DEVELOPMENTAL FEATURES OF THE SPERMATOGENOUS CELL IN GINKGO BILOBA

A "double-blepharoplast" originates de novo in the spermatogenous cell of Ginkgo biloba L. Initially, the double-blepharoplast consists of two opposing hemispherical bodies comprised of densely staining material. The two blepharoplasts seemingly are pushed apart by the formation of densely packed fibrils which are oriented perpendicular to the distal, rounded edges of the two future blepharoplasts. As the latter move apart, each one develops lightly staining channels which are often organized in a hub and spoke configuration (procentrioles). Microtubules extend from the blepharoplasts as the latter move to their final position in the cell, and centrioles (probasal bodies) become organized at the periphery of each blepharoplast. Two large "osmiophilic globules," conspicuous entities close to the nucleus of the mature spermatogenous cell, arise de novo. A fibrillogranular body in the cytoplasm, always closely associated with the nucleus, also arises de novo. CYCADS and Ginkgo biloba are the only known seed plants that produce motile sperms. One of the interesting features of these plants is the occurrence of distinctive organelles (blepharoplasts) in the spermatogenous cell (the cell which gives rise to the sperms; Sterling, 1963). Blepharoplasts form probasal bodies (centrioles) at their periphery, each of which becomes differentiated into a basal body of a flagellum at the time motile sperms are formed. In bryophytes and lower vascular plants (e.g., Equisetum, ferns), a de novo origin of centrioles has generally been accepted because they do not appear in cells of the vegetative body. In a recent study, Hepler (1976) has shown that the blepharoplast is formed de novo in Marsilea and is a precursor organelle for the production of procentrioles which will become the centrioles (basal bodies) of flagella of the motile sperms. The Marsilea blepharoplast arises first during the 7th mitotic division of the male gametophyte, soon degenerates and then reappears during the 8th division, near the nuclear membrane. Paolillo (1974) concluded that centrioles (two per sperm) arise de novo in the moss Polytrichum during early prophase of the ultimate mitotic division of each spermatogenous cell. There are other examples of de novo formation in plants and animals (see Paolillo, 1974; Hepler, 1976). For the cycad Zamia, Mizukami and Gall (1966) described the ultrastructure of the blepharoplast and the transformation of procentrioles into basal bodies of flagella. Norstog (1967, 1968, 1974) described in detail the ultrastructure of the sperm in Zamia, and Gifford 1 Received for publication 28 March 1979; revision accepted 17 July 1979. and Lin (1974, 1975) reported on the organization of the blepharoplast as well as on other features of the fully enlarged spermatogenous cell in Ginkgo. In this paper we describe the de novo origin of the blepharoplasts and the origin of other distinctive structures in the cytoplasm of the young spermatogenous cell of Ginkgo biloba. MATERIALS AND METHODS-Ovules of Ginkgo biloba L. with enclosed pollen grains were collected at intervals of every three or four days from trees in Sonoma, Sonoma County, and Davis, Yolo County, California, from the time of pollination (generally first two weeks in April) through liberation of sperms (generally first two weeks in September). When the ovules were small, a plug of tissue was excised at the micropylar end which included a portion of the integument and the underlying nucellus. As the ovules became larger, it was necessary to remove the integument and fix only a segment of the nucellus to which the developing male gametophytes were attached. Approximately 250 gametophytes were examined. The material was fixed in 4% glutaraldehyde, phosphate buffer (0.15 M), pH 6.8, for 3 hr at room temperature. In some instances the tissue remained in the buffered fixative overnight. No detectable differences were evident in the two procedures. After several rinses with the buffer, the material was then post-fixed in buffered 1% OS04 for 1 hr, after which the tissue segments were rinsed in buffer, dehydrated in a graded series of ethyl alcohol and then embedded in Spurr's low-viscosity resin. The grids were stained with uranyl acetate-lead citrate or with KMnO4 (30 min in aqueous KMnO4