Synthesis and characterization of Lanthanum Oxide nanoparticles using Citrus aurantium and their effects on Citrus limon Germination and Callogenesis | Scientific Reports

Scientific Reports volume 14, Article number: 21737 (2024) Cite this article

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The plant extract-mediated method is eco-friendly, simple, safe, and low-cost, using biomolecules as a reducing agent to separate nanoparticles. Lanthanum (La) is a rare earth metal that positively affects plant growth and agriculture. Citrus limon is a leading citrus fruit with many varieties. Conventional vegetative propagation methods depend on season, availability of plant material and are time-consuming. It is the main reason for limiting the acceptance of new varieties. So, In-vitro propagation of the lemon method is practiced overcoming all these problems. Lanthanum oxide nanoparticles (La2O3-NPs) were synthesized using plant extract of C. aurantium. Ultraviolet (UV)-Visible Spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FTIR) spectroscopy, and Thermal Gravimetric Analysis (TGA) were used to characterize the synthesized La2O3-NPs. Fabricated La2O3-NPs were oval and spherical, with an average size of 51.1 nm. UV-visible absorption spectra of La2O3-NPs were shown at a sharp single peak at 342 nm and FTIR showed stretching frequency at 455 cm−1-516 cm−1. In the TGA outcome, mass loss was 9.1%. In vitro experiments demonstrated that La2O3-NPs significantly enhanced the germination and growth of C. limon seeds, achieving an 83% germination rate at 5 mg/L concentration, with uncoated seeds showing root initiation at 10 days and shoot formation at 15 days. Furthermore, La2O3-NPs effectively stimulated callus induction and maturation, with optimal responses observed in media containing MS and 2 mg/L 2,4-D, resulting in a maximum callus frequency of 100% from leaves and 87.5% from shoots at 5 mg/L concentration. These findings underscore the potential of La2O3-NPs to improve seed germination rates, seedling vigor, and callogenesis efficiency, suggesting their promising integration into agricultural practices for sustainable crop production, especially in suboptimal growing conditions. Future research is recommended to explore the mechanisms and broader applications of La2O3-NPs across various plant species and environments.

Nanotechnology is a rapidly rising field for manufacturing materials at the nanoscale level with dimensions of 1–100 nm. Nanotechnology has many applications in the fields of biology and pharmacology1. Different metallic nanoparticles were synthesized from natural resources such as gold, silver, platinum, zinc, copper, magnetite, and nickel2. Different conventional physical and chemical processes are used to synthesize metal nanoparticles. It includes ion sputtering, solvothermal synthesis, reduction, and sol-gel methods3. These widely used methods allow one to obtain particles with the desired characteristics4. Simplicity, rapid rate of synthesis with diverse morphologies, elimination of elaborate maintenance of cell cultures, and eco-friendliness make plants an attractive platform for the synthesis of nanoparticles5,6. Plant-mediated synthesis is most exciting because of biochemicals and specific yield. Plants have genetic variation and possess many biomolecules like coenzymes, vitamin-based intermediates, and so many others. These can reduce metal ions to nanoparticles in a single step, at room temperature and pressure, without any hard and fast technical rules7.

The mechanism for the synthesis of nanoparticles in principle for plants is that metal salts comprising of the metal ion are first reduced to atoms using a reducing agent. This is done by mixing a sample of plant extract with a metal salt solution in a reaction mixture indicated by color change. Metal ions are converted to zero-valent states from their mono or divalent oxidation states. The obtained atoms then nucleate in small clusters that grow into particles. Smaller neighboring particles combine to form larger nanoparticles in different shapes cubes, spheres, triangles, hexagons, pentagons, rods, and wires. The plant extracts determine the most stabilized structures7.

Nanoparticles have gained significant importance in the field of Biomedicine. The nanoparticles extracted from plants are used in many applications for the benefit of humans. These beneficial activities involve anti-bactericidal activities, anti-fungicidal activities, anti-plasmodial activity of metallic nanoparticles, anti-inflammatory action, anticancer studies, antiviral effects, and antidiabetic management. Nanomaterials can also be used as a remedy for various diseases such as malaria, cancer, HIV, hepatitis, and other acute diseases. Nanotechnology is applicable in agriculture and plant biotechnology due to some properties like small size, morphology, tunable size, very reactive nature, and high surface area. Nanoparticles facilitate the delivery of herbicides. It also serves as a nano-pesticide fertilizer in plants and agriculture. Nanoparticles help in gene delivery of target-specific cellular organelles of plants and in releasing the content. Studies have shown that nanoparticles affect plant growth and development8.

