Assessing the impact of copper oxide nanoparticles on growth attributes and regenerative potential in date palm (Phoenix dactylifera) micropropagation
Keywords:
antioxidant activity, carbohydrate and nitrogen metabolites, copper oxide nanoparticles, date palm, genetic stability, histological analysis, micropropagation, nutrient uptakeAbstract
Copper oxide nanoparticles (CuO-NPs; 0–40 mg L–1) were applied in MS medium to evaluate microbial contamination, shoot regeneration, and multiplication in date palm (Phoenix dactylifera L. cv. Barhee). Compared to the untreated control (0 mg L–1), copper oxide nanoparticles at 10 mg L–1 eliminated microbial contamination without phytotoxicity, yielding the highest regeneration frequency (88.60%) and maximum shoot number (14.25 shoots jar–1), whereas 40 mg L–1 caused 21.6% culture mortality. Supplementation with 10 mg L–1 CuO-NPs significantly increased shoot nutrient accumulation (N, P, K, Fe, Zn, Cu) relative to the control and other treatments (p ≤ 0.05). Antioxidant enzyme activities (SOD, CAT, POD), total soluble carbohydrates (152.10 mg g–1 DW), and free amino acids (8.05 mg g–1 DW) reached maximum values at 10 mg L–1, compared to the control, whereas soluble protein declined to 10.40 mg g–1 DW at 40 mg L–1, dropping below the untreated control. Root induction was highest at 10 mg L–1 CuO-NPs, while higher concentrations (≥ 20 mg L–1) suppressed rooting and induced root cellular modifications compared to the control. Plantlets derived from the 10 mg L–1 CuO-NPs treatment achieved the highest ex vitro acclimatization survival rate (88%). Genetic fidelity analysis using monomorphic RAPD and ISSR markers confirmed genetic stability among regenerated plantlets relative to mother plants and control cultures.
References
Al-Asadi, A.Z, Al-Mayahi, A.M.W., Awad, K.M., 2024.
Effects of dicamba and casein hydrolysate on in vitro
growth and shoot regeneration of date palm (Phoenix
dactylifera L.) cv. Barhee. Folia Oecologica, 51 (1):
56–65. https://doi.org/10.2478/foecol-2024-0006
Ali, S.S., Darwesh, O.M., Kornaros, M., Al-Tohamy, R.,
2021. Nano-biofertilizers: synthesis, advantages, and
applications. In Manni, A., El-Shanshoury, A.R., Met-
wally, M.A., Elsamahy, T., Sun, J., Biofertilizers: ad-
vances in bio-inoculants. Woodhead Publishing, p. 359–370.
https://doi.org/10.1016/B978-0-12-821667-5.00007-5.
Al-Mayahi, A.M.W., 2022a. The effect of phenyl acetic acid
(PPA) on micropropagation of date palm followed by
genetic stability assessment. Journal of Plant Growth
Regulator, 41: 3127–3137. https://doi.org/10.21203/rs.3.rs-
545347/v1
Al-Mayahi, A.M.W., 2022b. The effect of polyamines and sil-
ver thiosulphate on micropropagation of date palm fol-
lowed by genetic stability assessment. World Journal of
Microbiology and Biotechnology, 38: 124. https://doi.org/
10.1007/s11274-022-03305-5
Al-Mayahi, A.M.W., 2023. Combined efficiency of iron na-
noparticles (IONPs) and salicylic acid (SA) on in vitro
propagation of date palm (Phoenix dacytlifera L.) under
combined drought and salinity. South African Journal
288
of Botany, 162: 324–333. https://doi.org/10.1016/j.sajb.2023.
09.019.
Al-Mayahi, A.M.W., 2024. Effect of ancymidol and phloro-
glucinol on the number and quality of shoots and roots
in the micropropagation of date palm (Phoenix dactylife-
ra L ). Journal of Horticultural Research, 32 (2): 47–56.
https://doi.org/10.2478/johr-2024-0013.
