THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation. IN GERMINATING SPORES of the sensitive fern, Onoclea sensibilis L., rhizoid formation is preceded by nuclear movement and asymmetric division that partitions each spore into a large cell and a small lens-shaped cell. The large cell and its derivatives can divide repeatedly to form a filamentous protonemata and then a flattened prothallus, whereas the small cell elongates and differentiates into a rhizoid. Many of the details of germination have been described and the sole requirements appear to be water, oxygen, light, and optimum temperature (Towill and Ikuma, 1973; 1975; Towill, 1978). The onset of nuclear movement occurs about 16 hr after initiation of germination and several hours after nuclear DNA synthesis (Fisher and Miller, 1978). At this time, the nucleus migrates from the center to the proximal (flattened) side of the spore where the raphe is located. It then moves along the raphe to one end of the spore where cell division will occur (Bassel, Kuehnert and Miller, 1981). The nucleus migrates at about 0.26 ,um min-' and, during migration, undergoes changes in shape (Vogelmann and Miller, 1980). Movement appears to be mediated by microtubules (Bassel et al., 1981; Vogelmann, Bassel and Miller, 1982). Polar rhizoid formation can be prevented by 1 Received for publication 15 October 1979; revision accepted 8 June 1981. 2 Present address: Institute of Plant Physiology, Lunds Universitet, Box 7007, 220 07. Lund, Sweden. 3 Address J.H.M. for reprints. germinating spores in the presence of methanol which also inhibits nuclear movement (Miller and Greany, 1976). Under these conditions, a symmetric cell division occurred in place of the initial normally highly asymmetric one and each spore was partitioned into daughter cells of equal size. Neither cell differentiated into a rhizoid; rather, both cells elongated, divided, and produced "twin" filaments of cells that were joined at the base. Other presumably membrane-active compounds such as ethanol, chloroform, diethyl ether, and chloral hydrate have similar effects (J. H. Miller, unpubl.) but methanol is easier to work with because it is less toxic to the spores than ethanol and chloral hydrate and less volatile than chloroform or diethyl ether. Agents that destroy or alter cellular polarity are of interest because they can provide insight into the processes that control polar development (Weisenseel, 1979). This paper describes in more detail the effects of methanol on the kinetics of nuclear migration and cell division. It establishes that there is a specific period during germination when methanol inhibits subsequent rhizoid differentiation. The inhibitory effects of methanol appear to be completely and rapidly reversible when spores are removed from the alcohol. MATERIALS AND METHODS-Spores and culture conditions-Sporophylls of Onoclea sensibilis L. were collected near Syracuse, New York in November 1976 and were stored in plastic bags at 4 C. Spores were isolated using the methods described by Stockwell and