Agrobacterium Protocols

Agrobacterium

Protocols

Kan Wang Editor

Volume 2

Third Edition

Methods in

M

M

B

Series Editor

John M. Walker

School of Life and Medical Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes:

Agrobacterium Protocols

Volume 2

Third Edition

Edited by

Kan Wang

Center for Plant Transformation, Plant Sciences Institute, and Department of Agronomy,

Iowa State University, Ames, IA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1657-3 ISBN 978-1-4939-1658-0 (eBook) DOI 10.1007/978-1-4939-1658-0

Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014950243 © Springer Science+Business Media New York 2015

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law.

The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

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Springer is part of Springer Science+Business Media (www.springer.com)

Editor

Kan Wang

Center for Plant Transformation Plant Sciences Institute

Department of Agronomy Iowa State University Ames , IA , USA

To Marc Van Montagu and Jeff Schell (1935–2003), my Ph.D. mentors, for their inspira-tion and encouragement.

vii

Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium is able to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agricultural advancement.

The fi rst transgenic plants were reported a little over 30 years ago in 1983 by three independent research groups. Using disarmed Agrobacterium vectors, these groups pro-duced antibiotic-resistant transgenic tobacco Nicotiana tobaccum (Herrera-Estralla et al., 1983, Nature 303: 209), Nicotiana plumbaginifolia (Bevan et al., 1983, Nature 304: 184), and petunia ( Petunia hybrid , Fraley et al., 1983, Proceedings of the National Academy of Sciences 80: 4803). The three scientists who led the landmark work, Marc Van Montagu, Mary-Dell Chilton, and Robert Fraley, were the laureates for the 2013 World Food Prize ( http://www.worldfoodprize.org/en/laureates/2013_laureates/#StatementAchievem ent ). As the statement of achievement of the World Food Prize says, “… each conducted groundbreaking molecular research on how a plant bacterium could be adapted as a tool to insert genes from another organism into plant cells, which could produce new genetic lines with highly favorable traits.”

While other methods such as biolistic gun, electroporation or polyethylene glycol can also be used for introducing DNA molecules into plant cells, the Agrobacterium -mediated transformation method remains the method of choices for most plant species in many labo-ratories due to its effi ciency and its propensity to generate single or a low copy number of integrated transgenes with defi ned ends.

When the fi rst edition of Agrobacterium Protocols was published in 1995, exactly 20 years ago, only a handful of plants could be routinely transformed using Agrobacterium . The second edition, which was published in 2006, collected transformation protocols for 59 plant species. In this third edition, we have updated protocols for 32 plant species from the second edition and added protocols for 17 new species. Together with the fi rst and second editions, these two new volumes offer Agrobacterium -mediated genetic transforma-tion protocols for a total of 76 plant species.

The third edition of Agrobacterium Protocols contains 57 chapters (two volumes) divided into 9 parts. This edition emphasizes on agricultural crops or plant species with economic values. For a number of important plants such as rice, barley, wheat, and citrus, multiple protocols using different starting plant materials for transformation are included. Like the second edition, plants are grouped according to their practical uses rather than their botanical classifi cations.

Agrobacterium Protocols provides a benchtop manual for tested protocols involving Agrobacterium -mediated transformation. All chapters are written in the same format as that used in the Methods in Molecular Biology series. Each chapter is contributed by authors who are leaders or veterans in their respective areas. The “Abstract” and “Introduction” sections provide outlines of protocols, the rationale for selection of particular target tissues, and information regarding overall transformation effi ciency. The “Materials” section lists the host materials, Agrobacterium strains and vectors, stock solutions, media, and other

viii

supplies necessary for carrying out these transformation experiments. The “Methods” section is the core of each chapter. It provides a step-by-step description of the entire transforma-tion procedure from the preparatransforma-tion of starting materials to the harvest of transgenic plants. To ensure the reproducibility of each protocol, the “Notes” section lists possible pitfalls in the protocol and suggests alternative materials or methods for generating transgenic plants. Typically, most laboratories only work on one or a few plant species. Each laboratory or individual researcher has his or her own favorite variation or modifi cation of any given plant transformation protocol. The protocols presented in this edition represent the most effi -cient methods used in the laboratories of the contributors. They are by no means the only methods for successful transformation of your plant of interest.

The broad range of target tissue selection and in vitro culture procedures indicate the complexity in plant transformation. It is the intention of this book to facilitate the transfer of this rapidly developing technology to all researchers for use in both fundamental and applied biology. I take this opportunity to thank all my colleagues whose time and effort made this edition possible. Special thanks go to my family for their unconditional love and support during the process of editing this book.

Ames, IA, USA Kan Wang

ix

Contents

Preface. . . vii

Contributors . . . xiii

P

I I

P

1 Brassica rapa. . . 3

Tom Lawrenson, Cassandra Goldsack, Lars Ostergaard, and Penny A. C. Hundleby née Sparrow

2 Cotton (Gossypium hirsutum L.) . . . 11

Keerti S. Rathore, LeAnne M. Campbell, Shanna Sherwood, and Eugenia Nunes

3 Jatropha (Jatropha curcas L.). . . 25

Devendra Kumar Maravi, Purabi Mazumdar, Shamsher Alam, Vaibhav V. Goud, and Lingaraj Sahoo

4 Sesame (Sesamum indicum L.) . . . 37

Sonia Kapoor, Sanjay S. Parmar, Manju Yadav, Darshna Chaudhary, Manish Sainger, Ranjana Jaiwal, and Pawan K. Jaiwal

5 Sunflower (Helianthus annuus L.). . . 47

Laura M. Radonic, Dalia M. Lewi, Nilda E. López, H. Esteban Hopp, Alejandro S. Escandón, and Marisa López Bilbao

P

II R

P

6 Carrot (Daucus carota L.) . . . 59

Owen S. D. Wally and Zamir K. Punja

7 Cassava (Manihot esculenta Crantz) . . . 67

Simon E. Bull

8 Potato (Solanum tuberosum L.) . . . 85

Venkateswari J. Chetty, Javier Narváez-Vásquez, and Martha L. Orozco-Cárdenas

9 Taro (Colocasia esculenta (L.) Schott) . . . 97

Xiaoling He, Susan C. Miyasaka, Maureen M. M. Fitch, and Yun J. Zhu

P

III N

F

10 Apricot (Prunus armeniaca L.) . . . 111

César Petri, Nuria Alburquerque, and Lorenzo Burgos

11 Blueberry (Vaccinium corymbosum L.). . . 121

x

12 Cherry . . . 133

Guo-Qing Song

13 Chestnut, American (Castanea dentata (Marsh.) Borkh.) . . . 143

Charles A. Maynard, Linda D. McGuigan, Allison D. Oakes, Bo Zhang, Andrew E. Newhouse, Lilibeth C. Northern, Allison M. Chartrand, Logan R. Will, Kathleen M. Baier, and William A. Powell

14 Chestnut, European (Castanea sativa) . . . 163

Elena Corredoira, Silvia Valladares, Ana M. Vieitez, and Antonio Ballester

15 Grapevine (Vitis vinifera L.) . . . 177

Laurent Torregrosa, Sandrine Vialet, Angélique Adivèze, Pat Iocco- Corena, and Mark R. Thomas

16 Melon (Cucumis melo) . . . 195

Satoko Nonaka and Hiroshi Ezura

17 Peach (Prunus persica L.) . . . 205

Silvia Sabbadini, Tiziana Pandolfini, Luca Girolomini, Barbara Molesini, and Oriano Navacchi

