William G. Hopkins Plant Biotechnology PDF

Plant
Biotechnology
Ethnobotany
Forestry
Horticulture
Photosynthesis and Respiration
Plant Biotechnology
Plant Cells and Tissues
Plant Development
Plant Diversity
Plant Ecology
Plant Genetics
Plant Nutrition
Plant Biotechnology
William G. Hopkins
Plant Biotechnology
Copyright 2007 by Infobase Publishing

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Hopkins, William G.
Plant biotechnology / William G. Hopkins.
p. cm. (The green world)
Includes bibliographical references and index.
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Introduction vii
1 What Is Biotechnology? 2
2 The Early Days of Biotechnology 12
3 Plants and Biotechnology

Before Recombinant DNA 30
4 DNA, Genes, and Protein 48
5 Engineering Plants 68
6 Genetically Modified Plants

From Herbicides to Vaccines 84
7 Putting Genetically Modified

Organisms in Perspective 104

Glossary 127
Bibliography 133
Further Reading 134
Index 136
By William G. Hopkins
Have you thanked a green plant today? reads a popular bumper sticker.
Indeed we should thank green plants for providing the food we eat, fiber for
the clothing we wear, wood for building our houses, and the oxygen we breathe.
Without plants, humans and other animals simply could not exist. Psycholo-
gists tell us that plants also provide a sense of well-being and peace of mind,
which is why we preserve forested parks in our cities, surround our homes
with gardens, and install plants and flowers in our homes and workplaces. Gifts
of flowers are the most popular way to acknowledge weddings, funerals, and
other events of passage. Gardening is one of the fastest growing hobbies in
North America and the production of ornamental plants contributes billions
of dollars annually to the economy.
Human history has been strongly influenced by plants. The rise of agri-
culture in the fertile crescent of Mesopotamia brought previously scattered
hunter-gatherers together into villages. Ever since, the availability of land
and water for cultivating plants has been a major factor in determining the
location of human settlements. World exploration and discovery was driven
by the search for herbs and spices. The cultivation of new world cropssugar,
vii
viii Introduction
cotton, and tobaccowas responsible for the introduction of slavery to

America, the human and social consequences of which are still with us. The
push westward by English colonists into the rich lands of the Ohio River
valley in the mid-1700s was driven by the need to increase corn production
and was a factor in precipitating the French and Indian War. The Irish Potato
Famine in 1847 set in motion a wave of migration, mostly to North America,
that would reduce the population of Ireland by half over the next 50 years.
As a young university instructor directing biology tutorials in a classroom
that looked out over a wooded area, I would ask each group of students to
look out the window and tell me what they saw. More often than not the
question would be met with a blank, questioning look. Plants are so much
a part of our environment and the fabric of our everyday lives that they
rarely register in our conscious thought. Yet today, faced with disappearing
rainforests, exploding population growth, urban sprawl, and concerns about
climate change, the productive capacity of global agricultural and forestry
ecosystems is put under increasing pressure. Understanding plants is
even more essential as we attempt to build a sustainable environment for
the future.
The Green World series opens doors to the world of plants. The series
describes what plants are, what plants do, and where plants fit into the
overall scheme of things. Plant Biotechnology traces the development of
biotechnology from prehistory to the present. It shows how plants are
genetically engineered, weighs the risks and benefits of this new technology,
and discusses the present impact and future potential of genetically modi-
fied plants.
What Is Biotechnology?

All problems are finally scientific problems.
George Barnard Shaw (18561950)
Irish playwright
It was late when he returned to the village. The moon had risen,
and the other villagers were asleep in their huts. He had been
hunting for meat and now he was hungry. Searching for some-
thing to eat, he spotted a bowl of grain that had been left sit-
ting beside his hut for several days. It had rained recently and
water had collected in the bowl. The grain was sprouting, but he
took a handful anyway. It had an unfamiliar but pleasant taste,
so he ate the rest of the grain and, to slake his thirst, he drank
the liquor in which it had sat. As he sat resting by the remains
of the cooking fire, he thought of the tales told by the elders in
the villagetales of how their ancestors had moved constantly
from place to place as they gathered the wild grains and of
how there was never more than just enough to eat. He thought
of how they now saved the fattest and healthiest of the grains
and sowed them in the moist ground near the river so they no
longer had to search for grain but simply waited for the crop
to mature. They no longer had to move about, and there was
always more than enough grain to feed the village. And as he
sat and thought, he began to feel strange sensations. His vision
began to blur and he felt dizzy. He thought, I should go into
the hut and sleep, but he had difficulty trying to rise and fell
asleep where he sat.
Beer, Cheese, and Freshly Baked Bread

Is it possible that early man discovered beer in this way? Perhaps,
but we dont really know, because the origins of brewing beer
and other intoxicating beverages have been lost in antiquity. We
do know that beer or a similar form of fermented beverage has
been brewed and drunk by various civilizations for thousands
of years. Archeological evidence tells us that as early as 11,000
years ago, there were agricultural villages established in the
eastern Mediterranean area known as the Fertile Crescent, an
area extending from the flood plains of the Tigris and Euphrates
rivers in what is now Iraq, across Syria, and down the eastern

coast of the Mediterranean to the Nile Valley of Egypt. Among

the crops being domesticated were wheat and barley (used
to produce beer) and grapes (for wine). It seems that bread-
making and brewing were both early technologies associated
with the beginnings of agriculture. It is known, for example,
that the ancient Sumerians were producing a beer made from
fermented, moistened bread more than 9,000 years ago. Wine,
another fermented beverage, was also produced by the Sumeri-
ans as well as the ancient Egyptians.
The production of beer, wine, and bread involves the fer-
mentation of the sugar glucose by a single-celled fungus called
yeast, usually a strain of Saccharomyces cerevisiae. The yeast
converts the glucose (C6H12O6) to ethyl alcohol (ethanol,
C2H5OH) and the gas carbon dioxide (CO2):
C6H12O6
> 2C2H5OH + 2CO2
In the process of brewing beer, the germinating grain (usu-

ally barley) secretes an enzyme called amylase. The amylase acts
on the starch in the endosperm (the nutritive tissue) of the seed,
breaking down the starch into its component glucose, or sugar,
units. The yeast that ferments the glucose is found naturally in
the environment and, in the beginning at least, fermentation
was the result of chance contamination. In other primitive
societies, beerlike brews were made from other starchy plant
products, and the amylase was often provided by chewing some
of the plant material and spitting into the brew. Amylase is
a natural component of human saliva. Wine is a little easier
to come by since the yeasts used to ferment the juices grow
naturally on the surface of the fruit. The first wines would have
been easily produced by simply allowing grape juices to stand
for a few days.
Another early development was the production of cheese
(Figure 1.1). Cheese, like wine, is a means for preserving food
Plant Biotechnology
Figure 1.1 A cheese maker stirs a cauldron of curd and the watery part of milk known
as whey. Cheesemaking is one of the oldest examples of biotechnology.
and bears the same relationship to milk as wine does to grapes.

The origin of cheese is equally obscure, although countless leg-
ends seem to involve a rider setting out on a horseback journey
and carrying with him some milk in a pouch made from the
stomach of a young cow. He later discovers, so the legend goes,
that the milk has turned into a slightly sour mixture of watery
fluid and curds. The reason, as we now know, is that the lining
of a calf s stomach produces rennin, an enzyme that coagulates
the milk proteins (albumin and casein). Cheese was well known
to the Sumerians at least as far back as 6,000 years ago and has
been made wherever domesticated animals produced more
milk than could be immediately used by the people.
Making Organisms Work for Us

What do beer, wine, bread, and cheese have to do with bio-
technology, the topic of this book? The common thread is that
all four involve harnessing living organisms or the products of
living organisms to process food and make a specific product
for humans.
Beer- and winemakers, of course, rely on the production
of alcohol for their characteristic beverages. In early societies,
there was probably little value, other than perhaps ceremonial,
to the production of beerlike beverages. Wine production, on
the other hand, has value as a means for preserving grapes or
other fruits.
The breadmaker is not interested in alcohol production but
takes advantage of the carbon dioxide gas to provide texture.
When preparing bread, the dough is kneaded, which causes
the flour proteins, called glutens, to form an elastic network.
It is this gluten network that traps the carbon dioxide bubbles
produced as the yeast ferments the glucose and maltose sugars
present in the flour. When the dough has finally risen and is
ready to bake, it also contains a significant amount of alcohol
(as much as 0.5%), but this is driven off during baking and
contributes to the enticing aroma that we associate with freshly
baked bread.
Coagulation of milk proteins is a natural step in the diges-
tion of milk by calves, young goats, and other young mam-
mals. Humans have taken advantage of the enzymes involved
to process and preserve the milk as food. Although rennin
was formerly obtained from calf stomachs, worldwide cheese
production has outstripped the supply of slaughtered calves.
Fortunately, there are some fungi that produce extracellular
Plant Biotechnology
(secreted into the organisms environment) enzymes, including

rennin, that will coagulate milk. Fungi are now the principal
commercial source of rennin for the cheese-making industry.
The use of living organisms to process foods and make other
products that are useful to humans is what we generally refer to
as biotechnology. Because the word has only recently entered
popular usage, most people think biotechnology is a very recent
invention. The truth is that humans have been using other
organisms to produce new products for a very long time. The
first humans to discover bread, beer, wine, and cheese were, in
fact, the worlds first biotechnologists and we have been eating
the products of biotechnology for thousands of years.
For most of history, biotechnology has focused on process-
ing food for humans and cattle. The silage, or fodder, that
becomes feed for livestock is stored by cattle and dairy farmers
in silos and also is a fermented product. More recently, engi-
neers have used fermentation to produce industrial feedstocks
such as acetic acid and citric acid; drugs such as penicillin;
ethanol for industrial purposes and as a gasoline additive; and
to treat sewage. Engineers use the term bioengineering when
engineering techniques are applied to biological processes, but
for most of us the terms bioengineering and biotechnology are
interchangeable and generally refer to any application of tech-
nology to living systems.
Over the past 30 years, biotechnology has come to mean
something very different in the eyes of the general public.
We hear about genetic modification (GM) and geneti-
cally modified organisms (GMOs), but we also hear about
genetic engineering or recombinant DNA (rDNA). These
new technologies have taken us far beyond just using bio-
logical fermentation to process foods, for we now have the
ability to modify organisms at the most fundamental levels
and make them work for us in ways previously undreamed
of (Figure 1.2).
Figure 1.2 A scientist looks at root growth on wheat plants that have
been genetically modified to resist infection by Fusarium, a type offun-
gus. Scientists typically modify the genes of plants in order to increase
yields or improve the nutritional quality of a plant.
10 Plant Biotechnology
This new biotechnology has unleashed a storm of public

controversy. Proponents see the opportunity to help eradicate
malnutrition, starvation, and genetic diseases. Opposition activ-
ists see scientists playing God and unleashing unspeakable
monsters and ecological havoc. As with most new technologies,
there is both benefit and risk involved with biotechnology and
the truth will lie somewhere between these two extremes.
In the chapters that follow, we will trace the history of tradi-
tional biotechnology and how humans have manipulated micro-
organisms and plants over time. To help us better understand
recombinant DNA technology, we will present an overview of
What's in a Word?
Even though the origins of biotechnology can be traced back thousands of
years, it has only recently become part of the public consciousness. For
example, in the Websters Encyclopedic Dictionary of the English Language
published in 1988, the word biotechnology is not listed. By that same year,
however, the word biotechnology had already become well established as
part of the dictionary of the scientific and academic world. The first geneti-
cally modified plant, a tobacco plant resistant to an antibiotic, had been
created in 1983, and in 1986 the Environmental Protection Agency (EPA)
approved the release of an herbicide-resistant tobacco variety. In 1988, the
National Library of Medicine together with the National Institutes of Health
(NIH) established the National Center for Biotechnology Information, with
an emphasis, of course, on human and animal biotechnology.
Today, less than 20 years later, if you google biotechnology, you will
bring up over 119 million hits. There are thousands of biotechnology
companies around the world, and most major universities have formal bio-
technology courses or programs. A Google search for plant biotechnology
alone will bring up over 15 million hits, and scarcely a day goes by that you
cannot find a reference to biotechnology in major newspapers.
What Is Biotechnology? 11
DNA and genes and look at how this new biotechnology came
into being. We will show how plants are genetically engineered
and how this new technology compares with traditional methods
for producing new food plants. We will dispel some of the myths
surrounding genetic modification and review the present impact
and future potential of genetically modified plants. In the final
chapter, we will examine some of the moral and ethical questions
surrounding genetically modified organisms.
Summary
The discoveries of the fermentation process to make beer and
wine and the use of enzymes from animals to make cheese are all
lost in antiquity, but they harness the activities of living organ-
isms. Using living organisms to process foods and to manufac-
ture other products that are useful to humankind is commonly
referred to as biotechnology. Beginning in the 1980s, however,
the word biotechnology has taken on a whole new meaning.
Most people now understand the term biotechnology to mean
the manipulation of an organisms genes to create genetically
modified organisms (GMOs). This book will trace the develop-
ment of biotechnology from its early beginnings to the present,
explain the science behind biotechnology, and help the reader
make an informed judgment about the role of biotechnology in
the future.
The Early Days of Biotechnology
12
There are science and the applications of science, linked together
as fruit to the tree that has borne it.
Louis Pasteur (18221895)
French chemist and microbiologist
14 The Early Days of Biotechnology
Plant Biotechnology
Scientific Roots of Biotechnology

The origins of biotechnology as a science can be traced back to
1857, when French biologist Louis Pasteur (18221895) discov-
ered that the fermentation of lactic acid was due to the action of
live microbes, or microorganisms (Figure 2.1). Pasteurs discov-
ery not only gave rise to the science of microbiology and con-
tributed to the salvation of the French silk and wine industry, but
it also stimulated a general interest in brewing and other types
of industrial fermentation. Fermentation on an industrial scale
grew during the latter half of the nineteenth century and was used
to produce a number of important commercial products. The
industry represented a marriage of microbiology with chemi-
cal engineering that became known as zymotechnology (from
zymology, the scientific study of fermentation). The term zymo-
technology has since been replaced with the more encompassing
term biotechnology.
The first recorded use of the term biotechnology is credited to
a Hungarian agricultural engineer, Karl Ereky, nearly 100 years
ago. In an effort to modernize Hungarian agriculture in the
early years of the twentieth century, Ereky sought and obtained
funding to support agriculture on an industrial scale. In 1919,
Ereky published a tract with the German title Biotechnologie
der Fleisch-, Fett-, und Milcherzeugung im Landwirtschaftlichen
Grobetriebe (Biotechnology of Meat, Fat, and Milk Production
in Large-scale Agricultural Industry). Erekys enterprise became
one of the worlds largest and most productive meat operations.
His farm covered 50 hectares (110 acres) and turned out over
100,000 pigs a year. We tend to treat factory farms as a recent
North American innovation, but large-scale farms, especially for
pigs, were common in Germany and eastern Europe in the early
twentieth century.
Two manufacturing processes illustrate the state of the art
of biotechnology shortly after the turn of the twentieth century.
In 1911, German scientist C. A. Neuberg showed that significant
14
The Early Days of Biotechnology 15
Figure 2.1 Louis Pasteur was a pioneer in the field of biotechnology.

He is perhaps best known for inventing pasteurization, which is the
process of heating a beverage or other food in order to destroy harmful
microorgansims.
amounts of glycerol could be produced by fermentation of starch.

Four years later, German industry was able to produce more than
12,000 tonnes (a tonne is a metric ton = 1,000 kilograms or 2,200
pounds) of glycerol per year.
At the same time in Britain, a group of organic chemists at

Manchester University began working on the rubber problem.
At that time, Brazil held a monopoly on wild rubber trees,
although British plantations were being established in Malaysia.
With the rise of the automobile industry, a reliable supply of rub-
ber was becoming increasingly important. In order to break the
Brazilian monopoly on rubber, however, the world needed a good
synthetic rubber. The Manchester University group included a
young Russian immigrant, Chaim Weizmann (who would later
become the first president of the state of Israel). In 1912, Weiz-
mann was instrumental in developing a fermentation method to
produce butanol. Butanol was readily converted to butadiene, the
raw material for producing a superior synthetic butyl rubber.
Fortuitously, a second product of the so-called Weizmann
process was acetone. Acetone was another commercially impor-
tant chemical as an ingredient in the manufacture of explo-
sives. In addition, acetone and butanol together could be used
to make isobutyl acetate, the best solvent known for the new
plastic nitrocellulose. Interestingly, nitrocellulose was the film
on which the early stars of Hollywood were immortalized.
Nitrocellulose was also the source of gun cotton, a component
of high explosives. (Yes, the early films produced by Hollywood
were highly flammable!)
And what about all that glycerol being produced by fermenta-
tion in Germany at the same time? When glycerol is chemically
reacted with nitric acid, the product is nitroglycerin. Nitroglyc-
erin, which is extremely unstable and highly explosive, was the
explosive of choice for early miners. In 1867, Swedish inventor
Alfred Nobel discovered that the explosive power of nitroglycerin
could be stabilized by absorbing the liquid on an inert (non-reac-
tive) powder, thus producing dynamite. Dynamite made Nobel
a very rich man and, on his death, he endowed the international
prizes that bear his name. Rubber and explosivesit is not dif-
ficult to draw connections between the direction taken by bio-
technology in the years prior to 1918 and the developing political

climate of the time, which culminated in World War I.
The Weizmann process proved to be significant in many ways,
not just for its economic impact. Most industrial fermentations
up to then were carried out in oak caskscontinuing in the brew-
ing tradition, of courseand did not require aseptic (sterile)
conditions. The Weizmann process, on the other hand, required
that laboratory standards for sterility be maintained on an indus-
trial scale and production was carried out in modern aluminum
fermentation vessels. Weizmanns process sealed a partnership
between microbiology, chemical engineering, and modern mate-
rials that was to dominate biotechnology until the 1980s, when
recombinant DNA came on the scene.
What is Fermentation?
One of the more interesting things about nature is its extreme
conservatism. In spite of their striking differences, organisms as
diverse as fungi, oak trees, earthworms, and elephants all share
many of the same genes and do things, in a metabolic sense at
least, in much the same way. For example, when organisms break
down sugars, fats, and proteins to retrieve energy, the pathway
used is virtually identical in all living organisms. The end result,
however, is different depending on whether or not oxygen is
available. When oxygen is present, this pathway is called cellular
respiration. When oxygen is absent, the same pathway is called
fermentation.
The initial steps of respiration and fermentation, a process
called glycolysis, are the same. In preparation for respiration or
fermentation, complex storage molecules such as starch (plants)
or glycogen (animals) must first be broken down into their com-
ponent glucose molecules. Glucose, a simple sugar made up of six
carbon atoms, is further broken down through glycolysis (Figure
2.2). The net result of glycolysis is that one six-carbon mol-
ecule (glucose) is converted to two three-carbon molecules called
Figure 2.2 Glycolysis is the breakdown of glucose into pyruvic acid

inside cells. The pyruvic acid molecules are broken down further via two
different pathways depending on the presence or absence of oxygen.
pyruvic acid. A small amount of energy is released, and the carbon

atoms in the glucose have been slightly rearranged, but otherwise
not a lot has happened up to this point.
The difference between respiration and fermentation lies in the

fate of pyruvic acid. When oxygen is present, the pyruvic acid enters
a metabolic pathway called the citric acid cycle (or Krebs cycle),
where it is completely broken down into carbon dioxide and most
of the energy is retrieved for use by the cell. This is what normally
happens in your own cells to provide the energy the cells need to
function. In the absence of oxygen, however, the citric acid cycle
shuts down and the pyruvate undergoes more limited changes. This
is what we call fermentation. Depending on the organism and the
conditions of fermentation, a variety of end products are possible.
Lactic acid: The fermentation product in human muscle
when exercising under oxygen debt. Lactic acid is responsible
for muscle soreness, and it is also the fermentation product of
certain fungi and bacteria. Lactic acid can be converted to iso-
prene, which can be used in the manufacture of synthetic butyl
rubber.
Ethyl alcohol (ethanol): The fermentation product of several

fungi, especially Saccharomyces cerevisiae.
Glycerol: Under alkaline conditions, S. cerevisiae produces

glycerol instead of ethanol.
Acetic acid (vinegar): The fermentation product of the bacte-

rium Acetobacter. Contamination of wine with Acetobacter can
be a problem for wine producers because the acetic acid sours
the wine. Acetic acid is widely used as a raw material in the
manufacture of fibers, plastics, and other industrial products.
Processing Food and Drink

In modern systems of classification, fungi are no longer considered
plants, but since the beginnings of biotechnology are so intimately
associated with the fungi, we will consider them as close cousins to
the plants and include them in our discussions. In addition to beer,
wine, and bread, fungi are used in the processing of many different
foods, especially in Asia. As with beer and wine, the fungi are used
to improve the texture, flavor, and nutritional value of foods as well
as to delay spoilage.
In Japan, China, and other Asian countries, a large variety of
foods are prepared from soybeans (Glycine max). These include
tempeh, a solid food prepared by processing soybeans with the
fungus Rhizopus species; sufu, a Chinese cheese prepared from
soybeans with the help of the fungus Actinomucor elegans; and soy
sauce, a condiment prepared by fermenting soybeans and wheat
with Aspergillus oryzae.
In addition to fungi, bacteria have also proven useful in tradi-
tional biotechnology. As noted earlier, bacteria produce acetic acid,
which is used in preserving and flavoring foods and as an impor-
tant industrial feedstock. In Europe and North America, lactic acid
produced by the bacterium Lactobacillus has long been used to
preserve cabbage (sauerkraut) and naturally fermented pickles.
Industrial Production of Fungal Metabolites

We have seen how fungi have been used to process foods, chang-
ing both the flavor and the composition of foodstuffs. Fungi
also produce a wide range of metabolites, or chemical products
of metabolism, that have commercial use on their own. These
products include organic acids, alcohols, antibiotics, vitamins,
and enzymes (Table 2.1). In almost all cases, modern industrial
fermentation methods have made it possible to produce these
products in larger amounts, higher purity, and more often at
lower cost than by direct chemical synthesis.
Large-scale production of chemicals by fungi is usually carried
out in vats or tanks with a capacity of several thousand liters. The
vat is filled with a watery solution of some carbon source, such
as cane syrup or molasses, and supplemented with vitamins and
amino acids to ensure rapid, healthy growth of the microorgan-
isms. The tank is then inoculated with the appropriate organism.
The tank may be aerated, if the culture requires oxygen, or sealed
if anaerobic conditions are required. Either way, actively growing

organisms release a lot of heat, so the culture will be cooled by
circulating water through coils surrounding the tank. Once the
culture has run its course, the cells are separated from the culture
medium and the metabolite of interest is extracted by filtration
and chemical extraction. The culture tanks are often cylindrical
and, with various filters, pumps, and cooling coils, the entire set-
up may resemble a miniature oil refinery. Commercial production
may involve either batch culture, in which the vat is emptied after
the reaction runs its course, or continuous culture, in which the
culture medium flows through the tank and production of the
desired metabolite continues indefinitely. We will have more to
say about the continuous culture method later.
Table 2.1 Commercially Important Fungal Metabolites

