Abstract:

Genetic improvement of crops can be achieved by biotechnological approaches such as Agrobacterium-mediated transformation. Unfortunately, there were only a few successful transformation experiments on ramie reported to date. In order to confirm its practical value of the green fluorescent protein (GFP), a visual marker, in ramie transformation, Agrobacterium tumefaciens-mediated transformation of ramie with the strain EHA105: pBIN m-GFP5-ER using cotyledon as explants excised from ‘5041-3’ was investigated. Ramie seeds were germinated on MS medium for 4–6 days followed by excised cotyledons from seedlings and inoculated with Agrobacterium tumefaciens cells (OD 0.5–0.8) for 5–10 min. The infected cotyledons were co-cultured on MS medium supplemented with 0.5 mg L^-1 thidiazuron (TDZ), 0.01 mg L^-1 indoleacetic acid (IAA), and 50 mg L^-1 acetosyringone (AS). Three days later, explants were transferred to MS medium supplemented with 0.5 mg L^-1 TDZ, 0.01 mg L^-1 IAA, 500 mg L^-1 cefotaxime (cef), and 30 mg L^-1 kanamycin (km) for selection. Putative transformed shoots were obtained in 4–6 weeks, which elongated on MS medium supplemented with 1.0 mg L^-1 6-benzylaminopurine (6-BA), 200 mg L^-1 cef, and 30 mg L^-1 km, and then rooted on MS medium supplemented with 0.025 mg L^-1 α-naphthalene acetic acid (NAA), 100 mg L^-1 cef, and 30 mg L^-1 km. Well rooted putative transformed plants were transplanted into plastic pots for acclimatization. Transformation efficiency of ramie plants above was 3.6% and the average number of resistant shoots per regenerated explant was 1.3. GFP detection was conducted during the transformation and strong GFP expression was observed in putative transformants, which indicated that GFP could be used in ramie transformation. Stable integration and maintenance of the transgenes in the genome of putative transformants were confirmed by Polymerase chain reaction and genomic Southern blot analysis. The transformation strategy described in this study will provide a practical protocol to generate diverse transgenic ramie plants.

Key words: Boehmeria nivea (L.) Gaud., Agrobacterium tumefaciens, Transformation, Green fluorescent protein (GFP)