Les sucres, sont des signaux métaboliques, impliqués dans le développement des plantes et leurs réponses aux contraintes du milieu. Les transporteurs de sucres se révèlent à la fois acteurs de la répartition des sucres et cibles de leur signalisation. L'ASR (ABA, Stress and Ripening) de la Vigne, VvMSA, étant identifiée comme protéine régulatrice de l'expression génique du transporteur d’hexoses VvHT1, l'objectif de la thèse est d'appréhender ses rôles physiologiques dans une démarche de biologie intégrée.Le premier axe a été dédié à la mise en place des modèles biologiques, des cellules embryogènes et non embryogènes de Vigne, issues du même fond génétique mais cultivées sur deux sources de carbone différentes. La caractérisation des cinétiques de prolifération et l'analyse des métabolomes ont mis en évidence leur sensibilité/tolérance différentielle à la carence en sucres. Le deuxième axe a porté sur la régulation de VvHT1 dans les deux types cellulaires sauvages et leurs mutants de surexpression/répression de VvMSA. L'approche pharmacologique utilisant des analogues du glucose, l'analyse de l'expression génique, le transport du glucose et l'activité des enzymes de la glycolyse indiquent que VvMSA affecte l'expression de VvHT1 par la voie dépendante du métabolisme du glucose. Le troisième volet a été réalisé par une approche de protéomique quantitative et comparative des protéines nucléaires des cellules embryogènes sauvages et réprimées pour VvMSA. Les protéines à expression significativement affectée par l'absence de l'ASR, laissent entrevoir un nouveau rôle à l'interconnexion des réponses métaboliques aux stress et la régulation épigénétique de l'expression génique. / Sugars are metabolic signals involved in plant development and responses to environmental cues. Sugar transporters are both actors of sugar partitioning and targets of sugar signaling. As Grape ASR (ABA, Stress, Ripening), VvMSA, is identified as a regulatory protein controlling gene expression of the hexose transporter VvHT1, the aim of the PhD thesis is to assess its physiological functions by an integrative biology approach. The first part of the study consisted in the establishment of biological models, embryogenic and non embryogenic grape cells, sharing the same genetic background but growing on distinct carbon sources. The characterization of the proliferation kinetics and metabolomes of both cell types revealed differences in their sensitivity/tolerance to sugar starvation.The second objective was focused on VvHT1 expression regulation in both cell types and their mutants overexpressing or silenced for VvMSA. The pharmacological approach using glucose analogues, coupled to the analysis of gene expression, glucose transport and glycolytic enzymes activity, suggest that VvMSA affects VvHT1 expression through a glucose metabolism dependent pathway.The third research axis was carried out through a quantitative and comparative proteomic analysis of nuclear proteins in embryogenic wild type and VvMSA silenced cells. Proteins whose expression is affected by ASR repression suggest a new functional role of VvMSA at the interplay between metabolic responses to stress and epigenetic regulation of gene expression.

Made available in DSpace on 2017-07-10T17:37:42Z (GMT). No. of bitstreams: 1 Cleuza_Aparecida_da_Rocha_Montanucci.pdf: 1925062 bytes, checksum: 49db8be66a9a1189bfa31d0bd6b5f69d (MD5) Previous issue date: 2011-03-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The knowledge of morphological and ecophysiological features ofBrugmansia suaveolens (Willd.).Bercht.& J. Presl is the first step towards the use of this species with biotechnological purpose. This study aimed to: 1) the morphological characteristics of specimens of B. suaveolens, 2) evaluation of in vitro germination rates under different conditions of seed´s treatment. 