18 Strawberry (Fragaria × ananassa) . . . 217

Roberto Cappelletti, Silvia Sabbadini, and Bruno Mezzetti

19 Walnut (Juglans) . . . 229

Charles A. Leslie, Sriema L. Walawage, Sandra L. Uratsu, Gale McGranahan, and Abhaya M. Dandekar

P

IV T

P

20 Citrus Transformation Using Juvenile Tissue Explants. . . 245

Vladimir Orbović and Jude W. Grosser

21 Citrus Transformation Using Mature Tissue Explants . . . 259

Vladimir Orbović, Alka Shankar, Michael E. Peeples, Calvin Hubbard, and Janice Zale

22 Coffee (Coffea arabica L.) . . . 275

Eveline Déchamp, Jean-Christophe Breitler, Thierry Leroy, and Hervé Etienne

23 Pineapple [Ananas comosus (L.) Merr.] . . . 293

Gaurab Gangopadhyay and Kalyan K. Mukherjee

24 Sugarcane (Saccharum Spp. Hybrids) . . . 307

Hao Wu and Fredy Altpeter

P

V O

I

P

25 Hemp (Cannabis sativa L.). . . 319

Mistianne Feeney and Zamir K. Punja

26 Orchids (Oncidium and Phalaenopsis) . . . 331

Chia-Wen Li, Chia-Hui Liao, Xia Huang, and Ming-Tsair Chan

xi 27 Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch). . . 347

M. Ashraful Islam, Tage Thorstensen, and Jihong Liu Clarke

28 Populus trichocarpa . . . 357

Quanzi Li, Ting-Feng Yeh, Chenmin Yang, Jingyuan Song, Zenn-Zong Chen, Ronald R. Sederoff, and Vincent L. Chiang

29 Tall Fescue (Festuca arundinacea Schreb.). . . 365

Yaxin Ge and Zeng-Yu Wang

Index . . . 373

xiii

ANGÉLIQUE ADIVÈZE • Montpellier SupAgro-INRA , UMR AGAP–Genetic Improvement

and Adaptation of Mediterranean and Tropical Plants , Montpellier Cedex , France

SHAMSHER ALAM • Department of Biotechnology , Indian Institute of Technology Guwahati ,

Guwahati , India

NURIA ALBURQUERQUE • Grupo de Biotecnología de Frutales, Departamento de Mejora ,

CEBAS-CSIC , Murcia , Spain

FREDY ALTPETER • Plant Molecular and Cellular Biology Program, Agronomy Department,

Genetics Institute , University of Florida , Gainesville , FL , USA

KATHLEEN M. BAIER • Department of Environmental and Forest Biology , SUNY College

of Environmental Science and Forestry , Syracuse , NY , USA

ANTONIO BALLESTER • Instituto de Investigaciones Agrobiológicas de Galicia, IIAG , CSIC ,

Santiago de Compostela , Spain

JEAN-CHRISTOPHE BREITLER • Centre de Coopération Internationale en Recherche

Agronomique pour le Développement , UMR RPB , Montpellier , France

SIMON E. BULL • Plant Biotechnology Group , ETH Zurich , Universitaetstrasse,

Zurich , Switzerland

LORENZO BURGOS • Grupo de Biotecnología de Frutales, Departamento de Mejora ,

CEBAS-CSIC , Murcia , Spain

LEANNE M. CAMPBELL • Institute for Plant Genomics and Biotechnology , Texas A&M

University , College Station , TX , USA

ROBERTO CAPPELLETTI • Department of Agriculture, Food and Environmental Sciences ,

Università Politecnica delle Marche , Ancona , Italy

MING-TSAIR CHAN • Academia Sinica Biotechnology Center , Tannan , Taiwan ; Academia

Sinica Agricultural Biotechnology Research Center , Taipei , Taiwan

ALLISON M. CHARTRAND • Horn Performance and Environmental Science , Northwestern

University , Evanston , IL , USA

DARSHNA CHAUDHARY • Centre for Biotechnology , M. D. University , Rohtak , India

ZENN-ZONG CHEN • Division of Silviculture , Taiwan Forestry Research Institute ,

Taipei , Taiwan

VENKATESWARI J. CHETTY • Plant Transformation Research Center , University of California

Riverside , Riverside , CA , USA

VINCENT L. CHIANG • Forest Biotechnology Group, Department of Forestry , North Carolina

State University , Raleigh , NC , USA

JIHONG LIU CLARKE • Bioforsk- Norwegian Institute for Agricultural and Environmental

Research , Ås , Norway

ELENA CORREDOIRA • Instituto de Investigaciones Agrobiológicas de Galicia, IIAG ,

CSIC , Santiago de Compostela , Spain

ABHAYA M. DANDEKAR • Plant Science Department , University of California , Davis ,

CA , USA

EVELINE DÉCHAMP • Centre de Coopération Internationale en Recherche Agronomique

pour le Développement , UMR RPB , Montpellier , France

xiv

ALEJANDRO S. ESCANDÓN • Instituto de Genética Ewald A. Favret , Instituto Nacional

de Tecnología Agropecuaria , Castelar , Buenos Aires , Argentina

HERVÉ ETIENNE • Centre de Coopération Internationale en Recherche Agronomique pour

le Développement , UMR RPB , Montpellier , France

HIROSHI EZURA • University of Tsukuba , Tsukuba , Ibaraki , Japan

MISTIANNE FEENEY • School of Life Sciences , University of Warwick , Coventry , UK

MAUREEN M. M. FITCH • Hawaii Agriculture Research Center , Kunia , HI , USA

GAURAB GANGOPADHYAY • Division of Plant Biology , Bose Institute , Kolkata , India

YAXIN GE • Forage Improvement Division , The Samuel Roberts Noble Foundation ,

Ardmore , OK , USA

LUCA GIROLOMINI • Scienze Agrarie, Alimentari ed Ambientali D3A , Università

Politecnica delle Marche , Ancona , Italy

CASSANDRA GOLDSACK • Department of Crop Genetics , John Innes Centre,

Norwich Research Park , Norwich , UK

VAIBHAV V. GOUD • Center for Energy and Department of Chemical Engineering ,

Indian Institute of Technology Guwahati , Guwahati , India

JUDE W. GROSSER • Citrus Research and Education Center , University of Florida/IFAS ,

Lake Alfred , FL , USA

XIAOLING HE • Hawaii Agriculture Research Center , Kunia , HI , USA

H. ESTEBAN HOPP • Instituto de Biotecnología , Instituto Nacional de Tecnología

Agropecuaria , Castelar , Buenos Aires , Argentina

XIA HUANG • Sun Yat-sen University , Guangzhou , The People’s Republic of China

CALVIN HUBBARD • Citrus Research and Education Center , University of Florida/IFAS ,

Lake Alfred , FL , USA

PENNY A. C. HUNDLEBY NÉE SPARROW • Department of Crop Genetics , John Innes Centre,

Norwich Research Park , Norwich , UK

PAT IOCCO-CORENA • Horticulture Unit , CSIRO Plant Industry , Glen Osmond ,

SA , Australia

M. ASHRAFUL ISLAM • Department of Horticulture , Bangladesh Agricultural University ,

Mymensingh , Bangladesh

RANJANA JAIWAL • Department of Zoology , M. D. University , Rohtak , India

PAWAN K. JAIWAL • Centre for Biotechnology , M. D. University , Rohtak , India

SONIA KAPOOR • Centre for Biotechnology , M. D. University , Rohtak , India ; University

Institute of Engineering and Technology , M. D. University , Rohtak , India

TOM LAWRENSON • Department of Crop Genetics , John Innes Centre, Norwich Research

