Optimization of copper for the improvement of in vitro plant tissue growth of Solanum nigrum

  • Nasim A. R. M. Othman Biology Department, Faculty of Science, Taiz University, Taiz, Yemen
Keywords: Amino acids, Antioxidant enzymes, Carbohydrates, Copper, Lipid peroxidation, Potassium, Proteins


Here was investigated the incorporation of copper in MS medium on growth, and metabolic activities of Solanum nigrum callus. Copper up to 75 µM increased the growth, and thereafter a decline was observed. No considerable alteration in MDA, H2O2, bound phenolics, flavonoids, ascorbate, and copper content was observed with the existence of 25 µM copper, then levels of these parameters were raised with rising copper concentrations. Similarly, 25 µM copper didn't induce a considerable change in lipoxygenase, superoxide dismutase, catalase, peroxidase, phenylalanine ammonia lyase, and polyphenol oxidase activities, however, high levels stimulated these enzymes. Copper at 25 µM didn’t considerably reduce amino acids and soluble proteins, whereas higher concentrations reduced these parameters. Copper treatments reduced the soluble carbohydrates accumulation; only 75 µM enhanced this accumulation. Copper at 25 µM significantly increased the potassium accumulation, whereas higher concentrations reduced this accumulation. From these results, it might be contemplated the optimum effect concerning copper.

DOI: http://dx.doi.org/10.5281/zenodo.2848646


1. Rehman MZ, Rizwan M, Ali S, Ok YS, Ishaque, Saifullah W, et al. Remediation of heavy metal contaminated soils by using Solanum nigrum: A review. Ecotoxicol Environ Saf. 2017; 143: 236-248.

2. Ramage CM, Williams RR. Mineral nutrition and plant morphogenesis. In Vitro Cell Develop Biol Plant. 2002; 38: 116-124.

3. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15: 473-497.

4. Dahleen LS. Improved plant regeneration from barley callus cultures by increased copper levels. Plant Cell Tissue Organ Culture. 1995; 43: 267-269.

5. Li S, Zhang G, Gao W, Zhao X, Deng C, Lu L. Plant growth, development and change in GSH level in safflower (Carthamus tinctorius L.) exposed to copper and lead. Arch Biol Sci. 2015; 67: 385-396.

6. Adhikari T, Sarkar D, Mashayekhi H, Xing B. Growth and enzymatic activity of maize (Zea mays L.) plant: Solution culture test for copper dioxide nano particles. J. Plant Nutr. 2016; 39: 99-115.

7. Adrees M, Ali S, Rizwan M, Ibrahim M, Abbas F, Farid M, et al. The effect of excess copper on growth and physiology of important food crops: a review. Environ Sci Pollut Res. 2015; 22: 8148-8162.

8. Bouazizi H, Jouili H, Geitmann A, El Ferjani E. Copper toxicity in expanding leaves of Phaseolus vulgaris L.: antioxidant enzyme response and nutrient element uptake. Ecotoxicol Environ Saf. 2010; 73: 1304-1308.

9. Madhava Rao KV, Sresty TV. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000; 157: 113-128.

10. Mukherjee SP, Choudhuri MA. Implications of water stress‐induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol Plant. 1983; 58: 166-170.

11. Minguez-Mosquera MI, Jaren-Galan M, Garrido-Fernandez J. Lipoxygenase activity during pepper ripening and processing of paprika. Phytochemistry. 1993; 32: 1103-1108.

12. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972; 247: 3170-3175.

13. Aebi H. Catalase in vitro. Methods Enzymol. 1984; 105: 121-126.

14. Zaharieva T, Yamashita K, Matsumoto H. Iron deficiency induced changes in ascorbate content and enzyme activities related to ascorbate metabolism in cucumber roots. Plant Cell Physiol. 1999; 40: 273-280.

15. Havir EA, Hanson KR. L-phenylalanine ammonia-lyase. II. Mechanism and kinetic properties of the enzyme from potato tubers. Biochemistry. 1968; 7: 1904-1914.

16. Kumar KB, Khan PA. Peroxidase and polyphenol oxidase in excised ragi (Eleusine corocana cv PR 202) leaves during senescence. Indian J Exp Biol.1982; 20: 412-416.

