Magnetofection approach for the transformation of okra using green iron nanoparticles

Scientific Reports volume 12, Article number: 16568 (2022) Cite this article

1857 Accesses

4 Altmetric

Metrics details

Climate change, pesticide resistance, and the need for developing new plant varieties have galvanized biotechnologists to find new solutions in order to produce transgenic plants. Over the last decade scientists are working on green metallic nanoparticles to develop DNA delivery systems for plants. In the current study, green Iron nanoparticles were synthesized using leaf extract of Camellia sinensis (green tea) and Iron Chloride (FeCl3), the characterization and Confirmation was done using UV–VIS Spectroscopy, FTIR, SEM, and TEM. Using these nanoparticles, a novel method of gene transformation in okra plants was developed, with a combination of different Magnetofection factors. Maximum gene transformation efficiency was observed at the DNA to Iron-nanoparticles ratio of 1:20, by rotation of mixture (Plasmid DNA, Iron-nanoparticles, and seed embryo) at 800 rpm for 5 h. Using this approach, the transformation of the GFP (green fluorescent protein) gene was successfully carried out in Abelmoschus esculentus (Okra plant). The DNA transformation was confirmed by observing the expression of transgene GFP via Laser Scanning Confocal Microscope (LSCM) and PCR. This method is highly economical, adaptable, genotype independent, eco-friendly, and time-saving as well. We infer that this approach can be a potential solution to combat the yield and immunity challenges of plants against pathogens.

Advancement in medicine and the wide use of drugs has developed resistance in human and plant pathogens. Therefore, there is a need to develop new transgenic crop varieties that can sustain in the changing climate and can resist the pathogen attack. However, it requires a precise method that can successfully deliver foreign DNA into the plants. In this regard, over the nineteenth century, many methods of genetic transformation have been developed like the biolistic method (gene gun)1, electroporation2, sonoporation3, transfection4, magnetofection5, protoplast fusion6, microinjection7, vacuum infiltration8, and Agrobacterium-mediated transformation9, etc. Every genetic transformation method, whether the method is chemical, physical, or biological, has certain limitations10. For example, electroporation can damage the DNA or cause it to lose its integrity11. Similarly, Agrobacterium-mediated transformation is not a target-specific method12. All these limitations have urged biologists to purpose new solutions for DNA delivery.

All around the globe, there are high concerns regarding environment risk assessment of different genetically modified plants13. So, it's important to move towards the techniques that pose minimal risk. Since last decade, Green Iron-nanoparticles (GINPs) are being synthesized and used in different fields such as medicine, antimicrobial activity, pollution control, and Azo-dye degradation, etc.14,15,16,17. Green synthesis is an alternative method for chemical and physical methods as it provides economic and environmental benefits18. However, its use in gene transformation for treating human diseases is quite new19. GINPs have the DNA binding capability that can be used for developing DNA delivery systems20,21. Indeed, this system uses the Magnetofection technique for gene transformation in the plants as well as in tumor cells22.

Recently, plant extracts are extensively used in the synthesis of GINPs that provide more stability to the metallic nanoparticles23. Valentine V. et al. in their study of the biosynthesis of iron oxide nanoparticles, suggested that the presence of organic acids (such as citric or oxalic acids) aids mainly in the stabilization of iron nanoparticles and plant having these organic acids can produce highly stable iron nanoparticles24. Many organic compounds (such as flavonoids, alkaloids, and saponins, etc.) are present in plant extracts that provide more solubility and compatibility to the GINPs25. Besides these, GINPs reduce the risk of environmental toxicity; as the coating of metallic particles with other non-biodegradable polymers can be harmful26. Hence, the metallic-nanoparticles prepared by using plant extract and used as DNA delivery system in plants is much more efficient as compared to the Magnetic Nanoparticles (MNPs) prepared with synthetic polymers.

In the current study, we have prepared the GINPs using leaf extract of Camellia sinensis. The optimization of Magnetofection was done to enhance DNA delivery via GINPs. We have successfully transformed the GFP gene in the Abelmoschus esculentus (Okra). Green tea leaves were used in the study because they contain organic acids (such as citric or oxalic acids), which aids in the production of stable iron nanoparticles. To revolutionize agriculture, this novel method can be used to transfer genes in plants.

