Recent Advances in Plant Biotechnology
Ara Kirakosyan · Peter B. Kaufman
Recent Advances in Plant
Biotechnology
123
Ara Kirakosyan
University of Michigan
1150 W. Medical Center Dr.
Ann Arbor MI 48109-0646
USA

Peter B. Kaufman
University of Michigan
1150 W. Medical Center Dr.
Ann Arbor MI 48109-0646
USA

ISBN 978-1-4419-0193-4 e-ISBN 978-1-4419-0194-1
DOI 10.1007/978-1-4419-0194-1
Springer Dordrecht Heidelberg London New York
Library of Congress Control Number: 2009928135
c

Springer Science+Business Media, LLC 2009
All rights reserved. This work may not be translated or copied in whole or in part without the written
permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York,
NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in
connection with any form of information storage and retrieval, electronic adaptation, computer
software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if

use of genetically transferred cultures in order to understand the genetics of specific
plant traits. Such relevant methods can be used to determine the markers that are
retained in genetically manipulated organisms and to determine the elimination of
marker genes. As a result, a number of transgenic plants have been developed with
beneficial characteristics and significant long-term potential to contribute both to
biotechnology and to fundamental studies. These techniques enable the selection
of successful genotypes, better isolation and cloning of favorable traits, and the
creation of transgenic organisms of importance to agriculture and industry.
We start the book by tracing the roots of plant biotechnology from the basic
sciences to current applications in the biological and agricultural sciences, indus-
try, and medicine. These widespread applications signal the fact that plant biotech-
nology is increasingly gaining in importance. This is because it impinges on so
vii
viii Preface
many facets of our lives, particularly in connection with global warming, alternative
energy initiatives, food production, and medicine. Our book would not be complete
unless we also addressed the fact that some aspects of plant biotechnology may have
some risks. These are covered in the last section.
The individual chapters of the book are organized according to the following
format: chapter title and contributors, abstract, introduction to the chapter, chapter
topics and text, and references cited for further reading. This format is designed in
order to help the reader to grasp and understand the inherent complexity of plant
biotechnology better.
The topics covered in this book will be of interest to plant biologists, biochemists,
molecular biologists, pharmacologists, and pharmacists; agronomists, plant breed-
ers, and geneticists; ethnobotanists, ecologists, and conservationists; medical prac-
titioners and nutritionists; and research investigators in industry, federal labs, and
universities.
Ann Arbor, MI Peter B. Kaufman
Ann Arbor, MI Ara Kirakosyan

and Peter B. Kaufman
Part III Use of Plant Secondary Metabolites in Medicine
and Nutrition
10 Interactions of Bioactive Plant Metabolites: Synergism,
Antagonism, and Additivity ..................... 213
John Boik, Ara Kirakosyan, Peter B. Kaufman, E. Mitchell
Seymour, and Kevin Spelman
11 The Use of Selected Medicinal Herbs for Chemoprevention
and Treatment of Cancer, Parkinson’s Disease, Heart
Disease, and Depression ........................ 231
Maureen McKenzie, Carl Li, Peter B. Kaufman, E. Mitchell
Seymour, and Ara Kirakosyan
12 Regulating Phytonutrient Levels in Plants – Toward
Modification of Plant Metabolism for Human Health ....... 289
Ilan Levin
Part IV Risks and Benefits Associated with Plant Biotechnology
13 Risks and Benefits Associated with Genetically Modified
(GM) Plants .............................. 333
Peter B. Kaufman, Soo Chul Chang, and Ara Kirakosyan
14 Risks Involved in the Use of Herbal Products ............ 347
Peter B. Kaufman, Maureen McKenzie, and Ara Kirakosyan
15 Risks Associated with Overcollection of Medicinal Plants
in Natural Habitats .......................... 363
Maureen McKenzie, Ara Kirakosyan, and Peter B. Kaufman
16 The Potential of Biofumigants as Alternatives to Methyl
Bromide for the Control of Pest Infestation in Grain and
Dry Food Products .......................... 389
Eli Shaaya and Moshe Kostyukovsky
Index ..................................... 405
About the Authors