3) the establishment of an efficient regeneration protocol in B. suaveolens. We evaluated the rates of regeneration, the percentages of calli induction and calli size, germination of embryos, the number of embriogenic spots and the number of regenerated plantlets. As results, 22 individuals were collected and grouped into three different groups, differing only by flowers colors. The fruits showed mean values of: 975 mm long, 189 mm wide and 6.99 g of weight Seeds presented mean values of7 mm long, 6.00 mm width, 3.04 mm thicknessand mass of 0.39 g.The estimated period for initiation of the germination process without the presence of the tegument was 14 days. In all treatments there was an increased cumulative germination. Regarding the effect of pre-treatments can be observed that temperatures of 4 ºC and 50 ºC did not promote differences between treatments. Exposure to sulfuric acid and soaking the seeds in water for longer periods of reduced germination. The presence of gibberellic acid caused inhibition of embryo development in vitro. In the development of regeneration protocols, the establishment of plants from mature embryos of this species, the percentage of calli induction ranged from 26 to 100%, the ratio seedling / callus induced ranged from 0 to 0.84 and the efficiency of regeneration ranged from 0 to 32%. The dosages of 0.5 mg L-1 2.4 D and 1.0 mg L-1 KIN were more efficient regarding to calli induction, calli size, number of green spots points and regenerated plantlets. High concentrations of growth regulators (1 mg L-1 2-4 D and 1.0 mg L-1 KIN) were inhibitory to calli formation and regeneration, the absence of these regulators promoted the formation of calli but any plantlets of B.suaveolens were gotten / O conhecimento das características morfológicas e ecofisiológicas de plantas de Brugmansia suaveolens (Willd.). Bercht. & J. Preslé o primeiro passo para o uso desta espécie com finalidade biotecnológica. Este trabalho teve como objetivos: 1) a caracterização morfológica de acessos da B. suaveolens; 2) avaliação do comportamento germinativo das sementes in vitro sob diferentes condições de tratamento e 3) o estabelecimento de protocolo de regeneração eficiente de B. suaveolens. Foram avaliados os índices de regeneração, além das porcentagens de indução e tamanho de calos, da germinação de embriões, o número de pontuações verdes e o número de plântulas regeneradas. Como resultados obtidos, 22 indivíduos de B. suaveolens foram coletados e agrupados em três grupos, diferindo apenas nas cores das flores. Os frutos apresentaram valores médios de: 975mm de comprimento, 189 mm de largura e 6,99 g de massa, as sementes de 7mm de comprimento, 6 mm de largura, 3,04mm e espessura e massa de 0,39 g. O período estimado para o início do processo germinativo sem a presença do tegumento foi de 14 dias. Em todos os tratamentos observou-se uma germinação acumulada aumentada. Em relação ao efeito dos tratamentos pode-se observar que temperaturas de 4 ºC e 50 ºC não propiciaram diferenças estatísticas entre os tratamentos. A exposição ao ácido sulfúrico e a embebição das sementes em água por períodos maiores que 24 horas, reduziram a germinação. A presença do ácido giberélico promoveu a inibição do desenvolvimento dos embriões in vitro. No desenvolvimento do protocolo de regeneração para o estabelecimento de plantas a partir de embriões maduros para esta espécie, a porcentagem de indução de calos variou de 26a100%, a razão plântula/calo induzido variou de 0 a 0,84 e a eficiência de regeneração variou de 0a32%. As dosagens de 0,5mgL-1 de 2,4-D e 1,0mg L-1 KIN foram as mais eficientes nas respostas para indução de calos, no tamanho de calos, no número de pontos embriogênicos e plântulas regeneradas. Altas concentrações de reguladores de crescimento (1,0mgL-1 de 2,4-D e 1,0mg L-1 KIN) inibiram a calogênese e regeneração; a ausência destes reguladores promoveram a formação de calos mas não a regeneração de plantas de B. suaveolens

Conifer species are subjected to major time constraints in tree improvement because of their long regeneration cycle and large sizes. However, integration of developing biotechnologies could significantly reduce this time limitation in tree breeding programs. In this regard, somatic embryogenesis (SE) offers a great potential in commercially important Turkish red pine (Pinus brutia TEN.) for rapid production of larger number of clones as well as capture of greater genetic gains. In this study, seven collections were done to sample precotyledonar zygotic embryos for induction of embryogenic tissue (ET) from 15 clones located in Antalya. Afterwards, abscisic acid, carbohydrates, polyethylene glycol (PEG), and gellan gum were tested to obtain mature somatic embryos in maturation experiments. Analyses of variance showed a significant variation among collection dates (43.1% of total variance) and clones studied (18.8% of total variance) for induction of ETs. Overall initiation frequency of ET in this study was 11.6% with clonal range of 4.7 &amp / #8211 / 24.1%. Of those tested maturation treatments, 80&amp / #956 / M ABA, sucrose and maltose at 3 and 6%, 3.75% PEG combined with 1% gellan gum were found to be suitable for maturation of somatic embryos in Turkish red pine. Sixty nine somatic embryos were obtained from Clone 22, which was one of tested clones. Induction frequencies could be further improved by using different basal media and/or manipulating media components, such as plant growth regulators. For proper maturation of somatic embryos, embryogenic lines need to be screened to find suitable lines, which are developmentally responsive to ABA treatment.