Arabidopsis Protocols

M E T H O D S I N M O L E C U L A R B I O L O G Y "

John M. Walker, SERIES EDITOR

83. Receptor Signal Transduetion Protocols, edited by R. A. J. Challiss, 1997

82. Arabidapsis Protocols, edited by Josb M Martinez-Zapater and Julio Salinas, 1998

81. Plant Virology Protocols, edited by Gary D. Foster, 1998 80. lmmunncbemieal Protocols (2rid. ed.), edited by

John Pound, 1998 79. Polyamine Protocols, edited by DavidM. L. Morgan, 1998 78. Antibacterial Peptide Protocols, edited by William M.

Shafer, 1997 77. Protein Synthesis: Methods and Protocols, edited by Robin

Martin, 1998 76. Glycoanalysis Protocols, edited by Elizabeth F.

HounseI, 1998 75. Basic Cell Culture Protocols, edited by Jeffrey W. Pollard

and John M. Walker, 1997 74. Ribuzyme Protocols, edited by Philip C. Turner, 1997 73. Neuropeptide Protocols, edited by G. Brent lrvine and

Carve# H. Williams, 1997 72. Neurotransmitter Methods, edited by Richard C Rayne. 1997 71. PRINS and In Situ PCR Protocols, edited by John R.

Gosden, 1997 70. Sequence Data Analysis Guidebook, edited by Simon R.

Swindell, 1997 69. eDNA Library Protocols, edited by Inn G. Cowell

and Caroline A. Austin. 1997 68. Gene Isolation and Mapping Protocols, edited by

Jacqueline Boultwood. 1997 67. PCR Cloning Protocols: From Molecular Cloning to

Genetic Engineering, edited by Bruce A. White, 1996 66. Epitope Mapping Protocols, edited by Glenn E. Morris, 1996 6L PCR Sequencing Protocols, edited by Ralph Rapley, 1996 64. ProteinSequencingProtocols, editedbyBJyanJ. Smith, 1996 63. Recombinant Proteins: Detection and lsolation Protocols,

edited by Rocky S. Tuan, 1996 62. Recombinant Gene Expression Protocols, edited by Rocky

S. Tuan, I996 61. Protein and Peptide Analysis by Mass Spectrometry,

edited by John R. Chapman, 1996 60. Protein NMR Protocols, edited by David G. Reid, 1996 59. Protein Purification Protocols, edited by Shawn Doonan.

1996 58. Basic DNA and RNA Protocols, edited by Adrian J.

Harwood, 1996 57. in Vitro Mntagenesis Protocols, edited by Michael K.

Trower, 1996 56. CrystallographicMethodsandProtocols, editedbyChris-

topher Jones, Barbara Mulloy, and Mark Sanderson, 1996 55. Plant Cell Electroporation and Electrofusion Protocols,

edited by Jac A. Niekoloffl 1995 54. YAC Protocols, edited by David Markie, 1995

53. Yeast Protocols: Methods in Cell and Molecular Biology, edited by lvor H. Ew~ns, 1996

52. Capillary Eteetrophnresis: Principles, Instrumentation, and Applications, edited by Kevin D. AItria, I996

51. Antibody Engineering Protocols, edited bySudhir Paul, 1995 50. Species Diagnostics Protocols: PCR and Other Nucleic

Acid Methods, edited by Justin P. Clapp, 1996 49. Plant Gene Transfer and Expression Protocols, edited by

Heddwyn Jones, 1995 48. Animal Cell Electroporation and Electrofusion Proto-

cols, edited by Jac A. Nickoloff 1995 47. Electroporation Protocols for Microorganisms, edited by

Joe A. Nickoloff 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny

howard and David ~ Whitcombe, 1995 45. Monoclonal Antibody Protocols, edited by William C.

Davis, 1995 44. Agrobacterium Protocols, edited by Kevan M. A. Gartland

and Michael R. Dave),, 1995 43. In Vitro Toxicity Testing Protocols, edited by Sheila

0 'Hare and Chris K. Atterwill, 1995 42. ELISA: Theory and Practice, by John R. Crowther, 1995 41. Signal T ransduction Protncols, edited by David A. Kendall

and Stephen J. Hill, 1995 40. Protein Stability and Folding: Theory and Practice,

edited by Bret A. Shirley, 1995 39. Bacolovirus Expression Protocols, edited by Christopher

D. Richardson, 1995 38. Cryopreservatinn and Freeze-Drying Protocols,

edited by John G. Day and Mark R. McLellan, 1995 37. lu Vitro Transcription and Translation Protocols,

edited by Martin J. Tymms, 1995 36. Peptide Analysis Protocols, edited by Ben M. Dunn and

MichaeI W Pennington, 1994 35. Peptide Synthesis Protocols, edited by Michael W.

