7743.Genetically Modified Crops Their Development, Uses, And Risks by G.h. Liang

(1)

(2)

G. H. Liang, PhD

D. Z. Skinner, PhD

Editors

Genetically Modified Crops

Their Development,

Uses, and Risks

Pre-publication REVIEWS,

COMMENTARIES, EVALUATIONS . . .

“T

his book is a useful reference for all researchers working on the im-provement of crops through plant trans-formation. Breeders of all field crops (corn, wheat, rice, soybean, cotton, sor-ghum, and alfalfa), vegetable crops, horti-cultural crops, and turfgrasses can get an overview of the progress of genetic engi-neering efforts in a particular crop. Molec-ular biologists and cell/tissue culture spe-cialists will find Chapter 1 through Chap-ter 3 very informative for understanding the methods, strategies, and mechanisms of transgene integration. Both scientists and the general public need to carefully evaluate the benefits and risks of trans-genic crop plants by first understanding all facets of each one. This book covers those facets, from the source of the gene, compositions of a gene construct, method

of gene delivery, and result of gene inte-gration and expression, to effects of the transgene on plants and the ecology.”

Richard R.-C. Wang, PhD

Research Geneticist, USDA-ARS Forage & Range Research Laboratory, Logan, Utah

“T

his book will help the public gain a better understanding of agricultural biotechnology’s merits and risks, thus re-ducing the public’s fear and anxiety. In addition, it will also benefit plant breed-ers by helping them to decide where to fo-cus their research efforts and to gain more efficiency in breeding methodology.”

Paul Sun, PhD

Vice President, Research, Dairyland Seed Co., Inc., Clinton, Wisconsin

(3)

More pre-publication

REVIEWS, COMMENTARIES, EVALUATIONS . . .

“T

his book is useful to graduate and

advanced undergraduate students,

as well as the interested public for a general understanding of plant trans-formation and for experienced scien-tists in specific areas of interest.

It is excellent—full of high-quality in-formation, and illustrated with clear fig-ures and tables. It has an application-oriented explanation and up-to-date ref-erences. One exciting part of the book is the review of some of the plant transfor-mation protocols that include some vali-dated modifications to be considered as resources for advanced undergraduate and beginning graduate students who desire orientation on relevant topics and meth-ods used in plant transformation re-search. It also is appropriate as a textbook for advanced courses in plant transforma-tion and as supplemental references for undergraduate courses in plant breeding

or other related courses. Genetically Mod-ified Crops is useful as a tool to help stu-dents understand research results that are published in refereed journal articles. For students who desire to apply a method, the book provides many examples of pit-falls associated with a particular tech-nique. Such information is quite useful and often is difficult to pry from the de-tailed literature. The information listed in this book would enable the federal regu-latory agencies to contribute technical ex-pertise to the realm of national discus-sions and the making of science-based de-cisions on guidelines for commercial re-lease of other genetically modified crops.”

Suleiman S. Bughrara, PhD

Assistant Professor,

Department of Crop and Soil Sciences, Michigan State University,

East Lansing, Michigan

Food Products Press® An Imprint of The Haworth Press, Inc.

(4)

NOTES FOR PROFESSIONAL LIBRARIANS AND LIBRARY USERS

This is an original book title published by Food Products Press®, an imprint of The Haworth Press, Inc. Unless otherwise noted in specific chapters with attribution, materials in this book have not been previ-ously published elsewhere in any format or language.

CONSERVATION AND PRESERVATION NOTES All books published by The Haworth Press, Inc. and its imprints are printed on certified pH neutral, acid-free book grade paper. This paper meets the minimum requirements of American National Standard for Information Sciences-Permanence of Paper for Printed Material, ANSI Z39.48-1984.

(5)

Genetically Modified Crops

Their Development,

(6)

FOOD PRODUCTS PRESS

®

Crop Science

Amarjit S. Basra, PhD Senior Editor

Heterosis and Hybrid Seed Production in Agronomic Crops edited by Amarjit S. Basra Intensive Cropping: Efficient Use of Water, Nutrients, and Tillage by S. S. Prihar, P. R. Gajri,

D. K. Benbi, and V. K. Arora

Physiological Bases for Maize Improvement edited by María E. Otegui and Gustavo A. Slafer Plant Growth Regulators in Agriculture and Horticulture: Their Role and Commercial Uses

edited by Amarjit S. Basra

Crop Responses and Adaptations to Temperature Stress edited by Amarjit S. Basra Plant Viruses As Molecular Pathogens by Jawaid A. Khan and Jeanne Dijkstra In Vitro Plant Breeding by Acram Taji, Prakash P. Kumar, and Prakash Lakshmanan Crop Improvement: Challenges in the Twenty-First Century edited by Manjit S. Kang Barley Science: Recent Advances from Molecular Biology to Agronomy of Yield and Quality

edited by Gustavo A. Slafer, José Luis Molina-Cano, Roxana Savin, José Luis Araus, and Ignacio Romagosa

Tillage for Sustainable Cropping by P. R. Gajri, V. K. Arora, and S. S. Prihar

Bacterial Disease Resistance in Plants: Molecular Biology and Biotechnological Applications by P. Vidhyasekaran

Handbook of Formulas and Software for Plant Geneticists and Breeders edited by Manjit S. Kang

Postharvest Oxidative Stress in Horticultural Crops edited by D. M. Hodges

Encyclopedic Dictionary of Plant Breeding and Related Subjects by Rolf H. G. Schlegel Handbook of Processes and Modeling in the Soil-Plant System edited by D. K. Benbi

and R. Nieder

The Lowland Maya Area: Three Millennia at the Human-Wildland Interface edited by A. Gómez-Pompa, M. F. Allen, S. Fedick, and J. J. Jiménez-Osornio

Biodiversity and Pest Management in Agroecosystems, Second Edition by Miguel A. Altieri and Clara I. Nicholls

Plant-Derived Antimycotics: Current Trends and Future Prospects edited by Mahendra Rai and Donatella Mares

Concise Encyclopedia of Temperate Tree Fruit edited by Tara Auxt Baugher and Suman Singha Landscape Agroecology by Paul A Wojkowski

Concise Encyclopedia of Plant Pathology by P. Vidhyasekaran

Molecular Genetics and Breeding of Forest Trees edited by Sandeep Kumar and Matthias Fladung

Testing of Genetically Modified Organisms in Foods edited by Farid E. Ahmed

Agrometeorology: Principles and Applications of Climate Studies in Agriculture by Harpal S. Mavi and Graeme J. Tupper

Concise Encyclopedia of Bioresource Technology edited by Ashok Pandey

Genetically Modified Crops: Their Development, Uses, and Risks edited by G. H. Liang and D. Z. Skinner

(7)

Genetically Modified Crops

Their Development,

Uses, and Risks

G. H. Liang, PhD D. Z. Skinner, PhD

Editors

Food Products Press® An Imprint of The Haworth Press, Inc.

(8)

Published by

Food Products Press®, an imprint of The Haworth Press, Inc., 10 Alice Street, Binghamton, NY 13904-1580.

© 2004 by The Haworth Press, Inc. All rights reserved. No part of this work may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, microfilm, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Printed in the United States of America.

