PlantBiotechnology

Ethnobotany
Forestry
Horticulture
Photosynthesis and Respiration
Plant Biotechnology
Plant Cells and Tissues
Plant Development
Plant Diversity
Plant Ecology
Plant Genetics
Plant Nutrition
William G. Hopkins
PlantBiotechnology
Plant Biotechnology
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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
FurtherReading 134
Index 136
“Have you thanked a green plant today?” reads a popular bumper sticker.
Indeedweshouldthankgreenplantsforprovidingthefoodweeat,fiberfor
theclothingwewear,woodforbuildingourhouses,andtheoxygenwebreathe.
Withoutplants,humansandotheranimalssimplycouldnotexist.Psycholo-
giststellusthatplantsalsoprovideasenseofwell-beingandpeaceofmind,
whichiswhywepreserveforestedparksinourcities,surroundourhomes
withgardens,andinstallplantsandflowersinourhomesandworkplaces.Gifts
offlowersarethemostpopularwaytoacknowledgeweddings,funerals,and
other events of passage. Gardening is one of the fastest growing hobbies in
NorthAmericaandtheproductionofornamentalplantscontributesbillions
ofdollarsannuallytotheeconomy.
Human history has been strongly influenced by plants. The rise of agri-
culture in the fertile crescent of Mesopotamia brought previously scattered
hunter-gathererstogetherintovillages.Eversince,theavailabilityofland
andwaterforcultivatingplantshasbeenamajorfactorindeterminingthe
locationofhumansettlements.Worldexplorationanddiscoverywasdriven
bythesearchforherbsandspices.Thecultivationofnewworldcrops—sugar,
By William G. Hopkins
vii
cotton, and tobacco—was responsible for the introduction of slavery to
America,thehumanandsocialconsequencesofwhicharestillwithus.The
pushwestwardbyEnglishcolonistsintotherichlandsoftheOhioRiver
valleyinthemid-1700swasdrivenbytheneedtoincreasecornproduction
andwasafactorinprecipitatingtheFrenchandIndianWar.TheIrishPotato
Faminein1847setinmotionawaveofmigration,mostlytoNorthAmerica,
thatwouldreducethepopulationofIrelandbyhalfoverthenext50years.
Asayounguniversityinstructordirectingbiologytutorialsinaclassroom
thatlookedoutoverawoodedarea,Iwouldaskeachgroupofstudentsto
look out the window and tell me what they saw. More often than not the
questionwouldbemetwithablank,questioninglook.Plantsaresomuch
apartofourenvironmentandthefabricofoureverydaylivesthatthey
rarelyregisterinourconsciousthought.Yettoday,facedwithdisappearing
rainforests,explodingpopulationgrowth,urbansprawl,andconcernsabout
climate change, the productive capacity of global agricultural and forestry
ecosystems is put under increasing pressure. Understanding plants is
evenmoreessentialasweattempttobuildasustainableenvironmentfor
thefuture.
The GreenWorld 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
geneticallyengineered,weighstherisksandbenefitsofthisnewtechnology,
anddiscussesthepresentimpactandfuturepotentialofgeneticallymodi-
fiedplants.
viii IntroductIon
Z
What Is Biotechnology?
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 village—tales 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 don’t 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
4
What Is Biotechnology?
b What Is ß|ctechnc|cgyî
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 (C
6
H
12
O
6
) to ethyl alcohol (ethanol,
C
2
H
5
OH) and the gas carbon dioxide (CO
2
):
C
6
H
12
O
6
———> 2C
2
H
5
OH + 2CO
2
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
b F|ant ß|ctechnc|cgy
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
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.
7 What Is ß|ctechnc|cgyî
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
ß F|ant ß|ctechnc|cgy
(secreted into the organism’s 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 world’s 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).
9 What Is ß|ctechnc|cgyî 9
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.
I0 F|ant ß|ctechnc|cgy
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 |n a Wcrdî
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 Webster’s 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.
II What Is ß|ctechnc|cgyî
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 organism’s 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.
12 12
The Early Days of Biotechnology
The Early Days of Biotechnology
14 Plant Biotechnology
14
SciEnTific RooTS of BioTEchnology
The origins of biotechnology as a science can be traced back to
1857, when French biologist Louis Pasteur (1822–1895) discov-
ered that the fermentation of lactic acid was due to the action of
live microbes, or microorganisms (Figure 2.1). Pasteur’s 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
Großbetriebe (“Biotechnology of Meat, Fat, and Milk Production
in Large-scale Agricultural Industry”). Ereky’s enterprise became
one of the world’s 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
The Early Days of Biotechnology
15 The Early Days of Biotechnology
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.
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.
16 Plant Biotechnology
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 explosives—it is not dif-
ficult to draw connections between the direction taken by bio-
17 The Early Days of Biotechnology
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 casks—continuing in the brew-
ing tradition, of course—and 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. Weizmann’s 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
18 Plant Biotechnology
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.
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.
19 The Early Days of Biotechnology
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
20 Plant Biotechnology
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
21 The Early Days of Biotechnology
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)
22 Plant Biotechnology
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).
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
(continued from page 21)
Metabolite Use
23 The Early Days of Biotechnology
Still, the largest and most important products of commer-
cial bioengineering are antibiotics, especially penicillin. Penicillin
was discovered in 1929 when Alexander Fleming (1881–1955)
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
24 Plant Biotechnology
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 sugar—and therefore fewer calories—is 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
25 The Early Days of Biotechnology
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).
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.
26 Plant Biotechnology
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
27 The Early Days of Biotechnology
refineries and removal of perchlorate from groundwater. The
latter is a particularly serious problem in California, Utah,
Nevada, and Texas, where ammonium perchlorate (NH
4
ClO
4
)
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.
28 Plant Biotechnology
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 Pasteur’s 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
29 The Early Days of Biotechnology
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.
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.
30
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 don’t 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 plant—the 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 Plants and Biotechnology: Before Recombinant DNA
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 cell’s 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
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.
34 Plant Biotechnology
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.
35 Plants and Biotechnology: Before Recombinant DNA
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 reproduction—by seed, for exam-
ple—the 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
36 Plant Biotechnology
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.
37 Plants and Biotechnology: Before Recombinant DNA
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.
38 Plant Biotechnology
All this is made possible by the fact that virtually every plant cell
is totipotent—it 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 2–3 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
Apple (Malus)
Carnation (Dianthus)
Chrysanthemum
Grape (Vitis)
Kiwi fruit (Actinidia)
Orchids (Cattleya, Cymbidium)
Peach, Cherry (Prunus)
Poplar (Populus)
Rhododendron
(Rhododendron)
Rose (Rosa)
Strawberry (Fragaria)
table 3.1 CropsCommonlyPropagatedbyShoot-tipCulture
39 Plants and Biotechnology: Before Recombinant DNA
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
40 Plant Biotechnology
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 (CH
4
). 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 can’t do much about the cattle, but most landfill sites,
particularly in urban areas where they have been reclaimed
41 Plants and Biotechnology: Before Recombinant DNA
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 won’t 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.
42 Plant Biotechnology
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).
43 Plants and Biotechnology: Before Recombinant DNA
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 world’s 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
(continued on page 44)
44 Plant Biotechnology
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? Let’s do a simple calculation,
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.
(continued from page 43)
45 Plants and Biotechnology: Before Recombinant DNA
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
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.
46 Plant Biotechnology
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 alcohol–lye 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!
47 Plants and Biotechnology: Before Recombinant DNA
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.
48
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 acids—DNA 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 DNA, Genes, and Protein
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.
52 Plant Biotechnology
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
53 DNA, Genes, and Protein
“backbone.” The rungs of the ladder—and the key to the Watson
and Crick hypothesis—are 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 Chargaff ’s 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 DNA—in 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.
54 Plant Biotechnology
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 computer—its 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
55 DNA, Genes, and Protein
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.
56 Plant Biotechnology
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 organism’s
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 herbicides—the triazines—that 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
57 DNA, Genes, and Protein
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 DNA’s instructions and direct the synthesis, or
translation, of protein (Figure 4.3).
How can a code using only four “letters”—the four different
bases in a DNA molecule—specify 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 (4
3
), 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
58 Plant Biotechnology
Figure 4.3 During translation, transfer RNA (tRNA) carry amino acids
to the cell’s 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.
59 DNA, Genes, and Protein
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.
60 Plant Biotechnology
It turns out that a gene is more like a recipe—it 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 doesn’t
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
(–CH
3
) 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.
61 DNA, Genes, and Protein
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 A’s (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
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.
62 Plant Biotechnology
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. Berg’s
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” DNA—say from the chromosome of another
organism—that 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 bacteria—their 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
63 DNA, Genes, and Protein
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.
64 Plant Biotechnology
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
tetracycline—those are the colonies that have been transformed
with the recombinant DNA.
65 DNA, Genes, and Protein
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 Organism’s 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 individual’s 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.
(continued on page 66)
66 Plant Biotechnology
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.
(continued from page 65)
67 DNA, Genes, and Protein
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.
68
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 don’t
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 cell’s 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 Engineering Plants
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 don’t 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
72 Plant Biotechnology
a change—any change—in 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
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.
73 Engineering Plants
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 engineering—creating 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
74 Plant Biotechnology
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 95°C (200°F),
which separates the double-stranded DNA into single strands. The sample
is then cooled to about 60°C (140°F), 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 2
20
or more than 1 million copies from a single
starting molecule.
Most proteins are denatured or inactivated at temperatures near 95°C
(200°F), 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.
75 Engineering Plants
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
76 Plant Biotechnology
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 cell’s
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.
naturE’s 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.
77 Engineering Plants
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 unusual—they 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 5.2 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.
78 Plant Biotechnology
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.
79 Engineering 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,
80 Plant Biotechnology
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 (NO
3-
) and convert it to ammonium
ion (NH
4+
) 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 doesn’t 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
81 Engineering Plants
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).
82 Plant Biotechnology
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
83 Engineering Plants
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 gene—transforming plant cells—is
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 nature’s
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.
84
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 Genetically Modified Plants: From Herbicides to Vaccines
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 farmer’s 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 cell’s own gene and the transgene produce
RNA with the same base sequence, the plant’s 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
(continued on page 88)
88 Plant Biotechnology
resisTance To insecTs and disease
Weeds aren’t the only problem faced by farmers—they 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
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 Huntington’s disease, Lou
Gehrig’s 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.
(continued from page 87)
89 Genetically Modified Plants: From Herbicides to Vaccines
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-
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.
90 Plant Biotechnology
tors in the insect’s 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 group’s 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.
91 Genetically Modified Plants: From Herbicides to Vaccines
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 cm
2
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
cm
2
.
• 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.
92 Plant Biotechnology
neered crops don’t 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 don’t 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
93 Genetically Modified Plants: From Herbicides to Vaccines
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 world’s 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
94 Plant Biotechnology
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
95 Genetically Modified Plants: From Herbicides to Vaccines
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 temperature—a fat—while
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.
96 Plant Biotechnology
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.
97 Genetically Modified Plants: From Herbicides to Vaccines
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 vaccines—products 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
98 Plant Biotechnology
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 body’s
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.
99 Genetically Modified Plants: From Herbicides to Vaccines
immune cells to produce antibodies that fight off invading organ-
isms. Arntzen’s 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.