Pennington and Ben M. Dunn, 1994 34. lmmunocytochemical Methods and Protocols, edited by

Lorette C. Javois, 1994 33. In Situ Hybridization Protocols, edited by K. H. Andy

Choo, I994 32. Basic Protein and Peptide Protocols, edited by John M.

Walker, 1994 31. Protocols for Gene Analysis, edited by Adrian J. Harwood,

1994 30. DNA-Protein Interactions, edited by G. GeoffKneale, 1994 29. Chromosome Analysis Protocols, edited by John R.

Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive

Probes, edited by Peter G. 1sane, 1994 27. Biomembrane Protocols: IL Architecture and Function,

edited by John M. Graham and Joan A. Higgins, 1994

Arabidopsis Protocols

Edited by

Jose M. Martinez-Zapater and

Julio Salinas Centro de Investigaci6n y Tecnologia, Instituto Nacional

de Investigaci6n y Tecnologia Agraria y Alimentaria, Madrid, Spain

Humana Press ~ Totowa, New Jersey

© 1998 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

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Cover illustration: From Fig. 3 in Chapter 38, "13-Glucuronidase Enzyme Histochemistry on Semithin Sections of Plastic-Embedded Arabidopsis Explants," by Marc De Block and Mieke Van Lijsebettens.

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Methods in molecular biology TM.

Arabidopsis Protocols/edited by Jos6 M. Martinez Zapater and Julio Salinas p. cm.- - (Methods in molecular biologyVM; vol. 82)

Includes index. ISBN 0-89603-391-0 (alk. paper) 1. Arabidopsis--Laboratory manuals. 2. Arabidopsis---Molecular aspects--Laboratory manuals. 1. Martinez-Zapater, Jos6 M. I1. Salinas, Julio. IlL Series: Methods in molecular biology (Totowa, N J); 82.

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Preface

The use of Arabidopsis in genetics and biology laboratories can be traced back to the beginning of the century, when the small size of this plant species, its short generation time, and the thousands of seeds produced by each plant attracted the interest of geneticists. In the middle of the century, this plant had already gained a place in several genetics laboratories both in Europe and the United States, being widely used in mutagenesis experiments. However, exponential growth in the use of Arabidopsis as a model system did not get started until the 1980s, when the small size of its genome was realized, thus triggering the interest of geneticists and molecular biologists, not only from the plant field, but from other research fields needing a novel model system.

In recent years, high expectations for Plant Biotechnology in the next century, together with the need for basic information in every area of plant biology, have served to mobilize resources and orient much new research towards the plant field, where the special biological features of Arabidopsis have further focused attention on this useful species. The high level of interest in Arabidopsis has worked as a catalyst and, today, the system has been devel- oped to the point where any gene identified on the basis ofa phenotype can be cloned with a reasonable amount of time and effort. In little more than 10 years from the beginning of this exponential growth phase, the system has reached a self-sustaining stage that enhances its everyday utility as a result of the accumulating flow of powerful information and newly available tools. In short, it is now widely accepted that Arabidopsis has become the system of choice for approaching fundamental questions in plant biology.

Arabidopsis Protocols provides to both experienced researchers and beginners in the field a comprehensive set of up-to-date protocols covering the many methods developed during work with this species, from growing plants, to explants, to gene cloning strategies. The book also includes sections on genetic, transformation, and gene expression analyses that will be espe- cially helpful for scientists involved in mutation analysis or in producing and analyzing transgenic plants. In many cases, the protocols can also be applied to other plant species with minor adjustments. Although a few chapters go beyond the scope of a protocol, providing useful information for the arabidopsologist, the majority of them contain step-by-step protocols, in line

vi Preface

with other volumes in this series. Altogether, these Protocols form a useful reference for laboratories working not only with Arabidopsis, but also with other plant species

The editors want to thank C. R. Somerville, S. Somerville, and M. Koornneef for their helpful discussions and advice at the beginning of this project, and all colleagues who have contributed their protocols to this latest Humana Press publication.

