Gametophytes of Botrychium multifidum as Grown in Axenic Culture

Mature gametophytes of Botrychium from nature have been described for several species, including B. dissectum (Bierhorst, 1958), B. japonicum (Nozu, 1954; Nishida, 1955), B. lunaria (Bruchmann, 1906), B. obliquum (Campbell, 1921), B. simplex (Campbell, 1922), and B. virginianum (Jeffrey, 1898; Bierhorst, 1958). The gametophytes are subterranean and are tuberous or somewhat elongate to button-shaped. They vary from 1-3 mm to 5-6 mm long, and even reach 1.5-2 cm in some species. The possession of a "dorsal ridge" in which antheridia are embedded is a typical feature of all the known species. The gametophytes have an associated endophytic fungus, the presence of which presumably is essential for continued growth under natural conditions. Botrychium spores appear to have a dormancy that is not easily overcome. To date, only the spores of B. virginianum (Campbell, 1895), B. ternatum (du Buysson, 1889), and more recently of B. dissectum (Whittier, 1972, 1973a) have been germinated, the last species in axenic culture. The morphology of the B. dissectum gametophytes cultured by Whittier (1972) fits the description for B. dissectum from nature, except for the presence of the endophytic fungus. Sucrose and other additives in the medium presumably replaced the contribution of the fungus under natural conditions. The gametophytes, when sexually mature, were only a few millimeters long. Whittier (1973a) has shown that light inhibits spore germination in B. dissectum. He found that a minimum of 3-4 weeks in darkness is necessary for germination to occur after eight weeks in culture. Increasing the length of the dark period increased the percentage of germination. It is interesting to note that Whittier (1973b) showed that six months of darkness were required for the germination of Psilotum spores. In this paper we will describe the morphology of Botrychium multifidum (Gmel.) Rupr. gametophytes as grown on a defined medium in axenic culture. The gametophytes of B. multifidum have never been observed carefully in nature.