This study was carried out to investigate the optimal condition for multiple propagation through leaf tissue culture and to apply anther culture techniques to Pulsatilla koreana Nakai breeding. Leaf and anther of Pulsatilla koreana Nakai were cultured on MS, MT, LS and $B_5$ media supplemented with several growth regulators and nitrogen sources under various conditions. For callus induction and differentiation from the Pulsatilla koreana leaf segments were more effective in the combination of zeatin and auxin than auxin alone. The color of the callus was green when treated with IBA alone. Shoot differentiation was more effective when treated with zeatin than auxin alone, especially the best hormoal combination for shoot differentiation was zeatin 1.0mg/l +NAA 0.1mg/l, while 2,4-D inhibited shoot differentiation. The appeared rate of S pollen was 35% in vivo, while that of S pollen by low temperature$(4^{circ}C)$ pretreatment for 4 days was increased by 53% and the optimum culture time for callus induction from anther was uni-nucleate stage. $B_5$ basal medium supplemented with NAA 0.5mg/l and zeatin 1 mg/l was the most effective on callus formation and the best results of plant regeneration were obtained from combination of NAA 0.5mg/l and zeatin 0.5mg/l in anther culture. $NH_{4}NO_3$ as more effectives as the nitrogen source than $KNO_3$ and the combination with zeatin 2.0mg /L was the best effective. The best combination for plant regeneration in callus induced from anther was $NH_{4}NO_3$ 1650mg/l + $KNO_3$ 3800mg/l + zeatin 2.0mg/l. Ploidy level of anther-derived plants appeared 28% haploid, 47% diploid and the others were triploid, tetraploid and mixploid. In compare with E.S.T, M.D.H and P.X banding patterns were distinguished among callus, haploid and diploid plants in electrophoresis.