Arbuscular mycorrhizal (AM) fungi are obligate biotrophic organisms. Root organ culture (ROC) can be used to grow these fungi under in vitro conditions. The ROC technique was used with Gigaspora margarita and Ri T-DNA transformed carrot roots to examine the fungal growth and physiology under long-term axenic symbiosis. The fungus formed spores inside the host roots under in vitro conditions. Sporulation was a temporal phenomenon found in dual cultures more than 18–20 mo old. The spores were formed singly or in rare cases clusters of two or three. No preferential zone of formation was found. The spores formed intraradically were 10–15% of the total spores formed in a single culture. These intraradical spores were analysed for their morphology and DNA polymorphism pattern with spores which had formed conventionally in the medium. Both analyses showed no detectable variation. This is the first report of spore formation by Gigaspora inside the roots of a host.