利用改良的农杆菌培养液、侵染液、共培养培养基和筛选培养基等技术体系，对4个玉米基因型Hi-Ⅱ、H99、国内2个优良自交系R18-599和齐319的新鲜幼胚、预培养幼胚、胚性愈伤组织进行了农杆菌转化研究，并比较了不同共培养方式对农杆菌侵染不同玉米组织的影响。结果表明，Hi-Ⅱ新鲜幼胚在固体培养基上共培养和滤纸上共培养后GUS基因瞬间表达率分别为83.7%和4.4%，前法优于后法；Hi-Ⅱ新鲜幼胚、预培养3 d的幼胚愈伤组织经农杆菌侵染后GUS基因瞬间表达率分别为83.7%和12.1%，新鲜幼胚比预培养的幼胚愈伤组织更适合于农杆菌转化。用上述优化的条件对另外3个不同基因型的2种不同外植体H99、齐319(新鲜幼胚)和R18-599（胚性愈伤组织）进行遗传转化，共培养3 d后的GUS基因瞬间表达率分别为65.2%、52.6%和58%，表明该转化体系适合于上述4种基因型的幼胚和胚性愈伤组织的遗传转化。此外农杆菌侵染Hi-Ⅱ新鲜幼胚后经在含巴龙霉素25~100 mg L-1培养基上3轮选择后，抗性愈伤组织获得率为2.4%，近似反映了本研究的遗传转化率。其他3个基因型的抗性愈伤也正在筛选中。结果初步表明，本研究建立的农杆菌介导的玉米遗传转化体系可能对4种基因型均适用。
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.
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