100 Plant Biotechnology
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
101 Genetically Modified Plants: From Herbicides to Vaccines
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
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.
102 Plant Biotechnology
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
103 Genetically Modified Plants: From Herbicides to Vaccines
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.
104
Putting Genetically Modified
Organisms in Perspective
Perhaps you have seen them in the news—anti-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 Putting Genetically Modified Organisms in Perspective
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
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.
108 Plant Biotechnology
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 year’s 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 plant—yields 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
109 Putting Genetically Modified Organisms in Perspective
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
110 Plant Biotechnology
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.
Figure 7.2
111 Putting Genetically Modified Organisms in Perspective
25,000 or more genes that come along for the ride. Many of these
genes may be undesirable—they 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 breeder’s 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 10–12 generations—meaning
10–12 years—to 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 don’t 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
112 Plant Biotechnology
interest. Earlier, in chapter 5, we mentioned the use of mutations
to generate new traits. Let’s 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
113 Putting Genetically Modified Organisms in Perspective
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
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.
114 Plant Biotechnology
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, let’s address the question of “foreign” DNA and proteins.
In his book Pandora’s Picnic Basket, Alan McHughen relates an
anecdote about an anti-GMO activist who stormed out of a meet-
ing declaring that “You’ll 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 can’t avoid bacteria—they 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
115 Putting Genetically Modified Organisms in Perspective
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 gene—one that
codes for the enzyme that causes the tissue to soften—and reinserting
it backwards. The inverted gene could not be read by the cell’s 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 tried—with strawberries, not
tomatoes—and it didn’t 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.
116 Plant Biotechnology
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
117 Putting Genetically Modified Organisms in Perspective
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 body’s 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
mouth’s 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
118 Plant Biotechnology
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 agencies—the 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
119 Putting Genetically Modified Organisms in Perspective
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 resistance—traits that have yet to be developed.
120 Plant Biotechnology
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 Monsanto’s 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 prairies—more than 200,000 acres in
2005—without 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
121 Putting Genetically Modified Organisms in Perspective
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
BASF’s proprietary brand of imidazolinone herbicides.
So, we have two cases involving genetic modification at the level of a
single gene. In Monsanto’s 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
neighbor’s field.
Some would say that our concern should be focused on the product, not
the method. The question is whether the product—in this case, herbicide-
tolerance in wheat—presents a measurable risk, rather than how that toler-
ance was bred into the plant. What do you think?
122 Plant Biotechnology
involve the potential for gene spread through cross-pollination.
Many farmers still exercise the traditional practice of saving seed
from each year’s crop to use as seed for the following season. This is
called bin run seed because the seed is taken from the farmer’s 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 seeds—the crop is normal in all respects
123 Putting Genetically Modified Organisms in Perspective
Figure 7.4 Due to the public’s concern over the safety of GM foods,
some manufacturers have chosen to label their foods as being free of
genetically engineered ingredients.
124 Plant Biotechnology
except that the seeds produced by that crop will not germinate.
Terminator technology could seriously erode the farmer’s 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 public’s 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 recreation—Rollerblading, bicycling,
skiing, swimming in shark-infested waters—and, 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
125 Putting Genetically Modified Organisms in Perspective
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
126 Plant Biotechnology
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.
127
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 farmer’s 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.
128
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.
129
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.
130
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 (CH
4
) 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.
131
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.
132
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.
133
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Hart, K. Eating in the Dark. New York: Vintage Books, 2002.
Hopkins, W. G., and N. P. A. Hüner. Introduction to Plant Physiology, 3rd
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McHughen, A. Pandora’s Picnic Basket: The Potential and Hazards of
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Kreuzer, H., and A. Massey. Recombinant DNA and Biotechnology.
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Pringle, P. Food, Inc.: Mendel to Monsanto—The Promises and Perils of the
Biotech Harvest. New York: Simon & Schuster, 2003.
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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.
135
“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.
136
Accumulator species, 32–35
Acetic acid, fermentation and, 19
Acetyl-CoA, PHAs and, 100–102
Actinomucor elegans, 20
Adaptive advantages, 119–121
Advantages of GMO crops, 111
Adventitious shoots, 36
Aflatoxins, peanuts and, 117
Agrobacterium tumefaciens, 77–79, 108
Alaska, bioremediation in, 28
Alcohols, 5, 7
Alkali poisoning, 32
Alkaloids, in foods, 116–117
Amino acids, 56, 80–82
Ammonium perchlorate, 27
Amylase, 5, 22
Antibiotics, 10, 21, 23, 62, 64
Antibodies, 98–99
Antifreeze genes, 115
Antiparallel, defined, 54
Apical meristems, 38
Arabidopsis thaliana, 101
Arntzen, C.J., 98–99
Aseptic technique, 17, 36
Aspergillus niger, 22
Aspergillus oryzae, 20
Astralgus, 32
Auxins, crown gall and, 77–79
Axillary buds, 38–39
Bacillus thuringiensis (Bt) protein,
89–92, 116
Backcrossing, 111
Bacteria, 61–62, 114
Bacteriophages, 60–62
Base pairing, 52–55
Batch culture, defined, 21
Beer, 4–5
Berg, Paul, 61–62
Beta-carotene, 22, 96
Bialaphos, 80–82
Bin run seed, 122
Biodegradable plastics, 100–102
Biodiesel, 41–43, 44–46
Bioengineering, defined, 8
Biofuels, 23, 40–44, 47
Biogas, 41, 44
Biolistics, 76, 77
Bioreactors, 25, 26, 27, 41, 97–102
Bioremediation, 26–28, 29
Biotechnology, defined, 8, 10
Blind staggers disease, 32
Boyer, Herbert, 62
Brassica species, 93
Brazil, 24, 43–44
Breadmaking, 4–5, 7
Butanol, fermentation and, 16
Butterflies, Bt and, 90–91
Butyl rubber, 16
Caffeine, toxicity of, 117
Canadian Food Inspection Agency
(CFIA), 118, 121
Cane sugar, 24, 43–44
Canola, 93, 94, 122
Catalysts, enzymes as, 24
Cattails, bioremediation and, 26
Cattle, 32, 34, 40
CDC Imagine wheat, 120–121
cDNA, 70–71
Cellular respiration, 17–19
Cellulose, gasohol and, 43–44
Certification of organic crops, 122
Chargaff, Erwin, 50–52
Cheese, 5–7
Chelation, 33
Chemical mutagens, 73,
120–121
Chemicals, 20–22, 97–102
Chitinase, viral resistance and, 92
Citric acid, 21, 22
Citric acid cycle, 18, 19
Clones, defined, 35
Codons, protein synthesis and, 57
Cohen, Stanley, 62
Colorado potato beetles, 89, 90, 91
137
Competitive advantages, 119–121
Complementarity, 53–55, 59, 67
Complementary DNA (cDNA), 70–71
Contamination, seeds and, 122
Continuous culture, defined, 21
Conventional plant breeding
acceptance of, 106
disadvantages of, 109–111
herbicide tolerance and, 120–121
history of, 107–108
pesticide tolerance and, 119
process overview, 108–109
trait development by, 111–114
Cooking oils, 44, 46, 100
Corn, 76, 96, 108, 110
Corn steep liquor, 23
Cotton, 101–102
Crick, Francis, 52–53
Crossing, 108–110
Cross-pollination, 122
Crown gall, 76–79
CryIAb, 91
Cyanogenic glycosides, 116
Cytokinins, crown gall and, 77–79
Denitrifying bacteria, 26
Deoxyribonucleic acid (DNA). See
DNA
Department of Agriculture, U.S., 118,
119
Disease resistance, 89–92
DNA
genes and, 54–56
insertion into plant cells, 75–79
modern biotechnology and, 60–66
overview of, 50–54
presence of in all foods, 114, 116
proteins and, 56–60
DNA fingerprinting, 65–66, 74
DNA ligase, 54, 74
DNA polymerase, 55, 74
Domestication, 4–5
Double helix model, 52–54
EcoRI enzyme, 61–62, 63
Edible vaccines, 99–100
Effervescence, citric acid and, 22
Electrophoresis, 66
Electroporation, 75–76, 77
Embryo rescue, 112–113
Enolpyruvyl-shikimate-3-phosphate
synthase (EPSPS), 80, 82
Enterotoxins, 89–92
Environmental Protection Agency
(EPA), 10
Environmental safety, 119–124
Enzymes, 22, 24–25. See also specific
enzymes
Ereky, Karl, 14
Erucic acid, 93
Escherichia coli (E. coli), 28, 61–62, 99
Ethanol, 19, 21, 23–24, 43–44
Ethyl methane sulfonate (EMS), 73
Explants, micropropagation and, 38
Exxon Valdez, 28
Farming, molecular, 97–102, 120
Fatty acids, 41, 42, 46, 93–95
Feedstocks, 8
Fermentation
biodegradable plastics and, 102
butanol production and, 16
feedstock production and, 8
gasohol and, 44
glycerol production and, 14–15
industrial production and, 20–25
Louis Pasteur and, 14
overview of, 5, 17–19
Fingerprinting, DNA, 65–66, 74
Fish genes, tomatoes and, 115
Flavr-Savr tomatoes, 115
Fleming, Alexander, 23
Flounder genes, 115
Flower coloration, 87–88
Food and Drug Administration
(FDA), 118
Food safety, 114–117, 118
138
Forensics, 65–66
Franklin, Rosalind, 53
Fungi, 7–8, 9. See also specific species
Fungicides, 80–81
Fusarium infections, 9
Fusion of protoplasts, 75
Gall, 76–79
Gasohol, 43–44
Gels, 66
Gene guns, 76, 77
Genes, overview of, 54–56
Gene spread, 119–122
Genetically modified organisms
(GMOs), defined, 8
Genetic engineering, 8
Genetic modification (GM), 8
Genetic reassortment, 35
Genetic use restriction technology
(GURT), 122–124
Genetic variability, 109
Genomes, defined, 56
Gibberellin, 22, 112
Glucose, fermentation and, 5
Glucose isomerase, 24
Glucosinolates, 93, 117
Glufosinate, 80–82
Glycerin, biofuels and, 46
Glycerol, 14–15, 19, 21, 41, 42
Glycine max, 20, 44–46, 81
Glycolysis, 17–18
Glyphosate, 79–82, 86–87, 120–121
GM crops, criticisms of
bacterial consumption, 114
DNA consumption, 114, 116
gene spread, 119–122
myths, 115
patenting, 109
pesticide consumption, 116
precautionary principle, 118–119
risk assessment, 124–125
terminator technology, 122–124
toxin presence as, 116–118
Goitrogens, 117
Golden rice, 94–96
Greenpeace, 107
Griseofulvin, 21
Groundwater, 27
Gun cotton, 16
Haploid breeding, 113
Heavy metals, 26, 32
Helicase, 54, 55
Helices, 52–54
Hemophilus influenzae, 61
Hepatitis B antigen, 98–99
Herbicide tolerance
criticisms of GMOs and, 119
genetic engineering and, 79–82
protein structure and, 56–57
public resistance to, 120–121
tobacco, 10
value of, 86–87
Heritage varieties, 109
High-fructose syrups, 24–25
Hind III enzyme, 61
Human immunodeficiency virus
(HIV), 71
Human toxicity, 82
Hybridization, 75, 108–109
Hydrocarbons, 28
Hydrogenation, 93–95
Hydrogen bonds, 52–55
Immunizations, 98–100
Inbred lines, 108–109
Indicator species, 32–33
Insecticides, 88–89, 89–92
Insulin, 63
Invertase, 22
Ionizing radiation, 73
Jorgensen, Rich, 87–88
Krebs cycle, 18, 19
Labeling, GMOs and, 123
139
Lactic acid, 14, 19, 20
Lactobacillus, 20
Lake Ontario, 28
Landfills, methane and, 40–41
Landraces, 109
Lepidopterans, Bt and, 90–91, 116
Liberty, 80–82
Libraries, cDNA, 70–71
Ligase, 54, 74
Lipids, 95
Locoweed, 32
Lodging, 112, 113
Lysine, feedstock and, 96
Maize, transformation of, 76
Mannitol, protoplasts and, 73–75
Marker genes, 64, 65
Marquis wheat, 108
Marsh gas, 40–41
Measles, edible vaccines and, 100
Messenger RNA (mRNA), 57, 58, 59,
70–71, 87–88
Metabolites, production of, 20–25
Metals, 26, 32
Methane, biofuels and, 40–41
Methylation, 60
Microbiology, Pasteur and, 14
Micropropagation, 36–40, 113–114
Molecular farming, 97–102, 120
Monarch butterflies, Bt and, 90–91
mRNA, 57, 58, 59, 70–71, 87–88
Mutagens, chemical, 73
Mutation breeding, 112
Mutations, 71–73, 112
Nanotechnology, 76
National Center for Biotechnology
Information, 10
Natural gas, methane and, 40
Nature, 52–54, 90
Neuberg, C.A., 14–15
Nickel, phytomining and, 34
Nitroglycerin, 16
Nobel, Alfred, 16–17
Nobel Prize, 17, 61, 62
Norwalk virus, 99
Nucleotides, 50, 51, 59
Nutrition, improving, 93–96
Oil refineries, 26–27
Oils, 41–44, 46, 93–95, 100
Oilseed rape, 93, 94, 122
Open-pollination, 109
Orchids, 39
Organic farming, 116, 122
Origins of biotechnology, 4–7, 14
Osmosis, protoplasts and, 73–75
Overexpression, defined, 80
Oxygen, 18–19, 44
Pandora’s Picnic Basket, 114
Papain, 24
Paper mills, 34
Pasteur, Louis, 14, 15
Pasteurization, 114
Pathogens, 39–40
Pauling, Linus, 52–54
PCR. See Polymerase chain reaction
Peanuts, aflatoxins and, 117
Pectinase, 22
Penicillin, 21, 23
Penicillium chrysogenum, 23
Perchlorate, 27
Pesticides, 116, 119
Petroleum products, 28
Petunias, pigmentation and, 87–88
Phages, 60–62
Pharming, molecular, 97–102, 120
Phytoaccumulation, 34–35
Phytochelatins, 33
Phytoprospecting, 32–33
Phytoremediation, 32–35
Plant cell transformation, 73, 75–76
Plant oils, biofuels and, 41–44
Plasmids, 62–64, 65, 79
Plastics, biodegradable, 100–102
140
Pollination, 88–90, 109, 122
Polyhydroxyalkanoates (PHAs),
100–102
Polymerase chain reaction (PCR),
65–66, 74
Polymerases, 55, 74
Potatoes
alkaloids in, 116–117, 118
disease resistance and, 75
gasohol and, 44
micropropagation and, 40
molecular farming and, 99
somaclonal variation (SCV) and,
114
Precautionary principle, 118–119
Precision, as advantage, 111
Prince William Sound, 28
Probes, fingerprinting and, 66
Productivity, defined, 9, 92, 92–94
Proteases, 22, 117
Proteins, 56–60, 89–92
Protoplasts, 73–75
Pseudomonas, 28
Pyruvic acid, 18–19
Radiation, ionizing, 73
Rapeseed, 93
Reassortment, 35
Recombinant DNA (rDNA), 8, 61–62,
67
Rennet, industrial production of, 22
Rennin, 6, 7, 8, 24
Replica plating, 64
Replication, 54, 62–64, 74
Resistance, 88–92. See also Herbicide
tolerance
Respiration, 17–19
Restriction enzymes, 60–62, 62–64,
65–66, 67
Retroviruses, 70–71
Reverse engineering, 71
Reverse transcriptase, 70–71
Rhizopus, 20
Riboflavin, 22
Ribonucleic acid (RNA), 50, 57
Ribosomes, 59
Rice, Golden, 94–96
Risk, assessment of, 124–125
RNA interference (RNAi), 87–88
RNA silencing, 87–88
Roundup, 79–82, 86–87, 120–121
Rubber, synthetic, 16
Russet Burbank potatoes, 114
Saccharomyces cerevisiae, 5, 19
Safety, 114–117, 118, 119–124
Salix species, 35
Saturation, 93–95
Sedges, bioremediation and, 26
Seed oils, 41, 93–94
Selection, 64, 108
Selenium, 32, 34
Sequestration, 33
Serotonin, bananas and, 117
Sewage treatment plants, 40
Shoot-tip culture, 38
Shotgun biolistics, 76, 77
Silencing, 87–88
Silos, methane and, 41
Smith, Hamilton, 61–62
Solanine, 116–117, 118
Solanum tuberosum, 75
Somaclonal variations, 40, 113–114
Somatic hybridization, 75
Soybeans, 20, 44–46, 81
Soy sauce, 20
Starches, gasohol and, 43–44
Start codons, 59
Stearic acid, 94
Sterile technique, 17, 36
Sterility, 122–124
Sticky ends, 62
Streptomyces hygroscopicus, 80–82
Sufu, 20
Sugar cane, 24, 43–44
Sugar-phosphate backbone, 52–55
141
Suicide seeds, 122–124
Sunflower oil, biofuels and, 41
Suppression, RNA and, 87–88
Swamp gas, 40–41
Sweet potatoes, 44
Tempeh, 20
Temperature, PCR and, 74
Teosinte, 108
Terminator technology, 122–124
Thallium, phytomining and, 34
Thermal properties, 101–102
Thlaspi species, 33
Ti plasmids, 79
Tissue culture, 36
Tobacco, 10, 97–98
Tobacco mosaic virus (TMV), 72
Tomatoes, 115
Totipotency, 38
Toxicity, human, 82
Toxins, in all foods, 116–118
Trace elements, 34
Transcription, 57
Transfer RNA (tRNA), 58, 59
Transformation
of bacteria, 62–63, 64
of plant cells, 73, 75–76, 76–79
Translation, 58
Triazine resistance, 56–57
Triglycerides, 41, 42, 95
Uniformity, clones and, 35–36
Vaccines, 98–100
Variability, genetic, 109
Variations, somaclonal, 40
Vegetative reproduction, 36
Vetch, 32
Vinegar, 19
Viruses, 39–40, 60–62, 70–72, 92
Visualization, 66
Vitamins, 22, 96
Waste management, 26
Watson, James, 52–53
Weeds, control of, 86–87
Weizmann, Chaim, 16
Weizmann process, 16, 17
Wetlands, 26
Wheat, 108, 112, 120–121
White, Phillip, 36
Wilkins, Maurice, 53
Willows, 35
Wines, 5
Yeasts, fermentation and, 5
Yields, 9, 92–94
Zea mays, 76
Zymology, defined, 14
Zymotechnology, 14
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143
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 university’s 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.

