Abstract:The leaves of Eucalyptus grandis clone EG5 were used as explants for callus induction and shoot regeneration. The results indicated that the optimum medium for callus induction and adventitious shoot differentiation was modified MS + 0.12 mg·L-1TDZ + 0.25 mg·L-1NAA; and NH4NO3 played an important role in callus growth and differentiation, The best concentration in the medium was 0.412 5 g·L-1 .The optimum medium for shoot elongation was modified MS+0.3 mg·L-16BA+0.05 mg·L-1IBA+0.3 mg·L-1IAA; A ten-day’s darkness culture could improve the adventitious shoot induction and put off the aging process and the browning of callus. The highest rooting rate was 65.47% on medium with improved MS + 0.4 mg·L-1NAA, and the survival rate of transplanting was 90%.