Park , Norwich , UK

THIERRY LEROY • Centre de Coopération Internationale en Recherche Agronomique pour

le Développement , UMR AGAP , Montpellier , France

CHARLES A. LESLIE • Plant Sciences Department , University of California Davis , Davis ,

CA , USA

DALIA M. LEWI • Instituto de Genética Ewald A. Favret , Instituto Nacional de Tecnología

Agropecuaria , Castelar , Buenos Aires , Argentina

CHIA-WEN LI • Department of Biotechnology , TransWorld University , Douliu City ,

Yunlin , Taiwan

QUANZI LI • State Key Laboratory of Tree Genetics and Breeding , Chinese Academy of

Forestry , Beijing , China ; College of Forestry , Shandong Agricultural University, Taian , Shandong , China

CHIA-HUI LIAO • Academia Sinica Biotechnology Center in Southern Taiwan , Tainan ,

Taiwan ; Academia Sinica Agricultural Biotechnology Research Center , Taipei , Taiwan

xv MARISA LÓPEZ BILBAO • Instituto de Biotecnología , Instituto Nacional de Tecnología

Agropecuaria , Castelar , Buenos Aires , Argentina

NILDA E. LÓPEZ • Instituto de Biotecnología , Instituto Nacional de Tecnología

Agropecuaria , Castelar , Buenos Aires , Argentina

DEVENDRA KUMAR MARAVI • Center for Energy , Indian Institute of Technology Guwahati ,

Guwahati , India

CHARLES A. MAYNARD • Department of Forest and Natural Resources Management ,

SUNY College of Environmental Science and Forestry , Syracuse , NY , USA

PURABI MAZUMDAR • Center for Energy , Indian Institute of Technology Guwahati ,

Guwahati , India

GALE MCGRANAHAN • Plant Science Department , University of California , Davis , CA , USA

LINDA D. MCGUIGAN • Department of Forest and Natural Resources Management ,

SUNY College of Environmental Science and Forestry , Syracuse , NY , USA

BRUNO MEZZETTI • Department of Agriculture, Food and Environmental Sciences ,

Università Politecnica delle Marche , Ancona , Italy

SUSAN C. MIYASAKA • Department of Tropical Plant and Soil Sciences , University of Hawaii ,

Hilo , HI , USA

BARBARA MOLESINI • Dipartimento di Biotecnologie , Università di Verona , Verona , Italy

KALYAN K. MUKHERJEE • Division of Plant Biology , Bose Institute , Kolkata , India

JAVIER NARVÁEZ-VÁSQUEZ • Plant Transformation Research Center , University of California

Riverside , Riverside , CA , USA

ORIANO NAVACCHI • Vitroplant Italia , Cesena , Italy

ANDREW E. NEWHOUSE • Department of Environmental and Forest Biology , SUNY College

of Environmental Science and Forestry , Syracuse , NY , USA

SATOKO NONAKA • University of Tsukuba , Tsukuba , Ibaraki , Japan

LILIBETH C. NORTHERN • Department of Environmental and Forest Biology , SUNY College

of Environmental Science and Forestry , Syracuse , NY , USA

EUGENIA NUNES • Faculty of Science , The University of Porto , Porto , Portugal

ALLISON D. OAKES • Department of Environmental and Forest Biology , SUNY College

of Environmental Science and Forestry , Syracuse , NY , USA

VLADIMIR ORBOVIĆ • Citrus Research and Education Center , University of Florida/IFAS ,

Lake Alfred , FL , USA

MARTHA L. OROZCO-CÁRDENAS • Plant Transformation Research Center , University

of California Riverside , Riverside , CA , USA

LARS OSTERGAARD • Department of Crop Genetics , John Innes Centre, Norwich Research

Park , Norwich , UK

TIZIANA PANDOLFINI • Dipartimento di Biotecnologie , Università di Verona , Verona , Italy

SANJAY S. PARMAR • Centre for Biotechnology , M. D. University , Rohtak , India

MICHAEL E. PEEPLES • Citrus Research and Education Center , University of Florida/IFAS ,

Lake Alfred , FL , USA

CÉSAR PETRI • Grupo de Biotecnología de Frutales, Departamento de Mejora ,

CEBAS- CSIC , Murcia , Spain

WILLIAM A. POWELL • Department of Environmental and Forest Biology , SUNY College

of Environmental Science and Forestry , Syracuse , NY , USA

ZAMIR K. PUNJA • Department of Biological Sciences , Simon Fraser University , Burnaby ,

BC , Canada

LAURA M. RADONIC • Instituto de Biotecnología , Instituto Nacional de Tecnología

Agropecuaria , Castelar , Buenos Aires , Argentina

xvi

KEERTI S. RATHORE • Institute for Plant Genomics and Biotechnology , Texas A&M University ,

College Station , TX , USA ; Department of Soil and Crop Sciences , Texas A&M University , College Station , TX , USA

SILVIA SABBADINI • Scienze Agrarie, Alimentari ed Ambientali D3A , Università Politecnica

delle Marche , Ancona , Italy

LINGARAJ SAHOO • Center for Energy and Department of Biotechnology , Indian Institute

of Technology Guwahati , Guwahati , India

MANISH SAINGER • Centre for Biotechnology , M. D. University , Rohtak , India

RONALD R. SEDEROFF • Forest Biotechnology Group, Department of Forestry , North Carolina

State University , Raleigh , NC , USA

ALKA SHANKAR • Citrus Research and Education Center , University of Florida/IFAS ,

Lake Alfred , FL , USA

SHANNA SHERWOOD • Institute for Plant Genomics and Biotechnology , Texas A&M

University , College Station , TX , USA

GUO-QING SONG • Department of Horticulture, Plant Biotechnology Resource

and Outreach Center , Michigan State University , East Lansing , MI , USA

JINGYUAN SONG • Institute of Medicinal Plant Development, Chinese Academy of Medical

Sciences and Peking Union Medical College , Beijing , China

MARK R. THOMAS • Horticulture Unit , CSIRO Plant Industry , Glen Osmond , SA , Australia

TAGE THORSTENSEN • Bioforsk- Norwegian Institute for Agricultural and Environmental

Research , Ås , Norway

LAURENT TORREGROSA • Montpellier SupAgro-INRA , UMR AGAP—Genetic Improvement

and Adaptation of Mediterranean and Tropical Plants , Montpellier Cedex , France

SANDRA L. URATSU • Plant Science Department , University of California , Davis , CA , USA

SILVIA VALLADARES • Instituto de Investigaciones Agrobiológicas de Galicia, IIAG , CSIC ,

Santiago de Compostela , Spain

SANDRINE VIALET • Unité Expérimentale de Pech-Rouge , INRA, UEPR , Gruissan , France

ANA M. VIEITEZ • Instituto de Investigaciones Agrobiológicas de Galicia, IIAG , CSIC ,

Santiago de Compostela , Spain

SRIEMA L. WALAWAGE • Plant Science Department , University of California , Davis ,

CA , USA

OWEN S. D. WALLY • Department of Plant Sciences , University of Manitoba , Winnipeg ,

MB , Canada

ZENG-YU WANG • Forage Improvement Division , The Samuel Roberts Noble Foundation ,

Ardmore , OK , USA

LOGAN R. WILL • The American Chestnut Research and Restoration Project ,

SUNY College of Environmental Science and Forestry , Syracuse , NY , USA

HAO WU • Agronomy Department, Plant Molecular and Cellular Biology Program,

Genetics Institute , University of Florida , Gainesville , FL , USA

MANJU YADAV • Centre for Biotechnology , M. D. University , Rohtak , India

CHENMIN YANG • Forest Biotechnology Group, Department of Forestry , North Carolina

State University , Raleigh , NC , USA

TING-FENG YEH • School of Forestry and Resource Conservation , National Taiwan

University , Taipei , Taiwan

JANICE ZALE • Citrus Research and Education Center , University of Florida/IFAS ,

Lake Alfred , FL , USA

BO ZHANG • Therapeutic Proteins International, LLC , Chicago , IL , USA

YUN J. ZHU • Hawaii Agriculture Research Center , Kunia , HI , USA Contributors

Part I

3

Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224, DOI 10.1007/978-1-4939-1658-0_1, © Springer Science+Business Media New York 2015

Chapter 1

Brassica rapa

Tom Lawrenson , Cassandra Goldsack , Lars Ostergaard ,

and Penny A. C. Hundleby née Sparrow

Abstract

Within this chapter we outline an A. tumefaciens -mediated transformation method for B. rapa using 4-day-old cotyledonary explants and the genotype R-o-18. Transformation effi ciencies are typically achieved in the region of 1 % (based on 2 PCR-positive independent shoots from 200 inoculated explants). This system has been developed to work with gentamicin selection.