The growth and multiplication of totipotent cells, tissues, and organs of plants on defined solid or liquid media containing essential nutrients under a well-defined environment is termed in-vitro multiplication of plants or referred to as tissue culture, axenic, or sterile culture9. Its application is many commercial productions of identical individual plants. Rare or endangered plant species conservation is done with the help of tissue culture technique10,11. It facilitates in regeneration of a new hybrid by crossing off less related species by protoplast fusion. It is used to examine the effect of change in physiological, biochemical, and reproductive mechanisms in plants, e.g. for stress-tolerant plants examination12,13. Tissue and gene transformation is probable to do in micropropagation. It is also applicable in the growth of identical sterile hybrid species.

Lanthanum (La) occurs in the earth’s crust and igneous rocks. It possesses unique physical and chemical properties. It is mostly present in the form of oxide in ores such as cerite, parasite, allanite, orthite, and monazite sand14. La in a soluble form does have positive effects on plant growth, at low concentrations and increases dry weight with an increase in concentrations15. Citrus limon is the leading acid citrus fruit due to its appealing color, aroma, and taste. The tree reaches almost 10 to 20 ft in height with sharp thorns and alternative dark green leaves. The slightly scented flowers have 4 or 5 petals which are white on the upper surface, purplish beneath, and have 20–40 stamens with yellow anthers. The fruit is oval having aromatic light-yellow peel with dotted oil glands and the pulp is pale yellow with acidic juice, mostly whitish seeds are present and only a few are without seeds.

An eco-friendly technique can be used to synthesize nanoparticles of Lanthanum oxide (La2O3), using leaf extract of ‘Moringa Oleifera, Eucalyptus globulus, Ficus religiosa and Plectranthus amboinicus16,17,18. There are different applications of these eco-friendly synthesized La2O3-NPs in plant biotechnology, suppressing plant disease and increasing the shelf life of apples19, promoting S. nigrum growth20. It shows the potential to start seed germination, increases root and shoots, supports the photosynthetic process, and helps plants to survive environmental stresses.

This study presents a novel approach to synthesizing lanthanum oxide nanoparticles (La₂O₃-NPs) using Citrus aurantium leaf extract, marking a shift from conventional chemical methods to a more sustainable and eco-friendly green synthesis. The objectives are to develop and characterize La₂O₃-NPs via this green method, evaluate their effects on the germination and growth of rough lemon (Citrus jambhiri) seeds, and assess their impact on callus formation in tissue cultures. Additionally, the study aims to highlight the advantages of green-synthesized nanoparticles over traditional methods, demonstrating their potential for sustainable agricultural and biotechnological applications.

Fresh leaves of C. aurantium were collected from the pesticide and pollution-free area, a garden in Sector G-9/1, Islamabad (Pakistan). The collected plant specimen was identified using Flora of Pakistan. The voucher numbers were allotted to specimens. To obtain the taxonomic validation, the botanical names of collected plant species were confirmed with the aid of the International Plant Name Index (IPNI) (www.ipni.org). Collected plant samples were meticulously processed, including shade-drying, pressing, cataloging, and identification by Dr. Tauseef Anwar. Herbarium entries were carefully labeled and archived for future reference. Comprehensive records, from botanical names to voucher specimen numbers, were diligently maintained, encompassing growth characteristics and medicinal properties, and kept in the Botanical Garden for the Public via deposition number IIU-IBD-BOT-569. The collection of plant material abides by the relevant international, national, and institutional guidelines and legislation.

For extract preparation 50 g fine-grinded leaf powder was added in 400 mL of distilled water (H2O), left undisturbed for 3.5 h at room temperature, and then placed in a shaking incubator for 2 h at 50 °C at 60 rpm. The obtained extract was filtered with Whatman No. 1 filter paper and filtrate was stored at room temperature for further usage. 4.3 g of lanthanum nitrate [La2(NO3)3.6H2O] having a molar weight of 433.01 was added to 100 ml of plant extract. For 2 h, the solution was stirred with a magnetic stirrer on a magnetic hotplate at 50 °C, 1,500 rpm (Fig. 1). The solution was maintained at room temperature after removing from the magnetic hotplate machine.

