Influence of PVP/PEG impregnated CuO NPs on physiological and biochemical characteristics of Trigonella foenum‐graecum L

Abstract

Incorporation of nanoparticles into a number of manufacturing products raised the concern of environmental release via deliberate or accidental routes. Here, experiments were performed to examine the effect of copper oxide nanoparticles (CuO NPs), and polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) impregnated CuO NPs on seed germination and growth of Trigonella foenum‐graecum L. as well as on callus induction through tissue culture technique. Seed germination frequency, length, and weight parameters did not inhibit at higher extent by application of NPs; however, copper acetate, PVP, and PEG significantly decreased the values of all parameters. In all the cases, negative effects were observed concentration‐dependent. PVP and PEG impregnated CuO were found less toxic for calli fresh and dry weight induced from leaf and stem explants. The 2, 2‐diphenyl‐1‐picrylhydrazyl reagent‐free radical scavenging activity, total antioxidative potential, and total reducing power potential along with total flavonoid and phenolic contents are found elevated in root when compared with shoot. Furthermore, impregnation of PVP and PEG on CuO NPs increases the oxidative response. The results conclude that impregnation of organic molecules on nanoparticles does not reduce the toxicity though can be exploited for enhanced production of secondary metabolites for medicinal purposes.

1 Introduction

Nanoparticles are defined as the particles having <100 nm size and comparatively large surface area to volume ratio than its bulk counterparts. Increased use of engineered nanoparticles has raised concern about their release into the environment with potential adverse effects on ecosystems including human health [1]. Copper oxide nanoparticles (CuO NPs) have gained special attention due to simplicity and various physical properties including superconductivity, electron correlation effects, and spin dynamics. CuO NPs are progressively used in various applications such as in catalysis, batteries, gas sensors, heat transfer fluids, and solar energy [2]. A number of studies have been performed showing both beneficial and toxic effects of NPs on plant growth [3, 4]. Excessive production of CuO NPs may lead to environmental exposure, thus causing toxicity to bacteria, nematodes, and other organisms. Studies on the toxicity of nanomaterials are facing rapid emergence, and there are several evidences of negative effects on the growth and development of plants [5]. Polyethylene glycol (PEG) is often used to induce drought stress in seed germination studies and also influence growth of plant negatively as its concentration increases by reducing the oxygen availability [6]. Polyvinyl pyrrolidone (PVP) is a versatile polymer, and water‐soluble with excellent colloidal, stabilising, and complexing properties. PVP is metabolically and physiologically inert. Previous studies have shown that PVP has positive effect on plant growth at various concentrations.

Trigonella foenum‐graecum L. (fenugreek) is one of the oldest medicinal plants in the world, commonly known as ‘methi’. It is a self‐pollinating, leguminous crop native to Indian subcontinent and Eastern Mediterranean region. It is widely cultivated in central Asia, central Europe, Northern Africa, North America, India, and parts of Australia [7]. T. foenum‐graecum L. exhibits antioxidant activity due to the presence of certain phytochemicals including vitamins, flavonoids, terpenoids, carotenoids, cumarins, curcumins, lignin, saponins etc. Different studies proved that T. foenum‐graecum L. extracts exhibit scavenging activity against hydroxyl free radicals that are associated with the prevention of diseases like diabetes mellitus, atherosclerosis, cataract, rheumatism, and other autoimmune diseases. Studies have also shown anti‐bacterial activity possessed by T. foenum‐graecum L. [8, 9].

The current study reports the toxicity induced by CuO NPs that can be attributed to induction of oxidative stress by crossing the cellular barriers owing to smaller size. This study investigates the effects of CuO NPs on seed germination, morphology, callus induction, as well as biochemical screening of T. foenum‐graecum L. plant.

