METHODS IN MOLECULAR BIOLOGY™

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

For further volumes:

http://www.springer.com/series/7651
 Transgenic Plants
Methods and Protocols

Second Edition

Edited by

Jim M. Dunwell
School of Biological Sciences, University of Reading, Reading, UK

Andy C. Wetten
School of Biological Sciences, University of Reading, Reading, UK
Editors
Jim M. Dunwell Andy C. Wetten
School of Biological Sciences School of Biological Sciences
University of Reading University of Reading
Reading, UK Reading, UK

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-557-2 e-ISBN 978-1-61779-558-9
DOI 10.1007/978-1-61779-558-9
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2012930136

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Preface

In 2010, the global area of transgenic crops reached 148 million hectares, an 87-fold
increase since 1996, the first year of planting. This makes such technology the most rapidly
adopted one in the history of modern agriculture.
This success has been based on the scientific foundation developed in many international
laboratories since the 1980s, and it is the purpose of the present volume to provide access to
the continuous improvements being made for the production and analysis of transgenic
plants. Importantly, this volume is designed to complement and extend the information
published in this same series Transgenic Plants: Methods and Protocols in 2005.
The present collection of novel methods is divided into eight parts which cover a range
of protocols for both model and crop species. The first part covers various methods for the
selection and detection of transgenic plants, and this is followed by a series of sections that
describe specific methods for algae and then for higher plants. These latter sections are
divided into those devoted to monocots, with an emphasis on rice, and then to dicots such
as plum, grape, and cotton. Part VI covers a rapidly growing area of research, namely, the
specific targeting and directed silencing of plant genes. Many of these technologies will
cause considerable debate among the various regulatory regimes as they are not always
detectable by molecular methodology and therefore are indistinguishable from mutations
produced either spontaneously or by other methods. Part VII describes a range of applied
examples to generate plants with modified metabolism or the production of pharmaceuti-
cals. Finally, Part VIII describes the experience gained from the growth of transgenic
Arabidopsis in the field.
Throughout the preparation of this volume, stimulated by the enthusiasm of the series
editor John Walker, we have been aided by the continual high quality input from the large
number of authors and we thank them all for their respective contributions.

Reading, UK Jim M. Dunwell

Andy C. Wetten

v
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I SELECTION AND DETECTION METHODS

1 Employment of Cytokinin Vectors for Marker-Free and Backbone-Free

Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Craig M. Richael and Caius M. Rommens
2 Organophosphorus Hydrolase: A Multifaceted Plant Genetic Marker Which
Is Selectable, Scorable, and Quantifiable in Whole Seed . . . . . . . . . . . . . . . . . . . . . 11
T. Scott Pinkerton, James R. Wild, and John A. Howard
3 Use of Northern Blotting for Specific Detection of Small RNA Molecules
in Transgenic Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Basel Khraiwesh

PART II TRANSFORMATION TECHNOLOGY: LOWER PLANTS

4 Genetic Transformation of the Model Green Alga Chlamydomonas reinhardtii . . . . 35

Juliane Neupert, Ning Shao, Yinghong Lu, and Ralph Bock

PART III TRANSFORMATION TECHNOLOGY: RICE

5 A High-Efficiency Agrobacterium-Mediated Transformation System of Rice

(Oryza sativa L.). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Kenjirou Ozawa
6 Selection of Transgenic Rice Plants Using a Herbicide Tolerant Form
of the Acetolactate Synthase Gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Masaki Endo, Tsutomu Shimizu, and Seiichi Toki
7 Visual Selection in Rice: A Strategy for the Efficient Identification
of Transgenic Calli Accumulating Transgene Products . . . . . . . . . . . . . . . . . . . . . . 67
Hiroaki Saika, Haruko Onodera, and Seiichi Toki
8 Characterization of Rice Genes Using a Heterologous Full-Length
cDNA Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Mieko Higuchi, Youichi Kondou, Masaki Mori, Takanari Ichikawa,
and Minami Matsui
9 Bioactive Bead-Mediated Transformation of Plants with Large
DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Naoki Wada, Joyce A. Cartagena, Naruemon Khemkladngoen,
and Kiichi Fukui