Centrifugation of solution was performed at 10,000 rpm (GR BioTek, Orpington, England) for about 10 min. The pallet was collected with the help of double distilled water in a petri dish and the supernatant was discarded. Several washing rounds were done with the help of deionized water to avoid contamination. After washing, it is dried in a hot air oven for 6 h at a temperature of 60 °C. Dried nanoparticles in the petri dish were scratched by using a spatula and ground in mortar and pestle. La2O3-NPs were annealed at 700 °C in a tube furnace for a time of 2.5 h. Obtained La2O3-NPs were stored in an airtight jar at room temperature.

Schematic diagram of plant extract mediated synthesis of La2O3-NPs.

UV-visible spectroscopy of La2O3-NPs was performed in the range of 290–400 nm with a Shimadzu spectrophotometer (model UV-1800, Kyoto, Japan) functioning at a resolution of 1 nm. Three milligrams of La2O3-NPs dissolved in 10mL of deionized H2O. Sonication of the sample was done for 20 min. The size and shape of green fabricated La2O3-NPs were studied using Mira TESCAN SEM operating at 10 kV. For internal morphology, internal morphology of La2O3-NPs TEM analysis was performed by TEM (model no JEOL-1010) operating at 80 kV. For confirmation of La2O3-NPs and to find unknown bio-active compounds in plant extract and utilization of capping agents, FTIR was performed. FTIR spectroscopy is done by following the KBr pellet method (model SHIMADZU FTIR, Kyoto, Japan) in the wavenumber range 400–4,000 cm−1. Thermogravimetric Analyzer (TGA) gives information about the measurement of weight change as a function of time or temperature, to find thermal stability or composition of nanoparticles. An external gas switch (TAGS box) permits sample purge gases to be altered during the experiment. It is used in oxidation experiments in which the sample changes from an inert to an oxidizing environment. A 20-position auto-sampler permits the measurement of many samples and is specifically used for running samples overnight. To observe the thermal properties and capping action of bioactive compounds for tailoring La2O3-NPs, TGA (model Diamond TGA; PerkinElmer, Waltham, USA) under a nitrogen environment from 25 °C to 800 °C at 10 °C/minute was performed.

The stock solution of green synthesized La2O3-NPs was prepared. Nanoparticles were diluted in double distilled water. The dilution ratio is 4:1 as four parts nanoparticles in one part of double distilled water (ddH2O). Nanoparticles were sonicated every time before use, for about 20–25 min in an electric sonicator. MS (MURASHIGE and SKOOG 1962) medium was prepared by adding powdered MS synthetic salt at the concentration of 0.4% (w/v) in double-distilled water(ddH2O). Media was nourished with 3% sucrose, solidified with 0.8% gelrite, and heated for thorough mixing. The pH of the solution was maintained between 5.6 and 5.8 with the help of 0.1 M NaOH or 0.1 M HCL. Three different concentrations of La2O3-NPs viz. 1 mg/L, 3 mg/L, and 5 mg/L were added in the MS media separately to check its effect. Prepared media, with all apparatus, instruments, and glassware required for the experiment, was disinfected in an autoclave machine at 121 °C, 15 psi for 45 min, and then left in a cabinet laminar hood for 15 min in UV light. The fresh seeds of lemon variety called rough lemon (C. jambhiri) used in this study were obtained from the plant collection of the National Agricultural Research Centre (NARC), Islamabad (Pakistan). The seed coat was removed to break the dormancy of seeds so they could germinate under the provided favorable conditions (Fig. 2). After peeling, seeds were sterilized using 70% ethanol for a few seconds and 40% Clorox for 1 min. Uncoated seeds were rinsed 3 times with distilled and placed for 5 min in a cabinet laminar hood then dried on filter paper.

Dormancy breakage of rough lemon seeds (a) Coated seeds (b) Uncoated seeds.

The seeds were then inoculated separately in 25 × 150 mm culture tubes holding 25 ml of MS (MURASHIGE and SKOOG 1962) medium in a laminar flow hood. The flame was used to remove moisture and contamination of test tubes. Cotton plugs were used to cover the test tubes. For germination, the cultures were incubated for a dark period of 15 days in the growth room and 3 weeks in the chamber with 16 h of photoperiod at a constant 25 °C and 60% relative humidity. Based on daily visual observation, different parameters like first rooting, shooting, germination of fresh seeds versus stored seeds, and germination of coated versus uncoated seeds were recorded. Germination was observed for 30 days for seedlings’ growth. Four treatment groups were selected to check the effect of La2O3-NPs on the germination of the C. limon in MS media (Table 1).