2 Material and methods

2.1 Synthesis and characterisation of CuO, CuO‐PEG/PVP nanoparticles

The co‐precipitation method was employed for the synthesis of CuO, CuO‐PEG, and CuO‐PVP. About 600 ml of 0.2 M copper acetate monohydrate (Cu(CH₃ COO)₂ ·H₂ O; 98%) was mixed with 2 ml glacial acetic acid (CH₃ COOH) at 100°C during continuous stirring and freshly prepared 30 ml of 6 M NaOH solution was dropwise added. The black coloured CuO nanoparticles were filtered and washed by distilled water. The NPs were calcinated at 500°C for 4 h. The capping process of CuO nanoparticles with PEG and PVP was carried out separately during synthesis by mixing the capping agents with precursor salt, i.e. copper acetate monohydrate at 1:1 ratio, and later on following the similar procedure of CuO nanoparticles synthesis.

The crystalline nature of synthesised nanoparticles was found by X‐ray diffraction (XRD). The XRD pattern of CuO nanopowder was acquired with the help of a PANalytical Empyrean X‐ray diffractometer equipped with Ni filtered using Cu Kα (λ  = 1.54056 Å) radiations as an X‐ray source. FTIR spectra were recorded over 4000–500 cm⁻¹ from Tensor 27 Bruker (Germany) OPUS Data Collection Program with a resolution of 1 cm⁻¹. Morphological studies of nanoparticles were done by scanning electron microscope (SEM, Nova NanoSEM 450) operated at an accelerating voltage of 10 kV.

2.2 MS medium preparation containing CuO, CuO‐PEG, CuO‐PVP, Cu‐acetate, PEG, and PVP

The CuO NPs were suspended directly in distilled water and dispersed by ultrasonic vibration (100W, 40 kHz) for 30 min. Different doses of NPs suspensions, i.e. 50, 100, 200, and 400 mg/l, were prepared. PEG and PVP solutions were also prepared with concentrations; 10, 20, 40, and 80 mg/l. Along with these, salt solutions of Cu‐acetate, 0.5 and 1%, were prepared by dissolving in distilled water.

2.3 Seed germination experiment

Seeds of T. foenum‐graecum L. were purchased from National Agricultural Research Centre (NARC), Islamabad. Selection of seeds was based on ethnobotanical, traditional medicinal importance, and least exploration. Seeds were cleaned, freed from dust and foreign material, and then kept in dark and dry place before use.

Four different media, supplemented with 3% sucrose and 0.44% gelrite, were prepared containing: (i) half strength, i.e. 2.2 g/l of Murashige and Skoog medium (MS), containing 0, 50, 100, 200, and 400 mg/l CuO NP, CuO NP‐PEG, and CuO NP‐PVP, (ii) half strength MS media containing 10, 20, 40, and 80 mg/l of PEG and PVP, (iii) half strength MS media containing 0.5 and 1% Cu acetate, and (iv) simple half strength MS media (control). pH of all types of media was maintained at 5.7.

After heating, media were autoclaved at 121°C, 15 psi, and for 20 min. Under aseptic conditions, seeds were immersed for 3–4 min in freshly prepared 0.1% HgCl₂ solution for surface sterilisation. Subsequently, seeds were washed thrice with distilled water. Five seeds per flask were inoculated and kept in dark in growth room at 25°C. Seed germination parameters were recorded after 5 days and flasks were transferred at 16 h photoperiod condition for growth of seeds. Seedling growth parameters including root and shoot length were calculated after 15 days.

2.4 Callus induction experiment

For callus induction, seeds of T. foenum‐graecum L. were first inoculated on MS media and plantlets were collected after 15 days. Media were prepared for callus initiation containing 20 ml MS media in 100 ml flask supplemented with plant growth regulators (PGRs), i.e. 2 mg/l 1‐naphthaleneacetic acid, 0.5 mg/l 2,4‐dichlorophenoxyacetic acid (2,4‐D), and 0.5 mg/l 6‐benzylaminopurine. In addition to PGRs, CuO, CuO NP‐PEG, and CuO NP‐PVP were also added into the medium at concentrations of 0 (control), 2.5, 5, and 10 mg/l. The media were autoclaved prior to induction at 121°C for 20 min.

