以苎麻优良品系“5041-3”子叶作为受体，利用根癌农杆菌介导法进行了绿色荧光蛋白基因的遗传转化研究，农杆菌菌株为EHA105，重组质粒为pBIN m-GFP5-ER。经农杆菌浸染后的子叶外植体经过共培养、选择培养、伸长培养和生根培养后再生出抗性植株，对抗性植株的再生过程进行了GFP荧光检测，结果表明，GFP基因能在苎麻中强烈表达，证明GFP基因能够在苎麻遗传转化中得到应用。对抗性植株的Southern杂交检测证实外源基因已整合到苎麻基因组中。
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.
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