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Novel Approaches to Improving Qualities of Maize Endosperm Anastasia Lorellen Bodnar Iowa State University

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Novel Approaches to Improving Qualities of Maize Endosperm Anastasia Lorellen Bodnar Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2011 Novel approaches to improving qualities of maize endosperm Anastasia Lorellen Bodnar Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agronomy and Crop Sciences Commons Recommended Citation Bodnar, Anastasia Lorellen, "Novel approaches to improving qualities of maize endosperm" (2011). Graduate Theses and Dissertations. 10271. https://lib.dr.iastate.edu/etd/10271 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. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Novel approaches to improving qualities of maize endosperm by Anastasia Bodnar A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY Major: Genetics Program of Study Committee: M. Paul Scott, Co-Major Professor Manju Reddy, Co-Major Professor Philip Becraft Erik Vollbrecht Clark Wolf Iowa State University Ames, Iowa 2011 Copyright © Anastasia Bodnar, 2011. All rights reserved. ii Table of Contents List of Tables ......................................................................................................................... viii List of Figures .......................................................................................................................... ix Terms ....................................................................................................................................... xi Abstract .................................................................................................................................. xiii Preface.................................................................................................................................... xiv Rationale ............................................................................................................................ xiv Dissertation organization ................................................................................................... xiv Authors’ roles..................................................................................................................... xiv Chapter 1. General Introduction ............................................................................................... 1 Malnutrition .......................................................................................................................... 1 The problem of malnutrition ............................................................................................. 1 Potential solutions to malnutrition .................................................................................... 1 Biofortification ...................................................................................................................... 3 Iron deficiency and improvements in dietary iron ................................................................ 4 Iron deficiency and absorption.......................................................................................... 4 Inhibitors and enhancers of iron bioavailability ............................................................... 5 Iron forticants .................................................................................................................... 6 Biofortification of iron .......................................................................................................... 8 Hemoglobin....................................................................................................................... 9 Plant hemoglobin .............................................................................................................. 9 Protein deficiency and seed storage proteins in maize ....................................................... 11 Protein deficiency ........................................................................................................... 11 Dietary requirements for protein ..................................................................................... 12 iii Maize protein content and quality................................................................................... 13 Zein description and function ......................................................................................... 15 Green fluorescent protein and its use as a marker .............................................................. 16 Characteristics of green fluorescent protein ................................................................... 16 Uses of green fluorescent protein ................................................................................... 17 Breeding with transgenes .................................................................................................... 18 Recurrent selection.......................................................................................................... 18 Modifier genes ................................................................................................................ 18 Evolutionary breeding ..................................................................................................... 20 Transgene breeding ......................................................................................................... 22 Chapter 2. Iron Bioavailability of Maize Hemoglobin Using a Caco-2 Cell Culture Model . 23 Abstract ............................................................................................................................... 23 Introduction ......................................................................................................................... 23 Methods............................................................................................................................... 25 Expression vector construction ....................................................................................... 25 Plant transformation, tissue culture, and seed production .............................................. 27 Maize hemoglobin and antibody production .................................................................. 28 Transgenic kernel screening ........................................................................................... 28 Iron bioavailability of maize hemoglobin produced in E. coli ....................................... 29 Iron bioavailability of maize globin expressed in maize kernels .................................... 31 Results ................................................................................................................................. 31 The bioavailability of iron in maize flour fortified with maize hemoglobin is similar to iron in bovine hemoglobin and ferrous sulfate ............................................................... 31 Production of the ZmG-GFP fusion protein in transgenic maize kernels ....................... 32 iv Maize flour expressing ZmG-GFP has higher iron bioavailability than transgene negative flour .................................................................................................................. 34 Discussion ........................................................................................................................... 35 Acknowledgements ......................................................................................................... 37 Chapter 3. Recurrent Selection for Transgene Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter .................................................................................. 38 Abstract ............................................................................................................................... 38 Introduction ......................................................................................................................... 38 Materials and methods ........................................................................................................ 40 Transgenic seed development ......................................................................................... 40 Development of segregating populations and seed production ...................................... 40 Field design ..................................................................................................................... 42 GFP screening ................................................................................................................. 42 Quantification of seed storage proteins ........................................................................... 43 Phenotypic evaluation ..................................................................................................... 43 Statistical analysis ........................................................................................................... 44 Results ................................................................................................................................. 45 Expression of GFP was increased with selection ........................................................... 45 Expression of 27kDa gamma zein was higher in Sel3 than Ran3 .................................. 46 Selection for fluorescence had
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