Magnetic nanoparticle-mediated genetic transformation and gene editing system in loquat (Eriobotrya japonica)

Loquat (Eriobotrya japonica Lindl.) is a valuable subtropical fruit tree whose genetic improvement has been significantly constrained by the absence of an efficient genetic transformation system. Although Agrobacterium-mediated transformation is the most widely used method, it proves ineffective in loquat due to the species' recalcitrance to in vitro regeneration. Pollen-based transformation offers a promising alternative by bypassing the need for tissue culture. However, the pollen wall poses a major physical barrier to the uptake of exogenous DNA. In this study, we investigated magnetic nanoparticle (MNP)-mediated transformation as a novel strategy for loquat. We confirmed that loquat pollen contains tricolporate apertures with diameters ranging from 3.0 to 5.0 μm, which are structurally suitable for the entry of MNPs-DNA. Based on this finding, we developed and optimized a transformation protocol using polyethyleneimine-coated Fe₃O₄ nanoparticles to deliver genetic material into loquat pollen grains. Using this approach, we successfully generated stable transgenic loquat lines, including both overexpression and gene-edited mutants. To our knowledge, this is the first report of successful MNP-mediated pollen transformation in a woody plant species. This work establishes a robust and efficient genetic transformation platform for loquat, providing a valuable tool for functional genomics and molecular breeding, as well as a potentially applicable strategy for other recalcitrant woody plants.