Callus was initiated by cutting stem of a plantlet into pieces of 8 ± 1 mm, and leaf was also cut into size of 5 ± 1 mm using a sterilised scalpel. After inoculation, the flasks were transferred to growth room where temperature was maintained at 25°C with 16 h photoperiod. The calli were generated over a time period of 4 weeks from both stem and leaf explant. After 30 days, calli were separated from the media and their fresh weight was calculated using analytical balance. The callus was then subjected to drying in a vacuum oven at 45°C for 3 days. After drying, callus was once again weighed and readings were noted.

2.5 Phytochemical screening

2.5.1 Extract preparation

Fresh weight extracts of shoot and root were prepared in ethanol. The dried extracts were dissolved in dimethyl sulphoxide (DMSO) at 20 mg/ml concentration for antioxidant analysis. Dry weight callus extracts were prepared in ethanol and then dissolved in DMSO at concentration of 100 mg/ml.

2.5.2 Determination of total flavonoid content

Total flavonoid content (TFC) of the test samples were identified according to the method described by Ali et al. [10]. About 20 μl test sample, standard and blank were taken in 96‐well microplate followed by addition of 10 μl aluminium chloride solution and then 10 μl potassium acetate (1 M). About 160 μl of distilled water was added to attain a final volume of 200 μl. The plate was then incubated for 30 min at room temperature. The absorbance was measured by using microplate reader (Bioteck, USA) at 415 nm.

2.5.3 Determination of total phenolic content

Total phenolic content (TPC) of the test samples was determined by using the method described by Rehman et al. [11]. About 20 μl of the test sample, positive control (gallic acid) and negative control (DMSO) were added to 96‐well microplate followed by addition of 90 μl of Folin–Ciocalteu reagent and incubated for 5 min at room temperature. After incubation, 90 μl of sodium carbonate was added into the plate. Readings were taken at 630 nm wavelength of microplate reader.

2.6 Antioxidant activities

2.6.1 Total antioxidant capacity

Total antioxidant capacity (TAC) of the fresh weight extracts was determined by Zafar et al. [12]. In eppendorf tubes, 100 μl of test sample, standard and blank were taken and mixed by adding 900 μl of antioxidant reagent. The tubes were then incubated at 95°C for 90 min. After incubation, the reaction mixture was cooled to room temperature, and 200 μl of sample was transferred to microplate. Optical density was measured at 630 nm on microplate reader.

2.6.2 Total reducing power

Total reducing power (TRP) of the test samples was investigated according to the protocol described by Zafar et al. [13]. About 100 μl of test sample, positive control (ascorbic acid) and negative control (DMSO) were taken and then 200 μl of phosphate buffer was added, followed by addition of 250 μl of 1% potassium ferricyanide into the eppendorf tubes. The mixture was then incubated at 50°C for 20 min. After incubation, 200 μl of 10% trichloroacetic acid was added. The reaction mixture was centrifuged at 3000 rpm for 10 min. Supernatant layer of 150 μl was picked and poured into microplate well and then 50 μl of 0.1% ferric chloride was added. Readings were taken at 630 nm using microplate reader.

2.6.3 DPPH free radical scavenging assay

The free radical scavenging activity of the test samples against 2, 2‐diphenyl‐1‐picrylhydrazyl reagent (DPPH) was determined according to the protocol described by Fatima et al. [14]. In a 96‐well microplate, 10 μl of test sample, standard (ascorbic acid) and blank were added. Then 190 μl of DPPH reagent was added and incubated for 1 h at 37°C. Further readings were noted at 517 nm wavelength on microplate reader.

2.7 Statistical analysis

Each treatment was conducted with three replicates and the results were presented as mean with standard deviation. The means were further analysed by analysis of variance and least significant difference at 0.05 probability.