Produced by Biotechnology
Metabolite Use
Organic Acids
Citric acid Flavoring ingredient in candies, soft drinks,
medicines; used in inks and the dye industry
Alcohols
Ethyl alcohol Industrial solvent, medicines, raw material
in the manufacture of materials such as ether,
acetic acid, synthetic rubber
Glycerol Glycerin; explosives
Drugs
Penicillin Oral and injectible antibiotic
Griseofulvin Oral and topical antibiotic
(continued on page 22)

(continued from page 21)
Metabolite Use
Vitamins and Growth Factors
B vitamins Nutritional supplement and medical therapy
Riboflavin Nutritional supplement and medical therapy
Gibberellin Plant growth hormone with commercial
applications in floricultural
Beta-carotene Synthesis of vitamin A (provitamin A),
nutritional supplement, and coloring agent
for margarine
Enzymes
Amylase Conversion of starch to sugars prior to
fermentation
Rennet Milk coagulation in the manufacture of cheese
Invertase Hydrolyzes sucrose (table sugar) to glucose
and fructose
Pectinase Pretreatment of fruit juices to remove turbidity;
removal of pectins before concentrating fruit
juices
Proteases Hydrolysis of proteins during food processing
One of the most important organic acids produced commer-

cially from large-scale fungal cultures is citric acid. It is used as a
flavoring in beverages and foods (especially gelatin powders and
soft-drink crystals), desserts, jellies, candies, and frozen fruits. Cit-
ric acid is widely used in pharmaceuticals, effervescent products,
and blood transfusions. It is used in hair rinses, electroplating, and
leather tanning. Citric acid was originally obtained from lemons,
but now virtually all of it is produced industrially by the fungus
Aspergillus niger. Annual production worldwide exceeds several
hundred million kilograms (1 kg = 2.2 pounds).
Still, the largest and most important products of commer-

cial bioengineering are antibiotics, especially penicillin. Penicillin
was discovered in 1929 when Alexander Fleming (18811955)
observed that his cultures of a pathogenic (disease-causing) bac-
terium, Staphylococcus aureus, had been contaminated by a mold.
The mold, growing in a petri dish, was surrounded by a clear
ring where it had killed the bacteria in its immediate vicinity. The
contaminating fungus was identified as a species of Penicillium, so
the active principal was called penicillin. Although penicillin was
recognized as an effective antibiotic, it found little use because its
production was difficult and yields were low. The increased need
for effective antibiotics as a result of World War II stimulated the
search for efficient methods for large-scale production.
Penicillin is produced commercially using strains of Penicil-
lium chrysogenum cultured on a medium such as corn steep
liquor, a by-product of corn starch manufacturing. Corn steep
liquor is a concentrate of water-soluble materials from the corn
grain and is especially rich in nitrogen-containing chemicals that
are necessary for a good yield of the antibiotic. The corn steep
liquor is inoculated with the fungus and incubated for five or six
days, after which the fungus is filtered off and the penicillin is
chemically extracted and purified from the liquor that remains.
A third example of industrial bioengineering is the produc-
tion of ethanol. Ethyl alcohol production as an end product of
anaerobic fermentation in beer and wine production is, as we
have seen, the oldest form of biotechnology. Ethanol also has
many industrial uses as a solvent and, more recently, as a biofuel.
In the early part of the twentieth century, as much as 75% of the
industrial ethanol production was achieved by fermentation and
subsequent distillation of molasses, but molasses is comparatively
expensive and occasionally in short supply. It became cheaper to
produce ethanol from petroleum, which at the time appeared
to be relatively inexpensive and abundant. Now, with increasing
concerns about diminishing oil supplies and a focus on renewable
resources, attention has once more turned to fermentation as a

source of industrial ethanol.
Brazil has led the way in ethanol production because it has
abundant supplies of cane sugar, which is readily fermented. In
North America, the favored substrate (material being fermented)
is corn starch, but the starch must first be treated with enzymes
that break the starch into soluble sugars. Yeasts can then ferment
the soluble sugars into ethanol. Other potential sources of car-
bohydrate for ethanol production include starches and sugars
of sweet potatoes and the cellulose that makes up wheat straw.
These too must be treated with enzymes before beginning the
fermentation process.
Bacteria and fungi are also the source of numerous enzymes
that have commercial application. We have already mentioned
rennin, used in the production of cheese. Protein-degrading
enzymes such as papain are used to clarify beer. Glucose isom-
erase is an example of an enzyme that has given rise to a major
industry. When starch is broken down completely, the product
is the 6-carbon sugar glucose. Glucose isomerase is the enzyme
that plants and many other organisms use to convert glucose to
another 6-carbon sugar called fructose. Beginning in the 1960s,
high-fructose corn syrups derived from corn starch have com-
pletely replaced cane sugar (sucrose) in soft drinks. High-fructose
syrups are also used extensively in the baking, canning, and food-
preserving industries. The reason that high-fructose syrups are
so popular is that fructose is twice as sweet as conventional sugar
(sucrose, or table sugar). The high sweetness of fructose means
that much less sugarand therefore fewer caloriesis required.
The industrial use of enzymes again illustrates the synergies
between biotechnology and chemical engineering. Enzymes are
expensive to produce and use. To perform enzyme conversions by
batch culture can be inefficient and costly. Because enzymes are
organic catalysts, organisms do not need to make large quantities
of them. Instead, they use the same enzyme over and over again in
Figure 2.3 The components of a bioreactor are seen in this illustration.

A bioreactor is a vessel in which microorganisms, cells, or enzymes are
used to break down harmful substances or create useful products.
order to produce large amounts of product. In the early 1960s, it

was found that industry could follow the same strategy. Enzymes
such as glucose isomerase are now sealed to the surface of an inert
bed, such as glass beads or cellulose, to form a bioreactor. Fruc-
tose is then produced continuously simply by passing a stream of
glucose solution through the reactor. This flow-through process
continually renews the supply of substrate processed by the same
enzymes and the reactor produces a continuous stream of effluent
that is rich in fructose (Figure 2.3).
Waste Management and Bioremediation

One area where traditional biotechnology has proven particu-
larly effective is in waste management and remediation of waste
water. Home and municipal septic systems are the classic exam-
ple, where bacteria have long been used to degrade solid wastes.
Small-scale pilot projects have also demonstrated that plants can
also play a role in sewage management. After a primary treat-
ment in a septic tank, the effluent enters an artificial wetland
planted with a mixture of sedges (Carex spp.), reeds (Phragmites
spp.), bulrushes (Juncus spp.), and cattails (Typha spp.). The
wetland provides for additional purification of the wastewater
by aerobic bacteria. The plants serve two primary functions: they
hold the bacteria in place and, through the process of transpira-
tion, maximize the evaporation of water into the atmosphere. If
the effluent contains heavy metals, these may also be removed by
plants that grow in the artificial wetland. We will return to this
interesting property of plants in chapter 3.
Bacteria are now being used extensively to treat industrial
wastes other than sewage and to clean up contaminated ground-
water. The use of living organisms such as bacteria to clean up
environmental sites contaminated with chemical pollutants is
known as bioremediation (bio meaning living and remedia-
tion meaning to correct a fault). The systems used are generally
like that described above for enzymes. The bacteria are cultivated
on an inert bed (activated carbon is commonly used) in a cylin-
drical reactor chamber and the water to be treated is pumped
through from the bottom. The contaminant is broken down by
the bacteria as it passes through the reactor and purified water
comes out the other end. This kind of system is now being used
with denitrifying bacteria for removal of nitrates from industrial
and municipal wastewater and for remediation of contaminated
groundwater.
Other areas where this general approach has proven ben-
eficial include decontamination of soils at decommissioned oil
refineries and removal of perchlorate from groundwater. The

latter is a particularly serious problem in California, Utah,
Nevada, and Texas, where ammonium perchlorate (NH4ClO4)
was used for decades as an oxidizing agent in solid propellants
and explosives. Discharge from manufacturing operations and
from replacement of outdated fuels in military missiles and
rockets was and remains a major source of groundwater contam-
ination. Bacteria are now being used extensively to clean up the
groundwater by breaking the perchlorate down into harmless
chloride and water. Similar innovative strategies are being used
to clean up a wide range of toxic chemicals in various environ-
mental situations (Figure 2.4). Different strains of bacteria such
Bioreactors
A term that has begun to permeate the bioengineering field is bioreactor,
used broadly to indicate any vessel or container where organisms are used
to produce a product. The organism may be microorganisms, plant cells, or
animal cells. In that sense, fermentation vats used to produce citric acid
or penicillin would be considered bioreactors. Indeed, an entire industry
has developed around the design and manufacture of bioreactors using
batch culture or immobilized cells for the production of enzymes, vaccines,
hormones, pharmaceuticals, and a host of other useful chemicals. Most
bioreactors consist of tanks surrounded by pumps and pipes that move
fluids and gases into the reaction chamber, provide cooling, and remove
effluent for downstream chemical processing. Bioreactors may range in
size from small, bench-top devices for laboratory experimentation and
testing up to large industrial versions that process several thousand liters.
The concept of bioreactor has even been extended to include the use of
bacteria to assist in the breakdown of materials deposited into landfills and
the use of genetically modified plants to produce chemicals with industrial
or pharmaceutical value.
as Pseudomonas and Escherichia coli are capable of degrading

hundreds of different toxic chemicals.
Soils near oil refineries are often contaminated with hydro-
carbons, such as unrefined petroleum oil, diesel fuel, or gaso-
line. In one such situation, several bioreactors were used to
decontaminate the soil around a decommissioned refinery near
Lake Ontario, where an area covering 15 acres was contami-
nated with hydrocarbons to a depth of three feet. Nutrients were
supplied to encourage the growth of microorganisms already
present in the soil, and within two years the hydrocarbon levels
were reduced by 97%.
Finally, one of the most well-known examples of bacteria in
bioremediation efforts is the follow-up of the Exxon Valdez oil
spill in Prince William Sound off the coast of Alaska in 1989.
Initially, physical cleaning measures, such a steam cleaning and
rinsing, were put in place to contain and remove large volumes
of oil. The beaches were then treated with nitrogen and phos-
phorus fertilizers to accelerate the growth of oil-degrading
bacteria that lived among the rocks and sand. Although the
process is not yet complete, careful monitoring of the site has
shown that the bacteria have degraded a significant amount of
the oil and have even restored some sections of the shoreline to
pre-spill conditions.
Summary
The origins of biotechnology as a science can be traced back
to Pasteurs discovery that fermentation was caused by micro-
organisms. This stimulated the development of fermentation
on an industrial scale to produce a variety of feed stocks that
supplied the manufacturing industry. The term biotechnology
was first used by Karl Ereky in the early twentieth century in
the context of large-scale agricultural meat and milk produc-
tion. In addition to producing products for the manufacturing
Figure 2.4 This bioremediation system purifies groundwater that has

been contaminated with gasoline. The polluted groundwater is pumped
into a bioreactor that contains oxygen, nutrients, and bacteria. The
microbes degrade the gasoline and the clean water is then pumped
back into the ground.
industry, biotechnology and bioengineering have been used

throughout the twentieth century to produce drugs such as
penicillin, process food and drink, control waste management,
and clean up contaminated soils.
Plants and Biotechnology
Before Recombinant DNA
30
Any sufficiently advanced technology is indistinguishable from magic.
Arthur C. Clarke (1917)
British author and inventor
Plants and Biotechnology
Before Recombinant DNA
Traditional biotechnology has not been limited to applications

involving microorganisms. Even before genetic modification
arrived on the scene, plants had been enlisted to serve human
needs in a variety of ways beyond their traditional role of provid-
ing food and fiber.
Phytoremediation: Cleaning Up With Plants

Have you ever heard of locoweed? As a child, I watched a lot of B-
grade movie Westerns and one thing I learned was that, if you are
a rancher, you dont want your cattle grazing on locoweed! Loco-
weed is the common name given to several species of Astragalus, a
genus in the Fabaceae or pea family. Also known as milk vetch or
poison vetch, many species of Astragalus take up unusually large
quantities of selenium from the alkaline soils of the western plains.
The high selenium content contributes to a disease known as alkali
poisoning or blind staggers in cattle unfortunate enough to graze
on this plantthe cattle literally behave as though they are crazy.
There are many regions where natural geochemical processes
have produced soils that are rich in metals such as nickel, chro-
mium, gold, cadmium, selenium, and arsenic. Normally high
levels of heavy metals would be toxic to plants, just as they are to
humans, yet many plants actually thrive on soils rich in such met-
als. For some plants, the metals are not a problem simply because
the cell membranes surrounding the root cells prevent the metals
from entering the root. Other plants actually take up the metals
and accumulate them to levels that would be toxic to most other
plants (Figure 3.1). In Astragalus, for example, selenium may
account for as much as 10% of the dry weight of the seeds. In soils
that are rich in nickel, some plants may contain 200,000 times
more nickel than plants growing in normal soils.
Many years ago, such plants were known as indicator species,
and prospectors would take the presence of such plants as an early
indication that the soils may have contained a mineral of interest,
such as gold. This was called phytoprospecting. We now call these
32
Plants and Biotechnology: Before Recombinant DNA 33
Figure 3.1 An agronomist examines the roots of a Thlaspi plant in a growth chamber.
Thlaspi and other metal-accumulating plants can be used to remove heavy metal
contamination from soils.
plants accumulator species, which are not injured by high con-

centrations of heavy metals because they sequester (isolate) the
metals with small proteins called phytochelatins. The sequestered
metals are then stored in the large central vacuole of the plant cell,
where they cannot interfere with the cells metabolism.
There has recently been a renewed interest in accumulator
species because these plants may have the potential to assist in
cleaning up soils contaminated with heavy metals as a result
of twentieth-century industrial activities. Using plants to clean

up soils is called phytoremediation (phyto meaning plant and
remediation meaning to correct a fault). The idea is to grow
accumulator species on mine tailings and wastes from paper mills,
for example, where they would extract the heavy metals. Plants
will naturally take longer to do the job, but plants are much more
cost-effective and would not create even more ecological problems
as engineering-based technologies often do. Phytoremediation
would also help to stabilize contaminated sites because the plants
help to control erosion.
An additional benefit of accumulator species is that they begin
the revegetation of barren industrial sites and assist in the recovery
of useful metals. Phytomining, as it is called, has proven effective
in the recovery of both nickel and thallium in demonstrations. In
Selenium and Alkali Poisoning

It is interesting that selenium would poison cattle because selenium is
actually an essential trace mineral for mammalian metabolism and is widely
incorporated into cattle feed. This is because selenium is part of a twenty-
first amino acid. All the textbooks will tell you that proteins are synthesized
from twenty standard amino acids specified by the standard genetic
code. But several enzymes require a twenty-first amino acid called seleno-
cysteine which, of course, contains selenium. It is often the case, however,
that trace elements (those required by organisms in very small amounts)
become toxic when there is too much available. Plants, for example,
require several trace elements, including boron, zinc, manganese, copper,
nickel, and molybdenum. Most trace elements serve necessary functions
as enzyme cofactors, non-protein molecules that must be present for an
enzyme to function. Although plants require very small amounts of these
trace elements in order to function properly, any one of them can become
toxic at relatively low concentrations.
other trials, various species of willows (Salix) have shown prom-

ise for extraction of heavy metals from soils treated with sewage
sludge. The advantage of using plants is that they can be harvested
and burned. The heavy metals remain concentrated in the ash,
which makes their disposal much easier.
Micropropagation: Starting Out Small

Cloning is one of the buzzwords of the new biotechnology (not
to mention medical mystery stories and science fiction), but gar-
deners, farmers, and plant scientists have been cloning plants long
before the first cloned sheep, Dolly, arrived on the scene in 1996.
A clone can be defined as a genetically uniform assemblage of
individuals derived originally from a single organism by asexual
reproduction. That may sound just a bit imposing, but the two
keys concepts here are genetically uniform and asexual repro-
duction. All organisms contain at least two copies, called alleles,
for every gene. During sexual reproductionby seed, for exam-
plethe alleles from each parent are randomly reassorted when
they are passed to the offspring. The result is that, except in the case
of identical twins in mammals, no two offspring resulting from
sexual reproduction have exactly the same genetic constitution.
The offspring are not genetically uniform. Genetic reassortment
is a distinct advantage in the natural world because it is the only
way to ensure that at least some of the offspring have the genetic
constitution to be more competitive when meeting new challenges
in the environment.
In the world of horticulture and agriculture, however, there is
profit to be had in genetically uniform lines that exhibit a particu-
lar trait or traits. It may be size, flower color, yield, or some other
marketable characteristic. It is an advantage for the producer to be
able to provide uniform plants on a predictable basis and that is
possible only if the genetic makeup of the plants can be maintained
without change from one generation to the next. The only way to
maintain a line of genetically uniform individuals, or clones, is to
resort to asexual reproduction. In plants, asexual or vegetative

reproduction, is very common. One often sees clumps or small
groups of poplar trees, for example, that are in fact a single tree.
Each individual arises as an adventitious shoot from the roots
of an original tree that may have started from seed. Each tree in
the group is therefore a genetically identical clone.
Gardeners and plant breeders routinely reproduce plants
vegetatively by simply making cuttings, inducing the cuttings to
form roots, and planting them. Another common method for
vegetative reproduction is a form of biotechnology called micro-
propagation, which uses plant tissue culture (Figure 3.2). Today,
micropropagation is a multimillion-dollar business. Many com-
mon houseplants, fruit trees, and forest trees began life in a test
tube or petri dish. By the early 1980s, growers in the Nether-
lands alone were producing over 21 million plants annually
by micropropagation.
Some 70 years ago, Phillip White gave birth to the science of
plant tissue culture when he successfully maintained cut tomato
roots in vitro. In vitro means literally in glass, but the expres-
sion refers to the culture of organisms in test tubes, petri dishes,
or other laboratory glassware. In tissue culture, a small piece of
plant is cut out and placed into aseptic, or bacteria-free, culture
on a synthetic medium in a flask or petri dish. The medium
usually contains agar, a semisolid gel obtained from certain
species of algae, supplemented with sucrose as a carbon source
plus some vitamins, essential minerals, and hormones. Under
these conditions, the cells begin to divide and grow and form a
clump of undifferentiated cells called a callus. By adjusting the
culture conditions, often by modifying the hormone balance, it
is possible to stimulate the callus to form plantlets with roots and
shoots. Once the roots and shoots are established, these plantlets
can be removed from the flask and planted in the greenhouse,
where they will continue to grow, flower, set seed, and do all the
other things that plants normally do.
Figure 3.2 A plant biologist examines papaya plantlets raised in a petri

dish by micropropagation. The plantlets will undergo further testing to
see whether they will yield flowers, which will then develop into fruit.
All this is made possible by the fact that virtually every plant cell
is totipotentit has the genetic potential to reproduce the entire
organism. All one has to do to make use of this potential is to find
the conditions that allow the potential to be expressed. Even cells
that have matured and already assumed specific functions can be
made to reverse the differentiation process and differentiate along
a different developmental path. Unlike plant cells, animal cells are
not generally totipotent: Most animal cells become highly differ-
entiated early in their development and this differentiation cannot
be reversed. The major exception is animal stem cells, which retain
some capacity to differentiate down different paths. But even stem
cells are not capable of producing an entire animal.
The most important commercial technique for micropropa-
gating plants is known as shoot-tip culture (Table 3.1). One sim-
ply cuts out a small tip of a shoot (called the explant) that includes
the growing region of the shoot (the apical meristem) and a few
of the most recently formed leaves. This explant may be as little
as 23 millimeters long. The explant is first washed with a dilute
solution of household bleach to remove any contaminating fungi
or bacteria. It is then placed on the culture medium under sterile
conditions. The shoot tip will continue to grow and, as it grows,
numerous microshoots will appear. Microshoots arise from small
groups of dividing cells that remain trapped where the young
leaves join the stem. These are called axillary buds. This is the same
Table 3.1 Crops Commonly Propagated by Shoot-tip Culture
Apple (Malus) Peach, Cherry (Prunus)

