Abstract:Safe and efficient microbial mutagenesis and high-throughput screening techniques are important for discovering microbial functions,endowing microbes with new functions,and developing new technology for biological industry.Thus,effective breeding and high-throughput screening techniques have attracted the interest of researchers.Atmospheric and room temperature plasma (ARTP),characterized by rich reactive species,ease of operation,severe DNA damage,high efficiency,moderate operation conditions,and high safety,has been used for the mutation breeding of over 100 microorganisms,animals,and plants,particularly the efficient breeding of microorganisms.Microbial microdroplet culture (MMC) generates a large number of microdroplets in a short time,with independent control of individual droplets and each droplet as an independent micro-cultivator.Attributing to the small volume,high throughput,good controllability,and real-time monitoring,the modularized system allows high-throughput cultivation and adaptive evolution of microbes,demonstrating unique advantages in high-throughput culture of microbes.This review summarized the application of ARTP to the breeding of edible and medicinal mushrooms and MMC system to high-throughput sorting of microbes,which is expected to serve as a reference for the breeding of edible and medicinal fungi.