3 Results

3.1 Synthesis and characterisation of nanoparticles

The reduction in copper acetate by sodium hydroxide as shown by chemical reaction below results in formation of different structured nanomaterials. The synthesised nanoparticles are further capped with organic polymers; PEG and PVP. The powder patterns were recorded with the use of Empyrean PANalytical X‐ray diffractometer with Bragg–Brentano geometry using Cu Kα radiation (λ  = 1.54 Å). The diffraction data (Fig. 1) reveals that the material is composed of crystalline monoclinic cubic cupric oxides. The crystallite size is determined using the Scherrer equation

$$D=\frac{k\lambda }{\beta \cos {\theta }_B}$$

where D is the crystallite size, k a constant (∼0.94 assuming that the particles are spherical), λ the wavelength of the X‐ray radiation, β the line width at half maximum intensity of the peak, and θ_B the angle of diffraction. The engineered CuO nanoparticles; uncapped CuO NPs (47 nm in size), CuO NP‐PEG (27 nm in size), and CuO NP‐PVP (27 nm in size) were synthesised. The size of the nanoparticles decreases due to capping with PEG and PVP.

Fig. 1.

XRD, FTIR, and SEM results of synthesised CuO and CuO‐PEG/PVP nanoparticles

FTIR spectroscopy is an effective technique to reveal the composition of sample. Fig. 1 shows the FTIR spectra of CuO nanoparticles. The absorption peak around 3419 cm⁻¹ is assigned to the O–H stretching vibration, and the peak around 2094 cm⁻¹ is due to the existence of CO₂ molecules in air. The bond at 1052 cm⁻¹ is due to the C –O stretching vibration. The origin of two well‐defined absorption bands at 1411 cm⁻¹ is due to CH₃ group, and CH₃ asymmetrical stretching mode present on the surface of CuO nanostructures. The bands at 2847 and 2920 cm⁻¹ are assigned to –CH₂ and C–H stretching mode. Typical SEM micrographs for prepared nanoparticles without/with capping agent (CuO, CuO‐PEG, and CuO‐PVP) are shown in Fig. 1. The SEM micrographs of CuO and CuO‐PEG clearly show irregular shaped morphologies of nanoparticles (Fig. 1). The average size of the nanoparticles lies between 40 and 100, 25 and 70, and 25 and 90 nm for CuO, CuO‐PEG, and CuO‐PVP nanoparticles, respectively.

Table 1 shows that seed germination frequency was 100% at all concentrations of CuO NPs and their derivatives (CuO NP‐PEG, CuO NP‐PVP), Cu‐acetate, PEG, and PVP. Hence, the NPs, salt, and polymers did not negatively affect the seed germination of T. foenum‐graecum L plant. Regarding shoot and root growth from seedlings, the data (Fig. 2, Table 1) showed that Cu‐acetate salt, PEG, and PVP inhibited the shoot and root growth. The NPs also resulted in inhibition but only at higher concentrations. The shoot and root response of the plant was not inhibited up to 50 mg/ml of CuO NPs where maximum response was obtained. The average fresh and dry weight of plantlets were also recorded, and it was observed that the maximum average fresh weight (0.47 g) and average dry weight (0.05 g) were shown by CuO NPs at 50 mg/ml concentration (Table 1). Callus induction was conducted from stem and leaf explants (Fig. 3), and the maximum average fresh and dry weight of callus obtained from stem and leaf explants were obtained in control (Table 2). Stem and leaf explant extracts also showed the presence of flavonoids and phenolics after treatment with NPs (Table 3).

Table 1.

