This study established an efficient in vitro callus formation and plant regeneration protocol for a prevıously reported Cu accumulator, Brassica nigra, black mustard collected from Diyarbakir (Station site). Node explants from 10-day old mature plants were used for callus formation and shoot regeneration. The highest callus formation frequency (100%) was observed on Murasige Skoog (MS) medium containing 0.1 mg/L Benzylaminopurine (BAP) + 0.5 mg/L Naphthylacetic acid (NAA) (MS 2), 0.6 mg/L BAP + 0.2 mg/L NAA (MS 7), the highest shoot regeneration frequency (100%) was achieved on MS medium containing 0.6 mg/L BAP + 0.05 mg/L Indole butyric acid (IBA) (MS 8), 0.2 mg/L IBA + 0.2 mg/L NAA (MS 10) and the highest number of shoots per explant (3,25) was obtained on MS medium supplemented 0.6 mg/L BAP + 0.05 mg/L IBA (MS 8). After root, stem, and leaf formation from explants in MS medium, these plants were transferred to soil and grown in the plant growth room for one month. A dependable and effective shoot regeneration procedure was developed, laying the groundwork for genetic transformation in Brassica nigra.