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.

Submitted by Luis Ricardo Andrade da Silva (lrasilva@uefs.br) on 2016-02-22T22:50:07Z No. of bitstreams: 1 Disserta??o_Alex_RGV_Final2.pdf: 2862200 bytes, checksum: e222cf5496c76484ec34f8fdd9012d23 (MD5) / Made available in DSpace on 2016-02-22T22:50:07Z (GMT). No. of bitstreams: 1 Disserta??o_Alex_RGV_Final2.pdf: 2862200 bytes, checksum: e222cf5496c76484ec34f8fdd9012d23 (MD5) Previous issue date: 2015-11-16 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / In Brazil, research on Arecaceae pollen grains are related to melissopalynology, the palynotaxonomy, being scarce the physiology of pollen. Reproductive biology studies pollination are of great importance in different cultures, from their results can obtain parameters to be used to obtain a considerable increase in the production of croops, as well as the size and quality of fruits, helping for good economic income of the producers. In vitro pollen germination also allows you to check its viability and reproductive strength, and are important tools in breeding programs of plants, assisting in the selection of the most effective genotypes and training hybrids. The chapter one of this work consists of evaluation of the type of culture medium and implications on the choice of micronutrients which form part of its composition, as well as the pretreatment of specimens. It was observed that oil palm pollen (Elaeis guineensis Jacq.) Showed an increase in germination rates after being dissected for a period of four hours and the culture medium with only boric acid and calcium nitrate, in addition to the power source (sucrose) are efficient to display their germination potential. Chapter two contained in the evaluation glycoconjugate (lectins) on the pollen germination of oil palm, which was discussed metabolic interactions of these processes. Lectins are important regulators and operate in recognition and acknowledgment of pollen on the pistil and can act as genetic incompatibility factors. Germination was evaluated in BCa means (boron and calcium) with the addition of lectins: Crotalaria pallida lectin (CPL), Concanavalin A (ConA) and Jacalin (JAC) in two concentrations. An increase of the germination rate in the presence of ConA and CPL lectins and inhibition in the presence of JAC. The results of this study were valuable, since aggregate informative values and extend discussions on the processes involving the germination of pollen grains and formation of pollen tubes of oil palm. / No Brasil, pesquisas com gr?os de p?len da fam?lia Arecaceae est?o relacionados ? melissopalinologia e palinotaxonomia, sendo escassos aqueles voltados ? fisiologia do p?len. Estudos de biologia reprodutiva relacionados ? poliniza??o s?o de grande import?ncia nas diversas culturas, visto que a partir dos seus resultados pode se obter par?metros a serem utilizados visando o aumento consider?vel da produ??o de plantas cultivadas, bem como no tamanho e na qualidade dos frutos, contribuindo para bons rendimentos econ?micos dos produtores. A germina??o de gr?os de p?len in vitro tamb?m permite verificar seu vigor reprodutivo, sendo importante ferramentas em programas de melhoramento gen?tico de plantas, auxiliando na sele??o de gen?tipos mais eficazes para cruzamentos e forma??o de h?bridos. O cap?tulo um deste trabalho teve como prop?sito avaliar o tipo de meio de cultura e as implica??es da escolha dos micronutrientes que fazem parte da sua composi??o, bem como do tratamento pr?vio das amostras. Foi observado que o p?len de dendezeiro (Elaeis guineensis Jacq.) apresentou aumento nas taxas de germina??o depois de ser desidratado por um per?odo de quatro horas e que o meio de cultura com apenas ?cido b?rico e nitrato de c?lcio, al?m da sacarose como fonte de energia, s?o eficientes para exibir o potencial germinativo. O cap?tulo dois consta da avalia??o de prote?nas glicoconjugadas (lectinas) sobre a germinabilidade pol?nica do dendezeiro. Foram discutidas as intera??es metab?licas desses processos, bem como o papel das lectinas que s?o importantes reguladores sobre a recogni??o e reconhecimento do p?len no pistilo, podendo atuar como fatores de incompatibilidade gen?tica. Foi avaliada a germina??o em meio BCa (boro e c?lcio) com adi??o das lectinas: Lectina de Crotalaria pallida Aiton (CPL), Concanavalina A (ConA) e Jacalina (JAC), em duas concentra??es. Verificou-se aumento das taxas de germina??o na presen?a das lectinas CPL e ConA e inibi??o da germina??o na presen?a da JAC. Os resultados deste trabalho agregam valores informativos sobre a compreens?o da biologia reprodutiva de dendezeiro (Arecaceae) ocorrente na ?Costa do Dend??/Bahia e ampliam as discuss?es sobre os processos que envolvem a germina??o de gr?os de p?len e forma??o dos tubos pol?nicos.