Seed germination frequency and growth parameters of T. foenum‐graecum L

Treatment	Conc.	FG, %	MPFG	Average shoot length, cm	Average root length, cm	Average fresh weight, g	Average dry weight, g
CuO NP (mg/l)	50	90b	0.5	7.7a	6.6a	0.47a	0.05a
	100	80c	0.5	6.5c	5.1c	0.42ab	0.03b
	200	100a	0.5	6.4c	4.4d	0.13c	0.01c
	400	80c	0.5	6.3c	4.3d	0.11c	0.01c
CuO NP‐PEG (mg/l)	50	100a	0.5	7.0b	6.8a	0.34b	0.04ab
	100	100a	0.5	6.9b	6.1b	0.32b	0.03b
	200	100a	0.5	6.8bc	6.0b	0.20bc	0.02bc
	400	100a	0.5	5.4bc	3.0f	0.31b	0.03b
CuO NP‐PVP (mg/l)	50	90b	0.5	7.0b	5.9b	0.43ab	0.04ab
	100	100a	0.5	6.7bc	5.8b	0.31b	0.03b
	200	100a	0.5	6.6bc	4.6d	0.32b	0.03b
	400	90b	0.5	6.4c	3.4ef	0.31b	0.02bc
PEG (mg/l)	10	100a	0.5	6.2cd	3.4ef	0.28bc	0.04ab
	20	100a	0.5	6.1cd	3.3ef	0.26bc	0.03b
	40	100a	0.5	6.0cd	2.2g	0.24bc	0.03b
	80	100a	0.5	5.9d	2.1g	0.22bc	0.03b
PVP (mg/l)	10	90b	0.5	6.6bc	3.7e	0.32b	0.04ab
	20	100a	0.5	6.5c	3.6e	0.29bc	0.04ab
	40	100a	0.5	6.2cd	3.6e	0.27bc	0.03b
	80	100a	0.5	5.3e	3.5e	0.13c	0.01c
Cu‐acetate, %	0.5	100a	0.5	4.9f	2.3g	0.22bc	0.02bc
	1	100a	0.5	4.3g	1.6h	0.19c	0.01c
control	0	100a	0.5	7.2b	6.6a	0.28bc	0.04ab

*The same superscript letters within the column are showing similar values that otherwise differ significantly at P  < 0.05.

Fig. 2.

Effect of CuO NPs and Cu‐acetate on root and shoot length of T. foenum‐graecum L. at different concentrations

Fig. 3.

Callus induction from stem and leaf explants of T. foenum‐graecum L. having CuO NPs

( a ) Control of stem explant; ( b–d ) CuO NP; 2.5, 5, and 10 mg/l; ( e ) control of leaf explant; ( f–h ) CuO NP; 2.5, 5, and 10 mg/l

Table 2.

Average fresh and dry weight of T. foenum‐graecum L. callus treated with CuO NPs

Treatment	Conc. (mg/l)	Average fresh weight/explant, g		Average dry weight/explant, g
		Stem	Leaf	Stem	Leaf
CuO NP	2.5	0.67c	0.85ab	0.05b	0.07ab
	5	0.25c	0.12c	0.02c	0.01c
	10	1.33ab	0.13bc	0.07ab	0.01c
CuO NP‐PEG	2.5	1.25b	0.11c	0.08ab	0.01c
	5	0.99bc	0.33b	0.05b	0.02b
	10	0.82bc	0.21b	0.06b	0.02b
CuO NP‐PVP	2.5	1.13b	1.24a	0.07ab	0.08a
	5	1.31ab	0.14bc	0.08ab	0.02b
	10	1.42a	0.12c	0.09a	0.01c
control	0	1.65a	1.33a	0.10a	0.09a

*The same superscript letters within the column are showing similar values that otherwise differ significantly at P  < 0.05.

Table 3.

Phytochemical and antioxidant activity of T. foenum‐graecum L. stem and leaf callus extracts treated with CuO NPs

Treatment	Conc., mg/l	TFC, µg QE/mg DW		TPC, µg GAE/mg DW		TAC, µg AAE/mg DW		TRP, µg AAE/mg DW		DPPH, % inhibition
		Stem	Leaf	Stem	Leaf	Stem	Leaf	Stem	Leaf	Stem	Leaf
CuO NP	2.5	2.8c	2.7bc	3.2b	3.0b	3.6f	5.6h	5.9g	10.3a	40c	48e
	5	3.0c	3.0b	4.0a	2.6c	5.2d	9.5b	7.7e	7.2f	43bc	54d
	10	2.8c	3.1b	3.3b	3.1b	4.3e	7.6d	9.4b	5.5g	36d	40f
CuO NP‐PEG	2.5	3.1c	2.6c	3.9a	3.5a	5.2d	9.9a	9.6b	9.7b	38cd	69b
	5	2.6cd	1.8d	3.1b	3.0b	5.9c	7.2e	7.9de	8.2d	56a	72a
	10	1.6e	2.0d	2.1d	2.6c	4.3e	5.9g	6.4f	8.1d	42bc	58c
CuO NP‐PVP	2.5	3.5b	3.2ab	3.9a	2.2d	4.1e	3.3i	8.2d	9.2c	44b	46e
	5	3.8a	3.5a	4.0a	3.1b	6.4b	6.4f	10.3a	7.5e	45b	59c
	10	3.9a	3.6a	3.9a	3.6a	6.5b	6.5f	10.5a	7.6e	56a	60c
control	0	2.4d	3.4ab	2.6c	3.5a	7.1a	8.1c	9.0c	8.2d	55a	58c