17. Kofalvi S, Nassuth A. Influence of wheat streak mosaic virus infection on phenylpropanoid metabolism and the accumulation of phenolics and lignin in wheat. Physiol Mol Plant Pathol. 1995; 47: 365-377.

18. Chang C, Yang M, Wen H, Chern J. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal. 2002; 10: 178-182.

19. Ordoñez AA, Gomez JG, Vattuone MA, Isla MI. Antioxidant activities of Sechium edule (Jacq.) Swart extracts. Food Chem. 2006; 97: 452-458.

20. Jagota SK, Dani HM. A new colorimetric technique for the estimation of vitamin C using Folin phenol reagent. Anal Biochem. 1982; 127: 178-182.

21. Moore S, Stein WH. Photometric ninhydrin method for use in the chromatography of amino acids. J Biol Chem. 1948; 176: 367-388.

22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.

23. Fales FW. The assimilation and degradation of carbohydrates by yeast cells. J Biol Chem. 1951; 193: 113-118.

24. Schlegel HG. Die Verwertung organischer Säuren durch Chlorella im licht. Planta. 1956; 47: 510-526.

25. Havre GN. The flame photometric determination of sodium, potassium and calcium in plant extracts with special reference to interference effects. Anal Chim Acta. 1961; 25: 557-566.

26. Shenkin A. The key role of micronutrients. Clin Nutr. 2006; 25: 1-13.

27. Jain P, Kachhwaha S, Kothari SL. Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) by using high copper levels in the culture medium. Sci Horticul. 2009; 119: 315-319.

28. Fatima N, Ahmad N, Anis M. Enhanced in vitro regeneration and change in photosynthetic pigments, biomass and proline content in Withania somnifera L. (Dunal) induced by copper and zinc ions. Plant Physiol Biochem. 2011; 49: 1465-1471.

29. Shahid A, Ahmad N, Anis M, Alatar AA, Faisal M. Morphogenic responses of Rauvolfia tetraphylla L. cultures to Cu, Zn and Cd ions. Rendiconti Lincei Scienze Fisiche Naturali. 2016; 27; 369-374.

30. Liu JJ, Wei Z, Li JH. Effects of copper on leaf membrane structure and root activity of maize seedling. Bot Stud. 2014; 55: 47.

31. Chen J, Shafi M, Li S, Wang Y, Wu J, Ye Z, et al. Copper induced oxidative stresses, antioxidant responses and phytoremediation potential of Moso bamboo (Phyllostachys pubescens). Sci Rep. 2015; 5: 13554.

32. Cuypers A. The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings. J Plant Physiol. 2011; 168: 309-316.

33. Yruela I. Copper in plants: acquisition, transport and interactions. Funct Plant Biol. 2013; 36: 409-430.

34. Hippler FW, Mattos-Jr D, Boaretto RM, Williams LE. Copper excess reduces nitrate uptake by Arabidopsis roots with specific effects on gene expression. J Plant Physiol. 2018; 228: 158-165.

35. Karimi P, Khavari-Nejad RA, Niknam V, Ghahremaninejad F, Najafi F. The effects of excess copper on antioxidative enzymes, lipid peroxidation, proline, chlorophyll, and concentration of Mn, Fe, and Cu in Astragalus neo-mobayenii. Sci World J. 2012; 2012: 615670.

36. Islek C, Unal BT. Copper toxicity in Capsicum annuum: superoxide dismutase and catalase activities, phenolic and protein amounts of in-vitro-grown plants. Polish J Environ Stud. 2015; 24: 2441-2445.

37. Zhang X, Liu CJ. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids. Mol Plant. 2015; 8: 17-27.

38. Ibrahim MH, Chee Kong Y, Mohd Zain NA. Effect of cadmium and copper exposure on growth, secondary metabolites and antioxidant activity in the medicinal plant Sambung Nyawa (Gynura procumbens (Lour.) Merr). Molecules. 2017; 22: E1623.

39. Boeckx T, Winters AL, Webb KJ, Kingston-Smith AH. Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization? J Exper Bot. 2015; 66: 3571-3579.

40. Dalfard AB, Khajeh K, Soudi MR, Naderi-Manesh H, Ranjbar B, Sajedi RH. Isolation and biochemical characterization of laccase and tyrosinase activities in a novel melanogenic soil bacterium. Enzyme Microb Technol. 2006; 39: 1409-1416.

