meristemáticas do câmbio vascular, caracterizando-se em organogênese direta. Desenvolveram-se, também, experimentos para estudar a transformação genética, via Agrobacterium, em segmentos de epicótilo de laranja 'Natal', laranja 'Valência' e limão 'Cravo'. Neste caso, estudou-se o tempo de inoculação com Agrobacterium, o período de co-cultivo, a utilização de acetoseringona e a temperatura de incubação durante o co-cultivo e o seccionamento do explante. Plantas transgênicas foram obtidas utilizando-se um período de inoculação de 20 minutos com Agrobacterium tumefaciens (EHA-105), co-cultivo por 3 dias na ausência de acetoseringona no meio de cultura e temperatura de co-cultivo de 23-27 °C. Finalmente, desenvolveu-se um estudo da indução da organogênese, análise histológica e transformação genética, a partir de segmentos internodais de plantas adultas das laranjas 'Hamlin', 'Pera', 'Valência' e 'Natal'. A organogênese foi induzida em meio de cultura DBA3, modificado, suplementado com 1,0 mg.L -1 de BAP e 0,5 mg.L -1 de NAA. Em função dos cortes histológicos concluiu-se que as gemas adventícias formaram-se na superfície do calo, o qual originou-se de sucessivas divisões celulares do câmbio, caracterizando-se em organogênese indireta. Plantas transgênicas de tecido adulto de laranja 'Hamlin' e laranja 'Valência' foram obtidas utilizando-se um dia de co-cultivo a 24 ºC, com ou sem o seccionamento do explante para Hamlin e apenas com o seccionamento do explante para Valência. / Genetic transformation has been more frequently associated with conventional genetic breeding programs of different species. It allows for the introduction of exogenous gene(s) into the plant genome, with the possibility of altering specific characteristics. Thus, it can be an important tool for Citrus conventional breeding programs, which present several limitations imposed by the characteristics of the reproductive biology of this genus. For the success of the transformation system, however, the previous establishment of an efficient in vitro regeneration system is required. The objective of this research was to define in vitro culture conditions for the organogenesis and genetic transformation of Hamlin, Pera, Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck) with the anatomical characterization of the process. Initially, the optimization of the in vitro organogenesis process and plant recovery was attempted using epicotyl segments as explants. For organogenesis induction, the explants were placed in MT culture medium supplemented with BAP (0.0; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0 or 4.5 mg.L -1 ). The percent of responsive explants and the number of adventitious shoots per explant (> 1.0 cm) were evaluated. The shoots were transferred to rooting media, consisting of MT medium supplemented with NAA or IBA, or absence of auxin. The best BAP concentration for organogenesis induction was 1.0 mg.L -1 for the sweet oranges and between 0.5 and 2.5 mg.L -1 for Rangpur lime. The best rooting medium was MT with 1.0 mg.L -1 IBA for all the cultivars. Anatomical analysis was done to describe the optimized culture conditions. Adventitious buds originated endogenously from meristematic regions of the vascular cambium, characterizing a direct organogenesis. Studies were also done to analyze the genetic transformation of the sweet orange cultivars Natal and Valencia and Rangpur lime via Agrobacterium. Several experiments were installed to define the period of Agrobacterium inoculation and co-cultivation, the presence or absence of acetoseryngone, the temperature of incubation and the explant condition (with or without a longitudinal cut). Transgenic plants were obtained using an inoculation period of 20 minutes and co-cultivation for 3 days at 23-27 °C in absence of acetoseryngone in the culture medium. Finally, a study of organogenesis induction, histological characterization and genetic transformation from internodal segments of mature plants of sweet orange cultivars Hamlin, Pera, Valencia and Natal was conducted. Organogenesis was induced in DBA3 modified medium supplemented with 1.0 mg.L -1 BAP and 0.5 mg.L -1 NAA. Histological analysis showed that the adventitious buds formed indirectly from the callus formed by successive cell divisions from the vascular cambium. Transgenic plants from mature tissue of Hamlin and Valencia sweet oranges were obtained using one day of co-cultivation at 24°C with or without the longitudinal sectioning of the explant for Hamlin and only when the explant was longitudinally sectioned for Valencia.

