Pollen Mediated Transgenic Technology Of Cotton By Magnetofection System Based On Fe₃O₄ Nanoparticles

Modified magnetic nanoparticles are widely used as non-viral gene carriers in biological applications. To achieve successful gene delivery, it is critical that nanoparticles effectually assemble with nucleic acids. However, relatively little work has been conducted on the assembly mechanisms between nanoparticles and DNA, and its effects on transfection efficiency. Using biophysical and biochemical characterization, along with atomic force microscopy(AFM) and transmission electron microscopy(TEM), we investigated the morphologies, assembling structures and gene delivering abilities of the polyethyleneimine(PEI) modified magnetic nanoparticles(MNPs) gene delivery system. In this gene delivery system, MNP/DNA complexes are formed via binding of DNA onto the surface of MNPs. MNPs are favorable to not only increase DNA concentration but also prevent DNA degradation. Magnetofection experiments showed that MNPs have low cytotoxicity and afford highly stable and long-lasting transfection of foreign genes in mammalian somatic cells. In addition, different binding ratios between MNPs and DNA result in various morphologies of MNP/DNA complexes. Dose–response profile indicated that transfection efficiency positively correlate with MNP/DNA ratio. Furthermore, intracellular fluorescence tracking imging demonstrated that MNPs are internalized through penetrating cell membranes, deliver and release exogenous DNA into the nucleus. Here, we developed a stable, targetable and convenient system for delivering multiple genes into the nuclei of porcine somatic cells using MNPs as gene carriers, in which, stable and efficient co-expression of GFP and Ds Red in porcine kidney PK-15 cells was successfully achieved by magnetofection. The results presented here demonstrate the potential application of magnetic nanoparticles as an attractive delivery system for animal genetics and breeding studies.Genetic modification plays a vital role in breeding new crops with excellent traits. Almost all of the current genetic modification methods require regeneration from tissue culture, involving complicated, long, and laborious processes. In particular, many crop species such as cotton and soybean are difficult to regenerate. Here, we report a novel transformation platform technology, pollen magnetofection, to directly produce transgenic seeds without regeneration. This was the first success of magnetofection in plant genetic transformation. In this system, exogenous DNA loaded with magnetic nanoparticles were delivered into pollen in the presence of a magnetic field. Through pollination with magnetofected pollen, transgenic plants were successfully generated from transformed seeds. Fused insecticide transgenic cotton were created. Exogenous DNA were successfully integrated into the genome, effectively expressed, and stably inherited in the offsprings. Our system is culture-free and genotype independent. In addition, it is exceedingly simple, fast, and capable of multi-gene transformation. We envision that pollen magnetofection can transform almost all crops, greatly facilitating breeding processes of new varieties of transgenic crops.