Vacuum and sonication treatment enables efficient transient gene expression in various monocot and eudicot plant seedlings

Transient gene expression in intact plants is essential for rapidly addressing biological questions. However, current methods are inefficient and have limited applicability across species, with no routine approach beyond the Agro-infiltration system in Nicotiana benthamiana leaves. Here, we introduce VAST (Vacuum and Sonication-Assisted Transient expression): a transient transformation method that substantially enhances gene expression efficiency, reproducibility, and versatility across diverse monocot and eudicot intact seedlings. By systematically optimizing plant growth conditions and incorporating vacuum infiltration and sonication pre-treatments prior to seedling co-culture with Agrobacterium tumefaciens , we significantly improved transient gene expression efficiency while minimizing tissue damage compared to existing methods such as AGROBEST and Fast-TrACC in Arabidopsis thaliana . We further demonstrated the broad applicability of VAST by successfully transforming key model and crop species, including tomato, Brassica rapa , Medicago sativa , Setaria italica (foxtail millet), switchgrass, and wheat. We also demonstrated a case study using the VAST-mediated transient transformation, where a cross-species analyses of nitrate-responsive gene expression highlighted both the conserved and diverged biological responses between the two model crops, A. thaliana and S. italica . VAST’s simplicity, versatility, efficiency, and reduced damage provide a powerful tool for functional genomics, synthetic biology, and biotechnology research, opening new avenues for the rapid exploration of gene function, regulation, and editing in diverse plant systems.