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

Authors

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

Abstract

Objective
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.
Results
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.
Conclusions
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.

Keywords

References

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Agricultural Biotechnology Journal
Volume 15, Issue 1
April 2023
Pages 61-80

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