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 All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage or retrieval systems, without permission in writing from the publisher. For information, contact: Chelsea House An imprint of Infobase Publishing 132 West 31st Street New York NY 10001 Library of Congress Cataloging-in-Publication Data Hopkins, William G. Plant biotechnology / William G. Hopkins. p. cm. — (The green world) Includes bibliographical references and index. ISBN 0-7910-8964-9 (hardcover) 1. Plant biotechnology—Juvenile literature. I. Title. II. Green world (Philadelphia, Pa.) SB106.B56H67 2006 630—dc22 2006008157 Chelsea House books are available at special discounts when purchased in bulk quantities for businesses, associations, institutions, or sales promotions. Please call our Special Sales Department in New York at (212) 967-8800 or (800) 322-8755. You can find Chelsea House on the World Wide Web at http://www.chelseahouse.com Text and cover design by Keith Trego and Ben Peterson Printed in the United States of America Bang FOF 10 9 8 7 6 5 4 3 2 1 This book is printed on acid-free paper. All links, web addresses and Internet search terms were checked and verified to be correct at the time of publication. Because of the dynamic nature of the web, some addresses and links may have changed since publication and may no longer be valid.

Introduction

vii 2 12

1 What Is Biotechnology? 2 The Early Days of Biotechnology 3 Plants and Biotechnology
Before Recombinant DNA

30 48 68

4 DNA, Genes, and Protein 5 Engineering Plants 6 Genetically Modified Plants
From Herbicides to Vaccines

84

7 Putting Genetically Modified
Organisms in Perspective
Glossary Bibliography Further Reading Index

104 127 133 134 136

.

and the oxygen we breathe. and install plants and flowers in our homes and workplaces. World exploration and discovery was driven by the search for herbs and spices. Gifts of flowers are the most popular way to acknowledge weddings. and other events of passage. humans and other animals simply could not exist. Psychologists tell us that plants also provide a sense of well-being and peace of mind. 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. The rise of agriculture in the fertile crescent of Mesopotamia brought previously scattered hunter-gatherers together into villages. Ever since. Indeed we should thank green plants for providing the food we eat. vii . the availability of land and water for cultivating plants has been a major factor in determining the location of human settlements.By William G. fiber for the clothing we wear. which is why we preserve forested parks in our cities. Human history has been strongly influenced by plants. funerals. The cultivation of new world crops—sugar. Hopkins “Have you thanked a green plant today?” reads a popular bumper sticker. surround our homes with gardens. wood for building our houses. Without plants.

viii IntroductIon cotton. The Irish Potato Famine in 1847 set in motion a wave of migration. what plants do. Plant Biotechnology traces the development of biotechnology from prehistory to the present. Plants are so much a part of our environment and the fabric of our everyday lives that they rarely register in our conscious thought. and discusses the present impact and future potential of genetically modified plants. and where plants fit into the overall scheme of things. As a young university instructor directing biology tutorials in a classroom that looked out over a wooded area. More often than not the question would be met with a blank. It shows how plants are genetically engineered. Yet today. questioning look. faced with disappearing rainforests. and concerns about climate change. exploding population growth. weighs the risks and benefits of this new technology. the human and social consequences of which are still with us. I would ask each group of students to look out the window and tell me what they saw. that would reduce the population of Ireland by half over the next 50 years. the productive capacity of global agricultural and forestry ecosystems is put under increasing pressure. 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 series describes what plants are. urban sprawl. mostly to North America. and tobacco—was responsible for the introduction of slavery to America. 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. .

