Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
Plant Cell Rep. 2011 Dec;30(12):2281-92. doi: 10.1007/s00299-011-1133-8. Epub 2011 Aug 19.
An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.
一种改良的豇豆农杆菌介导转化方法，采用超声处理和真空渗透处理。以 4 天大的子叶节作为与携带二元载体 pSouv-cry1Ac 的根癌农杆菌菌株 EHA105 共培养的外植体。在测试的不同损伤处理中，超声处理 20 秒，然后用根癌农杆菌真空渗透 5 分钟，导致瞬时 GUS 表达效率最高（再生部位有 93%的外植体表达 GUS）。共培养 3 天后，将外植体在含有 150mg/L 卡那霉素的选择培养基中培养，并回收可能转化的植株。通过聚合酶链反应（PCR）、基因组 Southern 和定性逆转录（RT）-PCR 分析，证实了 T0 转基因植物中 nptII 和 cry1Ac 基因的存在、整合和表达。Western blot 杂交和酶联免疫吸附测定（ELISA）检测并证明了Cry1Ac 蛋白在转基因植物中的积累。Cry1Ac 基因以孟德尔方式传递。与简单的农杆菌介导转化相比，超声辅助农杆菌介导转化（SAAT）和真空渗透处理使豇豆的稳定转化效率提高了 88.4%。
