Growth of Pteridium aquilinum (L.) Kuhn Gametophytes in Submerged Culture

Abstract

The culture of fern gametophytes under aseptic conditions is commonly carried out on various mineral solutions with or without agar. In these methods the ferns develop on the surface of the culture medium. In order to obtain a higher yield of gametophyte tissue, an attempt was made to develop a method for growing these plants in submerged, agitated, liquid cultures, thereby utilizing the entire volume of the medium instead of the surface alone. Since submerged gametophytes develop very slowly, the plants were aerated by bubbling sterile air through the medium. This paper will describe the method developed and will compare the development of gametophtyes grown by this method to that of ferns grown by more standard culture procedures. Two papers were found in the literature that described the development of lower archegoniates in agitated cultures: Nakazawa (1956) reported on the germination of Equisetum spores in aerated submerged cultures, and Machlis and Doyle (1962) described a technique for the growth of liverworts, mosses, and Equisetumn in flasks agitated on a rotary shaker. Voeller (1964) has described the growth of fern gametophytes in submerged cultures that were not agitated; the development was stimulated by the addition of coconut milk to the medium. Spores of the Bracken Fern, Pteridium aquilinum (L.) Kuhn, were collected in September, 1963, in Pulaski County, Indiana, and stored under refrigeration. The spores were filtered through six layers of lens paper to remove sporangia. For agitated cultures or cultures using a medium solidified with agar,