.

What Is Biotechnology?  .

—George Barnard Shaw (1856–1950) Irish playwright .All problems are finally scientific problems.

It had an unfamiliar but pleasant taste. His vision began to blur and he felt dizzy. because the origins of brewing beer and other intoxicating beverages have been lost in antiquity. he thought of the tales told by the elders in the village—tales 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. We do know that beer or a similar form of fermented beverage has been brewed and drunk by various civilizations for thousands of years. As he sat resting by the remains of the cooking fire.What Is Biotechnology? It was late when he returned to the village. he drank the liquor in which it had sat. and there was always more than enough grain to feed the village. It had rained recently and water had collected in the bowl. to slake his thirst. And as he sat and thought. there were agricultural villages established in the eastern Mediterranean area known as the Fertile Crescent. across Syria. Searching for something to eat. “I should go into the hut and sleep. Beer. he began to feel strange sensations. 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. He had been hunting for meat and now he was hungry. so he ate the rest of the grain and. but we don’t really know.” but he had difficulty trying to rise and fell asleep where he sat. and Freshly Baked Bread Is it possible that early man discovered beer in this way? Perhaps. Archeological evidence tells us that as early as 11. an area extending from the flood plains of the Tigris and Euphrates rivers in what is now Iraq. He thought. The grain was sprouting. but he took a handful anyway. and the other villagers were asleep in their huts. Cheese.000 years ago. and down the eastern  . The moon had risen. They no longer had to move about. he spotted a bowl of grain that had been left sitting beside his hut for several days.

wine. fermentation was the result of chance contamination. and the amylase was often provided by chewing some of the plant material and spitting into the “brew. like wine. The yeast converts the glucose (C6H12O6) to ethyl alcohol (ethanol. beerlike brews were made from other starchy plant products. It seems that breadmaking and brewing were both early technologies associated with the beginnings of agriculture. units.000 years ago. In other primitive societies.What Is Biotechnology?  coast of the Mediterranean to the Nile Valley of Egypt. breaking down the starch into its component glucose. and bread involves the fermentation of the sugar glucose by a single-celled fungus called yeast. in the beginning at least. It is known. Cheese. Wine is a little easier to come by since the yeasts used to ferment the juices grow naturally on the surface of the fruit. or sugar. another fermented beverage. The production of beer. C2H5OH) and the gas carbon dioxide (CO2): —> C6H12O6 — — 2C2H5OH + 2CO2 In the process of brewing beer.1). Another early development was the production of cheese (Figure 1. usually a strain of Saccharomyces cerevisiae. for example.” Amylase is a natural component of human saliva. is a means for preserving food . Among the crops being domesticated were wheat and barley (used to produce beer) and grapes (for wine). Wine. moistened bread more than 9. The yeast that ferments the glucose is found naturally in the environment and. the germinating grain (usually barley) secretes an enzyme called amylase. that the ancient Sumerians were producing a beer made from fermented. The amylase acts on the starch in the endosperm (the nutritive tissue) of the seed. was also produced by the Sumerians as well as the ancient Egyptians. The first wines would have been easily produced by simply allowing grape juices to stand for a few days.

 Plant Biotechnology Figure 1. The reason. an enzyme that coagulates the milk proteins (albumin and casein). is that the lining of a calf ’s stomach produces rennin. He later discovers. although countless legends 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.1 A cheese maker stirs a cauldron of curd and the watery part of milk known as whey. and bears the same relationship to milk as wine does to grapes. so the legend goes. as we now know. The origin of cheese is equally obscure. Cheese was well known . Cheesemaking is one of the oldest examples of biotechnology. that the milk has turned into a slightly sour mixture of watery fluid and curds.

bread.5%).000 years ago and has been made wherever domesticated animals produced more milk than could be immediately used by the people. Fortunately. When preparing bread. there was probably little value. wine. young goats. When the dough has finally risen and is ready to bake. other than perhaps ceremonial. there are some fungi that produce extracellular . 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. rely on the production of alcohol for their characteristic beverages. Coagulation of milk proteins is a natural step in the digestion of milk by calves. on the other hand.What Is Biotechnology?  to the Sumerians at least as far back as 6. worldwide cheese production has outstripped the supply of slaughtered calves. which causes the flour proteins. Although rennin was formerly obtained from calf stomachs. to the production of beerlike beverages. The breadmaker is not interested in alcohol production but takes advantage of the carbon dioxide gas to provide texture. MakIng OrganIsMs WOrk FOr Us What do beer. Wine production. to form an elastic network. In early societies. but this is driven off during baking and contributes to the enticing aroma that we associate with freshly baked bread. and other young mammals. and cheese have to do with biotechnology.and winemakers. Beer. of course. 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. called glutens. Humans have taken advantage of the enzymes involved to process and preserve the milk as food. the dough is kneaded. has value as a means for preserving grapes or other fruits. it also contains a significant amount of alcohol (as much as 0.

These new technologies have taken us far beyond just using biological fermentation to process foods. that will coagulate milk. and cheese were. beer. Engineers use the term bioengineering when engineering techniques are applied to biological processes. in fact. ethanol for industrial purposes and as a gasoline additive. biotechnology has focused on processing food for humans and cattle. The first humans to discover bread. but for most of us the terms bioengineering and biotechnology are interchangeable and generally refer to any application of technology to living systems. The silage. 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. The truth is that humans have been using other organisms to produce new products for a very long time. most people think biotechnology is a very recent invention. but we also hear about genetic engineering or recombinant DNA (rDNA). engineers have used fermentation to produce industrial feedstocks such as acetic acid and citric acid. We hear about genetic modification (GM) and genetically modified organisms (GMOs). the world’s first biotechnologists and we have been eating the products of biotechnology for thousands of years. including rennin. wine. Over the past 30 years.2). More recently. biotechnology has come to mean something very different in the eyes of the general public. . For most of history. 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. drugs such as penicillin. that becomes feed for livestock is stored by cattle and dairy farmers in silos and also is a fermented product.” Because the word has only recently entered popular usage. and to treat sewage. Plant Biotechnology (secreted into the organism’s environment) enzymes. Fungi are now the principal commercial source of rennin for the cheese-making industry. or fodder.

Scientists typically modify the genes of plants in order to increase yields or improve the nutritional quality of a plant. .What Is Biotechnology?  Figure 1. a type offungus.2 A scientist looks at root growth on wheat plants that have been genetically modified to resist infection by Fusarium.

10 Plant Biotechnology This new biotechnology has unleashed a storm of public controversy. In the chapters that follow. A Google search for plant biotechnology alone will bring up over 15 million hits. in the Webster’s Encyclopedic Dictionary of the English Language published in 1988. of course. there is both benefit and risk involved with biotechnology and the truth will lie somewhere between these two extremes. Today. the word biotechnology had already become well established as part of the dictionary of the scientific and academic world. Proponents see the opportunity to help eradicate malnutrition. and most major universities have formal biotechnology courses or programs. the word biotechnology is not listed. had been created in 1983. if you “google” biotechnology. and in 1986 the Environmental Protection Agency (EPA) approved the release of an herbicide-resistant tobacco variety. starvation. As with most new technologies. a tobacco plant resistant to an antibiotic. . Opposition activists see scientists “playing God” and unleashing unspeakable monsters and ecological havoc. on human and animal biotechnology. with an emphasis. In 1988. however. it has only recently become part of the public consciousness. For example. we will present an overview of What's in a Word? Even though the origins of biotechnology can be traced back thousands of years. By that same year. you will bring up over 119 million “hits. and scarcely a day goes by that you cannot find a reference to biotechnology in major newspapers. and genetic diseases. less than 20 years later.” There are thousands of biotechnology companies around the world. To help us better understand recombinant DNA technology. we will trace the history of traditional biotechnology and how humans have manipulated microorganisms and plants over time. The first genetically modified plant. the National Library of Medicine together with the National Institutes of Health (NIH) established the National Center for Biotechnology Information.

however. Most people now understand the term biotechnology to mean the manipulation of an organism’s genes to create genetically modified organisms (GMOs). but they harness the activities of living organisms. We will show how plants are “genetically engineered” and how this new technology compares with traditional methods for producing new food plants. the word biotechnology has taken on a whole new meaning. In the final chapter.What Is Biotechnology? 11 DNA and genes and look at how this new biotechnology came into being. This book will trace the development of biotechnology from its early beginnings to the present.” Beginning in the 1980s. . 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. we will examine some of the moral and ethical questions surrounding genetically modified organisms. We will dispel some of the myths surrounding genetic modification and review the present impact and future potential of genetically modified plants. and help the reader make an informed judgment about the role of biotechnology in the future. explain the science behind biotechnology. Using living organisms to process foods and to manufacture other products that are useful to humankind is commonly referred to as “biotechnology.

The Early Days of Biotechnology 12 .

linked together as fruit to the tree that has borne it. —Louis Pasteur (1822–1895) French chemist and microbiologist .There are science and the applications of science.

when French biologist Louis Pasteur (1822–1895) discovered that the fermentation of lactic acid was due to the action of live microbes. but it also stimulated a general interest in brewing and other types of industrial fermentation. The industry represented a marriage of microbiology with chemical engineering that became known as zymotechnology (from zymology. the scientific study of fermentation). We tend to treat “factory farms” as a recent North American innovation. In 1911. In an effort to modernize Hungarian agriculture in the early years of the twentieth century. und Milcherzeugung im Landwirtschaftlichen Großbetriebe (“Biotechnology of Meat. Karl Ereky. Fat. nearly 100 years ago. The first recorded use of the term biotechnology is credited to a Hungarian agricultural engineer. were common in Germany and eastern Europe in the early twentieth century. The term zymotechnology has since been replaced with the more encompassing term biotechnology. 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.000 pigs a year. Ereky’s enterprise became one of the world’s largest and most productive meat operations. Ereky sought and obtained funding to support agriculture on an industrial scale. German scientist C.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. Ereky published a tract with the German title Biotechnologie der Fleisch-. A. His farm covered 50 hectares (110 acres) and turned out over 100. In 1919.1). Two manufacturing processes illustrate the “state of the art” of biotechnology shortly after the turn of the twentieth century. especially for pigs. Neuberg showed that significant 14 . and Milk Production in Large-scale Agricultural Industry”). Fett-. but large-scale farms. Pasteur’s discovery not only gave rise to the science of microbiology and contributed to the salvation of the French silk and wine industry. or microorganisms (Figure 2.

Four years later. which is the process of heating a beverage or other food in order to destroy harmful microorgansims. He is perhaps best known for inventing pasteurization. German industry was able to produce more than 12.1 Louis Pasteur was a pioneer in the field of biotechnology.The Early Days of Biotechnology 15 Figure 2.200 pounds) of glycerol per year.000 kilograms or 2.000 tonnes (a tonne is a metric ton = 1. amounts of glycerol could be produced by fermentation of starch. .

