A stable and reproducible transformation system for the wetland monocot Juncus accuminatus (bulrush) mediated by Agrobacterium tumefaciens

Abstract

A highly reproducible Agrobacterium-mediated transformation system was developed for the wetland monocot Juncus accuminatus. Three Agrobacterium tumefaciens binary plasmid vectors, LBA4404/pTOK233, EHA105/pCAMBIA1201, and EHA105/pCAMBIA1301 were used. All vectors contained the 35SCaMV promoter driven, intron containing, β-glucuronidase (gus), and hygromycin phosphotransferase (hptII) genes within their T-DNA. After 48 h of cocultivation, 21-d-old seedling derived calli were placed on medium containing timentin at 400 mg l−1, to eliminate the bacteria. Calli were selected on MS medium containing 40 or 80 mg l−1 hygromycin, for 3 mo. Resistant calli were regenerated and rooted on MS medium containing hygromycin, 5 mg l−1(22.2 μM) of 6-benzylamino-purine (BA) and 0.1 mg l−1(0.54 μM) of alpha-naphthaleneacetic acid (NAA), respectively. Seventy-one transgenic cell culture lines were obtained and 39 plant lines were established in the greenhouse. All the plants were fertile, phenotypically normal, and set viable seed. Both transient and stable expression of the gus gene were demonstrated by histochemical GUS assays of resistant calli, transgenic leaf, root, inflorescence, seeds, and whole plants. The integration of gus and hptII genes were confirmed by polymerase chain reaction (PCR) and Southern analysis of both F0 and F1 progenies. The integrated genes segregated to the subsequent generation in Mendelian pattern. To our knowledge, this is the first report of the generation of transgenic J. accuminatus plants.