Abstract: Zhangqiu spring onion was used as experimental materials. We studied the optimum medium and hormone combination of callus induction from cotyledons, subculture of callus, shoot regeneration from callus, shoot-extension induction, root induction and identified the chromosome number of the plantlet. The results showed that: MS+2,4-D 2.0mg/L+KT 0.5mg/L, MS＋2,4-D 2.5mg/L＋KT 0.5mg/L, MS+2,4-D 0+KT 1.0mg/L, MS+KT 0.5 mg/L and basal MS medium. One plantlet among twenty two was determined as a tetraploidy based on the plant morphology, observation of the leaf stoma and chromosome number of the root, other two plantlets represented morphological variation.