Green tea plant leaves were kingly provided by Arif Ahmed (Botanical Garden, University of the Punjab) and the Okra seeds were kindly provided by Dr. Atif Kamran (Seed Centre, Department of Botany, University of the Punjab). All the experimental work on plant material described in this study complies with the relevant institutional, national, and international guidelines and legislation.

The Iron-nanoparticles were synthesized and characterized using the method described by Wang et al. with minor modifications27. For the preparation of GINPs, dried leaves (2 g) of green tea plant (C. sinensis) were mixed/soaked in 100 mL of de-ionized autoclaved water. The mixture was heated at 80 °C in the water bath for 20 min, and filtered via Whatman filter paper No.1. Later, 0.1 M aqueous solution of Iron Chloride (FeCl3) in 100 mL of de-ionized autoclaved water was prepared. After that, the filtrate of green tea solution and FeCl3 solution was mixed together with equal proportion by volume. Finally, to obtain Iron-nanoparticles, the mixture was centrifuged at 15,000 rpm for 15 min. For application, the pellet was washed with de-ionized autoclaved water (Fig. 1a–c).

Preparation of GINPs. (a) Green tea leave extract, 0.1 M FeCl3 solution, and iron nanoparticles. (b, c) synthesized iron-nanoparticles pellet after centrifugation.

For the initial identification, the obtained nanoparticles were subjected to UV–Vis spectroscopy (Ultraviolet–Visible Spectroscopy)28. For confirmation, nanoparticles were analyzed using FTIR (Fourier Transform Infrared Spectroscopy), SEM (Scanning Electron Microscope), and TEM (Transmission Electron Microscope).

MTT Assay was performed for cytotoxicity analysis of iron nanoparticles. For this purpose, J774 cells were grown in 96-well plates. Tween 80-coated iron nanoparticles were added to the cells at defined concentrations (25, 100, 200, 300, 400, and 500 μg/mL) and incubated for three and six hours. After incubation, the media was discarded and 90 μL fresh media was added per well to the cells after thorough washing with sterile phosphate-buffered saline. 10 μL (5 mg/mL stock) of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was then added in wells and the plate was incubated for six hours in an incubator. After incubation, the media was discarded from the wells and dimethyl sulfoxide 100 μL was added to solubilize the formazan crystals formed. Readings were then taken in an enzyme-linked immunosorbent assay reader at 490 nm, with subtraction for plate absorbance at 650 nm29. Percentage viability of the cells was calculated as the ratio of mean absorbance of triplicate readings concerning mean absorbance of control wells:

In the presented study, the pBIN.35 s-mgfp5-ER (Figure S1) plasmid was used30 carrying a modified GFP gene. GFP is a reporter gene of 717 bp and can be visualized at UV (395 nm) or blue light of wavelength 437 nm. Its promoter-reporter name is CaMV: GFP and its selection is kanamycin (SnapGene).

Okra seeds were soaked overnight in water and then were subjected to surface sterilization with 5% sodium hypochlorite for 20 min. To obtain the embryo, the peel of seed was removed (Fig. 2a–c).

Schematic view of transformation steps. (a) Okra seed germination; (b) Isolation of Embryo; (c) Isolated embryo; (d) Plasmid-Iron Nanoparticle Complex formation; (e) Incubation of embryos in MS medium; (f) Embryo in shooting media; (g) plants in rooting media; (h) plants After 15 days; (i) and Different stages of plant.

For the formation of a plasmid (pBIN.35 s-mgfp-ER)-iron nanoparticles complex, plasmid, and nanoparticles were taken in eppendorf, mixed, and kept at room temperature for 10 min. The isolated embryos dipped into a 1.5 mL tube having a plasmid-iron nanoparticle complex (Fig. 2d). For providing a magnetic field, the eppendorf was hanged in a beaker containing magnetic beads (Thermo Scientific™ Nalgene™-2.125 inches magnetic bead, catalog number: DS6630-0250). The beaker was placed in the magnetic stirrer at certain rpm and time (Fig. 3).

Magnetofection: illustration of DNA delivery using GINPs.

