Regeneration and genetic transformation in melon (Cucumis melo L.) cv. Gaucho

O presente trabalho teve como objetivo desenvolver um protocolo eficiente de regenera????o para o mel??o (Cucumis melo L.), cv. Ga??cho, visando posterior transforma????o gen??tica do mesmo. Para tal fim foram realizados tr??s experimentos. No primeiro, os cotil??dones foram divididos em 2, 3, 4 e 5 partes iguais e, inoculados em meio MS contendo 30 g L-1 de sacarose, 0,9 mg L-1 BAP, 0,3 mg L-1 ABA e 2,2 g L-1 CaCl2. No segundo experimento, utilizando o mesmo meio de cultura do experimento anterior, os cotil??dones foram submetidos a diferentes densidades de fluxo de f??tons. No terceiro procurou-se estabelecer as condi????es para germina????o das sementes e sua influ??ncia na regenera????o cotiledonar sob diferentes concentra????es de BAP. Observou-se que cotil??dones seccionados em 5 partes apresentaram maior m??dia de explantes regenerantes. J?? os cotil??dones que permaneceram sob intensidade luminosa de 2 ??mol m-2 s-1 apresentaram maior m??dia de explantes regenerantes e os que ficaram no escuro apresentaram maior m??dia de explantes com calos e raiz. Observou-se tamb??m que quanto maior o per??odo germinativo e maiores concentra????es de BAP no meio regenerativo, menor ?? a taxa de explantes regenerantes. Ap??s a obten????o do protocolo de regenera????o, foi realizado teste de sensibilidade dos explantes ao antibi??tico de sele????o, determinando-se a concentra????o de 75 mg L-1 de canamicina como ideal para selecionar as c??lulas transformadas. Para a regenera????o de brota????es os explantes foram cultivados em meio MS contendo 0,9 mg L-1 BAP, 0,3 mg L-1 ABA, 2,2 g L-1 CaCl2, 75 mg L-1 canamicina e 250 mg L-1 cefotaxima. Para a transforma????o gen??tica, foi utilizado Agrobacterium tumefaciens LBA 4404, com um clone da ACC oxidase em orienta????o antisense, denominado pAP4as. Entretanto, n??o foi confirmada a inser????o da seq????ncia, pelo teste de PCR, nos tecidos analisados. Verificou-se que estes estudos descritos com Agrobacterium n??o foram eficientes para a obten????o de pl??ntulas contendo o gene antisense da ACC oxidase.The objective of this work is to seek a protocol for regeneration of the melon (Cucumis melo L.) cv. Gaucho and subsequent genetic transformation of this fruit. To this end were conducted three experiments. In the first, the cotyledons were divided into 2, 3, 4 and 5 equal parts, and inoculated with MS medium containing 30 g L sucrose, 0.9 mg L-1 BAP, 0.3 mg L-1 ABA and 2.2 g L-1 CaCl2. It was observed that the number of cuts not influenced the percentage of explants regenerated. In the second experiment, using the same means of cultivation of the previous experiment, the cotyledons were submitted to different densities of flow of photons. It was found that the regenerative process was not affected by the absence or presence of light (2 and 21 ??mol m-2 s-1), but when the explants remained in the dark had greater formation of callus. In the third experiment sought to establish conditions for seed germination and its effect on the regeneration under different concentrations of BAP. It was the highest rate of explants regenerated lower when the period of germination in the middle and lower the concentration of BAP in the regenerative. After obtaining the protocol for regeneration, was conducted test of sensitivity of explants the selection of antibiotic, setting up the concentration of 75 mg L-1, kanamycin as ideal to select the cells transformed. For the regeneration of the shoots explants were cultivated in MS medium containing 0.9 mg L-1 BAB, 0.3 mg L-1 ABA, 2.2 g L-1 CaCl2, 75 mg L-1 kanamycin and 250 mg L-1 cefotaxime. For the genetic transformation was used Agrobacterium tumefaciens LBA 4404, with a clone of ACC oxidase in antisense orientation, called pAP4as. However, it was not confirmed by inserting the sequence, the PCR test, the tissue analyzed. It was found that these studies were not described with Agrobacterium efficient to obtain seedlings containing the antisense gene of ACC oxidase. It was found that these studies were not described with Agrobacterium efficient to obtain seedlings containing the antisense gene of ACC oxidase