Work

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

Year: 2025

Type: article

Abstract: 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 T 3 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. • We demonstrate a series of evidence for a tissue culture-free, ultrasound–nanoparticle method that delivers and integrates plasmid DNA directly into mature wheat seeds. • Transgenes were stably inherited to the T₃ generation across three genotypes, supporting levels of genotype-flexible applicability. • DNA-loaded mesoporous silica nanoparticles combined with optimized sonication enabled efficient in-planta uptake and integration of plasmid fragments, containing GUS and the resulting plants expressed GUS unevenly. • PCR, GUS assays, qPCR, and whole-genome sequencing collectively confirmed presence of target DNA in samples and multigenerational stability. • This simple, scalable platform, if developed to a mature technology, offers a low-cost alternative to conventional monocot transformation and shows promise for future trait engineering including marker-free traits and genome editing applications.

Source: New Biotechnology

DOI: 10.1016/j.nbt.2025.12.003

Authors: Fariba Rafiei, Nirman Nepal, Trever Thurgood, Roland Wilhelm, Steve Scofield, Mohsen Mohammadi

Institutions: Purdue University West Lafayette, Agricultural Research Service, United States Department of Agriculture

Language: English

Topic: Plant tissue culture and regeneration

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Zero hunger

Open Access status: gold

Estimated APC paid: $3,670