Key words Agrobacterium tumefaciens , Brassica rapa , Diploid , Gentamicin selection , Oilseed , Transformation

1

Introduction

Brassica rapa has historically been the most recalcitrant of Brassica species to transform, with published work focusing on Chiffu, the genotype of the sequencing program, and other B. rapa pekinensis genotypes (Chinese cabbage). However, one signifi cant downside to these genotypes is that they are vegetable types and also highly self-incompatible. As a transformation resource, the economic cost of generating enough seed for routine transformation, as well as the time to hand-pollinate transgenic lines to obtain next- generation material, makes these undesirable candidates for rou-tine transformation studies. The B. rapa variety R-o-18 was chosen as the target genotype for studies in our laboratories as its plant architecture is similar to B. napus oilseed rape. R-o-18 is derived from a B. rapa oilseed crop grown in Pakistan and India and is therefore already a crop in its own right. Moreover, the genotype is rapid cycling and self-compatible, enabling the production of large seed stocks to use in transformation studies, as well as gener-ating next-generation transgenic material cost-effectively without the need for laborious hand pollination. This genotype is also used as a model B. rapa genotype by a number of research labs, in par-ticular complementing the R-o-18 TILLING resource available via

4

the John Innes Centre ( http://revgenuk.jic.ac.uk ). In the current chapter, we describe our protocol for the routine transformation of R-o-18 using a gentamicin-based selection system. Typically, transformation effi ciencies in the region of 1 % are achieved (based on 2 PCR-positive independent rooted shoots from 200 inocu-lated explants).

2

Materials

1. Vitamins: 10 g/L thiamine-HCl, 1 g/L pyridoxine, 1 g/L nicotinic acid, and 100 g/L myoinositol. All vitamins are made up in sterile distilled water (SDW), fi lter sterilized, and stored individually at 4 °C, with the exception of myoinositol, which is stored at room temperature.

2. 1-Naphthaleneacetic acid (NAA): 1 mg/mL stock solution. Prepare by dissolving the powder in 1 M NaOH (50 mg in 100 μL). Make to fi nal volume with sterile distilled water (SDW) and fi lter sterilize. Store at 4 °C.

3. 6-Benzylaminopurine (BAP): 4 mg/mL stock solution. Dissolve the powder in 1 M NaOH (100 mg in 200 μL). Make to fi nal volume with SDW and fi lter sterilize. Store at 4 °C. 4. Indole-3-butyric acid (IBA): 1 mg/mL stock solution. Prepare

by dissolving the powder in 1 M NaOH (50 mg in 100 μL). Make to fi nal volume with SDW and fi lter sterilize. Store at 4 °C. 5. Gentamicin: 50 mg/mL stock solution. Store at 4–8 °C. 6. Timentin: 160 mg/mL stock solution. Dissolve in SDW, fi lter

sterilize, and store at −20 °C.

7. AgNO 3 : 20 mg/mL stock solution. Filter sterilize and store

foil-wrapped at 4 °C.

8. Germination medium: 4.3 g/L Murashige and Skoog [ 1 ] (MS) basal salts only, 30 g/L Sucrose, pH 5.7, 8 g/L phytagar. Autoclave at 120 °C for 20 min. Prior to pouring add 1 mL of each of the four vitamin stocks. One liter typically pours 20 Magenta™ boxes with 50 mL/Magenta™.

9. Callus induction media for cocultivation (CIM-C): As germi-nation medium plus 500 mg/L 2-( N -morpholino)ethanesul-fonic acid (MES). MES is a buffering agent, therefore ensure the pH is adjusted after adding. After autoclaving and before pouring, add 1 mL each of the four vitamin stocks, 4 mg/L BAP (1 mL of BAP at 4 mg/mL), and 0.1 mg/L NAA (100 μL of 1 mg/mL NAA). One liter typically pours 20 petri dishes (20 × 90 mm).

10. Callus induction medium for selection (CIM-S): As CIM-C with the addition after autoclaving and before pouring of 160 mg/L Timentin (1 mL of 160 mg/mL stock), 4 mg/L

2.1 Plant Culture Media and Components

5 AgNO 3 (200 μL of the 20 mg/L AgNO 3 stock), 10 mg/L

gentamicin (200 μL of 50 mg/mL stock). One liter typi-cally pours 20 petri dishes (20 × 90 mm). Selection is not included in the control plates (a single control plate can be poured before gentamicin is added to the medium for the other plates).

11. Shoot induction selection media: 1 L MS basal medium plus 500 mg/L MES. After autoclaving and before pouring, add 4 mg/L BAP (1 mL of BAP stock at 4 mg/mL), 160 mg Timentin (1 mL of 160 mg/mL stock), 4 mg/L AgNO 3

(200 μL of AgNO 3 at 20 mg/mL), 10 mg/L gentamicin

(200 μL gentamicin at 50 mg/mL), 1 mL each of the four vitamin stocks. No gentamicin is added to control plates. 12. Shoot elongation selection medium: 3.05 g/L Gamborg’s B5

salts, 30 g/L sucrose, pH 5.7, 8 g/L phytagar. Autoclave at 120 °C for 20 min. Prior to pouring add 1 mL each of the four vitamin stocks, 0.05 mg/L BAP (12.5 μL of 4 mg/mL stock), 160 mg/L Timentin (1 mL of 160 mg/mL stock), 4 mg/L AgNO 3 (200 μL of 20 mg/mL stock), and 10 mg/L

gentami-cin (200 μL of 50 mg/mL stock). No gentamigentami-cin in control plates.

13. Rooting media based on Gamborg’s B5 medium [ 2 ]: 3.05 g/L Gamborg’s B5 salts, 10 g/L sucrose, pH5.7, 8 g/L phytagar. Autoclave at 120 °C for 20 min. Prior to pouring add 1 mg/L IBA (1 mL of 1 mg/mL stock), gentamicin 10 mg/L (200 μL of 50 mg/mL stock), and Timentin 160 mg/L (1 mL of 160 mg/mL stock). No gentamicin is added to control jars. 1. LB medium: 5 g/L yeast extract, 10 g/L NaCl, 10 g/L

pep-tone ±15 g/L Bacto agar (for solid/liquid medium); auto-clave at 120 °C for 20 min.

2. 20× AB salts: To prepare 1 L, add 20 g NH 4 Cl, 6 g

MgSO 4 · 7H 2 O, 3 g KCl, 0.26 g CaCl 2 · H 2 O, and 0.05 g

FeSO 4 · 7H 2 O in SDW. Store at −20 o C in 50 mL aliquots.