Nitrocellulose was also the source of gun cotton. a group of organic chemists at Manchester University began working on the rubber problem. (Yes. thus producing dynamite. nitrocellulose was the film on which the early stars of Hollywood were immortalized. Chaim Weizmann (who would later become the first president of the state of Israel). Rubber and explosives—it is not difficult to draw connections between the direction taken by bio- . although British plantations were being established in Malaysia. the best solvent known for the new plastic nitrocellulose. the world needed a good synthetic rubber. the early films produced by Hollywood were highly flammable!) And what about all that glycerol being produced by fermentation in Germany at the same time? When glycerol is chemically reacted with nitric acid. a second product of the so-called Weizmann process was acetone. In order to break the Brazilian monopoly on rubber. Brazil held a monopoly on “wild” rubber trees. Swedish inventor Alfred Nobel discovered that the explosive power of nitroglycerin could be stabilized by absorbing the liquid on an inert (non-reactive) powder. Dynamite made Nobel a very rich man and. on his death. At that time. Nitroglycerin. In 1912. Interestingly. With the rise of the automobile industry. In 1867. a reliable supply of rubber was becoming increasingly important. In addition. a component of high explosives. which is extremely unstable and highly explosive. the raw material for producing a superior synthetic butyl rubber. the product is nitroglycerin. was the explosive of choice for early miners. Fortuitously. acetone and butanol together could be used to make isobutyl acetate. Butanol was readily converted to butadiene. he endowed the international prizes that bear his name.16 Plant Biotechnology At the same time in Britain. however. The Manchester University group included a young Russian immigrant. Acetone was another commercially important chemical as an ingredient in the manufacture of explosives. Weizmann was instrumental in developing a fermentation method to produce butanol.

The initial steps of respiration and fermentation. and elephants all share many of the same genes and do things. When oxygen is present. Glucose. of course—and did not require aseptic (sterile) conditions. the same pathway is called fermentation. not just for its economic impact. however.2). a process called glycolysis. and modern materials that was to dominate biotechnology until the 1980s. Weizmann’s process sealed a partnership between microbiology. the pathway used is virtually identical in all living organisms. when recombinant DNA came on the scene. complex storage molecules such as starch (plants) or glycogen (animals) must first be broken down into their component glucose molecules. oak trees. In spite of their striking differences. required that laboratory standards for sterility be maintained on an industrial scale and production was carried out in modern aluminum fermentation vessels. in much the same way. Most industrial fermentations up to then were carried out in oak casks—continuing in the brewing tradition. In preparation for respiration or fermentation. For example.The Early Days of Biotechnology 17 technology in the years prior to 1918 and the developing political climate of the time. and proteins to retrieve energy. which culminated in World War I. organisms as diverse as fungi. is further broken down through glycolysis (Figure 2. this pathway is called cellular respiration. are the same. chemical engineering. when organisms break down sugars. on the other hand. The Weizmann process proved to be significant in many ways. is different depending on whether or not oxygen is available. The Weizmann process. When oxygen is absent. earthworms. WhaT iS fERmEnTaTion? One of the more interesting things about nature is its extreme conservatism. fats. The end result. a simple sugar made up of six carbon atoms. in a metabolic sense at least. The net result of glycolysis is that one six-carbon molecule (glucose) is converted to two three-carbon molecules called .

. The pyruvic acid molecules are broken down further via two different pathways depending on the presence or absence of oxygen. and the carbon atoms in the glucose have been slightly rearranged. pyruvic acid. A small amount of energy is released. but otherwise not a lot has happened up to this point.18 Plant Biotechnology Figure 2.2 Glycolysis is the breakdown of glucose into pyruvic acid inside cells.

When oxygen is present. Lactic acid can be converted to isoprene. the pyruvic acid enters a metabolic pathway called the citric acid cycle (or Krebs cycle). a variety of end products are possible. cerevisiae produces glycerol instead of ethanol. and it is also the fermentation product of certain fungi and bacteria. Contamination of wine with Acetobacter can be a problem for wine producers because the acetic acid sours the wine. S. but since the beginnings of biotechnology are so intimately associated with the fungi. PRocESSing fooD anD DRink In modern systems of classification. and other industrial products. which can be used in the manufacture of synthetic butyl rubber. This is what we call fermentation. Depending on the organism and the conditions of fermentation. where it is completely broken down into carbon dioxide and most of the energy is retrieved for use by the cell. • Lactic acid: The fermentation product in human muscle when exercising under oxygen debt. the citric acid cycle shuts down and the pyruvate undergoes more limited changes. In addition to beer. • Ethyl alcohol (ethanol): The fermentation product of several fungi. • Glycerol: Under alkaline conditions. plastics. however. fungi are no longer considered plants. and bread.The Early Days of Biotechnology 19 The difference between respiration and fermentation lies in the fate of pyruvic acid. In the absence of oxygen. Lactic acid is responsible for muscle soreness. we will consider them as close cousins to the plants and include them in our discussions. This is what normally happens in your own cells to provide the energy the cells need to function. fungi are used in the processing of many different . wine. especially Saccharomyces cerevisiae. • Acetic acid (vinegar): The fermentation product of the bacterium Acetobacter. Acetic acid is widely used as a raw material in the manufacture of fibers.

that have commercial use on their own. higher purity. The tank is then inoculated with the appropriate organism. and supplemented with vitamins and amino acids to ensure rapid. and nutritional value of foods as well as to delay spoilage. a condiment prepared by fermenting soybeans and wheat with Aspergillus oryzae. As noted earlier. or chemical products of metabolism. flavor.1). and enzymes (Table 2. In addition to fungi. and more often at lower cost than by direct chemical synthesis. sufu. alcohols. vitamins. the fungi are used to improve the texture. or sealed .20 Plant Biotechnology foods. changing both the flavor and the composition of foodstuffs. In almost all cases. China. a solid food prepared by processing soybeans with the fungus Rhizopus species. inDuSTRial PRoDucTion of fungal mETaBoliTES We have seen how fungi have been used to process foods. bacteria have also proven useful in traditional biotechnology. In Europe and North America. In Japan. As with beer and wine. and other Asian countries. healthy growth of the microorganisms. a large variety of foods are prepared from soybeans (Glycine max). especially in Asia. antibiotics. Fungi also produce a wide range of metabolites. The tank may be aerated. if the culture requires oxygen. a Chinese cheese prepared from soybeans with the help of the fungus Actinomucor elegans. bacteria produce acetic acid. These include tempeh. such as cane syrup or molasses. which is used in preserving and flavoring foods and as an important industrial feedstock. These products include organic acids. Large-scale production of chemicals by fungi is usually carried out in vats or tanks with a capacity of several thousand liters. and soy sauce. lactic acid produced by the bacterium Lactobacillus has long been used to preserve cabbage (sauerkraut) and naturally fermented pickles. The vat is filled with a watery solution of some carbon source. modern industrial fermentation methods have made it possible to produce these products in larger amounts.

Either way. used in inks and the dye industry Alcohols Ethyl alcohol Industrial solvent. acetic acid. with various filters.The Early Days of Biotechnology 21 if anaerobic conditions are required. Commercial production may involve either batch culture. medicines. soft drinks. and cooling coils. synthetic rubber Glycerol Drugs Penicillin Griseofulvin Oral and injectible antibiotic Oral and topical antibiotic (continued on page 22) Glycerin.1 Commercially Important Fungal Metabolites Produced by Biotechnology Metabolite Organic Acids Citric acid Use Flavoring ingredient in candies. in which the culture medium flows through the tank and production of the desired metabolite continues indefinitely. medicines. The culture tanks are often cylindrical and. or continuous culture. explosives . Table 2. actively growing organisms release a lot of heat. Once the culture has run its course. so the culture will be cooled by circulating water through coils surrounding the tank. in which the vat is emptied after the reaction runs its course. raw material in the manufacture of materials such as ether. We will have more to say about the continuous culture method later. the cells are separated from the culture medium and the metabolite of interest is extracted by filtration and chemical extraction. pumps. the entire setup may resemble a miniature oil refinery.

but now virtually all of it is produced industrially by the fungus Aspergillus niger. . and blood transfusions. electroplating. and frozen fruits. Citric acid was originally obtained from lemons. jellies. Citric acid is widely used in pharmaceuticals. nutritional supplement. It is used in hair rinses. Annual production worldwide exceeds several hundred million kilograms (1 kg = 2. effervescent products.2 pounds). removal of pectins before concentrating fruit juices Proteases Hydrolysis of proteins during food processing One of the most important organic acids produced commercially from large-scale fungal cultures is citric acid. and leather tanning. It is used as a flavoring in beverages and foods (especially gelatin powders and soft-drink crystals). desserts. and coloring agent for margarine Vitamins and Growth Factors Enzymes Amylase Rennet Invertase Pectinase Conversion of starch to sugars prior to fermentation Milk coagulation in the manufacture of cheese Hydrolyzes sucrose (table sugar) to glucose and fructose Pretreatment of fruit juices to remove turbidity.22 Plant Biotechnology (continued from page 21) Metabolite B vitamins Riboflavin Gibberellin Beta-carotene Use Nutritional supplement and medical therapy Nutritional supplement and medical therapy Plant growth hormone with commercial applications in floricultural Synthesis of vitamin A (provitamin A). candies.

as a biofuel. Now. had been contaminated by a mold. Penicillin was discovered in 1929 when Alexander Fleming (1881–1955) observed that his cultures of a pathogenic (disease-causing) bacterium. so the active principal was called penicillin. after which the fungus is filtered off and the penicillin is chemically extracted and purified from the liquor that remains. The contaminating fungus was identified as a species of Penicillium. The increased need for effective antibiotics as a result of World War II stimulated the search for efficient methods for large-scale production. was surrounded by a clear ring where it had killed the bacteria in its immediate vicinity. the oldest form of biotechnology. the largest and most important products of commercial bioengineering are antibiotics. but molasses is comparatively expensive and occasionally in short supply. a by-product of corn starch manufacturing. as much as 75% of the industrial ethanol production was achieved by fermentation and subsequent distillation of molasses. Although penicillin was recognized as an effective antibiotic. especially penicillin. The corn steep liquor is inoculated with the fungus and incubated for five or six days. It became cheaper to produce ethanol from petroleum. with increasing concerns about diminishing oil supplies and a focus on renewable . it found little use because its production was difficult and yields were low. The mold. Ethyl alcohol production as an end product of anaerobic fermentation in beer and wine production is. A third example of industrial bioengineering is the production of ethanol. as we have seen. which at the time appeared to be relatively inexpensive and abundant. Ethanol also has many industrial uses as a solvent and. In the early part of the twentieth century. Penicillin is produced commercially using strains of Penicillium chrysogenum cultured on a medium such as corn steep liquor. Staphylococcus aureus. growing in a petri dish. more recently. 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 Early Days of Biotechnology 23 Still.

