Vacuum and Sonication Treatment Enable Efficient Transient Gene Expression in Various Monocot and Eudicot Plant Seedlings

Transient gene expression in intact plants is essential for rapidly addressing biological questions, and the current toolkit can be improved to achieve higher efficiency and a broader range of plant species. Here, we introduce VAST (Vacuum and Sonication-Assisted Transient transformation): a transient transformation method that substantially enhances gene expression efficiency and versatility across diverse monocot and eudicot seedlings. By systematically optimizing plant growth conditions and incorporating vacuum infiltration and sonication pretreatments prior to seedling coculture with <i>Agrobacterium tumefaciens</i>, we significantly improved transient gene expression efficiency while minimizing tissue damage compared to existing methods in <i>Arabidopsis thaliana</i>. We further demonstrated the broad applicability of VAST by successfully transforming key crop species, including tomato, <i>Brassica rapa</i>, <i>Medicago sativa</i>, <i>Setaria italica</i> (foxtail millet), switchgrass, maize, and wheat. We also presented a case study using VAST-mediated transient transformation, in which a cross-species analysis of nitrate-responsive gene expression highlighted both conserved and divergent biological responses between <i>A. thaliana</i> and <i>S. italica</i>. VAST's simplicity, versatility, and efficiency make it a powerful tool for functional genomics, synthetic biology, and biotechnology research, opening new avenues for rapid exploration of gene function, regulation, and editing across diverse plant systems.