Introduction

Nanotechnology can improve the yield of various crops with the help of innovative tools, thereby revolutionizing the agricultural and food industry1,2. It can deliver food safety by improving crop production via accurate planting, resourceful use of water, insect and disease protection, providing diverse implements for molecular and cellular biology, and innovative materials for the identification of pathogens and environmental protection3,4. Nanoparticles cause many morphological and physiological changes within the plants, depending on their properties. Scale, chemical composition, surface cover, reactivity and, most importantly, their effective dose can decide nanoparticle’s efficiency5,6. Nanotechnology offers several sustainable solutions to many traditional farming practices such as nanofertilizers and nanopesticides. Application of NPs promoted plant growth parameters such as germination, morphology of root, increasing productivity, physiochemical and biochemical composition7. It has been reported that NPs based materials have gained astounding intentions in providing solutions for eliminating the effects of metals from atmosphere8. Exposure of nanoparticles to environment has raised concern on ecology, soil microbes and plants9. Therefore, insight analysis is needed for the filling of the knowledge gap of NPs interaction with plants on molecular basis in near future.

Brassica napus L. (belonging to Brassicaceae family of Rhoedales order) is an imperative crop mostly grown for oil purposes. Globally it ranks 2nd after soybean (due to increased oil content with improved quality) and supplies over 13.2% of edible oil in the world10. During the last 20 years, the annual production of canola in major oil producing countries has increased 2–4 folds11. It is one of the most significant oil seed crops which can be grown successfully in many areas of Pakistan12. During 2019-20 it was grown on an area of about 128,000 acres with the production of 81,000 tons of seed which yields 31,000 tons of oil10. Brassica napus L. is basically a form of rapeseed with particularly reduced characteristics in oil such as erucic acid (≤ 2%) and low glucosinolate in oil seed cake i.e. less than 30 µg13. It could be a good candidate for investigating the impact of metal-based NPs on plants because in phytoremediation it is used as metal accumulator species2,14,15.

Recent years have seen a large rise in the use of silver nanoparticles for various agricultural and industrial applications16. They have pronounced impact on overall growth of plants like germination, growth of root and shoot, root to shoot ratio, root biomass as well as on root elongation and senescence inhibition. Silver nanoparticles enhanced the growth profile (root and shoot length, leaf area) and biochemical properties (chlorophyll, carbohydrates and protein content, antioxidant enzymes) of Brassica juncea L17. If definite concentration of silver nanoparticles is applied to plants, their growth might be significantly enhanced as compared to un-treated plants, while their elevated or lesser concentration could vigorously affect their growth18.

Copper nanoparticles have been documented to improve the parameters of production, yield, and quality of many crops3. They have positive impact on dry weight of shoot, plant height and reproduction coefficient19. CuNPs influence several mechanisms such as physiological, morphological, molecular & cellular and processes20. Conversely, very few research work has been done on discovering the possible role of silver and copper nanoparticles in crop growth. Seeing the prominence of nanoparticles in agricultural sector most probably towards enhanced crop production present study was planned to discover the potential role of silver and copper nanoparticles in enhancing the overall growth, yield, and quality of Brassica napus L.

Materials and methods

Synthesis of nanoparticles and their characterization

Silver nanoparticles (Ag-NPs) were synthesized using silver nitrate (AgNO₃) obtained from Sigma-Aldrich, USA, and copper nanoparticles (Cu-NPs) were synthesized using copper sulfate pentahydrate (CuSO₄) purchased from Merck, Germany. The synthesis process involved using onion extract as a reducing agent. Specifically, 50 mL of 0.1 M AgNO was mixed with 5 mL of onion extract for the silver nanoparticles, and similarly, 50 mL of 0.1 M CuSO₄ was mixed with 5 mL of onion extract for the copper nanoparticles. The reaction mixtures were maintained at a pH (9), under constant stirring at 60 °C for 2 h to ensure the complete reduction of metal ions to nanoparticles. Reaction was completed in the formation of nanoparticles when there is no ions present in the reaction mixture. The nanoparticles were then collected by centrifugation at 10,000 rpm for 15 min, washed with deionized water multiple times to remove any unreacted precursors, and subsequently dried at 50 °C to obtain the nanoparticle in powder form.

