Influência de antibióticos na morfogênese in vitro, transformação genética e isolamento de RNA de urucum (Bixa orellana L.)

• Autor(a) principal: Rodrigues, Simone de Miranda
• Data de Publicação: 2007
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1422
• topic: Urucum; Transformação genética; Bixina; Annatto; Genetic transformation; Bixa orellana; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL

The manipulation of genes involved in carotenoid biosynthesis in Bixa orellana has been attempted with the objective of generating plants producing high content of a natural natural colorant, bixin, and studying the biosynthetic pathway for the accumulation of this unique carotenoid in annatto seeds. The biotechnology, by means of plant tissue culture techniques, offers an alternative tool to produce annatto plants with high bixin content. The present study aimed to evaluate the influence of antibiotics on in vitro morphogenesis and to establish a protocol of genetic transformation and RNA isolation to support further gene isolation and cloning studies of the species. It was observed that hygromycin 4 mg L-1 effectively inhibited morphogenic responses of hypocotyl explants of both M1 and M6 sources of Bico de Pato variety, cultured in MS medium containing 4.56 μM zeatin. However, the same antibiotic concentration was ineffective to inhibit shoot regeneration from hypocotyls precultured for 3 days in the same medium lacking hygromycin. Hygromycin 2.5 mg L-1 lessened up to 98 % of the shoot regeneration in root segments of Peruana . Also, despite the absence of shoot regeneration, when root explants were cultured onto MS liquid medium containing 12.5 and 25 mg L-1 kanamycin, there was a marked root elongation; concentrations higher than 50 mg L-1 kanamycin led to a tissue browning. All antibiotics used to control bacteria, namely timentin, meropenem, augmentin, and cefotaxime somehow lessened regeneration frequencies of hypocotyl and root explants of Bico de Pato and Peruana varieties. The later, presented the lowest average for all the appraised treatments. Those results indicate that the morphogenic responses of the annatto explants are not only genotype-dependent, but also dependent of the source of the explants used. An efficient system of genetic transformation mediated by Agrobacterium rhizogenes R1601, carrying the chimerical construction nos-nptII-nos, was adapted for annatto hypocotyl explants. For the transformation assays two annatto sources were used, M2 and M6 . Differentiated roots from the inoculated distal ends of the explants were isolated and transferred to a semi-solid MS medium added with 100 mg L-1 and 300 mg L-1 timentin. Shoots spontaneously differentiated from roots growing in kanamycin-supplemented medium, however it was verified that the use of a 10 days induction period in liquid medium added with 4.56 μM zeatin, followed by cultivation in semi-solid medium, accelerated the callus formation and, or roots hairy root appearance, followed by adventitious shoot-bud formation. Finally, due to the difficulty of isolation good quality nucleic acid from annatto tissues, very rich in polyphenol and polysaccharide, a protocol using MATAB 2 % in NaCl 3 M was proposed. It was efficient for obtaining a high quality annatto RNA, that will favor future analyses on the isolation, cloning and expression of genes involved in carotenoid biosynthetic pathway, and the introduction of other genes of agronomic interest.