Histological Features of Anthers from Normal and ms3 Mutant Soybean

Anthers of male-sterile mutant (ross) and normal soybean [Glycine max (L.) Merr. ] were compared by using a triple*staining procedure for DNA, polysaccharides and proteins, and a singlestaining procedure for sporopollenin in conjunction with brightfield and fluorescent microscopy. The purpose of this study was to determine at what stage of anther development histoogical differences were detectable between male-sterile (mss) and normal soybean. At the tetrad stage, a prominent greenish-blue material, visible with the triple-staining procedure, filled some of the enlarged tapetal cells of anthers of mss plants. This material was also autofluorescent and had an affinity for the fluoo rochrome primuline which identified sporopollenin. By the early microspore stage, microspores of mss plants were unusually enlarged and the tapetum surrounding them prematurely degenerated. This abnormality of tapetal behavior seems to be associated with microspore abortion and serves as an early marker for sterility. Other differences noted between the anthers of mss and normal plants with the staining procedures used, were variations in the endothecium and the thickness and degree of staining of the microspore and pollen walls. All of these differences suggest that the effect of the mss gene in the homozygous condition is expressed during early anther development, probably beginning at meiosis and directly influences the malfunctioning of the tapetum. The results of this study support numerous other published reports on both genetic and cytoplasmic male sterility implicating the tapetum as the primary tissue responsible for male sterility. Additional index words: Genetic male sterility, Microsporogenesis, Tapetum, Sporopollenin. T HF~ causal mechanisms of cytoplasmic (Edwardson, 1970; Laser and Lersten, 1972) and genetic (Jain, 1959; Gottschalk and Kaul, 1974) male sterility in plants are not understood. Male sterility has been of practical use for plant breeding and commercial seed production in certain crops such as maize (Zea mays L.), sugarbeet (Beta vulgaris L.), onion (Allium cepa L.), sorghum [Sorghum bicolor (L.) Moench], and wheat (Triticum aestivum L.). Breeders widely employ male sterility in self-pollinated crops as well as in cross-pollinated crops. Recently, five types of male-sterile soybean [Glycine max (L.) Merr. ] plants (Brim and Young, 1971; Bernard and Cremeens, 1975; Palmer et al., 1980; Delannay and Palmer, 1982; Buss, 1983) and two partial male steriles (Caviness et al., 1970; Stelly and Palmer, 1980) have been reported. Their inheritance and cytological features have been noted. Palmer et al. (1980) reported the ~. enetic and cytological characteristics of male sterifity caused by the mss gene. The present study was conducted to clarify some features of male sterility in soybean associated with the ms~ gene, especially to determine the time that abortion occurs and to identify any significant histological features that would easily distinguish the sterile plants during early development. This work is part of a larger study being conducted at the cellular and molecular levels on mss and other soybean sterility mutants. MATERIALS AND METHODS Normal and mss mutant plants (genetic type collection number T273) of soybean were grown in the glasshouse and the field at Iowa State University, Ames. The same mutant has been studied previously by Buntman (1983), Buntman and Horner (1983), and Palmer et al. (1980). mss mutant is controlled by a single recessive gene. Maturing flowers were examined to make sure of the fertility of the plants used. Buds from both lines were fixed at various stages of development in formalin-acetic acid-alcohol at room temperature and stored in the refrigerator until examined. After ethanol dehydration and xylene infiltration, buds were embedded in paraffin. Sections were cut at 8 ~m and stained by either of the following two procedures. The triple-staining procedure of Himes and Moriber (1956) includes azure-A and 1N hydrochloric acid hydrolysis that stains nuclear DNA blue to green, leucofuchsin and periodic acid hydrolysis that stains water-insoluble polysaccharides red, and naphthol yellow S that stains proteins yellow. Sections stained as just described were observed with brightfield microscopy. The second staining procedure employs primuline in a 0.1 M phosphate buffer (pH 7.0) for 5 rain (Waterkeyn Bienfait, 1971). This stain localizes sporopollenin via fluorescence. Deparaffinized sections were brought to water and either were stained with primuline or left unstained. Sections were observed with phase contrast and fluorescence microscopy (heat-BG 38, UV-UG 1, and barrier 430 filters were used in conjunction with a mercury-vapor light source). A Leitz Ortholux compound microscope with an Orthomat camera system were used to view and photograph the material.