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

Area of Science:

• Mycology
• Molecular Biology
• Genetics

Background:

• Flammulina filiformis is an economically important edible mushroom.
• Functional genomics studies are hindered by low transformation efficiency and limited selectable markers in F. filiformis.

Purpose of the Study:

• To establish a high-efficiency Agrobacterium tumefaciens-mediated transformation (ATMT) system for Flammulina filiformis.
• To optimize parameters for successful transformation in both dikaryotic and monokaryotic strains.
• To develop tools for efficient identification and validation of transformants.

Main Methods:

• Systematic optimization of culture medium, Agrobacterium strain, recipient genetic background, and promoters.
• Utilized minimal medium (MM), Agrobacterium tumefaciens AGL1, and the endogenous F. filiformis glyceraldehyde-3-phosphate dehydrogenase promoter (Ffgpd) with nptII for selection.
• Employed the endogenous hyd13 promoter to drive enhanced green fluorescent protein (eGFP) for visual screening.

Main Results:

• Achieved high transformation efficiencies: 53.36% in dikaryotic strain 0747 and 29.82%-39.11% in monokaryotic derivatives.
• Confirmed random, single-copy T-DNA integration and mitotic stability via Southern blot and PCR.
• Demonstrated stable expression of nptII throughout vegetative growth and fruiting body development.

Conclusions:

• Established an efficient, stable, and scalable ATMT system for Flammulina filiformis.
• The developed system facilitates high-throughput functional genomics research.
• Enables precise, molecular-level improvement of agronomic traits in F. filiformis.