Effect of silver nitrate (AgNO3) and copper sulphate (CuSO4) on callus formation and plant regeneration from tow pepper varieties (Chile Ancho and Misraty) in vitro


  • Noaman Enfeshi Libyan Biotechnology Research Center Department tissue culture plant
  • Elshaybani Abdulali Libyan Biotechnology Research Center Department tissue culture plant
  • Mustafa Salama Libyan Biotechnology Research Center Department tissue culture plant
  • Zaineb Geath Libyan Biotechnology Research Center Department tissue culture plant
  • Ahmed Shaaban Libyan Biotechnology Research Center Department tissue culture plant
  • Zuhear Ben saad University of Tripoli, Factually of agriculture Department of Horticulture.
  • Elmundr Abughnia Libyan Biotechnology Research Center Department Tissue Culture Plant




Callus formation, copper sulfate, growth , plant tissue, silver nitrate, vegetative


This study was conducted at the Libyan center for biotechnology research laboratories in order to study the effect of different concentrations of silver nitrate and copper sulfate on the callus formation and plant regeneration of two varieties of pepper plant (Misraty and Chile Ancho). Silver nitrate (AgNO3) was added at a concentration of 10, 20, 30, 40 and 50 µM/L /L in the culture media, while copper sulphate (CuSO4) was added at concentrations of 0.1 (concentration of copper sulfate in MS standard medium), 0.5, 1, 2, 3 and 4 µM/L  with presence of 17.8 µM/L  of Kin and 1.7 µM/L  of NAA. The results indicated that adding silver nitrate to the culture medium did not have a positive effect on the rate of callus formation, while it increased the number of plant growths, especially at a concentration of 30 µM/L .As for copper sulfate, the results showed that, for the plant growth parameter there were no significant differences among the treatments of 2.0, 3.0 and 4.0 µM/L  compared to the control treatment in Chile Ancho variety, while the number of seedlings and leaves of the obtained plants improved by increasing the concentration of copper sulfate to record the best average of 4.5 and 7.2, respectively, at a concentration of 3 µM/L . Furthermore for the Misraty variety, the results proved that addition of copper sulfate did not have a positive effect on the rate of callus formation, but it effectively affected the plant growth parameter the percentage of plant growth increased from 15% in the control treatment to 45% in treatment of 3 µM/L. The results showed also that the number of leaves increased by increasing the concentration of copper sulfate by recording average of 7.5 leaves at the treatment of 3 µM/L.


Alejo, N.O. and R.R.Malagone. 2001. In Vitro chili pepper Biotechnology,In Vitro Cellular and Developmental Biology – Plant. 37:701-729.

Ashwani, S.; Ravishankar, G.A.; Giridhar, P. 2017. Silver nitrate and 2-(N-morpholine) ethane sulphonic acid in culture medium promotes rapid shoot regeneration from the proximal zone of the leaf of Capsicum frutescens Mill. Plant. Cell Tissue Organ. Cult., 129, 175–180.

Bais, H.P., G. sudha., B. suresh and G. A. ravishankar. 2000. AgNO3 influences in vitro root formation in Decalepishamiltonii Wight, Arn. Current Science, 79: 894-898.

Beyer, E. M. 1976. A potent inhibitor of ethylene action in plants. Plant Physiology, 58 (3): 268-271.

Bora,G. H., K. Gogoi and P. J. Handique. 2014. Effect of silver nitrate and gibberellic acid on in vitro regeneration, flower induction and fruit development in Naga Chile.Asia-Pacific Journal of Molecular Biology and Biotechnology. 22 (1): 137-144.

Caldas LS, Haridasan P, Ferreira ME .1990. Meios nutritivos. In: Torres AC, Caldas LS (Eds) Técnicas eAplicações da Cultura de Tecidos de Plantas (pp 37–70). ABCTP/EMBRAPA-CNPH, Brasília.

Ciardi J, Klee H .2001. Regulation of ethylene mediated responses at the level of the receptor. Ann Bot 88(5):813–822. doi:10.1006/anbo.2001.1523

Chi, G. L. andE. C. pua.1989. Ethylene inhibitors enhanced de novo shoot regeneration from cotyledons of Brassica campastris spp. in vitro. Plant Science 64: 243-250.

Christopher, T. and M. V.Rajam . 1996. Effect of genotype explant andmedium on In vitro regeneration of red pepper. Plant Cell, Tissue and Organ Culture.46: 245-250.

Dabauza, M.; Peña, L.2001. High efficiency organogenesis in sweet pepper (Capsicum annuum L.) Tissues from different seedling explants. Plant. Growth Regul., 33, 221–229.

Dahleen, L. S. 1995. Improved plant regeneration frombarley callus cultures by increased copper levels.Plant Cell, Tissue and Organ Culture. 43: 267-269.

DeWitt, D.;P.W. Bosland.1993.The pepper garden.Berkeley,CA:Ten Speed Press;:240 pp.

Duncan, D. R., M. E. Williams, B. zehr and J. M. Widholm. 1985. The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes. Planta 165 (3): 322-332.

Fuentes, S. R. L., M. B. P. Calleiros, J. Manetti-Filho and G. E. Vieira. 2000. The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffeacanephora. Plant Cell, Tissue and Organ Culture, 60: 5-13.

Gammoudi, N.; Pedro, T.; Ferchichi, A.; Gisbert, C. 2018. Improvement of regeneration in pepper: A recalcitrant species. Plant. Vitr.Cell. Dev. Biol. Plan, 54, 145–153

Garcia, R. A. and M. A. R .Bahillo. 1990. Tissue and cell culture of pepper (Capsicum annuum L. cv. Pico and cv. Piquillo). ActaHorticulturae.:1 249-254.