vii
viii Contents

PART IV TRANSFORMATION TECHNOLOGY: CEREALS AND OTHER MONOCOTS

10 Agrobacterium-Mediated Transformation of Sorghum bicolor Using

Immature Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Songul Gurel, Ekrem Gurel, Tamara I. Miller, and Peggy G. Lemaux
11 Split-Transgene Expression in Wheat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Mario Gils, Myroslava Rubtsova, and Katja Kempe
12 Agrobacterium-Mediated Transformation of Brachypodium distachyon . . . . . . . . . . . 137
Vera Thole and Philippe Vain
13 Transformation of Barley (Hordeum vulgare L.) by Agrobacterium tumefaciens
Infection of In Vitro Cultured Ovules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Inger Bœksted Holme, Henrik Brinch-Pedersen, Mette Lange,
and Preben Bach Holm
14 Biolistic-Mediated Production of Transgenic Oil Palm . . . . . . . . . . . . . . . . . . . . . . 163
Ghulam Kadir Ahmad Parveez and Bohari Bahariah
15 Transformation of Oil Palm Using Agrobacterium tumefaciens . . . . . . . . . . . . . . . . 177
Abang Masli Dayang Izawati, Ghulam Kadir Ahmad Parveez,
and Mat Yunus Abdul Masani

PART V TRANSFORMATION TECHNOLOGY: DICOTS

16 Highly Efficient Transformation Protocol for Plum (Prunus domestica L.). . . . . . . . 191
César Petri, Ralph Scorza, and Chinnathambi Srinivasan
17 Co-transformation of Grapevine Somatic Embryos to Produce Transgenic
Plants Free of Marker Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Manjul Dutt, Zhijian T. Li, Sadanand A. Dhekney, and Dennis J. Gray
18 Initiation and Transformation of Grapevine Embryogenic Cultures . . . . . . . . . . . . . 215
Sadanand A. Dhekney, Zhijian T. Li, Manjul Dutt, and Dennis J. Gray
19 Development of Highly Efficient Genetic Transformation Protocols
for Table Grape Sugraone and Crimson Seedless . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Mercedes Dabauza and Leonardo Velasco
20 Cotton Pistil Drip Transformation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Tianzhen Zhang and Tianzhi Chen
21 Enhanced Agrobacterium-Mediated Transformation of Embryogenic Calli
of Upland Cotton. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Tianzhen Zhang and Shen-jie Wu
22 Targeted Biolistics for Improved Transformation of Impatiens balsamina . . . . . . . . 255
Andy C. Wetten, Jean-Luc Thomas, Alina Wagiran, and Tinashe Chiurugwi
23 A Protocol for Transformation of Torenia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Ryutaro Aida
24 Efficient Modification of Floral Traits by Heavy-Ion Beam Irradiation
on Transgenic Torenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Norihiro Ohtsubo, Katsutomo Sasaki, Ryutaro Aida,
Hiromichi Ryuto, Hiroyuki Ichida, Yoriko Hayashi, and Tomoko Abe
 Contents ix

PART VI GENE TARGETING, SILENCING AND DIRECTED MUTATION

25 Expression of Artificial MicroRNAs in Physcomitrella patens . . . . . . . . . . . . . . . . . . 293

Isam Fattash, Basel Khraiwesh, M. Asif Arif, and Wolfgang Frank
26 High Frequency of Single-Copy T-DNA Transformants Produced After Floral
Dip in CRE-Expressing Arabidopsis Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Annelies De Paepe, Sylvie De Buck, Jonah Nolf, and Ann Depicker
27 A Developmentally Regulated Cre-lox System to Generate Marker-Free
Transgenic Brassica napus Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Lilya Kopertekh, Inge Broer, and Joachim Schiemann
28 Exploiting MultiSite Gateway and pENFRUIT Plasmid Collection
for Fruit Genetic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Leandro H. Estornell, Antonio Granell, and Diego Orzaez
29 A One-Time Inducible Transposon to Create Knockout Mutants in Rice . . . . . . . . 369
Yuh-Chyang Charng
30 Marker-Free Gene Targeting by Recombinase-Mediated Cassette Exchange . . . . . . 379
Hiroyasu Ebinuma, Kazuya Nanto, Saori Kasahara, and Atsushi Komamine
31 Targeting DNA to a Previously Integrated Transgenic Locus
Using Zinc Finger Nucleases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
Tonya L. Strange and Joseph F. Petolino
32 Double-Strand Break-Induced Targeted Mutagenesis in Plants . . . . . . . . . . . . . . . . 399
L. Alexander Lyznik, Vesna Djukanovic, Meizhu Yang, and Spencer Jones