Cover design by Jennifer M. Gaska.

Library of Congress Cataloging-in-Publication Data

Genetically modified crops : their development, uses, and risks / G. H. Liang, D. Z. Skinner, editors. p. cm.

Includes bibliographical references and index.

ISBN 1-56022-280-8 (case : alk. paper)—ISBN 1-56022-281-6 (soft : alk. paper) 1. Transgenic plants. 2. Crops—Genetic engineering. I. Liang, G. H. (George H.) II. Skinner, D. Z. (Daniel Z.)

SB123.57.G479 2004 631.5'233—dc22

(9)

Genetics at Kansas State University in Manhattan, Kansas. He has been teaching Plant Genetics and Quantitative Genetics at the graduate level for the past 34 years. As a plant geneticist, he has been working on crop transformation using various means since 1993, including pio-neer work with Agrobacterium-mediated transformation in bentgrass and sorghum. Together with his colleagues, he has published 12 book chapters, 120 refereed journal articles, and two books: Plant Genetics, Second Edition and Experiment Principles and Methods.

Dr. Liang is recipient of the American Society of Agronomy Fel-low award in 1997, the Crop Science FelFel-low in 2002, and the distin-guished faculty award by Gamma Sigma Delta in 2002. He was con-sultant for the World Bank and the United Nations Development Programme in China. He was elected honorary professor by China Agricultural University, Chinese Academy of Agricultural Sciences, Henan Agricultural University, and Shandong Academy of Agricul-tural Sciences.

Daniel Z. Skinner, PhD, currently works for the USDA-ARS, and

the Wheat Genetics, Quality, Physiology and Disease Research Unit and Crop and Soil Sciences Department at Washington State Univer-sity as a research leader and supervisory research geneticist (plants). His adjunct professorships include Associate Professor at the Depart-ment of Microbiology, Faculty of Science, at Kasetsart University in Bangkok, Thailand; Associate Professor of Agronomy and Associate Professor of Genetics at Kansas State University; and Associate Pro-fessor in the Department of Crop and Soil Sciences at Washington State University. His work has been published in many journals, in-cluding Weed Science, Journal of Industrial Microbiology and

(16)

Ap-plied Genetics, Plant Science, Molecular Breeding, and Trends in Agronomy.

Dr. Skinner has worked with plant and fungal genetics and bio-technology for more than 20 years. He has conducted studies de-signed to assess the potential spread of insect-borne alfalfa pollen from fields of transgenic plants using rare, naturally occurring mo-lecular markers to simulate transgenes. He also investigated the for-mation of interspecific hybrids among plant species, and found that mobile genetic elements are a factor in genetic rearrangements that occur in the hybrid genome.

(17)

Contributors

CONTRIBUTORS

Tom Clemente is associated with the Plant Science Initiative,

De-partment of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska.

Hui Duan is associated with the Department of Plant Science,

Uni-versity of Connecticut, Storrs, Connecticut.

Mistianne Feeney is Graduate Research Assistant, Centre for

Envi-ronmental Biology, Department of Biological Sciences, Simon Fra-ser University, Burnaby, British Columbia, Canada.

J. Jayaraj is affiliated with the Department of Agronomy, Kansas

State University, Manhattan, Kansas.

J. M. Jeoung is affiliated with the Department of Agronomy, Kansas

State University, Manhattan, Kansas.

Junda Jiang is associated with the University of Arkansas Rice

Re-search and Extension Center, Stuttgart, Arkansas.

Karl J. Kramer is Research Chemist, Grain Marketing and

Produc-tion Research Center, Agricultural Research Service, U.S. Depart-ment of Agriculture, Manhattan, Kansas.

Qi Li is associated with the Department of Plant Science, University

of Connecticut, Storrs, Connecticut.

Yi Li is associated with the Department of Plant Science, University

of Connecticut, Storrs, Connecticut.

Keming Luo is associated with the Biotechnology Center,

South-west Agricultural University, Beipei, Chongqing, People’s Republic of China.

Irina F. Makarevitch is associated with the Department of

Agron-omy and Plant Genetics, University of Minnesota, St. Paul, Minne-sota.

(18)

Richard J. McAvoy is associated with the Department of Plant

Sci-ence, University of Connecticut, Storrs, Connecticut.

Amitava Mitra is associated with the Department of Plant

Pathol-ogy, University of Nebraska, Lincoln, Nebraska.

S. Muthukrishnan is Professor, Department of Biochemistry,

Kan-sas State University, Manhattan, KanKan-sas.

James Oard is associated with the Department of Agronomy,

Loui-siana State University, Baton Rouge, LouiLoui-siana.

Paula M. Olhoft is associated with the Department of Agronomy

and Plant Genetics, University of Minnesota, St. Paul, Minnesota.

David W. Ow is associated with the Plant Gene Expression Center,

Agricultural Research Service, U.S. Department of Agriculture, Al-bany, California, and the Department of Plant and Microbial Biol-ogy, University of California, Berkeley, California.

Sung Hun Park is associated with the Vegetable and Fruit

Improve-ment Center and the DepartImprove-ment of Entomology, Texas A&M Uni-versity, College Station, Texas.

Yan Pei is associated with the Biotechnology Center, Southwest

Agri-cultural University, Beipei, Chongqing, People’s Republic of China.

Zamir K. Punja is Professor and Director, Centre for Environmental

Biology, Department of Biological Sciences, Simon Fraser Univer-sity, Burnaby, British Columbia, Canada.

Deborah A. Samac is associated with the U.S. Department of

Agri-culture, Agricultural Research Service, and the University of Minne-sota, St. Paul, Minnesota.

James W. Smith is associated with the Vegetable and Fruit

Improve-ment Center and the DepartImprove-ment of Entomology, Texas A&M Uni-versity, College Station, Texas.

Roberta H. Smith is associated with the Vegetable and Fruit

Im-provement Center and the Department of Entomology, Texas A&M University, College Station, Texas.

David A. Somers is associated with the Department of Agronomy

(19)

Paul St. Amand is affiliated with the Agronomy Department,

Sergei K. Svitashev is associated with the Department of Agronomy

and Plant Genetics, University of Minnesota, St. Paul, Minnesota.

Stephen J. Temple is associated with the U.S. Department of

Agri-culture, Agricultural Research Service, and the University of Minne-sota, St. Paul, Minnesota.

M. R. Tuinstra is affiliated with the Department of Agronomy,

Xiaoling Wang is associated with the Department of Plant Biology

and Pathology at Rutgers University, New Brunswick, New Jersey.

J. T. Weeks is affiliated with the Simplot Plant Sciences, J. R.

Simplot Company, Boise, Idaho.

Jack M. Widholm is Professor of Plant Physiology, Department of

Crop Sciences, Edward R. Madigan Laboratory, University of Illi-nois, Urbana, Illinois.

Yan H. Wu is associated with the Department of Plant Science,

John Wurst is associated with the Department of Plant Science,

Degang Zhao is associated with the Department of Plant Science,

University of Connecticut, Storrs, Connecticut.

Barbara A. Zilinskas is associated with the Department of Plant

Bi-ology and PathBi-ology at Rutgers University, New Brunswick, New Jersey.