Jos~ M. Martinez-Zapater

Julio Salinas

Contents

Preface ............................................................................................................. v

Contributors ..................................................................................................... xi

List of Color Plates ......................................................................................... xv

PART I. ARABIDOPSIS CULTURE

1 Growth of Plants and Preservation of Seeds

Randy Scholl, Luz Rivero-Lepinckas, and Deborah Crist ................ 1

2 Sterile Techniques in Arabidopsis Peter McCourt and Kallie Keith .......................................................... 13

3 Control of Pests and Diseases of Arabidopsis Mary Anderson ..................................................................................... 19

4 Establishment and Maintenance of Cell Suspension Cultures Jaideep Mathur and Csaba Koncz ..................................................... 27

5 Callus Culture and Regeneration

Jaideep Mathur and Csaba Koncz ..................................................... 31

6 Protoplast Isolation, Culture, and Regeneration Jaideep Mathur and Csaba Koncz ..................................................... 35

PART II. PURIFICATION OF SUBCELLULAR ORGANELLES AND MACROMOLECULES

7 Preparation of Physiologically Active Chloroplasts from Arabidopsis Ljerka Kunst ......................................................................................... 43

8 Purification of Mitochondria from Arabidopsis Mathieu Klein, Stefan Binder, and Axel Brennicke ......................... 49

9 Preparation of DNA from Arabidopsis Jianming Li and Joanne Chory .......................................................... 55

10 High Molecular Weight DNA Extraction from Arabidopsis David Bouchez and Christine Camilleri ............................................ 61

11 Chloroplast DNA Isolation

George S. Mourad ................................................................................ 71

12 Purification of Mitochondrial DNA from Green Tissues of Arabidopsis Mathieu Klein, Rudolf Hiesel, Charles Andre,

and Axel Brennicke ......................................................................... 79

viii Contents

13 Preparation of RNA Clifford D. Carpenter and Anne E. Simon ........................................ 85

PART III. MUTAGENESIS AND GENETIC ANALYSIS

14 Seed Mutagenesis of Arabidopsis Jonathan Lightner and Timothy Caspar ........................................... 91

15 Genetic Analysis Maarten Koornneef, Carlos Alonso-Blanco, and Piet Stam ......... 105

16 Cytogenetic Analysis of Arabidopsis John S. Heslop-Harrison .................................................................. 119

17 PCR-Based Identification of T-DNA Insertion Mutants Rodney G. Winkler and Kenneth A. Feldmann .............................. 129

PART IV. GENE MAPPING IN ARABIDOPSIS

18 The Use of Recombinant Inbred Lines (RILs) for Genetic Mapping Carlos Alonso-Blanco, Maarten Koornneef, and Piet Stam ......... 137

19 AFLP TM Fingerprinting of Arabidopsis Pieter Vos ........................................................................................... 14 7

20 Building a High-Density Genetic Map Using the AFLP TM Technology Martin T. Ft. Kuiper ............................................................................ 157

21 Use of Cleaved Amplified Polymorphic Sequences (CAPS) as Genetic Markers in Arabidopsis thaliana

Jane Glazebrook, Eliana Drenkard, Daphne Preuss, and Frederick Ausubel .................................................................. 173

22 Mapping Mutations with ARMS Anton R. Sch~ffner ........................................................................... 183

23 Mapping Cloned Sequences on YACs Francis D. Agyare, Gus Lagos, Deval Lashkari, Ronald W. Davis,

and Bertrand Lemieux .................................................................... 199

PART V. TRANSIENT AND STABLE TRANSFORMATION

24 Transient Gene Expression in Protoplasts of Arabidopsis thaliana Steffen Abel and Athanasios Theologis .......................................... 209

25 Transient Expression of Foreign Genes in Tissues of Arabidopsis thaliana by Bombardment-Mediated Transformation

Motoaki Seki, Asako lida, and Hiromichi Morikawa ...................... 219

26 Root Transformation by Agrobacterium tumefaciens Annette C. Vergunst, Ellen C. de Waal,

and Paul J. J. Hooykaas ................................................................ 227

Contents ix

27 Transformation of Arabidopsis thaliana C24 Leaf Discs by Agrobacterium tumefaciens

Eric van der Graaf and Paul J. J. Hooykas .................................... 245 28 In Planta Agrobacterium-Mediated Transformation

of Adult Arabidopsis thaliana Plants by Vacuum Infiltration Nicole Bechtold and Georges Pelletier ........................................... 259

