Abstract: For the establishment of tissue culture regeneration system of four new varieties of Hosta, we chose MS as the basic culture medium, and added in different concentrations of 6-BA and NAA to study the optimum growth medium of callus induction and subculture propagation of explant tissue culture. The results showed that high concentrations of 6- BA (greater than or equal to 3.0 mg/L) with 2,4- D (greater than or equal to 0.1 mg/L) were conducive to generate callus about the callus induction. MS+ 1.0 mg/L 6-BA+ 0.3 mg/L NAA+ 35 g/L sucrose + 12 g/L agar could make the proliferation multiple of Hosta Big Daddy (No. H1) and the ‘Eagle’(No. H2) up to 2.54 and 2.78, and the MS+ 1.0 mg/L 6-BA+ 0.2 mg/L NAA+30 g/L sucrose+11 g/L agar could make the‘little Huang Jinye’(No. H6) proliferation multiple up to 3.01, and the most suitable medium on proliferation of H15 was MS+ 3.0 mg/L 6-BA+ 0.1 mg/L NAA+ 30 g/L sucrose+ 12 g/L agar. The study results can provide favorable data for the induction and tissue culture of four new varieties of Hosta callus, callus induction rate of this method is the maximum, and the proliferation multiple of propagation is also the maximum.