tion Research Unit, US Plant, Soil, and Nutrition Laboratory, Ithaca, New York,
USA. In 2002, he was a Fulbright Visiting Research Fellow at the University of
Michigan, Department of Molecular, Cellular, and Developmental Biology in the
Laboratory of Prof. Peter B. Kaufman. Dr. Kirakosyan is principal author of over
50 peer-reviewed research papers in professional journals and several chapters in
xi
xii About the Authors
books dealing with plant biotechnology and molecular biology. He is second author
of the best-selling book, Natural Products from Plants, 2nd edition (2006). Ara
Kirakosyan is a full member of the Phytochemical Society of Europe and European
Federation of Biotechnology. He serves as an editorial board member in the Open
Bioactive Compounds Journal, Bentham Science Publishers, and as an editor as
part of the editorial board of 19 scientific domains journals, Global Science Books
(GSB), Isleworth, UK. He has received several awards, fellowships, and research
grants from the United States, Japan, and the European Union.
Peter B. Kaufman, Ph.D., is a professor of biology emeritus in the Department
of Molecular, Cellular, and Developmental Biology (MCDB) at the University
of Michigan and is currently senior scientist, University of Michigan Integrative
Medicine Program (UMIM). He received his B.Sc. in plant science from Cornell
University in Ithaca, New York, in 1949 and his Ph.D. in plant biology from the Uni-
versity of California, Davis, in 1954 under the direction of Prof. Katherine Esau. He
did post-doctoral research as a Muelhaupt Fellow at Ohio State University, Colum-
bus, Ohio. He has been a visiting research scholar at University of Calgary, Alberta,
Canada; University of Saskatoon, Saskatoon, Canada; University of Colorado, Boul-
der, Colorado; Purdue University, West Lafayette, Indiana; USDA Plant Hormone
Laboratory, BARC-West, Beltsville, Maryland; Nagoya University, Nagoya, Japan;
Lund University, Lund, Sweden; International Rice Research Institute (IRRI) at Los
Banos, Philippines; and Hawaiian Sugar Cane Planters’ Association, Aiea Heights,
Hawaii. Dr. Kaufman is a fellow of the American Association for the Advance-
ment of Science and received the Distinguished Service Award from the American

ımicas
yFarmac
´
euticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK
Rosario, Argentina,
Soo Chul Chang University College, Yonsei University, Seoul 120-749, Korea,

Leland J. Cseke Department of Biological Sciences, The University of Alabama
in Huntsville. Huntsville, AL 35899, USA,
Rainer Fischer Fraunhofer Institute for Molecular Biology and
Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany,
fi
Mohammad-Reza Hajirezaei Leibniz-Institute of Plant Genetics and
Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany,

Hiroaki Hayashi School of Pharmacy. Iwate Medical University. 2-1-1
Nishitokuta, Yahaba, Iwate 028-3603, Japan,
Peter B. Kaufman University of Michigan, Ann Arbor MI 48109-0646, USA,

Ara Kirakosyan University of Michigan, Ann Arbor, MI 48109-0646, USA,

Moshe Kostyukovsky ARO, the Volcani Center, Department of Food Science, Bet
Dagan, 50250, Israel,
Ilan Levin Department of Vegetable Research, Institute of Plant Sciences, The
Volcani Center, Bet Dagan, Israel 50250,
xiii
xiv Contributors
Carl Li Department of Social and Preventive Medicine, State University of
New York at Buffalo, Buffalo, NY 14214, USA,
Casey R. Lu Department of Biological Sciences, Humboldt State University,

York, YO10 5DD, UK,
Matias D. Zurbriggen Instituto de Biolog
´
ıa Molecular y Celular de Rosario
(IBR, UNR/CONICET), Divisi
´
on Biolog
´
ıa Molecular, Facultad de Ciencias
Bioqu
´
ımicas y Farmac
´
euticas, Universidad Nacional de Rosario, Suipacha 531,
S2002LRK Rosario, Argentina,
Chapter 1
Overview of Plant Biotechnology from Its Early
Roots to the Present
Ara Kirakosyan, Peter B. Kaufman, and Leland J. Cseke
Abstract In this chapter, we first define what is meant by plant biotechnology.
We then trace the history from its earliest beginnings rooted in traditional plant
biotechnology, followed by classical plant biotechnology, and, currently, modern
plant biotechnology. Plant biotechnology is now center stage in the fields of alter-
native energy involving biogas production, bioremediation that cleans up polluted
land sites, integrative medicine that involves the use of natural products for treatment
of human diseases, sustainable agriculture that involves practices of organic farm-
ing, and genetic engineering of crop plants that are more productive and effective
in dealing with biotic and abiotic stresses. The primary toolbox of biotechnology
utilizes the latest methods of molecular biology, including genomics, proteomics,
metabolomics, and systems biology. It aims to develop economically feasible pro-