For stabilization, Polyvinyl pyrrolidone (PVP) was used, as stabilizer for the preservation from photo-oxidation of nanoparticles and maintaining their size and shape. Freshly prepared nanoparticles were characterized through UV-visible spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and X-ray Diffraction (XRD) techniques to confirm their size and morphology.

Experimental procedure and plant material

To explore the effect of Ag and Cu NPs on brassica, seeds of Brassica (Brassica napus L. variety (CON-1) were used in the experiment followed by Completely Randomized Design (CRD) design with three replications. The experiment was conducted at research area of PMAS-Arid Agriculture University Rawalpindi, Pakistan (33° 39′ 06.7″ N 73° 04′ 49.6″ E). Pots were filled with (8 kg) fertile soil properly mixed farmyard manure for making a homogenous mixture. Seeds of brassica were sown in the pots at the depth of 3–5 cm. For maintaining the proper plant population of brassica (10–15 plants) per pot, thinning was done. After the establishment of plants of brassica treatments of nanoparticles Silver and Cu nanoparticles (0, 5, 10, 15, 20, 25, 30, 35) mg/L, at trifoliar stage and before initiation of the flowering of brassica plants were foliarl applied. Distilled water was used as control treatment. Data on growth attributes such as plant height (cm), Number of primary branches plant−1, Number of secondary branches plant−1 were collected. Similarly, yield parameters such as number of silique plant−1, Number of seeds siliqua−1, Siliqua length (cm), 1000-seed weight (g), Biological yield plant−1 (g), Seed yield plant−1 (g), Oil yield plant−1, Harvest index (%) were collected at the time of maturity.

2.3 Determination of oil and lipid Contents.

To checking the effect of Ag and Cu nanoparticles on quality attributes of brassica seed such as oil content %, Protein content %, Glucosinolate content (µmol g−1), Oleic acid (%), Linolenic acid (%) and Erucic acid (%) were determined. Oil contents of the seed were determined through Soxhelt extraction with n-hexane solvent. After the extraction of oil, fatty acid profile was analyzed through gas chromatography-mass spectrometry (GC-MS). Identification and quantification of fatty acid were determined in comparison with the retention time of known standards.

Statistical analysis

The data was subjected to statistical analysis and was carried out using the computer based statistical package MSTATC21. Means were compared by employing Least Significant Difference (LSD) Test at 5% significance level of probability.

Results

Characterization

UV-visible analysis

Absorption spectra of Ag NPs is presented in (Fig. 1) show broad absorption band at 430 nm approximately. The color change of Cu II indicated the formation of copper nanoparticles which was as a result of reduction to Cuo from Cu II as characterized by UV-Vis spectrum. The UV-visible results for copper nanoparticles (CuNPs) showed a maximum absorbance at 580 nm (Fig. 2.)

Fig. 1
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UV-visible spectrum of silver nanoparticles.

Fig. 2
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UV spectrum for copper nanoparticles.

FTIR analysis

The results for FTIR analysis of silver nanoparticles (AgNPs) are shown in Fig. 3. The FTIR spectra revealed various peak values observed at 3404.47 which corresponds to N-H stretch, peaks at 2920.32 and 2378.31 relates to single aldehyde and O–H bonds respectively. Whereas 2345.52 peak represent C≡C and peak at 1647.20 is assigned for C=C while peak at 1384.94 for C=O stretch. The peak near 902.41 cm−1 allocated to C=CH2. Moreover, the spectra also show absorption bands at 599.32 cm−1 and 547.90 cm−1, that were assigned to CH stretching vibration respectively. The FTIR measurements for Cu-nanoparticles are presented in Fig. 4, peak values at 3440.91, 2902.76, 2920.32, 2850 and 1647.26 cm−1 were observed.