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-05-17T12:51:55Z No. of bitstreams: 1 Lecio Leone de Almeida.pdf: 4818559 bytes, checksum: c904471979bd6f907fbbae1681e53e45 (MD5) / Made available in DSpace on 2016-05-17T12:51:55Z (GMT). No. of bitstreams: 1 Lecio Leone de Almeida.pdf: 4818559 bytes, checksum: c904471979bd6f907fbbae1681e53e45 (MD5) Previous issue date: 2015-10-01 / The inadequate use of herbicides may cause serious and sometimes chronic poisoning due to long exposure to low levels. Herbicides may also be teratogenic, mutagenic, cancerigenous agents and endocrine disruptors, with the occurrence of neurodegenerative diseases and reproduction disorders. Several studies have shown that melatonin has antioxidant, anti-inflammatory and immune-modulating qualities, besides affecting the reproduction system. It is a beneficent agent in the intoxication by herbicides even though no reports are extant on the use of melatonin against intoxications by Glyphosate-Roundup® alone or associated to Paraquat. Current analysis investigates the effect of melatonin on the reproduction and hepatic aspects in female rats exposed to sub-lethal doses of the herbicide Paraquat (50 mg/kg) and/or Glyphosate-Roundup® (500 mg/kg) of body weight, alone or associated. Thirty-five pregnant rats were used, randomly divided into 7 groups with 5 animals each. Group I (control/saline), groups (II and III) exposed to Paraquat and Glyphosate-Roundup® respectively, the IV group exposed to the associated herbicide, while the rats of the groups (V and VI ) were exposed to the herbicides alone and group (VII) above the associated herbicides, however the groups (V, VI and VII) were simultaneously treated with melatonin (10 mg/kg body weight). Both exposure as treatment with melatonin was during the first third of pregnancy. Thus, the hematological profile, function and hepatic oxidative stress, reproduction parameters such as body weight gain, ovaries weight, number of implantation sites, corpora lutea, pre-implantation losses, implantation rates and histopathology of implantation sites and liver were analyzed. Results post-exposure to associated or single herbicides revealed decrease in body and ovary weights, number of implantation sites, implantation rate (the implantation rate (number of implanted embryos compared with the number of corpora lutea), total number of corpora lutea, percentage increase of pre-implantation losses. The hematological profile revealed a reduction of erythrocytes in hemoglobin concentrations, in hematocrit rates, increase in total counts of leucocytes, in the number of segmented neutrophils, eosinophils and decrease in lymphocytes. Moreover, decrease in serum levels of albumin, increase in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AP), high levels of reactive compounds to thiobarbituric acid (TBARS) and reduction in the levels of reduced glutathione (GSH) were reported in biochemical and hepatic oxidative stress analyses. The histopathological analysis of the implantation sites revealed the disorganization of blastocyte and degeneration of cytotrophoblasts within the blastocyte cavity, whilst in histochemical analysis was observed reduction number of uterine natural killer cells (uNKs). Morphometry revealed decrease endometrial parameters of superficial and glandular epithelium, as well as the diameter of the endometrial glands. In the histopathological analysis of liver samples was observed the presence of necrotic hepatocytes, portal and central lobular inflammatory infiltrate and congestion of sinusoid capillaries. Treatments with melatonin provided results significantly similar to those in control animals, with the exception of serum albumin levels in rats associated group (Paraquat 50 mg/kg) and/or Glyphosate-Roundup® 500 mg/kg of body weight), which