| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 97 === Transformation of multiple genes into tobacco by Agrobacterium rhizogenes-mediated method was performed in this study. Nicotiana tabacum Wisconsin 38 was selected as the material for hairy root induction. Two reporter genes, gfp from pCAMBIA 1302 and gus from pCAMBIA 1201, were used for co-transformation study. Two strategies were developed. The first one, two reporter genes were carried by two respective plasmids. And then, transformation was conducted either by one transformed A. rhizogenes with two binary vectors (2BV), or conducted by co-cultured at the same time with two separate transformed A. rhizogenes which harboring one reporter gene (2AR). The second one was, constructing the two reporter genes in one binary vector. Thus, two reporter genes located in one T-DNA region or separated in two T-DNA regions were constructed, and they were named pCGG-1TD and pCGG-2TD, respectively. The above plasmids were transformed into A rhizogenes, and then inoculated tobacco leaf discs. The root inducing rate of 2AR and 2BV were 81 % and 75 %, respectively. On the other hand, those induced by pCGG-1TD and pCGG-2TD caused less hairy roots. Regarding expression of reporter genes, in 2BV and pCGG-1TD groups, we have observed GFP expression by fluorescence microscopy and GUS expression by tissue staining. Nowadays, the construction of 2BV was the better strategy to gain co-transformation and co-expression hairy roots. In this study, if all strategies can succeed to transform multiple genes into plants, we can integrate those strategies and transform multi-genes into plants at the same time to induce hairy roots for secondary metabolite production.
|
|---|