Data was observed and collected after every 10 days for experimental analysis. The percentage of germination, referred to as germinability G was calculated by using the formula:

Germinability G = (total seeds germinated/ total number of seeds sown) *100.

Trends of germinability over time using different concentrations of nanoparticles were observed for one month. Treatment groups mentioned in Table 1 were used for analysis. Different concentrations of La2O3-NPs on mean germination were checked by using the same treatment groups. After 30 days mean germination of all treatment groups was calculated.

Stock solutions of powdered form growth hormones were prepared. Two types of hormones were used auxins (2,4-D and IAA) and cytokinin (BAP). The ratio of stock solutions was 1:1 as 1 mg of growth regulator per 1 ml of ddH2O. The prepared stock solutions were stored at 4oC and used by sterilized micropipette when required within their shelf lifetime. Media was prepared by the same procedure as mentioned earlier for germination. Different concentrations of hormones and La2O3 were added to the media before adding a gelling agent. Sterilization of the apparatus was done. The 4-5-week-old in-vitro grown seedling was used as a source of different explants. The seedling was washed under running water and sanitized with 40% Clorox and 70% ethanol for a few minutes followed by 3 times rinsing with ddH2O then dried. Pieces from seedling’s roots stems, and leaves were excised to use as explants for further research. The size of fragments ranges from 0.5 to 1 cm. Pieces of leaf were cut perpendicularly to the midrib. Cutting was done in a slanting manner for more exposure of cells. Explant fragments were inoculated in 25 × 150 mm culture tubes and cultures were maintained. Callus induction was initiated in 25 × 150 mm test tubes containing 25 ml of prepared MS medium with callus-inducing growth hormone. MS medium was supplemented with different concentrations of 2,4-D callus growth-regulating hormone and other hormones in combination to find the best callus-inducing hormone and suitable concentration (Table 2).

To check the effect of La2O3 on the in-vitro multiplication of callus, its different concentrations were used in MS media with a callus-inducing hormone (Table 3). For interpreting results data was recorded after every 10 days for one month.

To check the effect of different explant responses against different mediums for callus induction of rough lemon, 4 treatment groups were selected. Roots stems and leaves were selected as explants to check their response for callus induction in different mediums. Callus frequency was calculated after 30 days using the formula:

Callus frequency = (callus induced/ total inoculated cultures) *100.

To check different medium responses for callus induction four treatment groups as in Table 3 were utilized. Data was recorded to measure mean values after 10 days for a month. MS media was supplemented with the best callus-inducing hormone in all treatment groups. Four treatment groups were selected. Data was analyzed every 10 days, and the mean callus induction response of different concentrations of La2O3-NPs on rough lemon was calculated after 30 days of the callus’s maturity. Using the same treatment groups, the effect of La2O3-NPs on callus formation was checked in the experiment and different explant responses were analyzed after 30 days.

Collected fresh leaves of C. aurantium were dried for a week and converted into fine powder form. Filtration of a solution made by mixing ground leaves in double distilled water left undisturbed for 3.5 h and 2 h in a shaking incubator, resulted in a blackish-brown extract. The solution stirred on a magnetic hotplate for 2 h showed a slight color change from blackish brown to light brown which indicates nanoparticle fabrication. Particles settled at the bottom of the flask were visible. Centrifugation of solution resulted in a pallet which was collected with the help of double distilled water in a petri dish and the supernatant was discarded. After several washing rounds, it was dried at 60 0C, and ground into fine powder. La2O3-NPs which were in fine powdered form were annealed at 700 °C for 2.5 h and showed transformation in color. Mud brown colored nanoparticles were changed into whitish La2O3-NPs.

TEM micrography showed that the green synthesized La2O3-NPs have oval spherical morphology. Symmetry is homogenous and the average particle size is 51.41 nm (Fig. 3A and B).

TGA of biosynthesized La2O3-NPs was performed to study the capping action of biomolecules and the thermal behavior of the synthesized La2O3-NPs. At 600 °C weight loss is due to oxygen molecules decomposition. According to TGA results total weight loss was 9.1% of the La2O3-NPs (Fig. 3C).

The graph explains the UV-visible absorption spectra of La2O3-NPs, which show a single peak at 342 nm (Fig. 3D).

