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.