Ghaemi, M., A. Sarrafi and G. Alibert. 1994. The effects of silver nitrate, colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat (Triticumturgidum). Plant Cell, Tissue and Organ Culture, 36: 355-359.

Giridhar, P., E. P. Indu, K. Vinod, A. Chandrashekar and G. A. Ravishankar. 2004.

Direct somatic embryogenesis from Coffeaarabica L and Coffeacanephora under the influence of ethylene action inhibitor silver nitrate. Acta Physiologia Plantarum 26 (3): 299 -305.

Joshi, A. S. and L. Kothari. 2007. High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledonsofCapsicum annuum L. Plant Cell, Tissue and Organ Culture 88:127–133.

Kim DH, Gopal J, Sivanesan I (2017) Nanomaterials in plant tissue culture: the disclosed and undisclosed. RSC Adv 7:36492–36505.

Kong, L. and E. C. Yeung. 1994. Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation. Plant Science, 104: 71- 80.

Kothari, S.; Joshi, A.; Kachhwaha, S.; Ochoa-Alejo, N.2010. Chilli peppers—A review on tissue culture and transgenesis. Biotechnol. Adv., 28, 35–48.

Kowalska., U., R. GóreckI, K. Janas and K. Górecka. 2009. Effect of increased copper concentrations on deformations of the regenerates of carrot obtained from androgenetic embryos. vegetable crops research bulletin, 71: 15 -23.

Lolkema, P. C. 1985. Copper resistance in higher plants.Doctoral Thesis, Vrije University, Amsterdam.

Mohiuddin, M., M. Chowdhury, C. A. Zaliha and S. Napis .1997. Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell, Tissue and Organ Culture, 51: 75-78.

Malik WA, Mahmood I, Abdul Razzaq, Afzal M, Shah GA, Iqbal A, Zain M, Ditta A, Asad SA, Ahmad I,Mangi N, Ye W.2021. Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.). GM Crops Food 1–22.

Monteiro do Rêgo, M.; Ramalho do Rêgo, E.; Barroso, P.A.. 2016. Tissue Culture of Capsicum spp. In Production and Breeding of Chilli Peppers (Capsicum spp.); Springer International Publishing: Cham, Switzerland, pp. 97–127..

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

Podlesna, A. and U. Wojcieska-Wyskupajtys. 1996. Collection and use of copper by cereals. Zesz.Nauk.Komit. Człow. iŚrod., 14: 129 -133.

Purnhauser, L., P. medgysey, M. czako, J. P. dix and L. marton. 1987. Stimulation of shoot regeneration in Triticumaestivum and Nicotianalumbaginifolia Viv tissue cultures using the ethylene inhibitor silver nitrate, Plant Cell Reports, 6 (1): 1- 4.

Orli ´ nska, M.; Nowaczyk, P.2015. In Vitro plant regeneration of 4 Capsicum spp. Genotypes using different explant types. Turk. J. Biol. 39, 60–68.

Sanatombi, K.; Sharma, G.2008. In Vitro plant regeneration in six cultivars of Capsicum spp. using different explants. Biol. Plant., 52, 141–145.

Sharma, A., K. Vinod, G. Parvatam and A. R. Gokare. 2008. Induction of in vitro flowering in Capsicum frutescensunder the influence of silver nitrate and cobalt chloride and pollen transformation. Electronic Journal of Biotechnology, DOI: 10.2225/vol11-issue2: 1- 8.

Songstad, D. D., D. R. Duncan and J. M. Widholm. 1988. Effect of 1-aminocycopropane-1-carboxilic acid silver nitrate and norbornadiene on plant regeneration from maize callus cultures. Plant Cell Reports, 7(4): 262 -265.

Steinitz B, Barr N, Tabib Y, Vaknin Y, Bernstein N .2010. Control of in vitro rooting and plant development in Corymbia maculata by silver nitrate, silver thiosulfate and thiosulfate ion. Plant Cell Rep 29(11):1315–1323.

Szasz, A., G. Nervo and M. Fasi. 1995. Screening for In vitro shoot forming capacity of seedling explants in bell pepper (Capsicum annuum L.) genotypesand efficient plant regeneration using thidiazuron. Plant Cell Reports, 14: 666 - 669.

Takasaki T., Hatakeyama K., Hinata K. 2004. Effect of silver nitrate on shoot regeneration and Agrobacterium-mediated transformation of turnip. Brassica rapaL. var. rapifera. Plant Biotechnology Journal, 21: 225 -228

Valadez-Bustos, M.; Aguado-Santacruz, G.; Carrillo-Castañeda, G.; Aguilar-Rincón, V.; Espitia-RangeL, E.; Montes-Hernández, S.; Robledo-Paz, A. 2009. In vitro propagation and agronomic performance of regenerated chili pepper (Capsicum spp.) plants from commercially important genotypes. Vitr. Cell. Dev. Biol. Plant, 45, 650–658.




How to Cite

Enfeshi, N., Abdulali, E., Salama, M., Geath, Z., Shaaban, A., Ben saad, Z., & Abughnia, E. (2023). Effect of silver nitrate (AgNO3) and copper sulphate (CuSO4) on callus formation and plant regeneration from tow pepper varieties (Chile Ancho and Misraty) in vitro. Scientific Journal for Faculty of Science-Sirte University, 3(1), 150–157. https://doi.org/10.37375/sjfssu.v3i1.943