关键词: 根癌农杆菌, 玉米, 基因型, 遗传转化, 瞬间表达, 抗性愈伤组织

Abstract:

Plant transformation offers opportunities for advancing biological research and genetic improvement in crops. With the progress of plant genomic studies, high throughput transformation systems are one of the critical technologies for basic scientific research and production of commercial genetically engineered crops. Agrobacterium-mediated plant transformation has the potential of high transformation frequencies and the advantage that T-DNA integration into the plant genome often occurs in single or low copy number. These putative advantages make it to be one of the popular methods for producing transgenic maize.
It has been known that many factors especially plant genotypes have a large impact on transformation results. Setting up variety-independent transformation system mediated by Agrobacterium is one of the most promising strategies to obtain high throughput transformation of plants. In this study we used an improved transformation system including mediums for Agrobacterium growth, inoculation, co-culture and selection to study the effect of Agrobacterium-mediated transformation with four maize genotypes (Hi-Ⅱ, H99, R18-599, and Qi 319). For two different co-culture methods with Hi-Ⅱ, it was observed that transit GUS expression in solid co-culture medium and filter paper was 83.7% and 4.4%, respectively, indicating that solid co-culture medium is better than paper. In the comparison of the effect of fresh isolated immature embryos (FIIEs) and pre-culture immature embryos (PCIEs) from Hi-Ⅱ on the transformation, we noted that transit GUS expression in FIIEs and PCIEs was 83.7% and 12.1%, respectively, demonstrating that FIIEs is more suitable for maize transformation than PCIEs. In addition, the optimized system was also used for transformation of the other three inbred lines H99, Qi 319(FIIEs) and R18-599 (embyrogenic calli). As a result, high transit GUS expression was also detected in all of these three genotypes after 3-day co-culture, reaching 65.2%, 52.6%, and 58.0%, respectively. When Hi-Ⅱ FIIEs infected with Agrobacterium was inoculated on selection medium containing 25–100 mg L^-1 paromomycine for three rounds of selection, 2.4% resistant calli were obtained, which approximately reflected the transformation efficiency of this system. The selection of resistant calli for the other three genotypes is still underway. Our preliminary results suggested that the Agrobacterium-mediated system we have set up has the potential of applicability for the four maize genotypes studied, and a further variety-independent transformation system mediated by Agrobacterium may be achieved by optimizing culturing conditions.

Key words: Agrobacterium tumefaciens, Maize, Genotype, Transformation, Transient expression, Resistant callus