(20)

(21)

Preface

Agricultural biotechnology is a major issue today, facing not only scientists but consumers and producers as well. A large number of people in the United States and Canada consume food and beverages produced from crops and enzymes that are modified through biotech-nology processes. Canola oil, soybean oil, corn oil, tofu, potato and potato chips, cheese, milk, fruits, and even beer are examples of ge-netically modified products. In addition, fiber crops, such as cotton, and vegetables, such as tomato, produced from genetically modified plants also are on the market. Biotechnology-modified foods, a suc-cess of the scientific community, clearly are a reality. On the one hand, these crop plants do provide advantages and are beneficial to society. On the other hand, the production and marketing of geneti-cally modified food has created fear and anxiety among the public. There is a definite need for this fear and anxiety to be addressed.

Many meetings and workshops have been organized to discuss the methodologies, advantages, and disadvantages of biotechnologically processed food products. Chapters in this book are a continuation of these efforts. Topics under discussion range from safety to evolution, ethics to ecology, and environment to legality. All contributors are genuinely concerned with the processes and the products. Regardless of the differences in personal viewpoints, the basic tenet of using bio-technology in food production is to resolve the problem of ever-increasing world population versus food production with limited crop land. Food insecurity and malnutrition still exist, even in the twenty-first century when humans have already landed on the moon, communication-satellite technology is in use by every household where there is a phone, human genome mapping is nearly completed, cloning of livestock and even humans is no longer a dream, and a computer has been installed in nearly every home, classroom, or of-fice in schools, governmental agencies, and private companies in many countries. Yet one of the most basic requirements of human life—food—still is not in adequate supply for every human being on

(22)

earth. Do those who are hungry or suffer from malnutrition have the right to express their desire and need? Should we hear from them? Conversely, in order to fulfill such needs, should we not attempt to provide some answers?

The processes of genetically modifying crops or livestock have been in existence since antiquity, although the techniques used now are revolutionary. The production of a mule by crossing a horse and donkey is an example of an old technique. Triticale, a product of crossing wheat and rye, is another. Many scientists working on live-stock, crops, fish, poultry, forest trees, enzymes, and even insects such as silkworm have changed the genetic constitution of certain species by adding genetic material from other species to produce a more adapted form for human consumption and utilization. Transfor-mation, a branch of agricultural biotechnology, simply expands the existing knowledge and skill from our ancestors to produce more adapted forms of crops that are environmentally friendly and benefi-cial to both producers and consumers.

This book is intended for use not only by scientists but also by the general public who are interested in being informed of the methods used and risks of introducing genetically modified organisms into the environment. To this end, the following questions are posed and an-swers are attempted:

• What are the techniques and goals of producing genetically modified crops?

• What genetic materials have been, or are going to be, used in transformation?

• What are the results of such endeavors?

Those who have contributed to the chapters in this book are all re-searchers who have hands-on experience in transformation and are willing to provide and share their firsthand information with their colleagues and the public.

(23)

Chapter 1

Transformation: A Powerful Tool for Crop Improvement

Transformation: A Powerful Tool

for Crop Improvement

D. Z. Skinner S. Muthukrishnan

G. H. Liang

INTRODUCTION

The demand for producing transgenic crops is the direct result of an ever-increasing world population. The World Bank reported that enough staple food is produced to feed everyone in the world, and yet 800 million people are food insecure and 180 million of them are pre-school children (Weeks, 1999). Clearly, equitable food distribution presents a challenge. In addition, production could become limiting in the future, with a projected addition of 1 billion people in Asian countries and an 80 percent increase in population in sub-Saharan Africa by 2020. Distribution of people throughout the world is highly uneven, and the countries with the largest gains in population are of-ten the ones without the resources to accommodate greater masses.

To meet the food demand by the year 2020, it is predicted that a 60 percent increase in the food supply will be necessary. Also, a 200 per-cent increase was projected for meat and dairy products, both of which must be converted from grain and forage, driven in part by in-creasing wealth in Asia and Southeast Asia. To compound the prob-lem, the increase in food supply will not come from an increase in cropland. Rather, it must come from increases in productivity per unit area of land and unit volume of water (Pinstrup-Andersen and Pandya-Lorch, 1999). Increases in cultivated area will contribute less than 20 percent to the increase in global cereal production between

(24)

1993 and 2020. Most of the growth in land for cereal production will be concentrated on the land with relatively low production potential in sub-Saharan Africa; some expansion of cultivated land will occur in South America, but cultivated area for cereals in Asia will remain stagnant.

If increase in cropland area does not contribute much to increased cereal production, then meeting the demand for cereals will depend on yield improvement. However, rate of increase in yield is declining because prices of cereals are low, forcing some farmers to abandon cereal crops for more profitable crops, which in turn reduces continu-ous investment in research, infrastructure, and irrigation facilities. Also, sustaining the same rate of yield gain will be difficult in certain regions where input of water and fertilizer is already high and opti-mum profitable economic return has been approached. The fore-casted yield increase will drop to an annual rate of 1.5 percent from 1993 to 2020 in contrast to an increase of 2.3 percent from 1982 to 1994 (Pinstrup-Andersen and Pandya-Lorch, 1999).

Much of the yield increase in cereals will likely go toward meeting projected increases in demand for meats. Due to population growth, coupled with increased income and changes in lifestyle in developing countries, demand for meat is projected to increase by 2.8 percent per year from 1993 to 2020. While per capita demand for cereals is pro-jected to increase by 8 percent, demand for meat will increase by 43 percent (Pinstrup-Andersen and Pandya-Lorch, 1999). The increase in demand for meat will make it necessary to produce more feed from already limited land resources.

Without proper management, availability of fresh water will emerge as the most important constraint to global food production. Further-more, water often is poorly distributed among countries, within coun-tries, and between seasons. The second longest river in China, the Yellow River, for example, was without water in downstream areas for more than 200 days in 1997, and many of the water springs in the northern provinces are out of water due to heavy use of underground water. Today, 28 countries with a total population exceeding 300 mil-lion people face water stress. By 2025, the number of people facing a water shortage could increase to 3 billion across 50 countries (Rose-grant et al., 1997).

(25)

Agriculture is by far the largest user of water, accounting for 72 percent of global water withdrawals and 87 percent of withdrawals in developing countries. Establishing sound policies or reforming the current ones to reduce wasteful use of water will provide improved efficiency in water use and increase crop production per unit of land. Nevertheless, global demand for irrigation water will continue to grow as demand for cereal production increases. The cost of develop-ing new sources of water will be high, and special sources of water supplies, such as desalination, reuse of wastewater, and water har-vesting, are unlikely to provide universal availability of water, al-though these methods may be established and helpful for certain lim-ited localities.

Crops also need protection from pests. Naturally occurring pests inflict tremendous yield and storage losses. For example, stalk rot of sorghum incited by Fusarium species could cause an $80 million loss each year in Kansas alone. Other pathogen threats also exist. There are scab, rust, and virus disease in cereals; leaf and stem diseases in forage crops; and root diseases in almost all the crops. All of these are common occurrences in crop fields. Likewise, insect damage is equally serious. In addition to pests, abiotic stresses, such as drought and extreme temperatures, could cause more damage not just to field crops but also to turfgrasses. For example, there are 23 million acres of turfgrass or lawn in the United States, and abiotic stress is known to lower the yield and reduce turfgrass quality.