remained low. Treatment with melatonin at a dose of 10 mg/kg of body weight is a protecting agent against experimentally induced maternal and embryo poisoning by herbicides. Body weight, reproduction, hematological, biochemical parameters maintenance and hepatic oxidative stress was reduced. / O uso inadequado de herbicidas pode resultar em intoxicações agudas e, às vezes crônicas por exposição em longo prazo a baixos níveis, podendo atuar ainda como agentes teratogênicos, mutagênicos, cancerígenos e disruptores endócrinos, levando ao aparecimento de doenças neurodegenerativas e distúrbios reprodutivos. Estudos têm revelado que a melatonina tem propriedades antioxidantes, anti-inflamatórias e imunomoduladoras e atua na reprodução. Essa indolamina está entre os agentes que têm se mostrado benéfico em intoxicações por herbicidas, porém não há relatos de seu uso contra intoxicações por Glifosato-Roundup® e/ou Paraquat associado com Glifosato-Roundup®. Assim, investigou-se o efeito da melatonina sobre indicadores reprodutivos e parâmetros hepáticos em ratas prenhes expostas a doses subletais dos herbicidas Paraquat (50 mg/kg do peso corporal) e/ou Glifosato-Roundup® (500 mg/kg do peso corporal). Foram utilizadas 35 ratas prenhes, divididas aleatoriamente em 7 grupos com 5 animais cada, sendo o grupo I (controle/salina), os grupos (II e III) expostos aos herbicidas Paraquat e Glifosato-Roundup® respectivamente, o grupo IV exposto aos herbicidas associados, enquanto as ratas dos grupos (V e VI) foram exposta aos herbicidas individualmente e o grupo (VII) exposto aos herbicidas associados, porém os grupos (V, VI e VII) foram tratados simultaneamente com melatonina (10 mg/kg do peso corporal).Tanto a exposição como o tratamento com melatonina foram durante o terço inicial da gestação. Assim, após esses procedimentos foram analisados o perfil hematológico, função e estresse oxidativo hepático, parâmetros reprodutivos como peso das fêmeas, peso dos ovários, número de sítios de implantação, de corpos lúteos, perdas pré-implantação, taxa de implantação, histopatologia do fígado, histopatologia e histoquímica dos sítios de implantação e morfometria dos epitélios superficial e glandular do endométrio. A aplicação experimental dos herbicidas Glifosato-Roundup e/ou Paraquat proporcionaram toxicidade materno-embrionário e hepática, onde podem-se detalhar os seguintes efeitos: redução nos seguintes parâmetros: ganho de peso corporal, peso dos ovários, no número de sítios de implantação, na taxa de implantação, no número total de corpos lúteos e aumento do percentual de perdas pré-implantação. Quanto ao perfil hematológico observou-se redução no número de eritrócitos, na concentração de hemoglobina, nos valores do hematócrito, aumento na contagem total de leucócitos, no número de neutrófilos segmentados, de eosinófilos e redução no número de linfócitos. Além disso, nas análises bioquímicas e do estresse oxidativo hepático foram observadas redução nos níveis séricos de albumina, aumento dos níveis séricos de alanina aminotransferase (ALT), aspartato aminotransferase (AST) e fosfatase alcalina (FA), níveis altos de substâncias reativas ao ácido tiobarbitúrico (TBARS) e redução dos níveis de glutationa reduzida (GSH). A análise histopatológica dos sítios de implantação revelou desorganização do blastocisto e degeneração do citotrofoblasto localizado dentro da cavidade blastocística, enquanto na análise histoquímica observou-se redução no número de células natural killer uterinas (nNK). A análise morfométrica de parâmetros do endométrio revelou redução dos epitélios superficial e glandular, bem como no diâmetro das glândulas endometriais. Na análise histopatológica de amostras de fígado foi observada a presença de hepatócitos necróticos, infiltrado inflamatório portal e centrolobular, congestão dos capilares sinusoides. Após tratamento com melatonina verificou-se que os parâmetros supracitados foram similares aos do controle, exceto os níveis de albumina sérica em ratas do grupo associado (Paraquat 50 mg/kg e Glifosato-Roundup® 500 mg/kg de peso corporal) que permaneceram baixos. Dessa forma pôde-se concluir que a melatonina na dose de 10 mg/kg de peso corporal atuou como um agente protetor contra a toxicidade materno/embrionária induzidas experimentalmente por herbicidas, favorecendo a manutenção do peso corporal, de parâmetros reprodutivos, hematológicos, bioquímicos e redução do estresse oxidativo hepático.

