In vitro effect of zinc oxide nanoparticles on Nicotiana tabacum callus compared to ZnO micro particles and zinc sulfate (ZnSO4)

  • Maryam Mazaheri-TiraniEmail author
  • Soleyman Dayani
Original Article


Nano-technology has changed the properties of metal elements’ delivery into and effect on living systems. The current study, first, evaluated different combinations of plant growth regulators NAA, 2,4-d and KIN on Nicotiana tabacum callus induction from root, internode, petiole and leaf explants. Two mg L−1 NAA with 0.1 mg L−1 KIN on Murashige and Skoog (MS) medium induced calli in all explant sources. Then, the effect of different concentrations of nP-ZnO (0.015, 0.03, 0.06, 0.12, 0.24 mM) and µP-ZnO (0.03, 0.06, 0.12, 0.24 mM) compared to ZnSO4 (0.03 mM) on cell toxicity was investigated. SEM microscopy, XRD and EDX analyses were used to determine particles characteristics. Higher zinc content was accumulated in calli under nP-ZnO concentrations compared with µP-ZnO, which was positively correlated with calli fresh and dry weights. The nP-ZnO induced oxidative stress more significantly than µP-ZnO. Protein content did not increase under µP-ZnO, while it was raised at all concentrations of nP-ZnO that had positive correlation with MDA and ROS. SOD enzyme activity increased at all levels of µP-ZnO but remained unchanged under all levels of nP-ZnO compared to control. Ferric reducing antioxidant power (FRAP) was more sensitive than α, α-diphenyl-β-picrylhydrazyl (DPPH) for determining plant antioxidant capacity under nP-ZnO stress. The result showed that nP-ZnO induced a combined growth promoting and stress-induction effect in tobacco callus cells in a dose-dependent manner. The toxicity level of nP-ZnO was ameliorated compared to similar µP-ZnO concentrations. The chemical bio-reactivity of nP-ZnO-based Zn2+ could cause its fast-responsive and modified influence in tobacco cells.

Key message

ZnO nanoparticles increased Zn2+ bioavailability in tobacco callus but ameliorated metal toxicity. Zinc nanoparticles induced dose-dependent and combined oxidative and growth promoting effects. FRAP was recommended for nP-ZnO oxidative studies.


Tobacco plant Oxidative stress Callus induction Nano metal toxicity FRAP DPPH 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Supplementary material

