Agrobacterium tumefaciens-Mediated Transformation of the Halophyte Leymus chinensis (Trin.)

摘要

An Agrobacterium tumefaciens-mediated transformation system along with regeneration of transgenic plants of the halophyte Leymus chinensis (Trin.) was developed. A. tumefaciens strain EHA105 containing the binary vector pCAMBIA2300 was used for transformation, along with an Ipomoea batatas 2-cysteine peroxiredoxin (Ib2-Cys prx) gene under the control of the stress-inducible sweet potato anionic peroxidase 2 (SWPA2) promoter or the cauliflower mosaic virus (CaMV) 35S promoter. Among different pre-culture periods, 7-day pre-culture promoted the highest frequency of transformation. Among the different cocultivation periods, 20 min of cocultivation with bacterial cells (OD600 = 0.4) promoted the highest frequency of transformation. Acetosyringone at 100 mu M was added to increases virulence induction. Selection of transgenic shoots was done in the presence of 150 mg l(-1) kanamycin. Polymerase chain reaction (PCR) analysis of putative transgenic plants showed the presence of Ib2-Cys prx and nptII genes. The expression of transgene, Ib2-Cys prx was also confirmed in non-stressed and various stressed plants by RT-PCR. The SWPA2::prx transformants showed very low prx gene expression under non-stressed conditions but higher prx gene expression than the CaMV 35S::prx transformants, when exposed to various oxidative stresses. The highest transformation efficiency was found to be 8.97 %. The protocol provides a direct opportunity for improvement of the quality traits of L. chinensis via genetic transformation.