Al-Mayahi, A.M.W., 2025a. Effect of sodium nitroprusside
with plant growth regulators on the in vitro propagation
and genetic stability of Barhee date palm (Phoenix dac-
tylifera L). Journal of Horticultural Research, 33 (1):
1–14. https://doi.org/10.2478/johr-2025-0003
Al-Mayahi, A.M.W., 2025b. Micropropagation of Phoenix
dactylifera L using phloroglucinol, melatonin and in-dole
-3-acetic acid. Vegetos. https://doi.org/10.1007/s42535-
025-01350-7
Al-Mayahi, A.M.W., 2026a. Rooting of aerial offshoots of date
palm (Phoenix dactylifera L.) “Braim” by air layering
method. The Journal of Horticultural Science and Bio-
technology, 101: 1: 201–210. https://doi.org/10.1080/14
620316.2025.2520360
Al-Mayahi, A.M.W., 2026b. The effect of 24-epibrassinolide
and plant growth regulators on in vitro propagation, bio-
chemical changes and genetic stability of Phoenix dac-
tylifera 'Barhee'. Plant Biosystems, 160: 91. https://doi.org/
10.1007/s44473-026-00101-z
Al-Mayahi, A.M.W., 2026c. Effect of meta-topolin on mor-
phological, physiological, biochemical, and molecular
dynamics during in vitro propagation of Phoenix dacty-
lifera L. “Barhee”. South African Journal of Botany, 196
(2026): 796–806. https://doi.org/10.1016/j.sajb.2026.06.039
Amin, F., Khattak, B., Alotaibil, A., Qasim, M., Ahmad, I.,
Ullah, R., 2021. Green synthesis of copper oxide na-
noparticles using Aerva javanica leaf extract and their
characterization and investigation of in vitro antimicro-
bial potential and cytotoxic activities. Evidence-Based
Complementary and Alternative Medicine, 2021: 5589703,
1–12. https://doi.org/10.1155/2021/5589703
Anwaar, S., Maqbool, Q., Jabeen, N., Nazar, M., Abbas,
F., Nawaz, B., Hussain, T., Hussain, S., 2016. The ef-
fect of green synthesized CuO nanoparticles on calloge-
nesis and regeneration of Oryza sativa L. Frontiers in
Plant Science, 7: 1330 https://doi.org/10.3389/fpls.2016.01330
Asgari-Targhi, G., Iranbaksh, A., Ardebili, Z.O., 2018. Poten-
tial benefits and phytotoxicity of bulk and nano-chitosan
on the growth, morphogenesis, physiology, and micro-
propagation of Capsicum annuum. Plant Physiology and
Biochemistry, 127: 393–402. https://doi.org/10.1016/j.
plaphy.04.013
Black, C.A. (ed.), 1965. Methods of soil analysis. Part 1:
Physical and mineralogical properties, icluding statis-
tics of measurement and sampling. Madison, Wisconsin,
USA: American Society of Agronomy. 770 p.
Bradford, M.M., 1976. A rapid and sensitive method for
the quantitation of microgram quantities of protein uti-
lizing the principle of protein-dye binding. Analytical
Biochemistry, 38 (1-2): 248–252. https://doi.org/10.1016/
0003-2697(76)90527-3
Bukhari, S.I., Hamed, M.M., Al-Agamy, M.H., Gazwi, H.
S.S., Radwan, H.H., Youssif, A. M., 2021. Biosynthe-
sis of copper oxide nanoparticles using Streptomyces
MHM38 and its biological applications. Hindawi Journal
of Nanomaterials, 2021: 6693302, 16 p. https://doi.org/
10.1155/2021/6693302
Cakmak, I., Marschner, H., 1992. Magnesium deficiency
and high light intensity enhance activities of superoxide
dismutase, ascorbate peroxidase, and glutathione redu-
ctase in bean leaves. Plant Physiology, 98 (4): 1222–
1227. https://doi.org/10.1104/pp.98.4.1222
Chen, P., 2024. Copper oxide nano-particles effects on plant
growth. Journal of Plant Genetics and Breeding, 8 (2): 198.
Cresser, M.S, Parsons, J.W., 1979. Sulphuric perchloric acid
digestion of plant material for the determination of ni-
trogen, phosphours, potassium, calcium and magnesium.