Durante esse trabalho de pesquisa verificou-se que a aquisição de competência para conversão de ápices radiculares de Catasetum fimbriatum em gemas caulinares aumentava à medida que as plantas envelheciam. Esse processo esteve relacionado ao estabelecimento do crescimento determinado das raízes e com a parada da atividade e re-organização estrutural do meristema apical radicular (MAR). Este, quando ainda jovem e destituído de competência para a conversão em gemas, apresentava uma organização do tipo fechada, ao passo que em estágios avançados do envelhecimento este padrão transformou-se em um tipo aberto, marcado pela diferenciação e predominância de células parenquimáticas. Tais alterações, aparentemente, ocorreram com a concomitante perda das características e funções do centro de quiescente (CQ). De maneira complementar, constatou-se que a aquisição de competência do MAR para conversão em gemas estava correlacionada a uma série de alterações metabólicas, as quais, supostamente, participaram de uma condição fisiológica favorável a esse processo. Com base no conjunto de dados obtidos, pode-se observar que os teores endógenos de importantes participantes na progressão de divisões celulares, tais como auxinas, citocininas e formas reduzidas de ascorbato e glutationa tenderam a diminuir durante o envelhecimento das raízes. Por outro lado, durante esse mesmo período, o conteúdo de alguns hormônios envolvidos na sinalização de condições de estresse ou diferenciação celular, tais como etileno, ácido abscísico e giberelinas tenderam a aumentar. As concentrações relativas de importantes sinalizadores secundários, tais como óxido nítrico e cálcio citossólico também apresentaram aumento conspícuo na região do MAR durante o envelhecimento. Agregando elementos a estas constatações, verificou-se que o transporte polar de auxina seria um importante sinal posicional para a manutenção das características e função do MAR, uma vez que o seu bloqueio em plantas jovens foi suficiente para causar a aquisição da competência do MAR, no entanto, o processo de conversão não era consolidado enquanto os ápices radiculares permaneceram ligados às plantas. A aplicação de etileno em plantas jovens, por sua vez, desencadeou efeitos similares; no entanto, além de induzir a competência, esse hormônio também proporcionou a conversão dos MARs em gemas via aumento nos teores endógenos de citocininas. O tratamento de ápices radiculares jovens com diferentes tipos de citocininas revelaram que citocininas do tipo isopenteniladenina (iP e iPR) mostraram-se mais de perto relacionadas à retenção de características radiculares, ao passo que as do tipo zeatina (Z e ZR) apresentou maior influência e presença em condições em que as características radiculares foram perdidas. Por outro lado, a aplicação de substâncias moduladoras do balanço redox em ápices radiculares jovens mostrou que o estresse oxidativo proporcionou a aquisição de competência do MAR para conversão em gemas. Essa mesma tendência foi observada com a aplicação de concentrações relativamente elevadas de substâncias indutoras da elevação dos teores de óxido nítrico e cálcio citossólico nos tecidos. Os ápices radiculares com competência parcialmente estabelecida, analisados logo nas primeiras horas após o isolamento, revelaram que sua separação da planta-mãe acelerava as mudanças morfológicas que naturalmente ocorrem no MAR em estágios avançados do envelhecimento. Durante esse mesmo período, verificou-se uma queda rápida nos teores endógenos de citocininas (principalmente do tipo iP), proporcionando a predominância de citocininas do tipo Z durante a maior parte do primeiro dia de isolamento dos explantes, a qual coincidiu com a mudança no padrão de organização do MAR do tipo intermediário-aberto para o totalmente aberto. O avanço das modificações no ápice radicular após esse período desencadeou o estabelecimento do meristema caulinar, cujo evento esteve relacionado a uma tendência de aumento nos teores de citocininas e de ascorbato após o primeiro dia de isolamento. Dessa forma, os estágios mais avançados do envelhecimento radicular, bem como a separação de ápices radiculares com competência parcialmente estabelecida, parecem desencadear e aumentar a competência do MAR para conversão por meio de modificações morfológicas e fisiológicas muito similares nos ápices radiculares. Essas alterações envolveram a perda das características radiculares, a qual parece depender de alterações no controle exercido pelo CQ sobre o desenvolvimento das demais células no MAR. Esses eventos na região do CQ se revelaram condição sine qua non para a complementação da aquisição de competência do MAR, sendo esta dependente da intensidade das perturbações sobre o controle da organização do MAR. Dessa forma, a conversão do meristema apical radicular de C. fimbriatum em gemas caulinares parece decorrer da formação de um novo grupo de células na antiga região do CQ do MAR alterado. Essas células pareceram competentes para responder a