Keeping in mind the three factors: DNA-Nanoparticle ratio (DNR), Magnetic field time (used for magnetofection), and revolution per minute (rpm), 27 treatments with 10 replicates were designed. Each treatment was subjected to a different combination of DNR, rpm, and magnetic field time (Table 1).

The eppendorf (containing GINPs and Okra embryo) was hanged in a beaker. The beaker was placed on a magnetic stirrer for providing a magnetic field and stirring at a specific rpm and time. The illustration was prepared using the online BioRender tool (https://biorender.com/).

The MS (Murashige and Skoog) medium, containing Kanamycin, was used to grow the embryo using the tissue culture tubes. The embryo was placed in the basal media under sterilized conditions of laminar airflow (Fig. 2e). After that, the tubes were incubated at 28 °C for 21 days in a plant growth chamber (Fig. 2f–i). After germination, the presence of GFP in the okra plant was checked using a Laser Scanning Confocal Microscope (LSCM) (Fig. 4a,b) and by the amplification of the GFP (Fiqure 4c and Fig. S2) gene via polymerase chain reaction (PCR).

The confirmation of GFP gene transformation via Laser Scanning Confocal Microscope and PCR (a, b). The green fluorescence in transgenic Okra plants confirmed the gene delivery and its successful expression of GFP gene. (c) The PCR amplicons also corresponded to 200 bp as compare to the DNA ladder that confirms the delivery of GFP gene.

For the amplification of DNA, specific primers (Forward: 5′-ATGAGTAAAGGAGAAGAA-3′, Reverse: 3′-CATAAGAGAAAGTAGTG-5′) were designed to amplify the first 200 bp of the mgfp5 gene. For PCR, a reaction mixture of 25 µL was prepared to have 3 µL (20 ng/μL) template DNA, 2.5 µL 10 × Taq-polymerase buffer (Fermentas, MA, USA), 2.5 µL dNTPs (2 mM), MgCl2 (1.5 mM), 2 µL of forward and reverse primers, and 0.25 µl Taq-polymerase (Fermentas, MA, USA).

The thermocycler was programmed for initial degradation at 94 °C for 2 min followed by 35 cycles (94 °C for 30 s, 58 °C for 30 min, and 72 °C for 45 s). The final extension was done for 10 min at 72 °C. For confirmation, the PCR amplicons were run on 1% agarose gel (ethidium bromide; 0.5 μg/mL) and the DNA bands were observed using the digital Gel documentation system (Fig. 4c).

The data of 270 embryos were subjected to analysis of variance (ANOVA) (Fig. 5) followed by Fisher's protected least significant (LSD) difference test at the 5% probability level by using software Statistix 8.1 (https://www.statistix.com/). The GINPs size was measured using a TEM image with the help of ImageJ (https://imagej.net) and the frequencies of the size were calculated using OriginLab (https://www.originlab.com).

The effect of different treatments on gene delivery. Three factors: DNA to nanoparticles ratio (DNR), time of magnetofection (h), and revolution per minute (rpm). The treatment No. 14 yielded the highest number of transgenic plants that include 1:20 ration DNR, 5 h of magnetofection with 800 rpm.

Under the examination of UV–VIS Spectroscopy (Fig. 6a), the Iron-nanoparticles showed spectra between 350 and 450 nm that validate the formation of Iron-nanoparticles. The FTIR (Fourier Transformation Infrared Spectroscopy) spectrum of green Iron-nanoparticles is shown in (Fig. 6b).

The UV–VIS (a) and FTIR (b) spectrum of the Green Iron-nanoparticles. (a) The absorbance of Green Iron-nanoparticles was maximum at 395 nm and that range confirms the presence of Fe–Cl complex within the sample16. (b) The FTIR spectrum of nanoparticles indicates the presence of bands at 3410, 2924, 2050, 1633, 1041, 671, and 599 cm−1.

The FTIR spectrum of nanoparticles indicates the presence of bands at 3410, 2924, 2050, 1633, 1041, 671, and 599 cm−1. The mentioned bands may be due to N–H stretch, –CH, N=C, C=C, and C–Cl bonds. The presence of C–Cl bonds in the fingerprint region of the spectrum (1041, 671, and 599 cm−1) confirms the bonding of FeCl3 with the extract of green tea. The highest frequencies of GINPs size were between 7.5 and 12.5 nm as shown in Fig. 7c. The micrograph of SEM and TEM are shown in (Fig. 7a,b).