Carnation (Dianthus) Poplar (Populus)
Chrysanthemum Rhododendron
Grape (Vitis) (Rhododendron)
Kiwi fruit (Actinidia) Rose (Rosa)
Orchids (Cattleya, Cymbidium) Strawberry (Fragaria)
thing that happens on a much larger scale when gardeners prune

plants in order to encourage the proliferation of axillary or lateral
shoots and produce bushier plants.
After two to four weeks, the mass of shoots is divided and
the pieces are transferred to fresh culture medium. This process
is repeated, and each time the cluster of shoots is divided and
subcultured, the number of microshoots increases exponentially.
Through shoot-tip culture, literally millions of genetically iden-
tical microshoots can be cloned from a single desirable parent.
When a sufficient number of microshoots have been generated,
they are transferred to a medium with an appropriate hormone
balance that stimulates root formation. The young plantlets can
then be planted in the greenhouse and grown to maturity.
There are a number of advantages to micropropagation over
traditional methods of plant propagation. One is that exception-
ally large numbers of seedlings can be produced in a very small
amount of space. In the floral industry, it is commonly used to pro-
duce clones of cultivars (varieties under cultivation) that are par-
ticularly popular because of flower color or other characteristics.
The technique is used extensively in the production of forest tree
species for planting in tree plantations and reforestation efforts
because it avoids the tedious process of collecting and germinating
seeds. Moreover, trees with superior characteristics can be cloned
for higher productivity. On the other hand, cloning trees for
commercial pulp and logging industries does reduce the genetic
diversity of the forest, which could have long-term detrimental
effects. Genetic diversity enables some members of a population
to survive stresses that might damage others. In a cloned forest,
however, if the clone turns out to be particularly susceptible to
drought or a new disease, the entire forest is at risk.
Micropropagation is also an effective way to eliminate viruses
and other pathogens. In fact, the first plants to be mass-pro-
duced by micropropagation were virus-free clones of Cymbid-
ium orchids. Potato is another plant that is often troubled with
virus infections. Potatoes are normally propagated vegetatively

by buds, or eyes, on the tubers, and any virus infection is read-
ily carried from one generation to the next. The most effective
way to eliminate potato viruses is by micropropagation of virus-
free lines through shoot-tip cultures.
Micropropagation, like any other technique, is not perfect but
is subject to the whims of nature. In spite of the fact that clones
are supposed to be identical, significant variations can some-
times arise. These variations, which probably involve spontaneous
mutations due to the culture conditions, are known as somaclonal
variations. Most somaclonal variants are discarded, but occasion-
ally one exhibits a particularly useful trait relating to field crops or
an interesting flower color, so all is not lost. The use of somaclonal
variation as a plant breeding technique is described in chapter 7.
Biofuels
It may sound like the stuff of folklore, but if you should hap-
pen to live near a swamp, you may occasionally have seen fires
dancing across the surface of the swamp. What actually causes
these fires is methane gas (CH4). In stagnant swamps, any dis-
solved oxygen is used up rather quickly and, since there is no
significant mixing of water and air, there is little opportunity
for the oxygen supply to be replenished. Under these condi-
tions, anaerobic bacteria, which thrive in the absence of oxy-
gen, will take over. Anaerobic bacteria break down the organic
material in the swamp and, in the process, generate methane
gas. Methane is commonly known as marsh gas or swamp gas.
It is also the principal constituent of natural gas, which is usu-
ally found in association with petroleum deposits.
There are many sources of methane in addition to swamps
and petroleum deposits. The principal sources today are cattle
flatulence, municipal sewage treatment plants, and landfill
sites. We cant do much about the cattle, but most landfill sites,
particularly in urban areas where they have been reclaimed
for parks or housing, are now vented by driving pipes through

the soil cover into the landfill. The pipes allow the methane
to escape into the atmosphere rather than accumulate in
the landfill where it poses a danger to human health. More
recently, we have come to recognize that this methane repre-
sents a potential energy source and the amount of gas that can
be generated in landfills is significant. Consequently, landfills
are now being engineered as giant bioreactors to capture the
methane gas for use as a fuel. Methane generated under these
circumstances is commonly referred to as biogas. Methane
is commonly produced on farms in areas such as silos and
manure storage tanks. It can become a real hazard, as every
year numerous farm workers are killed when overcome by
gases, including methane. Many farmers are now designing
bioreactors in order to recover biogas from agricultural wastes
for use as an on-farm source of energy.
Another source of biofuel that is gaining in popularity is the
oil that many plants store in their seeds. The energy content of
most plant oils compares favorably to that of petroleum-based
diesel fuel. The energy content of sunflower oil, for example,
is 37 megajoules (MJ) per kilogram compared with 42 MJ in a
kilogram of crude oil. Known as biodiesel, some plant oils can
be used directly in farm machinery and other diesel engines.
Most plant oils, however, are triglycerides. A triglyceride is a
fat or oil molecule that has three long carbon chains, called
fatty acids, attached to a 3-carbon molecule called glycerol (or
glycerin) (Figure 3.3). It wont do you much good to simply
pour triglycerides into your tank because the oils would clog
up the fuel injectors and could cause permanent damage to
the engine. Before they can be used as a fuel, triglycerides must
first be processed, which involves separating the fatty acids
from the glycerol. Once the glycerol is removed, the free fatty
acids may be used in place of ordinary petrochemical-based
diesel fuel.
Figure 3.3 A triglyceride consists of three fatty acids attached to a

glycerol molecule. Fatty acids in which all the carbon atoms are joined
by single bonds are said to be saturated (such as the green fatty acid in
illustration). Fatty acids with one or more double bonds between carbon
atoms are classified as unsaturated (red and blue in illustration).
Although diesel engines can run on pure processed veg-

etable oil, it is usually mixed with regular diesel fuel to improve
combustion and performance. According to the U.S. Depart-
ment of Energy, more than 25 million gallons of biodiesel
were produced in 2004 and production in 2005 was expected
to approach 100 million gallons (Figure 3.4). Biodiesel con-
tains little in the way of nitrogen or sulfur compounds, so it is
less polluting than conventional diesel fuel. Biodiesel is also a
renewable resource because it is produced from corn, soybean,
Gasohol: Fuel Salvation or Snake Oil?

Petroleum fuels (natural gas, gasoline, diesel fuel) are the single most
important materials driving the worlds economy. North America has led the
pack in consumption of petroleum fuels but increasing demands are being
made on petroleum supplies as the populations of North America and Europe
increase and the economies of China and the Indian subcontinent continue
to modernize and grow. Alternative supplies of energy are being sought
because the extraction, refining, and burning of oil is directly responsible
for much of the pollution we experience in the developed world. In addition,
it is widely anticipated that oil and natural gas supplies will run out during
the twenty-first century.
One attractive alternative is ethyl alcohol (ethanol), also known as gasohol
when used as a fuel. Gasohol was pioneered in Brazil, where the ethanol is
produced from cane sugar. The sugary juices are expressed from the cane, fer-
mented, and the resulting ethanol is collected by distillation. This is a highly
economical process in Brazil, which has a warm climate and large land area
that can be devoted to growing sugar cane. Moreover, the debris that remains
after the sugary juices are expressed, called bagasse, can be used as a fuel to
supply steam for the distillation and to generate electricity.
The situation is not so simple in North America, where the favored
candidates for conversion to ethanol are starch and cellulose. Both require

enzymatic breakdown to release the sugars for fermentation, which requires
additional energy, and, unlike sugar cane, the residues from corn and wheat
straw have negligible fuel value. Additional energy is required to distill the
ethanol. For the immediate future, most of the energy required to produce
ethanol will likely come, either directly or indirectly, from crude oil and
natural gas or coal-fired generating stations.
However, the hydrocarbons in gasoline contain no oxygen, but ethanol
does. Because ethanol is already partially oxidized, it produces 15% to
25% less energy than an equivalent volume of gasoline. This means that
a vehicle gets lower mileage with ethanol in the tank than with gasoline.
There is concern in some quarters that the energy cost of ethanol produc-
tion coupled with its lower energy content may mean that there is little or
no real energy gain.
A partial solution to the problem of fossil fuel dependency, however, may
be in the wings. A current project eyes sweet potatoes as the carbon source
for ethanol production. Sweet potatoes are rich in both starch and sugars.
This project proposes two parallel fermentations, anaerobic and aerobic.
The first fermentation would be anaerobic and would produce methane gas
(biogas). The methane would then be used as the fuel for the aerobic fer-
mentation and distillation process that separates the ethanol. It would be
effectively independent of fossil fuels and, if successful, could provide the
model for future fuel-ethanol production in North America.
and other oilseed crops such as sunflower and canola. Many

environmentally conscious celebrities, such as singers Neil
Young and Willie Nelson, are encouraging the use of biodiesel
by running their tour buses on the fuel.
In these days of concern over diminishing oil supplies, this
may sound too good to be true. Could there be a downside
to widespread use of biodiesel fuel? How much land do you
think would have to be devoted to crops just to supply the fuel
needs of the United States alone? Lets do a simple calculation,
Figure 3.4 An entrepreneur stands by the truck he uses to collect vegetable oil from
restaurants to make biodiesel fuel. Biodiesel has become increasingly popular with
energy-conscious people over the past few years.
using soybeans as an example. Soybeans are about 20% oil and

a bushel of soybeans will yield about 1.5 gallons of biodiesel
fuel. In 2005, 72 million acres of soybeans were planted in the
United States, with a yield of about 3 billion bushels. At 1.5 gal-
lons per bushel, the entire soybean crop could provide about
4.5 billion gallons of biodiesel fuel. That may sound like a lot
of diesel fuel, but in 2005 farm and highway vehicles alone
consumed approximately 40 billion gallons of diesel fuel. At

best, then, the entire soybean crop could supply a little more
than 10% of the diesel fuel required for farm and highway
vehicles. And that does not take into account the fuel and fer-
tilizer inputs, both obtained from fossil fuels, that are required
to produce the soybean crop.
One solution, of course, would be to grow more soybeans,
but virtually all of the arable land in North America is already
under cultivation, primarily in corn, soybeans, and wheat, to
provide food and raw materials used in manufacturing. The
diversion of soybean and other oilseed crops away from their
traditional markets would also drive up the price of food and
other products. Another solution might be to identify high-oil
crops that can be grown in marginal lands that are not suit-
able for conventional crops, but it is still unlikely that biodiesel
could make more than a small dent in our consumption of
petrochemical-based fuels.
Would You Like Fries or Biodiesel with Your Order?

Processing plant fats and oils to produce biodiesel is easy and relatively
inexpensive, although it does require some corrosive chemicals that must
be handled very carefully. In fact, biodiesel is probably the only vehicle fuel
that can actually be produced at home or on the farm, and many people do
just that. They start by collecting used cooking oils from their local fast-food
restaurant. The fatty acids are separated from the glycerol by stirring the
cooking oils with a methyl alcohollye mixture. After standing for a while,
the mixture separates into two distinct layers, with the biodiesel (or methyl-
ated fatty acids) on top and glycerin on the bottom. An added benefit is that
once the biodiesel has been siphoned off, the glycerol can be saved for use
as soap or a cleaning agent. This is a great way to recycle used cooking oils,
although your vehicle will smell faintly of french fries!
Summary
The role of plants in biotechnology was established long before
genetic modification brought plant biotechnology to the atten-
tion of the general public. Accumulator species of plants are
used to remove heavy metals from contaminated soil, a process
called phytoremediation. Various forms of plant tissue culture
have been used extensively to clone commercially valuable plant
varieties and to eliminate viruses and other pathogens.
Plants have also been used to generate environmentally sound
biofuels. Fuel ethanol is produced by fermentation of cane sugar,
corn, sweet potatoes, and other plants. Biogas, or methane, is
generated by the bacterial breakdown of agricultural wastes
for use as an on-farm energy supply, and plant fats and oils are
processed as biodiesel, a renewable fuel for buses, trucks, and
automobiles.
The advantage of biofuels over fossil fuels is that the plants
or plant oils used in the production of biofuels are a renewable
resource, but the diversion of large amounts of crops into biofu-
els of any kind will compete with traditional markets and drive
up the price of food and other products.
DNA, Genes, and Protein
48
We wish to suggest a structure for the salt of deoxyribonucleic
acid (DNA). The structure has novel features which are
of considerable biological interest.
James Watson (1928) and Francis Crick (19162004)
from Nature, 1953
DNA, Genes, and Protein
Biotechnology, in the sense that most people understand it today,
involves a manipulation of genes, thus tricking plants into pro-
ducing novel proteins. This is a controversial technology for a
variety of reasons, but we cannot make informed judgments about
the technology and its application without at least a rudimentary
understanding of the science behind it. Both the nature of the gene
and the synthesis of proteins are intimately related to the heredi-
tary DNA. Thus, in order to understand how this new technology
works, we should first have a basic understanding of DNA and of
the relationship between DNA and proteins.
A DNA Primer
Deoxyribonucleic acid (DNA) was first isolated from the nuclei of
cells in 1869, but it was not until early in the twentieth century that
it became clear DNA was the hereditary material that passed unique
characteristics from one generation to the next. Chemical analyses
eventually revealed that nucleic acidsDNA and its companion
ribonucleic acid (RNA)were very large molecules constructed
from only five simple building blocks called nucleotides. A nucleo-
tide is composed of three elements: a nitrogen-containing base, a
5-carbon (pentose) sugar, and a phosphate group (Figure 4.1). The
sugar in RNA is called ribose, hence ribonucleic acid. The sugar in
DNA is missing one oxygen atom and is thus called deoxyribose.
The four bases that make up DNA are adenine, guanine, cytosine,
and thymine, while in RNA uracil takes the place of thymine.
Biologists had long known that heredity was controlled by genes
and that genes were in some way related to DNA. Scientists knew
that it was important to understand the structure of DNA because
knowing the structure would lead to further understanding of how
the hereditary material worked in the cell.
The first clue to the structure of DNA came in the 1940s when
Erwin Chargaff analyzed DNA from several different species and
found that, regardless of the source, the DNA always contained
roughly equal proportions of adenine and thymine and that the
50
DNA, Genes, and Protein 51
Figure 4.1 DNA is made out of a basic unit called a nucleotide. Each
nucleotide is made out of a sugar molecule, a phosphate group, and
a base.
proportions of cytosine and guanine were also similar. The puzzle

was finally solved in 1953 by James Watson and Francis Crick, who
published their results in a remarkably brief letter to the scientific
journal Nature entitled The Molecular Structure of Nucleic Acids:
A Structure for Deoxyribose Nucleic Acid. In this letter, Watson
and Crick noted that their structure has novel features which are
of considerable biological interest. Given the impact that their dis-
covery has had on biology, agriculture, and medicine over the past
50 years, this may go down as one of the greatest understatements
in the history of science.
The Watson and Crick model for DNA is often described as a
ladder, twisted about its long axis to form a helix. Because there
are two paired DNA molecules, it is called a double helix. The
sides of the ladder are formed from alternating sugar molecules
and phosphate groups. This is referred to as the sugar-phosphate
The Double Helix

The famous paper describing the structure of DNA by James Watson and
Francis Crick was published in the April 25, 1953, issue of the prestigious
British science journal Nature. At the time, Watson was a postdoctoral fellow
(a recent Ph.D.) from Harvard University working with Crick at the Cavendish
Laboratories in Cambridge, England. Discovering the structure of DNA was
considered to be a holy grail of biology and several laboratories were known
to be working on the problem. It was understood that knowing the structure
of DNA would more than likely reveal to scientists how DNA was able to store
and pass on hereditary information.
One of those working on DNA was Linus Pauling at the California Institute
of Technology. Pauling was already well known for his work on the nature of
the chemical bond and would go on to be awarded the Nobel Prize in Chem-
istry in 1954 for his earlier work on the structure of another complicated
backbone. The rungs of the ladderand the key to the Watson

and Crick hypothesisare the paired bases: adenine (A) is always
paired with thymine (T) and guanine (G) is always paired with
cytosine (C). The bases pair up in these combinations because of
the way they bond to each other. The opposing nucleotides are held
together by weak hydrogen bonds; cytosine and guanine are held
together by three hydrogen bonds but only two bonds are formed
between adenine and thymine. In addition to the bonding, the
shape and the space occupied by the four molecules means this
is the only way they can fit together without distorting the sugar-
phosphate backbone. The consistent pairing of A with T and G with
C also explains Chargaffs earlier data.
There is a second important consequence of the pairing of
A with T and G with C that is fundamental to the way DNA
works. The two halves, or strands, of the DNA molecule are
macromolecule, protein. However, Watson and Crick won the DNA race pri-
marily because they had access to one critical piece of evidence: an X-ray
diffraction pattern of DNA provided by Rosalind Franklin and Maurice Wilkins
of the University College of London. When an X-ray beam is fired at a crystal
structure, the beam is scattered (or diffracted) in a particular pattern, depend-
ing on the arrangement of molecules in the crystal lattice. The diffraction pat-
tern obtained by Franklin and Wilkins indicated that their DNA crystals were
arranged in a helical pattern.
Watson and Crick put this information together with what was then known
about the chemistry of DNAin particular, the very consistent ratios of adenine
(A) plus thymine (T) and cytosine (C) plus guanine (G)and began to build mod-
els as big as themselves from pieces of wire and bits of brass. The one model
in which everything finally fit together was the now-famous double helix.
complementary. Wherever one strand has an A, the comple-

mentary strand will have a T. Where the strand has a G, the
complementary strand will have a C, and so forth. Each strand
of nucleotides also has polarity and the two strands are antipar-
allel. Antiparallel means that when the two strands are paired,
their polarity is reversed. If one strand can be said to run north
to south, the complementary strand runs south to north.
Complementarity provides the key to how DNA is able to rep-
licate itself so precisely during cell division. The hydrogen bonds
that hold complementary base pairs together are relatively weak
chemical bonds, so the two strands can easily be separated. When
DNA replicates itself, an enzyme called helicase simply splits the
molecule down the middle, separating the two strands of paired
nucleotides (Figure 4.2). Once the two strands are separated, the
base sequence in each strand then serves as a template (pattern)
to reconstruct its complementary strand. Free nucleotides pair up
with their complement on the single strand and an enzyme called
DNA ligase connects the adjacent sugar and phosphate groups to
form the new backbone. The result is two exact copies of the origi-
nal double-stranded DNA.
What is a Gene?
DNA is a lot like the hard drive on your computerits primary
function is to store information. Like the information in your
hard drive, the information in DNA is stored in discrete blocks or
addresses. These blocks or addresses are called genes. In a com-
puter, the information needed to perform specific operations is
downloaded into the RAM, which is where the instructions in
the software are actually carried out. In the cell, the information
contained in the gene (the DNA) is downloaded into ribonucleic
acid (RNA) which, in turn, directs the synthesis of the proteins that
make the cell function.
To put it in biochemical terms, DNA is a sequence of nucleotides
and a protein molecule is a sequence of amino acids. A gene is a
Figure 4.2 DNA replication results in the formation of two identical

double-stranded DNA molecules. Enzymes, such as helicase and DNA
polymerase, are essential to the process.
particular sequence of nucleotides within the DNA that specifies the

sequence of amino acids in a particular protein. By controlling which
proteins are produced by an individual cell, the gene controls the
characteristics of that cell and, consequently, the characteristics of
tissues, organs, and organisms. Proteins, and consequently genes,
vary in length, but most genes are approximately 1,000 to 4,000
nucleotides long. The entire complement of genes in an organisms
DNA is called the genome.
Proteins and the Genetic Code

There are more proteins in a cell than any other single organic
molecule, and they perform a vast variety of functions. The big-
gest single class of proteins is the enzymes, proteins that catalyze
biochemical reactions (including the synthesis of other proteins).
A typical cell may contain as many as 3,000 enzymes. Other pro-
teins carry messages between cells or contribute to the structure of
membranes and other cell components.
Proteins are long chains of amino acids. There are 20 standard
amino acids that can be assembled in a nearly infinite number of
combinations to produce this large array of proteins. The amino
acid sequence determines the structure of the protein and the
structure determines its function in the cell. In most proteins, the
amino acid chain twists, coils, and folds back on itself to form an
intricately shaped molecule. The shape of a protein is very sensitive
to the amino acid composition, and a change of a single amino acid
can have a profound effect on the ability of a protein to assume its
proper shape and carry out its function.
One example is herbicide resistance in plants. There is a family
of herbicidesthe triazinesthat kills plants by interfering with
photosynthesis. It does this by binding to a small protein in the
chloroplast. The change of one amino acid in that protein changes
the protein just enough so that the herbicide can no longer bind,
but the change does not impair the normal photosynthetic func-
tion of the protein. So, it takes only a single amino acid change
in one small protein to confer triazine resistance on the plant.

Triazine-resistance is a common occurrence in weeds because this
kind of subtle mutation occurs often in nature.
As noted above, DNA is not directly involved in protein synthe-
sis. Instead, the DNA is unzipped and the information in the gene
is transcribed into messenger RNA (mRNA). That is, rather than
making a complementary DNA strand as in DNA replication, the
DNA template is used to make a complementary strand of RNA.
Messenger RNA is identical to a single strand of DNA except that it
contains a ribose sugar and thymine is replaced by uracil. The RNA,
which now contains a copy of the genetic code, then moves out of
the nucleus into the cytoplasm, where it binds to a protein aggregate
called the ribosome. The mRNA-ribosome complex is now ready
to carry out the DNAs instructions and direct the synthesis, or
translation, of protein (Figure 4.3).
How can a code using only four lettersthe four different
bases in a DNA moleculespecify the assembly of at least 20 differ-
ent amino acids into literally thousands of different proteins? DNA
actually uses a three-letter, or triplet, code based on a sequence of
three nucleotides. Each triplet, or codon, is the code that specifies
one amino acid. The number of possible combinations for com-
bining 4 bases into groups of 3 is 4 cubed (43), or 64, so there are
at least three times as many codons as are necessary to encode only
20 amino acids. In fact, most amino acids are encoded by multiple
codons. In addition, there is also one codon that signals the starting
point for protein translation and three stop codons that signal the
end of translation.
The genetic code is universal: the same code is used by bacteria,
plants, fruit flies, and humans. This means that the gene encoding
human insulin, for example, can be inserted into a bacterium and
the bacterium will transcribe the gene and translate it into insulin
the same way that human pancreatic cells do.
There is, however, a bit more to a gene than just the amino
acid sequence of a protein. For example, the DNA sequence that
Figure 4.3 During translation, transfer RNA (tRNA) carry amino acids
to the cells ribosomes. Each tRNA molecule recognizes a sequence
of three nucleotides, known as a codon, on a strand of messenger
RNA (mRNA). A protein is created when the amino acids are chained
together.
codes for human insulin is over 4,000 bases long. With a code of
three bases for one amino acid, there is enough DNA to code for
over 1,300 amino acids, but insulin is a small protein that contains
just over 100 amino acids. What is the purpose of that extra DNA?
The Genetic Code

Each amino acid in a protein is specified by a sequence of three nucleotides in
the DNA of the gene. When cellular conditions call for the synthesis of a particu-
lar protein, one strand of the DNA is transcribed into messenger RNA (mRNA).
Messenger RNA is a single-stranded nucleic acid that carries the message of
the gene into the cytoplasm, where the protein is actually synthesized. So, the
three-letter codes (codons) for the various amino acids are expressed in terms of
the sequence of nucleotides in the mRNA rather than DNA. This distinction is
important because (a) the nucleotide composition of RNA is complementary to
the coding strand of DNA and (b) where the DNA contains an adenine molecule,
the complementary strand of RNA will incorporate uracil rather than thymine.
As a result, the codons in the mRNA will include U in place of T.
A simplified version of how the code directs protein synthesis is illustrated
in Figure 4.3. First, mRNA binds to a cytoplasmic protein unit called the ribo-
some. Then, transfer RNA (tRNA) binds to a specific amino acid and carries
(or transfers) it to the mRNA. Each molecule of tRNA includes a sequence of
nucleotides called an anti-codon, which is complementary to the codon for the
amino acid that it carries. The anti-codon binds to the codon on the mRNA, lin-
ing up the tRNA in the proper position so that its amino acid can be added to the
growing amino acid chain. Each time a new amino acid is added to the chain,
the ribosome shifts one codon, or three nucleotides, downstream on the mRNA,
preparing it to receive the tRNA for the next amino acid in the sequence.
As shown in the illustration, the codon for the amino acid alanine (Ala) is
GCC, so the anti-codon on the tRNA is complementary, or CGG. Similarly, for
tyrosine (Tyr) the codon is UAU, so the anti-codon is AUA. The start codon, AUG,
is also the codon for the amino acid methionine, so all proteins begin with a
methionine unit.
It turns out that a gene is more like a recipeit contains not only
the list of ingredients (the amino acids), but also the instructions
for making the protein. Sectors of the gene (promoters, introns,
etc.) contain the instructions that tell the cell when to make the
protein, how much to make, and so on.
The Birth of Modern Biotechnology

