Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2017-10-05T21:58:20Z No. of bitstreams: 1 TESE- MARIA MAIANY DE OLIVEIRA.pdf: 1486275 bytes, checksum: 077fcda867394bf1737e7758d79af6a2 (MD5) / Made available in DSpace on 2017-10-05T21:58:20Z (GMT). No. of bitstreams: 1 TESE- MARIA MAIANY DE OLIVEIRA.pdf: 1486275 bytes, checksum: 077fcda867394bf1737e7758d79af6a2 (MD5) Previous issue date: 2017-03-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Banana (Musa spp.) is considered one of the most important fruits in world trade due to its nutritional and economic potential.But although there is a large number varieties on the market, the banana is still affected by many diseases.The application of the method of crosses in this species is very difficult because the majority of cultivated varieties is triploid presenting low fertility. In this case, it is necessary to use biotechnology and its tools applied to non-conventional genetic improvement to develop new varieties that have resistance to their different types of pathogens.This work was carried out with the objective of adapting the technique of obtaining embryogeniccallus of banana by means of the Scalp method in cultivars Brazilian Ma?? and Pacovan, adjusting the processes of induction of meristematic structures and multiplication of shoots and induce increased genetic variability by in vitro mutagenesis using the chemical agent ethylmethanesulfonate and, of the genetic transformation by the bombardment of microparticles. Was evaluated the callus formation, the effect of mutagenic in the in vitro cultivation of shoots and the effects of genetic transformation on shoot resistance in selection medium containing the herbicide Imazapyr. The results showed that the merystematic structures obtained have the capacity to origin callus with only one month of cultivation, and both cultivars developed friable callus with average values above 90%.The efficiency of this method was evidenced by the high capacity of induction of friable callus in the two evaluated cultivars but also by the rapidity in the process of obtaining calluses, being the first study of adaptation of the methodology for Brazilian banana cultivars.On the other hand, the evaluation of the mutation induction allowed to conclude that the survival and the capacity of bud formation decreased as a function of the increase of the concentration and the immersion time in the ethylmethanesulfonate.The surviving plants underwent a sorting with the fusaric acid selective agent in which it was possible to regenerate in vitro plants of the cultivars submitted to treatment with the mutagen and to select possible mutants with fusaric acid resistance for cultivarsMa?? and Pacovan.And the genetic transformation method proved efficient in the regeneration of shoots resulting in high values of survival and multiplication, where possible transgenic plants of banana cv. Ma?? were obtained after selection of resistance to the herbicide.Therefore, it is concluded that all the material produced, both in the mutagenic phase and in the genetic transformation, presents a greater genetic variability potentially applicable to the banana improvement. / A banana (Musa spp.) ? considerada um dos mais importantes frutos no com?rcio mundial em virtude seu potencial nutritivo e econ?mico. Mas, apesar de existir um grande n?mero de variedades no mercado, a bananeira ainda ? acometida por muitas doen?as. A aplica??o do m?todo de cruzamentos nesta esp?cie ? muito dif?cil, pois a maioria das variedades cultivadas ? triploide apresentando baixa fertilidade. Nesse caso, faz-se necess?rio o uso da biotecnologia e de suas ferramentas aplicadas ao melhoramento gen?tico n?o convencional para desenvolver novas variedades que tenham resist?ncia aos seus diferentes tipos de pat?genos. Este trabalho foi realizado com os objetivos de adaptar a t?cnica de obten??o de calos embriog?nicos de bananeira por meio do m?todo Scalp nas cvs. Ma?? e Pacovan, ajustando