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Penelitian ini menyelidiki respon fisiologis, anatomis, dan morfologis tanaman tembakau (Nicotiana tabacum L.) terhadap cekaman logam berat Cu, Cd, dan Pb. Sampel yang digunakan adalah tembakau umur 3-4 minggu yang dikecambahkan secara in vitro dan kemudian dipapar logam berat selama 14 hari. Desain penelitian yang digunakan yaitu rancangan acak lengkap dengan satu faktor, yaitu konsentrasi logam Cu (0 µM, 50 µM, 100 µM, 150 µM & 200 µM), Cd (0 µM, 50 µM, 100 µM, 200 µM & 300 µM), dan Pb (0 µM, 5 µM, 20 µM, 50 µM & 100 µM). Parameter yang digunakan: pertambahan panjang akar, pertambahan jumlah akar, akumulasi logam dalam akar, lokalisasi penimbunan dalam akar, dan warna daun. Bertambahnya konsentrasi logam menghambat pertumbuhan akar dan menyebabkan deposit logam pada jaringan akar dan gejala klorosis. Hasil uji Atomic Absorbtion Spectrophotometry (AAS) menunjukkan semakin besar konsentrasi semakin banyak akumulasi logam pada jaringan akar. Akan tetapi, akumulasi Cd pada konsentrasi 200 µM lebih besar dibanding pada konsentrasi 300 µM. Analisis kualitatif membuktikan bahwa cekaman Cu tidak berpengaruh signifikan terhadap warna daun, sedangkan pada cekaman Cd (100, 150 dan 200 µM) dan Pb (150 µM) daun mengalami klorosis. Pada konsentrasi logam yang rendah seperti 50 µM Cu, 50 µM Cd, dan 5 µM Pb tidak berbeda nyata dengan kontrol. Dengan demikian disimpulkan bahwa tembakau mampu mentoleransi cekaman logam pada konsentrasi yang rendah. This research investigated the physiological, anatomical, and morphological responses of tobacco (Nicotiana tabacum L.) on stresses of heavy metals Cu, Cd, and Pb. The samples were 3- to 4-week tobacco plants germinated in vitro and then were exposed to heavy metals for 14 days. This study used a completed random design with single factor, i.e. the concentrations of Cu (0 µM, 50 µM, 100 µM, 150 µM & 200 µM), Cd (0 µM, 50 µM, 100 µM, 200 µM & 300 µM), and Pb (0 µM, 5 µM, 20 µM, 50 µM & 100 µM. Further, stress response was analyzed based on several parameters including root elongation, root number, metal accumulation and localization in roots, and leaf color. The increasing metals had caused root growth inhibition, metal deposit in root tissues, and chlorosis symptom. Atomic Absorption Spectrosphotometry (AAS) analysis results showed constant trend that the higher concentration of heavy metals the higher accumulation of the metals. Yet 200 µM of Cd accumulated more than 300 µM Cd, so was suggested that the concentration was not the absolute factor in determining metal absorption. On the other hand, qualitative analysis has proven that chlorosis was not found in Cu treatment but consistently observed in high concentrations of Cd (100 µM up to 200 µM) and Pb (100 µM). However, lower metal concentration such as 50 µM Cu, 50 µM Cd and 5 µM Pb as well as the control treatment were not significantly different at the level of 5%. Therefore, it can be concluded that tobacco was capable to tolerate low concentration of metal stress.