PART VII METABOLIC ENGINEERING AND PHARMING

33 Combinatorial Genetic Transformation of Cereals and the Creation

of Metabolic Libraries for the Carotenoid Pathway . . . . . . . . . . . . . . . . . . . . . . . . . 419
Gemma Farre, Shaista Naqvi, Georgina Sanahuja, Chao Bai,
Uxue Zorrilla-López, Sol M. Rivera, Ramon Canela, Gerhard Sandman,
Richard M. Twyman, Teresa Capell, Changfu Zhu, and Paul Christou
34 Production of a His-Tagged Canecystatin in Transgenic Sugarcane . . . . . . . . . . . . . 437
Flavio Henrique-Silva and Andrea Soares-Costa
35 Plastid Transformation as an Expression Tool for Plant-Derived
Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Nunzia Scotti and Teodoro Cardi
36 Use of a Callus-Specific Selection System to Develop Transgenic
Rice Seed Accumulating a High Level of Recombinant Protein . . . . . . . . . . . . . . . . 467
Yuhya Wakasa and Fumio Takaiwa

PART VIII FIELD TESTS

37 How to Grow Transgenic Arabidopsis in the Field. . . . . . . . . . . . . . . . . . . . . . . . . . 483

Hanna Johansson Jänkänpää and Stefan Jansson

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Contributors

TOMOKO ABE • National Institute of Floricultural Science, National Agriculture

and Food Research Organization, Tsukuba, Ibaraki, Japan
RYUTARO AIDA • National Institute of Floricultural Science, National Agriculture
and Food Research Organization, Tsukuba, Ibaraki, Japan
M. ASIF ARIF • Institute for Genetics, Heinrich-Heine-University Düsseldorf,
Düsseldorf, Germany
BOHARI BAHARIAH • Advanced Biotechnology and Breeding Centre, MPOB, No 6,
Persiaran Institusi, Kajang, Selangor, Malaysia
CHAO BAI • Department of Plant Production and Forestry Science, ETSEA,
University of Lleida, Lleida, Spain
RALPH BOCK • Max-Planck-Institut für Molekulare Pflanzenphysiologie,
Potsdam-Golm, Germany
HENRIK BRINCH-PEDERSEN • Department of Genetics and Biotechnology,
Research Centre Flakkebjerg, Aarhus University, Slagelse, Denmark
INGE BROER • Julius Kuehn Institute, Federal Research Centre for Cultivated
Plants (JKI), Institute for Biosafety of Genetically Modified Plants,
Quedlinburg, Germany
SYLVIE DE BUCK • Department of Plant Systems Biology, VIB, Department of
Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium
RAMON CANELA • Department of Chemistry, ETSEA, University of Lleida,
Lleida, Spain
TERESA CAPELL • Department of Plant Production and Forestry Science, ETSEA,
University of Lleida, Lleida, Spain
TEODORO CARDI • CRA-ORT, Research Centre for Vegetable Crops,
Pontecagnano, SA, Italy
JOYCE A. CARTAGENA • Department of Material and Life Science, Graduate School
of Engineering, Osaka University, Osaka, Japan
YUH-CHYANG CHARNG • Department of Agronomy, National Taiwan University,
Taipei, Taiwan, Republic of China
TIANZHI CHEN • National Key Laboratory of Crop Genetics and Germplasm
Enhancement, Cotton Research Institute, Nanjing Agricultural University,
Nanjing, China
TINASHE CHIURUGWI • Rothamsted Research, Broom’s Barn, Bury St Edmunds,
Suffolk, UK
PAUL CHRISTOU • Department of Plant Production and Forestry Science, ETSEA,
University of Lleida, Lleida, Spain; Institució Catalana de Reserca i
Estudis Avançats, Barcelona, Spain
MERCEDES DABAUZA • Instituto Murciano de Investigación y Desarrollo
Agrario y Alimentario (IMIDA), Murcia, Spain