*The same superscript letters within the column are showing similar values that otherwise differ significantly at P  < 0.05. TFC, total flavonoid content; TPC, total phenolic content; TAC, total antioxidant capacity; TRP, total reducing power; DPPH; 1,1‐diphenyl‐2‐picryl‐hydrazyl; QE, quercetin equivalent; GAE, gallic acid equivalent; AAE, ascorbic acid equivalent.

Phytochemical screening results showed the presence of flavonoids and phenolics in the fresh weight extracts of shoot and root treated with CuO NPs, CuO NP‐PEG, CuO NP‐PVP, Cu‐acetate salt, PEG, and PVP (Figs. 4 and 5). Total antioxidant assay results showed maximum activity in shoot extracts treated with PVP at 80 mg/l and 1% Cu‐acetate as compared to CuO NP and capped CuO NPs‐derived shoot extracts. On the other hand, root extracts data revealed maximum TAC in PVP and CuO NP‐PEG treatments (Figs. 4 and 5). TAC was found maximum in control as compared to CuO NP treatments in shoot explants (Table 3). The trend of TAC derived from leaf explants was found maximum in CuO NP and CuO NP‐PEG‐treated explants. The maximum total reducing power (TRP) was found in shoot extracts obtained from the treatment of PVP at 80 mg/l and Cu‐acetate at 1%. In the case of treated root extracts, highest TRP was observed in CuO NP‐PEG and PVP (Figs. 4 and 5). The maximum reducing power was observed in both shoot and leaf explants treated with CuO NPs (Table 3). The maximum free radical scavenging activity was found in plantlets treated with Cu‐acetate (1%), CuO NP‐PEG (50 mg/l), and CuO NP‐PVP (100 mg/l) in both root and shoot extracts as compared to control (Figs. 4 and 5). DPPH free radical scavenging activity was found maximum in both shoot and leaf explants treated with capped CuO NPs (Table 3).

Fig. 4.

Effect of CuO NP, CuO NP‐PEG, CuO NP‐PVP, Cu‐acetate, PEG, and PVP on DPPH, TAC, TRP, TFC, and TPC derived from shoot extracts of T. foenum‐graecum L. The same superscript letters are showing similar values that otherwise differ significantly at P  < 0.05

Fig. 5.

Effect of CuO NP, CuO NP‐PEG, CuO NP‐PVP, Cu‐acetate, PEG, and PVP on DPPH, TAC, TRP, TFC, and TPC derived from root extracts of T. foenum‐graecum L. The same superscript letters are showing similar values that otherwise differ significantly at P  < 0.05

4 Discussion

Seed germination is a physiological process that starts with water imbibition by means of seeds and ends with the emergence of roots. It is widely used as phytotoxicity test because it is sensitive, simple, and low cost, and it depends on nanoparticle–plant physical interactions [10, 15, 16]. In this study, seeds showing emergence of radical or cotyledon out of seed coat were recorded as being germinated. The findings of this study showed 100% seed germination without any significant adverse effect at any concentration. This data agrees with a study conducted by Adhikari et al. [5] showing 100% germination of seeds of soyabean and chickpea grown under CuO NPs. In addition to this, in another study, fenugreek seeds were treated with Ag NPs and results showed increased seed germination enhancing the seed potential by increasing the characteristics of seed germination [17]. It is probably due to the seed coat which acts as protector for the embryo and plays important role in selective permeability.

