Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1979 Microscopy of soybean seeds: cellular and subcellular structure during germination, development and processing with emphasis on lipid bodies Craig William Bair Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Food Science Commons Recommended Citation Bair, Craig William, "Microscopy of soybean seeds: cellular and subcellular structure during germination, development and processing with emphasis on lipid bodies " (1979). Retrospective Theses and Dissertations. 7188. https://lib.dr.iastate.edu/rtd/7188 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. 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Requests can be made to our Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases we have filmed the best available copy. Universi^ Microfilms International 300 N. ZEEB ROAD, ANN ARBOR, Ml 48106 18 BEDFORD ROW, LONDON WC1R 4EJ, ENGLAND 8000112 I BAIR, CRAIG WILLIAM MICROSCOPY OF SOYBEAN SEEDS: CELLULAR AND SUBCELLULAR STRUCTURE DURING GERMINATION, DEVELOPMENT AND PROCESSING WITH EMPHASIS ON LIPID BODIES. IOWA STATE UNIVERSITY, PH.D., 1979 UniversiV Micrdnlms Intematkxvd 300 N. 2EEB ROAD. ANN ARBOR. Ml 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark . 1. Glossy photographs 2. Colored illustrations 3. Photographs with dark background "4. Illustrations are poor copy 5. Print shows through as there is text on both sides of page 6. Indistinct, broken or small print on several pages throughout 7. Tightly bound copy with print lost in spine a. Computer printout pages with indistinct print 9. Page(s) lacking when material received, and not available from school or author 10. Page(s) seem to be missing in numbering only as text follows 11. Poor carbon copy 12. Not original copy, several pages with blurred type 13. Appendix pages are poor copy 14. Original copy with light type 15. Curling and wrinkled pages 16. Other Universi^ MicrdFilms International 300 N. ZEES no., ANN ARBOR. Ml *8106 <3131 761-4700 Microscopy of soybean seeds: cellular and subcellular structure during germination, development and processing with emphasis on lipid bodies by Craig William Bair A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major: Food Technology Approved; Signature was redacted for privacy. Signature was redacted for privacy. 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Iowa State University Ames, Iowa 1979 ii TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 4 Soybean Seed Anatomy 4 Subcellular Components 8 Protein bodies 8 Starch granules 13 Calcium oxalate crystals 15 Lipid Bodies 17 Soybeans 17 The controversy 20 Appearance 22 Light microscopy 22 Electron microscopy 26 Membrane 32 Cellular distribution and association 36 Chemical composition 38 Function 40 Cellular origin 46 Germination and Developmental Studies of Soybean Cotyledons 49 Germination 49 Maturation 53 Relationship of Processing and Soybean Ultrastructure 55 Conversion of soybeans to several food products 55 Processing of soybeans to desolventized-toasted flakes 59 Solvent extraction of lipids 61 iii Page MATERIALS AND METHODS 63 Soybeans 63 Processed Soybean Samples 63 Proximate Analysis 63 Moisture determination 63 Protein determination 63 Crude lipid 64 Ash determination 65 Carbohydrate 65 Protein Dispersibilify Index (PDI) 65 Microscopy Studies 67 Light microscopy (LM) 67 Paraffin embedding 67 Resin embedding 67 Transmission electron microscopy (TEM) 68 Scanning electron microscopy (SEM) 68 Energy dispersive X-ray (EDX) analysis 69 Photography: (LM), (TEM) and (SEM) 70 Germination and Maturation Studies 70 Germination 70 Cytochemical studies 71 Acid phosphatase localization 71 Catalase localization 72 Lipase localization 73 Maturation 73 îv Page Processing Methods 74 Solvent extraction of soybean cotyledons 74 Physical treatments 74 Heat treatment (boiling) 75 Freezing-thawing treatment 75 Sonication treatment 75 Enzyme treatment (trypsin) 75 Lipid Body Isolation 75 Density Gradient Centrifugation 76 RESULTS AND DISCUSSION 78 Soybean Cytology with Emphasis on Protein Bodies 78 Starch Granules 97 Calcium Oxalate Crystals 105 Soybean Lipid Bodies 113 Appearance and distribution 113 Light microscopy 113 Electron microscopy 114 Isolation and chemical characterization of lipid bodies 127 Isolation of lipid bodies 127 Chemical composition 128 Effect of isolation medium 131 Effect of soybean cotytedon:isolation medium ratio 131 Effect of centrifugation temperature 134 Effect of heating 134 V Page Microscopic examination of isolated lipid bodies 135 Membrane 143 Effect of trypsin treatment 150 Examination of centrifugation pellet 153 Problems associated with microscopical preparation of 158 isolated lipid bodies Density gradient centrifugation of lipid bodies 158 Germination and Developmental Studies of Soybean Cotyledons 159 Germination 159 Maturation 189 Relationship of Processing and Soybean Ultrastructure 211 Ultrastructural changes in soybeans during processing to yield 211 desolventized-toasted meal Effect of boiling 231 Effect of trypsin treatment 236 Effect of freezing-thawing 241 Effect of solvent extraction 244 Effect of son ication 251 FIGURE ABBREVIATIONS 252 SUMMARY 253 LITERATURE CITED 259 ACKNOWLEDGMENTS 276 1 INTRODUCTION The soybean is a virtual storehouse of two nutritionally and economically important substances, protein and oil. With the advent of world food shortages and the demand for lower cost consumables, considerable research has been directed towards characterization of these two components with hope of increasing their utilization. Research has dealt primarily with chemical and functional properties of soybean proteins and oil. In contrast, very little is known about ultrastructural characteristics of soybeans. Studies on soybean ultrastructure lag considerably behind similar research on wheat, com, cottonseed and peanuts (Wolf, 1972). Although the profile of soybean structure based on optical microscopy was described more than 60 years ago, little additional work was done until the early 1960's when more sophisticated tools and techniques, such as the electron microscope, became readily available. Since then, a few investigators have filled in some of the details of soybean structure, but much more research is needed. The soybean seed is made-up of four basic parts: a seed coat or hull (8%), the plumule and hypocotyl-axis (2%) and two large cotyledons (90%). The cotyledons are of primary interest, since they contain the storage tissue of the seed. Microscopy studies have revealed that the cotyledons are comprised of palisade-like cells that are filled predominantly with protein and oil. The electron microscope shows that most of the protein is packaged in subcellular inclusions referred to as protein bodies which are large (2 to 10 ^m in diameter) and nearly 2 spherical in shape, while the oil is located in much smaller (approximately 0.2 to 0.5^m in diameter) particles designated spherosomes (Wolf, 1972). Isolation, chemical characterization and electron microscopy studies have been directed mainly at protein bodies, whereas the spherosomes of soybeans have not been isolated nor characterized. Consequently, their structure, composition, susceptibility to