Evidence for nanoparticle-enhanced ultrasound-based In-Planta transformation and transgenerational inheritance in wheat

Here, we describe a series of evidence that most parsimoniously explains DNA delivery and integration of transgene into wheat genome by using an ultrasound-mediated, nanoparticle-based method without the need for tissue culture. This approach offers a potentially high-throughput, amenable to automation, and a cost-effective strategy for transforming mature wheat seeds and stably transmitting transgenes across generations, with stable inheritance confirmed through the T₃ generation. The method successfully delivered transgenes, via the pCAMBIA1305.1 plasmid, to multiple wheat varieties, including 'Apogee', 'Sonora-64' and 'Opata-85', indicating its potential as a genotype-independent platform for plant genetic engineering. Ultrasound treatment transiently increases the permeability of seed cell walls and membranes, facilitating the uptake of mesoporous silica nanoparticles coated with polyethyleneimine that are loaded with plasmid DNA. Quantitative PCR, segregation analysis, and whole-genome sequencing confirmed variable copy number and stable integration of segments of plasmid DNA across multiple loci. Transgene expression was validated using GUS staining and PCR assays across successive generations, supporting robust transgenerational inheritance. By eliminating the need for callus induction or genotype-specific regeneration protocols, this seed-transformation method offers a scalable, cost-effective, and potentially game-changing approach for the genetic transformation of wheat and other recalcitrant crop species.