Meeting the demand of a rapidly growing population, reducing the widening social and economic disparities both within and among countries, and at the same time safeguarding the earth’s natural re-sources, the quality of the atmosphere, and other critical components of the environment—all represent tremendous challenges facing us today. Genetically modified crops have the potential to contribute to solving these challenges.

INCORPORATION OF TRANSGENES INTO CROPS

Most of the methods currently used for plant transformation em-ploy a technique for delivering the DNA into the cell without regard

(26)

to its ultimate intracellular location. Once inside the appropriate cel-lular location by a chance process, the DNA is integrated into the chromosomal (or organellar) DNA, usually by a nonspecific recom-bination process. The exception is Agrobacterium-mediated transfor-mation, which delivers the DNA specifically into the nuclear com-partment with high efficiency and also provides a mechanism for its integration.

In plant transformation, two physical barriers prevent the entry of DNA into a nucleus, namely the cell wall and the plasma membrane. Most plant transformation methods overcome these barriers using physical and/or chemical approaches. In protoplast-mediated trans-formation, the cell wall is digested with a mixture of enzymes which attack cell wall components, yielding individual protoplasts that can be maintained intact using appropriate osmoticum in the medium. Entry of DNA is then facilitated by the addition of permeabilizing agents, such as polyethylene glycol, that allow DNA uptake, presum-ably by coating the negatively charged DNA inside the cell and vari-ous cellular compartments in a random process.

A second method, often referred to as biolistic transformation, uti-lizes fine metal particles (typically tungsten or gold) coated with DNA that are usually accelerated with helium gas under pressure. Other methods, such as microinjection, silicon carbide fiber treat-ment, vacuum infiltration, dipping of floral parts, sonication, and electroporation, cause transient microwounds in the cell wall and the plasma membrane, allowing the DNA in the medium to enter the cy-toplasm before repair or fusion of the damaged cellular structures. However, many of these methods are tedious and result in variable transformation efficiencies. In the following sections, we provide de-tails and relative merits of several, but not all, available protocols.

In all transformation techniques, the desired transgene is placed under the control of a promoter which produces high-level constitu-tive or inducible expression of the gene in specific or all tissues. In addition, another gene that allows detection or selection of the trans-formed cells is introduced in the same or different vector DNA. The presence of a screenable or selectable marker gene greatly simplifies the identification of transformed plants and increases the efficiency of recovery of transgenic plants. Typical screenable markers are the

(27)

green fluorescent protein. Selectable markers that have been most useful are those conferring resistance to antibiotics such as kana-mycin, paromokana-mycin, and hygrokana-mycin, or to herbicides such as bial-aphos, glyphosate, or cyanamide. In addition, selection also can be achieved by including metabolites that require special enzymes for their utilization as a carbon source, such as phosphomannose iso-merase and xylose isoiso-merase.

METHODOLOGY OF PRODUCING TRANSGENIC PLANTS

Most of the important crop species have been successfully trans-formed, at least in the laboratory. Two major approaches have been widely used to produce transgenic crop plants, both monocots and di-cots. One is biolistic bombardment and the other is Agrobacterium-mediated transformation. In the biolistic protocol, the primary deliv-ering systems are the BioRad PDS 1000/He helium-powered gun and similar designs, or the particle inflow gun. The second approach involves various strains of Agrobacterium tumefaciens, such as LBA4404, EHA105, and C58, carrying a T-DNA vector. The trans-gene and the selectable marker are inserted in the vector between two unique sequences, called the left border and the right border, which are utilized in insertion of the T-DNA into the host chromosomal DNA (Kado, 1998; Zupan and Zambryski, 1995).

Parameters involved in the biolistic gun include pressure (ranging from 900 to 1300 psi), particle size (0.6 to 1.1 m), and type of mate-rial (gold and tungsten), target distance (7.5 to 10 cm), and target ma-terial (cell suspension, callus, meristem, protoplast, immature em-bryo). These parameters vary somewhat with the crop involved. Disadvantages of using the biolistic gun are low success frequency, high copy numbers that often are correlated with gene silencing, pat-ent issues, and cost.

Agrobacterium-mediated transformation may correct some of the

weaknesses encountered with the biolistic approach and has been successful in rice, maize, sorghum, bentgrass, and other crops; its ef-fectiveness with other crops, especially wheat, remains questionable. Further, some cultivar versus Agrobacterium strain specificity may limit the range of cultivars that can be successfully transformed. De-velopment of reliable and efficient protocols are needed to improve

(28)

the efficiency and range of both approaches in transforming crop plants.

For Agrobacterium-mediated transformation, calli are induced from the explants, such as immature embryos from wheat and sorghum and mature seed from creeping bentgrass. To illustrate, the procedure used for sorghum is briefly described. Calli are induced from zygotic imma-ture embryos (1 to 2 mm in diameter). Agrobacterium culimma-ture carrying the desired transgene/selectable marker is grown overnight and resus-pended to reach an OD₆₀₀= 0.5 in I₆liquid medium (a modification of I₃medium [Casas et al., 1993]) containing 100 M acetosyring-one. The calli are cocultured with the Agrobacterium for 30 min and blotted dry for a few seconds. Then the calli are placed on semisolid I₆medium containing 2.5 g·L–1_{phytagel and 100} _M_{acetosyringone}

and cultured for three to four days at 25ºC in darkness. The calli should be washed three times with cefotaxime (300 to 500 mg·L–1₎

solution and cultured in I₆medium without selection for one week in darkness. The calli will then be transferred to fresh medium contain-ing a selection agent, e.g., 3 mg·L–1_{bialaphos or 12.5 mg·L}–1

cyana-mide hydratase. They will be cultured at 25 to 27ºC for six weeks with subculture to fresh medium once every two weeks. Next, shoots formed from bialaphos-resistant calli will be selected and transferred to rooting medium (half-strength MS medium with 0.2 mg·L–1_IAA,

0.2 mg·L–1_{NAA) containing 3 mg·L}–1_{bialaphos or 12.5 mg·L}–1

cya-namide hydratase. Finally shoots with well-developed root systems may be transferred to soil.

The efficiency of tissue culture methods is affected by species, surfactant use, and presence of acetosyringone. DNA delivery effi-ciency was significantly decreased in the absence of acetosyringone when embryo-derived calli were used in transformation of rice (Hiei et al., 1994) and maize (Ishida et al., 1996). However, when wheat cell suspension culture was used in transformation with

Agrobac-terium, acetosyringone was not essential (Cheng et al., 1997),

sug-gesting that different tissues may have different competence for

Agrobacterium infection. Use of a surfactant, such as Silwet, proved

to be essential to DNA delivery, possibly indicating that surface-tension-free cells favored Agrobacterium attachment.