Bacteria have to contend with viruses just as humans do. Bacterial
viruses, called bacteriophages or simply phages, are little more
than a piece of DNA surrounded by a coat of protein. The phage
infects the bacterium by attaching to the surface of the host cell
and injecting its DNA into the cell. Once inside, the viral DNA
takes over the synthetic machinery of the bacterium and directs the
unlucky host to replicate more viral DNA and protein. Eventually,
the host cell ruptures and the new generation of viruses is released
into the environment.
It is difficult to say exactly when genetic engineering began, but
certainly the discovery of restriction enzymes was an important
first step. Some bacteria are protected from phages because they
contain enzymes that can cut foreign DNA into shorter pieces
that are unable to replicate. Because these enzymes restrict viral
Restricting Restriction Enzymes

You may wonder why a bacterium that produces restriction enzymes doesnt
digest its own DNA. It is because some of the bacterial DNA nucleotides have
been methylated. The addition of a few strategically placed methyl groups
(CH3) at the restriction site prevents the restriction enzymes from digest-
ing the bacterial DNA. Of course, nature is a constant battle with one group
trying to get ahead of the other, so it is not too surprising that some phages
have evolved methylation as a way of gaining an edge over the bacterium
and avoiding being attacked by restriction enzymes in the host cell.
Figure 4.4 Restriction enzymes, such as HindIII and EcoR1, act as

biological scissors. They allow scientists to cut DNA and recombine it
into new configurations.
replication, they were called restriction enzymes. The first restric-

tion enzyme (called HindIII) to be discovered was isolated and
characterized by Hamilton Smith, a researcher at Johns Hopkins
University in 1970. In 1978, Smith shared the Nobel Prize with two
others for their discoveries of restriction enzymes.
There are now hundreds of restriction enzymes available from
a diverse array of bacteria. Each one cuts DNA at a unique point
identified by a particular sequence of base pairs. The enzyme EcoR1,
for example, recognizes the sequence GAATTC and cuts the DNA
between G and A, while HindIII recognizes the sequence AAGCTT
and cuts between the two As (Figure 4.4). EcoR1 has a preeminent
place in the history of bioengineering. In the early 1970s, Paul Berg
at Stanford University isolated DNA from two sources: the bacte-
rium Escherichia coli and a primate virus called SV40. He cut both
samples of DNA with EcoR1 and then mixed the two in a test tube.
The result was a hybrid molecule of SV40 and E. coli DNA. Because
the new DNA was created by splicing together (or recombining)
DNA from two different sources, it was called recombinant DNA
(rDNA). With this experiment, Berg created the first recombinant
DNA molecule, thus making genetic modification possible and
providing the foundation for all of modern biotechnology. Bergs
work was recognized with the Nobel Prize in 1980.
At the same time, Stanley Cohen and Herbert Boyer, also at
Stanford, were studying bacterial plasmids. Plasmids are inter-
esting little pieces of DNA that are found primarily in bacteria.
Plasmids are very small, circular pieces of DNA that are separate
from the normal chromosomal DNA of the bacterium. One of the
principal functions of plasmids appears to be to carry genes that
confer resistance to antibiotics. Cohen and Boyer found that if they
cut plasmids from two different sources with the enzyme EcoR1,
the two plasmids would readily join to form a hybrid plasmid. This
is because when EcoR1 cuts the DNA, it leaves overhanging, or
sticky ends. They are called sticky ends because when any two
pieces of DNA cut by this enzyme are brought together, they natu-
rally anneal, or stick together. The same thing will happen with
a piece of foreign DNAsay from the chromosome of another
organismthat has been cut out with the same enzyme. All pieces
of DNA cut by EcoR1 will anneal with a similarly treated plasmid
to form a hybrid plasmid (Figure 4.5).
Cohen and Boyer then took advantage of another peculiar trait
of bacteriatheir capacity to take up bits of DNA from their envi-
ronment and incorporate it into their own genome. This process is
called transformation, and the bacterium that takes up the foreign
DNA is said to be transformed. Cohen and Boyer found they could
induce bacteria to take up hybrid plasmid DNA. Once inside the
cell, the plasmid (including any newly introduced genes) is repli-
cated normally as the bacteria divide. Under optimal laboratory
conditions, E. coli may divide every 20 to 30 minutes, so that after
Figure 4.5 Restriction enzymes are used to create recombinant, or

hybrid, plasmids. In the illustration above, the restriction enzyme
EcoR1 is used to insert the gene for insulin into a bacterial plasmid.
The recombinant plasmid is incorporated into an E. coli cell by a pro-
cess known as transformation.
8 hours of laboratory culture, a single E. coli will give rise to over

16 million progeny. Each cell contains an exact copy of the plasmid
with the newly introduced gene. This replication of the genes to
produce zillions of exact copies is called gene cloning. (No, zillions
is not an official numerical unit, but it is an easy way to express a
really big number.)
Transformation is not 100% efficient, so there has to be some
method for selecting the cells that were successfully transformed
and contain the desired DNA. For this, Cohen and Boyer devel-
oped another little trick. They used a plasmid that contained
two different antibiotic resistance genes: one gene for ampicillin
resistance (amp R) and one for tetracycline resistance (tet R). The
DNA fragment containing the gene to be cloned was then inserted
at a restriction site within the tet R gene. This disrupts that gene
and prevents the synthesis of the resistance protein. Genes such as
amp R and tet R are called marker genes because they identify, or
mark, the cells that have been successfully transformed.
Using a technique called replica plating, the culture is plated
out on a medium that contains ampicillin. This technique spreads
a dilute bacterial culture on the surface of an agar medium in a
petri dish. After a period of incubation, each live bacterium will
give rise to a small, visible colony of cells but, because the medium
contains ampicillin, only transformed cells having the ampicillin
resistance gene will produce colonies. A sterile pad is then pressed
against the colonies on the ampicillin plate. Some of the cells from
each colony will be picked up by the pad, which is then pressed on
the surface of a second plate containing tetracycline. This transfers
a few cells from each colony to the tetracycline medium in the exact
image of the ampicillin plate. Cells that contain the recombinant
plasmid will form colonies on the ampicillin plate but will not
grow and form colonies on the tetracycline plate. It is then easy
enough to identify which colonies grew on ampicillin but not on
tetracyclinethose are the colonies that have been transformed
with the recombinant DNA.
With these experiments, the stage was now set. We have:

Restriction enzymes that allow scientists to cut DNA in
specific locations and insert new DNA to make recombinant
DNA (rDNA). The new DNA inserted could be a specific
sequence of nucleotides, that is, a gene.
Plasmids that, through the process of bacterial transforma-

tion, provide a vehicle, or vector, for inserting the new DNA
into another organism.
Marker genes that enable the scientists to screen a culture

and select for the cells that have been transformed with the
rDNA.
These experiments heralded the arrival of an entirely new way

to modify cells.
DNA Fingerprinting: An Organisms Bar Code

DNA fingerprinting is one of the latest technologies to help forensic
investigators bring the truth to light. In the world of criminal investiga-
tion, DNA fingerprinting has helped to convict guilty parties and has
led to the release of falsely convicted individuals. Just what is this
new technology?
DNA fingerprinting is a way of looking at the unique signature found
in an individuals genetic makeup, and it is made possible by the use of
restriction enzymes. First, the investigator needs to obtain a sample of
DNA. A milliliter of fresh whole blood is an ideal sample, but the poly-
merase chain reaction (PCR) can be used to amplify the quantity of DNA
if the original sample is very small (for example, saliva on an envelope, a
hair follicle, or a blood stain). Detergents are used to extract and purify
the DNA or it can be mechanically forced out of the cell through a syringe.
The purified DNA is then cut with restriction enzymes.

After the restriction enzymes have done their job, the mixture of DNA
fragments of various sizes must be separated. This is done using a technique
known as electrophoresis. The sample containing the DNA fragments is
applied in a narrow well at one end of thin layer of a gelatinous medium and
an electrical current is applied. The DNA fragments are negatively charged,
so they move through the gel toward the positively charged electrode. The
fragments are sorted according to size as they move through the gel because
their movement is restricted by pores in the gel. Smaller fragments are less
restricted by the pores and so will travel further through the gel than the
larger fragments.
The next step is to visualize the position of the different fragments on
the gel. This is done by first heating the gel to split the double-stranded
DNA into single strands and then transferring the strands to a nitrocel-
lulose membrane. The fragments are permanently fixed to the surface of
the membrane where they can be located with a probe. A probe is a small,
single-stranded fragment of DNA that has been made radioactive and that
contains the complementary code for a specific sequence of bases. The
probe will bind with the DNA on the membrane wherever the complementary
base sequence is found.
Finally, a sheet of X-ray film is placed against the membrane. Because
the probes are radioactive they emit energy that exposes the film. When the
film is developed, the positions of the various DNA fragments are indicated
by a series of black lines that vary by position and intensity (amount), just
like the bar codes on manufactured products. The bar code is the DNA
fingerprint. The image on the X-ray film may include 40 or more bands, and
because different samples are run side by side on the same gel, they may be
compared directly. The odds that DNA restriction fragments from two people
will match all 40 bands, unless they are identical twins, are extremely low.
DNA fingerprinting is not limited to forensic situations or humans. DNA
fingerprinting can be used to establish the relatedness of virtually any organ-
ism, including plants.
Summary
DNA is a relatively uncomplicated, double-stranded molecule,
consisting of only four building blocks called deoxyribonucleo-
tides. The key to DNA structure is that opposing nucleotides
in the two strands pair in a complementary relationship: the
adenine nucleotide (A) pairs only with the thymine nucleotide
(T) and the guanine nucleotide (G) pairs only with the cytosine
nucleotide (C). The geometry of these pairings is responsible for
maintaining the parallel spacing of the sugar-phosphate back-
bone of the molecule as it twists to form a helix.
The complementarity of nucleotide pairing is also the key to
both DNA replication and RNA synthesis. As the two strands
separate, free nucleotides naturally pair with each single strand
to form a new double-stranded molecule. In the same way,
ribonucleotides pair with complementary bases in the DNA
template to form a strand of messenger RNA that moves into
the cytoplasm of the cell where it directs protein synthesis. The
DNA code is based on the sequence of nucleotides, with each
sequence of three nucleotides coding for a specific amino acid in
the protein. The entire sequence of DNA nucleotides that speci-
fies a complete protein is known as a gene.
Limited sequences of DNA, some containing a complete gene,
can be isolated by cutting the DNA with restriction enzymes. Pieces
of DNA can then be recombined in different combinations to form
recombinant DNA (rDNA). Bacteria have the capacity to take up
pieces of DNA from their environment and incorporate it into their
own genome. A bacterium that has incorporated a foreign gene
is said to have been transformed. As the transformed bacterium
reproduces, it produces many copies of that gene, a process called
gene cloning.
Engineering Plants
68
Science can amuse and fascinate us all, but it is engineering
that changes the world.
Isaac Asimov (19201992)
American author and biochemist
Engineering Plants
A noted cookbook writer of the Victorian era began her recipe for
jugged hare (rabbit stew) with the instructions: First, catch your
hare. The recipe for genetic engineering is not a lot different.
Before you can start moving genes around in order to create a
transgenic plant, you have to first identify and isolate the gene of
interest. Random DNA fragments are of little value if you dont
know which fragment contains the gene you want to transfer. It
would be like looking for that needle in the haystack. In fact, if
the restriction enzymes you used to break up the DNA happened
to cut the DNA in the middle of the gene you want, then no frag-
ments will contain an intact copy of the gene.
So, we are now faced with two questions. How do we find and
clone a gene that we want to use to transform a plant? And once
we have the gene, how do we insert that gene into a plant? In
other words, how do we actually go about genetically engineer-
ing a plant?
Gene Hunters
One approach to finding a particular gene is to create a comple-
mentary DNA (cDNA) library; cDNA is made by running RNA
transcription in reverse. First, you have to isolate the messenger
RNA (mRNA) from cells that contain the gene you are interested
in. Then, an enzyme called reverse transcriptase is mixed with
this isolated mRNA. Reverse transcriptase uses the mRNA as a
template to make a single strand of DNA in the same way that
DNA serves as a template for making the mRNA. Remember that
making a copy of mRNA from DNA is called transcription, so
making a copy of DNA from RNA is going the other way; that is,
reverse transcription.
The enzyme reverse transcriptase is isolated from retroviruses.
Most animal viruses contain DNA as their genetic material. When
a virus invades a cell, it takes over the cells genetic machinery in
order to make copies of its own DNA. But there are some viruses,
called retroviruses, that contain RNA instead of DNA. When a
70
Engineering Plants 71
retrovirus invades a host cell, it uses a portion of its RNA to direct

the synthesis of the enzyme reverse transcriptase. The reverse tran-
scriptase then catalyzes the reverse transcription of DNA, using the
viral RNA as a template. Once the DNA has been synthesized, it is
then replicated using the host cell machinery. The viral DNA then
instructs the host cell to produce the other bits and pieces neces-
sary for virus multiplication. The human immunodeficiency virus
(HIV) is a familiar example of an animal retrovirus. Most plant
viruses are also RNA viruses (Figure 5.1).
After the cDNA strand has been synthesized, it must be cloned
and the bacterial colonies grown as described in the previous
chapter. The individual colonies are then stored, usually in a
freezer, and this is the cDNA library. Unfortunately, there is no
card catalog for this library, so you must test each colony until
you find those that contain the gene you want.
There is, however, a unique advantage to using cDNA for
building this library. Because cDNA is made from mRNA, it
ignores inactive genes and represents only genes that were actively
being expressed in the cell at the time the mRNA was isolated.
This means that if you know that the protein you are interested
in was being synthesized at the time (and you have selected the
right restriction enzyme or enzymes), there is a much better
chance that the gene of interest will be located on one of the
cDNA strands.
Another method for obtaining a gene is called reverse
engineering. If you dont know anything about the sequence of
a particular gene, you can often work back from the protein that
it encodes. Most proteins are easily isolated and purified, and the
method for determining the amino acid sequence of a protein
is now a pretty routine laboratory exercise. Once you know the
amino acid sequence, it is relatively easy to synthesize a DNA
strand that encodes for those amino acids in that sequence.
A third technique for coming up with new genes is to alter the
DNA by inducing mutations. A mutation is nothing more than
Figure 5.1 The tobacco mosaic virus (TMV) infects the leaves, flow-
ers, and fruit of many plants, including tobacco. TMV is an extremely
persistent plant virus and has been known to survive for many years in
dried plant parts.
a changeany changein the DNA of an organism. Mutations

occur naturally with a much greater frequency than you might
imagine, although the truth is that most mutations are not
terribly beneficial. Occasionally, however, a mutation appears

that gives the organism an edge and evolution takes a tiny
step forward.
It is not difficult to induce mutations in plants. Mutations can
easily be induced by exposing seeds or very young seedlings to
ionizing radiation or chemical mutagens. One popular chemi-
cal mutagen is ethyl methane sulfonate (EMS)a very nasty and
highly toxic chemical. Of course, if the level of radiation or EMS
dose is too high, the seedlings will probably die from radiation or
chemical poisoning. At lower doses, however, a few seedlings will
survive and can be screened for a new and desirable trait.
There is one more method and that is to find another organism
that has the gene you want. This is one of the most controversial
aspects of genetic engineeringcreating transgenic plants.
Transforming Plant Cells: Protoplasts and Gene Guns

Once you have found the gene you are interested in and have
cloned it, the next trick is to get that piece of DNA into a plant cell.
Unlike bacteria, plants do not spontaneously take up bits of DNA
from their environment. So how, then, does the genetic engineer
transform plants?
To begin with, whole plants are not transformed. Instead, one
transforms either cells in tissue culture (described in the micro-
propagation section in chapter 3) or protoplasts. Protoplasts are
naked plant cells that have been treated with a mixture of enzymes,
including cellulase, that strip away the cell walls and leave only the
protoplasm surrounded by a cell membrane. Removing the cell
wall, however, creates certain problems for plant cells, because they
depend on the strength and rigidity of the cell wall to keep intact.
Without the cell wall, water will continue to diffuse into the pro-
toplast by osmosis and the protoplast will swell until it ruptures.
In order to get around this problem, plant protoplasts must be
maintained in a medium containing a high concentration of solute
such as mannitol, a sugar alcohol. The high solute concentration
Polymerase Chain Reaction

It is much easier to transform a plant or other organism if you have many
copies of a gene to work with. Bacteria can be used to clone or make
multiple copies of genes, but now it is more common to use a method
called the polymerase chain reaction (PCR). A PCR machine is sort of like
a photocopier for genes.
The basis for a PCR machine is a temperature-regulated block that
can both heat and cool very rapidly. The sample chamber in the block is
loaded with a sample of DNA that contains the gene, some enzymes (DNA
polymerase and ligase), and a supply of each of the four nucleotides that
make up DNA. Also in the mix are primers, short pieces of DNA that have
been constructed to bind with the DNA at either end of the gene. Primers
select the target gene to be amplified and help to avoid copying the entire
genome.
The reaction is started by heating the sample to about 95C (200F),
which separates the double-stranded DNA into single strands. The sample
is then cooled to about 60C (140F), but before the two strands can
reunite, the primers get in the way by binding to the DNA. DNA polymerase
then fills in the gaps between the two primers and DNA ligase zips the
newly inserted nucleotides together to form a new DNA strand. There are
now two exact copies of the original DNA. The heating-cooling cycle is
repeated to produce 4 copies, then 8, then 16, and so forth. Every time
the cycle is repeated, the number of copies is doubled; 20 cycles will
produce approximately 220 or more than 1 million copies from a single
starting molecule.
Most proteins are denatured or inactivated at temperatures near 95C
(200F), but the DNA polymerase used in PCR is a special form obtained
from bacteria that are adapted to life in hot springs. You may also have
wondered how forensic scientists can do DNA fingerprinting with DNA from
a single hair, spot of blood, or other microscopic sample. The answer is
that they use PCR to amplify the DNA.
balances the osmotic properties of the surrounding medium with

the osmotic properties of the protoplast so that the protoplast does
not take up excess water. Incidentally, animal cells, which do not
have a cell wall, avoid this problem because they have mechanisms
for pumping water out of the cell to control cell volume. Plant cells
lack such pumping mechanisms.
Protoplasts can be isolated from virtually any plant tissue,
although leaf cells are most commonly used. Most protoplasts are
capable of continued cell division when cultured in an appropri-
ate medium. However, once the enzymes are removed from the
medium, protoplasts will quickly regenerate cell walls. The cells
can then be stimulated to form embryo-like cell clusters and, even-
tually, shoots and roots and intact, fertile plants.
Under the right conditions, protoplasts from two different
sources can be induced to fuse, forming single hybrid cells called
somatic hybrids. The term somatic hybrid refers to hybrids formed
by fusion of vegetative cells rather than normal reproductive cells
(sperm and eggs). Somatic hybridization is an important tool for
combining useful characteristics from different organisms. For
example, many wild species of the genus Solanum have resistance
to major diseases of the potato (Solanum tuberosum). However,
genetic improvement of the potato is hindered because the wild
species are sexually incompatible with the potato and cannot be
crossbred with potato plants in the normal manner. Protoplast
fusion is one way to circumvent pollen sterility or other incom-
patibility barriers and introduce new characteristics into crop
species. Protoplast fusion as a way to improve crops is also being
explored in citrus (oranges and grapefruits) and cereal grains
(rice and wheat) and to produce new floral colors in horticultural
species such as petunias.
Protoplasts are particularly useful to the bioengineer because
the absence of a cell wall makes it easier to insert DNA directly
into the cell. One technique is called electroporation. In this
technique, the protoplasts are suspended in a liquid medium along
with microscopic gold beads that have been coated with pieces of
DNA. The protoplasts are then subjected to a brief pulse of elec-
trical current (typically measured in milliseconds). The electrical
pulse opens up small holes in the cell membrane that allow the
DNA-coated beads to enter the protoplast. The membrane quickly
reseals itself and the DNA remains trapped within the protoplast.
Some of the DNA then becomes incorporated into the host cells
genome and is reproduced along with the rest of the DNA when
the cell divides.
Another way of transforming plant cells is literally a shotgun
approach. In this technique, known as biolistics, the DNA is first
coated on gold or tungsten beads approximately 1 micrometer
(0.000039 inch) in diameter. The particles are then loaded into a
gene gun, which fires them at a suspension of cultured cells (Fig-
ure 5.2). The gene gun was originally developed in the early 1980s
by a group of plant biologists and nanotechnologists (technolo-
gists who work with very small things) at Cornell University. The
particles, fired by a discharge of high-pressure helium gas, travel at
about 400 meters per second. This is fast enough to penetrate the
cell membranes and carry the DNA into the cells but not so fast
that the discharge destroys the cells. Believe it or not, in the early
days of the gene gun technique, some laboratories actually used
modified 0.22-caliber rifles! The gene gun technique has been par-
ticularly useful for transforming corn or maize (Zea mays) cells,
which do not lend themselves to protoplast-based techniques.
Natures Own Genetic Engineer

The most widely used vector for introducing foreign genes into
plants takes us back to bacteria and plasmids. A curious patho-
logical condition that affects many species of plants is a cancerous
growth that commonly appears on stems (Figure 5.3). Perhaps you
have seen one in a local garden. This growth, called crown gall,
shows us that nature discovered genetic engineering long before
we did.
Figure 5.2 A gene gun can be used to inject foreign DNA into a plant
cell. The foreign DNA is coated on metal beads and then fired at a col-
lection of plant cells. Once inside the cell, the DNA releases from the
bead and becomes incorporated into the plant chromosome.
Crown gall is caused by a common soil bacterium called Agro-

bacterium tumifaciens (we will call it Agrobacter, for short). The
genes that enable Agrobacter to cause crown gall are not par-
ticularly unusualthey are genes that encode enzymes for the
synthesis of two naturally occurring plant hormones called auxin
and cytokinin. In the plant, auxin normally causes cells to enlarge
and cytokinin normally stimulates cells to divide. The auxin and
cytokinin genes are not expressed in the bacterium. However, when
the bacterium invades a plant, normally at the site of a wound, it
transforms the plant by integrating the hormone genes into the
DNA of the infected plant cell. The transformed plant cell then
Figure 5.3 A crown gall is the result of an infection by a common

soil bacterium, Agrobacterium tumifaciens. The unique properties
of this bacterium have made it a useful tool in genetic engineering
of plants.
synthesizes higher-than-normal amounts of the two hormones.

