Transgenic Maize Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

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1 Transgenic Maize Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to

2 M ETHODS IN MOLECULAR BIOLOGY Transgenic Maize Methods and Protocols Edited by M. Paul Scott USDA-ARS, Corn Insects and Crop Genetics Research Unit, Ames, IA, USA

3 Editor M. Paul Scott USDA-ARS Corn Insects and Crop Genetics Research Unit 1407 Agronomy Hall Ames, IA ISBN: e-isbn: ISSN: e-issn: DOI: / Library of Congress Control Number: Humana Press, a part of Springer Science+Business Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper springer.com

4 Preface Maize is unique among plants because it is one of the most important crops in the world and it is one of the foremost model species for genetic studies. Maize grain is important as a staple food crop in many parts of the world and is also a major component of livestock and poultry diets. Maize is also grown as a forage crop for ruminant animals. In addition, maize is emerging as an important contributor to fuel markets. Maize grain is used to make ethanol and maize stover has been proposed to be a source of biomass for lignocellulosic biofuel production. As a genetic model, experiments in maize led to the rediscovery of Mendel s law s of inheritance (1), quantitative genetics theory (2), characterization of effect of X-rays on chromosomes (3), and the discovery of transposable elements (4). Increasing global population and economic development ensure continued need for production of maize and other crops. Several features of maize have led to its success as a model species and crop plant. First, it has tremendous genetic diversity. It is in part this diversity that has allowed plant breeders to develop maize varieties that are adapted to a broad range of environmental conditions. Maize is monoecious, meaning each plant has separate male and female flowers. This aids the making of controlled pollinations, facilitating breeding and genetic experiments. Finally, maize possesses C4 physiology, enabling it to fix carbon very efficiently. Transgenic technology allows the transfer of genetic material across species boundaries or the use of genetic material that does not exist in nature. This ability is valuable in genetic research. It allows genetic complementation studies and manipulation of maize systems on a molecular level. It has also been used as a crop improvement tool. Transgenic maize that is resistant to herbicides reduces crop management effort and expense, while transgenic maize producing insecticidal proteins increases yield and quality of maize grain. While not in widespread commercial production yet, the scientific literature contains examples of successful manipulation of maize nutritional quality and functional properties. It is possible that transgenic technology will play a role in improving draught tolerance and nitrogen use efficiency, two traits that limit crop production in many areas. One of the challenges of working with transgenic maize is that it requires expertise in a large number of disciplines. Transgenic maize projects are often carried out as cooperative efforts between research teams or use fee-for-service laboratories to accomplish parts of the project objectives. This book is roughly divided by the disciplines required to take a transgenic maize experiment from idea to evaluation of transgenic plants. Thus, design of transgenes requires a thorough understanding of gene expression and cell biology and is addressed in the first chapter. In the next section, several methods of maize transformation are described. Following this, examples of the use of transgenic maize in research are given. Expression of reporter genes, down-regulation of gene activity, and gene isolation are broadly applicable research objectives. A chapter covers each of these areas, focusing on the fluorescent proteins, RNAi, and plasmid rescue technologies that have emerged from several other technologies as being broadly effective. The next section is devoted to methods of analysis of transgenic maize plants at levels ranging from DNA to transgene function. The final section addresses issues encountered at the level of whole plants and v

5 vi Preface describes experiments that would typically be carried out in a field or greenhouse setting. In the United States of America, the Animal and Plant Health Inspection Service (APHIS) regulates the release of experimental transgenic varieties to the environment. Appendices are included with examples of the information required by APHIS. While it is unlikely that any given reader will use all of the procedures in this book, it is hoped that the reader will benefit from the procedures in this book that they use and that other procedures are helpful for understanding parts of the experiments being performed by cooperators or fee-for-service labs. M. Paul Scott Ames, IA References 1. Correns, C. G. (1900) Mendel s Regel über das Verhalten der Nachkommenschaft der Rassenbastarde. Berichte Deut. Bot. Ges. 18, Emerson, R. A. and East, E. M. (1913) The inheritance of quantitative characters in maize. Bull. Agric. Exp. Stn. Nebr. 2, McClintock, B. (1950) The origin and behavior of mutable loci in maize. Proc. Natl Acad. Sci. USA 36, Stadler L.J. (1928) Genetic effects of x-rays in maize. Proc. Natl Acad. Sci. USA, 14,

