Germinating Spores and Growing Sporelings of Aquatic Isoetes

Abstract

Several techniques have been described recently for germinating spores of aquatic species of Isoetes which grow in lakes, ponds, and streams in eastern North America. Boom (1980) used glass vials containing sterile sand and pond water in which he successfully raised sporelings of I. engelmannii, I. flaccida, I. macrospora, and I. riparia. Kott and Britton (1982) germinated spores of I. acadiensis, I. echinospora, I. macrospora, I. riparia, and I. tuckermanii in small vials half filled with distilled water. However, many of their vials became contaminated with fungi that destroyed the viability of the megaspores. Sam (1982) germinated megaspores of I. engelmannii in an inorganic nutrient medium supplemented with Nystatin to inhibit fungi. Webster (1979) described a technique for germinating spores of Selaginella that is similar to the following procedure we use for Isoetes. We have been culturing plants from spores of Isoetes acadiensis, I. echinospora, I. engelmannii, I. hieroglyphica, I. macrospora, I. riparia, and I. tuckermanii for use in germination and hybridization experiments for several years. The aquatic species of Isoetes are ideal pteridophytes for germination and hybridization experiments. Their sporangia are large and contain many spores. Megaspores and microspores are borne in different sporangia on separate leaves. If necessary, unopened sporangia can be excised from sporophylls intact and surface cleansed to eliminate extraneous spore contamination. Thus, megaspores and microspores can be completely isolated for controlled breeding experiments. Also, their spores readily germinate in demineralized water and gametophytes and sporophytes develop normally for several months without supplemental nutrients. Spores are obtained from plants collected in September and October, since plants harvested earlier in the year may not have mature, viable spores. Isoetes hieroglyphica and I. macrospora retain their spores through the winter, so mature spores of these species can also be gathered in the spring. Megaspores are cleansed of microspores in a sieving apparatus made of plastic tubing approximately 10 cm long and 2.5 cm in diameter. An 8 cm square of 0.27 mm sifter mesh (available from Carolina Biological Supply Company) is fitted to one end of the tubing with a rubber band. The sieving apparatus, containing megaspores, is attached to a water faucet using plastic strapping tape. Spores are washed with a steady flow of cold water for 30 minutes, followed by a one minute rinse with sterile, demineralized water. Although megaspore surfaces appear clean after the above treatment, unopened sporangia are preferred for crossing experiments because there is less chance of contamination from microspores. Intact sporangia can be excised from