Transformation techniques not involving tissue culture are desir-able for crop species in which many cultivars or inbred lines do not respond to tissue culture. These techniques include soaking and

(29)

vac-uum infiltration transformation of Arabidopsis inflorescence with

Agrobacterium (Bechtold, 1994; Bent et al., 1994), transformation

via the pollen tube pathway (Luo and Wu, 1988; Zhang, personal com-munication, 1999), and pollen transformation via biolistics (Ramaiah and Skinner, 1997). The dipping method adopted in Arabidopsis must be modified for cereal crops by altering the plant stage of infil-tration, the concentration of bacterial culture, and the duration of treatment. Further, each tiller (for wheat, barley, etc.) of the same plant must be kept separate. If this technique works, selection for transformants is made directly from seed-derived seedlings and tis-sue culture is avoided. Thus, genotypes that respond negatively to callus induction and plant regeneration will not present a problem; however, it is likely some genotypes will be more amenable to infil-tration transformation than others.

CONFIRMATION OF PUTATIVE TRANSGENIC PLANTS AND TRANSFORMATION EFFICIENCY

Commonly used methods to confirm the putative transgenic plants are polymerase chain reaction (PCR), Southern blotting, Western blotting, Northern blotting, functional assay (testing the presence of the selectable marker and the target gene), in situ hybridization, and progeny analysis (segregation of the target gene). Not all transgenic plants produce the same amount of protein from the target gene and selection based on the Western blot is necessary. This is because a positive correlation usually exists between the effectiveness of the gene in the bioassay and the amount of protein it produces. For exam-ple, the level of rice chitinase accumulating in transgenic sorghum plants with the chi11 gene was positively correlated with resistance to sorghum stalk rot (Krishnaveni et al., 2001).

Polymerase Chain Reaction

PCR, a simple and rapid procedure, is utilized to confirm whether a putatively transgenic plant that has survived selection is indeed transgenic. Usually, two primers (one forward and one reverse) spe-cific for the selectable marker (bar or cah gene, for example) are used in a PCR reaction with genomic DNA extracted from the transgenic plants. A thermostable DNA polymerase amplifies the region

(30)

be-tween the two primers during the multiple amplification cycles of the PCR, which yields a DNA fragment of predicted size (the length equal to the number of base pairs between the two primers in the transgene). This fragment is easily detected on an agarose gel by staining with ethidium bromide. PCR is a very sensitive and rapid method for identification of transgenic plants in the seedling stage and requires only a small amount of plant tissue. The presence of additional transgenes in the same plants carrying the selectable marker can be de-tected with a different set of primers using the same template DNA.

Southern Blotting/Hybridization Analysis

In Southern blotting, DNA fragments from transgenic plants gen-erated by digestion with restriction enzyme(s) are first separated ac-cording to fragment size by electrophoresis through an agarose gel. The DNA fragments then are transferred to a solid support, such as a nylon membrane or a nitrocellulose sheet. The transfer is effected by simple capillary action, sometimes assisted by suction or electric cur-rent. The DNA binds to the solid support, usually because the support has been treated to carry a net positive charge, or some other means of binding, such as inducing covalent binding of the DNA to the sup-port. DNA fragments maintain their original positions in the gel after transfer to the membrane. Hence, larger fragments will be localized toward the top of the membrane and smaller fragments toward the bottom. The positions of specific fragments can then be determined by “probing” the membrane. The probe consists of the DNA frag-ments of interest, such as a cloned gene, which has been labeled with a radioactive isotope or some other compound that allows its visual detection. Under the proper set of conditions, the denatured single-stranded probe will hybridize to its complementary single strands of genomic DNA affixed to the membrane. In this way, the size of the fragment on which the probe resides in the genomic DNA can be de-termined. In transgenic plant experiments, the Southern blot often is used to determine whether an introduced gene is indeed present in the plant DNA and whether multiple transgenic plants carry the intro-duced gene on the same size of DNA fragment (suggesting a single transformation event) or on different sized fragments (suggesting in-dependent transformation events). The results of Southern blots also

(31)

indicate whether a single copy of the gene has been inserted or if mul-tiple copies are likely to be present. The Southern blot derives its name from its inventor, E. M. Southern (Southern, 1975).

Northern Blotting

Northern blotting—the name was derived as a play on words from the Southern blot—is very similar to the Southern blot, except that in-stead of restriction enzyme-digested DNA, native RNA is separated according to size by electrophoresis through an agarose gel and then transferred to a solid support. The rest of the Northern blot procedure is very similar to that of the Southern blot and it is used to determine whether the introduced gene has been transcribed into messenger RNA and accumulates in the transgenic plant.

Western Blotting

The Western blotting procedure detects the protein of the gene in an extract of proteins prepared from various parts of the trans-genic plants and is, therefore, an assay for a functional transgene. In this technique the proteins are first electrophoresed in an SDS-polyacylamide gel and the proteins are then transferred to nitro-cellulose of polyvinylidene difluoride (PVDF) membrane by electro-phoretic transfer. The membrane is then treated with an antibody specific for the protein encoded by the transgene followed by a sec-ond antibody coupled to an enzyme, which can act on a chromogenic (or fluorogenic) substrate leading to visualization of the transgene protein with increased sensitivity. The expression level of the protein can be quantified using known amounts of the transgene-encoded protein.

Functional Assay

When the selectable markers used are antibiotic-resistant or herbi-cide-resistant genes, a function assay can be made by spraying antibi-otics or smearing herbicide on the leaves of those putative transgenic seedlings or plants in later segregating populations. Sensitive plants typically will turn brown and shrivel up whereas resistant (transgen-ic) plants stay healthy and green. Such an assay provides the initial

(32)

screening of large numbers of putative transgenic plants and reduces the work load by eliminating escapes during selection.

Progeny Test

With stable transformed genes, progeny testing should show the presence and activity of the selectable marker and target genes, such as the gene gfp encoding green fluorescence protein, or bar and dis-ease resistance. However, segregation does not always follow the typ-ical Mendelian fashion. For example, among the progeny in the

Agro-bacterium-mediated wheat transformation experiments reported by

Cheng and colleagues (1997), segregation in the T₁generation had ratios of 32:0, 1:34, 0:40, and 74:0 in addition to the expected 1:1, 3:1, or 15:1 ratios. This variability indicates aberrant segregation. However, in other cases, segregation follows the normal Mendelian pattern. For example, among six sorghum T₀transgenic plants pro-duced by biolistic bombardment, all showed typical 3:1 segregation ratios in the T₁generation (Zhu et al., 1998).

CROP SPECIES AMENABLE TO TRANSFORMATION

Any crop that is able to produce calli from explants and is capable of callus regeneration into plants with high efficiency is amenable to transformation using biolistic bombardment and Agrobacterium

tum-efaciens. However, it is known that response to tissue culture is highly

genotype dependent. Furthermore, somaclonal variation, spontaneous genetic variation occurring in cells growing in vitro (Larkin and Scowcroft, 1981), could occur during tissue culture processes. Thus, to confirm that the improved phenotype of the transgenic plants is due to the transgene, two controls should be included—one from seed-derived plants and the other from nontransformed tissue cul-ture-derived plants.

For those crops or genotypes that show a negative response to tis-sue culture, a transformation procedure independent from tistis-sue cul-ture should be considered and tested. It will be a great accomplish-ment to perfect a procedure bypassing tissue culture, because many

(33)

cultivars of wheat and rice and inbred lines of maize and sorghum do not respond to tissue culture operations.