Osmotic stress can be achieved by growing plants in a media containing varying concentrations of PEG. It modifies the osmotic potential of nutrient solution culture inducing plant water deficit in a relatively controlled manner [18]. Results of current research have not shown inhibitory effects of PEG on seed germination frequency. These findings are in accordance with the research of Hardegree and Emmerich [19] in which PEG contact with seed had no detrimental effects on seed germination in four plant species. However, in 1994, another study by Hardegree and Emmerich showed that germination is affected by increase in immersion in PEG solution depth. The imbibition path of the bare membrane treatment is limited to the solution volume associated with the capillary interface between seed and membrane surface [6].

Shoot and root elongation results are varying at different concentrations of NPs and phytotoxicity is evident. CuO NPs show maximum shoot elongation, i.e. 7.7 cm at 50 mg/l as compared to control, but the trend gradually decreases as concentration of nanoparticles increases. A similar trend is followed in plantlets treated with CuO NP‐PEG and CuO NP‐PVP. However, significant shoot inhibition has been observed in seedlings treated with Cu‐acetate salt, i.e. 4.3 cm at 1% concentration. Thus, it indicates minimal toxicity of CuO NPs on shoot growth with an increase in concentration in contrast to Cu‐acetate salt. In MS media containing PEG, the average shoot length decreases at higher concentrations and it is in accordance with the previous study in which PEG significantly reduces shoot growth in two populations of Anth xanthum odoratum performed by Anwer et al. [20]. The average shoot elongation is decreased under lower to higher concentrations of PVP.

Plantlets grown under capped CuO NPs and Cu‐acetate salt evidently inhibit root length as compared to plantlets obtained from media containing uncapped CuO NPs. At lower concentrations, CuO NPs show maximum root length, but it is inhibited with an increase in concentration of NPs in the media showing phytoxicity and markedly reduced root development. As roots are the first target tissue to confront the excess concentrations of pollutants, hence toxic symptoms seem to appear more in roots as compared to shoots. Therefore, comprehensive phytotoxicity profile should be investigated in higher plants for nanoparticles [21]. Although mechanism of nanoparticles toxicity is yet not clear, but it can be postulated that NPs possess ability to cross permeable membranes including seed coat and cellular membranes of roots, NPs may coagulate on the root surface and alter root chemistry. Cu ions can be released from NPs that can change ionic balance, pH, and cellular components, and it also depends on mass to size ratio, shape, and surface properties of nanoparticles. It is also observed in some reports that it also depends on species of plant and type of nanoparticles employed [22, 23, 24].

Exposure to excess copper salt had detrimental effects on plant growth. Copper ions tend to accumulate in the root tissues with little translocation to the shoot, thus principle effect of Cu toxicity on root growth [25]. Similar results were found in a study conducted by Thounaojam et al. [26], in which reduction in root and shoot length was observed due to accumulation of Cu in seedlings of rice, directly co‐related with toxicity in the plant. Results of the current study reconfirmed the earlier report showing co‐relation of Cu tolerance and its greater accumulation in roots as compared to shoots due to poor translocation.

Root length is an important feature against drought stress in plant varieties; in general, variety with longer root growth has resistant ability for drought [27]. In the present study, seedlings grown on PEG showed reduction in root length that attributes to the water stress induced by PEG at higher concentrations which affected root growth negatively. Similar results were reported by Kaydan and Yagmur [28], stating that PEG and NaCl affected seedlings growth of Presto negatively and showed reduction in shoot and root length. Whereas, plantlets treated with PVP also showed reduced shoot and root length as concentration were increased (Table 1).

The average fresh and dry weight were found to be maximum in CuO NPs at 50 mg/l as compared to control but gradual decreasing trend was observed (Table 2). The increase in biomass at certain concentration suggests the optimum dose limit for the growth of T. foenum‐graecum L. seedlings. However, a decrease in biomass beyond this concentration suggested the toxic effect of CuO NPs. Plantlets treated with PEG and PVP also showed a decrease in biomass with an increase in stress. The reduction in biomass can be due to reduced availability of water and other nutrients and disturbance in normal cellular function required for proper growth.

