Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca

• Autor(a) principal: Vaez, Juliana Rocha
• 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/123456789/286
• topic: Manihot esculenta; Cultura de tecidos; Manihot esculenta; Tissue culture; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

Cassava (Manihot esculenta Crantz) is one of the most important crops for human consumption. It is considered the fourth most important source of carbohydrates in the tropics. The increasing importance of the culture achieved during the last years, raised the interest in the improvement and development of new cassava varieties. Therefore, the aims of the present work were to evaluate factors associated to the in vitro regeneration and genetic transformation of five cassava cultivars. Explants from different genotypes were evaluated for their embryogenic response to two growth regulators, picloram and 2,4 D. The growth regulator 2,4 D led to the best embryogenic response and the concentrations of this regulator for cultivars Mantiqueira, Parazinho, Vassourinha and Urubu were two, one, two and two mg/L, respectively. Somatic embryogenesis is a process by which haploid cells develop through different embryogenic states, originating a plant. Embryos in the cotyledonary stage may have their development induced by several combinations of growth regulators. Aiming to evaluate which combination promotes the best stretching frequency, the following combinations were tested: 0.4 mg/L BAP; 0.25 mg/L BAP + 0.25 mg/L AIB; 0.4 mg/L GA3 and 0.25 mg/L GA3 + 0.25 mg/L AIB. Among the evaluated media, the most adequate was the one which contained 0.4 mg/L GA3. In order to induce organogenesis, the following combinations of growth regulators were tested: 1 mg/L BAP + 0.5 mg/L AIB and 1.6 mg/L BA + 1.6 mg/L AIB were evaluated. The highest organogenic frequency was obtained with the use of the first combination mentioned. The organogenic process may be influenced by many factors, such as genotype, type of explants and developmental stage of the explants. Different cotyledonal regions and maturation periods of somatic embryos at the cotyledonal stage which produced the greatest amount of shoots were evaluated. In the case of cultivar Rosinha, the central cotyledonal region was the most adequate for organogenesis induction and the 15-day maturation period was the one which produced the highest amount of in cotyledons. Various protocols for manihot transformation were developed, however, none of them have universal applicability and the selection systems used still either generate many escapes or are deleterious. There are more than 1,500 manihot cultivars reported, however, only a few was successfully used for transformation. Among the transformation systems used, there is a preference for the transformation via Agrobacterium tumefaciens for the production of transgenic plants. To increase the transformation efficiency by A. tumefaciens, parameters which could decrease histodifferentiation were evaluated in cultivar Rosinha. The parameters that interfered in the transformation were paramomycin at a concentration of 500 mg/L and the bacterial strain EHA 105. Os fatores que interferem no processo foram a paramomicina na concentração de 500mg/L e a estirpe bacteriana EHA105. A major factor to be defined during the transformation process is the antibiotic dosage. The concentration defined should prevent false positives without interfering with the regeneration potential of the explants used in the transformation process. The effects of different concentration of kanamycin and paramomycin on somatic embryos were evaluated in globular and cotyledonal stages. The concentrations of 10mg/L (kanamycin) and 20 mg/L (paramomycin) were chosen for embryo selection in globular stage of cultivar Rosinha. For embryo selection in cotyledonal stage of cultivar Rosinha the concentration of 10mg/L paramomycin was chosen. The transformation via SAAT (Sonication-assisted Agrobacterium-mediated transformation) uses ultrasound pulses to wound the tissue and this factor can affect transformation efficiency. In order to evaluate the efficiency of this transformation system, experiments were conducted which evaluated the regenerative response of explants that had undergone the SAAT procedure as well as the frequency of the GUS gene expression. It was verified that SAAT caused an increase on the transient expression of the GUS gene and the regeneration of explants were slightly affected by the process.