41. Dicko HM, Gruppen H, Traore AS, Voragen AG, Berkel WJ. Phenolic compounds and related enzymes as determinants of sorghum for food use. Biotechnol Mol Biol Rev. 2006; 1: 21-38.

42. Gautam S, Anjani K, Srivastava N. In vitro evaluation of excess copper affecting seedlings and their biochemical characteristics in Carthamus tinctorius L. (variety PBNS-12). Physiol Mol Biol Plant. 2016; 22: 121-129.

43. Jung C, Maeder V, Funk F, Frey B, Sticher H, Frossard E. Release of phenols from Lupinu salbus L. roots exposed to Cu and their possible role in Cu detoxification. Plant Soil. 2003; 252: 301-312.

44. Iwasaki Y, Hirasawa T, Maruyama Y, Ishii Y, Ito R, Saito K, Umemura T,Nishikawa A, Nakazawa H. Effect of interaction between phenolic compounds and copper ion on antioxidant and pro-oxidant activities. Toxicol In Vitro. 2011; 25: 1320-1327.

45. Panche AN, Diwan AD, Chandra SR. Flavonoids: an overview. J Nutr Sci. 2016; 5: e47.

46. Boguszewska D, Zagdańska B. ROS as signaling molecules and enzymes of plant response to unfavorable environmental conditions. InTech Rijeka Croatia. 2012; 341-362.

47. Horemans N, Foyer CH, Asard H. Transport and action of ascorbate at the plant plasma membrane. Trend Plant Sci. 2000; 5: 263-267.

48. Akram NA, Shafiq F, Ashraf M. Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front Plant Sci. 2017; 8: 613.

49. López-Vargas ER, Ortega-Ortíz H, Cadenas-Pliego G, Romenus K, Fuente MC, Benavides-Mendoza A, Juárez-Maldonado A. Foliar application of copper nanoparticles increases the fruit quality and the content of bioactive compounds in tomatoes. Appl Sci. 2018; 8: 1020.

50. Hildebrandt TM, Nesi AN, Araújo WL, Braun HP. Amino acid catabolism in plants. Mol Plant. 2015; 8: 1563-1579.

51. Zhang FQ, Wang YS, Lou ZP, Dong JD. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemosphere. 2007; 67: 44-50.

52. Hasan MK, Cheng Y, Kanwar MK, Chu XY, Ahammed GJ, Qi ZT. Responses of plant proteins to heavy metal stress - a review. Front Plant Sci. 2017; 8: 1492.

53. Wu QS, Srivastava AK, Zou YN. AMF-induced tolerance to drought stress in citrus: A review. Sci Horticul. 2013; 164: 77-87.

54. Mishra BS, Singh M, Aggrawal P, Laxmi A. Glucose and auxins signaling interaction in Arabidopsis thaliana seedling root growth and development. PloS One. 2009; 4: e4502.

55. Rodrigo-Moreno A, Andrès-Colas N, Poschenrieder C, Gunse B, Penarrubias L, Shabala S. Calcium- and potassium permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production. Plant Cell Environ. 2013; 36: 844-855.

56. Palm E, Nissim WG, Giordano C, Mancuso, S, Azzarello E. Root potassium and hydrogen flux rates as potential indicators of plant response to zinc, copper and nickel stress. Environ Exp Bot. 2017; 143: 38-50.

57. Demidchik V, Sokolik A, Yurin V. Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+. Planta. 2001; 212: 583-590.

58. Demidchik V, Straltsova D, Medvedev SS, Pozhvanov GA, Sokolik A, Yurin V. Stress induced electrolyte leakage: the role of K+-permeable channels and involvement in programmed cell death and metabolic adjustment. J Exp Bot. 2014; 65: 1259-1270.

59. Festa RA, Thiele DJ. Copper: an essential metal in biology. Curr Biol. 2011; 21: R877-R883.

60. Da Costa M, Sharma P. Effect of copper oxide nanoparticles on growth, morphology, photosynthesis, and antioxidant response in Oryza sativa. Photosynthetica. 2016; 54: 110-119.
How to Cite
Othman, N. (2019). Optimization of copper for the improvement of in vitro plant tissue growth of Solanum nigrum. European Journal of Biological Research, 9(2), 77-92. Retrieved from http://journals.tmkarpinski.com/index.php/ejbr/article/view/168
Research Articles