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 Agrobacterium
tumefaciens, we significantly improved transient gene
expression efficiency while minimizing tissue damage compared to existing
methods in Arabidopsis thaliana. We
further demonstrated the broad applicability of VAST by successfully
transforming key crop species, including tomato, Brassica
rapa, Medicago sativa, Setaria italica (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 A. thaliana and S. italica. 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.