Gossypium barbadense L. “algodón nativo” es oriundo de la costa norte del Perú y se caracteriza por presentar fibras de colores naturales. La evaluación del efecto de diferentes concentraciones de fitorreguladores de crecimiento en la inducción de callo embriogénico se realizó en explantes de hipocotilo de G. barbadense L. “algodón nativo” color pardo, bajo dos condiciones lumínicas distintas. La desinfección de semillas se llevó a cabo utilizando NaOCl al 2.5% en distintos tiempos de exposición (5-20 min) para obtener plántulas in vitro. Para la iniciación y proliferación de callos, se introdujeron explantes de hipocotilo (con 5 réplicas) en medios Murashige-Skoog (MS) suplementados con diferentes concentraciones de ácido 2,4-diclorofenoxiacético (2,4-D), kinetina (Kin) y agua de coco. Los callos friables de mayor proliferación fueron transferidos a medios MS con distintas concentraciones de Kin, 2,4-D y ácido indol-3-butírico (IBA) para la inducción de callo embriogénico. Los cultivos fueron mantenidos en condiciones de fotoperiodo 16 h luz/ 8 h oscuridad y oscuridad continua. Se logró el 100% de desinfección de semillas en 10 min de exposición al desinfectante. En 85% a 100% de explantes de hipocotilo se obtuvo la formación de callo en todos los tratamientos de iniciación de callo incubados en ambas condiciones lumínicas durante 21 días. La mayor proliferación de callo friable se obtuvo en 82.5% de explantes cultivados en medio MS enriquecido con 0.1 mg/l de 2,4-D y 100 ml/l de agua de coco e incubados en fotoperiodo 16 h luz/ 8 h oscuridad. En los medios MS suplementados con distintas concentraciones de Kin, 2,4-D e IBA no se logró la inducción de callo embriogénico. Sin embargo, en el medio MS sin reguladores de crecimiento y en el suplementado con 0.05 mg/l de Kin y 0.3 mg/l de IBA, en fotoperiodo 16 h luz/ 8 h oscuridad, se inició la organogénesis radical.Gossypium barbadense L. “native cotton” is originally from the northern coast of Peru and is characterized by its naturally colored fibers. The evaluation of the effect of different concentrations of plant growth regulators on embryogenic callus induction was performed in hypocotyl explants of Gossypium barbadense L. “native cotton” brown, under two different lighting conditions. Seed disinfection was carried out using 2.5% NaOCl in different exposure times (5-20 min) in order to obtain in vitro plants. For initiation and proliferation of callus, hypocotyl explants (with 5 replicates) were placed in Murashige-Skoog (MS) medium supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), kinetin (Kin) and coconut water. Friable calli with the highest proliferation were transferred to MS medium with different concentrations of Kin, 2,4-D and indole-3-butyric acid (IBA) for induction of embryogenic callus. The cultures were maintained under conditions of 16 h light/ 8 h dark photoperiod and continuous darkness. 100% of seed disinfection was achieved at 10 min of exposure to disinfectant. In 85% to 100% of hypocotyl explants, callus formation was obtained in all callus induction treatments, incubated in both lighting conditions during 21 days. The highest proliferation of friable callus was obtained in 82.5% of hypocotyl explants cultivated in MS medium enriched with 0.1 mg/l 2,4-D and 100 ml/l coconut water, in 16 h light/ 8 h dark photoperiod. In MS medium supplemented with different concentrations of Kin, 2,4-D and IBA, induction of embryogenic callus was not achieved. However, in MS medium without growth regulators and supplemented with 0.05 mg/l Kin and 0.3 mg/l IBA, in 16 h light/ 8 h dark photoperiod, root organogenesis was initiated.

No description available.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-07T17:17:10Z No. of bitstreams: 1 Deivid Almeida da Costa.pdf: 3034904 bytes, checksum: a3834e78a3b01627180c99a9fddc24a9 (MD5) / Made available in DSpace on 2017-02-07T17:17:10Z (GMT). No. of bitstreams: 1 Deivid Almeida da Costa.pdf: 3034904 bytes, checksum: a3834e78a3b01627180c99a9fddc24a9 (MD5) Previous issue date: 2007-02-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The somatic embryogenesis is a technique of plant tissue culture with several application potentials, as in the clonal propagation of plants, in the regeneration of transformed cells and in the basic studies of the molecular, biochemical and morphologic events that happen duringthe embryogenesis. In spite of the several works reporting somatic embryogenesis in cotton, several problems still persist in the regeneration through embryogenesis. The most of the works is with the line cocker, due to the fact of the cotton to be recalcitrant for the somatic embryogenesis. This way, the present study was conceived with the intention of analyzing the morphogenic response of the cotton to several growth regulators and stressful agents during the induction of embryogenic callus and somatic embryos, correlating the callus production with the antioxidant enzymes expression. For induction of the embryogenic callus, hypocotyl of germinated cotton (BRS – 187 – 