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  1. Adhikari T, Kundu S, Rao AS (2016) Zinc delivery to plants through seed coating with nano-zinc oxide particles. J Plant Nutr 39:136–146CrossRefGoogle Scholar
  2. Akanbi-Gada MA, Ogunkunle CO, Vishwakarma V, Viswanathan K, Fatoba PO (2019) Phytotoxicity of nano-zinc oxide to tomato plant (Solanum lycopersicum L.): Zn uptake, stress enzymes response and influence on non-enzymatic antioxidants in fruits. Environ Technol Innov 14:100325CrossRefGoogle Scholar
  3. Alharby HF, Metwali EM, Fuller MP, Aldhebiani AY (2016) Impact of application of zinc oxide nanoparticles on callus induction, plant regeneration, element content and antioxidant enzyme activity in tomato (Solanum lycopersicum Mill.) under salt stress. Arch Biol Sci 68:723–735CrossRefGoogle Scholar
  4. Andresen E, Peiter E, Küpper H (2018) Trace metal metabolism in plants. J Exp Bot 69:909–954. CrossRefPubMedGoogle Scholar
  5. Augusto O, Miyamoto S (2011) Oxygen radicals and related species. Princ Free Radic Biomed 1:19–42Google Scholar
  6. Bindschedler LV, Minibayeva F, Gardner SL, Gerrish C, Davies DR, Bolwell GP (2001) Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells involve cAMP and Ca2+. New Phytol 151:185–194CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  8. Chanu TT, Upadhyaya H (2019) Zinc oxide nanoparticle-induced responses on plants: a physiological perspective. Nanomaterials in plants, algae and microorganisms. Elsevier, Amsterdam, pp 43–64CrossRefGoogle Scholar
  9. Chutipaijit S, Sutjaritvorakul T (2017) Application of nanomaterials in plant regeneration of rice (Oryza sativa L.). Mater Today-Proc 4:6140–6145CrossRefGoogle Scholar
  10. Czyżowska A, Barbasz A (2019) Effect of ZnO, TiO2, Al2O3 and ZrO2 nanoparticles on wheat callus cells. Acta Biochim Pol 66:365–370PubMedGoogle Scholar
  11. Dimkpa CO et al (2012) CuO and ZnO nanoparticles: phytotoxicity, metal speciation, and induction of oxidative stress in sand-grown wheat. J Nanopart Res 14:1–15CrossRefGoogle Scholar
  12. Geethalakshmi S, Hemalatha B, Saranya N (2016) Optimization of media formulations for callus induction, shoot regeneration and root induction in Nicotiana benthamiana. J Plant Sci Res 3:150Google Scholar
  13. Giannopolitis CN, Ries SK (1977) Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol 59:309–314PubMedGoogle Scholar
  14. Hou J, Wu Y, Li X, Wei B, Li S, Wang X (2018) Toxic effects of different types of zinc oxide nanoparticles on algae, plants, invertebrates, vertebrates and microorganisms. Chemosphere 193:852–860. CrossRefPubMedGoogle Scholar
  15. Hussain PR, Wani AM, Meena RS, Dar MA (2010) Gamma irradiation induced enhancement of phenylalanine ammonia-lyase (PAL) and antioxidant activity in peach (Prunus persica Bausch, Cv. Elberta). Radiat Phys Chem 79:982–989CrossRefGoogle Scholar
  16. Itroutwar PD, Govindaraju K, Tamilselvan S, Kannan M, Raja K, Subramanian KS (2019) Seaweed-based biogenic ZnO nanoparticles for improving agro-morphological characteristics of rice (Oryza sativa L.). J Plant Growth Regul. CrossRefGoogle Scholar
  17. Javed R, Yucesan B, Zia M, Gurel E (2018) Elicitation of secondary metabolites in callus cultures of Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress. Sugar Tech 20:194–201CrossRefGoogle Scholar
  18. Jeevanandam J, Barhoum A, Chan YS, Dufresne A, Danquah MK (2018) Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations. Beilstein J Nanotechnol 9:1050–1074. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kalra Y (1997) Handbook of reference methods for plant analysis. CRC Press, Boca RatonCrossRefGoogle Scholar
  20. Kumar S, Patra AK, Datta SC, Rosin KG, Purakayastha TJ (2015) Phytotoxicity of nanoparticles to seed germination of plants. Int J Adv Res 3:854–865Google Scholar
  21. Latef AAHA, Alhmad MFA, Abdelfattah KE (2017) The possible roles of priming with ZnO nanoparticles in mitigation of salinity stress in lupine (Lupinus termis) plants. J Plant Growth Regul 36:60–70CrossRefGoogle Scholar
  22. Mahdieh M, Sangi MR, Bamdad F, Ghanem A (2018) Effect of seed and foliar application of nano-zinc oxide, zinc chelate, and zinc sulphate rates on yield and growth of pinto bean (Phaseolus vulgaris) cultivars. J Plant Nutr 41:2401–2412CrossRefGoogle Scholar
  23. Marslin G, Sheeba CJ, Franklin G (2017) Nanoparticles alter secondary metabolism in plants via ROS burst. Front Plant Sci 8:832CrossRefGoogle Scholar