The SEM and TEM analysis of GINPs and size distribution of GINPs. (a) Depicts the SEM and (b) highlights the TEM analysis of GINPs. (c) The Histogram indicates that the size of GINPs was between the range of 5–40 nm. However, the highest frequencies were between 7.5 and 12.5 nm.

The results of the MTT assay demonstrated that cells exposed to GINPs of mean size 30 nm for three and six hours resulted in time-dependent as well as concentration-dependent cytotoxicity. At 25 μg/mL concentration, the viability of cells at three and six hours was 100% and 95%, respectively. With increasing concentration of GINPs (25, 100, 200, 300, 400, and 500 μg/mL), the percentage viability was decreased from 100% to approximately 75% in 3 h. When the cells were incubated with the same concentration of GINPs for 6 h at 25 and 100 μg/mL, the cell viability was similar to that at three hours. In contrast, at 200 μg/mL and higher concentrations, the viability decreased significantly, ranging from 55 to 65% (Fig. 8).

The cytotoxicity analysis of GINPs. The effects of superparamagnetic iron oxide nanoparticles on cell proliferation and viability of J774 cells as determined by MTT assay. Concentration-dependent cytotoxic effects of nanoparticles evaluated after three and six hours of incubation. Results are represented as means ± standard error of the mean. *Significant difference from control (P < 0.05).

The delivery of the GFP gene in plants from treatment no. 14 was confirmed as the size of PCR amplicons corresponded to the target size (200 bp) as compared to the DNA ladder (Fig. 4c). On expression in plants, the GFP produces green fluorescence when observed under LSCM. In our study, LSCM confirmed the green fluorescence in okra plants (Fig. 4b). However, there was no green fluorescence in control plants when investigated under LSCM (Fig. 4a).

The statistical analysis showed that treatment no. 14 (1 µL DNA per 20 µL Iron nanoparticle solution, given the magnetic field for 5 h at 800 rpm) produced the maximum number of transgenic plants i.e., 9 out of 10 were transgenic (Fig. 5). The high Magnetofection time and rpm caused low production of transgenic plants. Even large DNR also did not increase the gene delivery into the plants.

After observing the best transformation from treatment no. 14, the experiment was reperformed with 30 okra plants subjected to treatment no. 14 i.e., 1 µL DNA per 20 µL Iron nanoparticles solution, given the magnetic field for 5 h at 800 rpm and following the same procedure of germination as mentioned above. The results obtained showed a 90% success rate as 27 out of 30 plants were transgenic.

Genetic engineering of plants has paved a new direction for improved crops, accelerating the progress of the global agricultural industry. Previously, many methods have been introduced for gene transformation but each method has its own limitations31,32. Gene delivery, with the help of metallic nanoparticles, is a new approach in the field of biotechnology. However, these metallic nanoparticles were previously synthesized using polymers or any other coating materials33,34. Several reports have indicated the toxicity caused due to the use of polymers in nanoparticle-mediated drug delivery systems35,36,37. In the present study, we used the green synthesis method for the production of nanoparticles as it is eco-friendly, less toxic, more stable, and cost-effective (as instead of high energy machines and expensive chemicals, plants are used). Above all, it is time-saving as green synthesis is an one step bio-reduction process (Fig. 9)38.

Schematic diagram showing the uptake of plasmid-nanoparticles complex by plant.

The use of nanoparticles as molecular transporter in animals, plants, and human; nanomaterials for gene therapy and cancer therapy has been reported in previous studies. Some of the highlighted examples include; the use of Au-NP (Gold-nanoparticles) for silencing polo-loke-kinase-1(PKL1) that causes apoptosis of the damaged cell and protects the surrounding cells to get affected. In another study, to enhance the Au-NP delivery to bone marrow-derived mesenchymal cells (MSC)39,40,41. Peng et al. used antimicrobial peptides extracted from lactoferrin for coating Au-NP42. Zhi et al. used graphene oxide (GO) nanoparticles for delivery of microRNA21 (mir21) and Adriamycin (an anti-cancer drug) for overcoming tumor multidrug resistance, in vitro43. Recently, the "green approach" was used to coat the super paramagnetic iron oxide nanoparticles (SPION) with stevia plant extracts44. The nanoparticles genearted by green approaches are low in toxicity, biocompatible and are superior in function.

