A CYTOCHEMICAL STUDY OF EMBRYO SAC DEVELOPMENT IN STELLARIA MEDIA

The caryophyllad type of embryogenesis in Stellaria media was investigated using topological cytochemical methods for the demonstration of nucleic acids, proteins and polysaccharides. The primary suspensor cell initially contained high levels of cytoplasmic RNA, but these declined rapidly after the first few cell divisions. Cytoplasmic protein levels were high throughout the existence of the primary suspensor cell, becoming concentrated into proteinoplasts at the time of cotyledon initiation in the embryo. These plastids were RNAand DNA-negative, and only slightly positive with methods for polysaccharides and lipids. Cytoplasmic and nucleolar RNA and protein levels were high in the embryo throughout its development. The cotyledons and endosperm also showed high RNA and protein levels. Cytoplasmic DNA was present in the embryonic apical meristematic cells in the form of Feulgen-positive, deoxyribonuclease-removable granules. Cytoplasmic histones were present in the embryo and endosperm. Polysaccharides were demonstrable in the cotyledons, cortex, endosperm, and nucellus. ALTHOUGH the embryology of a great number of plants has been described in considerable morphological detail, very little information is currently available concerning the physiological and biochemical aspects of plant growth and differentiation. Since higher plant embryos are enclosed in seeds, they have been, with few exceptions, technically less amenable to experimental manipulation than many of the lower animal forms. For this reason, comparatively little experimental embryological work has been done with plants. Two main technological developments in experimental biology have made functional investigations of plant embryos feasible: cytochemistry and in vitro embryo culture methods. In spite of the fact that botanical cytologists made early use of cytochemical techniques, the application of this technology to problems of plant physiology and biochemistry has lagged considerably behind its use in zoology. Gifford and Tepper's (1962a,b) study of the apical meristem, and Jensen's (1963) study of the cotton embryo represent 2 principal exceptions to this. I Received for publication September 12, 1963. This investigation formed a part of a Ph.D. dissertation submitted to Lehigh University, Bethlehem, Pennsylvania. The work was completed at the Department of Pathology, J. Hillis Miller Health Center, University of Florida, under support from University of Florida Grant # 6294H03, Cancer Training Grant # CT 5068 (C-6) and NIH research grant #RG-10003-02 to Dr. R. R. Cowden. The author is indebted to Dr. R. R. Cowden for his invaluable assistance, encouragement and advice in the experimentation and preparation of the manuscript, and to Dr. J. L. Edwards, Head of the Department of Pathology, for use of the facilities of that department. The previous portion of this study (Pritchard, 1964) was concerned with the distribution of 3 major classes of macromolecular compounds, i.e., nucleic acids, proteins, and polysaccharides during the development of the megaspore of Stellaria media (L.) Cyrill. The present study represents an extension of this investigation to include embryogenic development. Stellaria media, the common chickweed of the family Caryophyllaceae, was selected for this study because of its cosmopolitan distribution and year-long blooming period which made ovaries easily available at any time. According to the M\Jaheshwari (1950) classification, S. media undergoes a caryophyllad type of embryonic development. Development in this genus has previously been described by Crete (1947) for S. uliginosa Murr. and by Pal (1953) for S. media. Embryos of the latter, moreover, have been grown in vitro by White (1954), this offering further avenues for experimental studies of its development. MATERIALS AND METHODS-Specimens of S. media were grown in the greenhouse. After the plants had become established and in full bloom, all the blossoms were clipped off so that the following day would yield all new "first-day" blooms. These blossoms were pollinated using a camel's hair brush, and marked with colored threads. In this manner, several series of ovaries were obtained varying from 1-10 days old. Ovaries were killed and fixed in 3 different fixatives: FAA, Carnoy's, and 10%o neutral formalin for histone staining. Tissues fixed in FAA remained in that fluid for 3 days to 3 weeks before they were dehydrated