Păcurar Daniel Ioan, Thordal-Christensen Hans, Nielsen Klaus Kristian, Lenk Ingo
University of Agricultural Sciences and Veterinary Medicine, Mănăştur Street 3-5, Cluj Napoca, 400372, Romania.
Transgenic Res. 2008 Oct;17(5):965-75. doi: 10.1007/s11248-007-9159-y. Epub 2007 Dec 7.
In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.
在将二穗短柄草开发成温带谷类作物和饲草模式植物的过程中，我们为一个二倍体材料建立了一种高通量农杆菌介导的转化系统。从BDR018材料的未成熟胚诱导出的胚性愈伤组织，用携带两个T-DNA质粒pDM805和pWBV-Ds-Ubi-bar-Ds的根癌农杆菌AGL1菌株进行转化。通过改变预培养时间优化瞬时和稳定转化效率，预培养时间对稳定转化效率有很大影响。平均而言，17日龄与农杆菌共接种的愈伤组织中有55%再生出稳定的转基因植株。观察到稳定转化频率高达80%，据我们所知，这是禾本科物种中报道的最高转化效率。在一项对用pDM805转化的177个转基因株系的研究中，所有再生的转基因株系都对草丁膦具有抗性，而gusA基因在88%的转基因株系中表达。Southern杂交分析表明，35%的被测植株有单个T-DNA整合。对10个选定的T(0)植株的后代进行的分离分析表明，两个转基因呈简单孟德尔遗传。此外，pWBV-Ds-Ubi-bar-Ds的T-DNA上存在两个选择标记基因bar和hpt，这使我们能够根据全长整合率来表征所建立的转化方案。即使不进行选择，当使用双丙氨膦作为选择剂时，97%的转化体中发生了全长整合。