Overproduction of auxin and cytokinin causes the infected cells
to enlarge and divide more rapidly than normal and leads to an
uncontrolled cancerous type of growth. The bacterium Agrobacter
is thus a natural genetic engineer.
Scientists have learned to use the engineering skills of Agro-
bacter as an efficient way to introduce new genes into plants. The
bacterium carries the hormone genes on a Ti (tumor-inducing)
plasmid. The first step is to disarm the plasmid by removing the
hormone genes. A bacterium carrying the disarmed plasmid can
still invade plant cells, but without the hormone genes it is unable
to cause tumors. The second step is to replace the hormone genes
with a foreign gene of choice. When the bacterium carrying the
engineered plasmid infects cells of the target plant, the new gene
becomes incorporated into the host cell DNA and is expressed
along with all of the other host cell genes.
Agrobacter is most commonly used to transform disks of tissue
cut from leaves, where the bacteria infect the wound cells at the
edge of the disk. Once transformation is complete, the bacteria are
killed off with an antibiotic and the leaf disks can be manipulated
in culture to produce shoots and roots. Unfortunately, Agrobacter
infects only dicotyledenous plants (plants with two cotyledons or
seed leaves in the seed) such as beans and peas. This means that
plants with only one cotyledon (monocotyledonous plants), such
as corn and wheat, are most commonly transformed by using the
gene gun technique.
Herbicide Tolerance: How Genetic Engineering Works

You may have heard of the herbicide Roundup. Plants engi-
neered to tolerate (or resist) this herbicide have received a lot
of publicity and are probably the most widely known example
of GMOs worldwide. Plants engineered for herbicide tolerance
were the first genetically engineered plants to be released for two
reasons. First, herbicide tolerance usually involves only one gene,
so the engineering is relatively simple. Second, as we will see in

chapter 6, there appeared to be both an obvious benefit and a ready
market for the product.
Roundup is a trade name for a chemical compound called
glyphosate, an herbicide that kills plants by blocking the synthesis
of the so-called aromatic amino acids: tryptophan, phenylalanine,
and tyrosine (Figure 5.4). Without those amino acids, the plant
is unable to synthesize proteins and it dies of protein starvation.
Glyphosate acts by inhibiting the activity of an enzyme, enolpyr-
uvyl-shikimate-3-phosphate synthase (EPSPS), that catalyzes a
critical step in the synthesis of those three amino acids. Scientists
looked at EPSPS in a variety of species and found a variant in
petunia cells that was several times less sensitive to glyphosate
inhibition than the normal enzyme. Using rDNA technology,
they coupled this enzyme to a particularly active promoter (the
piece of DNA that activates or turns on the gene). Plants that are
transformed with this new combination of gene and promoter
will overexpress the gene for the more tolerant enzyme. The term
overexpression means that the cells produce higher-than-normal
amounts of the messenger RNA for the resistant enzyme. Cells with
more of the resistant enzyme will naturally tolerate higher doses of
the enzyme inhibitor.
Another example is the herbicide glufosinate, marketed under
the trademark Liberty. Glufosinate also inhibits an enzyme, but
this enzyme is involved in a complex set of reactions that incorpo-
rates nitrogen into amino acids. Plants take up nitrogen from the
soil in the form of nitrate (NO3-) and convert it to ammonium
ion (NH4+) which, under normal circumstances, is incorporated
immediately into amino acids. Glufosinate blocks this last step,
leaving the ammonium ion to accumulate in the cells. Unfor-
tunately, free ammonium ion is highly toxic and it doesnt take
too much to kill the cells. Here, a fungus comes to the rescue:
Streptomyces hygroscopicus is a common soil fungus that secretes
a chemical inhibitor, or fungicide, called bialaphos. But how do
Figure 5.4 Two test plots of soybeans are pictured side by side. The soy-
beans treated with Roundup herbicide (right) show less weed growth
than the soybeans that have not been treated with Roundup (left).
organisms protect themselves against toxic substances that they

themselves secrete? S. hygroscopicus resolves this problem by also
producing an enzyme that deactivates bialaphos. Fortunately, at
least for genetic engineers, this enzyme also deactivates other
chemicals, such as glufosinate, that have a chemical structure simi-
lar to bialaphos. This means that plants engineered with the fungal
gene for the enzyme may continue to take up glufosinate but will
deactivate the herbicide before it can do any damage to the plant.
The question of human toxicity naturally arises when discuss-
ing pesticides in general and extensive use of herbicides such as
glyphosate and glufosinate in particular. Glyphosate and glufos-
inate are not generally toxic to animals and humans because they
target specific enzymes that are not present in humans and other
animals. While humans and other animals make some amino
acids, they do not have the enzyme EPSPS and, consequently, do
not make the aromatic amino acids. As a result, these so-called
essential amino acids must be present in our diet. Nor do we get
our organic nitrogen from nitrate, as high nitrate levels are toxic
to humans, especially infants. We get our nitrogen from organic
compounds in our diet.
Keep in mind, however, that the same is not necessarily true
of other herbicides. The common lawn weed killer 2,4-D, for
example, is a chlorinated hydrocarbon, a family of highly reactive
chemicals that are known to have nasty effects on human genes
and metabolism. On the other hand, even though glyphosate and
similar chemicals are considered relatively safe, common sense
tells us to always treat all synthetic chemicals with caution.
Summary
The first step in producing a transgenic plant is to find the
gene for a trait that you want to insert into the plant. There are
several ways to obtain a gene. The first is to isolate messenger
RNA and, using the enzyme reverse transcriptase, construct a
complementary DNA (cDNA) library. Another method is to

reverse engineer DNA, based on the amino acid sequence of
the desired protein. A third method is to induce mutations by
treating seeds with ionizing radiation or chemical mutagens.
Inserting the cloned genetransforming plant cellsis
accomplished by treating protoplasts (naked plant cells) with
a brief pulse of electricity, by shooting the gene into pro-
toplasts with a gene gun, or by enlisting the aid of natures
own genetic engineer, the crown gall bacterium (Agrobacterium
tumifasciens).
Engineering herbicide tolerance in plants involves inserting
genes that encode a less sensitive version of a critical enzyme or a
gene that encodes a protein that deactivates the herbicide before
it can interfere with a critical metabolic process.
Genetically Modified Plants
From Herbicides to Vaccines
84
whoever could make two ears of corn, or two blades of grass,
to grow upon a spot of ground where only one grew before,
would do more essential service to his country, than the whole
race of politicians put together.
Jonathan Swift (16671745)
Anglo-Irish author of Gullivers Travels
Genetically Modified Plants
From Herbicides to Vaccines
In the previous chapter, we saw how easily plants are transformed,

so it may not be too surprising that most of the first genetically
engineered products to enter the market involved agriculture.
Crop plants engineered for resistance to herbicides, insects, and
disease have been widely accepted by farmers around the world.
Other plants have been engineered for enhanced food quality
and nutrition. Anticipated products include crop plants better
able to withstand the stresses of drought, flooding, salty soils,
and low temperature. Also on the horizon are plants engineered
as bioreactors to produce vaccines, plastics, and other useful
consumer products.
Herbicide Tolerance
Herbicide tolerance was one of the first traits to be engineered in
plants because it involves only a single gene and there appeared
to be a ready market for the product. Glyphosate tolerance is a
good example. Unlike most other herbicides, when glyphosate
is sprayed on the leaves, it rapidly moves to the roots. Because it
kills the roots as well as the aboveground portion of the plant,
glyphosate is particularly effective against perennial plants,
such as dandelions. This is one of the reasons for the immense
popularity of glyphosate herbicides.
What is the value in having genetically modified herbicide-
tolerant crops? The value in herbicide tolerance is most directly
for the farmer. Left unattended, weeds reduce crop yields by $12
billion annually or more. Additional billions are spent every
year attempting to control weeds. In normal practice, weed
control involves several passes over the field with different her-
bicides because various weeds germinate at different times. But
once the crop itself has germinated, spraying for weeds without
damaging the crop plants themselves is tricky at best. Thus, the
traditional approach requires complex mixtures of herbicides
and multiple sprayings with a consequent heavy chemical load
on the soil. When the crop plants themselves have the capacity
86
Genetically Modified Plants: From Herbicides to Vaccines 87
to resist herbicides, the weeds can be killed with a single pass

after the crop has emerged, without any lasting damage to the
crop plants. The result is that less diesel fuel is consumed, fewer
herbicides are sprayed on the fields, and the farmers costs go
down. A recent study by a British firm, for example, surveyed
18 countries and found that, by reducing the number of trips
across the field for spraying, genetically modified crops have
saved farmers 475 million gallons of fuel over the last nine
years and reduced pesticide use by 14%. In the end, consumers
benefit as well because of the reduction in greenhouse gas emis-
sions and lower pesticide load on the environment.
The Mystery of White Petunias

Dr. Rich Jorgensen, a plant biologist at the University of Arizona, had the
idea of using recombinant DNA to develop new colors in petunia flowers.
In the original experiment, extra copies of the gene for purple pigment were
inserted into petunia plants. The expectation was that the extra genes would
cause the petals to make more pigment than normal and thus produce flow-
ers with a more intense purple color. Unexpectedly, the transgenic plants
produced white flowers instead!
Jorgensen eventually discovered that the inserted genes produced an
unusual double-stranded messenger RNA rather than the single-stranded
messenger RNA that plants normally produce. But remember that plant
viruses are RNA viruses and their RNA happens to be double-stranded.
Apparently, petunia plants have a mechanism that allows them to recognize
viral RNA and destroy it. When the transgene produced double-stranded
RNA, the cell apparently mistook it for virus RNA and set about destroy-
ing it. However, because the cells own gene and the transgene produce
RNA with the same base sequence, the plants defense system could not
discriminate between the two and the RNA produced by the normal gene
was destroyed as well. The gene for purple color remained intact, but

the messenger RNA that it normally produces failed to accumulate. With
no intact messenger RNA left in the cytoplasm, the cell was unable to
make any of the purple pigment. Jorgenson called this phenomenon RNA
interference (RNAi).
It did not take long for scientists to recognize that RNAi was not a prop-
erty solely of plant cells but could be used to explain similar phenomena
in organisms from nematodes (roundworms) to fruit flies to humans. These
phenomena are now referred to more broadly as RNA silencing because
they prevent the gene from being expressed. It appears that virtually any
gene can be suppressed at will simply by knowing its DNA sequence and
constructing the appropriate double-stranded RNA to trigger the immune
response. RNAi and other RNA silencing techniques have great potential
for treating diseases with a genetic component. RNAi therapy has been
used successfully to treat diseases such as Huntingtons disease, Lou
Gehrigs disease, hepatitis, and breast cancer in mice and age-related
macular degeneration causing loss of vision in humans. And it all started
with transgenic petunias.
Resistance to Insects and Disease

Weeds arent the only problem faced by farmersthey must
constantly fight insects and diseases caused by bacteria and
fungi as well. Crop losses due to insects and plant diseases far
exceed those of weeds, amounting to economic losses of more
than $100 billion and significant losses in food production
(Figure 6.1). Traditional insecticides are not the answer because
they kill beneficial insects and crop pests equally well. Nowhere
is this more obvious than in orchards across North America.
Most orchard growers now find it necessary to import hives of
bees during the pollinating season because natural bee popula-
tions have been decimated by years of widespread insecticide
use. In fact, beekeeping is a growth industry as beekeepers fol-
low the pollinating seasons from one crop to the next. Crops
Figure 6.1 A Colorado potato beetle feeds on its favorite food, a potato leaf. A major
focus of the biotechnology industry is the creation of insecticides that will reduce
the impact of destructive insects, such as the Colorado potato beetle.
engineered to control insects eating on plants could be benefi-

cial because they would target only the insects that feed on the
crop plants themselves. They could possibly even stimulate a
resurgence in natural bee populations.
Genetic modifications to control insects exploit naturally
occurring bacterial poisons called enterotoxins (from the
Greek word enteron meaning intestine). Enterotoxins are
toxic chemicals that act in the gut. One of the most common
enterotoxins is the protein produced by Bacillus thuringiensis
(Bt), a common soil bacterium. When the protein is ingested
by the larvae of susceptible insects, the protein binds to recep-
tors in the insects gut and interferes with the absorption of

nutrients. Sprays containing dried Bt bacteria have been used
for several decades by organic gardeners as a way to control
the Colorado potato beetle and other garden insect pests. But
these products are expensive and they are rapidly inactivated in
Bt Corn and the Monarch Butterfly

You may have heard the claim that corn dusted with pollen from Bt corn could
wipe out monarch butterflies. It began in 1999 when a group of researchers
at Cornell University in New York State reported in the journal Nature the
results of an experiment they conducted on feeding corn pollen to monarch
larvae. After the monarchs migrate north in the summer, they lay their eggs
on milkweed plants. When the larvae emerge, they feed on milkweed as their
primary food source.
The Cornell group reported that larvae of the monarch butterfly grazing on
milkweed dusted with pollen from Bt corn apparently suffered higher mortality
than larvae grazing on milkweed dusted with non-Bt corn pollen. Should this
be a surprise? Not really. The Bt protein is toxic to lepidopteran insects, and
monarch butterflies are, after all, lepidopteran insects. However, this report
was quickly picked up by the news media and promoted as evidence for an
unexpected result indicating that genetic modification would have negative
effects on the environment.
At issue is how the Cornell groups laboratory results extrapolate to
the real world. As it turns out, the answer is not very well. Consider the
following points:
Farmers treat milkweed plants as weeds, so they are not generally

found in the cornfields themselves.
Although milkweed plants do occur in the vicinity of cornfields,

many, if not most, milkweed plants that are potential hosts for
monarch larvae are some distance from cornfields.
the field, so their use in large-scale agriculture is not practical.

However, the gene that encodes for the Bt protein (the protein is
called CryIAb) has been cloned into crops such as corn, cotton,
and potatoes as an effective control against the European corn
borer, cotton boll weevil, and Colorado potato beetle. The engi-
Corn pollen does not travel well. Only 10% travels farther than 3 to
5 meters (9 to 15 feet) from the edge of the field.
Corn pollen is normally shed over a period of 8 to 10 days. For the

pollen to harm the monarch larvae, they would have to emerge and
be feeding during that period. Typically, the feeding periods of mon-
arch larvae do not coincide with pollen shed.
Other studies have shown that for the pollen to be toxic to monarch
larvae, the pollen density must be greater than 135 to 150 pollen
grains per cm2 of leaf surface. The average density on milkweed
plants within 3 m (9 ft) of a corn field is only 20 to 30 grains per
cm2.
The Cornell study itself indicated that, given the option, monarch
larvae prefer to graze on leaves that are free of corn pollen. If a larva
encounters a pollen-coated leaf, it will usually avoid it and
move on.
It is now possible to control the expression of genes in specific

tissues, and new genetically modified lines that produce the Bt toxin
in leaves, but not in the pollen, are coming available.
Does Bt corn pollen place monarch butterflies at risk? Based on all of the avail-
able evidence, it seems that the risk to monarch butterflies in the wild is virtually
nil. This illustrates the fear with which many people approach genetic modifica-
tion and how one should not jump to conclusions until all the facts are in.
neered crops dont need to be sprayed with insecticides because

plants that carry the gene make the protein and the insects
ingest the protein as they graze. Beneficial insects and others
that dont graze on the engineered plants are not affected.
Genetic engineering has also proven successful in protecting
plants against fungi and viruses. Chitin is a major constituent
of fungal cell walls. Many plants, when invaded by fungi, pro-
duce chitinase, an enzyme that degrades chitin and prevents
further growth of the fungus. Plants engineered to produce
chitinase as a normal constituent exhibit increased resistance to
infection by fungal pathogens. In the case of viruses, it appears
possible to actually vaccinate plants against virus infections
by transforming the plant with a cDNA clone for the virus coat
protein. How the presence of the coat protein in the cell invokes
immunity is not known, but the strategy has proven successful
for several crops, including tobacco, tomato, alfalfa, and rice.
Engineering for Enhanced Productivity and Nutrition

Herbicide tolerance was one of the first genetic modifica-
tions to come onto the market for the simple reason that it
was a straightforward trait that involved only a single gene.
Many other desirable plant traits are more difficult to engineer
because they involve multiple genes. For example, one holy
grail for plant breeders and biotechnologists alike is to increase
the total amount of carbon that ends up in the leaves and seeds
of traditional crop plants due to photosynthesis. The amount
of carbon accumulated by the plant, called productivity, is one
of the keys to increasing food production for an ever-expanding
world population. It may sound relatively simple, but increas-
ing productivity through biotechnology is more easily said than
done. Productivity is a complicated process, dependent on the
balance between the amount of carbon taken up through pho-
tosynthesis and the amount of carbon lost through respiration.
Both photosynthesis and respiration are governed by a large
number of genes that interact with each other in complex ways.

The bottom line is that any improvements to plant productivity
through biotechnology are not likely to be achieved until sci-
entists understand much more about which genes are involved
and how these complex pathways interact.
Changing the oils in seeds, however, is another story. The
production of oilseed crops such as canola, safflower, and
sunflower is a large component of the North American agricul-
tural economy. These oils are used extensively as cooking and
salad oils and in manufactured food products. One popular
source of oil is rapeseed, a collection of Brassica species in the
mustard family. The oils of native rapeseed, however, contain
high amounts of glucosinolates and erucic acid. Glucosino-
lates are organic sulfur compounds that are easily converted
to mustard oils, flavor constituents that give the pungent taste
to mustard and horseradish as well as the distinctive flavors
of other members of the same family, such as cabbage, broc-
coli, and cauliflower. If that is not enough to discourage the
use of rapeseed oil, the erucic acid content adds its own dis-
tinctively unpleasant taste. Almost all the worlds rapeseed oil
now comes from a single line of rapeseed in which the content
of both glucosinolate and erucic acid has been reduced to
virtually zero. Known commercially as canola, this rapeseed
was developed in Canada not by genetic modification, but by
traditional plant breeding.
Scientists are now looking to recombinant DNA techniques
in order to further improve canola and other oilseed crops. One
objective, of course, is to simply increase the yield of oil. This
might be accomplished by engineering overexpression of certain
enzymes involved in the biosynthesis of oils. Another objective
is to change the fatty acid composition of plant oils to better
match different food or industrial applications (Table 6.1). For
example, unsaturated fatty acids are considered healthier than
saturated fatty acids, so engineering plants to produce oils with
a higher proportion of unsaturated fatty acids for use in cook-

ing and salad oils would benefit health-conscious consumers.
Soybean, canola, and flax have all been engineered to produce
more polyunsaturated and fewer saturated fatty acids. On the
other hand, oils with a higher proportion of stearic acid (a
natural saturated fatty acid) could be used to solidify marga-
rine and shortenings at room temperature. This would bypass
the need for hydrogenation and the associated generation of
unhealthy trans fats.
The development of golden rice is one of the greatest biotech
success stories. While North Americans and Europeans tend to
favor potatoes, white rice is the staple food throughout much of
Table 6.1 Fatty Acid Composition of Common Plant

and Animal Fats and Oils
Fatty Acid (% of total lipid)
Oil Saturated Unsaturated Ratio (Unsaturated/

Saturated
Safflower oil 8 87 10.9
Corn oil 11 85 7.7
Olive oil 11 84 7.6
Soybean oil 15 79 5.3
Peanut oil 18 76 4.2
Margarine 26 70 2.7
Fish 15 78 5.2
Beef fat 48 47 1.0
Butter 55 39 0.7
Fatty Acids and the Trans-fat Problem

Plant fats and oils belong to a large and diverse group of molecules called
lipids. Lipids are molecules that are soluble in lipid solvents. This may be a
confusing bit of circular reasoning, but it does emphasize that lipids share an
aversion to water (hydrophobic). Fats and oils have a similar chemical compo-
sition; the only difference is that fats are solid at room temperature and oils
are liquid.
The principal components of fats and oils in plants are triglycerides, in which
three long-chain fatty acids are attached to a three-carbon alcohol, glycerol.
Fatty acids are long chains of carbon and hydrogen atoms containing anywhere
from 8 to 22 carbon atoms with an acid group (called a carboxyl group) at one
end. A fatty acid is referred to as either saturated or unsaturated, depending on
the number of hydrogen atoms it contains. A fatty acid is considered saturated
when all of the hydrogen-binding sites are filled and unsaturated when one or
more pairs of hydrogen atoms are missing. Where a pair of hydrogen atoms is
missing, an extra bond (a carbon-carbon double bond) forms between the two
carbon atoms. A fatty acid with more than one double bond is often referred
to as polyunsaturated.
The key to the composition of fats and oils lies in the fatty acid mix that
makes up the triglyceride. Saturated fatty acids, for example, have a higher
melting point than unsaturated fatty acids. Thus, a triglyceride made up of two
or three saturated fatty acids will be solid at room temperaturea fatwhile
oils have a high proportion of unsaturated fatty acids. Another interesting
feature of the double bond is that it can occur in two configurations called cis
and trans. In the fatty acids that occur naturally in plants, the double bond is
always in the cis configuration. When margarine and shortening, both fats, are
manufactured from plant oils, the oils are cooked in the presence of hydrogen
at high temperature and under pressure. This process, called hydrogenation,
saturates some of the double bonds in order to give the product the required
melting properties. Unfortunately, hydrogenation also converts some of the cis
double bonds to trans double bonds, or trans fats. Trans fats have lately been
linked to heart disease and other human health problems.
the world. Unfortunately, polished white rice does not contain

