Sexual development of Onoclea sensibilis on agar and soil media without the addition of antheridiogen

Onoclea sensibilis gametophytes grown on agar, unsterilized soil or ashed soil without the addition of antheridiogen developed antheridia after the notch meristem was formed. There was a linear increase in maleness through time on all three media. Antheridial production on agar was delayed nearly 3 wk. Agar had a significantly higher proportion of females than ashed soil or unsterilized soil. It was concluded that agar is an inappropriate medium for experiments on the mechanism of sexual development in Onoclea gametophytes. ONOCLEA SENSIBILIS HAS BEEN used as an assay organism for antheridiogens of the polypodiaceous type. According to the literature, Onoclea does not become male in agar cultures without the addition of antheridiogens (Naif 1956, 1979). However, Klekowski and Lloyd (1968) report that 45 days after sowing, a small percentage of males and bisexuals appear spontaneously in cultures of Onoclea on agar. In agar culture, Onoclea produces a substance that, upon autoclaving or exposure to low pH, acts as an antheridiogen (Naf, 1965). The current concept of sexuality in homosporous ferns indicates that an antheridiogen is secreted by the faster growing (non-male) individuals and induces the slower growing individuals to become male (Miller, 1968; Naf, 1979), although it is known that antheridia can form without the intervention of antheridiogen (Naif, 1979; Schedlbauer, 1976; Shraudolf, 1967). Antheridiogens appear to act in naturally occurring populations of gametophytes in some species (Tryon and Vitale, 1977). The results with Onoclea grown on agar lead one to wonder how Onoclea reproduces sexually in nature. With this question in mind, the sexual development of Onoclea on soil vs. artificial media, was investigated. MATERIALS AND METHODSCulture conditionsSporophylls of Onoclea sensibilis L. were collected from the town of Danby, Tompkins ' Received for publication 24 September 1982; revision accepted 29 January 1983. The authors gratefully acknowledge Dr. D. S. Robson, Biometrics Unit, Cornell University, for assistance with experimental design and statistical analysis. The authors are indebted to Dr. A. W. Blackler, Section of Genetics and Development, Cornell University, for the generous loan of his drawing apparatus. This work was supported in part by NSF Grant PCM-771854. County, New York in October 1981 and were stored in polyurethane bags at 5 ? 1 C. Spores from 25 sporophylls were pooled. Fifty-mg aliquots were pretreated with 0. 1% Triton X-405 for 10 min and then were surface sterilized with a 10% (v/v) solution of Clorox for 90 sec. Spore collection and sterilization were patterned after Cooke and Paolillo (1979). Spore suspensions were diluted with sterile distilled water such that approximately 400 spores (in 2 drops) were inoculated onto sterile 35 X 10 mm optically clear polystyrene petri plates containing medium. Three media were used: 1) Agar-Voth's No. 5 medium (Voth, 1943) was solidified with 0.8% Difco Bacto agar; 2) Soil-an Erie channery silt loam was dug from an Onoclea population site, air dried and sieved with a 2 mm soil sieve; 3) Ashed-soil prepared as above was ashed at 510 C for 8 hr (Mitchell, 1932). Soil was rehydrated in the petri plates with sterile distilled water. Ashed was rehydrated with sterile Voth's No. 5 to compensate for the loss of the organic fraction with its readily available mineral elements, and thereby improve the growth rate. Supplementation of ashed did not change the pattern of development (see RESULTS). Ashing gave more consistent germination than gamma irradiation, autoclaving and steam sterilization of soils (Rubin, 1983). The pattern of development was the same on all soils supporting growth. All petri plates were sealed with surface sterilized Parafilm. The plates were placed in growth chambers with constant light (Sylvania cool white fluorescent lamps, 200 ft-c) and constant temperature (25 ? 1 C). Sampling and statistical analysis-Each petri plate was considered a replicate. For each plate, a field seen through a Wild M5 stereomicroscope at 1 2X was mapped using a draw-