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EMBRYO DEVELOPMENT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hongchang Ma, B.S., M.S. + + * * + The Ohio State University 1994 Dissertation Committee: Approved by J.G. Streeter J.J. Finer L.M. Lagrimini / ] V. S — ° X J Advisor K.A. Campbell Department of Agronomy ACKNOWLEDGMENTS I express my sincere appreciation to Dr. John J. Finer and Dr. John G. Streeter for their guidance, encouragement, and consistent support throughout my research. My gratitude goes to other members of my advisory committee, Dr. Mark Lagrimini for his invaluable suggestions in my research and for his help in analyzing some of my research data, Dr. Kimberly A. Campbell for her valuable comments on my manuscripts and providing laboratory space and equipment. I am especially grateful to Dr. Michael D. McMullen, who first suggested isolation of homeobox genes from soybean. The technical assistance of Barbara L. Norris, Mark W. Jones, Brenda S. Schult, and Kevin Simcox is gratefully acknowledged. I also thank Thomas G. Lanker, Seppo O. Salminen, Dimple R. Abernathy, and Marie E. Semko for their technical and instrument supports. I thank to my fellows, Masood Z. Hadi and Tai-Sheng Cheng for sharing exciting scientific information with me and making my graduate studies more enjoyable and fruitful. I also wish to thank Dr. Sarah Hake for providing theK nl cDNA clone, Dr. David P. Jackson and Dr. Sarah Hake for sharing their unpublished information and for their technique assistance in in situ hybridization. The supply of soybean ubiquitin gene from Dr. Desh-Pal Verma is also appreciated. To my wife, Huiling, I offer sincere thanks for her understanding, support, and encouragement throughout this journey. To my daughter, Li, 1 thank for her willingness to endure my frequent absences in her normal life. VITA April 14, 1955 .............................................................. Bom in Dongguang, Hebei, P.R. China March, 1982 ................................................................. B.S., Hebei Agricultural University, Baoding, Hebei, P.R. China 1982-1987 ..................................................................... Research Scientist, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang, Hebei, P.R. China December, 1989 ............................................................M.S., Kansas State University, Manhattan, Kansas March, 1990-Present .................................................. Graduate Research Associate, Ohio Agricultural Research and Development Center/The Ohio State University, Wooster, Ohio PUBLICATIONS 1. Ma H, McMullen MD, Finer JJ: Identification of a homeobox-containing gene with enhanced expression during soybean (Glycine max L.) somatic embryo development. Plant Mol. Biol. 24: 465-473 (1994). 2. Ma H, McMullen MD, Finer JJ: A pyruvate kinase cDNA from soybean somatic embryos. Plant Physiol. 102: 1345 (1993). 3. Ma H, Liang GH, Wassom CE: Effects of growth regulators and genotypes on callus and embryoid induction from maize anther culture. Plant Breeding 106: 47-52 (1991). 4. Ma H, Wassom CE, Liang GH: Direct generation of maize haploids via anther culture. Cytologia 56: 103-106(1991). 5. Gao K, Ma H, Liu A: Study on population size of hand-pollinated plants in seed renewal of maize cultivars. ACTA Agronomica Sinica 17: 314-319 (1991) [in Chinese]. FIELDS OF STUDY Major Field: Agronomy Studies in Tissue Culture and Cell Biology, Dr. John J. Finer Studies in Plant Genetics, Dr. Kimberly A. Campbell Studies in Plant Physiology, Dr. John G. Streeter Studies in Molecular Biology, Dr. Michael D. McMullen Dr. Mark Lagrimini TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................................................................. ii VITA .................................................................................................................................. iv LIST OF FIGURES ........................................................................................................viii INTRODUCTION ............................................................................................................ 1 CHAPTER PAGE I. ISOLATION AND CHARACTERIZATION OF A PYRUVATE KINASE cDNA FROM SOYBEAN .................................... 21 Abstract ....................................................................................................... 21 Introduction ................................................................................................ 22 Materials and Methods ................................................. 28 Results and Discussion ............................................................................. 31 II. IDENTIFICATION OF HOMEOBOX-CONTAINING GENES FROM SOMATIC EMBRYOS AND CHARACTERIZATION OF THE FIRST SOYBEAN HOMEOBOX-CONTAINING GENE .......................................................... 48 Abstract ...................................................................................................... 48 Introduction ................................................................................................ 49 Materials and Methods ............................................................................. 60 Results and Discussion ............................................................................. 62 CHAPTER PAGE III. TEMPORAL AND SPATIAL EXPRESSIONS OF SBH1 DURING PLANT DEVELOPMENT .................................................... 73 Abstract ................................................................................................ 73 Introduction ......................................................................................... 75 Materials and Methods ...................................................................... 79 Results .................................................................................................. 83 Discussion ........................................................................................... 87 GENERAL DISCUSSION ................................................................................... 99 LIST OF REFERENCES ....................................................................................... 102 APPENDIX .............................................................................................................. 119 LIST OF ABBREVIATIONS ............................................................................... 146 LIST OF FIGURES FIGURE PAGE 1.1. The development of soybean somatic embryos.................................... 38 1.2. DNA dot hybridizations of selected clones with other clones and themselves ............................................................................................ 39 1.3. RNA dot blots showing the expression patterns of the selected clones during soybean somatic embryogenesis.................................... 40 1.4. RNA dot blots to detect the effects of auxin on expression of the selected clones ..................................................................................... 41 1.5. Nucleic acid sequence of Sbpk and deduced amino-acid sequence of SBPK. ...................................................................................... 42-43 1.6. Alignment of the deduced amino acid sequence of soybean pyruvate kinase [S] with that of potato [P] and tobacco [T] pyruvate kinases.............................................................. 44 1.7. Southern hybridization analysis of soybean DNA using Sbpk cDNA as a probe ................................................................................ 45 1.8. Expression ofSbpk in somatic embryos developed under anaerobic or aerobic conditions .................................................