Stable Agrobacterium-mediated transformation system for Flammulina filiformis using Geneticin (G418) as efficient...

Flammulina filiformis is a commercially significant edible basidiomycete; however, functional genomics in this species have been impeded by low transformation efficiency and the limited selectable markers. In this study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation (ATMT) system applicable to both dikaryotic and monokaryotic F. filiformis strains through systematically optimizing key parameters, including culture medium composition, Agrobacterium strain, recipient genetic background, and promoters driving resistance gene expression. Specifically, using minimal medium (MM) for recipient culture, A. tumefaciens AGL1 as the donor, and the endogenous F. filiformis glyceraldehyde-3-phosphate dehydrogenase promoter (Ffgpd) to drive expression of the nptII under G418 selection, we attained an average transformation efficiency of 53.36% in the dikaryotic strain 0747 and efficiencies ranging from 29.82% to 39.11% in its monokaryotic derivatives. To facilitate rapid and convenient identification of resistant transformants, we utilized the endogenous hyd13 promoter, which exhibits high activity during mycelial growth, to drive enhanced green fluorescent protein (eGFP) expression. This strategy permitted efficient visual screening of transformants using a handheld fluorescence lamp. Southern blot and PCR analyses confirmed random, single-copy integration of T-DNA and demonstrated mitotic stability of the G418-resistant transformants; moreover, nptII was consistently detected throughout both vegetative growth and fruiting body development. Together, these findings establish an efficient and stable ATMT system for F. filiformis using the nptII gene as a selectable marker. This scalable tool has accelerated high-throughput functional genomics and enabled precise, molecular-level improvement of key agronomic traits in this economically important fungus.