Fig. 3
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FTIR spectrum of silver nanoparticles.

Fig. 4
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FTIR spectrum of copper nanoparticles.

The band at 3460 represents C=O stretching of amides while the peak at 1647.26 cm−1 was related to O-H stretching of phenolic compound. The rest of peak values obtained through FTIR spectrum of copper nanoparticles were at 1261.40, 1099.46, 1024.26 cm−1 which corresponds O-H stretching of phenols and alcohols covalently bonded with hydrogen.

XRD analysis

The XRD pattern of dried silver and copper nanoparticles is shown in Fig. 5. Three prominent peaks were observed at an angle 2 Theta of 28°, 35° and 48°, which corresponds to plane (220), (311) and (420) demonstrating ‘fcc’ phase of silver. Henceforth, the XRD analysis confirms crystalline structure of silver nanoparticles. Correspondingly, the XRD analysis for copper nanoparticles showed highest peaks at 2 angle theta at two points viz. 28° and 52° representing (800) and (550) plane respectively. The average size of silver and copper nanoparticles was determined by a classic equation proposed by Debye Scherrer.

\({\text{D}}={\text{K}}\lambda /\beta {\text{cos}}\theta\)where; D = characteristic crystalline size of nanoparticle; K = Scherer constant (value ranges between 0.9 and 1); β = Total width to a maximum of half the diffraction peak; θ = Bragg’s or diffraction angle.

Fig. 5
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XRD diffractogram of silver (Ag) and copper (Cu) nanoparticles.

SEM analysis

The Scanning Electron Microscopy was used to obtain surface morphology as well as the size of both nanoparticles (Figs. 6 and 7). Analysis confirmed that silver and copper nanoparticles synthesized through organic reduction were in nano range as fine nanomaterial. These analysis figs substantiate that the nanoparticle’s approximate spherical form, and most of the particles show some faceting. SEM analysis revealed that size of Ag NPs was < 10 nm (Fig. 6) while Cu nanoparticles were in uniform shape and their particle size within the range of 60–100 nm (Figs. 6 and 7).

Fig. 6
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SEM image of silver nanoparticles.

Fig. 7
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SEM image for copper nanoparticles.

Effect of combined application of Ag and CuNPs on Yield and associated attributes

The foliar application of Ag and CuNPs resulted in significant enhancement in yield and yield components of brassica napus L. Combined application of Ag and Cu nanoparticles @ 25 mg/L resulted in increased plant height, number of primary branches plant−1, number of secondary branches plant−1, number of silique plant−1, number of seeds siliqua−1, siliqua length, 1000-seed weight, biological yield plant−1, seed yield plant−1 as compared to untreated control plants. A positive response was shown by the application of Ag and Cu nanoparticles. 25 mg/L dosage of Ag and Cu nanoparticles resulted in improved yield in brassica napus L. Highest plant height (152.01 cm) as depicted in Table 1 was recorded in 25 mg/L combined concentration of Ag and Cu nanoparticles, while shortest plant height was observed for control (113.51 cm). The increase in the plant height might be due to effective absorption of nanoparticles directly from the leaves as they were foliarly applied. The number of silique per plant is the most responsive of all yield components in brassica napus L. is determined by the survival of branches, buds, flower and young pods. The highest number of silique (225.87) at 25 mg/L (Table 1) were due to an increased number of secondary branches per plant (1.70). It seems that increased photosynthates production increased photo-assimilate demand created by secondary branching and pod formation came at the expense of photo-assimilate translocation to seed. Seed yield per plant is a function of population density, number of siliqua plant−1, number of seeds per siliqua and seed weight. The highest seed yield (9.10 g) recorded with combined application of Ag and Cu nanoparticles was due to increase no of pods or siliqua per plant (225.87), improved no of seeds per siliqua (21.41) and enhanced seed weight (3.30 g).

