Morfogênese in vitro e transformação genética de citros mediada por Agrobacterium tumefaciens

• Autor(a) principal: Oliveira, Maria Luiza Peixoto de
• Data de Publicação: 2008
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/970
• topic: Transformação genética; Citros; Agrobacterium tumefaciens; Genetic transformation; Citrus; Agrobacterium tumefaciens; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL

Current protocols used for citrus genetic transformation have resulted in a low number of transgenic plants, furthermore, it used juvenile material which source of explants. Plants regenerated from these sources of explants have long juvenile periods for initial fruit production and are necessary many years for evaluation of the introduced characteristics of interest in the target plant. A reliable and ideal protocol for citrus plant transformation would be based on the use of mature tissue as sources of explant, because the juvenility problem could be lessened and the transgenic trait could be analyzed in a relatively short period of time. Therefore, the initial objective of the present work was to establish promotive conditions for a reliable in vitro regeneration system from internodal segments of mature tissue of sweet orange and rangpur lime to further adequate an efficient and reproducible transformation methodology. For organogenesis induction, we investigated the effects of different culture media associated, growth regulator (BAP and NAA) and β-lactams antibiotics, (timentim, cefotaxime, meropenen and augmentin) on the morphogenic response from mature tissue of Citrus sinensis and C. limonia. We have demonstrated that in vitro shoot induction from mature tissue of Citrus was affected mainly by the genotype, growth regulators, media formulation and antibiotics incorporated to the culture medium. Furthemore, an improved protocol for genetic transformation of epicotyl and immature cotyledons were developed via Agrobacteirum tumefaciens. For protocol involving epicotyl explants some factors were investigated, such as: the use of sonication, vacuum infiltration and sonication in association with vacuum infiltration, comparing with conventional Agrobacterium-mediated transformation method ( dipping method) during cocultivation of Pineapple sweet orange (Citrus sinensis L. Osbeck) and citrumelo Swingle (Citrus paradisi Macf. X Poncirus trifoliate L. Raf.) explants. The use of sonication for 2 seconds, followed by 10 minutes of vacuum infiltration had a positive effect on putative transgenic efficiency, resulting in the highest transformation efficiency (8.4%) for Pineapple sweet orange. For citrumelo Swingle the transformation efficiency also was enhanced with the combination of sonication and vacuum infiltration (9.6%), however power than the highest scores reached by ordinary co-cultivation protocol, which rended a transformation efficiency of 11.2%. Finally, we studied factors that might influence morphogenesis and Agrobacterium-mediated transformation efficiency of immature cotyledons from Duncan grapefruit . For organogenesis induction, the combination of 2 mg l-1 BAP, 1 mg l-1 KIN and 1 mg l-1 IAA led to the highest frequency of cotyledons forming shoots (96%) and number of shoots per explant (5.8), using explants cultured upside down for 3 weeks in the darkness, followed by 3 weeks in 16/8 h (light/dark) regime. Thus, optimization of Agrobacterium-mediated parameters showed that the highest transformation efficiency was achieved when explants were submitted to vacuum infiltration (15 min) in presence of agrobacteria (OD600nm 0.5); co- cultivation for 3- days on 100 μM acetoseryngone- supplemented medium; and finally transferred to a selective MS-based medium, added with 30 mg l-1 kanamycin, 12.5 mg l-1 de Meropenen®, 2 mg l-1 BAP, 1 mg l-1 KIN and 1 mg l-1 IAA.