Optimization of transient gene delivery to Catharanthus roseus L. using introduction of green synthesized superparamagnetic iron oxide nanoparticles and carbon nanotubes nanocarrier

Document Type : Research Paper


1 Ph.D. Student, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

2 Department of Agricultural Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

3 Associate Professor, Department of Agricultural Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

4 Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran


Conventional gene delivery to plant cells approaches have limitations such as narrow host range in Agrobacterium-mediated gene delivery method, removal of cell wall using polyethylene glycol and electroporation methods, and the higher cell damage in the use of gene gun. Recently, nanotechnology-based gene delivery methods have been developed for plant genetic transformation, and this nanostrategy shown the efficiency of gene transfer and biocompatibility and the target DNA protection.
Materials and methods
Superparamagnetic iron oxide nanoparticles (SPIONs) were fabricated via green route using Catharanthus roseus leave aqueous extract. SPIONs nanoparticles and carboxylated single-walled carbon nanotubes (SWCNTs-COOH) were functionalized with polyethyleneimine (PEI). For gene delivery to Catharanthus roseus leave, the pDNA@SPIONs and pDNA@SWCNTs nanocarriers were prepared by pDNA loading on the surface of cationic nanoparticles. Two methods were used to accelerate and increase the pass of the nanocarrier through the plant cells, immersion in nanocarrier suspension and infiltration by syringe. Tracking the mGFP5 protein fluorescent signal and RT-PCR reaction was evaluated to affirm mgfp5 gene delivery and its expression in plant cells.
The color change of the iron chloride salts solution, the absorption of the synthesized nanoparticles to a magnet, while the iron chloride salts do not have this property, and the results of the performed analysis confirmed the green synthesis of SPIONs nanoparticles. The functionalized nanoaprticles with PEI shown good ability to interact with DNA and effectively DNA protection against nuclease enzymes degradation. The mGFP5 protein fluorescent signal confirmed the gene delivery to plant cells ability of pDNA@SPIONs and pDNA@SWCNTs nanocarriers.
So the application of nanobiotechnology and nanocarriers for gene delivery to plant cells can be promising for plant genetic engineering. By nanocarriers, it is possible efficient gene delivery to almost all plant species regardless dicotyledonous or monocotyledonous through a method with features such as simple and low cost, elimination of Agrobacterium and its limitations, and without the specialized laboratory equipment.


