image: Magnetofected pollen gene delivery system applied to C. sativus. (A) Schematic illustration of pollen magnetofection. (B) Steps of production of magnetofected pollen gene delivered cucumber. 0.5 μg of MNPs (PolyMag200, Chemicell, Germany)) and 2 μg of plasmid DNA (200–250 ng/μg) were combined at a 1:4 ratio and allowed to stand for 15 minutes at room temperature for one magnetofection reaction. The MNP–DNA complexes were then added to a magnetofection buffer (15% sucrose, 0.01% boric acid, 1 mM Ca(NO3)2) containing pollen grains (Approximately 22 000 pollen grains, collected from 20 male flowers, were magnetofected with the MNP–DNA complexes. This corresponds to around 1100 pollen grains per male flower. [6]), and placed in a magnetic field (MagnetoFACTOR-24, Chemicell, Germany) for 30 minutes. Subsequently, the magnetofected pollen grains were carefully spread onto filter paper to remove the buffer and allowed to dry at room temperature overnight, followed by storage at 4°C. The next day, the magnetofected pollen grains were manually pollinated onto the stigma of female flowers. (C) Viability test of processed pollen. Dried magnetofected pollen grains were observed after the drying process and germination of magnetofected pollen grains which had dried 1 day before (Scale bar = 100 μm). (D) Increase in exogenous gene expression activity over time in the magnetofected pollen. Pollen-specific promoter (OsMTD2 promoter) showed stronger GUS activity than the for constitutive promoter (Scale bar = 100 μm). (E) Statistical analysis of GUS expression was conducted with T1 seedlings (n > 130 seedlings for each group). Error bars represent the standard error of three repeats. No significant difference was observed between the presence and absence of treatment. Images of non-treated and pre-treated cucumber pollen grains were captured using scanning electron microscope. (Scale bar = 30 μm, 5 μm) The third row of the figure exhibits the cotyledon of the seedling. (F) Transgenic seeds were germinated and T1 seedlings were analyzed for GUS assay, DNA examination, and transferred to the soil. P is positive control, and wild type (WT) is not amplified for transgene, as negative control. (G) PCR analysis of T1 transgenic plants of Cas9 vector delivered experiments. P, pKIR1.1 plasmid; WT, wild type cucumber DNA. (H) Schematic illustration of putative model of DNA migration through pollen magnetofection. N, Vegetative nucleus; Gc, Generative cell; Mt, Mitochondria; Pl, Plastid.
Credit: Horticulture Research
Researchers have achieved a groundbreaking advancement in plant biotechnology by using a magnetofected pollen gene delivery system to genetically transform cucumbers. This cutting-edge method uses DNA-coated magnetic nanoparticles to introduce foreign genes into pollen, producing genetically modified seeds without the need for traditional tissue culture or regeneration steps. This technique significantly streamlines and accelerates crop genetic modification, opening up new avenues to boost agricultural productivity and resilience.
Genetic modification in horticultural crops, particularly within the Cucurbitaceae family, is often hindered by complex tissue culture requirements and environmental pressures such as climate change. Traditional transformation techniques, like Agrobacterium-mediated gene transfer, frequently encounter barriers that limit their success in certain plant species. Magnetofection, a novel DNA delivery method using magnetic nanoparticles, offers a promising alternative to these conventional approaches. Given these persistent challenges, innovative gene delivery systems are urgently needed to advance crop genetic engineering.
Conducted by scientists at Pusan National University and published (DOI: 10.1093/hr/uhae179) in Horticulture Research on June 27, 2024, this study unveils an advanced pollen magnetofection technique for developing genetically modified cucumbers. Using magnetic nanoparticles, researchers successfully delivered exogenous DNA into cucumber pollen, effectively bypassing the limitations of traditional tissue culture methods. This breakthrough in genetic engineering provides a more direct and efficient way to produce transgenic plants, heralding a new era in agricultural biotechnology.
The research centered on refining a pollen magnetofection method tailored for cucumbers. By employing positively charged Fe3O4 magnetic nanoparticles as DNA carriers, the exogenous genes were introduced into the pollen apertures. Post-magnetofection, the treated pollen was manually applied to the stigma of female cucumber flowers, resulting in the generation of transgenic seeds. Notably, the pollen's viability was maintained throughout the process, and gene expression was observed in the transformed pollen over time. Key results demonstrated that gene expression efficiency varied significantly with different promoters, with the OsMTD2 (Mitochondrial Targeting Domain, MTD) promoter outperforming the p35S promoter. The transgenic seeds exhibited robust gene expression in the cotyledons and roots of the T1 seedlings. Despite challenges such as lower gene integration rates, the study validated the feasibility of this technique for cucumber transformation and underscored its potential application in other crop species.
Dr. Yu-Jin Kim, lead researcher at Pusan National University, highlighted the revolutionary potential of this gene delivery system: “Our findings underscore pollen magnetofection as a flexible and efficient approach to genetic transformation in cucumbers. This technique circumvents the challenges of conventional tissue culture, offering a quicker and more accessible method to produce transgenic plants. Future research could extend its applicability to other key crops, driving innovative solutions in sustainable agriculture.”
The successful implementation of pollen magnetofection in cucumbers opens up new possibilities for crop enhancement and genetic studies. This technique serves as a practical alternative to traditional transformation methods, enhancing genetic modification processes across a range of plant species. The broader implications of this technology extend well beyond cucumbers, offering pathways to develop more resilient and nutritionally fortified crops, vital for tackling global agricultural challenges like climate change and food security. Further refinement could expand its potential, making it applicable to more complex plant genomes and traits.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhae179
Funding information
This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (RS-2023-00217064 to Y.J.K.).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number two in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.
Journal
Horticulture Research
Subject of Research
Not applicable
Article Title
Magnetofected pollen gene delivery system could generate genetically modified Cucumis sativus
Article Publication Date
27-Jun-2024
COI Statement
The authors declare that they have no competing interests.