Analytica Chimica Acta, 109: 43–436. https://doi.org/
10.1016/S0003-2670(01)84273-2
Das, K, Samanta, L., Chainy, G.B.N., 2000. A modified spec-
trophotometric assay of superoxide dismutase using ni-
trite formation by superoxide radicals. Indian Journal of
Biochemistry and Biophysics, 37 (3): 201–204.
Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation pro-
cedure for small quantities of fresh leaf tissue. Phyto-
chemical Bulletin, 19 (1): 11–15.
Carleton, H.M., Drury, R.A.B., Wallington, E.A., Came-
ron, S.R, 1967. Carleton’s histological technique. 4th
ed. New York, Toronto: Oxford University Press. 432 p.
Elsayh, S.A.A. Arafa, R.N., Ali, G.A., Abdelaal, W.B.,
Sidky, R.A., Ragab, T.I.M., 2022. Impact of silver nano-
particles on multiplication, rooting of shoots and bio-
chemical analyses of date palm Hayani cv. by in vitro.
Biocatalysis and Agricultural Biotechnology, 43: 102400.
https://doi.org/10.1016/j.bcab.2022.102400
Farz, A., Faizan, M.D., Rajput, V., Minkina, T., Hayat, S.,
Faisal, M., Alatar, A.A., Abdel-Salam, E.M., 2023.
CuO nanoparticle-mediated seed priming improves
physio-biochemical and enzymatic activities of Brassica
juncea. Plants, 12: 803. https://doi.org/10.3390/plants
12040803
Feigl, G., 2023. The impact of copper oxide nanoparticles on
plant growth: a comprehensive review. Journal of Plant
Interactions, 18:1: 2243098. https://doi.org/10.1080/17
429145.2023.2243098
Fedorova, O., Grodetskaya, T., Evtushenko, N., Evlakov,
P., Gusev, A., Zakharova, O., 2021. The impact of copper
oxide and silver nanoparticles on woody plants obtained
by in vitro method. IOP Conference Series: Earth and
Environmental Science, 875: 012048. https://doi.org/
10.1088/1755-1315/875/1/012048
Giannousi, K., Pantazaki, A., Dendrinou-Samara, C., 2017.
Copper-based nanoparticles as antimicrobials. In Ficai,
A., Grumezescu, A.M. (eds). Nanostructures for anti-
microbial therapy Amsterdam: Elsevier, p. 515–529.
https://doi.org/10.1016/B978-0-323-46152-8.00023-8
Grigore, M.E., Biscu, E.R., Holban, A.M., Gestal, M.C.,
Grumezescu, A.M., 2016. Methods of synthesis, pro-
perties and biomedical applications of CuO nanopar-
ticles. Pharmaceuticals, 9 (4): 75. https://doi.org/10.3390/
ph9040075
Hasanin, M., HASHEM, A.H., Lashin, I., Hassan, S. A.M.,
2023. In vitro improvement and rooting of banana plant-
lets using antifungal nanocomposite based on myco-
synthesized copper oxide nanoparticles and starch. Bio-
mass Conversion and Biorefinery, 13: 8865–8875. https://
doi.org/10.1007/s13399-021-01784-4
Hameed, A., Ahmed, M.Z., Hussain, T., Aziz, I., Ahmad, N.,
Gul, B., Nielsen, B.L., 2021. Effects of salinity stress
on chloroplast structure and function. Cells, 10 (8): 23.
https://doi.org/10.3390/cells10082023
289
Jahan, M.S., Li, G., Xie, D., Farag, R., Hasan, M.M., Alab-
dallah, N.M., Al-Harbi, N.A., Al Qahtani, S.M., Zee-
shan, M., Nasar, J., 2023. Melatonin mitigates salt-in
duced growth inhibition through the regulation of carbo-
hydrate and nitrogen metabolism in tomato seedlings.
