Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration

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 T-0 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.