SOME ASPECTS OF FERTILIZATION IN VALLISNERIA

FOR ANGIOSPERMS the interpretation of sperms within the embryo sac presents difficulties. The nucleus of the male gamete at the time of fertilization is obvious, but the question as to the presence of accompanying cytoplasm may not be easy to answer. Direct observation of living material is ruled out because of opaque coverings, and conditions in the developing pollen tube, while suggestive, do not give definite information about the morphology of the sperm at the functional stage. The brevity of the interval between the emergence of a sperm from synergid or pollen tube and its junction with the egg greatly increases the difficulty of securing material which will show its structure at the time of gametic union. But progress along other lines only stresses the need for more precise information as to what type of sperm enters into syngamy in a given case. Meanwhile there is increasing interest in plasmatic fertilization on both theoretical and practical grounds, since certain genetical phenomena find their explanation in the assumption that male cytoplasm enters the egg with the sperm nucleus. There have been many critical studies of pollen mother cells; as favorite material for research on meiotic phenomena, they have been widely investigated. The generative cell has likewise received critical attention, and its division, whether in the microspore or in the pollen tube, has been carefully traced. Male cells have been demonstrated in the pollen tubes of a considerable number of angiosperms. Finn and his students have been especially active in these studies and some of their results, with those of other workers, were briefly summarized by him (Finn, 1935). More recently, Cooper (1936) and Anderson (1939) have reviewed the later literature dealing with the sperms of angiosperms. Obviously, in many species, the sperms approach the ovule as complete cells. A few workers have presented evidence that sperms may in certain cases retain their cytoplasmic investments after entering the ovule. Nawaschin and Finn (1913) figured sperms as male cells within the ovules of Juglans regia and J. nigra which had been derived from both primary pollen tubes and from secondary tubes entering after fertilization. Tchernoyarow (1915) working with Myosurus minimus showed paired sperms surrounded by cytoplasm within the embryo sac. Most of those figured were from secondary pollen tubes, but in certain cases male cells were shown near or in contact with an egg membrane; however, the author held that only a naked nucleus united with the egg. Ishikawa (1918) showed paired sperms emerging as male cells from synergids of Oenothera but stated that only a nucleus entered into fertilization. Wylie (1923) traced the sperms of Vallisneria as male cells to the egg and suggested that some or all of the male cytoplasm probably enters into the zygote. Finn (1925) figured 1 Received for publication November 4, 1940. male cells entering the embryo sac of Asclepias cornuti, their cytoplasm being confirmed both by its structure and staining qualities. Cooper (1936) traced the development of the sperms of Lilium and figured a second male cell in the tip of a pollen tube within the embryo sac with the companion sperm as an elongated nucleus near the egg nucleus. Anderson (1936) attempted to identify the male cytoplasm by its extra-nuclear inclusions. For Antirrhinum majus, he believed that some male cytoplasm passed into the embryo sac with the male nuclei, even though male cells were not established for that stage. In a later paper Anderson (1939) demonstrated mitochondria within the cytoplasm of the male cells of three species of Lilium, at least as long as they remain in the pollen tube. While the actual union of male and female cells has not been described for any of those plants, some of the workers, the writer included, believe that in certain species male cytoplasm probably enters the egg with the sperm nucleus. In an earlier paper, the writer (1923) traced the sperms of Vallisneria, as male cells, from the microspore into the embryo sac. Many preparations slhowed sperms emerging from the synergids as complete cells, with evident cytoplasm bordered by an external membrane. A few male cells from primary tubes were observed after release from the synergid and lying near its tip. One case was figured in that paper (Wylie, 1923) showing the sperms as male cells in contact with the egg membrane. Increasing interest in the morphology of the functioning male gamete of angiosperms particularly as related to plasmatic fertilization has invited further study of this form. The results here presented afford some additional information regarding the sperms within the embryo sac of Vallisneria and support the probability that in this species male cytoplasm enters the egg cell at the time of fertilization. In all such studies there is the common problem of finding sperms from primary pollen tubes inside the embryo sac; this difficulty is in part due to the short time between their emergence and fertilization. For Vallisneria fixation also involves physical problems because of the heavv, viscous material that fills the ovarian chamber about the ovules and through which reagents penetrate slowly. As outlined in the earlier paper (Wylie, 1923), favorable fixation was achieved only after dissolving this slime, otherwise all ovules with pollen tubes had completed fertilization before penetration of the fixing agent. To retard growth, material was cooled as soon as collected; subsequently, the gelatinous matrix including the numerous ovules was removed from a large number of ovaries and placed in cold water for several hours. This slowly dissolved the matrix and after repeatedly decanting, and adding fresh volumes of cooled water, enough slime was removed to permit favorable fixation by the use of Flemming's stronger solution. Material handled in this way gave