Journal of Soil Science and Plant Nutrition, 23: 4290–
4308. https://doi.org/10.1007/s42729-023-01348-7
Javed, R., Mohamed, A., Yücesan, B., Gürel, E., Kausar, R.,
Zia, M., 2017. CuO nanoparticles significantly influence
in vitro culture, steviol glycosides, and antioxidant acti-
vities of Stevia rebaudiana Bertoni. Plant Cell, Tissue
and Organ Culture. 131: 611–620. https://doi.org/10.1007/
s11240-017-1312-6
Ibrahim, I.A., Abul-Soad, A.A., Bakr, E.I., El-Sherbeny, N.R.,
1999. Effect of charcoal and light intensity on rooting
and acclimatization stages of regenerated date palm
(Phoenix dactylifera L.) Plantlets. In Proceedings of the
First International Conference on Plant Tissue Culture
and Its Applications. University of Zagazig, Egypt, 12–14
September 1999, p. 219–226.
Lee, Y.P., Takahashi, T., 1966. An improved colorimetric de-
termination of amino acids with the use of ninhydrine.
Analytical Biochemistry, 14: 71–77. https://doi.org/10.1016/
0003-2697(66)90057-1
Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids:
pigments of photosynthetic biomembranes. Methods in
Enzymology, 148: 350–382. https://doi.org/10.1016/
0076-6879 (87)48036-1
Murphy, T., Riley, J.P., 1962. A modified single solution met-
hod for the determination of phosphate in natural waters.
Analytica Chimica Acta, 27: 31–36. https://doi.org/10.1016/
S0003-2670(00)88444-5
Murashige, T., Skoog, F., 1962. A revised medium for rapid
growth and bioassay of tobacco tissue cultures. Physiologia
Plantarum, 18: 100–127. https://doi.org/10.1111/j.1399
-3054.1962.tb08052.x
Murshed, R., Lopez-Lauri, F., Sallanon, H., 2014. Effect
of salt stress on tomato fruit antioxidant systems depends
on fruit development stage. Physiology and Molecular
Biology of Plants, 20: 15–29. https://doi.org/10.1007/
s12298-013-0209-z
Nazir, S., Jan, H., Zaman, G., Khan, T., Ashraf, H., Meer, B.,
Zia, M., Drouet, S., Hano, C., Abbasi, B.H., 2021.
Copper oxide (CuO) and manganese oxide (MnO) nano-
particles induced biomass accumulation, antioxidants
biosynthesis and abiotic elicitation of bioactive compou-
nds in callus cultures of Ocimum basilicum (Thai basil).
Artificial Cells, Nanomedicine, and Biotechnology, 49:,
626–634. https://doi.org/10.1080/21691401.2021.1984
935
Page A.L., Miller, R.H., Keeny, D., 1982. Methods of soil
analysis. Part 2: Chemical and microbiological proper-
ties. 2nd ed. Madison. WI: American Society of Agrono-
my and Soil Science Society of America. 1159 p..
Pelegrino, M.T., Kohatsu, M.Y., Seabra, A.B., Monteiro,
L.R., Gomes, D.G., Oliveira, H.C., Lange, C.N., 2020.
Effects of copper oxide nanoparticles on growth of le-
ttuce (Lactuca sativa L.) seedlings and possible impli-
cations of nitric oxide in their antioxidative defense. En-
vironmental Monitoring and Assessment, 192: 232.https://
doi.org/10.1007/s10661-020-8188-3
Pharmawati, M., Wrasiati, L.P., Yowani, S.C., 2021. ISSR
and RAPD primers selection for assessing genetic di-
versity of Enhalus acoroides (L.f.) Royle. IOP Conferen-
ce Series: Earth and Environmental Science, 709: 012054,
7 p. DOI: 10.1088/1755-1315/709/1/012054
Rajput, V., Minkina, T., Suskova, S., 2018. Effects of cop-
per nanoparticles (CuO-NPs) on crop plants: a mini
review. Bionanoscience, 8: 3642. https://doi.org/10.
1007/s12668-017-0466-3
Sawant, R., Kamat, S., Salvi, P., Vagare, R., Dalvi, R., 2023.
Effect of CuO and FeO nanoparticles on seed germina-
tion, micropropagation, and callus culture. Journal of Emer-
ging Technologies and Innovative Research, 10 (6): 160–
169.