any of the orange pigment beta-carotene, a pro-vitamin that
the body converts to vitamin A. Vitamin A deficiency, which
leads to blindness, especially in young children, is a chronic
problem throughout much of Southeast Asia and other parts of
the developing world. Rice does, however, produce a precursor
to vitamin A. A team of Swiss and German scientists success-
fully modified rice, using genes from daffodils, to convert the
precursor to beta-carotene. The transgenic rice is known as
golden rice because of the color imparted by the accumulated
beta-carotene. The team was also able to add additional genes
that increased the iron content of rice, potentially reducing the
incidence of iron deficiency that affects an estimated 3.7 bil-
lion people worldwide. Golden rice differs from most GMOs
in that it was developed with the assistance of philanthropic
and government agencies rather than private multinational
companies and has been made freely available to rice breeders
and growers.
Nutritional deficiencies can affect farm animals as well.
Of the 20 amino acids that go into making protein, 8 of them
are synthesized only in microorganisms and plants. These
are the essential amino acids, mentioned earlier in chapter 5,
that must be included in the diet of all vertebrates. Corn is a
popular feed for livestock such as cattle, hogs, and poultry,
but corn protein contains exceptionally low amounts of the
essential amino acid lysine. Lysine is expensive to produce,
and it is estimated that the livestock feed industry purchases
$1 billion worth of lysine every year to supplement animal
feeds. High-lysine corn varieties have been available through
conventional breeding for more than 30 years, but yields were
too low to be profitable. One biotech company has genetically
modified high-yielding varieties of corn for high lysine con-
tent and is expected to bring the new varieties into commercial
production by 2007 or 2008.
Molecular Farming
Plants make a lot of strange chemicals. In most plants, a sig-
nificant proportion of their assimilated carbon and energy is
diverted to the synthesis of molecules that have no obvious role
in their growth and development. Many of these molecules have
found use in antiquity as folk remedies, soaps, and essences.
Others have found use as dyestuffs and feedstocks for chemical
industries (gums, resins, rubber, and others) and even more have
found use as therapeutic drugs. Even though the recent trend has
been toward chemical synthesis of drugs designed to target spe-
cific illnesses more effectively, plants traditionally have been the
principal source of therapeutic drugs. With the advent of rDNA
technology, however, plants are being viewed as production
vehicles with the potential to not only stock your local pharmacy
but to produce a variety of other useful molecules as well.
Using transgenic plants as bioreactors to produce useful
molecules is called molecular farming (Figure 6.2) Because
many of these molecules are intended to have use as therapeu-
tic drugs (or pharmaceuticals), the technique is often referred
to as molecular pharming. Most of the therapeutic products
produced in transgenic plants are proteins, such as antibodies
and antigens or vaccinesproducts that have previously been
extracted directly from other animals, cultured in chicken eggs,
or simply not produced at all.
Why engineer plants to produce these products? The answer
is that traditional methods are very costly. Plants, on the other
hand, are easily transformed and they can produce huge quanti-
ties of soluble protein at relatively low cost. Among plants that
are being used for molecular pharming are alfalfa, bananas,
carrots, potatoes, and tomatoes. One of the favored crops for
molecular pharming, however, is tobacco. Tobacco is one of the
easiest plants to transform, it produces a large leaf mass, and,
perhaps most important, it is a non-food crop. The significance
of this last attribute will be addressed in chapter 7. And the
Figure 6.2 A researcher inoculates a plant with an engineered virus that produces
proteins for human therapeutic use. This technique is often used in molecular farm-
ing, also known as molecular pharming.
purified pharmaceuticals are not contaminated with nicotine or

any other nasty tobacco chemicals.
Pioneering work in molecular pharming has been carried out
in the laboratory of C. J. Arntzen, a plant biologist at Arizona
State University. Arntzen began his work in the mid-1980s and by
1992 had cloned the gene for an antigen of the hepatitis B virus
into tobacco. An antigen is a protein that stimulates the bodys
immune cells to produce antibodies that fight off invading organ-

isms. Arntzens group showed that the antigens isolated from the
transgenic plants did indeed invoke an immune response when
injected into mice.
But Arntzen was primarily interested in developing a plant-
based system for producing low-cost vaccines that could be used
to immunize people in developing countries. These are countries
where high cost and logistical problems such as lack of transpor-
tation, trained medical personnel, and refrigeration can thwart
conventional vaccination programs. Arntzen believed that a
more cost-effective method would be an oral vaccine that could
be delivered in a locally grown food product. So, he switched
from tobacco to potatoes and showed that the cloned hepatitis
B antigens could also stimulate an immune response simply by
feeding the potatoes to mice. They had successfully produced
an oral vaccine! Prior to this, the only successful oral vaccine
has been the oral polio vaccine, and polio has been effectively
eradicated worldwide.
Arntzen and his group also successfully cloned into potatoes
the genes for the toxin produced by the bacterium Escherichia coli
(E. coli) and the coat protein for the Norwalk virus. Both E. coli
and the Norwalk virus are common agents for severe diarrhea in
humans and are a serious problem in developing countries. The
transgenic potatoes were also successful in invoking immune
responses in mice, so, in 1998, the first human trials were con-
ducted. Eleven volunteers were fed potatoes transformed with
the E. coli antigen with positive results: 10 volunteers showed a
fourfold increase in serum antibodies and 6 showed a fourfold
increase in intestinal antibodies as well. The results of this trial
clearly illustrated the potential for transgenic plants to be used
as production vehicles for edible vaccines. In 2001, the cost of
injecting a patient in remote areas with a conventional vaccine
was estimated to be about $120. The estimated cost for delivery
of an edible vaccine was as low as $0.02.
There is, however, one problem with using potatoes as a vac-

cine delivery vehicle. They must be eaten raw. Cooking, unfortu-
nately, denatures the protein and renders it inactive. While mice
may not object to eating raw potatoes, most humans do, especially
those in countries where potatoes are not a normal part of the
diet. It is likely that other, more palatable plant sources, such as
bananas or tomatoes, would be more acceptable.
In the meantime, scientists around the world were getting into
molecular pharming. In fact, by 2002, there were an estimated 400
plant-derived genetically engineered pharmaceutical products
(drugs and vaccines) in clinical development in the United States
and Canada alone. One example is an Australian group that has
cloned the gene for measles vaccine into tobacco. They are now
trying to clone the gene into rice. Measles is responsible for almost
a million deaths each year and they hope that mixing transgenic
rice flour with breast milk will be a simple and inexpensive way
to vaccinate children in poor, remote communities.
Although production of therapeutic proteins is an important
step forward and captures the imagination, molecular farming
is in no way limited to pharmaceuticals. One of the more inter-
esting applications is the production of biodegradable plastics.
Although corn oil has long been used as a substitute for petroleum
in the manufacture of traditional plastics, the product is no more
biodegradable than those made from petroleum. On the other
hand, there are many species of bacteria that naturally produce
fully biodegradable plastics known by the somewhat daunting
name of polyhydroxyalkanoates (PHAs). In bacterial cells, the
plastic accumulates as large bodies or inclusions, apparently as
a means for storing carbon, in much the same way that plant
cells store carbon as starch and animals store carbon as glycogen.
Plants could also be used to produce these plastics. PHAs are
synthesized from a metabolic intermediate, called acetyl-CoA,
that is found in virtually all cells. Acetyl-CoA is also the primary
building block for fats and oils, so plants produce it in fairly large
Figure 6.3 The cotton plant is a promising candidate for future genetic engineering.
By altering the genes of the cotton plant, scientists may be able to produce a cotton
fiber that has qualities superior to non-genetically engineered cotton fiber.
amounts. Recently, the enzymes necessary to produce PHAs have

been cloned into Arabidopsis (a favorite experimental plant for
plant biologists) and the transgenic plants accumulated PHA
inclusions virtually identical to those found in the bacteria.
One plant that has interesting possibilities is cotton (Gos-
sypium hirsutum) (Figure 6.3). In genetically engineered cotton
plants, the genes for biodegradable plastics were expressed in the
seed hair (fiber) cells. The amount of PHA (in this case, polyhy-
droxybutarate, or PHB) produced was not large, but it was enough
to alter the thermal properties of the cotton fiber. The transgenic

fibers conducted less heat than normal fibers, suggesting that
transgenic fibers could have enhanced insulation properties. One
of these winters you just might be wearing clothing made from a
cotton-polyester blend harvested directly from the cotton field!
Where is the value in engineering plants to produce plastic? As
you know, plastics are a mainstay of our modern consumer econ-
omy and because of that they also represent about one-fifth of the
municipal waste across North America. The plastics made from
petroleum do not break down very readily. Some biodegradable
PHAs are currently being produced by bacterial fermentation,
but fermentation requires large industrial facilities and consumes
substantial amounts of energy. Producing biodegradable plastics
by fermentation is currently about five times more expensive than
the cost of conventional plastics made from petroleum. Plants, on
the other hand, which are known for producing large quantities
of products such as starch and oils, might also be adapted to pro-
duce PHAs on a commercial scale. Since there are many bacteria
capable of using PHAs as a carbon source, PHA-based consumer
products discarded into landfills would be 100% biodegradable.
This would be a real benefit for the environment.
Summary
The first generation of genetically modified plants has focused on
herbicide tolerance and resistance to insects and disease. The pri-
mary beneficiaries of these products are the farmers, who benefit
by reduced production costs. Consumers benefit indirectly by the
reduction of greenhouse gas emissions from farming operations
and lowered pesticide load in the environment.
Another objective of using rDNA in plant breeding is to pro-
duce plants with enhanced nutrition. The improvement of quan-
tity and composition of oils from oilseed crops is one area under
development. It may be possible to change the oil composition to
serve particular needs. For example, increasing the proportion of

stearic acid in plant oils could help to solidify shortenings at room
temperature. This would avoid the need to hydrogenate oils, a
process that creates unhealthy trans fats. Golden rice is another
example of a genetically modified plant with improved nutrition
that could benefit young people in parts of the world where rice
is a staple product.
Molecular farming is the use of plants as bioreactors to pro-
duce a variety of useful molecules. The primary focus is presently
on using plants to produce vaccines and other protein-based
pharmaceutical products. Producing edible vaccines in plants
would drastically lower the cost of delivery and improve access to
immunizations in the developing world. It may also be possible
to engineer plants to produce biodegradable plastics and other
useful products.
Putting Genetically Modified
Organisms in Perspective
104
Every business and every product has risks. You cant get around it.
Lee Iacocca (1924)
American business executive
Putting Genetically Modified
Organisms in Perspective
Perhaps you have seen them in the newsanti-GMO protesters
dressed like a cob of corn or a tomato with a fish head. GMO
supporters might be tempted to laugh at such antics, but it is
important to remember that genetic modification (GM) is a
new technology. There are, no doubt, some protesters who are
simply anti new technology, but for others there are legiti-
mate concerns about the safety of the food they eat and the
impact this new technology may have on their lives and the
environment (Figure 7.1).
Most consumers accept without question foods produced
through what is now called conventional plant breeding. Such
foods are considered natural, safe, and acceptable. Many feel
that GMOs, however, are not natural, safe, or acceptable. GMOs
should be vigorously debated, as the product of any new tech-
nology should be, but the debate must be based on an informed
understanding of the facts. The debate should not be distorted
by scientific misinformation and misinterpretation or, worse yet,
personal invective or political objectives. Informed debate on the
GMO issue can only come about when the public has a broader
understanding of the underlying scientific foundation.
You should now be able to appreciate that the scientific con-
cepts behind genetically modified foods are not all that difficult
to comprehend. But how does rDNA technology stack up against
traditional plant breeding? Are GM foods any more or less safe
to eat than conventionally bred foods? Are GMOs potentially
harmful to the environment? Are there real benefits to GM foods
and other products for consumers in North America as well as
in developing nations?
Conventional Plant Breeding

We can be quite certain that someone who says that they prefer
natural foods does not have in mind going back to collecting
nuts and berries as their distant ancestors once did. What this
statement usually implies is that they do not want some scientist
106
Putting Genetically Modified Organisms in Perspective 107
Figure 7.1 A Greenpeace activist wears a mask and shows a hand full of peas in
front of the European Council building in Brussels, Belgium. Worldwide, many
organizations are calling for governments to restrict or ban the sale of GMOs and
GM-foods out of concern over the safety of these products.
or large multinational company messing with their food. But

how natural is natural?
The truth is that humans have been messing around with
their food for a very long time. We have, in fact, been practicing
genetic modification since the dawn of agriculture some 10,000
years ago. The wheat that goes into your bread and breakfast
cereal is the product of three different species, and the hybrid
corn of today bears little resemblance to its ancestor (a some-

what scrawny tropical grass called teosinte). Gene transfer by
Agrobacterium is not limited to the laboratory: A. tumifascium
is a common soil bacterium that goes about subtly transform-
ing plants on a daily basis. We have all occasionally, if unknow-
ingly, consumed these naturally occurring GMOs for as long as
humans have been eating plants. Along the way, humans began
cross-breeding plants to combine traits and produce crops such
as pumpkins, potatoes, oats, rice, tomatoes, and other food plants
that probably would not have existed as we know them without
human intervention.
Conventional plant breeding begins with selection. A farmer
selects the biggest and best of each years crop and reserves it for
use as seed for the following year. Farmers are practicing a form
of Darwinian evolution, because selection slowly changes the
characteristics of the plant until they may no longer resemble the
original plantyields are higher and the fruit is larger, sweeter,
and tastier. Corn is an excellent example. Under the influence
of human selection, the short floral head of the tropical grass
teosinte, with one or two rows of small seeds, has become the
modern version of sweet corn with a cob 8 to 10 inches long
and 16 to 18 rows of sweet-tasting fruits.
Over time, selection was combined with crossing. Unwilling to
leave the development of foods to the whims of nature, early farm-
ers (the first plant breeders) began transferring pollen from one
plant with superior characteristics to the pistil of another superior
plant. The hope was that at least some of the resulting offspring,
called hybrids, would possess the best traits of each parent. Mar-
quis wheat, until recently the world standard for breadmaking
quality, was developed in the early 1900s by crossing other wheat
varieties and carefully selecting for the best breadmaking seeds.
Hybridization has been developed to a fine art by modern corn
breeders. Hybrid corn is produced by crossing two or more inbred
lines. An inbred line is a genetically uniform variety of plant that
produces true by seed. In other words, the seeds from successive

generations can be planted with no significant changes in their
characteristics. Most hybrid corn seed sold to farmers today is actu-
ally double cross seed produced from four inbred lines (Figure 7.2).
Hybridization has brought many new vegetable cultivars (varieties
under cultivation) to farmers and gardeners. In addition to corn,
hybrid vegetables include tomatoes, melons, cucumbers, cabbage,
carrots, onions, cauliflower, broccoli, peppers, and squash.
Before hybrid crops appeared on the scene, farmers grew open-
pollinated cultivars and traditionally saved a portion of the grain
from the current crop to use as their seed for the next season. In
many cases, the farmers continued to select, perhaps inadvertently,
each time they saved seed. Over time, this practice gave rise to local
populations that were genetically suited to the microclimate of a
particular farm. These populations are called landraces, referring
to a breed or race that is highly adapted to local conditions. One
of the criticisms of GM crops is that they are patented, and farmers,
in their contract with the seed producer, are prohibited from saving
seed. However, most conventional hybrids do not breed true, so
new hybrid seed must also be purchased each year as well or the
performance of the crop deteriorates. While both conventional
hybrids and GM crops have led to increased yields, they both also
tend to decrease genetic variability through the loss of landraces. In
either case, the farmer must weigh the advantages of higher yields
against cost and any other disadvantages. In recent years, numer-
ous far-sighted organizations have sprung up with the purpose
of maintaining open pollinated heritage varieties of vegetables,
such as tomatoes, in order to preserve genetic diversity.
One disadvantage of crossing as a breeding technique is that
each parent in a cross contributes 50% of the genes. For example,
suppose you want to improve the disease resistance of a cultivar
of superior bread wheat by crossing it with a wild relative that has
a much higher disease resistance. Not only does the wild relative
provide the desired disease resistance gene, but there are another
Figure 7.2
Double cross seed
is produced by the
careful breeding
of four different
inbred lines. Two
of the inbred lines
must be detas-
seled in order
to prevent them
from pollinating
themselves.
25,000 or more genes that come along for the ride. Many of these
genes may be undesirablethey may cause the stem to grow long
and weak, reduce the yield, or lower the quality of the flour.
A large amount of the conventional breeders effort is spent
simply getting rid of this genetic garbage. This is usually accom-
plished by crossing the hybrid back to the original commercial
cultivar, a process called backcrossing. This means that all of
the progeny, which may number in the thousands, must be care-
fully screened and those with undesirable traits discarded. The
remaining progeny are again backcrossed to the original culti-
var. This screening-selection-backcrossing routine is repeated
generation after generation until the breeder is at last convinced
that he has eliminated the undesirable traits and has regenerated
the superior bread wheat with the single addition of the desired
disease resistance gene. It may take 1012 generationsmeaning
1012 yearsto eliminate the undesirable genes. Even then, the
new commercial strain will still be contaminated with unknown
foreign genes contributed by the wild relative. It is impractical, if
not impossible, to remove them all.
One advantage cited by proponents of GM crops is that pro-
ducing a transgenic hybrid is far more precise. Recombinant
DNA technology allows the breeder to select a single gene and
insert only that gene into the genome of the commercial cultivar.
The resulting progeny are then identical to the parent with the
exception of that one additional gene. There are no undesirable
or unknown genes carried along, and the new cultivar with the
new trait can be available to growers within two to three years.
Developing New Traits for the Conventional Breeder

Suppose you are a plant breeder and would like to introduce a new
trait, but you are unable to find a compatible wild type or other
plant with that particular trait. If you dont want to resort to rDNA
technology, what are your options? A conventional plant breeder
actually has a variety of methods for creating the gene for a trait of
interest. Earlier, in chapter 5, we mentioned the use of mutations

to generate new traits. Lets use wheat as an example and see how
mutations can be used to advantage.
Traditional wheat varieties carry their grain at the top of long
stems. As breeders succeeded in producing plants with larger
numbers of heavier grains, the plants developed a tendency
to fall over and lie on the ground. This is known as lodging,
a condition commonly precipitated by heavy rains or winds
(Figure 7.3). Lodged plants are a real problem for the farmer.
They are more difficult to harvest, and the grains, now in contact
with the ground, are more susceptible to losses from soil-dwelling
insects, fungi, and bacteria. What is the solution to this problem?
Well, if you have ever tried to break a pencil you must know that
a stubby pencil is much more difficult to break than a long pen-
cil of the same diameter. The same is true of plant stems, so one
solution to the lodging problem would be a wheat plant with a
shorter stem. Great idea, but how do you go about it?
In most cases, stem length is regulated by a natural plant hor-
mone called gibberellin. Plants that make less of the hormone
generally have shorter stems. In fact, genetic dwarfs with reduced
gibberellin levels are quite common in nature. Bush beans or
peas, for example, are shorter than their tall vinelike climbing
relatives simply because they make less gibberellin. Thus, one
way to create a dwarf plant is to induce a mutation that reduces
gibberellin levels. Mutated seedlings can be screened for a desired
trait such as shortened stem length. Those seedlings are then
introduced into a conventional breeding program. Mutation
breeding is a common technique in conventional breeding. Over
the last half of the 1900s, more than 1,400 cultivars, including
several short-stemmed wheat and rice cultivars, have been devel-
oped through induced mutations. These varieties are accepted as
natural foods solely because they are not GMOs.
A variety of other techniques for manipulating genes are
also available to the conventional breeder. Embryo rescue is a
Figure 7.3 The toppling of plants before harvest is known as lodging. These corn
plants became lodged after the roots of the plants were damaged by insects.
technique used to mate two plants, often from different species,

that would not normally mate in nature. The resulting embryos
do not often survive naturally, but can be removed (or rescued)
from the ovary and encouraged to reproductive maturity by cul-
turing them artificially. Haploid breeding is a method in which
pollen or egg cells, which normally have only one set of chromo-
somes, are treated with chemicals that force them to make a copy
of their own chromosomes. The cells, which now have two exact
copies of every gene, can be induced to form plantlets in tissue
culture and grown to sexual maturity.
Finally, when plants are cloned by micropropagation, there
are often odd plantlets that arise apparently by spontaneous
genetic changes (that is, by mutation) in the vegetative cells. To the
micropropagationist, these sports are simply defective plants

to be discarded. Others, however, have recognized that these
genetic changes, now called somoclonal variation (SCV), might
be sources of new and useful traits. SCV has been exploited to
improve growth habit, maturity date, and tuber characteristics
in potatoes. In fact, the popular Russet Burbank variety of potato
arose as a somaclonal variant of the original Burbank variety.
As intrusive as mutational breeding and these other tech-
niques may sound, they illustrate the extent to which conven-
tional plant breeding involves genetic modification but without
involving rDNA techniques.
Food Safety: Would You Eat DNA?

An increasing concern of most consumers is food safety and
whether genetic modification by rDNA techniques compromises
that safety. But in what way could rDNA techniques compromise
food safety? Would it be the new or foreign DNA that is intro-
duced or would it be the new protein encoded by that DNA? And,
for comparison, can we evaluate the safety of conventional foods?
First, lets address the question of foreign DNA and proteins.
In his book Pandoras Picnic Basket, Alan McHughen relates an
anecdote about an anti-GMO activist who stormed out of a meet-
ing declaring that Youll never convince me to eat DNA. A recent
survey indicated that a surprising percentage of the public believe
that natural foods do not contain DNA but that GMOs do. All of
the foods we eat contain DNA because DNA is a constituent of all
living cells. Only highly processed foods such as soy protein, refined
cooking and salad oils, and similar products do not contain signifi-
cant amounts of DNA, but even those might contain traces. Many
natural foods such as buttermilk and yogurt also contain cultures of
harmless bacteria. You cant avoid bacteriathey are everywhere in
our environment. We take steps such as pasteurizing milk to destroy
harmful bacteria in the food we eat, but the bacteria are still there;
they are just dead. So, our diets contain a fair amount of bacterial
Fish Genes and GMO Myths