CURRENT AND FUTURE TRANSGENIC CROPS

The great majority of transgenic crops currently grown in the field are derived from the “magic bullet” type of transformation. That is, a single gene has been inserted into the crop species; the product of that gene causes a demonstrable change in phenotype of the plants. For example, many crop species express an endotoxin from various

Ba-cillus thuringiensis (Bt) strains, conferring resistance to various

in-sect pests. Numerous examples have been reported in the literature (Arpaia et al., 2000; Leroy et al., 2000; Breitler et al., 2000; Mand-aokar et al., 2000; Acciarri et al., 2000; Giles et al., 2000; Walker et al., 2000; Harcourt et al., 2000; and Zhao et al., 2000).

Padgette and colleagues (1995) determined that a cloned 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium

tume-faciens strain CP4, engineered to express in plants, substituted for the

endogenous gene that is inhibited by the nonspecific herbicide gly-phosate (N-phosphonomethyl-glycine). The bacterial gene was not inhibited by the herbicide, resulting in transgenic plants, now com-monly referred to as Roundup Ready, able to withstand applications of glyphosate that kill all surrounding plants without this gene. Other herbicide resistance genes have been introduced into crop plants as well, including resistance to L-phosphinothricin due to the phos-phinothricin-acetyl transferase (pat) gene from Streptomyces

virido-chromogenes, or resistance to the closely related bialaphos

(phos-phinothricyl-L-alanyl-L-alanine) due to the bar (bialaphos resistance) gene isolated from Streptomyces hygroscopicus. These genes are used routinely as selectable markers in plant transformation experi-ments, as well as the functional genes in the herbicide-resistant Lib-erty-Link series of field crops.

Other experiments of single transgenes conferring a trait of inter-est include constitutive expression of a bacterial citrate synthetase gene, which reportedly conferred tolerance to acidic soil in tobacco and papaya (de la Fuente et al., 1997). Attempts have been made to interfere with normal growth and nutrient assimilation by pests and parasites by expressing in plants enzymes such as chitinase, or

(34)

en-zyme inhibitors such as protease inhibitors. Some success has been reported with this approach. For example, several plant species have shown enhanced resistance to fungal diseases when transformed to constitutively express a chitinase gene isolated from rice (Lin et al., 1995; Tabei et al., 1998; Nishizawa et al., 1999; Yamamoto et al., 2000; Takatsu et al., 1999).

Recently, combinations of genes have been expressed in plants to confer multiple resistance. Examples include ‘Herculex I’ maize, en-gineered to express resistance to glufosinate herbicides and to ex-press a Bt toxin effective against lepidopterans, and ‘NewLeaf Plus’ potatoes, engineered to express a Bt toxin and genes that confer viral resistance to viruses. It is likely that many more “magic bullet” com-binations will be constructed and marketed in the future.

Potential and existing transgene combinations necessitate the study of the interactions of the transgenes to elucidate any effect one gene has on the efficacy of the others. Some investigation has been con-ducted into the expression of multiple transgenes in the same plant (e.g., Maqbool and Christou, 1999; Maqbool et al., 2001). However, these combinations of genes are not interdependent. That is, the Bt gene in ‘Herculex I’ maize does not rely on the glufosinate herbicide resistance gene for expression, nor vice versa. In the future, entire metabolic pathways will be engineered to express several intermedi-ate gene products to gain expression of a particular desired metabo-lite, increasing the potential for transgene interactions.

The first successful attempt to engineer a system into plants in-volved expressing a form of the mammalian antiviral 2-5A system in tobacco plants (Mitra et al., 1996). Two genes were built into the same vector and transformed into tobacco plants. Some of the result-ing plants expressed both genes, resultresult-ing in a resistance response to three different plant pathogenic viruses (Mitra et al., 1996). A more complex system was developed in the construction of a novel form of rice (Oryza sativa L.). Through careful characterization of metabolic products naturally occurring in rice, it was deduced that if four en-zymes—phytoene synthase, phytoene desaturase, zeta-carotene desa-turase, and lycopene cyclase—were expressed in rice, provitamin A would result. After a relatively short eight years of work, “Golden Rice” was developed and found to express provitamin A in quantities theoretically sufficient to be pharmacologically therapeutic (Potry-kus, 2001). Genes cloned from ornamental flowers were used in the

(35)

construction of “Golden Rice.” As the biochemical pathways of in-creasingly complex traits are elucidated, we can expect to see more engineered pathways to gain expression of nutritionally beneficial metabolites in high-yielding crops, with the genes cloned from model systems that lend themselves to the isolation of the necessary genes. The knowledge and techniques gained from the comparatively sim-ple transformation studies done to date are just the beginning of what may develop into a second “green revolution.”

FOOD SAFETY AND RISK ANALYSIS

It is only natural for humans to be concerned with food safety. This concern is increasing, at least in industrialized countries, as evi-denced by the growing demand for organic foods, public objections to producing genetically modified organisms, requiring labeling of food to show the origin and mode of producing and processing, and establishment of regulations for producing, processing, storing, and transporting foods. In spite of regulation and precaution, recalls of manufactured food products and outbreaks of food poisoning are not uncommon. The extensive recall of food products containing Star-Link corn in 2001 is an example. In addition, the proposed use of ter-minator seed technology to protect the transgenic crops from illegal and unauthorized use of patented seed has met with strong objection. Chapter 14 discusses the risk analysis.

Clearly, transgenic crops will play a major role in modern agricul-ture. The efficient and safe use of this technology will depend on the free and open sharing of the developing technology and the rigorous examination of the productivity and safety of the resulting crops. In the chapters that follow, authors at the forefront of this developing technology present reviews of the current state of the art for field and horticultural crops. It is hoped that this collection will serve as a plat-form for the continued exchange of results and ideas for improving the productivity of the world’s supply of food, feed, and fiber.

REFERENCES

Acciarri, N., G. Vitelli, G. Mennella, F. Sunseri, and G.L. Rotino (2000). Transgen-ic resistance to the Colorado potato beetle in Bt-expressing eggplant fields. HortScience 35:722-725.

(36)

Arpaia, S., L. Demarzo, G.M. Di-Leo, M.E. Santora, G. Mennella, and J.J.A. van Loon (2000). Feeding behaviour and reproductive biology of Colorado beetle adults fed transgenic potatoes expressing the Bacillus thuringiensis Cry3B endotoxin. Entomologia Experimentalis et Applicata 95:31-37.

Bechtold, N., J. Ellis, and G. Pelletier (1994). Agrobacterium mediates gene trans-fer by infiltration of adult Arabidopsis thalina. Plant Journal 5:421-427. Bent, A., B.N. Kunkel, D. Kahlbeck, K.L. Brown, R. Schmidt, J. Giraudat,

J. Leung, and B.J. Staskawicz (1994). RPS2 of Arabidopsis thalina: A leucine-rich repeat class of plant diseases resistance genes. Science 265:1856-1860. Breitler, J.C., V. Marfa, M. Royer, D. Meynard, J.M. Vassal, B. Vercambre, R.

Frutos, J. Messeguer, R. Gabarra, and E. Guiderdoni (2000). Expression of a Ba-cillus thuringiensis cry1B synthetic gene products Mediterranean rice against the striped stem borer. Plant Cell Reports 19:1195-1202.