We have successfully optimized the magnetofection method for gene delivery using the GINPs. Without causing any damage to cells, Iron(III) can easily bind to the DNA and this property is useful for biotechnologists for developing a gene transformation method45. However, for this purpose, it requires proper magnetofection time and a specific number of revolutions and fixed DNR. Significantly, our results showed that 1:20 DNR, 800 rpm for 5 h is optimum for producing maximum number of transgenic plants via gene delivery. Contrary to this, higher rpm and time have reduced the number of transgenic plants. In a study, Lai and Singh46 concluded that Iron, in the presence of a magnetic field, can start a fenton reaction resulting in DNA damage. Hence, this could be the reason for yielding lesser number of transgenic plants when exposed to the magnetic field for more time.

The size of nanoparticles is also important for transfection and small-sized nanoparticles are more efficient for gene delivery47,48. Parallely, Wang et al.49 used an ionic gelation method for producing small-sized (100–200 nm) Chitosan nanoparticles for gene delivery. However, in our study, the average size of GINPs is 6 nm to 40 nm, from which the highest frequency was observed between 7.5 nm and 12.5 nm that can be more reliable for transfection.

Previously, there has been a lot of research regarding the use of nanoparticles for gene delivery in plants for crop improvement50,51. Demirer et al. in their study presented a nanomaterial-based delivery system that allows DNA delivery with high efficiency and nontoxicity or tissue damage and without transgene integration in plants52. Indeed, It can easily replace the traditional gene delivery methods for plants53. This method of gene delivery via GINPs not only shows promising consistent results and produce stable lines with higher viability than traditional gene delivery systems, but it also possesses the capability to overcome the restrictions of recalcitrant gene transmission of various plant species. So, our newly developed method could easily be used for improving crops safely in very short amount of time as it is non-toxic and cost-effective. This approach can also enable us to address abiotic limitations of the plants, ultimately making them more adaptable for cultivation on unfavourable environmental conditions.

Nanoparticles-mediated gene delivery can play an important role in the establishment of a new and stable platform for genetic engineering. In the presented study, the magnetofection approach proved to be a promising method for the transformation using nanoparticles as a carrier. This study is based upon a proficient magnetofection approach with low cytotoxicity. The iron NPs were synthesized from C. sinensis using green synthesis, which is a more eco-friendly approach. This method of gene delivery via GINPs not only shows promising consistent results and produce stable lines with higher viability than traditional gene delivery systems, but it also possesses the capability to overcome the restrictions of recalcitrant gene transmission of various plant species. This technique can be used for transformation of current susceptible crop varieties into more resistant ones having high yield, coping with the challenges of pathogen attack and resulting yield losses in a more cost-effective and environment-friendly way by averting the use of toxic pesticides. Furthermore, it can also be applied to overcome the regional constraints due to abiotic limitations that prohibits the cultivation of economically important crops (Supplementary figures).

Klein, T. M. & Jones, T. J. Methods of Genetic Transformation: The Gene Gun in Molecular Improvement of Cereal Crops 21–42 (Springer, 1999).

Google Scholar

Fromm, M. E., Taylor, L. P. & Walbot, V. Stable transformation of maize after gene transfer by electroporation. Nature 319(6056), 791–793 (1986).

Article ADS CAS PubMed Google Scholar

Rivera, A. L. et al. Physical methods for genetic plant transformation. Phys. Life Rev. 9(3), 308–345 (2012).

Article ADS PubMed Google Scholar

Masani, M. Y. A. et al. Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. PLoS ONE 9(5), e96831 (2014).

Article ADS PubMed PubMed Central Google Scholar

Zhao, X. et al. Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers. Nat. Plants 3(12), 956–964 (2017).

Article CAS PubMed Google Scholar

Guo, W. et al. Protoplast Technology and Citrus Improvement in Biotechnology and Sustainable Agriculture 2006 and Beyond 2007 461–464 (Springer, 2007).