29 PEG-Mediated Protoplast Transformation with Naked DNA Jaideep Mathur and Csaba Koncz .................................................. 267

PART Vl. GENE CLONING STRATEGIES

30 Cloning Genes of Arabidopsis thaliana by Chromosome Walking Jeffrey Leung and Jer6me Giraudat ............................................... 277

31 Chromosome Landing Using an AFLpTM-Based Strategy Ann Van Gysel, Gerda Cnops, Peter Breyne, Marc Van Montagu,

and Maria Teresa Cervera .............................................................. 305 Transposon Tagging with Ac/Ds in Arabidopsis Deborah Long and George Coupland ............................................. 315 Transposon Tagging with the EN-I System Andy Pereira and Mark G. M. Aarts ................................................ 329

Cloning Genes from T-DNA Tagged Mutants Brian P. Dilkes and Kenneth A. Feldmann ..................................... 339

VII. GENE EXPRESSION ANALYSES

In Situ Hybridization Gary N. Drews .................................................................................... 353

36 Whole-Mount In Situ Hybridization in Plants Janice de Almeida Engler, Marc Van Montagu,

and Gilbert Engler .......................................................................... 373

37 Bacterial and Coelenterate Luciferases as Reporter Genes in Plant Cells

William H. R. Langridge and Aladar A. Szalay ................................ 385 38 13-Glucoronidase Enzyme Histochemistry on Semithin Sections

of Plast ic-Embedded Arabidopsis Explants Marc De Block and Mieke Van Lijsebettens ................................... 397

39 In Situ Hybridization to RNA in Whole Arabidopsis Plants Qingzhong Kong and Anne E. Simon .............................................. 409

40 In Vivo Footprinting in Arabidopsis Anna-Lisa Paul and Robert J. Ferl ................................................... 417

Index ............................................................................................................ 431

32

33

34

PART

35

Contributors

MARK G. M. AARTS ° Centrum voor Plantenveredelings en Reproduktieonderzoek (CPRO-DLO), Wageningen, The Netherlands

STEFFEN ABEL • Department of Plant Biology, Plant Gene Expression Center, Albany, CA

FRANCIS D. AGYARE ° Department of Biology, Faculty of Pure and Applied Sciences, York University, Ontario, Canada

CARLOS ALONSO-BLANCO ° Department of Genetics, Agricultural University of Wageningen, The Netherlands

MARY ANDERSON • Nottingham Arabidopsis Stock Centre, Department of Life Science, Nottingham University, UK

CHARLES ANDRE ° Universitat Ulm, Allgemeine Botanik, Germany FREDERICK M. AUSUBEL ° Department of Genetics, Harvard Medical School;

and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA

NICOLE BECHTOLD ° Laboratoire de GOnOtique et AmOlioration des Plantes, INRA-Centre de Versailles, France

STEFAN BINDER • Allgemeine Botanik, Universitat Ulm, Germany AXEL BRENNICKE ° Allgemeine Botanik, Universitat Ulm, Germany PETER BREYNE • Laboratorium voor Genetica, Universiteit Gent, Belgium DAVID BOUCHEZ ° Laboratoire de Biologie Cellulaire, INRA-Centre

de Versailles, France CHRISTINE CAMILLERI ° Laboratoire de Biologie Cellulaire, INRA-Centre

de Versailles, France CLIFFORD D. CARPENTER ° Department of Biochemistry and Molecular

Biology, University of Massachusettes, Amherst, MA TIM CASPAR ° Dupont Central Research and Development, Experimental

Station, Wilmington, DE MARIA TERESA CERVERA ° Laboratorium voor Genetica, Universiteit Gent,

Belgium JOANNE CHORY ° Plant Biology Laboratory, Salk Institute, La Jolla, CA GERDA CNOPS • Laboratorium voor Genetica, Universiteit Gent, Belgium GEORGE COUPLAND ° Department of Molecular Genetics, John Innes Centre,

Norwich, UK

xi

xii Contributors

DEBORAH CRIST • Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH

RONALD W. DAVIS • Stanford DNA Sequence and Technology Center, Department of Biochernistry, School of Medicine, Stanford University, CA

JANICE DE ALMEIDA ENGLER • Laboratorium voor Genetica, Universiteit Gent, Belgium