6 Contents Preface Contributors v ix PART I: INTRODUCTION 1 Design of Gene Constructs for Transgenic Maize Dong Liu PART II: TRANSFORMATION METHODS 2 Transient Expression of GFP in Immature Seed Tissues Colin T. Shepherd 3 Biolistic Gun-Mediated Maize Genetic Transformation Kan Wang and Bronwyn Frame 4 Agrobacterium-Mediated Maize Transformation: Immature Embryos Versus Callus Vladimir Sidorov and David Duncan 5 Whiskers-Mediated Maize Transformation Joseph F. Petolino and Nicole L. Arnold PART III: TRANSGENIC MAIZE IN RESEARCH 6 Methods for Generation and Analysis of Fluorescent Protein-Tagged Maize Lines Amitabh Mohanty, Yan Yang, Anding Luo, Anne Sylvester, and David Jackson 7 Use of Transgene-Induced RNAi to Regulate Endogenous Gene Expression Karen McGinnis 8 Plasmid Rescue: Recovery of Flanking Genomic Sequences from Transgenic Transposon Insertion Sites Guo-Ling Nan and Virginia Walbot PART IV: ANALYSIS OF TRANSGENIC PLANTS 9 Nonradioactive Genomic DNA Blots for Detection of Low Abundant Sequences in Transgenic Maize Guo-Ling Nan and Virginia Walbot 10 Tissue-Print Immunodetection of Transgene Products in Endosperm for High-Throughput Screening of Seeds M. Paul Scott 11 Determination of Transgene Copy Number by Real-Time Quantitative PCR Colin T. Shepherd, Adrienne N. Moran Lauter, and M. Paul Scott vii

7 viii Contents PART V: BREEDING WITH TRANSGENES 12 Herbicide Resistance Screening Assay Joan M. Peterson 13 Characterizing Transgene Inheritance Earl H. Bicar 14 Backcross Breeding Karla E. Vogel Appendix A Appendix B Index

8 Contributors NICOLE L. ARNOLD Discovery Research, Cell Biology, Dow Agrosciences LLC, Indianapolis, IN, USA EARL BICAR Monsanto, Kihei, HI, USA DAVID DUNCAN Monsanto, Chesterfield, MO, USA BRONWYN FRAME Center for Plant Transformation, Iowa State University, Ames, IA, USA DAVE JACKSON Plant Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA DONG LIU Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China ANDING LUO Plant Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA KAREN MCGINNIS Plant Sciences, University of Arizona, Tucson, AZ, USA AMITABH MOHANTY DuPont Knowledge Centre, Hyderabad, India ADRIENNE N. MORAN LAUTER Corn Insects and Crop Genetics Research Unit, USDA-ARS, Ames, IA, USA GUO-LING (GILLIAN) NAN Biological Sciences, Stanford University, Stanford, CA, USA JOAN PETERSON BASF, Ames, IA, USA JOSEPH PETOLINO Discovery Research, Cell Biology, Dow Agrosciences LLC, Indianapolis, IN, USA M. PAUL SCOTT Corn Insects and Crop Genetics Research Unit, USDA-ARS, Ames, IA, USA COLIN T. SHEPHERD AimsBio, Inc., Ames, IA, USA VLADIMIR SIDEROV Monsanto, Chesterfield, MO, USA ANNE SYLVESTER Department of Molecular Biology, University of Wyoming, Laramie, WY, USA KARLA VOGEL Monsanto, Ankeny, IA, USA VIRGINIA WALBOT Biological Sciences, Stanford University, Stanford, CA, USA KAN WANG Center for Plant Transformation, Iowa State University, Ames, IA, USA YAN YANG Plant Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA ix