Megasporogenesis and megagametogenesis in soybean (Glycine max)

Megasporogenesis and megagametogenesis were examined in Glycine max with light, fluorescence, and electron microscopy. Megasporogenesis results in a lineartetrad offour megaspores. Megagametophyte development is ofthe Polygonum type, with the functional chalazal megaspore undergoing three successive mitotic divisions to produce an eight-nucleate, seven-celled mature megagametophyte. The central cell becomes packed with starch. At fertilization, the antipodals are degenerate, the polar nuclei have fused, starch is diminished, and the egg occupies most of the micropylarportion ofthe megagametophyte. Several pollen tubes were occasionally observed at each micropyle, yet only one was involved in fertilization. Pollen tube entry occurs through a slightly reduced, viable synergid cell. Endosperm development precedes embryo growth. These results describing normal development allow important comparison with genetic mutants of soybean that affect female fertility. In the interest of comparing the influence of male-sterile genes on female fertility in certain genetic mutants of soybean (Kennell and Horner, 1985), it was important to document normal development using procedures that provided optimum preservation and resolution, as has been demonstrated with other species (e.g., maize; Russell, 1978). The present paper reports cytological and anatomical aspects of the soybean megagametophyte from the archesporial cell to the young embryo by utilizing an ovule-clearing technique and resin-embedded material. The latter material also allows for ultrastructural observations by using a reembedding procedure of selected thick sections. MATERIALS AND METHODS Plants of Glycine max (L.) Merr. (genetic type collection numbers T260H, T266H, T267H, and T268H) were grown in the field, glasshouse, or growth chamber. Whole-ovule clearings-Gynoecia were dissected from flower buds and fixed in chromium-acetic acid-formaldehyde overnight. The gynoecia were sliced open to expose the ovules, bulk stained with Mayer's Hemalum for 2030 min, and destained in 2% acetic acid for 2 min. Dehydration was carried out in an ethanol (EtOH) series to 100% EtOH and gradually replaced with methyl salicylate (Stelly et al., 1984). Ovules then were removed from the gynoecia and mounted on Raj slides (Raj and Herr, 1970) in methyl salicylate. Observations and photomicrographs were made by using interference-contrast optics. HAVING UNDERGONE a sevenfold production increase in the past 40 yr, soybeans (Glycine max L. Merr.) are currently second in value amongallU.S. crops (Anonymous, 1984). Their increased importance has led to a great number of genetic and physiological studies; however, few reports exist concerning the morphological and anatomical nature of the soybean flower particularly the female reproductive system. There are relatively few studies characterizing the various developmental stages of the soybean gynoecium (Table 1). These studies employed diffierent procedures associated with microscopy. The first and most complete study, including both megasporogenesis and megagametogenesis, was by Pamplin (1963), but these observations of paraffin-embedded material were never published. Other studies (Table 1) have concentrated on a particular stage or were limited by the resolution associated with paraffin-embedded material (Prakash and Chan, 1976). 1 Received for publication 23 November 1984; revision accepted 7 May 1985. Journal Paper No. J11688 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2539. This study was supported by a USDA Competitive Grant 59-2191 1 1 -760-0 awarded to H. T. Horner and R. G. Palmer, and the work was carried out in the Bessey Microscopy Facility. The authors thank Drs. R. A. Graybosch, N. R. Lersten, and R. G. Palmer for their helpful criticisms of the manuscript. We also thank Lillian Johnson for typing the manuscript. Address all correspondence to: Professor H. T. Horner Department of Botany, 3A Bessey Hall, Iowa State University, Ames, IA 50011; telephone 515/294-8635 or -3872. 2 Present address: Department of Plant Pathology, University of Florida, Gainesville, FL 32611.