os processos de indu??o de estruturas polimeristem?ticas ede multiplica??o de brotos e induzir o aumento da variabilidade gen?tica por meio da mutag?nese in vitro com o uso do agente qu?mico etilmetanosulfonato e, da transforma??o gen?tica pelo bombardeamento de micropart?culas.Foram avaliados os calos formados, o efeito do mutag?nico no cultivo in vitro de brotos e, os efeitos da transforma??o gen?tica quanto ? resist?ncia dos brotos em meio de sele??o contendo o herbicida Imazapyr. Os resultados mostraram que as estruturas polimeristem?ticas obtidas t?m capacidade de originar calos com apenas um m?s de cultivo e, ambas as cultivares desenvolveram calos fri?veis com rendimentos m?dios acima de 90%. A efici?ncia desse m?todo foi comprovada pela alta capacidade de indu??o de calos fri?veis nas duas cultivares avaliadas, como tamb?m pela rapidez no processo de obten??o de calos, sendo este o primeiro estudo de adapta??o da metodologia para as cultivares Ma?? e Pacovan. Por outro lado, a avalia??o da indu??o de muta??o permitiu concluir que a sobreviv?ncia e a capacidade de forma??o de brotos diminu?ram em fun??o do aumento da concentra??o e do tempo de imers?o no etilmetanosulfonato. As plantas sobreviventes passaram por uma triagem com o agente seletivo ?cido fus?rico na qual, foi poss?vel regenerar plantas in vitro das cultivares submetidas ao tratamento com o mutag?nico e selecionar poss?veis mutantes com resist?ncia ao ?cido fus?rico para as cvs. Ma?? e Pacovan. O m?todo da transforma??o gen?tica mostrou-se eficiente na regenera??o dos brotos resultando em altos valores de sobreviv?ncia e multiplica??o, onde poss?veis plantas transg?nicas de bananeira cv. Ma?? foram obtidas, ap?s a sele??o de resist?ncia ao herbicida. Portanto, conclui-se que todo material produzido, tanto na fase mutag?nica quanto na transforma??o gen?tica, apresenta uma maior variabilidade gen?tica potencialmente aplic?vel ao melhoramento da bananeira.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-04-20T13:03:58Z No. of bitstreams: 1 RaquelSampaiodeOliveiraTese2015.pdf: 10137790 bytes, checksum: c5825d13e8bc435cc264076585a21c29 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-04-20T13:04:12Z (GMT) No. of bitstreams: 1 RaquelSampaiodeOliveiraTese2015.pdf: 10137790 bytes, checksum: c5825d13e8bc435cc264076585a21c29 (MD5) / Made available in DSpace on 2017-04-20T13:04:12Z (GMT). No. of bitstreams: 1 RaquelSampaiodeOliveiraTese2015.pdf: 10137790 bytes, checksum: c5825d13e8bc435cc264076585a21c29 (MD5) Previous issue date: 2015-12-15 / Cotton is an economically important natural fiber produced in the world and it is highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton???s resistance through the expression of different transgenes, including Cry toxins, hydrolytic proteinase inhibitors, toxic peptides, dsRNA, among others. However, transformation methods remain limited by cotton variety due to the difficulties imposed by tissue culture and the steps necessary in Agrobacterium-mediated transformation and/or particle bombardment. The pollen tube transformation technique involves the introduction of an exogenous DNA into the plant genome being independent of tissue culture. This method can be applied into different cotton cultivars and it has been used successfully in generation of Bt cotton. In Chapter 2, the pollen-tube pathway technique was used to transform a Brazilian cotton cultivar. The putative transgenic plants derived from boll seeds injected with a binary expression vector, harboring the cry1Ia12 gene were initially selected with kanamycin. Selected plants were characterized using PCR, Southern blot, Western blot and ELISA techniques to confirm the genetic transformation. Western blot and ELISA data showed variable protein expression among the transgenic plants varying from 1,?? ??g g-1 to ??,??6 ??g g-1. An insect bioassay using T1 plants revealed the entomotoxic effects of Cry1Ia12 on Spodoptera frugiperda, as evidenced