3. Induction media: To prepare 200 mL, add 10 mL 20× AB salts, 4 g glucose, 1.18 g MES, 0.06 g NaH 2 PO 4 , and 0.06 g

Na 2 HPO 4 in SDW. Adjust pH to 5.6 with 1 M NaOH. Filter

sterilize and store at 4 °C for up to 1 month.

4. Acetosyringone (20 mg/mL in dimethyl sulfoxide). Aliquot into 20 μL volumes and store at −20 °C.

R-o-18 is an inbred line of the Brassica rapa subsp. trilocularis (yellow sarson) with transparent seed coat [ 3 ] closely related to B.

rapa oilseed crops grown in Pakistan [ 4 ]. Seeds can be obtained by contacting Lars Østergaard on email: [email protected].

2.2 Agrobacterium

Culture Media

2.3 Seed Source

6

1. Sterilizing solution: 15 % sodium hypochlorite (BDH Chemicals), plus 0.1 % of surfactant Tween 20.

2. Soil: John Innes No. 2 commercial compost.

3. Perforated “bread” bags: supplied by Cryovac (UK) Ltd. 4. Phytagar: supplied by Duchefa (P1003).

5. Timentin: sold as ticarcillin disodium/clavulanate potassium (Duchefa T0190).

6. Sterile peat pots: Sterile peat pots (Jiffy No. 7) are placed into Magenta™ pots (Sigma), and soaked in water until fully expanded. Excess water is poured off and Magentas™ auto-claved at 120 °C for 20 min.

3

Methods

The protocol described below is applicable to B. rapa genotype R-o-18. Transformation is based on a previously reported method for B. napus [ 5 ].

1. Seeds are sterilized by immersion in 70 % ethanol for 2 min prior to treating with 15 % sodium hypochlorite commercial bleach/0.01 % Tween 20 for 15 min, and washed in SDW three times.

2. Seeds are transferred to the surface of germination medium in Magentas™ at a density of 25 seeds per Magenta™ pot. Magentas™ are then maintained in a 23 °C growth room with 16/24 light hours regime at 40 μmol/m 2 _{/s for 4 days}

( see Note 1 ).

1. Agrobacterium strain AGL1 is transformed by electroporation with the construct of interest using a suitable bacterial select-able marker ( see Notes 2 and 3 ).

2. Single colonies are used to inoculate liquid cultures which are subsequently used to make glycerol stocks for use as inoculum (standard inoculum) and to confi rm integrity of the construct of interest. The latter is done via plasmid miniprep of the Agrobacterium suspension and transformation into E. coli in order to obtain a suffi cient quantity of DNA to confi rm by restriction digest.

3. Glycerol stocks are composed of 20 % glycerol and 80 % LB Agrobacterium culture. Sterility during all stages of prepara-tion as well as subsequent inoculaprepara-tion is of utmost importance to avoid contamination of tissue culture material.

4. The standard inoculum is stored at −70 °C and used to initiate fresh 10 mL LB cultures 48 h prior to explant isolation/ inoculation. 2.4 Other Supplies and Reagents 3.1 Seed Sterilization and Germination 3.2 Agrobacterium Preparation

7 5. Twenty-four hours after inoculation with standard inoculum,

the LB should be visibly turbid and ready to induce.

6. 5 mL of the resulting suspension is pelleted in a microfuge at full speed for 10 s, and then resuspended in 5 mL of induction medium (pre-warmed to room temperature). A fresh aliquot of acetosyringone (at 20 mg/mL) is defrosted, and 5 μL of this is added to give a concentration of 20 mg/L.

7. The suspension is then incubated in a shaker at 22 °C for 16–20 h in darkness.

8. Directly before the inoculation is made, cells are diluted to OD 650 = 0.3 using induction media.

1. Cotyledonary petioles are isolated from 4-day-old seedlings. Cotyledons are cut with a sharp scalpel at the point indicated in Fig. 1a–c maximizing the length of the petiole extending from the cotyledons, but not so much as to prevent the separa-tion of the two cotyledons with a single cut. If the cut is made too far from the cotyledons and they do not cleanly separate, then apical meristem tissue will be included in the explant leading to regeneration of escape shoots.

2. The explants are immediately moved to CIM-C plates with just the cut end of the petiole imbedded. Ten explants are held on each plate.

3. When all explants are held on CIM-C plates, inoculation with the Agrobacterium harboring the construct of interest is car-ried out by dipping the cut end of the petiole into an Agrobacterium suspension.

4. One plate should remain without inoculation to be used as a regeneration control. Explants are then returned to the same CIM-C plates which are then sealed with Micropore tape and moved to a growth room at 23 °C with 16/8 light hours regime at 40 μmol/m 2 _{/s for 72 h.}

1. After 72 h explants are moved to CIM-S plates with the excep-tion of the two control plates (one inoculated and the other not exposed to Agrobacterium ) which are moved to fresh

CIM-C plates.

2. Plates are returned to the same growth conditions for a further 48 h, before being transferred to shoot induction medium (with the two controls moved to shoot induction with no selection). At this point the shallow lid of the tissue culture dish is replaced with a deeper base of a fresh dish, and the two are joined with Micropore tape ( see Note 4 ).

3. On day 11 (after explant isolation), explants are moved to shoot elongation selection media, retaining the “base-lid” format. Explants are moved to fresh shoot elongation selection media

3.3 Explant Isolation, Inoculation, and Cocultivation

3.4 Selection

Fig. 1 Stages in Agrobacterium -mediated Brassica rapa transformation. Four-day-old R-o-18 seedlings ready

for explant isolation, showing ( a ) excision line, ( b ) scalpel about to make cut, and ( c ) explant with correct peti-ole length. Explant cultures 14 days after isolation/inoculation seen from the basal side of plate showing ( d ) control explants; no Agrobacterium inoculation and no gentamicin selection, ( e ) control explants; inoculated but maintained in the absence of selection and ( f ) inoculated explants maintained on gentamicin selection. Explant cultures 3 weeks after isolation/inoculation seen from the top side of plate showing ( g ) proliferation of shoots in control explants where no inoculation or gentamicin selection was used; ( h ) proliferation of shoots in control explants inoculated but where no gentamicin selection has been applied; and ( i ) inoculated explants on gentamicin selection with one putative transgenic shoot (circled); ( j ) an enlarged view of the marked shoot in ( i ). Shoots isolated and moved to rooting media at approximately 4 weeks postinoculation ( k ) and root develop-ment after a further 2 weeks ( l )

9 after a further 10–14 days (maximum). There is a gradual increase in the size of the petiole base around the cut surface, from the time of isolation, as callus begins to form.

4. Two weeks after isolation, inoculated petioles will be in the region of 5 mm in diameter when on selection media (larger and more developed on nonselective controls). Typically, viewing from the base, inoculated petioles have a green center with a white surround under selection conditions and in the nonselective controls will be all green (Fig. 1d–f ). A small amount of browning is normal between 14 and 21 days. 1. When using R-o-18, the emergence of green shoots on

con-trol plates (no selection) occurs between 14 and 21 days after explant isolation (Fig. 1g, h ), with 90–100 % of explants pro-ducing shoots. Transgenic (green) shoots can be seen from 3 weeks onward on selection plates (Fig. 1i, j ). From 3 weeks transgenic shoots can be isolated to rooting media (Fig. 1k ). The period when most transgenic shoots are likely to appear is between 21 days and 1 month after inoculation.