Table 1 Effect of silver and copper nanoparticles on growth & yield attributes of Brassica napus L.
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Seed protein content, oil content and oil yield

The combined use of silver and copper nanoparticles resulted in increased protein content of brassica in a concentration dependent manner. The highest protein content (29.23%) was observed for 25 mg/L (Table 2) followed by 20, 15 and 10 ppm concentration of both Ag and Cu nanoparticles. The enhanced uptake of N and P in Ag and CuNPs-treated plants probably optimized the protein content in plants. Moreover, the Ag and Cu nanoparticle treated plants showed more oil compared to control or untreated plants where maximum oil content (12.82%) was shown for combined application Ag and Cu NPs @ 25 mg/L. The nanoparticles application also resulted in enhanced oil yield per plant. The improved oil content was a result of the highest seed yield (Table 1).

Table 2 Effect of silver and copper nanoparticles on oil & lipid contents of Brassica napus L.
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Fatty acid profile

As compared to other seed quality parameters, the applied concentrations of both silver and copper nanoparticles resulted in improved oleic acid, linolenic acid while suppressing of erucic acid content in brassica oil (Table 2). The application of 25 m213g/L dosage of silver and copper nanoparticles resulted in 14.09% and 12.59% increase in oleic acid and linolenic acid respectively. The erucic acid content of Brassica is an important quality parameter. 25 ppm concentration of both silver and copper nano particles when foliarly applied resulted in reduction of percent erucic acid by 22.03% when compared to control. The reduction in the erucic acid might be due to suppression of erucic acid linked gene expression. The genes controlling the erucic acid might be unable to show their expression due to suppression resulting from nanoparticles application.

Discussion

Characterization of nanoparticles

Nanomaterials have gained greater attention for their wider applications. For the confirmation of nanomaterials, several characterization techniques have been used for the determination of size and structural properties of nanoparticles. XRD, FTIR and TEM analysis were performed for the surface area, structural topography and surface composition respectively. From these results it was confirmed that green synthesized nanoparticles were in nanosize fine particles with regular symmetry. Finer NPs of Ag and Cu have positive and influencing effect on growth and quality of the brassica. Plant based extracts are utilized for the bioreduction of bulk material. Nanoparticles synthesis from biological sources proved as eco-friendly in comparison with other reduction methods such as physical and chemical22.

Results of UV-visible analyzed biosynthesized Ag NPs absorption peaks were in the range of 430 nm are in accordance with the results reported by Banerjee et al.23, Krithiga et al.24 and Pirtarighat et al.25. Similarly, biosynthesized copper NPs analyzed through UV-visible techniques, findings of the experiment revealed peaks ranges in 580 nm while results reported by Fatma et al.26 and Makvana et al.27 at 322 nm absorption peaks which were lower from the results of experiment. Biosynthesized Ag and copper NPs analyzed through FTIR spectrum revealed distinctive peaks and presence of capping agent with NPs. This observation aligns with the findings of Rani et al.28.

Similarly, XRD peaks for biosynthesized Ag and Cu NPs confirm fine nanoparticles with regular symmetry. In a related study, Fe NPs were synthesized through onion extract and same results were reported by Yasmeen et al.29. Copper nanoparticles are concerned, their formation, stability as well as the reaction progress were governed by UV-spectroscopy30. Organic reduction of Ag and Cu was carried out, and similar results were reported by Hafeez et al.31. Therefore, organic reduction of NPs resulted fine NPs with positive effects on plant growth and development.

The findings of the current study demonstrate the positive effects of silver (Ag-NPs) and copper (Cu-NPs) nanoparticles on the phenotypic traits and oil quality of Brassica napus L. The incorporation of these nanoparticles, especially at the concentration of 25 mg/L, increased several growth attributes of the plant such as height, number of primary and secondary branches along with the number of siliques per plant. These findings are in line with studies revealing the impact of nanoparticles on plant growth and development32. El-Saadony et al.33 showed that the use of Ag-NPs has increased the growth and yield of wheat plants and indicated that the nanoparticles could affect the plant hormones or nutrient uptake with the objective of boosting the entire plant Vigor.