Casas, A.M., A.K. Kononowicz, U.B. Zehr, D.T. Tomes, J.D. Axtell, R.A. Bresan, and P.M. Hasegawa (1993). Transgenic sorghum plants via microprojectile bombardment. Proceedings of the National Academy of Sciences of the United States of America 90:11212-11216.

Cheng, M., J.E. Fey, S. Pang, H. Zhou, C.M. Hironaka, D.R. Duncan, T.W. Conner, and Y. Wan (1997). Genetic transformation of wheat mediated by Agrobac-terium tumefaciens. Plant Physiology 115:971-980.

de la Fuente, J.M., V. Ramirez-Rodriguez, J.L. Cabrera-Ponce, and L. Herrera-Estrella (1997). Alluminum tolerance in transgenic plants by alteration of citrate synthesis. Science 276:1566-1568.

Giles, K.L., R.L. Hellmich, C.T. Iverson, and L.C. Lewis (2000). Effects of trans-genic Bacillus thuringiensis maize grain on B. thuringiensis-susceptible Ploida interpunctella (Lepidoptera: Pyralidae). Journal of Economic Entomology 93:1011-1016.

Harcourt, R.L., J. Kyozuka, R.B. Floyd, K.S. Bateman, H. Tanaka, V. Decroocq, D.L. Llewellyn, X. Zhu, W.J. Peacock, and E.S. Dennis (2000). Insect- and her-bicide-resistant transgenic eucalypts. Molecular Breeding 6:307-315.

Hiei, Y.S., S. Ohta, T. Komari, and T. Kumasho (1994). Efficient transformation of rice (Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6:271-282.

Ishida, Y., H. Saito, S. Ohta, Y. Hiei, T. Komari, and T. Kumashiro (1996). High ef-ficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology 14:745-750.

Kado, C.I. (1998). Agrobacterium-mediated horizontal gene transfer. In Genetic Engineering, Volume 20 (1-24), Ed. Setlow, J.K. New York: Plenum Press. Krishnaveni, S., J.M. Jeoung, S. Muthukrishnan, and G.H. Liang (2001).

Transgen-ic sorghum plants constitutively expressing a rTransgen-ice chitinase gene show improved resistance to stalk rot. Journal of Genetics and Breeding 55:151-158.

Larkin, P.J. and W.R. Scowcroft (1981). Somaclonal variation—A novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics 60:197-214.

(37)

Leroy, T., A.M. Henry, M. Royer, I. Altosaar, R. Frutos, D. Duris, and R. Philippe (2000). Genetically modified coffee plants expressing the Bacillus thuringiensis Cry1Ac gene for resistance to leaf miner. Plant Cell Reports 19:382-389. Lin, W., C.S. Anuratha, K. Datta, I. Potrykus, S. Muthukrishnan, and S.K. Katta

(1995). Genetic engineering of rice for resistance to sheath blight. Biotechnology 13:686-691.

Luo, Z.X. and R. Wu (1988). A simple method for the transformation of rice via the pollen-tube pathway. Plant Molecular Biology Reporter 6:165-174.

Mandaokar, A.D., R.K. Goya, A. Shukla, S. Bisaria, R. Bhalla, V.S. Reddy, A. Chaurasia, R.P. Sharma, I. Altosaar, and P.A. Kumar (2000). Transgenic to-mato plants resistant to fruit borer (Helicoverpa armigera Hubner). Crop Pro-tection 19:307-312.

Maqbool, S.B. and P. Christou (1999). Multiple traits of agronomic importance in transgenic indica rice plants: Analysis of transgene integration patterns, expres-sion levels and stability. Molecular Breeding 5:471-480.

Maqbool, S.B., S. Riazuddin, N.T.L. Angharad, M.R. Gatehouse, J.A. Gatehouse, and P. Christou (2001). Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Molecular Breeding 7:85-93. Mitra, A., D.W. Higgins, W.G. Langenberg, H. Nie, D.N. Sengupta, and R.H.

Silverman (1996). A mammalian 2-5A system functions as an antiviral pathway in transgenic plants. Proceedings of the National Academy of Sciences of the United States of America 93:6780-6785.

Nishizawa, Y., Z. Nishio, K. Nakazono, M. Soma, E. Nakajima, M. Ugaki, and T. Hibi (1999). Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase. Theoretical and Ap-plied Genetics 99:383-390.

Padgette, S.R., K.H. Kolacz, X. Delannay, D.B. Re, B.J. LaVallee, C.N. Tinius, W.K. Rhodes, Y.I. Otero, G.F. Barry, and D.A.W. Eichholtz (1995). Develop-ment, identification, and characterization of a gluphosate-tolerant soybean line. Crop Science 35:1451-1461.

Pinstrup-Andersen, P. and R. Pandya-Lorch (1999). Securing and sustaining ade-quate world food production for the third millennium. In World Food Security and Sustainability: The Impacts of Biotechnology and Industrial Consolidation (pp. 27- 53), Eds. Weeks, D.P., J.B.Segelken, and R.W.F. Hardy. Ithaca, NY: National Agricultural Biotechnology Council.

Potrykus, I. (2001). Golden rice and beyond. Plant Physiology 125:1157-1161. Ramaiah, S.M. and D.Z. Skinner (1997). Particle bombardment: A simple and

effi-cient method of alfalfa [Medicago sativa (L.)] pollen transformation. Current Science 73:674-682.

Rosegrant, M.W., C. Ringler, and R.V. Gerpacio (1997). Water and land resources and global food supply. Twenty-Third International Conference on Agriculture Economy, Sacramento, California, August 10-16.

Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503-517.

(38)

Tabei, Y., S. Kitade, Y. Nishizawa, N. Kikuchi, T. Kayano, T. Hibi, and K. Akutsu (1998). Transgenic cucumber plants harboring a rice chitinase exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant Cell Reports 17:159-164. Takatsu, Y., Y. Nishizawa, T. Hibi, and K. Akutsu (1999). Transgenic

chrysanthe-mum (Dendranthema grandiflorum [Ramat] Kitamura) expressing a rice chiti-nase gene shows enhanced resistance to gray mold (Botrytis cinerea). Scientia Horticulturae 82:113-123.

Walker, D.R., J.N. All, R.M. McPherson, H.R. Boerma, and W.A. Parrott (2000). Field evaluation of soybean engineered with a synthetic cry1Ac transgene for re-sistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidop-tera:Noctuidae), and lesser corn stalk borer (Lepidoptera:Pyralidae). Journal of Economic Entomology 93:613-622.

Weeks, D.P. (1999). An overview. In World Food Security and Sustainability: The Impacts of Biotechnology and Industrial Consolidation (pp. 3-12), Eds. Weeks, D.P., J.B. Segelken, and R.W.F. Hardy. Ithaca, NY: National Agricultural Bio-technology Council.

