Genetic variation is closely correlated with crop improvement. Garlic is mostly propagated through vegetative means, hence there is a need to induce a significant amount of heterogeneity. Ploidy is induced In-vitro by colchicine, which improves allelic variation among individuals. We examined the optimum concentrations of BAP and TDZ for shoot proliferation. The study suggests that BAP (3 mg/L) and TDZ (0.25 mg/L) exhibit considerable growth in shoot regeneration. 21 days old calli were subcultured and the 2nd-generation passage was subjected to a culture medium with BAP, TDZ and Different concentrations of Colchicine. Results show that Colchicine at 0.05 mg/L promotes explant survival. Treatment improved the genetic potential of garlic varieties in vitro but resulted in a lower duplication rate at 0.75 mg/L due to higher toxicity. LC50 colchicine concentration is at 0.03–0.05 mg/L. The study suggests that an increase in ploidy level is expected to generate higher molecular diversity in garlic.