  24. Mosavat N, Golkar P, Yousefifard M, Javed R (2019) Modulation of callus growth and secondary metabolites in different Thymus species and Zataria multiflora micropropagated under ZnO nanoparticles stress. Biotechnol Appl Biochem 66:316–322. CrossRefPubMedGoogle Scholar
  25. Mousavi Kouhi S, Lahouti M (2018) Application of ZnO nanoparticles for inducing callus in tissue culture of rapeseed. Int J Nanosci Nanotechnol 14:133–141Google Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  27. Nalci OB, Nadaroglu H, Pour AH, Gungor AA, Haliloglu K (2019) Effects of ZnO, CuO and γ-Fe3O4 nanoparticles on mature embryo culture of wheat (Triticum aestivum L.). Plant Cell Tissue Organ Cult 136:269–277. CrossRefGoogle Scholar
  28. Priyanka N, Geetha N, Ghorbanpour M, Venkatachalam P (2019) Role of engineered zinc and copper oxide nanoparticles in promoting plant growth and yield: present status and future prospects. Adv Phytonanotechnol. CrossRefGoogle Scholar
  29. Radi AA, Farghaly FA, Al-Kahtany FA, Hamada AM (2018) Zinc oxide nanoparticles-mediated changes in ultrastructure and macromolecules of pomegranate callus cells. Plant Cell Tissue Organ Cult 135:247–261. CrossRefGoogle Scholar
  30. Rahman M, Alam M, Hossain M, Hossain A, Afroz R (2010) In vitro regeneration of popular tobacco varieties of Bangladesh from leaf disc. Bangladesh J Agric Res 35:125–134CrossRefGoogle Scholar
  31. Rajput V et al (2019) ZnO and CuO nanoparticles: a threat to soil organisms, plants, and human health. Environ Geochem Health. CrossRefPubMedGoogle Scholar
  32. Reddy Pullagurala VL, Adisa IO, Rawat S, Kalagara S, Hernandez-Viezcas JA, Peralta-Videa JR, Gardea-Torresdey JL (2018) ZnO nanoparticles increase photosynthetic pigments and decrease lipid peroxidation in soil grown cilantro (Coriandrum sativum). Plant Physiol Biochem 132:120–127. CrossRefPubMedGoogle Scholar
  33. Rizwan M et al (2019) Zinc and iron oxide nanoparticles improved the plant growth and reduced the oxidative stress and cadmium concentration in wheat. Chemosphere 214:269–277CrossRefGoogle Scholar
  34. Sanjay SS, Pandey AC, Singh M, Prasad MS (2015) Effects of functionalized ZnO nanoparticles on the phytohormones: growth and development of Solanum melongena L. (brinjal) plant. World J Pharm 5:1990–2009Google Scholar
  35. Shkryl YN et al (2018) Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae. Artif Cells Nanomed Biotechnol 46:1646–1658PubMedGoogle Scholar
  36. Siddiqi KS, Husen A (2017) Plant response to engineered metal oxide nanoparticles. Nanoscale Res Lett 12:92CrossRefGoogle Scholar
  37. Singh A, Singh N, Hussain I, Singh H, Yadav V, Singh S (2016) Green synthesis of nano zinc oxide and evaluation of its impact on germination and metabolic activity of Solanum lycopersicum. J Biotechnol 233:84–94CrossRefGoogle Scholar
  38. Sturikova H, Krystofova O, Huska D, Adam V (2018) Zinc, zinc nanoparticles and plants. J Hazard Mater 349:101–110. CrossRefPubMedGoogle Scholar
  39. Tirani M, Haghjou M (2019) Reactive oxygen species (ROS), total antioxidant capacity (AOC) and malondialdehyde (MDA) make a triangle in evaluation of zinc stress extension. J Anim Plant Sci 29:1100–1111Google Scholar
  40. Vianna DR et al (2012) Evaluation of the antioxidant capacity of synthesized coumarins. Int J Mol Sci 13:7260–7270CrossRefGoogle Scholar
  41. Wojdyło A, Oszmiański J, Czemerys R (2007) Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem 105:940–949CrossRefGoogle Scholar
  42. Yan B, Dai Q, Liu X, Huang S, Wang Z (1996) Flooding-induced membrane damage, lipid oxidation and activated oxygen generation in corn leaves. Plant Soil 179:261–268CrossRefGoogle Scholar
  43. Yoshihara S, Yamamoto K, Nakajima Y, Takeda S, Kurahashi K, Tokumoto H (2019) Absorption of zinc ions dissolved from zinc oxide nanoparticles in the tobacco callus improves plant productivity. Plant Cell Tissue Organ Cult 138:377–385. CrossRefGoogle Scholar
  44. Zhu Y et al (2019) Nanomaterials and plants: positive effects, toxicity and the remediation of metal and metalloid pollution in soil. Sci Total Environ 66:414–421CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Biology, Faculty of ScienceUniversity of JiroftJiroftIran
  2. 2.Department of Agricultural BiotechnologyPayame Noor University (PNU)TehranIran

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