Moghaieb Reda E A, El-Arabi Nagwa I, Momtaz Osama A, Youssef Sawsan S, Soliman Mohamed H
Department of Genetics and Genetic Engineering Research Center (GERC), Cairo University, Giza, Egypt.
GM Crops. 2010 Mar-Apr;1(2):87-93. doi: 10.4161/gmcr.1.2.11172.
The objective of the present study is to develop an efficient protocol for regeneration of transgenic wheat plants using Agrobacterium- mediated transformation of mature embryos of hexaploid bread wheat (Triticum aestivum) and tetraploid pasta wheat (Triticum durum). The data indicated that embryogenic calli were formed within 7 days in the presence of 2 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic calli in the presence of 2 mgl-1 BA. Shoot regeneration frequency varied between wheat cultivars according to their genetic background differences. Regeneration frequency was higher in the cultivar Gemmiza 10 (95 %) compared with the other cultivars tested. Mature embryos derived callus of the cultivars Gemmiza 10 and Gemmiza 9 were co-cultivated with A. tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (npt-II). The resulted putative transgenic plantlets were able to grow on kanamycin containing medium. A successful integration of the transgene was confirmed by analyzing the T0 plantlets using Southern hybridization and PCR amplification. The gus gene expression can be detected only in the transgenic plants. The reported protocol is reproducible and can be used to regenerate transgenic wheat plants expressing the genes present in A. tumifaciens binary vectors.
本研究的目的是开发一种高效的方案，用于通过农杆菌介导的六倍体面包小麦（普通小麦）和四倍体意大利面小麦（硬粒小麦）成熟胚转化来再生转基因小麦植株。数据表明，在2 mg/L 2,4 - D存在的情况下，7天内即可形成胚性愈伤组织。在2 mg/L BA存在的情况下，不定芽从胚性愈伤组织中长出。根据小麦品种的遗传背景差异，其芽再生频率有所不同。与其他测试品种相比，Gemmiza 10品种的再生频率更高（95%）。将Gemmiza 10和Gemmiza 9品种的成熟胚来源的愈伤组织与携带含有新霉素磷酸转移酶II基因（npt - II）的二元载体pBI - 121的根癌农杆菌菌株LBA4404共培养。所得的推定转基因幼苗能够在含有卡那霉素的培养基上生长。通过使用Southern杂交和PCR扩增分析T0幼苗，证实了转基因的成功整合。仅在转基因植物中可检测到gus基因表达。所报道的方案具有可重复性，可用于再生表达根癌农杆菌二元载体中存在的基因的转基因小麦植株。