At the beginning of this chapter, we mentioned anti-GMO activists who dress
as tomatoes with fish heads. This costume is based on the belief that toma-
toes were engineered with genes from a fish, specifically arctic flounder. This
is one of the myths of GMOs that arose probably out of a misinterpretation
by an overzealous and non-critical reporter.
GM tomatoes were the first GMO vegetable to be released for human
consumption in the early 1980s, but they were not modified with fish
genes. The tomato, called the Flavr-Savr, was engineered to delay
softening and prolong shelf life while continuing to develop normal color
and flavor. It was accomplished by removing a tomato geneone that
codes for the enzyme that causes the tissue to softenand reinserting
it backwards. The inverted gene could not be read by the cells genetic
machinery any better than you could read this sentence if it were printed
backwards. (!ees dna flesruoy ti yrT) The engineered tomato made less of
the softening enzyme and the shelf life of the tomato was prolonged.
Another unrelated idea being kicked around at the same time was
that of transferring an anti-freeze gene from the Artic flounder to a crop
plant. The idea was that this gene, which encodes an anti-freeze protein
that helps the fish to survive in icy waters, would also help crop plants
survive unseasonable frosts. In writing the story, the reporter apparently
combined the Flavr-Savr project with the anti-freeze concept. Although
the story has been thoroughly discredited, it has persisted with a life
of its own.
The flounder gene idea was eventually triedwith strawberries, not
tomatoesand it didnt work. This should not be too surprising since fish
and plants are very different organisms. They live in very different worlds
and what works in one will not necessarily work in the other. Moreover,
recent research has shown that winter strains of rye and other plants make
their own anti-freeze proteins, so it is not necessary to look to fish genes
for frost protection in plants.
DNA as well. We also eat lots of protein in the form of enzymes and
structural proteins in our food. But cooking denatures both DNA
and protein. DNA and protein are further denatured by the acids
in our stomachs, and intestinal enzymes break down these mol-
ecules into their basic building blocks, which are absorbed into the
bloodstream. These DNA and protein building blocks are the same
regardless of whether they came from plants, animals, or bacteria.
Some people express concern about eating a pesticide or
toxin when corn or potatoes are transformed with the gene
that encodes Bt protein. Remember that Bt protein is toxic only
to the larval stage of insects in the family Lepidoptera. It kills the
larva by interfering with the absorption of nutrients in the gut.
Bt protein does not affect humans, however, because the human
gut is fundamentally different from the insect gut. The human gut
does not have the receptors that are attacked by Bt protein, and
the strongly acidic environment of the human gut immediately
denatures the protein. The protein is then digested as any other
protein is. Keep in mind as well that Bt is an approved insecticide
for organic gardening. Finally, what is foreign DNA anyway?
You might be aware that recent research has shown that humans
share as much as 70% of their genes (and hence DNA) with earth-
worms and more than 95% with mice!
GM foods are not immune to safety and security problems, but
neither are conventional foods. After all, plants have had billions of
years to develop toxins that discourage creatures such as us from
eating them. For example, the seeds (but thankfully not the fruit) of
apples, cherries, and apricots, as well as some strains of lima beans,
contain cyanogenic glycosides. These are chemical compounds
that release cyanide when the cells are disrupted. The odd apple seed
eaten with a core will probably cause little harm, but a half cup of
dried seeds releases enough cyanide to kill an adult. Potatoes and
tomatoes are in the same family as belladonna, deadly nightshade,
and Datura (jimson weed), all of which contain deadly alkaloids.
The potato alkaloid solanine accumulates in the green plants but not
in the potato tubers themselves. However, potatoes that have turned

green following exposure to strong light will contain elevated levels
of solanine, which is why you should avoid eating green potatoes.
Numerous other foods contain various toxins as well. Here are
just a few examples of toxins that are natural constituents of com-
mon foods:
Banana peels and, to a lesser extent, the pulp, contain sero-
tonin and other chemicals that raise blood pressure in animals.
Kidney beans, soybeans, and peas contain lectins, molecules

that cause red blood cells to clump and in large doses can
interfere with the bodys immune system.
Brassicas (broccoli, brussels sprouts, cauliflower, cabbage, and

others) contain glucosinolates (mustard oils) that are responsi-
ble for the pungent taste of these vegetables. Glucosinolates are
also called goitrogens because they can interfere with iodine
uptake by the thyroid gland, resulting in goiters (an enlarge-
ment of the thyroid gland). This is not a concern if the diet
contains sufficient iodine.
Coffee contains caffeine, an alkaloid that is toxic to the central

nervous system.
Papaya and pineapple contain proteolytic enzymes (enzymes

that break down proteins) that will cause irritation of the
mouths mucous membranes.
Aside from allergenic protein that can lead to anaphylactic

shock in sensitive individuals, peanuts can become contami-
nated with molds that produce deadly aflatoxins.
This is not intended to put you off from eating fruits and
vegetables. What you have to remember is that, except in rare
circumstances, the concentrations of toxins in common food plants
are so low that they can be tolerated without ill effects. We have
also learned to avoid eating plants or parts of plants that produce

unacceptably high levels of toxins. The point is that some plants
do make things that are not necessarily good for you. We assume
a certain level of risk when we eat these foods, but we have learned
that the nutritional value of fruits and vegetables far outweighs the
risk of death by starvation. The risk is manageable, so we do not
use it as an excuse to stop eating fruits and vegetables.
Is it possible to create a toxic food by genetic modification? Of
course it is, but the same is true of conventionally bred foods. A
while back, for example, a new strain of potato produced by con-
ventional breeding was found to have unacceptably high levels of
solanine and had to be pulled from the market. There are two fac-
tors that help mitigate any concerns about food safety, not only for
GMOs but for conventional foods as well. First, it is unlikely that
any company would knowingly release an unsafe food, regardless
of its origin, into the marketplace. The attendant publicity would
likely be the death knell for the company and that is not what
investors in those companies want. The second factor is that, at
least in North America, new crop varieties are subjected to the
scrutiny of several government agenciesthe U.S. Department
of Agriculture (USDA) and the Food and Drug Administration
(FDA) in the United States and the Canadian Food Inspection
Agency (CFIA) in Canada. In general, any new crop variety,
whether produced by GM or conventional plant breeding, must
meet three criteria before it can be registered and approved for
release: it must be genetically distinct from other approved variet-
ies, it must be genetically stable, and the new variety must produce
a uniform population of plants. In addition, both GMOs and
conventionally bred foods are carefully scrutinized to ensure that
they are no more dangerous, especially with respect to allergenic
and toxic properties, than the food they replace.
Some opponents of GMOs insist that no transgenic plant
should be released until it is proven safe. This is called the
precautionary principle. Is this a reasonable approach? Can you
anticipate or test for every possible eventuality? Probably not.

However, after more than 25 years of experimentation, there have
been no unpredicted results from the release of any genetically
modified product. Moreover, the USDA has reported that in 2002
American farmers planted nearly 80 million acres of genetically
modified corn and soybeans. That acreage is without doubt much
higher at this time and there have been no ill effects reported.
What About the Environment?

Beyond food safety, there appears to be some concern that GM plants
might have an adverse impact on the environment. What kind of
an impact might be expected? The principal concern appears to be
that pesticide-resistant genes could spread into wild populations,
creating a race of super weeds. Is this a valid concern?
The spread of genes could happen in two ways: GM plants could
transfer genes by pollinating close relatives in the wild or they might
escape and establish themselves outside cultivated plots. Most of
the first generation GMOs are pesticide-resistant and should such
a plant or gene escape into the wild, it is not likely to be a major
problem. Why? A plant can succeed in the wild only when it enjoys
some advantage over its competitors. An herbicide-resistant plant,
for example, would enjoy no competitive advantage because it
would not be challenged with herbicide. Even if it were necessary
to use herbicide, a glyphosate-resistant plant could be killed with
any one of several other herbicides. In addition, we know that resis-
tance to herbicides arises naturally in wild and weed populations
based on selection pressure. Finally, pesticide-resistant plants have
been produced by conventional breeding, but no one seems unduly
concerned about their release. If pesticide resistance is a problem,
should it matter whether that resistance has been produced by
rDNA techniques or by conventional breeding?
There might be some reason to be concerned about second-
generation GMOs, however. These include traits such as cold tol-
erance or fungal resistancetraits that have yet to be developed.
In this case, escaped plants could suddenly acquire an adaptive

advantage that could be exploited if a sudden cold snap, for
example, killed off the competition. Another concern is molecular
pharming: Plants that are used as bioreactors to make pharma-
ceuticals are clearly not intended for widespread environmental
release or commodity food markets and must be carefully isolated
to ensure there is no gene transfer to food crops.
If there is a final lesson when it comes to using this new tech-
nology, it is this: if there are going to be problems with food safety
or the environment, they will arise because of the nature of the
A Tale of Two Wheats

In 2004, the U.S.-based chemical company Monsanto announced plans to
seek permission for the release of a genetically modified, herbicide-tolerant
wheat variety into the Canadian market. The wheat was modified by inserting
a gene from a soil bacterium, and it would be tolerant to Monsantos very
popular herbicide, glyphosate. The announcement was met with vocal resis-
tance from consumer groups, environmentalists, and farmers themselves.
Consumer groups were concerned about the release of yet another GMO
and corporate control over a major crop. Environmentalists were concerned
that the foreign gene would spread into native grasses, and farmers who
wanted to continue growing conventional wheat feared contamination of
their crops with wind-spread pollen. Even the Canadian wheat marketing
board expressed opposition, fearing the loss of European and other markets
where the import of GMOs has been banned. Fearing a public relations
disaster, Monsanto wisely decided to cancel its plans.
Meanwhile, thousands of acres of herbicide-tolerant wheat are already
being grown across the Canadian prairiesmore than 200,000 acres in
2005without a sound of protest. Consumers, environmentalists, and
farmers alike are conspicuous by their silence. Why the difference? Only
that this wheat, called CDC Imagine, was created by chemical mutagenesis
product itself, not because of the method that was used to generate
that product. It is more important that we be able to trust our gov-
ernment agencies and other organizations to carefully scrutinize
the safety and potential environmental impact of any new crop
variety, regardless of whether it was generated by conventional
breeding or genetic engineering, than it is to worry about the
breeding method that was used to arrive at that product.
This does not mean, however, that there are not potential
problems with GMOs. There are, in fact, two problems that are of
particular concern to a growing number of farmers. Both problems
rather than by insertion of a bacterial gene. A product of the European-based

chemical giant BASF, CDC Imagine was created by bathing wheat seeds with
a chemical mutagen and selecting the surviving seedlings for tolerance to
BASFs proprietary brand of imidazolinone herbicides.
So, we have two cases involving genetic modification at the level of a
single gene. In Monsantos case, the wheat was transformed with a gene
for herbicide tolerance, while in the BASF case an existing wheat gene was
mutated. Both methods end up with the same result, yet the Monsanto
wheat faced stiff opposition based on the perception that a modified plant
with herbicide tolerance posed a risk to human health and the environment.
The Canadian Food Inspection Agency ruled that the gene modification in
the BASF wheat posed no significant risk to either human health or the
environment and it was approved. Either variety has the same potential for
cross-pollinating with wild grasses or contaminating the wheat growing in a
neighbors field.
Some would say that our concern should be focused on the product, not
the method. The question is whether the productin this case, herbicide-
tolerance in wheatpresents a measurable risk, rather than how that toler-
ance was bred into the plant. What do you think?
involve the potential for gene spread through cross-pollination.

Many farmers still exercise the traditional practice of saving seed
from each years crop to use as seed for the following season. This is
called bin run seed because the seed is taken from the farmers own
storage bin. Saving seed reduces costs for the farmer who does not
have to purchase new seed each year and often results in the selec-
tion of seed that performs especially well within the conditions
of a particular farm. If a genetically modified crop is planted in a
neighboring field, there is the danger that pollen from the geneti-
cally modified crop will contaminate the non-modified plants.
Contamination can cause problems for the farmer who saves
seed. The genes used to modify crop plants are patented, and
farmers who wish to plant the modified crops must purchase new
seed each year. They sign agreements with the seed company and
are legally prohibited from saving seed. There are a number of
ongoing court cases between seed companies and farmers, based
on patented genes showing up in saved seed. The farmers claim the
seed was contaminated through no doing of their own, while the
seed companies claim the farmer is breaking patent law by saving
seed that contains their patented gene. Most of these court cases
have involved herbicide resistance in oilseed rape (or canola).
Oilseed rape is a particularly precocious plant that readily cross-
pollinates over long distances.
Organic farmers are also concerned about the spread of modi-
fied genes. Organic farmers must meet a number of conditions in
order to have their crops certified as organic. One of those condi-
tions prohibits the use of genetically modified crops (Figure 7.4).
Organic farmers risk loss of their certification if their crops are
contaminated with modified genes.
Both seed savers and organic growers are also concerned about
the possibilities of a new technology called genetic use restric-
tion technology (GURT), commonly referred to as terminator
technology or suicide seeds. This genetic technology causes
plants to produce sterile seedsthe crop is normal in all respects
Figure 7.4 Due to the publics concern over the safety of GM foods,
some manufacturers have chosen to label their foods as being free of
genetically engineered ingredients.
except that the seeds produced by that crop will not germinate.
Terminator technology could seriously erode the farmers abil-
ity to save seed and increase their dependence on multinational
corporations. This would be particularly serious in Third World
countries, where an estimated 1.4 billion people depend on seed
saving to feed themselves.
Terminator technology generated substantial protest when it
was first brought to the publics attention in 1998. As a result, the
technology was banned outright in several countries, and further
research on the technology was banned under a moratorium
issued by the United Nations convention of biological diversity in
2000. Although several large U.S. seed companies indicated they
would not use the technology in their products, there is concern
that research continues in the United States, which did not sign
on to the moratorium.
Terminator technology may help to focus farmers, consumers,
governments, and seed companies on the real risks and benefits
of genetic modification. On the one hand, terminator technology
would help seed companies protect their investment in modified
crops, which can be substantial. On the other hand, it offers no
agronomic benefit of any kind for farmers. It will likely be very
difficult to balance the rights of seed companies against those of
farmers and consumers, whose rights must also be protected.
A Question of Risk
Back in 1987, noted biochemist Daniel Koshland wrote in Science
that to be alive is to be at risk. We subject ourselves to risk in
hundreds of ways every day of our lives, every time we step off the
curb to cross a street, climb into an automobile or airplane, or eat
food in a restaurant. Our recreationRollerblading, bicycling,
skiing, swimming in shark-infested watersand, in fact, virtually
everything we do involves some level of risk. We accept that risk
because each activity in some way makes our lives easier or better
and we weigh the risks against the perceived benefit. But in each
case, we also take steps to minimize risk. We install traffic lights

at pedestrian crossings, we add more safety features to our auto-
mobiles and airplanes, we wear knee pads and helmets when we
Rollerblade. Moreover, we attempt to educate our children and
others about these risks and how to minimize our exposure.
In the end, we should approach biotechnology with the same
caution. Both as individuals and as a society, we have to weigh the
risks associated with biotechnology in all its forms, and especially
GMOs, against the risks associated with conventional food pro-
duction. And we have to take appropriate steps, including educa-
tion, to minimize any risk. That means we should continue the
necessary oversight to ensure that any new crop varieties, regard-
less of the method of production, meet acceptable guidelines for
food safety and environmental protection.
Summary
Very early in the development of agriculture, farmers began what
we now know as conventional plant breeding, simply by select-
ing the best seeds and crossing their best plants to improve the
quality of grains and other food crops. In the twentieth century,
however, a whole new arsenal of methods was developed by plant
breeders, including double-crossed hybrids, mutations, embryo
rescue, haploid breeding, and somaclonal variation. Each of these
new methods has allowed the introduction of new traits into our
food plants, and the products of these methods are considered
acceptable by the public. Conventional breeding, however, always
carries unknown genes into the new variety. By contrast, genetic
modification (GM) is a far more precise technique, allowing the
insertion of one specific gene expressing a single trait, yet this
method is unacceptable to many.
A strong argument can be made that concern over food safety
and protection of the environment should not be focused on the
method. Herbicide tolerance, for example, can be introduced
by both genetic modification and conventional breeding. If

herbicide-resistance trait is a potential problem, should it matter
how that trait was introduced into the crop? It would be more
productive to focus on the safety of the product, rather than
the method.
The question of food and environmental safety of any new
crop really comes down to a question of risk management. We
know that many foods contain potentially toxic substances and
that genes naturally spread through the environment, but over
the millennia we have learned to manage any potential risk and
produce foods that are high-yielding, healthy, and nutritious. The
real focus should be that all new crop varieties, regardless of how
they are produced, continue to meet acceptable guidelines for
food safety and environmental protection.
Accumulator species Plants that take up large amounts of heavy metal
ions from the soil without injury.
Adventitious shoot (root) A shoot (or root) that arises where it is not
normally expected, such as at the base of stem cuttings or from a clump of
callus tissue in culture.
Alkaloid A nitrogen-containing chemical produced by plants that is physi-
ologically active in vertebrates. Nicotine, caffeine, and morphine are exam-
ples of alkaloids.
Allele One of the two or more possible variants for a gene. A diploid
organism contains two alleles, one contributed by each parent.
Anti-parallel The condition in double-stranded DNA in which the two
strands with polarity run in opposite directions.
Apical meristem The growing point at the tip of a shoot or root.
Aseptic Free of contamination with microorganisms.
Backcross A cross between a hybrid and one of its parents (or a geneti-
cally equivalent organism).
Bacteriophage A virus that infects bacteria; also called simply a phage.
Base A type of molecule, such as one of the five bases that make up
nucleic acids: adenine, guanine, cytosine, thymine, and uracil.
Batch culture A bioreactor that is emptied and set up anew each time the
reaction has run to completion.
Bin run seed Seed saved from a farmers current crop to be used to plant
the fields the following year, as opposed to buying new seed each season.
Bioengineering The application of engineering techniques to biological
processes, such as large-scale cultures of fungi to produce drugs.
Biogas Methane gas that is generated by the microbial decomposition of
organic matter in the absence of oxygen.
Bioreactor Any system that uses living organisms or enzymes to effect
chemical changes, such as decomposition, decontamination, or production
of chemical products.
Bioremediation The use of living organisms, usually bacteria, to decon-
taminate polluted soils.
127
Biotechnology The use of living organisms to generate products for the use
and benefit of humans.
Butanol A type of alcohol consisting of four carbon atoms per molecule.
Cellular respiration A sequence of metabolic reactions that converts sugars
and other substrates to carbon dioxide and water in the presence of oxygen.
The principal energy-generating metabolism in a cell.
Chemical mutagen A substance that induces mutations, or genetic
changes, in the DNA of a living organism.
Codon A sequence of three adjacent nucleotides in DNA or RNA that com-
prises the genetic code for one amino acid in a protein.
Complementarity The property of base pairing in nucleic acid synthesis,
in which the nucleotide sequence in the original strand is preserved in the
newly formed complementary strand; a second round of copying restores
the sequence of the original strand.
Continuous culture A bioreactor that is fed by a stream of nutrients and
produces a continuous stream of effluent to be processed.
Conventional plant breeding Any method for producing new plant variet-
ies that does not involve recombinant DNA.
Crown gall A cancerous growth on plants due to transformation of the
host cells by the bacterium Agrobacterium tumifaciens.
Cyanogenic glycoside A chemical, found in some plants, that releases toxic
hydrogen cyanide when the cells are disrupted.
DNA ligase An enzyme that catalyses the formation of strong covalent
bonds along the sugar-phosphate backbone of DNA.
Edible vaccine A vaccine produced in a common food (such as bananas)
that can be administered simply by eating the food.
Effervescence The property of producing large numbers of bubbles that
rise to the surface with a fizzing sound, such as in beer or soda pop.
Electroporation The momentary induction of small pores in the membrane
of a cell or protoplast by a brief pulse of electric current. It enables plant
protoplasts to take up small pieces of DNA from the surrounding medium.
Embryo rescue A technique for culturing the hybrid embryo formed when
two plants that do not normally mate are crossed.
128
Enterotoxin A toxin that has its effect in the gastrointestinal
tract.
Fatty acid A long-chain hydrocarbon (composed of only carbon and
hydrogen) with an oxygen-containing carboxylic acid group at one
end. The principal component of fats and oils.
Feedstock The raw or starting material for an industrial manufac-
turing process. For example, crude oil is the feedstock for the produc-
tion of gasoline and diesel fuel in a refinery.
Fermentation The metabolic breakdown of sugars and other sub-
strates in the absence of oxygen. The product of fermentation is usu-
ally ethanol, lactic acid, or a similar chemical.
Fructose A six-carbon sugar molecule with a structure similar to
glucose. Common table sugar (sucrose) is made up of one molecule
each of glucose and fructose.
Gene The basic unit of heredity. A sequence of nucleotides in DNA
that encodes the sequence of RNA, amino acids in a protein, or car-
ries other instructions for the synthesis of proteins.
Gene gun A laboratory tool for shooting foreign DNA into plant
cells. The technique is known as biolistics.
Genetically modified organism (GMO) Any organism whose genetic
constitution has been successfully modified.
Genetic engineering The use of recombinant DNA technology to
alter the genetic constitution of an organism.
Genetic modification (GM) Any change to the genetic constitution
of an organism, although in common usage it refers specifically to
changes involving recombinant DNA technology.
Genome The sum of all the genes in an organism.
Glufosinate An herbicide that kills plants by blocking the incorpora-
tion of inorganic nitrogen into organic molecules.
Glycolysis A sequence of enzymatic reactions in a cell that convert
one molecule of glucose to two molecules of pyruvic acid. It is com-
mon to both cellular respiration and fermentation. The difference
between respiration and fermentation is in the fate of the
pyruvic acid.
129
Glyphosate A herbicide that kills plants by blocking the synthesis of the
aromatic amino acids, which are characterized by having a chemical ring as
part of their structure.
Helicase An enzyme that separates the two strands of double-stranded
DNA during replication.
Hybrid The offspring or progeny from two organisms differing by one or
more genes.
Hydrocarbon A molecule made up of only the elements carbon and hydro-
gen. Methane (CH4) is the simplest hydrocarbon. Diesel fuel and gasoline
are mixtures of several hydrocarbons.
Hydrogen bond Weak forces that hold molecules together by sharing a
hydrogen ion. The hydrogen is normally shared between two oxygen and/or
nitrogen atoms.
Inbred line A genetically pure line that breeds true. Inbred lines usually
arise through self-pollination and selection.
Ionizing radiation Radiation that has sufficient energy to cause permanent
changes in molecules.
Landrace A variety of any agricultural crop that has become adapted to the
microclimate of a particular limited area through generations of selection.
Marker gene A gene for a trait such as antibiotic resistance that enables
technicians to identify cells that have been successfully transformed.
Metabolite Any chemical that participates in the chemical reactions that
occur within a cell or organism.
Micropropagation The technique for reproducing plants asexually through
tissue culture.
Microshoot The small shoots that are produced by micropropagation.
Molecular farming (pharming) The use of genetically modified plants
as bioreactors to produce molecules that plants do not normally produce.
When the molecule has pharmaceutical value, the process is called molecu-
lar pharming.
Mutation breeding The introduction, via conventional breeding, of genes
modified by inducing mutations with ionizing radiation or chemical
mutagens.
130
Nanotechnologist Researchers who work at the nanometer scale (1 nano-
meter = 1 billionth of a meter).
Nucleotide A molecule composed of a nitrogen base, a sugar, and a phos-
phate group. The basic building block of nucleic acids.
Open pollinated Pollinated by natural means.
Osmosis The diffusion of water across a membrane.
Overexpression A term that describes what happens when a gene is engi-
neered to produce more than normal amounts of the protein that it codes
for.
Phytochelatins A small protein that prevents heavy metal toxicity by
sequestering the metal ions and storing them in the large central vacuole of
the cell.
Phytoprospecting The use of accumulator species of plants as an indica-
tion of the presence of particular metals in the environment.
Phytoremediation The use of plants to decontaminate polluted soils,
especially soils contaminated with heavy metals.
Plasmid A small piece of circular DNA found in bacteria and in the mito-
chondria and chloroplasts of animal and plant cells.
Polyhydroxyalkoanate (PHA) A biodegradable plastic synthesized by cer-
tain bacteria.
Protoplast A plant cell that has been stripped of its cell wall, leaving the
cell membrane intact. A naked plant cell.
Pyruvic acid A three-carbon molecule that serves as a precursor for fer-
mentation and respiration products such as ethanol and carbon dioxide.
Recombinant DNA A fragment of DNA containing the DNA from two
different species spliced together in the laboratory. A plant cell naturally
infected with Agrobacterium also contains recombinant DNA.
Restriction enzyme A substance that cuts double-stranded DNA at the
site of a particular base sequence.
Reverse engineering The synthesis, in the laboratory, of an artificial gene
or a strand of DNA matching the amino acid sequence of a known protein.
Reverse transcriptase An enzyme that transcribes RNA into DNA.
131
Selection A technique for plant improvement in which the best seed from
each crop representing a particular trait is saved for planting the next gen-
eration or making the next cross.
Sequester To withdraw or tie up.
Shoot tip culture A technique for culturing the growing region at the tip
of a plant stem in order to induce the formation of multiple microshoots.
Somaclonal variation Mutations that arise during micropropagation.
Somatic hybrid A hybrid formed asexually by the fusion of two or more
protoplasts.
Totipotent The capacity for any plant cell or tissue to develop into a fully
competent mature plant.
Transcription The assembly of an RNA molecule complementary to a
strand of DNA.
Transformation The transfer of genes from one organism to another.
Transgenic An organism that has been genetically transformed.
Translation The assembly of a protein on a ribosome in accordance with
the sequence of nucleotides in a strand of messenger RNA.
Triglyceride A fat or oil molecule consisting of three fatty acids attached
to a molecule of glycerol.
Vector In genetics, a means for carrying DNA into a cell. The
Agrobacterium plasmid is an example of a vector that is used to transform
plant cells.
Vegetative reproduction Plant reproduction that does not involve the
union of sperm and eggs.
132
Alberts, B., A. Johnson, J. Lewis, et al. Molecular Biology of the Cell. New
York: Garland Science, 2002.
Bud, R. The Uses of Life: A History of Biotechnology. Cambridge, England:
Cambridge University Press, 1993.
Cummins, R., and B. Lilliston. Genetically Engineered Food: A Self-Defense
Guide for Consumers. New York: Marlowe & Co., 2000.
Grace, Eric R. Biotechnology Unzipped. Toronto: Trifolium Books, 1997.
Hart, K. Eating in the Dark. New York: Vintage Books, 2002.
Hopkins, W. G., and N. P. A. Hner. Introduction to Plant Physiology, 3rd
ed. New York: John Wiley & Sons, 2004.
McHughen, A. Pandoras Picnic Basket: The Potential and Hazards of
Genetically Modified Foods. Oxford: Oxford University Press, 2000.
Kreuzer, H., and A. Massey. Recombinant DNA and Biotechnology.
Washington, DC: ASM Press, 2001.
Plasterk, R. H. A. RNA Silencing: The Genomes Immune System. Science
296 (2002): 12631265.
Pringle, P. Food, Inc.: Mendel to MonsantoThe Promises and Perils of the
Biotech Harvest. New York: Simon & Schuster, 2003.
Raven, P. H., R. F. Evert, S. E. Eichhorn. Biology of Plants. New York: Worth
Publishers, 1999.
Tagliaferro, L. Genetic Engineering: Progress or Peril? New York: Lerner
Publications, 1997.
Thieman, W. J., and M. A. Palladino. Introduction to Biotechnology. San
Francisco: Benjamin Cummings, 2004.
Turner, N. J., and A. F. Szczawinski. Common Poisonous Plants and
Mushrooms of North America. Portland, OR: Timber Press, 1991.
Watson, J. D. The Double Helix. New York: Atheneum, 1968.
133
Hart, K. Eating in the Dark. New York: Vintage Books, 2002.
Pringle, P. Food, Inc.: Mendel to MonsantoThe Promises and Perils of the
Biotech Harvest. New York: Simon & Schuster, 2003.
Tagliaferro, L. Genetic Engineering: Progress or Peril? New York: Lerner
Publications, 1997.
Watson, J. D. The Double Helix. New York: Atheneum, 1968.
Web Sites
Canadian Renewable Fuels Association
http://www.greenfuels.org
This site has many links to both Canadian and American renewable fuels sites.
Cold Spring Harbor DNA Learning Center

