Arabidopsis CBF1 gene transfer into tomato (Lycopersicon esculentum Mill) by Agrobacterium-mediated transformation

• Main Authors: Li-Hui Chiu, 邱麗慧
• Other Authors: Y.C. Wang
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/80854118905487264398

碩士 === 國立臺灣師範大學 === 生物研究所 === 87 === 英文摘要 Overexpression of the low-temperature transcriptional activator CBF1 (CRT/DRE-binding factor 1) can induce the expression of a pool of COR -related genes and enhance freezing tolerance of nonacclimated Arabidopsis plants (Jaglo-Ottosen et al., 1998). The objectives of this study were to investigate whether genes homologus to the Arabidopsis CBF1, COR15a and COR47 genes were present in the tomato genome, to establish an Agrobacterium-mediated transformation system for Taiwan local tomato cultivar, and to transfer a constitutively expressed Arabidopsis CBF1 gene into the particular tomato cultivar. The tomato cultivar CL5915-93D4-1-0-3 from AVRDC was chosen as the target plant due to its sensitivity to chilling, its commercial value, and its previous absence in transformation experiments. To investigate whether homologes of the Arabidopsis CBF1, COR15a and COR47 genes were present in the tomato genome, Southern and Northern blotting analyses were performed. Our results indicated that these counterpart genes did exist in the tomato genome. However, the CBF1-like gene in tomato showed a restricted developmental expression, only being detected in one-month-old seedlings. To establish an Agrobacterium-mediated transformation system for Taiwan local tomato cultivar, the binary vector pCAMBIA2301 was selected. This vector contains an intron GUS reporter gene and a kanamycin-resistant marker, and both were driven by CaMV35S promoter. Tomato cotyledons were infected by Agrobacterium strain LBA4404 and EHA105 containing pCAMBIA2301, which after kanamycin selection and GUS histochemical staining, resulted in 2 and 1 regenerated transformants, respectively. This study also transformed tomato with another binary vector, pJLM1 containing CaMV35S promoter and the Arabidopsis CBF1 gene. By optimizing the transformation conditions including, Agrobacterium concentration, numbers of explants per plate while co-culture, and the frequency of changing selection medium, we have obtained eighteen rooted transgenic plants. PCR amplifications showed that Kanamycin-resistant and intron GUS genes had been inserted to the transgenic tomato genome. Southern blotting analysis also demonstrated the existence of Arabidopsis CBF1 gene in the transgenic tomato genome. These transgenic plants should be useful for future chilling tolerance experiments.