Botany Publication and Papers Botany 10-1993 Nuclear Size and DNA Content of the Embryo and Endosperm During their Initial Stages of Development in Glycine max (Fabaceae) Mark A. Chamberlin Iowa State University Harry T. Horner Iowa State University, [email protected] Reid G. Palmer United States Department of Agriculture Follow this and additional works at: http://lib.dr.iastate.edu/bot_pubs Part of the Agronomy and Crop Sciences Commons, Botany Commons, Plant Biology Commons, and the Plant Breeding and Genetics Commons Recommended Citation Chamberlin, Mark A.; Horner, Harry T.; and Palmer, Reid G., "Nuclear Size and DNA Content of the Embryo and Endosperm During their Initial Stages of Development in Glycine max (Fabaceae)" (1993). Botany Publication and Papers. 37. http://lib.dr.iastate.edu/bot_pubs/37 This Article is brought to you for free and open access by the Botany at Iowa State University Digital Repository. It has been accepted for inclusion in Botany Publication and Papers by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Nuclear Size and DNA Content of the Embryo and Endosperm During their Initial Stages of Development in Glycine max (Fabaceae) Abstract A technique was developed for isolating embryo sacs from ovules of soybean and for separating embryo from endosperm. Image analysis and cytophotometry were used to determine the relative mass of DNA and size of nuclei of endosperm and embryo cells. Analyses were done at the globular through late heart-shaped embryo stages to correlate ploidy level or nuclear size, and differentiation in these tissues. Mean size of embryo nuclei was fairly constant through all stages studied. Ploidy condition of the embryo was stable, 950/o99% of the nuclei were distributed in a bipolar pattern by relative mass at 2C and 4C. Few embryo nuclei (3%) had ploidy levels above 4C at the late heart-shaped embryo stage. Variability in size ofendosperm nuclei seemed correlated with the morphological state of these nuclei (free-nuclear vs. cellular). Most endosperm cells did not show significant polyploidy with 840/o-92% of nuclei in the expected 3C-6C range, but some nuclei with elevated ploidy levels were noted during endosperm cellularization. Endosperm senescence was correlated with nuclear DNA loss over time. Polyploidy seems to have no direct role in the early differentiation of the soybean embryo and endosperm, but these stable conditions may be necessary for the early establishment of the embryo. Disciplines Agronomy and Crop Sciences | Botany | Plant Biology | Plant Breeding and Genetics Comments This article is from American Journal of Botany 80 (1993): 1209. Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/bot_pubs/37 Nuclear Size and DNA Content of the Embryo and Endosperm During their Initial Stages of Development in Glycine max (Fabaceae) Author(s): Mark A. Chamberlin, Harry T. Horner and Reid G. Palmer Source: American Journal of Botany, Vol. 80, No. 10 (Oct., 1993), pp. 1209-1215 Published by: Botanical Society of America, Inc. Stable URL: http://www.jstor.org/stable/2445550 Accessed: 14-06-2016 19:56 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. Botanical Society of America, Inc. is collaborating with JSTOR to digitize, preserve and extend access to American Journal of Botany This content downloaded from 129.186.1.55 on Tue, 14 Jun 2016 19:56:21 UTC All use subject to http://about.jstor.org/terms American Journal of Botany 80(10): 1209-1215. 1993. NUCLEAR SIZE AND DNA CONTENT OF THE EMBRYO AND ENDOSPERM DURING THEIR INITIAL STAGES OF DEVELOPMENT IN GLYCINE MAX (FABACEAE)1 MARK A. CHAMBERLIN,2 HARRY T. HORNER,2,4 AND REID G. PALMER3 2 Department of Botany and Bessey Microscopy Facility; and 3USDA-ARS FCR, Departments of Agronomy, and Zoology and Genetics, Iowa State University, Ames, Iowa 50011-1020 A technique was developed for isolating embryo sacs from ovules of soybean and for separating embryo from endosperm. Image analysis and cytophotometry were used to determine the relative mass of DNA and size of nuclei of endosperm and embryo cells. Analyses were done at the globular through late heart-shaped embryo stages to correlate ploidy level or nuclear size, and differentiation in these tissues. Mean size of embryo nuclei was fairly constant through all stages studied. Ploidy condition of the embryo was stable, 950/o99% of the nuclei were distributed in a bipolar pattern by relative mass at 2C and 4C. Few embryo nuclei (3%) had ploidy levels above 4C at the late heart-shaped embryo stage. Variability in size ofendosperm nuclei seemed correlated with the morphological state of these nuclei (free-nuclear vs. cellular). Most endosperm cells did not show significant polyploidy with 840/o-92% of nuclei in the expected 3C-6C range, but some nuclei with elevated ploidy levels were noted during endosperm cellularization. Endosperm senescence was correlated with nuclear DNA loss over time. Polyploidy seems to have no direct role in the early differentiation of the soybean embryo and endosperm, but these stable conditions may be necessary for the early establishment of the embryo. The endosperm is a unique tissue formed during double suspensor basal cells contained large polytene chromo- fertilization. As the endosperm develops, its nuclei may somes with ploidy levels of 2C-64C and 256C-8,192C, become polyploid and have maximum levels of 384C in respectively (Brady, 1973). Other taxa have demonstrated maize (Tschermak-Woess and Enzenberg-Kunz, 1965), cytological distinction between embryo proper and sus- 96C in Phaseolus vulgaris (Nagl, 1970), 24C in Triticum pensor such as in Tropaeolum majus (Nagl, 1976), various aestivum (Chojecki, Bayliss, and Gale, 1986), 96C in Bo- species of Phaseolus (Nagl, 1974), and also, between cot- rassusflabellifer (Stephen, 1974) and 3,072C in Echino- yledons and embryo axes in Brassica napus (Silcock et cystis lobata (Turala, 1966). Not all nuclei within a given al., 1990), Phaseolus vulgaris (Johnson and Sussex, 1990), endosperm exhibit these high ploidy levels inasmuch as and other papilionaceous legumes (Bryans and Smith, cytological differentiation within this tissue exists. The 198 5). These results suggest a division of labor of embryo aleurone endosperm of Oryza sativa exhibited no poly- and endosperm tissues that may be a result ofthe increased ploidization of its nuclei, retaining the base 3C-6C ploidy ploidy level of their cells. D'Amato (1952) suggested fur- level, but greater than 20% of the central endosperm cells ther that polyploidy in the embryo may directly affect attained the 24C level (Ramachandran and Raghavan, differentiation and, therefore, function of these tissues. 1989). A similar pattern existed in Zea mays, in which The gradient ploidy levels in the epidermal cells (2C), the central endosperm cells evidenced a mean DNA con- hypodermal cells (8C-32C), and central storage cells (64C) tent up to 20-fold greater than that of peripheral endo- of the cotyledons of Pisum sativum (Scharpe and Van sperm cells (Kowles and Phillips, 1985, 1988). Numerous Parijs, 1973) seem to support this premise. examples also have been reported in the differential poly- Therefore, polyploidy in endosperm and embryo tissues ploidization of the endosperm proper and the haustoria is likely to play an important functional role due to its of various taxa (Chopra and Sachar, 1963; Turala, 1969). prevalence in a number of diverse genera. In this study The differentiation of the young angiosperm embryo we report on the relative mass of DNA and the size of into distinct organs may suggest a cytological differenti- nuclei in soybean endosperm and embryo at the globular ation of its tissues similar to that seen in the endosperm. through the late heart-shaped embryo stages of devel- The nuclei of the embryo proper of Phaseolus coccineus opment (6-35 d postfertilization, dpf), to determine contained only two base levels of DNA (2C and 4C), whether polyploidy has any relevance to morphogenesis whereas the nuclei of the suspensor proper and the large in these soybean tissues. The small size of the soybean ovules at these early stages and difficulties in obtaining a sufficient number of endosperm cells from this "nonen- dospermic" legume have challenged our abilities to study ' Received for publication 16 November 1992; revision accepted 5 May 1993. the endosperm and embryo tissues for ploidy changes. The authors thank Bruce Wagner for assistance with the image anal- Because of these difficulties, we have developed a tech- ysis. This research is a joint contribution of the Iowa Agriculture and nique for the isolation of embryo sacs that allows us to Home Economics Experiment Station, Ames, Joumal Paper no. J- 15117, obtain homogeneous samples of embryo and endosperm Project 2985 and the USDA-ARS FCR. The mention of a trademark nuclei for cytophotometric analysis. In the present study, or proprietary product does not constitute a guarantee or warranty of we utilize this new technique to analyze the embryo and the product by the Iowa State University or the United States Depart- ment of Agriculture and does not imply its approval to the exclusion endosperm nuclei in a normal system to serve as a model of other products that may be suitable.