ogy? With the advent of recombinant DNA technology and new approaches that
utilize genomics, metabolomics, proteomics, and systems biology strategies (Cseke
et al., 2006), it may now be possible to re-examine plant cell cultures as a reasonable
candidate for commercial production of high-value plant metabolites. This is espe-
cially true if natural resources are limited, de novo chemical synthesis is too com-
plex or unfeasible, or agricultural production of the plant is not possible to carry out
year-round. Indeed, a study of the biochemistry of plant natural products has many
practical applications. Thus, specific processes have now been designed to meet the
requirements of plant cell cultures in bioreactors. In addition, plant cells constitute
an effective system for the biotransformation involving the addition of various sub-
strates to the culture media in order to induce the formation of new products. The
specific enzymes participating in such biotransformation processes can furthermore
be isolated and characterized from cells immobilized on various solid support matri-
ces, such as fiber-reinforced biocers (e.g., aqueous silica nanosols and commercial
alumina fibers) that are now used in bioreactors.
Modern plant biotechnology research uses a number of different approaches that
include high-throughput methodologies for functional analyses at the level of genes,
proteins, and metabolites. Other methods are designed for genome modification
through homologous and site-specific recombination. The potential for including
plant productivity or agricultural trials is directly dependent upon the use of the new
molecular markers or DNA construct technology. Therefore, plant biotechnology
now allows for the transfer of an incredible amount of useful genetic information
in a much more highly controlled and targeted manner. This is especially important
for the use of GM (genetically modified) organisms, in spite of risks and limita-
tions that have been voiced by individuals and organizations not in favor of this
technology. It is noteworthy that a number of transgenic plants are being developed
for long-term potential use in fundamental plant science studies (Sonnewald, 2003).
Some of these transgenic plants also have significant and beneficial characteristics
that allow for their safe use in industry and agriculture. Biotechnological approaches
can selectively increase the amounts of naturally produced pesticides and defense

Industrial
Pharmaceuticals
Ornamental
plants
Natural dyes
Agronomic plants
Fig. 1.1 A schematic representation of plant biotechnology applications
6 A. Kirakosyan et al.
lite biosynthesis is regulated by particular enzymes, transcription factors, substrate
availability and end-products and to apply this understanding to the economically
feasible production of specifically designed plants that are grown in a safe envi-
ronment and brought forth for agricultural, medical, and industrial applications
(Fig. 1.1).
1.2 Tracing the Evolution of Classical Plant Biotechnology
Early in the twentieth century, plant cell culture was introduced (White, 1943, 1963).
It had applications in plant pathology (Braun, 1974), plant morphogenesis, plant
micropropagation, cytogenetics, and plant breeding. Then, protoplast culture was
discovered (Cocking, 1960). It had applications in studies on cell wall biosynthesis,
somatic cell hybridization, and genome manipulation (Power et al., 1970). Paral-
lel studies led to the discovery that the ratio of auxin and cytokinin type hormones
in tissue culture media largely determined whether one obtained shoots, roots, or
undifferentiated callus tissue using tobacco (Nicotiana tabacum) as the model sys-
tem (Miller and Skoog, 1953; Murashige and Skoog, 1962). These three discoveries
in the plant sciences became the cornerstones of classical plant biotechnology.
The earliest roots of classical plant biotechnology emanate from studies by
agronomists, horticulturists, plant breeders, plant physiologists, biochemists, ento-
mologists, plant pathologists, botanists, and pharmacists. Their primary aim has
been to solve practical problems associated with (1) the use of classical meth-
ods of plant breeding to develop new cultivars of plants that are resistant to plant
pathogens, insect pests, and environmental stresses due to cold, drought, or flood-