diferentes estímulos que as direcionariam a uma nova rota do desenvolvimento que, nesse caso, seria o estabelecimento de um meristema caulinar com conseqüente desenvolvimento de uma gema vegetativa. / During this research work, it was noticed that competence acquisition for the conversion of Catasetum fimbriatum root tips into buds was related to the plant ageing. This process seems to be coupled with the establishment of the determinate root growth and with the cessation in the activity and structural re-organization of the root apical meristem (RAM). Young and non-competent root tips showed a closed RAM architecture, and the ageing process stimulated the establishment of an open organization in the RAM, as indicated by a higher level of differentiation and a predominance of parenchymatic cells in the old root apices. These alterations were concomitant with the modifications on the characteristics and functions of quiescent center (QC). In agreement with these observations, the competence acquisition to the conversion of the MAR into buds was linked to a series of metabolic alterations, which probably play a role in this process. Based on the data obtained, it was observed that the endogenous levels of important components of the cell division progression, such as auxins, cytokinins and the reduced forms of ascorbate and glutathione showed a tendency of decrease during the root ageing. On the other hand, during this same period, the content of some hormones involved in signalling events of stress conditions or cellular differentiation, such as ethylene, abscisic acid and gibberellins exhibited a pattern of increase. The relative concentrations of important second messengers, such as nitric oxide and cytosolic calcium also displayed a marked increased in the RAM region during the ageing. Additionally, it was noticed that the auxin polar transport represents an important positional signal for the maintenance of the RAM characteristics and functions, once treatments that blocked the transport of this hormone promoted the MAR competence acquisition even in young plants, although, the conversion process did not complete while the root tips were maintained attached to the plants. The treatment of young plants with ethylene, on the contrary, caused similar effects; however, besides inducing the competence, this hormone also promoted the RAM conversion into buds via the elevation in the endogenous levels of cytokinins. The treatment of young root tips with different types of cytokinins indicated that iP-type cytokinins (iP and iPR) were more closely associated to the preservation of the root characteristics, while the Z-type cytokinins (Z and ZR) showed a higher importance when the root characteristics were lost. Furthermore, the treatment of young root apices with compounds that cause alterations in the cellular redox status indicated that the oxidative stress stimulated the competence acquisition for the RAM conversion into buds. This same tendency was observed with the application of relatively high concentrations of compounds that induce elevations in the levels of nitric oxide and cytosolic calcium in the tissues. The analyses carried out during the first hours after the isolation of partially competent root apices indicated that the detachment of the root tips from the original plants accelerated the morphological modifications that naturally occur at advanced stages of ageing. During this same period, it was observed a rapid decrease in the endogenous levels of cytokinins (specially of the iP-type), leading to a predominance of the Z-type cytokinins during the first day after the isolation of the explants, which coincided with the alteration of the RAM architecture from the intermediate-open type to the completely open type. After the first day of isolation, the progress in the root apices modifications resulted in the establishment of the shoot meristem, which was accompanied by an elevation in the endogenous levels of cytokinins and ascorbate. Therefore, advanced stages of root ageing, as well the isolation of the partially competent root apices, seem to increase the competence for the RAM conversion into buds via similar morphological and physiological changes in the root apices. These alterations involved the loss of the root characteristics, which possibly resulted from modifications in the control of the QC on the development of the other cells in the RAM. These events in the QC represent a sine qua non condition for the completion of the MAR competence acquisition, which is affected by the intensity of the perturbations on the control of the RAM organization. Therefore, the conversion of root apical meristem of C. fimbriatum into buds probably results from the formation of a new group of cells in the region of the QC of the altered RAM. These cells seem to be competent to respond to different stimulus that would directionate them to a new developmental route that, in this case, consists in the establishment of a shoot meristem.