FTIR results of La2O3-NPs illustrate significant absorption peaks in the wave number range 500–6,000 cm−1 as in graph 3. O–H and C–H stretch ranges between 2,500 and about 4,000 cm−1. The plant extract showing N–O symmetric stretch is at 1,355 cm−1. The onset of a broad intense band that can be observed as one tends toward higher vibrations (2,800 cm−1) can be attributed to O–H stretching range. The spectra because of La2O3-NPs stretching frequency are at 455 cm−1-516 cm−1. This indicates the formation of La2O3-NPs (Fig. 3E).

According to SEM results green synthesized La2O3-NPs show homogeneity and symmetry in morphology. The shape of nanoparticles is oval and spherical shape with a mean size of 51.41 nm (Fig. 4).

Characterization of La2O3-NPs TEM micrography of La2O3-NPs (A) Low-resolution image (B) High-resolution image (C) TGA analysis (D) UV-visible spectroscopy (E) FTIR spectroscopy.

Scanning Electron Microscopy analysis images of La2O3-NPs.

A 2 mm radicle appeared after 10 days when data was collected. Results showed that the germination capacity of fresh seeds from fruits is higher (as best within 7 days) and even after a month not even a single seed showed germination. Germination of seeds with coats is slower than uncoated seeds. Rooting from the uncoated sown seeds started after 10 days in all treatment groups and root length showed a slight increase with La2O3-NPs concentration increase. Seeds sown in media containing 0.03 mg/L La nanoparticles showed the earliest rooting. Shoots start appearing after 15 days. Young seedling with leaves starts appearing after 17–20 days from uncoated seeds and after 30–40 days from coated seeds. Data for germination was collected for a month. Germinability increases with increases in the concentration of La2O3-NPs (Figs. 5, 6, 7, 8 and 9).

Effect of different concentrations of La2O3-NPs on germinability of lemon plant seeds.

Rooting of rough lemon seeds starts in germination media after 10 days (A) control treatment group (B) 1 mg/L (La2O3) treatment group (C) 3 mg/L (La2O3) treatment group (D) 5 mg/L (La2O3) treatment group.

(A)Shooting initiation after 15 days (B) Young seedlings of rough lemon with leaves without seedcoat after 17 days (C) with seed coat after 30 days.

Seedlings of rough lemon growth after 30–40 days (A) Control group (B) 1 mg/L (La2O3) (C) 3 mg/L (La2O3) (D) 5 mg/L (La2O3).

Grown seedlings of rough lemon plant from in-vitro germination.

Invitro germinated seedlings were used as explants for callus induction and data was recorded after every 10 days for about a month. Callus formation started after 6–7 days in all treatment groups which is slightly green (Fig. 10). After a month, callus became mature and turned to a yellow color (Fig. 11).

Callus initiation in treatment groups with different hormones and with different concentrations for the best callus-inducing hormone (A) Callus initiation from the shoot in MS + 1.5 mg/L 2,4-D treatment group (B) Callus initiation from the shoot in MS + 2 mg/L 2,4-D treatment group (C) Callus initiation from leaves in MS + 4 mg/L 2,4-D treatment group (D) Callus initiation from shoot in MS + 2 mg/L IAA, 2 mg/L BAP, 1 mg/L NAA treatment group.

Callus maturation after 30 days in treatment groups with different hormones and with different concentrations for best callus-inducing hormone (A) the best callus from a shoot in MS + 1.5 mg/L 2,4-D treatment group (B) the best callus from a shoot in the MS + 2 mg/L 2,4-D treatment group (C) the best callus from leaves in the MS + 4 mg/L 2,4-D treatment group (D) best callus from shoot in MS + 2 mg/L IAA, 2 mg/L BAP, 1 mg/L NAA treatment group.

Different hormones with different concentrations were tested for callus initiation and maturation. According to which best response was shown in media MS + 2 mg/L 2,4-D as shown in Figs. 12 and 13 so it was selected for further research. Different explant responses against different mediums were checked and media with MS + 2 mg/L 2,4-D from shoot showed maximum response as 75% and leaves 50%.

Different explant responses against different medium.

Different medium response for callus induction.

The mean callus induction response of different concentrations of La2O3-NPsnanoparticles on rough lemon was calculated. According to the results, the concentration of nanoparticles increased in the number of calluses also increased in treatment groups (Figs. 14 and 15).

Mean callus induction response of different concentrations of La2O3-NPs.

Effect of different concentrations of La2O3-NPs on callus induction and different explant response.