2. Putative transgenic (green) shoots are excised and transferred to 100 mL jars containing 25 mL of rooting medium using the base of 50 mm petri dishes (Sterilin 124) as lids and maintained at 23 °C under 16-h day length of 40 μmol/m 2 _{/s ( see Note 5 ).}

3. After root elongation (Fig. 1l ) (to approximately 20 mm in length), plantlets are transferred to sterile peat pots to allow further root growth (approx. 2 weeks) before being trans-ferred to the glasshouse ( see Note 6 ).

1. Plants are transferred to soil in 9 cm pots (John Innes No. 2) and maintained under shade within a propagator for the fi rst week. This ensures that plants gradually adjust to reduced humidity and increased light intensity. Glasshouse light condi-tions; day/night temperatures of 18/12 ± 2 °C, 16-h day length, with supplementary lighting (“high pressure sodium lamps” with an average bench reading of 200 μmol/m 2 _/s).

Plants are fed weekly with a 2:1:1 NPK fertilizer.

2. After 3 weeks, plants are potted up into 2 L containers under long day conditions (16 h light, 8 h dark) and 18–20 °C. R-o- 18 will fl ower after 6–8 weeks, and seeds can be harvested after 20–25 weeks. The time can be shortened if grown in smaller pots but the seed yield is often reduced.

3. When in bud, plants are covered with clear, perforated “bread” bags to prevent cross-pollination and shaken daily once in fl ower to encourage seed set. Pods are allowed to dry on the plant, before being threshed.

3.5 Shoot Isolation

3.6 Transfer of Plants to Greenhouse

10

1. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays and tobacco tissue culture. Physiol Plant 15:437–497

2. Gamborg OL, Miller RB, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158 3. Rusholme RL, Higgins EE, Walsh JA, Lydiate

DJ (2007) Genetic control of broad-spectrum resistance to turnip mosaic virus in Brassica rapa (Chinese cabbage). J Gen Virol 88:3177–3186

4. Rana D, van den Boogaart T, O’Neill C, Hynes L, Bent E, Macpherson L, Park JY, Lim YP, Bancroft I (2004) Conservation of the microstructure of genome segments in Brassica

napus and its diploid relatives. Plant J 40:

725–733

5. Moloney MM, Walker JM, Sharma KK (1989) High-effi ciency transformation of Brassica napus using Agrobacterium vectors. Plant Cell

Rep 8:238–242

4

Notes

1. Typically, 1.2 g of seed is enough for a 300 explant transfor-mation. Seed should be placed onto the surface of the medium and not embedded.

2. AGL1 is the Agrobacterium tumefaciens strain routinely used in our lab.

3. We used a plant selection cassette consisting of a double 35S promoter driving the aacC1 coding region with a CaMV ter-minator to provide gentamicin resistance.

4. Using large petri dishes (20 × 90 mm) and the deeper base as a lid gives a larger vessel volume which helps prevent the accu-mulation of ethylene and water vapor. Sealing with Micropore tape also helps with better gas exchange. We have found that these changes result in a less stressful environment, leading to healthier shoots.

5. It may be possible to omit the inclusion of gentamicin at the root induction stage, as the number of escapes making it through to this stage will be few; however, we recommend that you confi rm transgene presence by PCR.

6. R-o-18 plants are often on the verge of fl owering during the rooting period in peat pots. To reduce the risk of fl owering, the time spent in vitro once roots are established and moving to the greenhouse should be kept as short as possible. Once established in greenhouse pots, early fl owers may fail to set, but plants should gather vigor to produce fruit.

Acknowledgments

The authors acknowledge the support of the Biotechnology and Biological Science Research Council (BBSRC) Strategic Tools and Resources Grant BB/I023763/1 “Development of an effi cient B. rapa transformation system to facilitate studies on fruit develop-ment in a diploid Brassica oilseed crop” and further support by BBSRC Strategic Programme Grant B/J004588/1 (GRO) and the John Innes Foundation.

References

11

Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224, DOI 10.1007/978-1-4939-1658-0_2, © Springer Science+Business Media New York 2015

Chapter 2

Cotton ( Gossypium hirsutum L.)

Keerti S. Rathore , LeAnne M. Campbell , Shanna Sherwood ,

and Eugenia Nunes

Abstract

Cotton continues to be a crop of great economic importance in many developing and some developed countries. Cotton plants expressing the Bt gene to deter some of the major pests have been enthusiastically and widely accepted by the farmers in three of the major producing countries, i.e., China, India, and the USA. Considering the constraints related to its production and the wide variety of products derived from the cotton plant, it offers several target traits that can be improved through genetic engineering. Thus, there is a great need to accelerate the application of biotechnological tools for cotton improvement. This requires a simple, yet robust gene delivery/transformant recovery system. Recently, a protocol, involving large-scale, mechanical isolation of embryonic axes from germinating cottonseeds followed by direct trans-formation of the meristematic cells has been developed by an industrial laboratory. However, complexity of the mechanical device and the patent restrictions are likely to keep this method out of reach of most academic laboratories. In this chapter, we describe the method developed in our laboratory that has under-gone further refi nements and involves Agrobacterium -mediated transformation of cotton cells, selection of stable transgenic callus lines, and recovery of plants via somatic embryogenesis.

Key words Agrobacterium , Regeneration , Somatic embryogenesis , Transformation , Transgenic cotton

1

Introduction

Cotton, the most important source of natural fi ber worldwide, is grown in more than 80 countries across fi ve continents. Compared to the synthetic fi bers, cotton provides some major environmen-tal/societal benefi ts. Firstly, unlike the petroleum-based synthetics, it is a renewable resource. Secondly, its cultivation, processing, and use in textile manufacturing provide for the livelihoods of a much higher number of people compared to the synthetic fi bers. Of course, the superiority of cotton clothing in terms of its com-fort level cannot be matched by the synthetic versions. With an unrelenting growth in global population and the resulting demand for food and feed, cottonseed is also becoming an increasingly precious commodity. A major by-product, it is used as cattle feed,

12

either as seed or as meal following the extraction of edible oil. Biotechnology-based solutions are playing a major role in improv-ing cotton yields in most cotton-growimprov-ing regions. Genetically engi-neered cotton developed to confer resistance against a class of insects through the expression of the Bt gene represents the fi rst successful application of genetic engineering for commercial pur-poses [ 1 , 2 ]. Bt-cotton has lowered production costs by reducing the use of chemical pesticides, fuel, and labor input [ 3 , 4 ]. In addi-tion, this biotechnology product has had a positive impact on the environment and health of farmers in poor countries by reducing pesticide use. In future, genetic engineering (GE) is likely to play a major role not only in increasing the production but also in improv-ing the quality of fi ber and the seed. One of the major hurdles in the application of GE for cotton improvement is our ability to generate transgenic cotton plants in a reliable, rapid, and effi cient manner.

The fi rst two reports on the generation of transgenic cotton were both published in 1987 [ 5 , 6 ], only 4 years following the suc-cessful transformation of the model species, tobacco. Despite these early successes, few reports on successful transformation of cotton followed for the next 15 years. This was because the production of transgenic cotton plants was extremely diffi cult and highly ineffi -cient and required a high degree of tissue culture skills. There are two critical steps in the production of transgenic plants in any spe-cies. The fi rst step entails transfer and stable integration of the transgene into the plant genome. The second step involves regen-eration of a transgenic plant from the stably transformed cell. Considering the diffi culties involved in the production of trans-genic cotton, our laboratory conducted a comprehensive study to investigate both of these aspects of transgenic cotton production [ 7 ]. Although it is possible to transform cotton with a gene gun [ 8 ,

9 ] as well as with the Agrobacterium method [ 5 , 6 ], our study [ 7 ] focused on the latter since it does not require specialized equip-ment, is relatively inexpensive, and is more likely to result in single- copy transgenic events.