High-fructose syrups are also used extensively in the baking. Other potential sources of carbohydrate for ethanol production include starches and sugars of sweet potatoes and the cellulose that makes up wheat straw. Bacteria and fungi are also the source of numerous enzymes that have commercial application. We have already mentioned rennin. These too must be treated with enzymes before beginning the fermentation process. which is readily fermented. The industrial use of enzymes again illustrates the synergies between biotechnology and chemical engineering. Because enzymes are organic catalysts. canning. the favored substrate (material being fermented) is corn starch.24 Plant Biotechnology resources. they use the same enzyme over and over again in . or table sugar). attention has once more turned to fermentation as a source of industrial ethanol. When starch is broken down completely. but the starch must first be treated with enzymes that break the starch into soluble sugars. organisms do not need to make large quantities of them. high-fructose corn syrups derived from corn starch have completely replaced cane sugar (sucrose) in soft drinks. In North America. Glucose isomerase is the enzyme that plants and many other organisms use to convert glucose to another 6-carbon sugar called fructose. used in the production of cheese. Protein-degrading enzymes such as papain are used to clarify beer. Instead. Enzymes are expensive to produce and use. Glucose isomerase is an example of an enzyme that has given rise to a major industry. and foodpreserving industries. Yeasts can then ferment the soluble sugars into ethanol. To perform enzyme conversions by batch culture can be inefficient and costly. The high sweetness of fructose means that much less sugar—and therefore fewer calories—is required. Beginning in the 1960s. Brazil has led the way in ethanol production because it has abundant supplies of cane sugar. The reason that high-fructose syrups are so popular is that fructose is twice as sweet as conventional sugar (sucrose. the product is the 6-carbon sugar glucose.

The Early Days of Biotechnology 25 Figure 2. to form a bioreactor. In the early 1960s. . or enzymes are used to break down harmful substances or create useful products. 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. 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. order to produce large amounts of product. A bioreactor is a vessel in which microorganisms. Fructose is then produced continuously simply by passing a stream of glucose solution through the reactor.3). cells. such as glass beads or cellulose.3 The components of a bioreactor are seen in this illustration.

The contaminant is broken down by the bacteria as it passes through the reactor and purified water comes out the other end. Home and municipal septic systems are the classic example. The bacteria are cultivated on an inert bed (activated carbon is commonly used) in a cylindrical reactor chamber and the water to be treated is pumped through from the bottom. the effluent enters an artificial “wetland” planted with a mixture of sedges (Carex spp. Bacteria are now being used extensively to treat industrial wastes other than sewage and to clean up contaminated groundwater. If the effluent contains heavy metals. reeds (Phragmites spp. 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 remediation meaning “to correct a fault”).26 Plant Biotechnology WaSTE managEmEnT anD BioREmEDiaTion One area where traditional biotechnology has proven particularly effective is in waste management and remediation of waste water. The plants serve two primary functions: they hold the bacteria in place and.).). where bacteria have long been used to degrade solid wastes.). and cattails (Typha spp.). 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. bulrushes (Juncus spp. these may also be removed by plants that grow in the artificial wetland. The systems used are generally like that described above for enzymes. Small-scale pilot projects have also demonstrated that plants can also play a role in sewage management. through the process of transpiration. We will return to this interesting property of plants in chapter 3. maximize the evaporation of water into the atmosphere. The wetland provides for additional purification of the wastewater by aerobic bacteria. After a primary treatment in a septic tank. Other areas where this general approach has proven beneficial include decontamination of soils at decommissioned oil .

The latter is a particularly serious problem in California. pharmaceuticals. Most bioreactors consist of tanks surrounded by pumps and pipes that move fluids and gases into the reaction chamber. Utah. bench-top devices for laboratory experimentation and testing up to large industrial versions that process several thousand liters. and Texas. Bioreactors may range in size from small.The Early Days of Biotechnology 27 refineries and removal of perchlorate from groundwater. an entire industry has developed around the design and manufacture of bioreactors using batch culture or immobilized cells for the production of enzymes. hormones. where ammonium perchlorate (NH4ClO4) was used for decades as an oxidizing agent in solid propellants and explosives. plant cells. vaccines. and a host of other useful chemicals. Discharge from manufacturing operations and from replacement of outdated fuels in military missiles and rockets was and remains a major source of groundwater contamination. provide cooling. used broadly to indicate any vessel or container where organisms are used to produce a product. Different strains of bacteria such Bioreactors A term that has begun to permeate the bioengineering field is bioreactor. The organism may be microorganisms. Nevada. Bacteria are now being used extensively to clean up the groundwater by breaking the perchlorate down into harmless chloride and water. 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. fermentation vats used to produce citric acid or penicillin would be considered bioreactors. Indeed. or animal cells. and remove effluent for downstream chemical processing. Similar innovative strategies are being used to clean up a wide range of toxic chemicals in various environmental situations (Figure 2.4). . In that sense.

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. In one such situation.28 Plant Biotechnology as Pseudomonas and Escherichia coli are capable of degrading hundreds of different toxic chemicals. Initially. Finally. such a steam cleaning and rinsing. Although the process is not yet complete. Nutrients were supplied to encourage the growth of microorganisms already present in the soil. The term biotechnology was first used by Karl Ereky in the early twentieth century in the context of large-scale agricultural meat and milk production. were put in place to contain and remove large volumes of oil. and within two years the hydrocarbon levels were reduced by 97%. 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. Summary The origins of biotechnology as a science can be traced back to Pasteur’s discovery that fermentation was caused by microorganisms. diesel fuel. This stimulated the development of fermentation on an industrial scale to produce a variety of feed stocks that supplied the manufacturing industry. physical cleaning measures. In addition to producing products for the manufacturing . such as unrefined petroleum oil. The beaches were then treated with nitrogen and phosphorus fertilizers to accelerate the growth of oil-degrading bacteria that lived among the rocks and sand. several bioreactors were used to decontaminate the soil around a decommissioned refinery near Lake Ontario. where an area covering 15 acres was contaminated with hydrocarbons to a depth of three feet. or gasoline. Soils near oil refineries are often contaminated with hydrocarbons.

industry. and bacteria. and clean up contaminated soils.The Early Days of Biotechnology 29 Figure 2. control waste management. . process food and drink. nutrients. The polluted groundwater is pumped into a bioreactor that contains oxygen. biotechnology and bioengineering have been used throughout the twentieth century to produce drugs such as penicillin.4 This bioremediation system purifies groundwater that has been contaminated with gasoline. The microbes degrade the gasoline and the clean water is then pumped back into the ground.

Plants and Biotechnology Before Recombinant DNA 30 .

—Arthur C. Clarke (1917–) British author and inventor .Any sufficiently advanced technology is indistinguishable from magic.

This was called phytoprospecting. such as gold.Plants and Biotechnology Before Recombinant DNA Traditional biotechnology has not been limited to applications involving microorganisms. some plants may contain 200. selenium may account for as much as 10% of the dry weight of the seeds. Also known as milk vetch or poison vetch. a genus in the Fabaceae or pea family.” and prospectors would take the presence of such plants as an early indication that the soils may have contained a mineral of interest. plants had been enlisted to serve human needs in a variety of ways beyond their traditional role of providing food and fiber. if you are a rancher. such plants were known as “indicator species.1). and arsenic. Many years ago. In Astragalus. For some plants. I watched a lot of Bgrade movie Westerns and one thing I learned was that. There are many regions where natural geochemical processes have produced soils that are rich in metals such as nickel. for example. We now call these 32 . selenium. yet many plants actually thrive on soils rich in such metals. gold. cadmium. many species of Astragalus take up unusually large quantities of selenium from the alkaline soils of the western plains. PhytoRemeDiAtioN: CleANiNg UP With PlANts Have you ever heard of locoweed? As a child. Other plants actually take up the metals and accumulate them to levels that would be toxic to most other plants (Figure 3. Even before genetic modification arrived on the scene. you don’t want your cattle grazing on locoweed! Locoweed is the common name given to several species of Astragalus. the metals are not a problem simply because the cell membranes surrounding the root cells prevent the metals from entering the root. In soils that are rich in nickel. The high selenium content contributes to a disease known as alkali poisoning or “blind staggers” in cattle unfortunate enough to graze on this plant—the cattle literally behave as though they are crazy. chromium. just as they are to humans.000 times more nickel than plants growing in normal soils. Normally high levels of heavy metals would be toxic to plants.

where they cannot interfere with the cell’s metabolism.Plants and Biotechnology: Before Recombinant DNA 33 Figure 3. Thlaspi and other metal-accumulating plants can be used to remove heavy metal contamination from soils. 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 . The sequestered metals are then stored in the large central vacuole of the plant cell. which are not injured by high concentrations of heavy metals because they sequester (isolate) the metals with small proteins called phytochelatins. plants accumulator species.1 An agronomist examines the roots of a Thlaspi plant in a growth chamber.

for example. and molybdenum. But several enzymes require a twenty-first amino acid called selenocysteine which. manganese. Although plants require very small amounts of these trace elements in order to function properly. . for example. nickel. zinc. Phytomining. This is because selenium is part of a twentyfirst amino acid. but plants are much more cost-effective and would not create even more ecological problems as engineering-based technologies often do. however. non-protein molecules that must be present for an enzyme to function. The idea is to grow accumulator species on mine tailings and wastes from paper mills. Using plants to clean up soils is called phytoremediation (phyto meaning “plant” and remediation meaning “to correct a fault”).34 Plant Biotechnology of twentieth-century industrial activities. that trace elements (those required by organisms in very small amounts) become toxic when there is too much available. require several trace elements. Most trace elements serve necessary functions as enzyme cofactors. has proven effective in the recovery of both nickel and thallium in demonstrations. An additional benefit of accumulator species is that they begin the revegetation of barren industrial sites and assist in the recovery of useful metals. contains selenium. Phytoremediation would also help to stabilize contaminated sites because the plants help to control erosion. It is often the case. as it is called. where they would extract the heavy metals. of course. Plants will naturally take longer to do the job. Plants. any one of them can become toxic at relatively low concentrations. All the textbooks will tell you that proteins are synthesized from twenty “standard” amino acids specified by the “standard” genetic code. 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. copper. including boron.

there is profit to be had in genetically uniform lines that exhibit a particular trait or traits. yield. That may sound just a bit imposing. various species of willows (Salix) have shown promise for extraction of heavy metals from soils treated with sewage sludge. for example—the alleles from each parent are randomly reassorted when they are passed to the offspring.Plants and Biotechnology: Before Recombinant DNA 35 other trials. and plant scientists have been cloning plants long before the first cloned sheep. or clones. The result is that.” All organisms contain at least two copies. which makes their disposal much easier. During sexual reproduction—by seed. The advantage of using plants is that they can be harvested and burned. Dolly. flower color. miCRoPRoPAgAtioN: stARtiNg oUt smAll Cloning is one of the buzzwords of the new biotechnology (not to mention medical mystery stories and science fiction). It may be size. no two offspring resulting from sexual reproduction have exactly the same genetic constitution. is to . however. A clone can be defined as a genetically uniform assemblage of individuals derived originally from a single organism by asexual reproduction. or some other marketable characteristic. but the two keys concepts here are “genetically uniform” and “asexual reproduction. The only way to maintain a line of genetically uniform individuals. farmers. but gardeners. 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. except in the case of identical twins in mammals. 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. for every gene. called alleles. The heavy metals remain concentrated in the ash. arrived on the scene in 1996. The offspring are not genetically uniform. In the world of horticulture and agriculture.

