A membrane-separated bi-compartmented root organ culture system enables scalable in vitro growth of Gigaspora sp. and Scutellospora reticulata.

Root Organ Cultures (ROC) of arbuscular mycorrhizal fungi (AMF) have greatly advanced the study of these obligate biotrophs and provide a reliable source of clean material for inoculum production. While ROC in bi-compartmented systems are well established for Rhizophagus and Glomus species, they are rarely successful for species within the Gigasporaceae which are generally grown in mono-compartmented in vitro cultures systems. Here, we developed an innovative membrane-separated bi-compartmented root organ culture system (MSB-ROC system), in which a 15 μm nylon mesh physically separates the root compartment from the hyphal compartment. The system was compared to the classical mono-compartmented in vitro culture system using two Gigasporaceae: Gigaspora sp. MUCL 52331 and Scutellospora reticulata CNPAB11. We demonstrated that, in both systems, auxiliary cell and spore production followed sigmoidal growth patterns (lag, log and stationary phase) but reached significantly higher abundances in the MSB-ROC system. Auxiliary cell abundance increased four-fold in Gigaspora sp. and two-fold in S. reticulata, while spore abundance increased seven-fold and two-fold (i.e., a mean of 131 and 82 spores of Gigaspora sp. and S. reticulata), respectively. These results indicate that physically separating the root and hyphal compartments enhances extraradical growth and sporulation, making the MSB-ROC system an efficient tool for non-destructive monitoring of fungal development, ecological and physiological studies, spore production, and related molecular studies.