Full text
| |
| Author(s): |
Solange Rocha Monteiro de Andrade
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Piracicaba. |
| Institution: | Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) |
| Defense date: | 2001-04-10 |
| Advisor: | Carlos Alberto Labate |
| Abstract | |
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. (AU) | |