xi
xii Contributors

ANN DEPICKER • Department of Plant Systems Biology, VIB, Department of

Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium
SADANAND A. DHEKNEY • Mid-Florida Research and Education Center,
University of Florida/IFAS, Apopka, FL, USA
VESNA DJUKANOVIC • Pioneer Hi-Bred International, Research Center,
Johnston, IA, USA
MANJUL DUTT • Citrus Research and Education Center, University of Florida/IFAS,
Lake Alfred, FL, USA
HIROYASU EBINUMA • R&D Division, Nippon Paper Industries Co. Ltd., Tokyo, Japan
MASAKI ENDO • Plant Genetic Engineering Research Unit, National Institute
of Agrobiological Sciences, Tsukuba, Japan
LEANDRO H. ESTORNELL • Instituto de Biología Molecular y Celular de Plantas,
Valencia, Spain
GEMMA FARRE • Department of Plant Production and Forestry Science, ETSEA,
University of Lleida, Lleida, Spain
ISAM FATTASH • Plant Biotechnology, Faculty of Biology, University of Freiburg,
Freiburg, Germany
WOLFGANG FRANK • Plant Biotechnology, Faculty of Biology, University of Freiburg,
Freiburg, Germany
KIICHI FUKUI • Department of Biotechnology, Graduate School of Engineering,
Osaka University, Osaka, Japan
MARIO GILS • Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung
(IPK) Gatersleben, Gatersleben, Germany
ANTONIO GRANELL • Instituto de Biología Molecular y Celular de Plantas,
Valencia, Spain
DENNIS J. GRAY • Mid-Florida Research and Education Center,
University of Florida/IFAS, Apopka, FL, USA
EKREM GUREL • Department of Plant and Microbial Biology, University of California,
Berkeley, CA, USA
SONGUL GUREL • Department of Plant and Microbial Biology, University
of California, Berkeley, CA, USA
YORIKO HAYASHI • National Institute of Floricultural Science National Agriculture
and Food Research Organization, Tsukuba, Ibaraki, Japan
FLAVIO HENRIQUE-SILVA • Laboratory of Molecular Biology, Department of Genetic
and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil
MIEKO HIGUCHI • RIKEN Plant Science Center, Tsurumi-ku, Yokohama,
Kanagawa, Japan
PREBEN BACH HOLM • Department of Genetics and Biotechnology, Aarhus University,
Research Centre Flakkebjerg, Slagelse, Denmark
INGER BÆKSTED HOLME • Department of Genetics and Biotechnology,
Aarhus University, Research Centre Flakkebjerg, Slagelse, Denmark
JOHN A. HOWARD • Applied Biotechnology Institute, California Polytechnic
State University, San Luis Obispo, CA, USA
HIROYUKI ICHIDA • National Institute of Floricultural Science, National Agriculture
and Food Research Organization, Tsukuba, Ibaraki, Japan
 Contributors xiii

TAKANARI ICHIKAWA • Accreditation and Evaluation Team for the New University,
Okinawa Institute of Science and Technology Promotion Corporation,
Okinawa, Japan
ABANG MASLI DAYANG IZAWATI • Advanced Biotechnology and Breeding Centre,
MPOB, Kajang, Selangor, Malaysia
HANNA JOHANSSON JÄNKÄNPÄÄ • Umeå Plant Science Center, Umeå, Sweden
STEFAN JANSSON • Umeå Plant Science Center, Umeå, Sweden
SPENCER JONES • Pioneer Hi-Bred International, Research Center, Johnston, IA, USA
SAORI KASAHARA • Forest Science Laboratory, Nippon Paper Industries Co. Ltd.,
Tokyo, Japan
KATJA KEMPE • Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung
(IPK) Gatersleben, Gatersleben, Germany
NARUEMON KHEMKLADNGOEN • Department of Biotechnology, Graduate School
of Engineering, Osaka University, Osaka, Japan
BASEL KHRAIWESH • Department of Plant Systems Biology, Flanders Institute
for Biotechnology (VIB), Flanders, Belgium; Department of Plant Biotechnology
and Genetics, Ghent University, Gent, Belgium
ATSUSHI KOMAMINE • The Research Institute of Evolutionary Biology, Tokyo, Japan
YOUICHI KONDOU • RIKEN Plant Science Center, Yokohama, Kanagawa, Japan;
Department of Applied Material and Life Science, Kanto Gakuin University,
Yokohama, Japan
LILYA KOPERTEKH • Julius Kuehn Institute, Federal Research Centre
for Cultivated Plants (JKI), Institute for Biosafety of Genetically Modified Plants,
Quedlinburg, Germany
METTE LANGE • Department of Genetics and Biotechnology, Aarhus University,
Research Centre Flakkebjerg, Slagelse, Denmark
PEGGY G. LEMAUX • Department of Plant and Microbial Biology,
University of California, Berkeley, CA, USA
ZHIJIAN T. LI • Mid-Florida Research and Education Center, University of Florida/
IFAS, Apopka, FL, USA
YINGHONG LU • Max-Planck-Institut für Molekulare Pflanzenphysiologie,
Potsdam-Golm, Germany
L. ALEXANDER LYZNIK • Pioneer Hi-Bred International, Research Center,
Johnston, IA, USA
MAT YUNUS ABDUL MASANI • Advanced Biotechnology and Breeding Centre, MPOB,
Kajang, Selangor, Malaysia
MINAMI MATSUI • RIKEN Plant Science Center, Yokohama, Kanagawa, Japan
TAMARA I. MILLER • Department of Plant and Microbial Biology,
University of California, Berkeley, CA, USA
MASAKI MORI • Disease Resistance Research Unit, National Institute
of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
KAZUYA NANTO • Forest Science Laboratory, Nippon Paper Industries Co. Ltd.,
Tokyo, Japan
SHAISTA NAQVI • Department of Plant Production and Forestry Science,
ETSEA, University of Lleida, Lleida, Spain
xiv Contributors

