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Morphological and Genetic Analysis of Embryo Specific Mutants in Maize

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Morphological and Genetic Analysis of Embryo Specific Mutants in Maize University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2018 Morphological And Genetic Analysis Of Embryo Specific utM ants In Maize Dale Cletus Brunelle Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Brunelle, Dale Cletus, "Morphological And Genetic Analysis Of Embryo Specific utM ants In Maize" (2018). Theses and Dissertations. 2392. https://commons.und.edu/theses/2392 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. MORPHOLOGICAL AND GENETIC ANALYSIS OF EMBRYO SPECIFIC MUTANTS IN MAIZE by Dale Cletus Brunelle Bachelor of Science, University of North Dakota, 2009 Master of Science, University of North Dakota, 2011 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota December 2018 i PERMISSION Title- Morphological and Genetic Analysis of Embryo Specific Mutants in Maize Department Biology Degree Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Dale C. Brunelle December 06, 2018 iii TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... VIII LIST OF FIGURES .......................................................................................................... IX ACKNOWLEDGMENTS ................................................................................................ XI ABSTRACT .................................................................................................................... XIII CHAPTER I. INTRODUCTION ........................................................................................................1 Maize ............................................................................................................................1 Normal Kernel Development .......................................................................................3 Double Fertilization .................................................................................................3 Proembryo Stage ......................................................................................................4 Transition Stage .......................................................................................................4 Coleoptile Stage .......................................................................................................5 Stage 1 ......................................................................................................................6 Stage 2 ......................................................................................................................7 Stage 3 ......................................................................................................................7 Stage 4 ......................................................................................................................8 Stage 5 and Stage 6 ..................................................................................................8 Defective Kernel Research ...........................................................................................8 Early Defective Maize Kernel Research ..................................................................8 The First Large Screening for Defective Kernel Mutants .....................................10 Reports for Select Defective Kernel Mutants .........................................................15 The Second Large Screening for Defective Kernel Mutants ..................................16 Current Defective Kernel Mutant Gene Identification ..........................................17 Embryo Specific Mutant Research .............................................................................18 First Screening for Embryo Specific Mutants ........................................................18 Reports for Select Defective Kernel Mutants .........................................................19 Current Embryo Specific Mutant Gene Identification ...........................................26 Fluorescent Protein Fusion Constructs .......................................................................28 Fluorescent Protein Tagging .................................................................................28 iv Expression Profiles of Protein Fusion Constructs.................................................31 pin1 .....................................................................................................................31 dr5 .......................................................................................................................33 tcs ........................................................................................................................33 wus ......................................................................................................................34 abphyll 1 .............................................................................................................35 bes1 .....................................................................................................................36 his1 ......................................................................................................................36 rab17 ...................................................................................................................37 pyabby ................................................................................................................38 prk ......................................................................................................................38 mre11b................................................................................................................39 zmperi .................................................................................................................40 II. MATERIALS AND METHODS ..............................................................................41 Genetic and Morphological Methods .........................................................................41 Mutant Production by EMS Treatment ..................................................................41 Screening for Embryo Specific Mutants ................................................................43 Morphological Examination of Embryo Specific Mutants.....................................45 Germination Test ...................................................................................................46 Complementation Test ...........................................................................................46 Confocal Microscope Methods...................................................................................48 Fusion Protein Construct Maintenance .................................................................48 Fusion Protein Constructs in Embryo Specific Mutant Lines ...............................49 Embryo Tissue Collection for Fresh and Paraformaldehyde Samples ..................49 Production of Slides ...............................................................................................50 vibratome sectioning .........................................................................................50 wholemounts ......................................................................................................54 Confocal Microscope Settings ...............................................................................54 laser specifications.............................................................................................54 zen software settings .........................................................................................55 III. RESULTS .................................................................................................................58 Production and Mutation Frequency of Embryo Specific Mutants ............................58 v Segregation Frequency ...............................................................................................59 Germination ................................................................................................................59 Complementation Test................................................................................................60 Extent of Mutant Embryo Morphogenesis .................................................................61
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