Stable <i>Agrobacterium</i>-mediated transformation of the halophytic <i>Leymus chinensis</i> (Trin.)

Abstract

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