8H) surface-sterilized seeds was used. The embriogenic callus induction medium (MICE) consisted of the MSB medium with different combinations and concentrations of 2,4-D, Kinetin, 2iP and Picloram. For conversion of embryogenic callus, it was used the embryo induction medium (MIE) that consistes of MSB medium and MSB supplemented with zeatin; L-glutamine, asparagine; espermine, espermidine or different concentrations of NH4NO3 and KNO3. The results show that among the MICE tested, the medium with 0,1 mg L-1 2,4-D and 0,3 mg L-1 Kinetin (MSB1 medium) and the MCIM medium showed the largest average in the production of embryogenic callus by explant. Explants in the MIE-1 presented the best proliferation of the embryogenic callus. However, the formation of globular and embryogenic structures was only visualized in the embryogenic callus formed in MSB1 medium, which were transferred for MSB medium and subcultivated in MIE-4 medium. It was concluded that different growth regulators can induce the formation of embryogenic callus in cotton cultivar BRS – 187 – 8H and the suppression of the growth regulators favors the appearance of globular and pre-embryogenic structures. In the experiments of the influence of the stress in cotton callus formation and the isozymic antioxidant system expression, it was used for induction of embryogenic callus, thecombination of MSB and MSB1 medium with two concentrations of FeEDTA: the concentration standard of the MS medium; and a concentration five times larger. It wasestablished three regimes of luminous intensity: darkness; 3,4 Klx and 6,0 Klx. Embryogenic callus were obtained on the MSB1 (3,4 Klx), MSB1 (6,0 Klx) and MSB1+FeEDTA (5X) medium. The electrophoretic system of the catalase and peroxidase showed variation in the intensity of the bands. The results show that the embryogenic callus formation dependent of the action of growth regulators and of the light presence, and the isozymic standards of catalase and peroxidase present similar pattern and they are related with the embryogenic aspect of the calluses. / A embriogênese somática é uma técnica de cultura de tecidos vegetais com diversos potenciais de aplicação, como na propagação clonal de plantas, na regeneração de células transformadas geneticamente e nos estudos básicos dos eventos moleculares e bioquímicos e morfológicos que ocorrem durante a embriogênese. Apesar dos diversos trabalhos relatando embriogênese somática em algodão, vários problemas ainda persistem na regeneração via embriogênese e a maioria dos trabalhos é com a linha cocker, devido a recalcitrância do algodão a embriogênese somática. Deste modo, o presente estudo foi concebido com o intuito de analisar a resposta morfogênica do algodão a vários reguladores de crescimento e agentes estressantes durante a indução de calos embriogênicos e embriões somáticos, correlacionando a calogênese com a expressão de isoformas do sistema antioxidativo. Para indução dos calos embriogênicos utilizou-se sementes desinfestadas da cultivar de algodão BRS – 187 – 8H. Os meios de indução de calos embriogênicos (MICE) utilizados consistiram no meio MSB acrescido de varias combinações e concentrações dos reguladores de crescimento 2,4-D; Cinetina, Picloram e 2-iP. Já para conversão dos calos embriogênicos em embriões, utilizouse o meio de indução de embiões (MIE) que consiste no meio MSB e MSB acrescido dezeatina, ou diferentes concentrações de NH4NO3 e KNO3, ou acrescido de L glutamina; asparagina; espermina; espermidina. Os dados obtidos mostram que entre os MICE testados, o meio contendo 0,1 mg L-1 de 2,4-D e 0,3 mg L-1 de cinetina (meio MSB1) e o meio MCIM mostraram as maiores médias na produção de calos embriogênicos por explante. Entre os MIE, o MIE-1 apresentou melhor proliferação dos calos embriogênicos. Contudo, a formação de estruturas globulares e embriogênicas só foi visualizada nos calos embriogênicos formados em meio MSB1 e transferidos para meio MSB e subcultivados em meio MIE-4. Concluiu-se então que diferentes reguladores podem induzir a formação de calos embriogênicos na cultivar BRS – 187 – 8H, e a supressão dos reguladores de crescimento favorece o surgimento de estruturas globulares e pré-embriogênicas. Nos experimentos da influência do estresse na calogênese e a expressão de isoenzimas do sistema antioxidativo, utilizou-se para a induçãode calos embriogênicos a combinação do meio MSB e MSB1 com duas concentrações de FeEDTA: a concentração padrão do meio MS; e uma concentração cinco vezes maior. E foiestabelecido três regimes de intensidade luminosa: escuro; 3,4 Klx e 6,0 Klx. Calos embriogênicos foram obtidos nos meios MSB1(3,4 Klx), MSB1(6,0 Klx) e MSB1+FeEDTA(5X). Analises de eletroforese de isoenzimas da catalase e peroxidase mostraram variação na intensidade das bandas. Os resultados obtidos permitiram concluir que formação de calos embriogênicos é dependente da ação de reguladores de crescimento e da presença de luz, e que as isoformas de catalase e peroxidase apresentam padrão similar e estão relacionadas com o aspecto embriogênico dos calos.

