Background and objectives: Anise Pimpinella anisum L. is a valuable medicinal plant that 2-3% of its seed weight is essential oils, and about 80-90% of its essential oil is trans-antol. This research was carried out to optimize the indirect organogenesis of P. anisum under in vitro conditions. Methodology: In the first experiment, different methods of seed germinating and disinfection treatments for decontamination of samples were investigated. In the second experiment, the effect of different combinations of BA, Kin and NAA on the direct regeneration of hypocotyl bud explants was investigated. In the third experiment, callus production was investigated from explants of root, hypocotyl, hypocotyl bud, cotyledon leaf and normal leaf, using different concentrations of 2,4-D. In the fourth experiment, the effect of a narrower range of 2,4-D concentration on the callus formation of petiole and root explants was evaluated. In the fifth experiment, the produced calli was exposed to different concentrations of BA and NAA for regeneration. All experiments were conducted in a completely randomized design with three replications. Data analysis was done using Minitab software, and the mean comparison was made using the Tukey method (p<0.05). Results: The best germination was obtained at a value of 70% in 24 hours of seed soaking in water (hydro priming) and disinfection with 70% alcohol and 1.5% sodium hypochlorite in 1/2 MS culture medium. In direct regeneration, stem-less leaves were produced. The best direct regeneration response in terms of petiole length was related to treatments of 9 (0.5 mg/L BA plus 0.5 mg/L Kin plus 0.5 mg/L NAA), 14 (0.5 mg per liter Kin plus 1mg/L NAA), 17 (0.25 mg/L BA plus 0.25 mg/L Kin plus 1mg/L NAA) and 19 (1mg/L BA plus 1mg/L Kin plus 1mg/L NAA) with about 4 cm. The highest leaf number (47 leaves) was obtained using the treatments of 14 and 17. In the third experiment, petiole and root explants had the best callus formation. In the fourth experiment, using the treatment of (0.5 mg/L BA +0.75 mg/L of 2, 4-D), the highest rate of 97.22% callus formation was observed in petiole explants. In the fifth experiment, the callus obtained from the previous stage induced the highest shoot with a value of 40% in the control treatment without hormones. Some branches were rooted, and the resulting seedlings were transferred to the greenhouse after adaptation. Conclusion: It was concluded that for the regeneration and breeding of anise plant, the indirect regeneration of the callus is more suitable than the direct regeneration method. Also, the petiole was a suitable organ as the initial explant. The 1.5% sodium hypochlorite was the best disinfectant for seed decontamination. Hormonal treatment of 0.75 mg/L of 2, 4-D +0.5 mg/L of BA induced the highest rate (97.22%) of callus in a petiole explant. Therefore, it could be used as a successful hormonal compound for callus formation in anise. The production of branches and roots from callus in the control treatment indicated that it was possible to obtain a whole plant without using any plant growth regulators. The results of this research can be used in micropropagation studies, genetic engineering, cell suspension culture and production of secondary metabolites and other projects related to anise improvement and molecular studies.

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