JULIANE NEUPERT • Max-Planck-Institut für Molekulare Pflanzenphysiologie,

Potsdam-Golm, Germany
JONAH NOLF • Department of Plant Systems Biology, VIB, Department of
Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium
NORIHIRO OHTSUBO • National Institute of Floricultural Science, National
Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan
HARUKO ONODERA • Division of Plant Sciences, National Institute
of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
DIEGO ORZEZ • Instituto de Biología Molecular y Celular de Plantas (IBMCP),
CSIC-UPV, Valencia, Spain
KENJIROU OZAWA • Transgenic Crop Research and Development Center,
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
ANNELIES DE PAEPE • Department of Plant Systems Biology, VIB, Department of
Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium
GHULAM KADIR AHMAD PARVEEZ • Advanced Biotechnology and Breeding Centre,
MPOB, Kajang, Selangor, Malaysia
JOSEPH F. PETOLINO • Discovery R&D, Dow AgroSciences, Indianapolis, IN, USA
CÉSAR PETRI • USDA-ARS-Appalachian Fruit Research Station,
Kearneysville, WV, USA
T. SCOTT PINKERTON • Crop Bio-Protection Unit, National Center for Agriculture
Utilization Research/ARS/USDA, Peoria, IL, USA
CRAIG M. RICHAEL • Department of Plant Sciences, JR Simplot Company,
Boise, ID, USA
SOL M. RIVERA • Department of Chemistry, ETSEA, University of Lleida,
Lleida, Spain
CAIUS M. ROMMENS • Department of Plant Sciences, JR Simplot Company,
Boise, ID, USA
MYROSLAVA RUBTSOVA • Leibniz-Institut für Pflanzengenetik
und Kulturpflanzenforschung (IPK) Gatersleben, Gatersleben, Germany
HIROMICHI RYUTO • National Institute of Floricultural Science National
Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan
HIROAKI SAIKA • Plant Genetic Engineering Research Unit, National Institute
of Agrobiological Sciences, Tsukuba, Japan
GEORGINA SANAHUJA • Department of Plant Production and Forestry Science,
ETSEA, University of Lleida, Lleida, Spain
GERHARD SANDMAN • Biosynthesis Group, Molecular Biosciences,
J.W. Goethe Universitaet, Frankfurt, Germany
KATSUTOMO SASAKI • National Institute of Floricultural Science,
National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan
JOACHIM SCHIEMANN • Julius Kuehn Institute, Federal Research Centre
for Cultivated Plants (JKI), Institute for Biosafety of Genetically Modified Plants,
Quedlinburg, Germany
RALPH SCORZA • USDA-ARS-Appalachian Fruit Research Station,
Kearneysville, WV, USA
NUNZIA SCOTTI • CNR-IGV, National Research Council of Italy, Institute of Plant
Genetics, Res. Division Portici, Portici, NA, Italy
 Contributors xv

