Garlic (Allium sativum) is propagated asexually. Since sexual cross breeding is almost impossible, means for effective breeding are not currently available and the available production cultivars are seriously aged and degenerated. A possible alternative for breeding is chemical induction. Trifluralin, a type of herbicide, has been reported to provoke chromosome doubling. However, this chemical had not been tested on garlic. We tested various trifluralin concentrations and treatment durations for efficiency in the induction of tetraploid garlic. A clove base of garlic with a stem cv. Gailiang was used as the ex-plant to induce calluses on Murashige and Skoog (MS) medium; the calluses were then inoculated onto MS medium containing different levels of trifluralin and cultured to induce chromosome number variation in vitro. Garlic calluses were effectively induced via the ex-plant and both shoots and roots differentiated well on MS medium containing 6-benzylaminopurine at 3.0 mg/L and indole-3-acetic acid at 0.1 mg/L. However, increases in trifluralin concentration and treatment duration reduced the survival rate and differentiation rate of calluses. Garlic callus cultured for 15 days on medium containing 100 μM trifluralin gave the highest rate of chromosome doubling. Through observation of chromosome number in the root apical cells and the morphologyof guard cells on the leaf epidermis of the regenerated plantlets, it was clear that chromosome number variation was induced and tetraploids were produced in vitro by trifluralin treatment.