Flavonoids and phenolics play an important role in detoxification of ROS. Total flavonoid and phenolic contents were determined and results showed that extracts of roots grown under NPs accumulated more flavonoids and phenols as compared to control root extracts. Whereas, the plantlets treated with Cu‐acetate salt had accumulated maximum phenolic contents indicating the elicitation of secondary metabolites production in the presence of abiotic stress. The same trend was seen in the shoot extracts and CuO NPs have shown variations in the flavonoid and phenolic content accumulation. These results depict that ROS generated inside the plant on exposure to CuO NPs.

ROS are proposed to be responsible for negative effects of NPs, but toxicity mechanism of NPs has not yet been clearly understood [29]. Antioxidant activity and reducing power of T. foenum‐graecum L. treated with CuO NPs was determined in the current study. According to results, it was shown that there was less significant difference on exposure to CuO NP, CuO NP‐PEG, and CuO NP‐PVP. NPs induce oxidative stress that damages cell [30]. DPPH results depict that CuO NPs are responsible for oxidative stress and interfere in normal growth through shoot and root length, and fresh weight and dry weight of plants. Therefore, plants activate their defence mechanism to protect themselves from damage, and failure of defence mechanism leads to lipid peroxidation, mitochondrial perturbation, DNA damage, and eventually apoptosis of cell [31]. So, antioxidants play an important role to combat stress.

Micropropagation is an interesting method that can be economically exploited for medicinal as well as ornamental purposes. Callus culture, a type of plant tissue culture producing undifferentiated tissues, constitutes an important tool in plant biotechnology. It can be used in numerous ways, for example, for indirect organogenesis, indirect somatic embryogenesis, and generation of somaclonal variations [32, 33]. T. foenum‐graecum L. is a plant with medicinal properties and callus culture is an alternative method to enhance the production of secondary metabolites like flavonoids, phenolics, and antioxidants in it.

The results of this research showed that best growth of callus was obtained in control, i.e. simple MS medium without supplementation of NPs as compared to explants grown under CuO NPs, and it was determined by fresh weight; 1.65 g (stem explant) and 0.10 g (leaf explant) after 30 days of growth. These results are contradictory to the study in which ZnO nanoparticles were used to observe the uptake of nanoparticles by Prosopisfarcta L. and results showed maximum growth at 100 mg/L as compared to lower concentrations of nanoparticles. However, results of the present study are in accordance with results obtained from callus cultures of banana that showed reduction in callus growth due to the presence of ZnO nanoparticles in the medium [34].

Although callus growth was decreased due to the presence of NPs in the medium, but flavonoid and phenolic contents were increased. Phytochemical screening of callus cultures showed more flavonoid and phenolic contents accumulation in explants of stem and leaf treated with capped CuO NP, i.e. CuO NP‐PVP as compared to other two forms of NPs and control. The DPPH free radical scavenging activity and antioxidant activity was evidently higher in explants treated with CuO NP‐PEG. These results indicate that capped nanoparticles have more phytotoxic effects on explants as compared to uncapped CuO NPs that lead to the formation of higher secondary metabolites in them.

5 Conclusion

Overall, experimental results demonstrated that the presence of CuO NPs affects the growth of T. foenum‐graecum L. seedlings at different concentrations. The seed germination experiments in the presence of NPs conclude that at some extent, germination process is not concentration‐dependent; however, capping of NPs with PVP and PEG reduces the toxicity. Furthermore, low concentration may act as fertiliser because increases in physiological characters were observed. The biochemical analysis of shoot and root extracts concludes that root biochemistry changes at higher extent as compared to shoot as TAC, TRP, TFC, and TPC were found many times elevated in roots extracts. The low toxicity of capped CuO NPs also derived from calli results. Callus is a delicate undifferentiated mass of cells and higher fresh and dry weight, in the presence of capped NPs, support the phenomenon of low toxicity. However, productions of phenolics and flavonoids provide better understanding towards application of such nanoparticles as abiotic elicitors for production of valuable components in bioreactors.