Shafiq, H., Shani, M.Y., Ashraf, M.Y., De Mastro, F.,
Coczza, C., Abbas, S., Ali, N., Zaib-un-Nisa, Tahir,
A., Iqbal, M., Khan, Z, Gul, N, Brunetti, G., 2024.
Copper oxide nanoparticles induced growth and physio-
biochemical changes in Maize (Zea mays L.) in saline
soil. Plants, 13, 1080. https://doi.org/10.3390/plants
13081080
Singh, D., Jain, D., Rajpurohit, D., Jat, G., Kushwaha,
H.S., Singh, A., Mohant, S.R., Al-Sadoon, M.K.,
Zaman, W., Upadhyay, S.K., 2023. Bacteria assisted
green synthesis of copper oxide nanoparticles and their
potential applications as antimicrobial agents and plant
growth stimulants. Frontiers in Chemistry, 11: 1154128.
https://doi.org/10.3389/fchem.2023.1154128
Sivanesan, I., Upadhyay, S., Keum, Y.S., Chun, S.C., Saini,
R.K., 2025. Impact of cCopper oxide nanoparticles on
adventitious shoot regeneration, axillary shoot multipli-
cation, rooting, and bioactive compounds in Ajuga mul-
tiflora Bunge. Plants, 14: 3807. https://doi.org/10.3390/
plants14243807
Tikendra, L., Potshangbam, A.M., Dey, A., Devi, T.R.,
Sahoo, M.R., Nongdam, P., 2021. RAPD, ISSR, and
SCoT markers based genetic stability assessment of
micropropagated Dendrobium fimbriatum Lindl. var.
oculatum Hk. f.- an important endangered orchid. Phys.
Mol. Biol. Plants. 27(2): 341–357. https://doi.org/
10.1007/s12298-021-00939-x
Wang, Y., Diao, P., Kong, L., Yu, R., Zhang, M., Zuo, T.,
Niu, Y., Fan, Y., Wuriyanghan, H., 2020. Ethylene en-
hances seed germination and seedling growth under sa-
linity by reducing oxidative stress and promoting chlo-
rophyll content via ETR2 pathway. Frontiers in Plant
Science, 11: 1066. https://doi.org/10.3389/fpls.2020.01066
Watanabe, S., Kojima, K., Ide, Y., Sasakii, S., 2000. Effect of
saline and osmotic stress on proline and sugar accumu-
lation in Populus euphratica in vitro. Plant Cell, Tissue
and Organ Culture, 63: 199–206. https://doi.org/10.1023/
A:1010619503680
Welsh, J., McClelland, M., 1990. Fingerprinting genomes
using PCR with arbitrary primers. Nucleic Acids Re-
search, 18:7213–7218. https://doi.org/10.1093/nar/18.24.
7213
Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A.,
Tingey, S.V. , 1990. DNA polymorphism amplified by
arbitrary primers are useful as genetic markers. Nucleic
Acids Research, 18 (22): 6531–6535. https://doi.org/
10.1093/nar/18.22.6531
Xiong, T., Zhang, S., Kang, Z., Zhang, T., Li, S., 2021.
Dose-dependent physiological and transcriptomic res-
ponses of lettuce (Lactuca sativa L.) to copper oxide
nanoparticles—insights into the phytotoxicity mecha-
nisms. International Journal of Molecular Sciences, 22
(7): 3688. https://doi.org/10.3390/ijms22073688
290
Yasmeen, F., Raja, N.I., Razzaq, A., Komatsu, S., 2017. Pro-
teomic and physiological analyses of wheat seeds ex-po-
sed to copper and iron nanoparticles. Biochimica et Bio-
physica Acta (BBA) - Proteins and Proteomics, 1865:
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Ahmed Madi Waheed Al-Mayahi, Taghreed Mahmood Kuder ALfarjawi (Author)
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
This journal provides immediate open access to its content under the Creative Commons BY-NC-ND 4.0 license. Authors who publish with this journal retain all copyrights except for commercial rights (transfer of commercial rights) and agree to the terms of the above-mentioned CC BY-NC-ND 4.0 license.