Google Scholar

Neuhaus, G. & Spangenberg, G. Plant transformation by microinjection techniques. Physiol. Plant. 79(1), 213–217 (1990).

Article CAS Google Scholar

Mahmoudian, M., Yücel, M. & Öktem, H. A. Transformation of lentil (Lens culinaris M.) cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens. Plant Mol. Biol. Rep. 20(3), 251–257 (2002).

Article Google Scholar

Shrawat, A. K., Becker, D. & Lörz, H. Agrobacterium tumefaciens-mediated genetic transformation of barley (Hordeum vulgare L.). Plant Sci. 172(2), 281–290 (2007).

Article CAS Google Scholar

Rivera, A. L. et al. Genetic transformation of cells using physical methods. J.Genet. Syndrom. Gene Ther. 5(4), 237–242 (2014).

Google Scholar

Kotnik, T. et al. Electroporation-based applications in biotechnology. Trends Biotechnol. 33(8), 480–488 (2015).

Article CAS PubMed Google Scholar

Mohammed, S., Abd Samad, A. & Rahmat, Z. Agrobacterium-mediated transformation of rice: Constraints and possible solutions. Rice Sci. 26(3), 133–146 (2019).

Article Google Scholar

Mimmi, S. F. H., Islam, A. & Islam, A. A comparative study of environment risk assessment guidelines for genetically engineered plants of developing and developed countries including Bangladesh. Sci. Herit. J. 5(2), 21–28 (2021).

Article Google Scholar

Lohrasbi, S. et al. Green synthesis of iron nanoparticles using plantago major leaf extract and their application as a catalyst for the decolorization of azo dye. BioNanoScience 9(2), 317–322 (2019).

Article Google Scholar

Murgueitio, E. et al. Green synthesis of iron nanoparticles: Application on the removal of petroleum oil from contaminated water and soils. J. Nanotechnol. 2018, 1–10 (2018).

Article Google Scholar

Saif, S., Tahir, A. & Chen, Y. Green synthesis of iron nanoparticles and their environmental applications and implications. Nanomaterials 6(11), 209 (2016).

Article PubMed Central Google Scholar

Turakhia, B., Chikkala, S. & Shah, S. Novelty of bioengineered iron nanoparticles in nanocoated surgical cotton: a green chemistry. Adv. Pharmacol. Sci. 2019, 1–10 (2019).

Google Scholar

Salem, S. S. & Fouda, A. Green synthesis of metallic nanoparticles and their prospective biotechnological applications: An overview. Biol. Trace Elem. Res. 199(1), 344–370 (2021).

Article CAS PubMed Google Scholar

Zuvin, M. et al. Magnetofection of green fluorescent protein encoding DNA-bearing polyethyleneimine-coated superparamagnetic iron oxide nanoparticles to human breast cancer cells. ACS Omega 4(7), 12366–12374 (2019).

Article CAS PubMed PubMed Central Google Scholar

Majidi, S. et al. Magnetic nanoparticles: Applications in gene delivery and gene therapy. Artif. Cells Nanomed. Biotechnol. 44(4), 1186–1193 (2016).

CAS PubMed Google Scholar

Riley, M. K. & Vermerris, W. Recent advances in nanomaterials for gene delivery: A review. Nanomaterials 7(5), 94 (2017).

Article PubMed Central Google Scholar

Prosen, L. et al. Magnetofection: A reproducible method for gene delivery to melanoma cells. BioMed Res. Int. 2013, 1–11 (2013).

Article Google Scholar

Ahmad, S. et al. Green nanotechnology: A review on green synthesis of silver nanoparticles: An ecofriendly approach. Int. J. Nanomed. 14, 5087 (2019).

Article CAS Google Scholar

Makarov, V. V. et al. Biosynthesis of stable iron oxide nanoparticles in aqueous extracts of Hordeum vulgare and Rumex acetosa plants. Langmuir 30(20), 5982–5988 (2014).

Article CAS PubMed Google Scholar

Singh, J. et al. ‘Green'synthesis of metals and their oxide nanoparticles: Applications for environmental remediation. J. Nanobiotechnol. 16(1), 1–24 (2018).

