APOGAMY AND SOMATIC RESTITUTION IN THE FERN CERATOPTERIS

A mutant stock of the fern Ceratopteris has been derived from an inbreeding study following an interspecific hybridization between two diploid species. The mutant is characterized by gametophytes that produce non-functional spermatozoids and are incapable of selfing. Sporophytes develop apogamously from the mutant gametophytes and, although they are initially haploid and sterile, portions of the fronds later become doubled somatically and behave like tissues of sexually derived homozygous sporophytes. The mutant segregates from sporophytes in a 1:1 ratio when crosses are made with wild type gametophytes. Certain aspects of the behavior are similar to those seen in some naturally occurring apomictic ferns. PREVIOUS studies of Ceratopteris by the author have documented various aspects of normal and mutant reproductive behavior within this polyploid homosporous fern genus. These studies have included the demonstration of temperaturesensitive meiotic pairing mutants (Hickok, 1977a), meiotic restitution and chromosome stickiness associated with the origin of an apomictic life cycle (Hickok, 1977c), and homoeologous chromosome pairing in homozygous, F1 hybrid, and hybrid-derived sporophytes (Hickok, 1978a, b). This genus continues to provide an experimental system within which various normal and aberrant aspects of the genetic system of a polyploid homosporous fern can be investigated. This capability is important in that homosporous ferns in general are difficult to work with cytologically and genetically and, as a result, our knowledge of their genetic potentialities is limited by a lack of experimental studies. This report documents irregular behavior within a diploid series that involves somatic restitution in sporophytic tissues. This behavior is associated with the apogamous production of sporophytes from gametophytes that are incapable of selfing because of non-functional spermatozoids. The somatic restitution converts apogamously produced haploid sporophyte tissue into fertile, diploid sporophyte tissue. MATERIALS AND METHODS-The mutant series was derived from an inbreeding study following a hybridization between two diploid species, C. richardii Brongn. and C. pteridoides ' Received for publication 5 February 1979; revision accepted 29 April 1979. This research was supported by NSF grants DEB76-09756 and DEB78-02809. I thank Mr. Jim Wiseman for excellent technical assistance. (Hook.) Hieron. (n = 39). Morphological and cytological characteristics of this hybrid and the techniques associated with inbreeding are referred to by Hickok (1977b). All culture methods and cytological techniques for meiotic material are described by Hickok (1977c). Somatic counts of gametophyte and sporophyte tissues were made by modification of the technique described by Gastony and Haufler (1976) using the enzyme "Glusulase" (Endo Laboratories, Garden City, N.Y.). In the present study the meristematic material to be examined was treated with 0.1% colchicine for 9 hours at 23 C and subsequently fixed in 3:1 ethanol/glacial acetic. The fixed material was rinsed in 45% acetic acid and soaked in 1% acetic acid for 10 min. This material was then treated for 3-5 hours in a 2:3 mixture of "Glusulase" and 1% acetic acid prior to squashing and staining. Except where noted, all mitotic counts in this paper are approximations that were made only to determine if the tissue was haploid (n = 39) or diploid (2n = 78). RESULTS AND DISCUSSION-In the F5 generation of the inbreeding study 25 gametophyte isolates were established from the spores of an individual sporophyte, HFIJ-2-2-8-8. These were watered weekly and by 100 days after the initial sowing of the spores all but one of the gametophyte isolates produced a sporophyte. During this time some apogamous activity was evident in the cultures in the form of thickened, vascularized projections from the gametophytes. However, although detailed examinations were not made, sporophyte production appeared to be completely sexual through self fertilizations. An examination of mitotic divisions within a portion of the single gametophyte without a sporophyte revealed that it was haploid. Later examinations (see below) indicated that this gametophyte was probably incapable of selfing because of non-