Standardized Protocol for Isolation and Cryopreservation of Cultivable Endophytes From Fresh and Dried <i>Citrus aurantium</i> Peels

ABSTRACT Citrus peels, particularly those of Citrus aurantium , are agro‐industrial and medicinal byproducts that harbor specialized endophytic communities with potential roles in fruit aroma, quality, and biocontrol. However, research studies on these communities have been limited by the lack of standardized, reproducible protocols for recovering cultivable endophytes from both fresh peel and processed materials such as “Zhike,” where desiccation creates a stringent selective environment. Here, we present a systematic workflow for isolating, molecularly identifying, and cryopreserving endophytic microorganisms from Citrus aurantium peel collected as fresh fruit and dried “Zhike” across production regions. The protocol integrates surface sterilization regimens tailored to distinct physical properties of fresh versus dried tissues, sterility validation, tissue homogenization, and multi‐medium cultivation under nutrient‐rich and low‐nutrient conditions to recover diverse bacterial and fungal isolates. Molecular identification based on 16S rRNA gene and ITS region sequencing resolves taxonomic identities and supports construction of a cryopreserved strain collection representing cultivable citrus peel endophytes. By standardizing key steps from sample preparation through long‐term storage, this protocol facilitates studies of endophytes from stressed, low‐moisture peel matrices and establishes a microbial resource library for future investigations into plant–microbe–metabolite interactions, bio‐aroma (biologically derived aroma compounds) enhancement, endophyte‐based biocontrol, and sustainable valorization of citrus processing byproducts.