Article Google Scholar

Rai, R., Alwani, S. & Badea, I. Polymeric nanoparticles in gene therapy: New avenues of design and optimization for delivery applications. Polymers 11(4), 745 (2019).

Article CAS PubMed Central Google Scholar

Wang, Y. et al. A magnetic nanoparticle-based multiple-gene delivery system for transfection of porcine kidney cells. PLoS ONE 9(7), e102886 (2014).

Article ADS PubMed PubMed Central Google Scholar

Pomposo, J. A. et al. Conductivity enhancement in raw polypyrrole and polypyrrole nanoparticle dispersions. Polym. Adv. Technol. 17(1), 26–29 (2006).

Article CAS Google Scholar

Mahmoudi, M. et al. Cell toxicity of superparamagnetic iron oxide nanoparticles. J. Colloid Interface Sci. 336(2), 510–518 (2009).

Article ADS CAS PubMed Google Scholar

Philips, J. G. et al. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence. PLoS ONE 12(2), e0171311 (2017).

Article PubMed PubMed Central Google Scholar

Sharma, K. K., Bhatnagar-Mathur, P. & Thorpe, T. A. Genetic transformation technology: Status and problems. In Vitro Cell. Dev. Biol. Plant 41(2), 102–112 (2005).

Article CAS Google Scholar

Kumar, A. et al. Methods, potentials, and limitations of gene delivery to regenerate central nervous system cells. Biol. Targets Ther. 3, 245 (2009).

CAS Google Scholar

Valdeperez, D. et al. Polymer-coated nanoparticles: Carrier platforms for hydrophobic water-and air-sensitive metallo-organic compounds. Pharmacol. Res. 117, 261–266 (2017).

Article CAS PubMed Google Scholar

Yang, J. et al. Iron oxide nanoparticles with different polymer coatings for photothermal therapy. J. Nanopart. Res. 19(10), 1–12 (2017).

Article ADS Google Scholar

Chervenkov, T. et al. Toxicity of Polymeric Nanoparticles with Respect to Their Application as Drug Carriers in Nanotechnology-Toxicological Issues and Environmental Safety and Environmental Safety 111–118 (Springer, 2007).

Book Google Scholar

Voigt, N. et al. Toxicity of polymeric nanoparticles in vivo and in vitro. J. Nanopart. Res. 16(6), 1–13 (2014).

Article CAS Google Scholar

Qi, L. et al. Polymer-coated nanoparticles for reversible emulsification and recovery of heavy oil. Langmuir 34(22), 6522–6528 (2018).

Article CAS PubMed Google Scholar

Parveen, K., Banse, V. & Ledwani, L. Green Synthesis of Nanoparticles: Their Advantages and Disadvantages (AIP Publishing LLC, 2016).

Google Scholar

Bae, K. H., Chung, H. J. & Park, T. G. Nanomaterials for cancer therapy and imaging. Mol. Cells 31(4), 295–302 (2011).

Article CAS PubMed PubMed Central Google Scholar

Barreto, J. A. et al. Nanomaterials: applications in cancer imaging and therapy. Adv. Mater. 23(12), H18–H40 (2011).

Article CAS PubMed Google Scholar

Son, S. et al. i-motif-driven Au nanomachines in programmed siRNA delivery for gene-silencing and photothermal ablation. ACS Nano 8(6), 5574–5584 (2014).

Article CAS PubMed Google Scholar

Peng, L.-H. et al. Integration of antimicrobial peptides with gold nanoparticles as unique non-viral vectors for gene delivery to mesenchymal stem cells with antibacterial activity. Biomaterials 103, 137–149 (2016).

Article CAS PubMed Google Scholar

Zhi, F. et al. Functionalized graphene oxide mediated adriamycin delivery and miR-21 gene silencing to overcome tumor multidrug resistance in vitro. PLoS ONE 8(3), e60034 (2013).

Article ADS CAS PubMed PubMed Central Google Scholar

Khatami, M. et al. Super-paramagnetic iron oxide nanoparticles (SPIONs): Greener synthesis using Stevia plant and evaluation of its antioxidant properties. J. Clean. Prod. 208, 1171–1177 https://doi.org/10.1016/j.jclepro.2018.10.182(2019).