Abstract Changes in climate will impose abiotic stress on plant species, and eventually affect their distributional range. This is a particular challenge especially to tree species with long generation times and slow rates of evolution. In the boreal zone, most of these species also hold high economic value. The aim of the study was to enhance understanding of the abiotic stress coping strategies of Scots pine (Pinus sylvestris L.) in its vulnerable early growth phase. In particular, the study evaluates the role of polyamine (PA) metabolism of Scots pine under abiotic stress. Polyamines (PAs) are small, ubiquitous nitrogenous compounds involved in fundamental biological processes of plants such as growth, organogenesis, embryogenesis and abiotic and biotic stress defence. The effects of different water availabilities and spring frost temperatures on shoots and roots of young seedlings were investigated in two growth chamber experiments. Proembryogenic liquid cell culture system was established and used as a controllable platform. Effects of polyethylene glycol (PEG) induced osmotic stress was investigated at the cellular level in liquid cultures. Responses to treatments were evaluated by quantifying changes in the expression of PA metabolism, stress, cell death and cell division -related genes, metabolite concentrations, as well as stress-induced physiology and morphology. The results revealed that PA metabolism is tissue specific and strictly regulated. Drought stress induced the accumulation of putrescine (Put) in photosynthesizing tissues, whereas the prime response to spring frost was a reorganization of root growth. Generally, drought and osmotic stress decreased the expression of PA catabolizing genes, leading to consistent PA levels in tissues even under severe stress. To conclude, PAs have a protective role in maintaining the growth and development of pine tissues subjected to abiotic stress. / Tiivistelmä Ilmastonmuutos vaikuttaa eri kasvilajien levinneisyyteen. Nopeasti muuttuva ilmasto on haaste erityisesti metsäpuulajeille, jotka pitkän sukupolvenvälin takia kehittyvät evolutiivisesti hitaasti. Erilaisten ympäristötekijöiden aiheuttamien puolustusaineenvaihdunnan muutosten tunteminen metsäpuilla on tulevaisuuden kannalta myös taloudellisesti tärkeää. Työn tavoitteena oli selvittää millainen merkitys polyamiineilla on metsämännyn (Pinus sylvestris L.) abioottisessa stressivasteessa. Polyamiinit (PAt) ovat pienimolekyylisiä typpiyhdisteitä, joiden tiedetään olevan mukana solujen perusaineenvaihdunnassa, kasvien kasvussa ja kehityksessä ja stressivasteissa. Tutkimus koostuu kolmesta kokeesta, joissa selvitettiin PA aineenvaihduntaa osana männyn taimien ja erilaistumattomien somaattisista alkioista peräisin olevien soluviljelmien stressivasteita. Kahdessa kokeellisessa tutkimuksessa männyn sirkkataimia altistettiin erilaisille kasvualustan vesipitoisuuksille ja keväthallan lämpötilaolosuhteille. Kolmannessa kokeessa PA aineenvaihdunnan reagointia osmoottiseen stressiin tutkittiin erilaistumattomissa somaattisissa epäkypsissä männyn alkiosta peräisin olevissa solukoissa kontrolloidussa nesteviljely-ympäristössä. Tutkimuksessa tarkastelin PA aineenvaihduntaa säätelevien geenien ilmenemistä ja PA pitoisuuksien muutoksia suhteessa kasvuun, kehitykseen ja stressissä indusoituvien geenien ilmenemiseen. Tulokset osoittavat, että PA aineenvaihdunta on tiukasti säädeltyä erilaisissa solukoissa. Taimien yhteyttävät solukot kerryttivät putreskiinia (Put) kuivuudessa. Ensimmäiset vasteet keväthallaolosuhteisiin havaittiin muutoksena juurten kasvun säätelyssä. Sekä kuivuus että osmoottinen stressi laskevat PA hajottavien geenien ilmenemistä, mikä on metsämännyn tapa ylläpitää tasaiset PA pitoisuudet solukoissa stressitekijöistä huolimatta. Johtopäätöksenä voidaan todeta, että PA aineenvaihdunta on tärkeä metsämännyn kasvun ja kehityksen kannalta.

