AN ELECTRON MICROSCOPE STUDY OF GERMINATING LYCHNIS ALBA POLLEN

The fine structure of germinating Lychnis alba pollen is described and correlated with some basic tests for chemical composition. The primary storage product in both pollen grain and pollen tube appears to be lipid. Pregermination synthesis of pollen tube wall material appears characteristic in this species, along with the presence of crystalloid structures having approximately 80 A periods. The crystalloid bodies are not found in the tip cytoplasm of the pollen tubes. Limited acid phosphatase activity is found associated with the crystalloid structures as well as within vesiculate structures of the pollen grain. The cytoplasmic structure of both the pollen grains and pollen tubes is characterized' by few dictyosomes and plastids and no microtubules, although mitochondria and polyribosomes are abundant. Pectins have not been verified in the pollen tube walls, their primary composition being cellulose. LYCHNIS ALBA AlVill. (family Caryophyllaceae) is a dioecious, long-day plant, bearing pollen typically in five anthers, each of which is two-celled. At anthesis the pollen is airborne to the styles of pistillate flowers where germination and intercellular penetration of the pollen tube occurs (Crang, 1966). Ultrastructural changes in the development of the pollen tube occur with considerable rapidity and must be intimately involved with the assumption of new physiological functions. In previous reports (Chambers and Godwin, 1961; Larson and Lewis, 1961, 1962; Rosen, 1964; Rosen et al., 1964; Sassen, 1964a, b; Larson, 1965a, b; Rosen and Gawlik, 1966; Kroh, 1967) a background literature has been established outlining basic ultrastructural organization of pollen grains and pollen tubes in a number of plant species. That the cytoplasm. and wall organization of Lychnis alba germinating pollen grown in vitro possesses variations in fine structure from previous accounts is to be presented in this paper. MATERIALS AND METHODS Greenhouse-grown Lychnis alba plants at anthesis were periodically harvested for pollen which was germinated at 24 C in sterilized nutrient solution (Brewbakei and Kwack, 1963). The tubes were allowed to achieve lengths up to 200,u. The germinating pollen tubes were either observed unstained by bright-field light microscopy or prepared for observation by electron microscopy. In the latter case gentle centrifugation preceded fixation in 1 Received for publication 12 August 1968. Study supported in part by a grant from the BrownHazen Fund of the Research Corporation. The authors gratefully acknowledge the assistance of Mr. Kenneth W. Bladh. 2 Present address: Philadelphia College of Osteopathic Medicine, Philadelphia, Pa. 6.5 % glutaraldehyde buffered to a pH of 7.2 with 0.05 M sodium cacodylate. The pollen tubes in the buffered fixative were cooled to 4 C over a period of 2 hr; the sample was then washed' with cold buffer and postfixed in cold 2 % Palade's osmium tetroxide (pH 7.2) for an additional 2 hr. In the authors' experience such alternate fixations as potassium permanganate and osmium tetroxide alone have not proved to present as good a fixation in pollen tubes as that outlined above and were therefore not employed. After washing and dehydration in ethanol and propylene oxide, the pollen material was embedded in Epon 812 (Luft, 1961). Sections were cut on a PorterBlum MT-2 ultramicrotome using a diamond' knife and were collected on 200-mesh formvarsupported grids and 400-mesh unsupported grids. Post-staining of the sections in uranyl acetate (Pease, 1964) and lead citrate (Reynolds, 1963) preceded examination with a Zeiss EM-9A electron microscope equipped with a 50,u objective aperture. Electron microscopical localization of Gomori acid phosphatase activity was performed after the method of Seeman and Palade (1967). Localization of other primary components of the pollen grain and pollen tube were performed at the light microscope level using living material and 1 % aqueous IKI for starch, Sudan IV for lipids, 0.02 % aqueous ruthenium red for pectins and IKI-H2SO. for the presence of cellulose (Jensen, 1962). RESULTS-Prior to germination swelling at the apertures of the pollen grains was prominent and the cytoplasm in the apertures was highly vesiculate (Fig. 1). The internal cytoplasm of the pollen grain was less vesiculate although it contained many lipid bodies. As pollen tube