The effect of La2O3-NPs on callus formation was checked in the experiment and different explant responses were analyzed as in the table. Overall maximum callus frequency was found in the 5 mg/L La2O3-NPssupplemented group as 100% from leaves. The 3 mg/L La2O3-NPssupplemented group had a high callus number of 87.5% from a shoot. (Figures 16 and 17).

Callus initiation in treatment groups with different concentrations of La2O3-NPs (A) Callus initiation from a shoot in the control treatment group (B) Callus initiation from the shoot in MS+1 mg/L La2O3NP, 2mg/L 2,4-D treatment group (C) Callus initiation from shoot in MS+3 mg/L La2O3 NP2mg/L 2,4-D treatment group (D) Callus initiation from leaves in MS+5 mg/L La2O3 NP, 2mg/L 2,4-D treatment group.

Callus maturation in treatment groups with different concentrations of La2O3-NPs (A) Callus maturation from a shoot in the control treatment group (B) Callus maturation from the shoot in MS + 1 mg/L La2O3NP, 2 mg/L 2,4-D treatment group (C) Callus maturation from shoot in MS + 3 mg/L La2O3 NP2mg/L 2,4-D treatment group (D) Callus maturation from leaves in MS + 5 mg/L La2O3 NP, 2 mg/L 2,4-D treatment group.

An emerging field of nanotechnology deals with materials at the nanoscale level having one of the dimensions in the range of 1–100 nm. There are two categories of nanoparticles, organic which is composed of carbon nanoparticles and inorganic composed of metal nanoparticles, magnetic nanoparticles, and semi-conductor nanoparticles. Several synthesized metallic nanoparticles are gold, silver, platinum, zinc, copper, magnetite, and nickel. Approaches for the synthesis of nanoparticles are top-down and bottom-up methods. Different conventional physical and chemical processes are used to synthesize metal nanoparticles. It includes ion sputtering, solvothermal synthesis, reduction, and sol-gel methods. These widely used methods allow one to obtain particles with the desired characteristics. Some difficulties are expense, labor, and potential hazards to the environment and living organisms so21 an alternative cost-effective and safe environment-friendly green synthesis method of nanoparticle was required22.

The alternative method is the green synthesis method, which is simple, rapid, elimination, eco-friendliness, low cost of cultivation, short production time, safe, and the ability to up production volumes making plants an attractive platform for the synthesis of nanoparticles. Some biological systems, including plants and algae23 diatoms bacteria24, yeast25 fungi, and human cells26 are used for nanoparticle synthesis. Plants have genetic variation, and they possess many biomolecules like coenzymes, vitamin-based intermediates, and so many others which can reduce metal ions to nanoparticles in a single step, at room temperature and pressure, without any hard and fast technical rules.

Lanthanum, a rare earth metal, occurs in the earth’s crust and igneous rocks. La in a soluble form does have positive effects on plant growth, at low concentrations and increased dry weight at increased concentrations. La2O3-NPs were synthesized in this research27,28. The formation of La2O3-NPs in our study is mediated by the hydrolysis of La3+ ions. When lanthanum nitrate is introduced into the plant extract solution, La3+ ions undergo hydrolysis, leading to the formation of La (OH)3. Upon further heating, La (OH)3 is converted to La2O3 through a dehydration process. We have conducted additional experiments and included further evidence in the revised manuscript to support this mechanism. FTIR confirm the presence of La2O3-NPs. The FTIR spectra indicate the absence of hydroxyl groups, ruling out the presence of La (OH)3. La₂O₃-NPs can be absorbed by C. limon primarily through the roots, where they enter root cells via endocytosis or pore spaces and are then transported to the aerial parts through the xylem. Once in the leaves and stems, these nanoparticles distribute throughout various tissues, potentially interacting with cellular components and influencing physiological processes such as nutrient uptake and oxidative stress27.

The synthesis of La2O3-NPs using C. aurantium leaf extract was successfully achieved, as evidenced by various analytical techniques. Characterization technique TEM micrograph displayed oval and spherical shapes of the green synthesized La2O3-NPs which were homogenous with an average particle size is 51.41 nm. This morphology is consistent with the expectations for NPs synthesized through green methods, as the biomolecules in plant extracts often act as reducing and capping agents, leading to uniform particle sizes and shapes. The SEM results corroborated the TEM findings, indicating that the synthesis method yielded uniformly shaped nanoparticles28.

