Objective To establish the tissue culture system of Cynanchum otophylum in genetic transformation. Methods Asepsis seedling was set up by using different explant parts, disinfectors, and disinfecting time. The callus was induced, breeded, and differentiated by using different media, different hormone categories and combinations, and different hormone concentrations of 2,4-D and KT. Results It was the best method that asepsis seeding was built up by using seed as the explant and using 10% NaClO to treat the seed for 20—30 min or using 0.2% HgCl2 to treat the stem for 3 min. The seed, root, stem, and leaf of C. otophylum can form callus easily. The 2,4-D was important in forming callus. C. otophylum was more sensitivity to KT. The effect was better when KT was used to induce callus or adventitious buds. MS+2,4-D 1.0 mg/L+KT 1.0 mg/L was the better medium to induce callus to form. The callus cant be induced to form adventitous buds without reference to using seed or root, stem, and leaf as the explant, without reference to using hormone 6-BA or KT, ZT, 2ip. Gentamicin 100 ?g/mL is the optimum pressure in genetic transformation of C. otophylum. Conclusion The seed or stem is the ideal material bombarded by particle gun that is induced to form callus for 14 d in the media MS+2, 4-D 1.0 mg/L+KT 1.0 mg/L and without forming adventitious buds.