Zhang Tianzhen, Wu Shen-Jie
National Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Research Institute, Nanjing Agricultural University, Nanjing, China.
Methods Mol Biol. 2012;847:245-53. doi: 10.1007/978-1-61779-558-9_21.
Agrobacterium tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. The binary vector pBI121 (containing a neomycin phosphotransferase II gene npt-II as a selection marker and a uidA gene as a reporter gene) was used to research transformation efficiency. After 48 h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse granule EC. It indicated that the efficiency of transient transformation was affected by EC morphology. Transient transformation efficiency also was improved by cocultivation on the medium by adding 50 mg/L acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density increased the production of kanamycin-resistant (Km-R) calli lines. From an original 0.3 g EC, an average of 20 Km-R calli lines were obtained from a selection dish, and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments, and the GUS-positive rate of regeneration plants was about 72.6%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and gus gene. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, was integrated into the cotton genome.
通过选择合适的棉花胚性愈伤组织（EC）并提高共培养、选择培养和植株再生效率，增强了根癌农杆菌介导的棉花胚性愈伤组织转化。使用二元载体pBI121（含有新霉素磷酸转移酶II基因npt-II作为选择标记和uidA基因作为报告基因）来研究转化效率。共培养48小时后，以黄色、疏松和细颗粒状的胚性愈伤组织为特征的β-葡萄糖醛酸酶（GUS）阳性愈伤组织数量比灰色、棕色和粗颗粒胚性愈伤组织多两倍。这表明瞬时转化效率受胚性愈伤组织形态的影响。在添加50 mg/L乙酰丁香酮的培养基上于19°C共培养48小时也提高了瞬时转化效率。在低细胞密度的选择培养基上继代培养胚性愈伤组织增加了卡那霉素抗性（Km-R）愈伤组织系的产生。从最初的0.3 g胚性愈伤组织开始，每个选择培养皿平均获得20个Km-R愈伤组织系，Km-R克隆的GUS阳性率为81.97%。通过脱水处理在分化培养基上快速再生出大量正常植株，再生植株的GUS阳性率约为72.6%。对GUS阳性植株的聚合酶链反应分析显示新霉素磷酸转移酶II基因和gus基因的阳性检测率为100%。对从不同Km-R愈伤组织系再生的转基因植株进行Southern杂交表明，目标基因大多以低拷贝数整合到棉花基因组中。