basis for a complex type of communication between plants and animals. Because
of their biological activities, some plant natural products have long been exploited
by human beings as pharmaceuticals, stimulants, and poisons. Therefore, there is an
immense interest in isolating, characterizing, and utilizing these metabolites. While
plant natural products hold a great deal of potential use for many human ailments,
they are often made in only trace amounts within the specific plant species that
produce them. Furthermore, the biosynthesis of the various metabolites proceeds
along metabolic pathways that are highly complicated and located in one or more
cell compartment(s) (e.g., cell walls, membrane systems, the cytosol, and various
cellular organelles) within tissues that are often specialized for particular tasks. The
specific enzymes that catalyze the respective steps in each metabolic pathway are
encoded in nuclear, chloroplast, or mitochondrial genomes by specific genes.
Plant scientists enthusiastically endorsed the idea that plant cell and protoplast
culture would eventually lead to the production of natural products using in vitro
plant cell suspension cultures in bioreactors, similar to those produced by microbial
and fungal cells cultivated in bioreactors. However, this expectation, in large part,
failed to materialize, even in spite of ingenious strategies that were developed (Zenk
et al., 1977). Only a few compounds were able to be successfully produced in plant
cell cultures scaled-up in bioreactors for industrial applications (Verpoorte et al.,
1994; Cseke et al., 2006). The main limitations were attributed to relatively slow
growth rates of plant cells in shaker or bioreactor cultures, low rates of synthesis of
desired products, and synthesis of compounds not present in intact plants. In fact,
it was discovered in the course of these studies that biosynthesis of many types of
plant metabolites occurs only in organized shoots or roots, but not in cell cultures
per se. Thus, in vitro shoot or root cultures became an alternative strategy for the
production of desired metabolites (Kirakosyan et al., 2004).
Many scientists have now combined extensive research experience using plant
cell cultures in order to develop the best strategies for biotechnological application.
This is enabling us to follow-up in greater detail points of interest, both theoretical
and practical. Consequently, the development of an information base on a cellular

supplied compounds offers a broad potential and can make an interesting contri-
bution toward the modification of natural and synthetic chemicals as well. This
attribute of plant cells is designated as in vivo enzymatic bioconversion.Inmany
cases, the enzymes involved in this process can be identified, purified, and immo-
bilized, and this accomplished by what is termed in vitro bioconversion. Then, the
enzymatic potential of the plants can be employed for bioconversion purposes. The
bioconversion process thus involves enzyme-catalyzed modification of added pre-
cursors into more desired or valuable products, using plant cells or specific enzymes
isolated from plants. This type of metabolite modification is particularly accurate
and is not so labor intensive. The biocatalyst may be free in solution, immobilized
on a solid support, or entrapped in a matrix. Systems applied for bioconversion can
consist of freely suspended cells, where precursors are supplied directly to cultures;
immobilized plant cells, which are useful especially for secondary metabolite pro-
duction but still need development to elicit an increase in the half-life of the cells;
and finally enzyme preparation and further usage, which take into account prob-
lems connected with enzyme stability and sufficiency. In bioconversions elicited by
whole cells or extracts, a single or several enzymes may be required for an action to
occur.
In the same context, as described above, two biocatalytic systems can be
employed in biotechnology. First, the catalysis of specific foreign substances, either
chemically prepared or isolated from nature, can be carried out by enzymatic con-
version outside the organisms. Second, bioconversion of a particular product uses
1 Overview of Plant Biotechnology 9
Transcriptiomics Proteomics Metabolomics
5.0 7.5
0e6
10e6
20e6
Metabolic and Gene Engineering
Application of Functional genomics