Caizer C (2016) Nanoparticle size effect on some magnetic properties. Handbook of Nanoparticles 475.
Chen G, Qiu J, Liu Y, et al. (2015) Carbon nanotubes act as contaminant carriers and translocate within plants. Sci Rep 5,1–9.
Cunningham FJ, Goh NS, Demirer GS, et al. (2018) Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol 36,882–897.
de Almeida MS, Susnik E, Drasler B, et al. (2021) Understanding nanoparticle endocytosis to improve targeting strategies in nanomedicine. Chem Soc Rev 50,5397–5434.
Demirer G, Zhang H, Goh N, et al. (2019) Nanotubes effectively deliver siRNA to intact plant cells and protect siRNA against nuclease degradation. Available SSRN 3352632.
Demirezen DA, Yılmaz Ş, Yılmaz DD, Yıldız YŞ (2022) Green synthesis of iron oxide nanoparticles using Ceratonia siliqua L. aqueous extract: improvement of colloidal stability by optimizing synthesis parameters, and evaluation of antibacterial activity against Gram-positive and Gram-negative bacteria. Int J Mater Res.
Farmanbar N, Mohseni S, Darroudi M (2022) Green synthesis of chitosan-coated magnetic nanoparticles for drug delivery of oxaliplatin and irinotecan against colorectal cancer cells. Polym Bull 1–19.
Goswami N, Saha R, Pal SK (2011) Protein-assisted synthesis route of metal nanoparticles: exploration of key chemistry of the biomolecule. J Nanoparticle Res 13,5485–5495.
Guan Z, Ying S, Ofoegbu PC, et al. (2022) Green synthesis of nanoparticles: Current developments and limitations. Environ Technol Innov 102336.
He X, Wang K, Tan W, et al. (2003) Bioconjugated nanoparticles for DNA protection from cleavage. J Am Chem Soc 125,7168–7169.
Khalid MK, Asad M, Henrich-Noack P, et al. (2018) Evaluation of toxicity and neural uptake in vitro and in vivo of superparamagnetic iron oxide nanoparticles. Int J Mol Sci 19,2613.
Khanna L, Verma NK, Tripathi SK (2018) Burgeoning tool of biomedical applications-Superparamagnetic nanoparticles. J Alloys Compd 752,332–353.
Kommareddy S, Amiji M (2007) Poly (ethylene glycol)–modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery. MBN J 3,32–42.
Kotakadi VS, Rao YS, Gaddam SA, et al. (2013) Simple and rapid biosynthesis of stable silver nanoparticles using dried leaves of Catharanthus roseus. Linn. G. Donn and its anti microbial activity. Colloids Surfaces B Biointerfaces 105,194–198.
Kulkarn R N, Baskaran K, & Jhang T (2016) Breeding medicinal plant, periwinkle [Catharanthus roseus (L) G. Don]: a review. Plant Genet. Res. 14, 283–302.
Kwak S-Y, Lew TTS, Sweeney CJ, et al. (2019) Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat Nanotechnol 14,447–455.
Lin Z, Weng X, Owens G, Chen Z (2020) Simultaneous removal of Pb (II) and rifampicin from wastewater by iron nanoparticles synthesized by a tea extract. J Clean Prod 242,118476.
Liu Q, Chen B, Wang Q, et al. (2009) Carbon nanotubes as molecular transporters for walled plant cells. Nano Lett 9,1007–1010.
Ma L, Zhuang HL, Wei S, et al. (2016) Enhanced Li–S batteries using amine-functionalized carbon nanotubes in the cathode. ACS Nano 10,1050–1059.
Ochoa-Olmos OE, León-Domínguez JA, Contreras-Torres FF, et al. (2016) Transformation of plant cell suspension cultures with amine-functionalized multi-walled carbon nanotubes. J Nanosci Nanotechnol 16,7461–7471.
Pouya S, Kazemi M, Pouya S, et al. (2022) Evaluation of CTAB-coated gold nanoparticles as a potential carrier for gene delivery. Trends Pharm Sci 8,3.
Rahmani R, Gharanfoli M, Gholamin M, et al. (2020) Plant-mediated synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) using aloe vera and flaxseed extracts and evaluation of their cellular toxicities. Ceram Int 46,3051–3058.
Safdar M, Kim W, Park S, et al. (2022) Engineering plants with carbon nanotubes: a sustainable agriculture approach. J Nanobiotechnology 20,1–30.
Schwartz SH, Hendrix B, Hoffer P, et al. (2020) Carbon dots for efficient small interfering RNA delivery and gene silencing in plants. Plant Physiol 184,647–657.
Sharma A, Verma P, Mathur A, & Mathur A K (2018) Genetic engineering approach using early Vinca alkaloid biosynthesis genes led to increased tryptamine and terpenoid indole alkaloids biosynthesis in differentiating cultures of Catharanthus roseus. Protoplasma 255, 425–435.
Sosa‑Acosta JR, Iriarte‑Mesa C, Ortega GA, Díaz‑García AM (2020) DNA-iron oxide nanoparticles conjugates: Functional magnetic nanoplatforms in biomedical applications. Surface-modified Nanobiomaterials Electrochem Biomed Appl 19–47.
Tang K, & Pan Q (2017) Strategies for enhancing alkaloids yield in Catharanthus roseus via metabolic engineering approaches. In Catharanthus roseus 1–16 Springer.
Tabasi H, Mosavian M T H, Darroudi M, et al. (2022). Synthesis and characterization of amine-functionalized Fe3O4/Mesoporous Silica Nanoparticles (MSNs) as potential nanocarriers in drug delivery systems. J Porous Mater, 1–12.
Torney F, Trewyn BG, Lin VS-Y, Wang K (2007) Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2,295–300.
Velsankar K, Parvathy G, Mohandoss S, et al. (2022) Celosia argentea leaf extract-mediated green synthesized iron oxide nanoparticles for bio-applications. J Nanostructure Chem 12,625–640.
Wang Q, Xing S, Pan Q, et al. (2012) Development of efficient Catharanthus roseus regeneration and transformation system using Agrobacterium tumefaciens and hypocotyls as explants.          BMC Biotechnol. 12, 1–12.
Wong MH, Misra RP, Giraldo JP, et al. (2016) Lipid exchange envelope penetration (LEEP) of nanoparticles for plant engineering: a universal localization mechanism. Nano Lett 16,1161–1172.
Zaboli M, Saeidnia F, Zaboli M, Torkzadeh-Mahani M (2021) Stabilization of recombinant d-Lactate dehydrogenase enzyme with trehalose: Response surface methodology and molecular dynamics simulation study. Process Biochem 101,26–35.
Zhang H, Demirer G S, Zhang H, et al. (2019) DNA nanostructures coordinate gene silencing in     mature plants. PNAS 116,15:7543-7548.
Zhang H, Goh NS, Wang JW, et al. (2022) Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves. Nat Nanotechnol 17,197–205. https://doi.org/10.1038/s41565-021-01018-8
Zhao X, Meng Z, Wang Y, et al. (2017) Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers. Nat plants 3,956–964.
Zhi H, Zhou S, Pan W, Shang Y, Zeng Z, & Zhang H (2022)  The Promising Nanovectors for Gene Delivery in Plant Genome Engineering. Int J Mol Sci 23,15:8501.
Zhou ML, Zhu XM, Shao JR, et al. (2012). An protocol for genetic transformation of Catharanthus roseus by Agrobacterium rhizogenes A4. Appl. Biochem. Biotechnol. 166, 1674–1684.