The choice of explant is critical in successfully obtaining trans-genic plants since the cells within these tissues must be susceptible to Agrobacterium infection and be able to regenerate into healthy, fertile plants. Most published studies had used either hypocotyl segments [ 1 , 5 , 10 – 13 ] or cotyledon pieces [ 6 , 10 ] derived from a young seedling as tissue explants for transformation. However, the use of these explants necessitates passage of transformed cells through either a callus phase or a combination of callus and sus-pension cultures prior to the induction of somatic embryogenesis for recovering transgenic plants. In addition to these tissue culture- based approaches, there are a few reports that claim the transfor-mation of cells within shoot apices in cotton [ 14 – 18 ]. Direct transformation of the “germline progenitor” cells within the shoot apex has the obvious advantages of avoiding laborious tissue Keerti S. Rathore et al.

13 culture passages and reducing the length of time for the recovery of transformants and, therefore, somaclonal variations. Another major advantage of this system is that regeneration from shoot api-ces is genotype independent.

Various systems for obtaining transgenic cotton were thor-oughly evaluated in our laboratory [ 7 , 19 ] using a reporter gene encoding the green fl uorescent protein (GFP) [ 20 ]. GFP expres-sion provided an excellent tool to measure the effi ciency of T-DNA transfer to cotton cells and was also useful in revealing the timing and localization of transient transgene expression [ 7 ]. Cells at the cut edge of the cotyledon segments proved to be the most suscep-tible to Agrobacterium -mediated transformation as indicated by the transient GFP activity. This was followed by conversion of some of these transiently transformed cells to stable transformation events. Fewer cells at the cut surface of the hypocotyl showed tran-sient GFP activity as compared to the cotyledonary tissue. The cells that showed GFP expression were seen as a ring of fl uorescent cells in the middle of the cut surface that appeared to be part of the vascular tissue; however, their true identity was masked by the hypersensitive response displayed by cells around them. Despite the lower rate of transient transformation, hypocotyl segments gave rise to several stable transgenic events that grew as small fl uo-rescent clusters at the cut surface of hypocotyls during the selec-tion on kanamycin-supplemented medium over 3–4 weeks. Thus, although hypocotyl segments showed a low level of transient activ-ity as compared to the cotyledons, these explants were still capable of producing several stable transgenic events [ 7 , 19 ]. Cotyledonary petiole segments are also as effi cient as the hypocotyl segments in terms of producing stable transformation events. Thus, the use of GFP as the reporter gene in combination with neomycin phos-photransferase II ( npt II) gene as a selectable marker showed that cotyledons, hypocotyls, and cotyledonary petioles are highly c ompetent explants for Agrobacterium -mediated transformation [ 7 , 19 ]. A single experiment involving 50 donor seedlings can yield several hundred independent transgenic events in the form of kanamycin-resistant calli. Although most of the experiments with GFP were conducted with cv. Coker 312, we have shown previ-ously that cotyledon segments of several Texas cultivars are also competent for Agrobacterium -mediated T-DNA transfer and are capable of yielding stable transgenic callus [ 7 ].

As mentioned earlier, there are a few studies that have reported transformation of cells within the shoot apical meristem via the Agrobacterium method [ 15 – 18 ]. However, these investigations relied heavily on the ability of explants to survive selection pressure as a measure of their transgenic status and/or did not provide con-vincing molecular and genetics data to support their claims. In our laboratory, the competence of shoot apices for Agrobacterium - mediated transformation was evaluated by cocultivating these with

14

Agrobacterium under conditions that were found to be optimal for the infection of the other three tissue explants. Both GFP and gus A reporter genes were used in these experiments. However, neither transient nor stable reporter gene expression in the “germline pro-genitor” cells within these tissues was observed in any of the exper-iments, each involving several hundred shoot apices. The results indicated that the effi ciency of Agrobacterium -mediated transfor-mation of apical meristems may be extremely low in cotton. Limitations and weaknesses of this method and some other alter-native methods have been described previously [ 21 ]. A recent patent issued to Monsanto [ 22 ] describes a mechanical device that can be used to isolate several thousand embryonic axes from germinating cottonseeds in a few hours. The investigators have used these explants to recover germline transformants following Agrobacterium -mediated transgene delivery. The patent further describes optimization of various experimental conditions to obtain transgenic regenerants. Thus, the low effi ciencies for direct trans-formation of germline cells appear to have been overcome by using brute force. However, patent restrictions on the device and the method will prevent most public sector laboratories to utilize this method to generate transgenic cotton plants.

There are a few reports in the literature that claim to obtain regeneration via adventitious organogenesis in cotton [ 23 , 24 ]. Over the past 18 years, a number of attempts have been made in our laboratory to obtain adventitious shoot organogenesis in cot-ton without success. In a majority of the laboratories involved in the generation of transgenic cotton, the mode of regeneration from callus or suspension cultures is via somatic embryogenesis [ 25 – 30 ]. However, embryogenesis in the cultured tissue occurs at a very low frequency and is highly genotype dependent (Table 1 ). In addition, only a small number of genotypes have been identifi ed that are competent for regeneration [ 10 , 30 – 35 ]. Even with geno-types that exhibit the best response, regeneration requires 6–8 sub-cultures on various media and can take as long as 6–9 months to

Table 1

Screening of fi ve different genotypes of G. hirsutum for their embryogenic response using the tissue culture protocol described in this chapter (# of lines undergoing somatic embryogenesis 8 months following culture initiation/# of cultured lines)

Coker 312 TM-1 Tamcot 22 Tamcot 73 DP50

Hypocotyl 67/86 1/16 0/7 0/7 0/5

Petiole 45/52 0/14 0/3 0/6 0/8

Cotyledon 5/6 0/9 0/6 0/11 0/14

Root 37/45 0/8 0/6 0/2 0/4

Combined total 154/189 1/47 0/22 0/26 0/31 Keerti S. Rathore et al.

15 obtain plants following transformation. Problems are encountered at every step during regeneration. These include (1) survival of transgenic events following excision from the hypocotyl, petiole, or cotyledon segments; (2) low effi ciencies of the excised events forming embryogenic calli, somatic embryogenesis, and germina-tion of the somatic embryo into a normal plantlet with a proper shoot and root; and (3) extreme fragility of the plantlets during the transition from culture to soil. Thus, while the production of stably transformed calli is an effi cient process in cotton as mentioned ear-lier, the recovery of healthy transgenic cotton plants from trans-formed cells was found to be highly ineffi cient.

We conducted a thorough examination of the factors impacting both transformation and regeneration and developed an effi -cient protocol for the production of transgenic cotton plants [ 7 ]. The percentage of transgenic events obtained from hypocotyl and cotyledonary petiole explants (number of kanamycin-resistant, transgenic events obtained/number of explants cocultivated with Agrobacterium strain × 100) ranged from 97 to 384 % in various experiments. Although regeneration is possible through suspension cultures in cotton, we prefer to recover plants from callus cultures because this culture system requires less labor and equipment, and the possibility of contamination is lower. By using the protocol described in this chapter, regeneration effi ciencies (number of trans-genic callus lines regenerating into healthy plants/number of kana-mycin-resistant culture lines × 100) have ranged from 0 to 24 %. The method refi ned over the last 12 years for producing transgenic cotton (cv. Coker 312) is presented in detail in this chapter.

2

Materials

All media are autoclaved at 121 °C for 20 min after adjusting the pH and after the addition of the gelling agent. Autoclaved media (with the gelling agent) should be cooled to <60 °C before adding fi lter-sterilized antibiotics or acetosyringone.