that are in fact a single tree. where they will continue to grow. Another common method for vegetative reproduction is a form of biotechnology called micropropagation. these plantlets can be removed from the flask and planted in the greenhouse. One often sees “clumps” or small groups of poplar trees. or other laboratory glassware. the cells begin to divide and grow and form a clump of undifferentiated cells called a callus. Many common houseplants. By adjusting the culture conditions. a semisolid gel obtained from certain species of algae. and forest trees began life in a test tube or petri dish.” but the expression refers to the culture of organisms in test tubes. essential minerals. Once the roots and shoots are established. In tissue culture. Phillip White gave birth to the science of plant tissue culture when he successfully maintained cut tomato roots in vitro. for example. and hormones.2). The medium usually contains agar. asexual or vegetative reproduction. petri dishes. it is possible to stimulate the callus to form plantlets with roots and shoots. In vitro means literally “in glass. fruit trees. By the early 1980s. which uses plant tissue culture (Figure 3. or bacteria-free. . Today. inducing the cuttings to form roots. Under these conditions. is very common. In plants. often by modifying the hormone balance. micropropagation is a multimillion-dollar business. Each individual arises as an adventitious shoot from the roots of an original tree that may have started from seed. culture on a synthetic medium in a flask or petri dish. a small piece of plant is cut out and placed into aseptic. and planting them. Each tree in the group is therefore a genetically identical clone. Gardeners and plant breeders routinely reproduce plants vegetatively by simply making cuttings.36 Plant Biotechnology resort to asexual reproduction. growers in the Netherlands alone were producing over 21 million plants annually by micropropagation. set seed. Some 70 years ago. flower. supplemented with sucrose as a carbon source plus some vitamins. and do all the other things that plants normally do.

. which will then develop into fruit. The plantlets will undergo further testing to see whether they will yield flowers.2 A plant biologist examines papaya plantlets raised in a petri dish by micropropagation.Plants and Biotechnology: Before Recombinant DNA 37 Figure 3.

The major exception is animal stem cells. All one has to do to make use of this potential is to find the conditions that allow the potential to be expressed. One simply 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. It is then placed on the culture medium under sterile conditions. This explant may be as little as 2–3 millimeters long. These are called axillary buds. numerous microshoots will appear. animal cells are not generally totipotent: Most animal cells become highly differentiated early in their development and this differentiation cannot be reversed. The most important commercial technique for micropropagating plants is known as shoot-tip culture (Table 3. 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. Cherry (Prunus) Poplar (Populus) Rhododendron (Rhododendron) Rose (Rosa) Strawberry (Fragaria) . which retain some capacity to differentiate down different paths. Unlike plant cells. Cymbidium) Crops Commonly Propagated by Shoot-tip Culture Peach. But even stem cells are not capable of producing an entire animal. as it grows.38 Plant Biotechnology All this is made possible by the fact that virtually every plant cell is totipotent—it has the genetic potential to reproduce the entire organism.1 Apple (Malus) Carnation (Dianthus) Chrysanthemum Grape (Vitis) Kiwi fruit (Actinidia) Orchids (Cattleya. This is the same table 3.1). The explant is first washed with a dilute solution of household bleach to remove any contaminating fungi or bacteria. The shoot tip will continue to grow and. Microshoots arise from small groups of dividing cells that remain trapped where the young leaves join the stem.

and each time the cluster of shoots is divided and subcultured. trees with superior characteristics can be cloned for higher productivity. In fact. if the clone turns out to be particularly susceptible to drought or a new disease. cloning trees for commercial pulp and logging industries does reduce the genetic diversity of the forest. There are a number of advantages to micropropagation over traditional methods of plant propagation. it is commonly used to produce clones of cultivars (varieties under cultivation) that are particularly popular because of flower color or other characteristics. This process is repeated. the first plants to be mass-produced by micropropagation were virus-free clones of Cymbidium orchids. which could have long-term detrimental effects. Moreover. they are transferred to a medium with an appropriate hormone balance that stimulates root formation. 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.Plants and Biotechnology: Before Recombinant DNA 39 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. the number of microshoots increases exponentially. On the other hand. In a cloned forest. literally millions of genetically identical microshoots can be cloned from a single desirable parent. the mass of shoots is divided and the pieces are transferred to fresh culture medium. the entire forest is at risk. The young plantlets can then be planted in the greenhouse and grown to maturity. Potato is another plant that is often troubled with . Micropropagation is also an effective way to eliminate viruses and other pathogens. In the floral industry. Through shoot-tip culture. however. Genetic diversity enables some members of a population to survive stresses that might damage others. When a sufficient number of microshoots have been generated. One is that exceptionally large numbers of seedlings can be produced in a very small amount of space. After two to four weeks.

It is also the principal constituent of natural gas. The use of somaclonal variation as a plant breeding technique is described in chapter 7. municipal sewage treatment plants. you may occasionally have seen fires dancing across the surface of the swamp. since there is no significant mixing of water and air. Methane is commonly known as marsh gas or swamp gas. Potatoes are normally propagated vegetatively by buds. so all is not lost. anaerobic bacteria. Anaerobic bacteria break down the organic material in the swamp and. Under these conditions. will take over. or “eyes. The most effective way to eliminate potato viruses is by micropropagation of virusfree lines through shoot-tip cultures.40 Plant Biotechnology virus infections. any dissolved oxygen is used up rather quickly and. Micropropagation. BiofUels It may sound like the stuff of folklore. There are many sources of methane in addition to swamps and petroleum deposits. in the process. The principal sources today are cattle flatulence. like any other technique. which thrive in the absence of oxygen. These variations. but most landfill sites. is not perfect but is subject to the whims of nature. there is little opportunity for the oxygen supply to be replenished. What actually causes these fires is methane gas (CH4). which is usually found in association with petroleum deposits. and landfill sites. generate methane gas. particularly in urban areas where they have been reclaimed . We can’t do much about the cattle. In stagnant swamps. significant variations can sometimes arise. Most somaclonal variants are discarded. but occasionally one exhibits a particularly useful trait relating to field crops or an interesting flower color. In spite of the fact that clones are supposed to be identical. which probably involve spontaneous mutations due to the culture conditions. and any virus infection is readily carried from one generation to the next. but if you should happen to live near a swamp. are known as somaclonal variations.” on the tubers.

It won’t 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. is 37 megajoules (MJ) per kilogram compared with 42 MJ in a kilogram of crude oil. are now vented by driving pipes through the soil cover into the landfill.3). for example. The energy content of most plant oils compares favorably to that of petroleum-based diesel fuel. called fatty acids. The energy content of sunflower oil. A triglyceride is a fat or oil molecule that has three long carbon chains. Before they can be used as a fuel. including methane. More recently. the free fatty acids may be used in place of ordinary petrochemical-based diesel fuel. some plant oils can be used directly in farm machinery and other diesel engines. The pipes allow the methane to escape into the atmosphere rather than accumulate in the landfill where it poses a danger to human health. as every year numerous farm workers are killed when overcome by gases. . however. landfills are now being engineered as giant bioreactors to capture the methane gas for use as a fuel. Known as biodiesel. Consequently.Plants and Biotechnology: Before Recombinant DNA 41 for parks or housing. Methane is commonly produced on farms in areas such as silos and manure storage tanks. Many farmers are now designing bioreactors in order to recover biogas from agricultural wastes for use as an on-farm source of energy. Once the glycerol is removed. Another source of biofuel that is gaining in popularity is the oil that many plants store in their seeds. attached to a 3-carbon molecule called glycerol (or glycerin) (Figure 3. which involves separating the fatty acids from the glycerol. are triglycerides. Methane generated under these circumstances is commonly referred to as biogas. Most plant oils. triglycerides must first be processed. we have come to recognize that this methane represents a potential energy source and the amount of gas that can be generated in landfills is significant. It can become a real hazard.

42 Plant Biotechnology Figure 3.3 A triglyceride consists of three fatty acids attached to a glycerol molecule. . Fatty acids with one or more double bonds between carbon atoms are classified as unsaturated (red and blue in illustration). 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).

In addition. and burning of oil is directly responsible for much of the pollution we experience in the developed world. refining. Biodiesel contains little in the way of nitrogen or sulfur compounds. Gasohol was pioneered in Brazil. Alternative supplies of energy are being sought because the extraction. Department of Energy. This is a highly economical process in Brazil. can be used as a fuel to supply steam for the distillation and to generate electricity.S. Moreover. Gasohol: Fuel Salvation or Snake Oil? Petroleum fuels (natural gas. it is widely anticipated that oil and natural gas supplies will run out during the twenty-first century. 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.Plants and Biotechnology: Before Recombinant DNA 43 Although diesel engines can run on pure processed vegetable oil. the debris that remains after the sugary juices are expressed. called bagasse. where the favored candidates for conversion to ethanol are starch and cellulose. Biodiesel is also a renewable resource because it is produced from corn. According to the U. One attractive alternative is ethyl alcohol (ethanol). soybean. fermented. Both require (continued on page 44) . where the ethanol is produced from cane sugar. and the resulting ethanol is collected by distillation. diesel fuel) are the single most important materials driving the world’s economy. more than 25 million gallons of biodiesel were produced in 2004 and production in 2005 was expected to approach 100 million gallons (Figure 3. so it is less polluting than conventional diesel fuel. also known as gasohol when used as a fuel.4). gasoline. which has a warm climate and large land area that can be devoted to growing sugar cane. it is usually mixed with regular diesel fuel to improve combustion and performance. The sugary juices are expressed from the cane. The situation is not so simple in North America.

from crude oil and natural gas or coal-fired generating stations. The methane would then be used as the fuel for the aerobic fermentation and distillation process that separates the ethanol. however. and other oilseed crops such as sunflower and canola. but ethanol does. it produces 15% to 25% less energy than an equivalent volume of gasoline. A current project eyes sweet potatoes as the carbon source for ethanol production. Sweet potatoes are rich in both starch and sugars. However. this may sound too good to be true. such as singers Neil Young and Willie Nelson. if successful. It would be effectively independent of fossil fuels and. either directly or indirectly. the residues from corn and wheat straw have negligible fuel value. For the immediate future. Because ethanol is already partially oxidized. A partial solution to the problem of fossil fuel dependency. In these days of concern over diminishing oil supplies. which requires additional energy. unlike sugar cane. The first fermentation would be anaerobic and would produce methane gas (biogas). . most of the energy required to produce ethanol will likely come. This project proposes two parallel fermentations. There is concern in some quarters that the energy cost of ethanol production coupled with its lower energy content may mean that there is little or no real energy gain. may be in the wings. could provide the model for future fuel-ethanol production in North America. are encouraging the use of biodiesel by running their tour buses on the fuel. Many environmentally conscious celebrities. and. the hydrocarbons in gasoline contain no oxygen. Additional energy is required to distill the ethanol. anaerobic and aerobic. This means that a vehicle gets lower mileage with ethanol in the tank than with gasoline.44 Plant Biotechnology (continued from page 43) enzymatic breakdown to release the sugars for fermentation. 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? Let’s do a simple calculation.