so-called chiral compounds. For example, the production of single left- or right-
handed forms is not easy, and it is apparent that no single approach is likely to
dominate. Scientists must continue to draw upon the entire range of chemical, enzy-
matic, and whole-organism tools that are available to produce chiral compounds.
Despite some duplication in activity amongst enzymes, there is a need to charac-
terize more of them in order to exploit their unique specificity and activity. In this
10 A. Kirakosyan et al.
regard, plant enzymes are able to catalyze regio- and stereo-specific reactions and
therefore can be used for the production of desired substances. Stereospecificity con-
cerns high optical purity (100% of one stereoisomeric form) of biologically active
molecules being catalyzed by plant enzymes. Regiospecificity allows for more pre-
cise conversion of one or more specific functional groups into others or, in the case
of precursor molecules, selective introduction of functional groups on nonactivated
positions.
In studies with the above-described plant cell cultures and their applications, we
must, however, emphasize that not all aspects are clear and well-studied. Fundamen-
tal and practical researches are ongoing because problems related to monitoring the
production of secondary metabolites in cell cultures still exist.
1.3 Modern Plant Biotechnology
Present-day studies are progressing in several different directions. It is notewor-
thy that each new plant gene, protein, or metabolite discovery may proffer sev-
eral applications for agricultural, food, or pharmaceutical industries. These studies
not only focus on the above topics but also utilize (1) genetically modified organ-
isms (GMOs), (2) molecular farming techniques, (3) sustainable agriculture strate-
gies, (4) production of green energy crops, (5) development of biological control
strategies that can replace or reduce the use of toxic pesticides via integrated pest
management schemes, (6) development of life-support systems in space, and (7)
development of plant-derived products for use in medicine. These are topics that
constitute the basis for recent advances in plant biotechnology. The current state
of plant biotechnology research, using a number of different approaches, includes

over other tomato cultivars: first, it has a longer shelf life in storage, and second, the
fruit of this tomato could be left on the plant until optimally ripe. Because of these
attributes, FlavR Saver tomatoes are sold for premium prices.
Another successful marketing initiative was concerned with oilseed crops.
Canola-producing laurate is the world’s first oilseed crop that has been genetically
engineered to modify oil composition. Similarly, Calgene isolated the gene responsi-
ble for laurate production from the California laurel (Umbellularia californica) tree.
This gene was then engineered into canola (Brassica napus and B. rapa), resulting
in the production of oil containing approximately 40% laurate – a fatty acid that is
found in the seed oils of only a few plant species, mostly coconut and palm ker-
nel oil from tropical regions. Laurate possesses unique properties, which make it
desirable in edible and industrial products. Lauric oil is ideal for use in the soap and
detergent industries, as it is responsible for the cleansing and sudsing properties of
shampoos, soaps, and detergents.
Other examples of transgenic agricultural crops include many plants, such as
potatoes with more starch and less water to prevent damage when they are mechan-
ically harvested, crops with low saturated oils, sweet mini-peppers, modified lignin
in paper pulp trees, pesticide-resistant plants, and frost-resistant fruits.
One of the important directions in plant biotechnology is the introduction of
genetically engineered organisms (GMOs) to the market. This is based on a desire
by consumers for more tasty and more healthy foods. It is also based on a prefer-
ence for products grown without using pesticides or other soil additives. However,
the choice of companies to keep the public ignorant of these genetic changes led to
a great scare in the public once people found out what was going on. It would have
been better if companies had informed the public prior to releasing any GMOs. As
a consequence of these events, the regulatory requirements and safety assessment
studies are far greater, not only in the United States but also worldwide.
An improvement in the quality or the composition of animal products has also
been achieved through modern plant biotechnology. This has resulted in increased
feed utilization and growth rate, improved carcass composition, improved milk pro-

to the production of biopharmaceuticals.
In summary, plant biotechnology concentrates much attention on the complex-
ity and interrelatedness of plant biology, with such targets as agricultural and
pharmaceutical biotechnology. Needless to say, and subject to clarification of cer-
tain ethical and public acceptance issues, plant biotechnology is also set to make
an indelible contribution to human health and welfare well into the foreseeable
future.
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and to introduce novel compound production into specific plants. Recent achieve-
ments have also been made in altering various metabolic pathways by use of spe-
cific genes encoding biosynthetic enzymes or genes that encode regulatory proteins.
Gene and metabolic engineering approaches are now being used to successfully
achieve highest possible levels of value-added natural products in plant cell cul-
tures. Applications through functional genomics and systems biology make plant
cell biotechnology much more straightforward and more attractive than through pre-
vious, more traditional approaches.
A. Kirakosyan (
B
)
University of Michigan, Ann Arbor, MI 48109-0646, USA
e-mail:
15
A. Kirakosyan, P.B. Kaufman, Recent Advances in Plant Biotechnology,
DOI 10.1007/978-1-4419-0194-1_2,
C

Springer Science+Business Media, LLC 2009