http://www.dnalc.org
A student-friendly resource with links to current topics in gene cloning and recom-
binant DNA techniques.
Gene Gun
http://www.nysaes.cornell.edu/pubs/press/1999/genegun.html
This site describes the origins of the helium-powered gene gun.
International Survey of Herbicide-Resistant Weeds

http://www.weedscience.org/in.asp
With extensive use of herbicides and herbicide-resistant crops, the development of
herbicide-resistant weeds is an increasing problem. This interesting Website tracks
the origin and distribution of herbicide-resistant weeds.
Molecular Farming
http://www.molecularfarming.com
Well-constructed site with extensive links to other sites both for and against
genetic engineering.
National Center for Biotechnology Information

http://www.ncbi.nlm.nih.gov
Website sponsored by the National Library of Medicine and the National Institutes
of Health that is useful for a general perspective on biotechnology (with an empha-
sis on human and animal applications).
Nobel Prize in Physiology or Medicine

http://www.nobel.se/medicine
Information on past recipients of the Nobel Prize.
134
RNAi. Nova (Public Broadcasting System)
http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html
Provides animated video and links to questions and answers.
Terminator Technology and Transgenic Crops

http://filebox.vt.edu/cals/cses/chagedor/terminator.html
A brief description of how the genetic restriction technology works. Includes a link
to the Transgenic Crops Homepage (http://filebox.vt.edu/cals/cses/chagedor/crops.
html), which provides access to other useful information about transgenic crops.
135
Accumulator species, 3235 Biodiesel, 4143, 4446
Acetic acid, fermentation and, 19 Bioengineering, defined, 8
Acetyl-CoA, PHAs and, 100102 Biofuels, 23, 4044, 47
Actinomucor elegans, 20 Biogas, 41, 44
Adaptive advantages, 119121 Biolistics, 76, 77
Advantages of GMO crops, 111 Bioreactors, 25, 26, 27, 41, 97102
Adventitious shoots, 36 Bioremediation, 2628, 29
Aflatoxins, peanuts and, 117 Biotechnology, defined, 8, 10
Agrobacterium tumefaciens, 7779, 108 Blind staggers disease, 32
Alaska, bioremediation in, 28 Boyer, Herbert, 62
Alcohols, 5, 7 Brassica species, 93
Alkali poisoning, 32 Brazil, 24, 4344
Alkaloids, in foods, 116117 Breadmaking, 45, 7
Amino acids, 56, 8082 Butanol, fermentation and, 16
Ammonium perchlorate, 27 Butterflies, Bt and, 9091
Amylase, 5, 22 Butyl rubber, 16
Antibiotics, 10, 21, 23, 62, 64
Antibodies, 9899 Caffeine, toxicity of, 117
Antifreeze genes, 115 Canadian Food Inspection Agency
Antiparallel, defined, 54 (CFIA), 118, 121
Apical meristems, 38 Cane sugar, 24, 4344
Arabidopsis thaliana, 101 Canola, 93, 94, 122
Arntzen, C.J., 9899 Catalysts, enzymes as, 24
Aseptic technique, 17, 36 Cattails, bioremediation and, 26
Aspergillus niger, 22 Cattle, 32, 34, 40
Aspergillus oryzae, 20 CDC Imagine wheat, 120121
Astralgus, 32 cDNA, 7071
Auxins, crown gall and, 7779 Cellular respiration, 1719
Axillary buds, 3839 Cellulose, gasohol and, 4344
Certification of organic crops, 122
Bacillus thuringiensis (Bt) protein, Chargaff, Erwin, 5052
8992, 116 Cheese, 57
Backcrossing, 111 Chelation, 33
Bacteria, 6162, 114 Chemical mutagens, 73,
Bacteriophages, 6062 120121
Base pairing, 5255 Chemicals, 2022, 97102
Batch culture, defined, 21 Chitinase, viral resistance and, 92
Beer, 45 Citric acid, 21, 22
Berg, Paul, 6162 Citric acid cycle, 18, 19
Beta-carotene, 22, 96 Clones, defined, 35
Bialaphos, 8082 Codons, protein synthesis and, 57
Bin run seed, 122 Cohen, Stanley, 62
Biodegradable plastics, 100102 Colorado potato beetles, 89, 90, 91
136
Competitive advantages, 119121 EcoRI enzyme, 6162, 63
Complementarity, 5355, 59, 67 Edible vaccines, 99100
Complementary DNA (cDNA), 7071 Effervescence, citric acid and, 22
Contamination, seeds and, 122 Electrophoresis, 66
Continuous culture, defined, 21 Electroporation, 7576, 77
Conventional plant breeding Embryo rescue, 112113
acceptance of, 106 Enolpyruvyl-shikimate-3-phosphate
disadvantages of, 109111 synthase (EPSPS), 80, 82
herbicide tolerance and, 120121 Enterotoxins, 8992
history of, 107108 Environmental Protection Agency
pesticide tolerance and, 119 (EPA), 10
process overview, 108109 Environmental safety, 119124
trait development by, 111114 Enzymes, 22, 2425. See also specific
Cooking oils, 44, 46, 100 enzymes
Corn, 76, 96, 108, 110 Ereky, Karl, 14
Corn steep liquor, 23 Erucic acid, 93
Cotton, 101102 Escherichia coli (E. coli), 28, 6162, 99
Crick, Francis, 5253 Ethanol, 19, 21, 2324, 4344
Crossing, 108110 Ethyl methane sulfonate (EMS), 73
Cross-pollination, 122 Explants, micropropagation and, 38
Crown gall, 7679 Exxon Valdez, 28
CryIAb, 91
Cyanogenic glycosides, 116 Farming, molecular, 97102, 120
Cytokinins, crown gall and, 7779 Fatty acids, 41, 42, 46, 9395
Feedstocks, 8
Denitrifying bacteria, 26 Fermentation
Deoxyribonucleic acid (DNA). See biodegradable plastics and, 102
DNA butanol production and, 16
Department of Agriculture, U.S., 118, feedstock production and, 8
119 gasohol and, 44
Disease resistance, 8992 glycerol production and, 1415
DNA industrial production and, 2025
genes and, 5456 Louis Pasteur and, 14
insertion into plant cells, 7579 overview of, 5, 1719
modern biotechnology and, 6066 Fingerprinting, DNA, 6566, 74
overview of, 5054 Fish genes, tomatoes and, 115
presence of in all foods, 114, 116 Flavr-Savr tomatoes, 115
proteins and, 5660 Fleming, Alexander, 23
DNA fingerprinting, 6566, 74 Flounder genes, 115
DNA ligase, 54, 74 Flower coloration, 8788
DNA polymerase, 55, 74 Food and Drug Administration
Domestication, 45 (FDA), 118
Double helix model, 5254 Food safety, 114117, 118
137
Forensics, 6566 Goitrogens, 117
Franklin, Rosalind, 53 Golden rice, 9496
Fungi, 78, 9. See also specific species Greenpeace, 107
Fungicides, 8081 Griseofulvin, 21
Fusarium infections, 9 Groundwater, 27
Fusion of protoplasts, 75 Gun cotton, 16
Gall, 7679 Haploid breeding, 113

Gasohol, 4344 Heavy metals, 26, 32
Gels, 66 Helicase, 54, 55
Gene guns, 76, 77 Helices, 5254
Genes, overview of, 5456 Hemophilus influenzae, 61
Gene spread, 119122 Hepatitis B antigen, 9899
Genetically modified organisms Herbicide tolerance
(GMOs), defined, 8 criticisms of GMOs and, 119
Genetic engineering, 8 genetic engineering and, 7982
Genetic modification (GM), 8 protein structure and, 5657
Genetic reassortment, 35 public resistance to, 120121
Genetic use restriction technology tobacco, 10
(GURT), 122124 value of, 8687
Genetic variability, 109 Heritage varieties, 109
Genomes, defined, 56 High-fructose syrups, 2425
Gibberellin, 22, 112 Hind III enzyme, 61
Glucose, fermentation and, 5 Human immunodeficiency virus
Glucose isomerase, 24 (HIV), 71
Glucosinolates, 93, 117 Human toxicity, 82
Glufosinate, 8082 Hybridization, 75, 108109
Glycerin, biofuels and, 46 Hydrocarbons, 28
Glycerol, 1415, 19, 21, 41, 42 Hydrogenation, 9395
Glycine max, 20, 4446, 81 Hydrogen bonds, 5255
Glycolysis, 1718
Glyphosate, 7982, 8687, 120121 Immunizations, 98100
GM crops, criticisms of Inbred lines, 108109
bacterial consumption, 114 Indicator species, 3233
DNA consumption, 114, 116 Insecticides, 8889, 8992
gene spread, 119122 Insulin, 63
myths, 115 Invertase, 22
patenting, 109 Ionizing radiation, 73
pesticide consumption, 116
precautionary principle, 118119 Jorgensen, Rich, 8788
risk assessment, 124125
terminator technology, 122124 Krebs cycle, 18, 19
toxin presence as, 116118 Labeling, GMOs and, 123
138
Lactic acid, 14, 19, 20 Nobel, Alfred, 1617
Lactobacillus, 20 Nobel Prize, 17, 61, 62
Lake Ontario, 28 Norwalk virus, 99
Landfills, methane and, 4041 Nucleotides, 50, 51, 59
Landraces, 109 Nutrition, improving, 9396
Lepidopterans, Bt and, 9091, 116
Liberty, 8082 Oil refineries, 2627
Libraries, cDNA, 7071 Oils, 4144, 46, 9395, 100
Ligase, 54, 74 Oilseed rape, 93, 94, 122
Lipids, 95 Open-pollination, 109
Locoweed, 32 Orchids, 39
Lodging, 112, 113 Organic farming, 116, 122
Lysine, feedstock and, 96 Origins of biotechnology, 47, 14
Osmosis, protoplasts and, 7375
Maize, transformation of, 76 Overexpression, defined, 80
Mannitol, protoplasts and, 7375 Oxygen, 1819, 44
Marker genes, 64, 65
Marquis wheat, 108 Pandoras Picnic Basket, 114
Marsh gas, 4041 Papain, 24
Measles, edible vaccines and, 100 Paper mills, 34
Messenger RNA (mRNA), 57, 58, 59, Pasteur, Louis, 14, 15
7071, 8788 Pasteurization, 114
Metabolites, production of, 2025 Pathogens, 3940
Metals, 26, 32 Pauling, Linus, 5254
Methane, biofuels and, 4041 PCR. See Polymerase chain reaction
Methylation, 60 Peanuts, aflatoxins and, 117
Microbiology, Pasteur and, 14 Pectinase, 22
Micropropagation, 3640, 113114 Penicillin, 21, 23
Molecular farming, 97102, 120 Penicillium chrysogenum, 23
Monarch butterflies, Bt and, 9091 Perchlorate, 27
mRNA, 57, 58, 59, 7071, 8788 Pesticides, 116, 119
Mutagens, chemical, 73 Petroleum products, 28
Mutation breeding, 112 Petunias, pigmentation and, 8788
Mutations, 7173, 112 Phages, 6062
Pharming, molecular, 97102, 120
Nanotechnology, 76 Phytoaccumulation, 3435
National Center for Biotechnology Phytochelatins, 33
Information, 10 Phytoprospecting, 3233
Natural gas, methane and, 40 Phytoremediation, 3235
Nature, 5254, 90 Plant cell transformation, 73, 7576
Neuberg, C.A., 1415 Plant oils, biofuels and, 4144
Nickel, phytomining and, 34 Plasmids, 6264, 65, 79
Nitroglycerin, 16 Plastics, biodegradable, 100102
139
Pollination, 8890, 109, 122 Riboflavin, 22
Polyhydroxyalkanoates (PHAs), Ribonucleic acid (RNA), 50, 57
100102 Ribosomes, 59
Polymerase chain reaction (PCR), Rice, Golden, 9496
6566, 74 Risk, assessment of, 124125
Polymerases, 55, 74 RNA interference (RNAi), 8788
Potatoes RNA silencing, 8788
alkaloids in, 116117, 118 Roundup, 7982, 8687, 120121
disease resistance and, 75 Rubber, synthetic, 16
gasohol and, 44 Russet Burbank potatoes, 114
micropropagation and, 40
molecular farming and, 99 Saccharomyces cerevisiae, 5, 19
somaclonal variation (SCV) and, Safety, 114117, 118, 119124
114 Salix species, 35
Precautionary principle, 118119 Saturation, 9395
Precision, as advantage, 111 Sedges, bioremediation and, 26
Prince William Sound, 28 Seed oils, 41, 9394
Probes, fingerprinting and, 66 Selection, 64, 108
Productivity, defined, 9, 92, 9294 Selenium, 32, 34
Proteases, 22, 117 Sequestration, 33
Proteins, 5660, 8992 Serotonin, bananas and, 117
Protoplasts, 7375 Sewage treatment plants, 40
Pseudomonas, 28 Shoot-tip culture, 38
Pyruvic acid, 1819 Shotgun biolistics, 76, 77
Silencing, 8788
Radiation, ionizing, 73 Silos, methane and, 41
Rapeseed, 93 Smith, Hamilton, 6162
Reassortment, 35 Solanine, 116117, 118
Recombinant DNA (rDNA), 8, 6162, Solanum tuberosum, 75
67 Somaclonal variations, 40, 113114
Rennet, industrial production of, 22 Somatic hybridization, 75
Rennin, 6, 7, 8, 24 Soybeans, 20, 4446, 81
Replica plating, 64 Soy sauce, 20
Replication, 54, 6264, 74 Starches, gasohol and, 4344
Resistance, 8892. See also Herbicide Start codons, 59
tolerance Stearic acid, 94
Respiration, 1719 Sterile technique, 17, 36
Restriction enzymes, 6062, 6264, Sterility, 122124
6566, 67 Sticky ends, 62
Retroviruses, 7071 Streptomyces hygroscopicus, 8082
Reverse engineering, 71 Sufu, 20
Reverse transcriptase, 7071 Sugar cane, 24, 4344
Rhizopus, 20 Sugar-phosphate backbone, 5255
140
Suicide seeds, 122124 Uniformity, clones and, 3536
Sunflower oil, biofuels and, 41
Suppression, RNA and, 8788 Vaccines, 98100
Swamp gas, 4041 Variability, genetic, 109
Sweet potatoes, 44 Variations, somaclonal, 40
Vegetative reproduction, 36
Tempeh, 20 Vetch, 32
Temperature, PCR and, 74 Vinegar, 19
Teosinte, 108 Viruses, 3940, 6062, 7072, 92
Terminator technology, 122124 Visualization, 66
Thallium, phytomining and, 34 Vitamins, 22, 96
Thermal properties, 101102
Thlaspi species, 33 Waste management, 26
Ti plasmids, 79 Watson, James, 5253
Tissue culture, 36 Weeds, control of, 8687
Tobacco, 10, 9798 Weizmann, Chaim, 16
Tobacco mosaic virus (TMV), 72 Weizmann process, 16, 17
Tomatoes, 115 Wetlands, 26
Totipotency, 38 Wheat, 108, 112, 120121
Toxicity, human, 82 White, Phillip, 36
Toxins, in all foods, 116118 Wilkins, Maurice, 53
Trace elements, 34 Willows, 35
Transcription, 57 Wines, 5
Transfer RNA (tRNA), 58, 59
Transformation Yeasts, fermentation and, 5
of bacteria, 6263, 64 Yields, 9, 9294
of plant cells, 73, 7576, 7679
Translation, 58 Zea mays, 76
Triazine resistance, 5657 Zymology, defined, 14
Triglycerides, 41, 42, 95 Zymotechnology, 14
141
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142
William G. Hopkins received a B.A. in biology from Wesleyan University
and a Ph.D. in botany from Indiana University. His post-doctoral train-
ing was conducted at Brookhaven National Laboratories. He has taught
at Bryn Mawr College and the University of Western Ontario, where he is
now professor emeritus (Biology). Dr. Hopkins has taught primarily in the
areas of plant physiology and cell biology, was responsible for design and
implementation of an honors program in cell biology, and served many
years as an undergraduate counselor. In 1988, Dr. Hopkins was awarded
the universitys Gold Medal for Excellence in Teaching. His research and
publications have focused on the role of light and temperature in plant
development, the organization of chlorophyll-protein complexes, and
energy transformations in chloroplasts. Dr. Hopkins has been a contribut-
ing author to two high school biology textbooks and is the senior author
of Introduction to Plant Physiology, a textbook published by John Wiley &
Sons and now in its third edition.
143

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How did the rise of agriculture influence human civilization?

How did the rise of agriculture influence human civilization?

What are some of the early techniques used in plant biotechnology before recombinant DNA?

What are some of the early techniques used in plant biotechnology before recombinant DNA?

Plant

Copyright 2007 by Infobase Publishing

Library of Congress Cataloging-in-Publication Data

Text and cover design by Keith Trego and Ben Peterson

Printed in the United States of America

This book is printed on acid-free paper.

2 The Early Days of Biotechnology 12

3 Plants and Biotechnology

4 DNA, Genes, and Protein 48

6 Genetically Modified Plants

7 Putting Genetically Modified

cotton, and tobaccowas responsible for the introduction of slavery to

Beer, Cheese, and Freshly Baked Bread

coast of the Mediterranean to the Nile Valley of Egypt. Among

In the process of brewing beer, the germinating grain (usu-

and bears the same relationship to milk as wine does to grapes.

Making Organisms Work for Us

(secreted into the organisms environment) enzymes, including

This new biotechnology has unleashed a storm of public

Scientific Roots of Biotechnology

Figure 2.1 Louis Pasteur was a pioneer in the field of biotechnology.

amounts of glycerol could be produced by fermentation of starch.

At the same time in Britain, a group of organic chemists at

technology in the years prior to 1918 and the developing political

Figure 2.2 Glycolysis is the breakdown of glucose into pyruvic acid

pyruvic acid. A small amount of energy is released, and the carbon

The difference between respiration and fermentation lies in the

Ethyl alcohol (ethanol): The fermentation product of several

Glycerol: Under alkaline conditions, S. cerevisiae produces

Acetic acid (vinegar): The fermentation product of the bacte-

Processing Food and Drink

Industrial Production of Fungal Metabolites

if anaerobic conditions are required. Either way, actively growing

Table 2.1 Commercially Important Fungal Metabolites

(continued on page 22)

(continued from page 21)

One of the most important organic acids produced commer-

Still, the largest and most important products of commer-

resources, attention has once more turned to fermentation as a

Figure 2.3 The components of a bioreactor are seen in this illustration.

order to produce large amounts of product. In the early 1960s, it

Waste Management and Bioremediation

refineries and removal of perchlorate from groundwater. The

as Pseudomonas and Escherichia coli are capable of degrading

Figure 2.4 This bioremediation system purifies groundwater that has

industry, biotechnology and bioengineering have been used

Traditional biotechnology has not been limited to applications

Phytoremediation: Cleaning Up With Plants

plants accumulator species, which are not injured by high con-

of twentieth-century industrial activities. Using plants to clean

Selenium and Alkali Poisoning

other trials, various species of willows (Salix) have shown prom-

Micropropagation: Starting Out Small

resort to asexual reproduction. In plants, asexual or vegetative

Figure 3.2 A plant biologist examines papaya plantlets raised in a petri