Article CAS Google Scholar

Netto, L. E., Ferreira, A. M. D. C. & Augusto, O. Iron (III) binding in DNA solutions: Complex formation and catalytic activity in the oxidation of hydrazine derivatives. Chem. Biol. Interact. 79(1), 1–14 (1991).

Article CAS PubMed Google Scholar

Lai, H. & Singh, N. P. Magnetic-field-induced DNA strand breaks in brain cells of the rat. Environ. Health Perspect. 112(6), 687–694 (2004).

Article CAS PubMed PubMed Central Google Scholar

Prabha, S. et al. Size-dependency of nanoparticle-mediated gene transfection: Studies with fractionated nanoparticles. Int. J. Pharm. 244(1–2), 105–115 (2002).

Article CAS PubMed Google Scholar

Prabha, S. et al. Effect of size on biological properties of nanoparticles employed in gene delivery. Artif. Cells Nanomed. Biotechnol. 44(1), 83–91 (2016).

Article CAS PubMed Google Scholar

Wang, L. et al. Establishing gene delivery systems based on small-sized chitosan nanoparticles. J. Ocean Univ. China 17(5), 1253–1260 (2018).

Article ADS CAS Google Scholar

González-Melendi, P. et al. Nanoparticles as smart treatment-delivery systems in plants: Assessment of different techniques of microscopy for their visualization in plant tissues. Ann. Bot. 101(1), 187–195 (2008).

Article PubMed Google Scholar

Nair, R. et al. Nanoparticulate material delivery to plants. Plant Sci. 179(3), 154–163 (2010).

Article CAS Google Scholar

Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14(5), 456–464 (2019).

Article ADS MathSciNet CAS PubMed Google Scholar

Cunningham, F. J. et al. Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol. 36(9), 882–897 (2018).

Article CAS PubMed Google Scholar

Download references

The authors are highly thankful to the University of the Punjab for providing funds to complete the research work and Lahore garrison university for support and providing lab facilities.

Department of Biotechnology, Lahore Garrison University, P.O BOX. 54000, Lahore, Pakistan

Naila Farooq, Laraib Ather & Qamar Abbas

Department of Horticulture, Faculty of Agricultural Sciences, University of the Punjab, P.O BOX. 54590, Lahore, Pakistan

Muhammad Shafiq

Virology Lab, CABB University of Agriculture, Faisalabad, Pakistan

Muhammad Shah Nawaz-ul-Rehman

Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, P.O BOX. 54590, Lahore, Pakistan

Muhammad Haseeb, Tehmina Anjum, Mujahid Hussain & Numan Ali

Department of Agronomy, Faculty of Agricultural Sciences, University of the Punjab, P.O BOX. 54590, Lahore, Pakistan

Syed Agha Armaghan Asad Abbas

Faculty of Agricultural Sciences, University of the Punjab, P.O BOX. 54590, Lahore, Pakistan

Sehrish Mushtaq & Muhammad Saleem Haider

Institute of Biochemistry, Biotechnology, and Bioinformatics (IBBB), The Islamia University of Bahawalpur, P.O BOX. 63100, Bahawalpur, Pakistan

Saleha Sadiq

North Florida Research and Education Center, 155 Research Rd., Quincy, FL, 32351, USA

Muhammad Adnan Shahid

You can also search for this author in PubMed Google Scholar

N.F., L.A., M.S., T.A., Q.A., S.A.A.A.A., N.A., Muj.H, Muh.H and S.M. were involved in the data analysis. M.S. and T.A. provided the overall direction and experimental design. N.F., L.A., Q.A., Muj.H, Muh.H, S.A.A.A.A., N.A. and S.M. wrote the manuscript. All authors read and approved the final manuscript.

Correspondence to Muhammad Shafiq or Muhammad Adnan Shahid.

The authors declare no competing interests.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

Farooq, N., Ather, L., Shafiq, M. et al. Magnetofection approach for the transformation of okra using green iron nanoparticles. Sci Rep 12, 16568 (2022). https://doi.org/10.1038/s41598-022-20569-x

Download citation

Received: 30 October 2021

Accepted: 15 September 2022

Published: 04 October 2022

DOI: https://doi.org/10.1038/s41598-022-20569-x

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.