NING SHAO • Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm,

Germany
TSUTOMU SHIMIZU • Life Science Research Institute, Kumiai Chemical
Industry Co. Ltd, Kikugawa, Shizuoka, Japan
ANDREA SOARES-COSTA • Laboratory of Molecular Biology, Department of Genetic
and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil
CHINNATHAMBI SRINIVASAN • USDA-ARS-Appalachian Fruit Research Station,
Kearneysville, WV, USA
TONYA L. STRANGE • Discovery R&D, Dow AgroSciences, Indianapolis, IN, USA
FUMIO TAKAIWA • National Institute of Agrobiological Sciences, Tsukuba,
Ibaraki, Japan
VERA THOLE • Department of Crop Genetics, John Innes Centre, Norwich, UK
JEAN-LUC THOMAS • Laboratoire de Biologie Moléculaire de la Cellule, Equipe
Différenciation Neuro-musculaire, ENS Lyon, Lyon, France
SEIICHI TOKI • Plant Genetic Engineering Research Unit, National Institute
of Agrobiological Sciences, Tsukuba, Japan; Kihara Institute for Biological Research,
Yokohama City University, Yokohama, Japan
RICHARD M. TWYMAN • Department of Biological Sciences, University of Warwick,
Coventry, UK
PHILIPPE VAIN • Department of Crop Genetics, John Innes Centre, Norwich, UK
LEONARDO VELASCO • Instituto Murciano de Investigación y Desarrollo
Agrario y Alimentario (IMIDA), La Alberca, Murcia, Spain
NAOKI WADA • Department of Material and Life Science, Graduate School
of Engineering, Osaka University, Osaka, Japan
ALINA WAGIRAN • Biology Department, Faculty of Biosciences and Bioengineering,
Technological University of Malaysia, Skudai, Johor, Malaysia
YUHYA WAKASA • Transgenic Crop Research and Development Center,
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
ANDY C. WETTEN • School of Biological Sciences, University of Reading, Reading, UK
JAMES R. WILD • Department of Biochemistry and Biophysics, Texas A&M University,
College Station, TX, USA
SHEN-JIE WU • National Key Laboratory of Crop Genetics and Germplasm
Enhancement, Cotton Research Institute, Nanjing Agricultural University,
Nanjing, China
MEIZHU YANG • Pioneer Hi-Bred International, Research Center, Johnston, IA, USA
TIANZHEN ZHANG • National Key Laboratory of Crop Genetics and Germplasm
Enhancement, Cotton Research Institute, Nanjing Agricultural University,
Nanjing, China
CHANGFU ZHU • Department of Plant Production and Forestry Science, ETSEA,
University of Lleida, Lleida, Spain
UXUE ZORRILLA-LÓPEZ • Department of Plant Production and Forestry Science,
ETSEA, University of Lleida, Lleida, Spain
 Part I

Selection and Detection Methods

 Chapter 1

Employment of Cytokinin Vectors for Marker-Free

and Backbone-Free Transformation
Craig M. Richael and Caius M. Rommens

Abstract
Marker-free methods of plant transformation sacrifice the advantages of a selectable marker during
regeneration or add work after regeneration to remove the marker. On the positive side, there is no stably
integrated marker gene in the plant genome to present regulatory hurdles or potential biosafety hazards
once the plant is released to the environment. A marker-free method that is simple and adaptable to
multiple crop species—even asexually propagated species—is presented herein. This method employs an
engineered vector that utilizes the isopentenyltransferase (ipt) to drive the regeneration of intragenic
cells containing the gene(s) of interest. The ipt gene also acts as a marker to screen against events where
the vector backbone is stably integrated.

Key words: Cytokinin, Isopentenyltransferase, Intragenic, Cisgenic, Transgenic, Marker-free,

Agrobacterium, Potato, Tomato

1. Introduction

At about the turn of the century, intragenic or cisgenic crops were

proposed as an alternative to the currently released transgenic crops
(1, 2). Unlike transgenics, intragenic crop plants harbor no foreign
DNA; all introduced DNA sequences, including gene and gene
regulatory elements, are of plant origin and derived from sexually
compatible species. Moreover, no extraneous vector DNA (vector
backbone) or foreign selection marker genes are permissible for
co-transfer. Using intragenesis, one can manipulate the expression
of native genes without providing the target plant with a new trait
that neither occurs in the recipient species in nature nor can be
introduced through traditional breeding. Intragenesis most
closely approximates traditional breeding in that no foreign DNA
is involved. The method is employed to correct deficits of elite

Jim M. Dunwell and Andy C. Wetten (eds.), Transgenic Plants: Methods and Protocols, Methods in Molecular Biology, vol. 847,
DOI 10.1007/978-1-61779-558-9_1, © Springer Science+Business Media, LLC 2012