The cultured tissues and organs of Duboisia myoporoides, an endemic medicinal plant of Australia, were investigated with the aim of establishing a relationship between organogenesis, differentiation and alkaloid localization. Histological analyses explained the relationship between cell arrangement in the cultured tissues and organs and the cytokinin/auxin combinations used at the callus induction stage. The cultured tissues and organs were analysed histochemically to localize alkaloids in different types of cells by using selected alkaloid colour reagents i.e., platinic chloride (5%) and iodoplatinate. The presence or absence of nicotine, hyoscyamine and scopolamine in the cultured tissues and organs was then confirmed by GC-MS analysis. This is the first work to show that tropane alkaloid formation in the separated cultured organs is related to xylem differentiation and tropane alkaloid formation in the calli cultured in suspension may allow commercial tropane alkaloid production without regenerating the organs. The roots of the D. myoporoides field-grown trees were colonized by the AM fungi and the mycorrhizal infection was ranged from 0-30% which indicates that the secondary metabolite atropine and scopolamine did not prevent AM fungal colonization. / Doctor of Philosophy (PhD)

Une caractéristique fondamentale des vertébrés est leur organisation métamerique, visible au niveau de la colonne vertébrale. Cette organisation se met en place au cours du développement embryonnaire et l'émergence des précurseurs des vertèbres, les somites, formés à partir du mésoderm paraxial présomitique (PSM). Depuis la découverte d'une activité transcriptionelle oscillatoire de la voie de signalisation Notch dans le PSM, on propose que cette activité oscillatoire représente l'action d'une horloge embryonnaire, l'horloge de segmentation, responsable de contrôler la formation des somites de façon périodique. Dans cette étude, je présente la découverte d'une nouvelle association de la voie Wnt avec l'horloge de segmentation en décrivant l'activité oscillatoire de Axin2, une cible de la voie Wnt, dans le PSM. Ensuite, j'ai mise en place un system d'imagerie biphoton qui nous permet d'observer l'action de l'horloge de segmentation en temps réel et in vivo dans les embryons de souris. Ce système permet de mesurer directement les paramètres des oscillations. Ultérieurement je décris la découverte d'un gradient d'expression de la protéine -caténine dans le PSM. A l'aide d'expériences de recombinaisons homologues conditionnelles nous avons déterminé que ce gradient de - caténine contrôle la maturation et différentiation des cellules dans le PSM. De plus, ce gradient constitue un signal essentiel et permissif pour les oscillations de l'horloge de segmentation. En conclusion, je propose un nouveau modèle de mise en place des somites incorporant les résultats présentés.

Many vertebrate organs form via the sequential, reciprocal exchange of signaling molecules between juxtaposed epithelial (E) and mesenchymal (M) tissues. For example, the instructive signaling potential for tooth development (odontogenesis) resides in the dental epithelium at the initiation-stage, and subsequently shifts to the dental mesenchyme one day later at the bud-stage. However, the properties of the gene regulatory networks (GRNs) that control the signaling dynamics during epithelial-mesenchymal (E-M) interactions in organogenesis are largely unknown. This dissertation describes an interdisciplinary effort between developmental and systems biology to elucidate the E-M GRN that controls early odontogenesis. The results provide a molecular mechanism for the longstanding paradigm of sequential, reciprocal E-M tissue interactions in development. We generated large-scale spatiotemporal gene expression data for the developing mouse tooth. Surprisingly, the shift in signaling molecule expression from E to M is accompanied by a striking concordance in genome-wide expression changes in both E-M compartments as development proceeds. We hypothesized that since diffusible signaling molecules can act on either E or M independent of their tissue site of synthesis, signaling molecules are uniquely able to simultaneously synchronize and couple the transcriptional dynamics and hence the developmental progression of E and M. To identify the unifying mechanism behind concordant E and M genome-wide expression changes in the face of the discordant expression changes in signaling molecule expression, we developed a novel probabilistic technique that integrates regulatory evidence from microarray gene expression data and the literature to determine the E-M GRN for early tooth development. This GRN contains a uniquely configured E-M Wnt/Bmp feedback circuit in which the Wnt and Bmp signaling pathways in E cross-regulate the expression of Wnt and Bmp4 signaling molecules, whereas both pathways jointly regulate Bmp4 expression in M. We validated the Wnt/Bmp feedback circuit in vivo using compound genetic mutations in mice that either short-circuit or break the circuit, and used mathematical modeling to show how the structure of the Wnt/Bmp feedback circuit can account for reciprocal signaling dynamics. Collectively, these results provide a simple mechanistic framework for how simultaneous signal transduction in E-M compartments can account for the signaling dynamics in organogenesis.

