Arbuscular mycorrhizal (AM) symbiosis is an important biological breakthrough which assisted plant land colonization over 400 million years ago. This widespread mutualistic interaction between fungi and plants enhances nutrient exchange, ecological sustainability, plant stress resistance, and host plant development. AM symbiosis improves plant nutrition by deriving nutrients through both mycorrhizal pathways and the Plant’s own pathways. AMF influence nutrient availability by altering soil properties, microbial populations, and nutrient cycling. Understanding the life cycle of AMF, spore germination, sporulation, colonization, and symbiosis formation are critical for large-scale agricultural applications. Root organ culture (ROC) techniques offer intriguing possibilities to mass producing AMF under in vitro. This review surveys the literature on these topics, focusing on methods for enhancing sporulation in in vitro. Enhancing in vitro sporulation can be achieved by supplementing growth media with phenolic compounds, fatty acids, and phytohormones, and optimizing the media and related factors. These compounds regulate fungal growth and development, leading to increased sporulation and improved AMF inoculant efficacy. Further research is needed to provide quality inoculum and develop crop-specific formulations and delivery methods to harness the potential of AMF in diverse agroecosystems.