1. YEP: 10 g/L Bacto™ peptone, 5 g/L NaCl, 10 g/L Bacto™ yeast extract, pH 7.0, 1.5 % Bacto™ agar. Aliquot in small batches in bottles, autoclave, and store at room temperature. When required, melt in microwave, add necessary antibiotics, and pour into Petri dishes.

2. YEP liquid: 10 g/L Bacto™ peptone, 5 g/L NaCl, 10 g/L Bacto™ yeast extract, pH 7.0. Autoclave and store at room temperature.

3. PIM (preinduction medium): 10 g/L glucose, 14.62 g/L morpholinoethanesulfonic acid (MES), 20 mL/L sodium phosphate buffer (0.1 M, pH 5.6), 50 mL/L AB salts stock (20×), pH 5.6. Filter-sterilize, aliquot in 10 mL portions, and store at 4 °C.

2.1 Agrobacterium

Media

16

4. AB salts stock (20×): 20 g/L NH 4 Cl, 6 g/L MgSO 4 · 7H 2 O,

3 g/L KCl, 0.264 g/L CaCl 2 · 2H 2 O, 0.05 g/L

FeSO 4 · 7H 2 O. Autoclave and store at 4 °C.

5. Sodium phosphate buffer (0.1 M, pH 5.6): 2.759 g NaH 2 PO 4 · H 2 O/200 mL water (solution A). 2.839 g

Na 2 HPO 4 /200 mL water (solution B). Add solution B to

solution A in a stepwise manner until the pH is brought to 5.6, autoclave, and store at room temperature.

1. Cotton seeds ( Gossypium hirsutum L., cv. Coker 312).

2. MSO: 4.31 g/L Murashige and Skoog (MS) salts [ 36 ] (MS basal salts mixture), 2 % glucose, pH 5.8, 0.2 % Phytagel. 3. P1-AS: 4.31 g/L MS salts, 100 mg/L myoinositol, 0.4 mg/L

thiamine HCl, 5 mg/L N 6 _{-(2-isopentenyl)adenine (2iP),}

0.1 mg/L α-naphthaleneacetic acid (NAA), 3 % glucose, 1 g/L MgCl 2 · 6H 2 O, pH 5.8, 0.2 % Phytagel, 50 μM

aceto-syringone (AS) (Sigma-Aldrich, Cat. #D134406; AS stock is made at 10 mg/mL in 70 % ethanol and stored at −20 °C; add 1 mL of AS stock to 1 L of P1).

4. P1-c4k50: 4.31 g/L MS salts, 100 mg/L myoinositol, 0.4 mg/L thiamine HCl, 5 mg/L 2iP, 0.1 mg/L NAA, 3 % glucose, 1 g/L MgCl 2 · 6H 2 O, pH 5.8, 0.2 % Phytagel,

400 mg/L carbenicillin, 50 mg/L kanamycin.

5. P7-c4k50: 4.31 g/L MS salts, 100 mg/L myoinositol, 0.4 mg/L thiamine HCl, 0.1 mg/L 2iP, 5 mg/L NAA, 3 % glucose, 1 g/L MgCl 2 · 6H 2 O, pH 5.8, 0.2 % Phytagel,

400 mg/L carbenicillin, 50 mg/L kanamycin.

6. MSEm-c4k50: 2.68 g/L modifi ed MS salts (Phytotechnology, #M571), 100 mg/L myoinositol, 1 mg/L nicotinic acid, 10 mg/L thiamine HCl, 1 mg/L pyridoxine HCl, 1.9 g/L KNO 3 , 825 mg/L NH 4 NO 3 , 2 g/L glutamine, 500 mg/L

asparagine, 3 % glucose, 1 g/L MgCl 2 · 6H 2 O, pH 5.8, 0.24 %

Phytagel, 400 mg/L carbenicillin, 50 mg/L kanamycin. 7. MSEm: same as above but without the antibiotics.

8. EG3: 2.16 g/L MS salts, 0.5 % glucose, 100 mg/L myoinosi-tol, 0.4 mg/L thiamine HCl, 0.01 mg/L NAA, pH 6.5, 0.2 % Phytagel.

9. MS3: 2.16 g/L MS salts, 0.5 % glucose, 0.14 mg/L thiamine HCl, 0.1 mg/L pyridoxine HCl, 0.1 mg/L nicotinic acid, pH 5.8, 0.08 % Phytagel, 0.6 % Bacto™ agar.

10. Sunshine LP5 soil mix: 70–80 % Canadian sphagnum peat moss + fi ne perlite + dolomite limestone + gypsum + wetting agent (Sun Grow Horticulture Canada Ltd.).

11. Metro-Mix 900 soil mix: 50–60 % bark + Canadian sphagnum peat moss + horticulture grade vermiculite + dolomite lime stone + wetting agent (Sun Grow Horticulture Canada Ltd.).

2.2 Cotton Tissue Culture

17

3

Methods

1. Streak the Agrobacterium tumefaciens strain, harboring a

binary vector with the gene of interest, on antibiotic-supple-mented YEP plates and incubate for 2–3 days at 28 o _C

( see Note 1 ).

2. Inoculate fi ve colonies into fi ve different test tubes each con-taining 2 mL of YEP liquid medium (supplemented with appropriate antibiotics). Grow cells at 28 °C for about 30 h on a shaker (200 rpm) in dark.

3. Pool bacterial cultures in a 15 mL centrifuge tube. Centrifuge at 2,060 × g for 20 min. Remove as much supernatant as pos-sible with a sterile pipette, and resuspend the bacterial pellet in 10 mL of PIM with 100 μM acetosyringone (add 20 μL of 10 mg/mL AS stock to 10 mL of PIM just before use). Be sure to break up bacterial clumps by gentle vortexing. Transfer the suspension to a 125-mL fl ask. Grow cells at 28 °C over-night on a shaker (200 rpm) in the dark.

4. Use 1 mL of suspension to check OD at 600 nm and adjust to ~0.6. Add 2 μL of acetosyringone stock for each milliliter of the fi nal suspension before using the Agrobacterium culture for cocultivation ( see Note 2 ).

1. Rinse 50 fuzzy cottonseeds under running tap water overnight.

2. Sterilize the seeds by shaking them in a fl ask containing 100 mL of 40 % commercial bleach (+2 drops of Tween 20) for 40 min under vacuum on a shaker. Rinse four times with sterile distilled or deionized water (DW) ( see Note 3 ).

3. Germinate one seed per jar (Phytotechnology, #C1770) or Magenta™ box on MSO (25 mL/jar; 50 mL/Magenta™ box) at 25 °C, under fl uorescent light (70 μmol/m 2 _{/s, 16-h}

pho-toperiod) for 10 days.

1. Cut 3- to 4-mm-long segments from either hypocotyl or coty-ledonary petiole. Place 10–12 segments horizontally on sterile fi lter paper over P1-AS medium in a Petri dish (100 × 15 mm). 2. Apply 5 μL of acetosyringone-induced Agrobacterium

suspen-sion to each cut surface. Keep the plates under light (70 μmol/ m 2 _{/s, 16-h photoperiod) at 25 °C for 3 days for cocultivation}

( see Note 4 ).

3. Transfer hypocotyl/petiole pieces to the P1-c4k50 medium. Keep the plates under light (70 μmol/m 2 _{/s, 16-h}

photope-riod) at 28 °C for 3–4 weeks without subculture ( see Note 5 ).

3.1 Preparation of Agrobacterium Inoculant 3.2 Aseptic Seed Germination 3.3 Transformation