Abstract: The tender leaf blade and stems of Populus deltoides×P.nigra cv. Chile were used as experiment materials for tissue culture research, and the effect of basic culture medium, auxin, cytokinin and other factors on the explants regeneration was studied. The results showed that the optimal medium for differentiation of adventitious roots from the leaf blades was MS + 6-BA 025mg/L + NAA009mg/L + Agar 5g/L + Sugar 30g/L, on which the differentiation rate could reach 9907%. The suitable medium for inducing the callus with tender stems was 1/2MS + 6-BA01mg/L + NAA01mg/L + IAA01mg/L + Agar 5g/L + Sugar 30g/L, many adventitious buds could differentiate from the callus when transferred to the differentiation medium. The best medium for rooting was 1/2MS + IBA005mg /L + IAA01mg/L + Agar5g/L + Sugar30g/L, by which the rooting rate was 100% within 15 days, and the rooted seedlings all survived after being transplanted.