Peculiarities of edible onions (Allium cepa L.) gynogenesis investigated in the dissertation work. Conditions for creation of dihaploid plants evaluated. Biological assumptions for preparation of methodical suggestions, that would enable improving of gynogenesis, determined. Stimulation effect of using TDZ and NAR in media on the formation of edible onion embryogenic tissue has been revealed for the first time. Characteristic higher frequency of gynogenesis of unfertilized flower of edible onion isolated from the flower stems soaked in 2,4-D solution has been evaluated. By using experimental method it has been established that 3 day length gynogenesis induction period, using media containing 2,4-D and BA, is enough to induce gynogenesis of edible onion. High heterogeneity of edible onion variety population ‘Lietuvos didieji’ has been demonstrated from the point of organogenesis in isolated unfertilized flower culture. It has been established that the efficiency of edible onion gynogenesis can be increased by decreasing plant donor growing temperature in the final phases of flower development and using of exogenous growth regulators TDZ and NAA in plant regeneration media as well as by flower stems soaking in 2,4-D solution for 14 days. Plants with characteristic high gynogenesis frequency have been determined according to evaluation of edible onion variety population ‘Lietuvos didieji’ organogenetic response. 10 homozygous lines of edible onion have been created. 2 lines with... [to full text]

To date, studies into sex determination and gonadal development have focused on the regulatory mechanisms governing development of the male or female phenotype. However, the formation of the testis and ovary from the bipotential gonad also present a fascinating model of tissue organisation which has been largely overlooked. When seeking to understand tissue organisation during gonadal development, the formation of testis cords takes center stage. However, despite a growing understanding of the cellular events in testis development, a number of key questions about the formation of testis cords remain unanswered. Specifically, I aimed to investigate the role of cell migration in testis organization, and the structure and morphogenesis of testis cords in three dimensions. To address these aims experimentally, I investigated the early morphogenesis of testis cords and the dependence of cord formation on cell migration from the mesonephros. I found that virtually all of the migrating cells express endothelial markers, indicating that endothelial, not peritubular myoid cells underlie the dependence of cord formation on cell migration. Further, disruption of endothelial cell migration and vascular organisation using a blocking antibody to VE-cadherin, also disrupted the development of testis cords. These data reveal that migrating endothelial cells are required for testis cord formation, consistent with increasing evidence of a broader role for vasculature in establishing tissue architecture during organogenesis. To address the question of cord structure and morphogenesis, I developed and applied a novel fluorescence-based three-dimensional modeling approach to show that Sertoli cells coalesce into irregular groups surrounding germ cells, and that these groups are remodeled to form highly regular toroidal loops, joined by a flattened plexus at the dorsal side. This plexus is punctured by blood vessels as they ingress from the mesonephros, and contracts during maturation to form part of the rete testis. Variation in cord number and position demonstrates that cord establishment is not a stereotypic process. However, a tightly regulated modeling mechanism must contribute to uniformity on cord diameter and orientation as these parameters remain consistent across samples of the same age. These data clarify questions of cord structure and organisation, establish that cord formation is a variable process, and demonstrate novel structural features within the network of testis cords. Finally, to investigate an in vivo model where vascularisation and cord formation may be disrupted, I analysed gonads from embryos lacking Cited2. Consistent with a previous study, I found that testis development was delayed in Cited2-/- gonads, but found that despite the reported transcriptional recovery after the delay, testis vascular and cord structure was permanently disrupted. To investigate the defects in cord formation I assayed cell migration and found that migration was not disrupted in XY gonads, or mesonephroi lacking Cited2. However, ectopic cell migration was observed in the XX gonad in a dose-dependent response to loss of functional Cited2 alleles. Correspondingly, the female pathway was initially delayed but rallied for a late recovery, implicating Sf1 in the initiation of ovarian differentiation. These data underscore the fragility of the molecular control of sex determination as absence of Cited2 in the male permanently disrupts testis morphology, whereas in the female, promoters of the male pathway are not sufficiently suppressed. From these studies I construct an integrated model of testis cord formation and conclude that testis cord formation is a novel form of tubulogenesis. This morphogenesis is unique and offers insights into cell and tissue organisation, vascular interactions in organogenesis, and mechanisms of tube formation. Further study of cord formation is likely to lead to a broader understanding of tissue morphogenesis during development.