Yamamoto, T., H. Iketani, H. Ieki, Y. Nishizawa, K. Notsuka, T. Hibi, T. Hayashi, and N. Matsuta (2000). Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Reports 19:639-646. Zhao, J.Z., H.L. Collins, J.D. Tang, J. Cao, E.D. Earle, R.T. Roush, S. Herrero,

B. Escriche, J. Ferre, and A.M. Shelton (2000). Development and characteriza-tion of diamondback moth resistance to transgenic broccoli expressing high lev-els of Cry1C. Applied and Environmental Microbiology 66:3784-3789. Zhu, H., S. Muthukrishnan, S. Krishnaveni, G. Wilde, J.M. Jeoung, and G.H. Liang

(1998). Biolistic transformation of sorghum using a rice chitinase gene. Journal of Genetics and Breeding 52:243-252.

Zupan, J.R. and P.C. Zambryski (1995). Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiology 107:1041-1047.

(39)

Chapter 2

Mechanism(s) of Transgene Locus Formation

Mechanism(s) of Transgene

Locus Formation

David A. Somers Paula M. Olhoft Irina F. Makarevitch Sergei K. Svitashev INTRODUCTION

The production of genetically engineered plants was first achieved in 1983. A large number of plant species have since been genetically engineered, primarily using two different strategies for DNA delivery into totipotent cells. The first strategy employed T-DNA delivery me-diated by disarmed strains of the soil bacterium Agrobacterium

tumefaciens (De Block et al., 1984; Horsch et al., 1984). Agrobac-terium tumefaciens was initially used in transformation of a broad

range of dicotyledonous crops (Horsch et al., 1987). At that time, it appeared that Agrobacterium would not be useful for transformation of the monocotyledonous cereal grains (Potrykus, 1990; Vain et al., 1995). This led to the concurrent development of direct DNA deliv-ery methods as the second strategy for transformation of the recalci-trant monocot species. Fertile, transgenic plants produced via direct DNA delivery methods were reported soon after the success with

A. tumefaciens (Paszkowski et al., 1984). Direct DNA delivery methods

were initially based on protoplasts as the target cells for DNA deliv-ery and employed polyethylene glycol (PEG)- and electroporation-mediated delivery of DNA (Paszkowski et al., 1984; Potrykus et al., 1987; Vain et al., 1995). In 1987, Klein and colleagues reported the development of microprojectile bombardment technology for

(40)

deliv-ery of DNA into intact plant cells (Klein et al., 1987, 1989; Klein and Jones, 1999; Sanford, 1988, 1990; Sanford et al., 1987, 1991, 1993). This development resulted in production of the first fertile transgenic plants in soybean (McCabe et al., 1988), maize (Fromm et al., 1990; Gordon-Kamm et al., 1990), wheat (Vasil et al., 1992, 1993), oat (Somers et al., 1992), and barley (Wan and Lemaux, 1994). Com-bined with improvements in plant tissue culture procedures, new se-lection systems for isolation of transgenic tissue cultures and plants, and optimization of DNA delivery parameters, microprojectile bom-bardment protocols have led to efficient transformation systems for a number of important crops (Christou et al., 1991; Christou, 1992; Walters et al., 1992; Weeks et al., 1993; Russell et al., 1993; Barcelo et al., 1994; Becker et al., 1994; Brar et al., 1994; Nehra et al., 1994; Koprek et al., 1996; Torbert et al., 1998). The development of trans-formation systems for cereal crops has been extensively reviewed in a book edited by Vasil (1999).

Agrobacterium-mediated genetic engineering is currently the most

widely used plant genetic engineering strategy. Agrobacterium-medi-ated transformation of monocots was first demonstrAgrobacterium-medi-ated in rice in 1994 (Hiei et al., 1994) and is now routinely used to transform cere-als, including rice, corn (Ishida et al., 1996), barley (Tingay et al., 1997), and wheat (Cheng et al., 1997). Researcher familiarity with

Agrobacterium-mediated transformation systems and their

simplic-ity and independence from DNA delivery devices are important ratio-nale for the major shift toward using Agrobacterium for crop genetic engineering (Songstad et al., 1995). The greatest motivation to use

Agrobacterium appears to be driven by molecular and cytogenetic

analyses showing that higher frequencies of transgene loci produced via microprojectile bombardment and other direct DNA delivery methods exhibit complex structures composed of multiple copies of whole and rearranged delivered DNA compared to transgene loci produced via Agrobacterium (Pawlowski and Somers, 1996). Com-plex transgene loci are associated with problems of transgene expres-sion (Finnegan and McElroy, 1994; Matzke and Matzke, 1995; Paw-lowski et al., 1998; Kohli, Gahakwa, et al., 1999) and inheritance (Christou et al., 1989; Choffnes et al., 2001) and pose considerable dif-ficulties in transgene locus characterization for regulatory approval be-fore commercialization of transgenic crops. Thus, transformation meth-ods that are simple and inexpensive and produce the simplest transgene

(41)

locus structures, such as Agrobacterium-based methods, are readily adopted for both basic and applied uses of plant genetic engineering.

Impressive progress has been achieved since 1983 in developing methods for genetically engineering a diverse range of plant species, in commercialization of transgenic traits in a number of crops that are planted on substantial acreages throughout the world, and in charac-terization of transgene locus structure. However, much remains to be learned about the mechanisms that result in the formation of transgene loci, why the main transformation systems differ in the proportions of complex transgene loci produced, and how to routinely produce sim-ple transgene loci.

A large body of literature describes the structure of transgene loci produced using both Agrobacterium-mediated and direct DNA deliv-ery transformation; however, a paucity of studies prospectively inves-tigate factors that affect transgene locus formation mechanism(s) and the resulting structure of transgene loci. The primary analytical method used to characterize transgene loci involves various restric-tion digesrestric-tions of genomic DNA from transgenic plants followed by Southern blot analysis (Southern, 1975). Although these studies have been useful in documenting several features of transgene loci, South-ern analyses are unable to provide detailed characterization of transgene loci or even good estimates of their size. More recently, fluorescent in situ hybridization (FISH) has been applied to studying transgene loci and has resulted in a number of new insights into their structure and size (Ambros et al., 1986; Moscone et al., 1996; Mouras et al., 1987; Mouras and Negrutiu, 1989; for review see Svitashev and Somers, 2001a). Polymerase chain reaction (PCR) has been used to isolate small portions of transgene loci (Kohli, Griffiths, et al., 1999) and genomic sequences flanking integrated delivered DNA; however, lit-tle direct DNA sequence data have been reported for complete or par-tial transgene loci. As transgene sequence data are reported, the real-ization emerging is that many features of transgene loci are more complex than previously thought. Moreover, these data are providing new insights into mechanism(s) of transgene locus formation. The aim of this chapter is to review the literature concerning the structure of nuclear transgene loci and relate these features to mechanism(s) leading to their formation in plants transformed via Agrobacterium-mediated T-DNA and direct DNA delivery.

7743.Genetically Modified Crops Their Development, Uses, And Risks by G.h. Liang

G. H. Liang, PhD

D. Z. Skinner, PhD

Editors

Genetically Modified Crops

Their Development,

Uses, and Risks

“T

“T

“T

Genetically Modified Crops

Their Development,

FOOD PRODUCTS PRESS

Genetically Modified Crops

Their Development,

Uses, and Risks

CONTENTS

Preface

Chapter 1

Transformation: A Powerful Tool

for Crop Improvement

Chapter 2

Mechanism(s) of Transgene

Locus Formation