by a decrease in the development of insects compared with untransformed controls. Entomotoxic effect with Anthonomus grandis was also demonstrated by a decrease in the number of emerging insect adults. In Chapter 3, the agrolistic transformation technique was used to transform the Coker 310 cultivar. The putative transgenic plants derived from transformed embryos with a vector harboring the cry8Ka5 and ??AI-C3 genes were selected in vitro, and acclimatized in a greenhouse. Acclimatized plants were characterized using PCR and ELISA techniques to confirm the genetic transformation. Collected seeds from positive plants were sown for T1 plants analysis, aiming the technique evaluation. It was observed the cry8Ka5 gene amplification, and the ELISA results showed variable protein expression between transgenic plants. It was possible to demonstrate with this work, the efficacy of GM cotton plants generation using alternative biotechnological approaches, efficiently applied to the lepidopterans and coleopterans control. / O algod??o ?? uma fonte de fibra natural de grande import??ncia econ??mica, sendo uma cultura altamente afetada por diferentes insetos-praga e pat??genos. Diversas estrat??gias de transgenia t??m sido desenvolvidas para melhorar a resist??ncia do algod??o, por meio da express??o de transgenes, incluindo toxinas Cry, inibidores de enzimas hidrol??ticas, pept??deos t??xicos, dsRNA, entre outros. No entanto, os m??todos de transforma????o ainda s??o limitados a algumas variedades de algod??o devido ??s dificuldades impostas pela cultura de tecidos e ??s etapas necess??rias de transforma????o, seja mediada por Agrobacterium e/ou bombardeamento de part??culas. A t??cnica de transforma????o via tubo pol??nico ?? uma t??cnica que envolve a introdu????o de DNA gen??mico ex??geno em plantas, sendo independente de cultura de tecidos. Este m??todo pode ser aplicado para diferentes cultivares de algod??o e tem sido utilizado com sucesso na gera????o de algod??o Bt. No Cap??tulo 2, a t??cnica de transforma????o via tubo pol??nico foi utilizada para transformar uma cultivar de algod??o brasileira. As potenciais plantas transg??nicas geradas, derivadas de sementes que tiveram o bot??o floral injetado com um vetor de express??o bin??rio, portador do gene cry1Ia12, foram inicialmente selecionadas com o antibi??tico canamicina. Plantas selecionadas foram caracterizadas por PCR, Southern blot, Western blot, e ensaio de ELISA, visando confirmar a transforma????o gen??tica. Os dados de Western blot e de ELISA mostraram uma express??o vari??vel da prote??na entre as plantas transg??nicas, com concentra????es variando de 1,?? ??g g-1 a ??,??6 ??g g-1. Ensaios biol??gicos com o inseto alvo utilizando plantas T1 revelaram os efeitos entomot??xicos da toxina Cry1Ia12 em Spodoptera frugiperda, evidenciado por uma diminui????o no desenvolvimento de insetos, em compara????o com as plantas controle n??o transformadas. Foi observado um efeito entomot??xico em Anthonomus grandis, demonstrado pela diminui????o no n??mero de adultos emergentes. No Cap??tulo 3, a t??cnica de transforma????o via agrol??stica foi utilizada para transformar uma cultivar de algod??o Coker 310 visando avaliar essa t??cnica. As plantas potencialmente transg??nicas geradas, derivadas dos embri??es transformados com o vetor portando os genes cry8Ka5 e ??AI-C3 foram selecionados in vitro e, posteriormente, aclimatizados em casa de vegeta????o. Estas plantas foram caracterizadas por PCR e ELISA para confirmar a transforma????o gen??tica. Sementes das plantas positivas foram semeadas para an??lise das plantas T1. A amplifica????o do gene cry8Ka5 foi observada e os resultados de ELISA mostraram uma express??o vari??vel da prote??na entre as plantas transg??nicas. Os estudos aqui apresentados demonstraram a efici??ncia na gera????o de plantas de algod??o GM, utilizando abordagens biotecnol??gicas alternativas e eficientes para controle de lepid??pteros e cole??pteros.