MARC DE BLOCK • Plant Genetic Systems NV, Gent, Belgium ELLEN C. DE WAAL • Clusius Laboratory, Institute of Molecular Plant Sci-

ences, Faculty of Mathematics and Natural Sciences, Leiden, The Netherlands

BRIAN P. DILKES • Department of Plant Sciences, University of Arizona, Tucson, AZ

ELIANA DRENKARD • Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA

GARY N. DREWS • Department of Biology, University of Utah, Salt Lake City, UT

GmBERT ENGLER • Laboratorium voor Genetica, Universiteit Gent, Belgium KENNETH A. FELDMANN • Department of Plant Sciences, University

of Arizona, Tucson, AZ ROBERT FERL • Horticultural Sciences Department, Institute of Food

and Agricultural Sciences, Gainesville, FL JER6ME GIRAUDAT • Institut des Sciences V~g~tales, France JANE GLAZEBROOK ° Center for Agricultural Biotechnology, University

of Maryland, Biotechnology Institute, College Park, MD RUDOLF HIESEL • Allgemeine Botanik, Universitat Ulm, Germany JOHN S . HESLOP-HARRISON • Department of Cell Biology, John Innes Centre,

Norwich, UK PAUL J. J. HOOYKAAS • Clusius Laboratory, Institute of Molecular Plant

Sciences, Faculty of Mathematics and Natural Sciences, Leiden, The Netherlands

ASAKO hSA • Biotechnology Laboratory, Sumitomo Chemical Co., Hyogo, Japan

KALLIE KEITH • Department of Botany, University of Toronto, Canada MATHIEU KLEIN • Allgemeine Botanik, Universitat Ulm, Germany CSABA KONCZ • Max-Planck-Institut fiir Zuchtungsforschung, Cologne,

Germany QINGZHONG KONG • Department of Biochemistry and Molecular Biology,

University of Massachusetts, Amherst, MA

Contributors xiii

MAARTEN KOORNNEEF • Department of Genetics, Agricultural University of Wageningen, The Netherlands

MARTIN T. R . KUIPER ° Keygene, Wageningen, The Netherlands LJERKA KUNST ° Department of Botany, University of British Columbia,

Vancouver, BC, Canada G u s LAGOS ° Department of Biology, Faculty of Pure and Applied Sciences,

York University, Ontario, Canada WILLIAM H . R. LANGRIDGE ° Center for Molecular Biology and Gene

Therapy, Loma Linda University, CA DEVAL LASHKARI ° Stanford DNA Sequence and Technology Center,

Department of Biochemistry, School of Medicine, Stanford University, CA BERTRAND LEMIEUX ° Department of Biology, Faculty of Pure and Applied

Sciences, York University, Ontario, Canada JEFFREY LEUNG • Institut des Sciences V6g6tales, France J1ANMING LI ° Plant Biology Laboratory, Salk Institute, La Jolla, CA JONATHAN LIGHTNER ° Dupont Central Research and Development,

Experimental Station, Wilmington, DE DEBORAH LONG ° Department of Molecular Genetics, John Innes Centre,

Norwich, UK JAIDEEP MATHUR • Max,Planck-Institut fiir Zuchtungsforschung, Cologne,

Germany PETER M c C O U R T ° Department of Botany, University of Toronto, Canada HIROMICHI MORIKAWA ° Laboratory of Plant Molecular Biology, The Institute

of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, Ibaraki, Japan

GEORGE MOURAD ° Department of Biology, Indiana University-Purdue University Fort Wayne, IN

ANNA-LISA PAUL ° Horticultural Sciences Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL

GEORGES PELLETIER ° Laboratoire de G6n6tique et Amelioration des Plantes, INRA-Centre de Versailles, France

ANDY PEREIRA • Centrum voor Plantenveredelings-en Reproduktieonderzoek (CPRO-DLO), Wageningen, The Netherlands

DAPHNE PREUSS ° Department of Molecular Genetics and Cell Biology, University of Chicago, IL

L u z RIVERO-LEPINCKAS ° Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH

ANTON R. SCHXFFNER ° GSF Research Center, Institute of Biochemical Plant Pathology, Oberschleissheim, Germany

xiv Contributors

RANDY SCHOLL ° Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH

MOTOAKI SEKI • Laboratory of Plant Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, Ibaraki, Japan