The embryogenic calli were grown on MS medium containing NaCl with concentrations from 50 to 300 mM. After 2 weeks of culture, salinity tolerance threshold was identified at 150 mM NaCl. Higher concentrations of NaCl stimulated a significant reduction in the calli survival rate and the highest rate was 78.67% at 50 mM. After subculturing callus to the embryo culture medium containing NaCl, the growth and embryogenesis were not affected at the concentrations of 50 – 100 mM. Especially, at 50 mM NaCl the embryogenesis rate reached 83.33%. In contrast, 150 mM NaCl inhibited the somatic embryogenesis. After 4 weeks, culturing somatic embryos on medium MS with addition of 0.07 mg/l spermidin at 50 – 100 mM NaCl, the embryogenesis was considered good and embryos developed through several stages: globular, heart, torpedo and cotyledonary. However, at 150 mM NaCl the globular stage appeared in the culture process. The process of morphohistology and using dye carmine – iod and acridine orange observed the structure of generative callus and embryos at several stages. / Mô sẹo có khả năng phát sinh phôi được nuôi cấy trong môi trường có chứa muối NaCl với nồng độ thay đổi từ 50 – 300 mM. Sau 2 tuần nuôi cấy, chúng tôi xác định được ngưỡng chịu mặn của mô sẹo có khả năng sinh phôi cây Cọc rào là 150 mM. Nồng độ muối NaCl càng cao thì tỷ lệ sống của mô sẹo giảm dần và đạt giá trị cao nhất là 78,67% tại nồng độ 50 mM NaCl. Khi chuyển mô sẹo sang môi trường phát sinh phôi có chứa muối NaCl với nồng độ thay đổi, chúng tôi thấy ở nồng độ muối NaCl 50 – 100 mM không ảnh hưởng đến khả năng sinh trưởng và phát sinh phôi, đặc biệt là tại nồng độ 50 mM NaCl giúp kích thích sự hình thành phôi từ mô sẹo với tỷ lệ hình thành phôi đạt 83,33%. Ngược lại, nồng độ từ 150 mM NaCl gây ức chế quá trình hình thành phôi soma từ mô sẹo. Tiếp tục khảo sát ảnh hưởng của muối đến khả năng phát triển và nảy mầm của phôi soma. Ghi nhận kết quả sau 4 tuần nuôi cấy phôi soma trong môi trường MS có bổ sung 0.07 mg/l spermidin, tại nồng độ 50 – 100 mM NaCl khả năng hình thành phôi tốt và phôi phát triển qua các giai đoạn phôi hình cầu, hình tim, hình thủy lôi và hình lá mầm. Đặc biệt ở nồng độ 50 mM số lượng phôi lá mầm đạt giá trị cao với 13,33 phôi. Nồng độ muối NaCl 150 mM chỉ xuất hiện phôi hình cầu trong suốt thời gian nuôi cấy. Quá trình giải phẫu hình thái phôi và sử dụng thuốc nhuộm 2 màu carmin – iod và acridine orange đã cho thấy rõ hơn về cấu trúc mô sẹo có khả năng sinh phôi và phôi hình thái.

Somatic embryogenesis (SE) represents potentially very suitable way of plant production - micropropagation - of plants in vitro. The process had been studied for more than thirty years and a lot of knowledge has been gained in the field though new ways based on new knowledge leading to improvement and increase of the effectiveness of cultivation protocols are still under focus in the field. Relatively recent discovery of aromatic cytokinins lead to new knowledge on very positive effects on different morphogenetic and physiological processes during both cultivation in vitro or in vivo treatments. This gave origin to the topic of the present thesis, which focused on study of the effect of meta-topolin (mT), aromatic cytokinin, on proliferation phase of SE on structural level of embryogenic lines of Norway spruce Picea abies (L.) Karst. The aim of the thesis is analysis of structural development of embryogenic lines with the use of anatomical preparations and quantitative stereological methods. The material represented three embryogenic lines: 107 and 34C were obtained from the Forestry and Game Management Research Institute from the lab of Dr. Jana Malá and the line AFO 541, which was used as a standard line. To compare the effect of mT with benzylaminopurine (BAP) on proportion of different...