Spore Germination and Young Gametophyte Development of Botrychium and Ophioglossum in Axenic Culture

Abstract

Spores sown in axenic culture required darkness, took weeks to germinate, and, at least for B. dissectum, produced a three-dimensional gametophyte immediately after germination (Whittier, 1973; Gifford & Brandon, 1978). None of the earlier reports by Campbell and du Buysson gave any indication that darkness was required for germination. In soil cultures, 0. moluccanum, 0. vulgatum, and B. ternatum germinated in a few days, which is a period comparable to that for spores of the Polypodiaceae (Campbell, 1907; du Buysson, 1889). Young gametophytes of O. vulgatum and B. ternatum were illustrated by du Buysson (1889) with filamentous and two-dimensional growth habits which are similar to those for polypodiaceous gametophytes. Consequently, the present study of additional species was carried out in axenic culture to determine how the requirements for germination and the type of early gametophyte development for these species compared with the results from previous studies. MATERIALS AND METHODS Spores of eight Ophioglossaceae species, Botrychium biternatum (Sav.) Underw., B. dissectum Spreng. var. dissectum, B. lunarioides (Michx.) Swartz, B. matricariifolium A. Braun, B. virginianum (L.) Swartz, Ophioglossum engelmannii Prantl, and 0. vulgatum var. pycnostichum Fern. and var. pseudopodum (Blake) Farw., were used in this study. With the exception of the spores of B. matricariifblium and 0. vulgatum var. pseudopodum, which were from Ontario, the spores were collected in the southeastern United States. Voucher specimens are on deposit in the Vanderbilt University Herbarium (VDB). The techniques of Whittier (1973) were employed. The spores were sown on 15 ml of nutrient medium in culture tubes with a diameter of 20 mm. The tubes had screw caps which were tightened to reduce moisture loss. The nutrient medium was composed of Knudson's solution of mineral salts, minor elements, FeEDTA, and 0.6% agar (Whittier, 1973). The medium was supplemented with 0.5% sucrose and had a pH of 6.3. The spores were cultured at 24+ 1?C in the light at an intensity of