Abstract:

Flammulina filiformis is an economically important edible and medicinal mushroom. However， its genetic transformation has historically relied solely on hygromycin resistance as a selectable marker and this limitation has impeded research into the coordinated regulation of multiple genes. In this study， we established an Agrobacterium tumefaciens-mediated genetic transformation system for F. filiformis using the herbicide resistance gene bar and glufosinate-ammonium as the selectable marker and agent， respectively. Two vectors were used， pAgbar， in which the bar gene is driven by the constitutive gpd promoter from Agaricus bisporus； and pAgbarG， which harbors a bar-egfp translational fusion expression cassette. Both constructs were delivered into the monokaryotic strain Dan3， yielding stable， glufosinate-ammonium-resistant transformants. Transformation efficiency was markedly higher on M-100 medium than on PDA. PCR and Southern blot hybridization confirmed the integration of the exogenous bar gene into the F. filiformis genome. Moreover， the bar-egfp fusion system enabled effective fluorescence signal expression， providing a rapid and visual means of identifying positive transformants. This study establishes a genetic manipulation platform for F. filiformis using bar as a resistance selectable marker， offering reliable technical support for functional gene analysis， molecular-assisted breeding， and mechanistic studies of secondary metabolic pathway regulation.

Key words: Flammulina filiformis, bar, selection marker, genetic transformation