TGA analysis technique was used to find out the capping molecules and the thermal behavior of the synthesized La2O3-NPs. Results depict the total weight loss as 9.1% of the La2O3 NPs. This weight loss indicates the thermal stability of the La2O3-NPs, an essential characteristic for their application in various fields. The UV-visible spectroscopy results, showing an absorption peak at 342 nm, confirmed the presence of La2O3-NPs, as this peak is characteristic of lanthanum oxide nanoparticles. UV-visible absorption spectra of La2O3-NPs were shown at sharp a single peak at 342 nm29,30,31. According to FTIR results, absorption peaks ranged from 500 to 6,000 cm−1. Between 2,500 and about 4,000 cm−1, the stretch was because of O–H and C–H bond vibration. The plant extract showing N–O symmetric stretch peaks at 1,355 cm−1 is due to N–O vibrations and further stretch up to 2,800 cm−1 was because of the O–H bond. The spectra because of La2O3-NPs stretching frequency are at 455–516 cm−1. This indicates the formation of La2O3-NPs. SEM results described that green synthesized La2O3-NPs were homogenous, oval, and spherical, with a mean size of 51.41 nm. These comprehensive characterizations collectively confirm the successful green synthesis of La2O3-NPs using C. aurantium leaf extract, highlighting the method’s efficacy and potential for scalability.

Tissue culture, a technique for growing and multiplication of totipotent cells, tissues, and organs of plants on defined solid or liquid media containing essential nutrients under a well-defined environment, was applied32. La2O3-NPs effect on in-vitro multiplication of the C. limon plant was checked. Two levels of in-vitro multiplication were performed to find the effect of La2O3-NPs. In-vitro, the germination of seed of rough lemon was analyzed and seedlings were grown in the lab and a controlled environment. The appearance of a 2 mm radicle is referred to as physiological germination. Germination results of rough lemon seeds showed that seeds from fresh fruit possess more germination capacity and the highest capacity of seeds to germinate is within 7 days. Seeds were not viable after a month. Germination of seeds with coats is slower than uncoated seeds. Rooting from the uncoated sown seeds was initiated after 10 days in all treatment groups, in the control treatment group,1 mg/L La2O3 NP treatment group, 3 mg/L La2O3 NP treatment group, and 5 mg/L La2O3 NP treatment group33. There was a minor root length increase with the increase in nanoparticle concentration. Seeds sown in media containing 3 mg/L La nanoparticles showed the earliest rooting. After 15 days, shoots started appearing and young seedlings with leaves formed after 17–20 days from uncoated seeds and after 30–40 days from uncoated seeds34.

The percentage of germination referred to as germinability increased with time. A rise in the concentration of La2O3 NP causes a rise in germinability. Seeds sown in 5 mg/L La2O3-NPs supplemented media showed maximum germinability as 83%. Mean germination of the Lemon plant increases as we increase the concentration of La2O3-NPs in MS media according to the mean calculated after 30 days. In short, La2O3-NPs have a positive effect on seed germination of Citrus jambhiri as reported in another plant for lanthanum metal13. The dose-response relationship observed, where higher concentrations of La2O3-NPs correlated with increased germinability, supports the hypothesis that La2O3-NPs may provide essential nutrients or enhance the biochemical processes involved in germination. This positive effect extended to the development of shoots and young seedlings, with uncoated seeds showing advanced growth stages more quickly than coated seeds. These findings suggest that Ff can be used to improve seed germination rates and seedling vigor, which could have significant implications for agricultural practices, particularly in regions with suboptimal growing conditions.

The La2O3-NPs demonstrated a notable impact on callus induction and maturation. The rapid initiation of callus formation within 6–7 days across all treatment groups indicates that La2O3-NPs can effectively stimulate cell division and callus formation. The maturation of the callus to a yellow color after a month signifies the progression to a more differentiated state, which is essential for further tissue culture applications. The media composition, specifically MS supplemented with 2 mg/L 2,4-D, was critical for optimal callus initiation and maturation, achieving the highest response rates from shoots (75%) and leaves (50%). The dose-dependent increase in callus number with higher concentrations of La2O3-NPs suggests that these nanoparticles can enhance the efficiency of tissue culture processes by providing additional stimuli for cell proliferation and differentiation. The maximum callus frequency of 100% in the 5 mg/L La2O3-NPs group and 87.5% in the 3 mg/L group underscores the potential of La2O3-NPs to significantly boost callogenesis11.

