Enhancing the Utility of Plant Associated Bacterium; Ensifer adhaerens Strain OV14, as a Vector for Plant Transformation

Historically, Agrobacterium species were considered to be unique for their ability to transfer and integrate foreign DNA into plant genomes. However, the increasing number of non-Agrobacterium species with attained capability to genetically engineer host cells, suggests that this is not the case. Recently, Ensifer adhaerens OV14 was reported to underpin the successful crop transformation protocol termed as Ensifer-mediated transformation (EMT) technology. The adaptability and efficiency of EMT technology to successfully transform dicots viz., Arabidopsis thaliana, Solanum tuberosum, Nicotiana tabacum, Manihot esculenta and the monocot Oryza sativa, has been previously reported. The goal of this study was to expand on this programme and investigate the potential of E. adhaerens strain OV14 to genetically transform Brassica napus, the host species from which it was originally isolated. By modifying current A. tumefaciens-based protocols to suit the growth parameters of E. adhaerens strain OV14, the successful transformation of the commercial B. napus cultivar Delight was achieved via EMT. The EMT protocol involves the co-cultivation of 5 days old cotyledonary petiole explants with E. adhaerens strain OV14 (OD_600nm = 0.8) for 5 days in the presence of 200 µM acetosyringone, that delivered transgenic plants of morphological equivalence to the original treated cv. Delight. A transformation frequency of 4.0 ± 0.2 % was attained based on stable integration patterns recorded for T1 individuals, which indicated transgene integrations of 1–3 copies/line. Segregation analysis based on the inheritance of the nptII transgene in the T2 generation showed Mendelian and non-Mendelian segregation patterns for the designated kanamycin resistance phenotype. To conclude, this practical study highlights the continued expansion of EMT’s host range and provides possibilities to further the EMT research as a promising technology for genetic engineering and genome editing of plants.