Discovery of an endophytic alga in Ginkgo biloba

Although intracellular associations with mycorrhizal fungi are known for Ginkgo biloba, no other endosymbiotic relationships have ever been reported for this living fossil. A protoplast culture derived from haploid explants has now revealed the existence of a green alga in vitro, whose eukaryotic status was confirmed by transmission electron microscopic studies. Phylogenetic 18S rDNA sequence analyses showed this alga to be closely related to the lichen photobiont Coccomyxa. Algae, which in host cells exist as more or less undifferentiated precursor forms, proliferated within necrosing G. biloba cells of a subculture derived from a zygotic embryo and were finally released into the medium. Light and electron microscopic observations showed that G. biloba cells rapidly filled up with countless green particles whose number increased up to the bursting of the hypertrophic host cells. At the beginning of reproduction no algae were visible in the nutritive medium, demonstrating that the proliferation started inside the G. biloba cells and excluding the possibility of an exogenous contamination. Occasionally, mature algae together with their precursor forms were detected by transmission electron microscopy in intact host cells of a green callus. The algae were easily identified by their similarity to the cultured algae. Eukaryotic algae have never been reported to date to reside inside higher plant cells, whereas several algal associations are well known from the animal kingdom.