ANNE E. SIMON ° Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA

PIET STAM ° Department of Genetics, Agricultural University of Wageningen, The Netherlands

ALADAR A . SZALAY ° Center for Molecular Biology and Gene Therapy, Loma Linda University, CA

ATHANAS1OS THEOLOGIS ° Department of Plant Biology, Plant Gene Expression Center, Albany, CA

ERIC VAN DER GRAAF ° Clusius Laboratory, Institute of Molecular Plant Sciences, Faculty of Mathematics and Natural Sciences, Leiden, The Netherlands

ANNE VAN GYSEL ° Laboratorium voor Genetica, Universiteit Gent, Belgium MIEKE VAN LIJSEBETTENS • Laboratorium voor Genetica, Universiteit Gent,

Belgium MARC VAN MONTAGU ° Laboratorium voor Genetica, Universiteit Gent,

Belgium ANNETTE C. VERGUNST • Clusius Laboratory, Institute of Molecular Plant

Sciences, Faculty of Mathematics and Natural Sciences, Leiden, The Netherlands

PIETER VOS ° Keygene, Wageningen, The Netherlands RODNEV G . WINKLER ° Department of Plant Sciences, University of Arizona,

Tucson, AZ

List of Color Plates

Color plates appear as an insert following p. 230

Plate 1 (Fig. 1 from Chapter 24). Histochemical localization of GUS activity in Arabidopsis mesophyll protoplasts transfected with translational GUS fusions. Purified protoplasts (A) were mock-transfected (D) or challenged with the following plasmid DNAs: pRTL2-GUS which encodes the nonfused, authentic GUS protein (B); pRTL2-GUS-IAA 1 encoding a GUS::IAA 1 nuclear fusion protein (C); pRTL2-GUS-PS-IAA6 encoding a GUS::PS-IAA6 nuclear fusion protein (E,F). Transfected protoplasts were cultured for 20 h, stained for GUS activity (B-E) and nuclei (F) as described (1O.

Plate 2 (Fig. 1 from Chapter 29). GUS and GFP reporter gene expres- sion assays with Arabidopsis protoplasts. (A) GUS staining of PEG-transformed protoplasts derived from roots ofArabidopsis ecotype Columbia after incuba- tion with X-gluc for 6 h at room temperature. (B) Leaf mesophyll protoplasts from Arabidopsis ecotype Columbia transformed with pCK-GFPs65c exhibit green fluorescence when illuminated with blue light. The chloroplasts emit red fluorescence, whereas the yellow fluorescence results from overlapping red and green areas.

Plate 3 (Fig. 1 from Chapter 36). (A) WISH on anArabidopsis thaliana seedling hybridized with an antisense cdc2a probe detected by gold-labeled antibodies. The signal is visible as a black precipitate resulting from the silver amplification reaction. (B) In situ localization ofcdc2a mRNA on a vibroslice of an Arabidopsis root infected with a root-knot nematode. The blue precipi- tate resulted from the histochemical reaction with the substrates X-phosphate and NBT. (C) Cotyledon hybridized with an antisense rhal probe. Hybrids were visulalized by silver amplification of specifically bound gold-labeled antibodies seen as a dark precipitate. (D) Chicory root hybridized with an antisense nitrate reductase probe detected by AP-labeled antibodies. A strong expression is visible in the vascular cylinder and in the root meristem. The sample was cleared in CLP for better visualization of the signal.

XV

xvi List of Plates

Plate 4 (Fig. 3 from Chapter 38). X-Gluc reactions on Arabidopsis tissue. (A) Transverse section through a 2-wk-old-root ofapRPS18A-gus trans- formed line (15). (B) Detail of the vascular tissue; main GUS activity in vas- cular tissue. (C) Transverse section through a young (stage 9) flower of a line transformed with a gus gene fused to a stomium-specific tobacco promoter (provided by T. Beals and P. Sanders, Plant Molecular Biology Laboratory, University of California, Los Angeles, CA). (D) Detail of mature anther. Main GUS activity located at the stomium, the site of anther dehiscence. Abbrevia- tions: al, anther locule; c, cortex; en, endodermis; ep, epidermis; g, gynoecium; po, pollen; rh, root hair; s, sepal; sc, stomium cells; se, septum; st, stamen; vt, vascular tissue. Visualization with Normansky interference microscopy. Bar = 50 gm (A), 20 ~tm (B,D), and 100 gm (C).