A key conclusion of this study is that the use of Ag-NPs and Cu-NPs improves both the oil yield and quality of Brassica napus L. The 25- mg/L concentration of nanoparticles enhanced the oil yield per plant and positively contributed to reducing erucic acid levels, a legally mandated quality standard for canola oil. Erucic acid, which is present in high concentrations, can lead to health issues. Therefore, one important application of nanoparticles is their ability to reduce erucic acid content, making edible oils healthier through crop improvement programs. This aligns with the findings of Siddiqui et al.5 and Wang et al.34, who demonstrated that nanoparticle treatments can alter the fatty acid profile of soybeans, enhancing the quality of the oil.

The exact process through which Ag-NPs and Cu-NPs enhance concentration of oil and decrease erucic acid is unknown, though it has been hypothesized to be as a result of interference with plant biochemical processes. These particles can also get inside plant tissues and may be in direct contact with enzymes and or proteins involved in lipid production. This study now indicates that these nanoparticles may be capable of putting into practice some genetic modifications in fatty acid metabolism that will see an increased proportion of the desired fatty acids like oleic and linolenic acids and a decreased proportion of erucic acid. In line with this hypothesis, Chen et al.35 conducted a study that showed that metal nanoparticles enhanced an alteration of the lipid metabolic genes that improved the quality of oil produced in plants.

The decrease in erucic acid in this study is impressive because it rectifies one of the biggest drawbacks of canola oil. The fact that low-erucic acid canola could be grown by deploying nanoparticles means that there is an approach to breed and genetically modify crops as the two conventional methods of growing low-erucic acid canola. The application of nanoparticles thus, could be a more economic and time-saving approach along with the advantages of other favorable quality attributes like disease resistance and tolerance to stress. This is in line with potential works where research conducted by Zhang et al.36 who established that nanoparticles have the ability to trigger systemic resistance in plants and at the same time are capable of protecting plants against several pathogens besides improving their growth and yield.

Ag-NPs and Cu-NPs have a beneficial impact on the enhancing yield and biomass of Brassica napus through increasing the plant’s height and the number of siliques per plant, portend enhanced rate in photosynthesis and nutrient absorption stemming from the interactions between the nanoparticles and root and leave tissues. Different research works indicate that nanoparticles increase bioaccessibility of the nutrients in the soil by altering the dynamics of nutrient release and nutrient uptake by plants37,38,39,40. This enhanced nutrient uptake could be the reason for biomass and yield component increase and also the increase in oil percentage.

Nevertheless, the present study shows that Ag-NPs and Cu-NPs enhance the growth of Brassica napus L. It is necessary to note the possible risks associated with application of nanoparticles in plant cultivation. It is still unknown how the nanoparticles will affect the environment in the long run and the groundwork for determining the effect of nanoparticles on the accumulation of nanoparticles in the soil and water ecosystems that need to be carried out. From the review of literature like Perez-de-Luque et al.38, it has also been found that nanoparticles can be rather permanent in the environment that can affect the soil microbiology as well as crop productivity. Thus, the results of the current research may be optimal; however, they should be taken with a pinch of salt and followed by another study that will help determine that nanoparticles used in agriculture practices are safe for usage in the food chain.

Therefore, the use of silver and copper nanoparticles proves to be effective for the improvement of growth, yield and the oil characteristics of Brassica napus L. especially through the reduction of erucic acid and promotion of beneficial fatty acid. These outcomes are helpful in understanding the novelties of nanoparticles’ application as one of the genetic engineering methods for plants. However, the potential environmental impacts and long-term effects of nanoparticle use must be carefully considered, and further research is needed to fully understand the mechanisms underlying these benefits and to ensure their safe application in agricultural practices.

Conclusion

Silver and copper nanoparticles have proven to be effective in improving the growth, yield, and seed quality of Brassica napus L. variety CON-1. The application of 25 mg/L of these nanoparticles resulted in significant increases in seed yield and oil content while reduced the undesirable fatty acid contents. Future research should focus on exploring the mechanisms behind these improvements, incorporating advanced techniques like TEM and ICP-OES for a deeper understanding.