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Doctoral Thesis
DOI
https://doi.org/10.11606/T.11.2019.tde-20191220-142316
Document
Author
Full name
Solange Rocha Monteiro de Andrade
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2001
Supervisor
Title in Portuguese
Otimização da técnica SAAT ("Sonication-Assisted Agrobacterium-Mediated Transformation") para transformação de Phaseolus vulgaris
Keywords in Portuguese
AGROBACTERIUM TUMEFACIENS
FEIJÃO
TRANSFORMAÇÃO GENÉTICA
VETORES
Abstract in Portuguese
Phaseolus vulgaris L. era considerado recalcitrante à transformação por Agrobacterium. Entretanto, alterações no meio de co-cultivo, utilização de linhagens hipervirulentas de Agrobacterium e de vetores binários contendo genes vir demonstraram que o feijoeiro é susceptível à Agrobacterium. No entanto, ainda não foram obtidas plantas transgênicas de feijoeiro por meio deste método. Em 1997, Trick & Finer descreveram a metodologia SAAT ("Sonication assisted Agrobacterium mediated transformation"). Esta técnica consiste em produzir microferimentos no explante através da sonicação por curtos períodos de tempo na presença do Agrobacterium. Por meio deste método foram obtidas plantas transgênicas de soja e trigo. O objetivo do presente trabalho foi ajustar a metodologia SAAT para a transformação de P. vulgaris. A variedade de feijoeiro utilizada foi a Olathe Pinto, a linhagem de Agrobacterium tumefaciens foi LBA4404:pTOK. Foram estudados o efeito do pré-tratamento, tempo de co-cultivo, duração do SAAT e presença de acetoceringona. Os resultados demonstram que a expressão transitória e estável da β-glucoronidase foi maior quando os eixos embrionários foram pré-tratados por 10 a 14 dias em meio de multibrotação. O antibiótico geneticina (G418) apresentou maior capacidade de seleção de brotos do que canamicina, nas concentrações estudadas. SAAT de 60 segundos dos explantes apresenta melhor expressão transitória e estável da β-glucoronidase. O tempo de co-cultivo líquido de 2 horas seguido de 48 horas em meio sólido, na ausência de acetoceringona, apresentou a maior expressão transitória. A presença de acetoceringona (20 mg/l) aumentou a expressão transitória. Pré-incubação por 24 horas não aumentou a eficiência do tratamento SAAT. As análises de microscopia óptica e de varredura demonstraram a presença de rupturas na epiderme, quebras da parede celular e invasão do Agrobacterium nos tecidos subepidérmicos. Os resultados demonstraram que o método SAAT é uma técnica viável para a transformação de P. vulgaris via Agrobacterium.
Title in English
Optimization of SAAT (Sonication-Assited Agrobacterium-Mediated transformation) technique to transform Phaseolus vulgaris
Abstract in English
Common bean was considered recalcitrant to Agrobacterium-mediated transformation. After some studies with co-cultivation medium, pre-treatment of explants, hipervirulent Agrobacterium strains and binary vectors with vir genes, it has been reported the susceptibility of Phaseolus vulgaris to some Agrobacterium strains. However, still no transgenic bean has been reported using this method. The Agrobacterium-mediated transformation of P.vulgaris presents several problems, such as the interaction Agrobacterium-host, explant maturity, pre-culture conditions, co-cultivation medium, and selection strategies. The technique SAAT consists to produce microwounds of the explant through sonication during short period of time in presence of Agrobacterium. Using this method Trick & Finer (1997; 1998) obtained transgenic wheat and soybean. The objective of the present work is to adjust the SAAT method to transform common bean. The bean variety used was Olathe Pinto, and Agrobacterium tumefaciens strain LBA4404:pTOK. Transient and stable transformation increased when mature embryos were pre-treated during 10 to 14 days in cytokinin-induced shoot organogenesis medium. Geneticin (G418) was observed to be better than kanamycin to select shoots. SAA T duration effect on transient and stable transformation shown, in both experiments, that 60 seconds presents the best results. Co-cultivation conditions, without acetosyringone, of 2 hours in liquid medium followed by 48 hours in solid medium presented the higher transient expression. Acetosyringone (20 mg/L) increases transient expression and pre-incubation for 24 hours didn't increase SAAT efficiency. Scanning and optical microscopy demonstrated that sonication produces microwounds in the explant epidermal surface, cell wall break and Agrobacterium invasion of subepidermal tissue. Results shown that SAAT is a promising method to transform of P. vulgaris.
 
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Publishing Date
2019-12-20
 
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