Magnetofected pollen gene delivery system applied to C. sativus. (IMAGE)
Caption
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
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