The study draws from existing literature to support the advantages of our green-synthesized nanoparticles. Previous research has demonstrated that plant-mediated synthesis of nanoparticles offers distinct benefits over conventional chemical synthesis, including reduced toxicity and enhanced biocompatibility1,2,4,35,36. Our results indicate that La2O3-NPs synthesized through this green method exhibit improved seed germination and callus formation compared to benchmarks in existing studies, emphasizing the potential of plant-based synthesis methods for enhanced agricultural and horticultural applications. These findings highlight the potential of La2O3-NPs to improve plant tissue culture protocols, particularly for species that are recalcitrant to traditional methods. The enhanced germination and callogenesis observed with La2O3-NPs application could lead to more efficient propagation techniques, thereby supporting sustainable agriculture and horticulture practices. Future research should focus on elucidating the mechanisms by which La2O3-NPs exert their beneficial effects and exploring their application across a broader range of plant species and environmental conditions. The integration of La2O3-NPs into agricultural practices holds promise for advancing crop production and resilience, particularly in challenging growing environments where traditional methods may fall short.

The study successfully synthesized lanthanum oxide nanoparticles (La2O3-NPs) using C. aurantium leaf extract, demonstrating their efficacy through comprehensive characterizations including TEM, SEM, TGA, UV-visible spectroscopy, and FTIR. The La2O3-NPs exhibited homogenous oval and spherical shapes with an average particle size of 51.41 nm. In vitro experiments revealed that La2O3-NPs significantly enhanced the germination and growth of C. limon seeds, with the highest germination rate of 83% observed at 5 mg/L. Additionally, La2O3-NPs effectively stimulated callus induction and maturation, with optimal responses in media containing MS and 2 mg/L 2,4-D, achieving a maximum callus frequency of 100% from leaves. These findings highlight the potential of La2O3-NPs to improve seed germination rates, seedling vigor, and callogenesis efficiency, suggesting their integration into agricultural practices could support sustainable crop production, particularly in challenging environments. Future research should explore the underlying mechanisms and broader applications of La2O3-NPs across various plant species and conditions.

The author confirms that all data generated or analyzed during this study are included in this published article.

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This research was funded by the Researchers Supporting Project No. (RSP2024R390), King Saud University, Riyadh, Saudi Arabia.

Applied Biotechnology and Genetic Engineering Lab, Department of Biological Sciences, International Islamic University, Islamabad, 44000, Pakistan

Zahra Hanif, Nyla Jabeen, Sadaf Anwaar & Ayesha Aftab

Department of Biological Sciences, Quaid e Azam University, Islamabad, 44000, Pakistan

Syed Zaheer Hussain

Department of Botany, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan

Tauseef Anwar

Department of Botany, University of Chakwal, Chakwal, 48800, Pakistan

Huma Qureshi

Department of Botany, Government College Women University, Sialkot, 51310, Pakistan

Mehmooda Munazir

Department of Life Sciences, Yeungnam University, Gyeongsan, 38541, Republic of Korea

Wajid Zaman

Plant Production Department, College of Food and Agriculture Sciences, King Saud University, Riyadh, 11451, Saudi Arabia

Walid Soufan

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ZH: Experimentation and data Curation NJ, SA: Methodology, supervision, Writing and drafting, and research design; AA, SZH: Validation and Software, writing, Investigation, drafting, statistical analysis, and validation; TA, HQ: writing, Software, Resource, research design, validation, data collection, drafting, statistical analysis; MM, WZ, WS: writing, statistical analysis, Resource, software, validation. All authors have read and approved the final manuscript and declare that they have no competitive interest.

Correspondence to Nyla Jabeen, Tauseef Anwar, Huma Qureshi or Wajid Zaman.

We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

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Table S1, Table S2, Table S3, Table S4, Table S5, Table S6.

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Hanif, Z., Jabeen, N., Anwaar, S. et al. Synthesis and characterization of Lanthanum Oxide nanoparticles using Citrus aurantium and their effects on Citrus limon Germination and Callogenesis. Sci Rep 14, 21737 (2024). https://doi.org/10.1038/s41598-024-73016-4

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Received: 10 June 2024

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Published: 17 September 2024

DOI: https://doi.org/10.1038/s41598-024-73016-4

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Synthesis and characterization of Lanthanum Oxide nanoparticles using Citrus aurantium and their effects on Citrus limon Germination and Callogenesis | Scientific Reports