Stable Agrobacterium-mediated transformation of the halophytic Leymus chinensis (Trin.)
In this study, an efficient procedure for stable Agrobacterium-mediated transformation of Leymus chinensis (Trin.) was established. Agrobacterium tumefaciens strain EHA105, harboring a binary vector pCAMBIA2300, was used for transformation, along with a sweet potato 2-cysteine peroxiredoxin (2-Cys Prx) gene under the control of the stress-inducible sweet potato anionic peroxidase 2 (SWPA2) promoter and the neomycin phosphotransferase (nptII) gene under the control of the cauliflower mosaic virus (CaMV) 35 S promoter. We found that a one-month-old callus derived from mature seeds could be efficiently transformed. Seven-day preculture followed by inoculation with the addition of 100 μmolL-1 acetosyringone (AS) and then a 3 day co-cultivation were performed before selection. Selection of transgenic shoots was done in the presence of 150 mgL-1 kanamycin (KM). An optical density at a wavelength of 600 nm (OD600) of approximately 0.4 for A. tumefaciens infection solution and 20 min of infection time gave the highest transformation efficiency. Polymerase chain reaction (PCR) analysis of KM-resistant plants and newly regenerated rhizomes revealed stable transformation of the 2-Cys Prx gene and the nptII gene, with the highest transformation frequency of 4.93%. RT-RCR analysis was conducted using salt stressed transgenic plants, and the results suggested that 2-Cys Prx had low transcription levels under non-stressed conditions, and increased transcription after 6 h of 200 mM NaCl stress. This gene continued to demonstrate high levels of transcription until 6 h after withdrawal of stress, with a slow recovery. The method reported herein provides a direct opportunity for improvement of the quality traits of L. chinensis via genetic transformation.
Keywords: Leymus chinensis, Agrobacterium-mediated transformation, 2-Cys peroxiredoxin, gene transformation