Soybeans are about 20% oil and a bushel of soybeans will yield about 1. That may sound like a lot of diesel fuel. 72 million acres of soybeans were planted in the United States.5 billion gallons of biodiesel fuel. Biodiesel has become increasingly popular with energy-conscious people over the past few years. In 2005. the entire soybean crop could provide about 4. At 1.5 gallons per bushel.4 An entrepreneur stands by the truck he uses to collect vegetable oil from restaurants to make biodiesel fuel.Plants and Biotechnology: Before Recombinant DNA 45 Figure 3. using soybeans as an example. but in 2005 farm and highway vehicles alone .5 gallons of biodiesel fuel. with a yield of about 3 billion bushels.

The diversion of soybean and other oilseed crops away from their traditional markets would also drive up the price of food and other products. both obtained from fossil fuels. but virtually all of the arable land in North America is already under cultivation. then. The fatty acids are separated from the glycerol by stirring the cooking oils with a methyl alcohol–lye mixture. but it is still unlikely that biodiesel could make more than a small dent in our consumption of petrochemical-based fuels. would be to grow more soybeans. Another solution might be to identify high-oil crops that can be grown in marginal lands that are not suitable for conventional crops. that are required to produce the soybean crop. the mixture separates into two distinct layers. An added benefit is that once the biodiesel has been siphoned off.46 Plant Biotechnology consumed approximately 40 billion gallons of diesel fuel. of course. and wheat. biodiesel is probably the only vehicle fuel that can actually be produced at home or on the farm. primarily in corn. They start by collecting used cooking oils from their local fast-food restaurant. Would You Like Fries or Biodiesel with Your Order? Processing plant fats and oils to produce biodiesel is easy and relatively inexpensive. In fact. although your vehicle will smell faintly of french fries! . And that does not take into account the fuel and fertilizer inputs. the entire soybean crop could supply a little more than 10% of the diesel fuel required for farm and highway vehicles. although it does require some corrosive chemicals that must be handled very carefully. the glycerol can be saved for use as soap or a cleaning agent. soybeans. with the biodiesel (or methylated fatty acids) on top and glycerin on the bottom. After standing for a while. At best. and many people do just that. One solution. This is a great way to recycle used cooking oils. to provide food and raw materials used in manufacturing.

Accumulator species of plants are used to remove heavy metals from contaminated soil. a process called phytoremediation. sweet potatoes. . and plant fats and oils are processed as biodiesel. Fuel ethanol is produced by fermentation of cane sugar. Various forms of plant tissue culture have been used extensively to clone commercially valuable plant varieties and to eliminate viruses and other pathogens. a renewable fuel for buses. corn.Plants and Biotechnology: Before Recombinant DNA 47 summary The role of plants in biotechnology was established long before genetic modification brought plant biotechnology to the attention of the general public. The advantage of biofuels over fossil fuels is that the plants or plant oils used in the production of biofuels are a renewable resource. and automobiles. or methane. is generated by the bacterial breakdown of agricultural wastes for use as an on-farm energy supply. and other plants. trucks. Biogas. but the diversion of large amounts of crops into biofuels of any kind will compete with traditional markets and drive up the price of food and other products. Plants have also been used to generate environmentally sound biofuels.

DNA. and Protein 48 . Genes.

The structure has novel features which are of considerable biological interest. 1953 .We wish to suggest a structure for the salt of deoxyribonucleic acid (DNA). —James Watson (1928–) and Francis Crick (1916–2004) from Nature.

and a phosphate group (Figure 4. Biologists had long known that heredity was controlled by genes and that genes were in some way related to DNA. Genes. involves a manipulation of genes. in order to understand how this new technology works. regardless of the source. Chemical analyses eventually revealed that nucleic acids—DNA and its companion ribonucleic acid (RNA)—were very large molecules constructed from only five simple building blocks called nucleotides. The first clue to the structure of DNA came in the 1940s when Erwin Chargaff analyzed DNA from several different species and found that. and Protein Biotechnology. 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.1). cytosine. A nucleotide is composed of three elements: a nitrogen-containing base. but we cannot make informed judgments about the technology and its application without at least a rudimentary understanding of the science behind it. a 5-carbon (pentose) sugar. hence ribonucleic acid. guanine. thus “tricking” plants into producing novel proteins. This is a controversial technology for a variety of reasons. while in RNA uracil takes the place of thymine. in the sense that most people understand it today. Both the nature of the gene and the synthesis of proteins are intimately related to the hereditary DNA. Thus. we should first have a basic understanding of DNA and of the relationship between DNA and proteins. The sugar in DNA is missing one oxygen atom and is thus called deoxyribose. A DNA Primer Deoxyribonucleic acid (DNA) was first isolated from the nuclei of cells in 1869. 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 four bases that make up DNA are adenine. and thymine.DNA. The sugar in RNA is called ribose. the DNA always contained roughly equal proportions of adenine and thymine and that the 50 .

Genes. and Protein 51 Figure 4. Each nucleotide is made out of a sugar molecule. and a base.1 DNA is made out of a basic unit called a nucleotide.DNA. . a phosphate group.

The Watson and Crick model for DNA is often described as a ladder. Watson and Crick noted that their “structure has novel features which are of considerable biological interest. One of those working on DNA was Linus Pauling at the California Institute of Technology. it is called a double helix. and medicine over the past 50 years. 1953. 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.52 Plant Biotechnology proportions of cytosine and guanine were also similar. 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. agriculture.” Given the impact that their discovery has had on biology. The puzzle was finally solved in 1953 by James Watson and Francis Crick. Watson was a postdoctoral fellow (a recent Ph. Because there are two paired DNA molecules. At the time.” In this letter. issue of the prestigious British science journal Nature. The sides of the ladder are formed from alternating sugar molecules and phosphate groups. England.D.) from Harvard University working with Crick at the Cavendish Laboratories in Cambridge. 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 Chemistry in 1954 for his earlier work on the structure of another complicated . 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. this may go down as one of the greatest understatements in the history of science. 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. twisted about its long axis to form a helix.

In addition to the bonding. . The one model in which everything finally “fit together” was the now-famous double helix.” The rungs of the ladder—and the key to the Watson and Crick hypothesis—are 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. of the DNA molecule are macromolecule. When an X-ray beam is fired at a crystal structure. the shape and the space occupied by the four molecules means this is the only way they can fit together without distorting the sugarphosphate backbone. The diffraction pattern obtained by Franklin and Wilkins indicated that their DNA crystals were arranged in a helical pattern. the very consistent ratios of adenine (A) plus thymine (T) and cytosine (C) plus guanine (G)—and began to build models as big as themselves from pieces of wire and bits of brass. or strands.DNA. Watson and Crick won the DNA race primarily 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. The two halves. The opposing nucleotides are held together by weak hydrogen bonds. Genes. cytosine and guanine are held together by three hydrogen bonds but only two bonds are formed between adenine and thymine. protein. 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. depending on the arrangement of molecules in the crystal lattice. The consistent pairing of A with T and G with C also explains Chargaff’s earlier data. the beam is scattered (or diffracted) in a particular pattern. and Protein 53 “backbone. Watson and Crick put this information together with what was then known about the chemistry of DNA—in particular. However.

Where the strand has a G. Once the two strands are separated. DNA is a sequence of nucleotides and a protein molecule is a sequence of amino acids. directs the synthesis of the proteins that make the cell function. Complementarity provides the key to how DNA is able to replicate itself so precisely during cell division. the information in DNA is stored in discrete blocks or addresses. and so forth. so the two strands can easily be separated. the information contained in the gene (the DNA) is downloaded into ribonucleic acid (RNA) which. the complementary strand runs south to north. the complementary strand will have a C.54 Plant Biotechnology complementary. These blocks or addresses are called genes. the complementary strand will have a T. If one strand can be said to run north to south. To put it in biochemical terms. the information needed to perform specific operations is downloaded into the RAM. WhAt is A GeNe? DNA is a lot like the hard drive on your computer—its primary function is to store information. the base sequence in each strand then serves as a template (pattern) to reconstruct its complementary strand. In a computer. Like the information in your hard drive. which is where the instructions in the software are actually carried out. A gene is a . In the cell. The result is two exact copies of the original double-stranded DNA. an enzyme called helicase simply splits the molecule down the middle. Each strand of nucleotides also has polarity and the two strands are antiparallel. in turn. The hydrogen bonds that hold complementary base pairs together are relatively weak chemical bonds. 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. When DNA replicates itself. separating the two strands of paired nucleotides (Figure 4. their polarity is reversed. Wherever one strand has an A. Antiparallel means that when the two strands are paired.2).

2 DNA replication results in the formation of two identical double-stranded DNA molecules. are essential to the process. Enzymes. . and Protein 55 Figure 4.DNA. Genes. such as helicase and DNA polymerase.

The shape of a protein is very sensitive to the amino acid composition.000 to 4. ProteiNs AND the GeNetic coDe There are more proteins in a cell than any other single organic molecule. and consequently genes. Other proteins carry messages between cells or contribute to the structure of membranes and other cell components. the characteristics of tissues.000 nucleotides long. but the change does not impair the normal photosynthetic function of the protein. The biggest single class of proteins is the enzymes. coils. It does this by binding to a small protein in the chloroplast. it takes only a single amino acid change . The amino acid sequence determines the structure of the protein and the structure determines its function in the cell. In most proteins. consequently. but most genes are approximately 1. So.000 enzymes. By controlling which proteins are produced by an individual cell. the gene controls the characteristics of that cell and. and they perform a vast variety of functions. proteins that catalyze biochemical reactions (including the synthesis of other proteins). the amino acid chain twists. There is a family of herbicides—the triazines—that kills plants by interfering with photosynthesis. There are 20 standard amino acids that can be assembled in a nearly infinite number of combinations to produce this large array of proteins. vary in length. The entire complement of genes in an organism’s DNA is called the genome. The change of one amino acid in that protein changes the protein just enough so that the herbicide can no longer bind.56 Plant Biotechnology particular sequence of nucleotides within the DNA that specifies the sequence of amino acids in a particular protein. Proteins. organs. 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. Proteins are long chains of amino acids. A typical cell may contain as many as 3. and organisms. and folds back on itself to form an intricately shaped molecule. One example is herbicide resistance in plants.

for example. The number of possible combinations for combining 4 bases into groups of 3 is 4 cubed (43). Genes. or translation. There is. or 64. then moves out of the nucleus into the cytoplasm. however. or codon. The mRNA-ribosome complex is now ready to carry out the DNA’s instructions and direct the synthesis. The genetic code is universal: the same code is used by bacteria.3). DNA is not directly involved in protein synthesis. Instead. the DNA sequence that . Each triplet. where it binds to a protein aggregate called the ribosome. there is also one codon that signals the starting point for protein translation and three stop codons that signal the end of translation. and Protein 57 in one small protein to confer triazine resistance on the plant. rather than making a complementary DNA strand as in DNA replication. Triazine-resistance is a common occurrence in weeds because this kind of subtle mutation occurs often in nature. plants. 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. fruit flies. Messenger RNA is identical to a single strand of DNA except that it contains a ribose sugar and thymine is replaced by uracil. is the code that specifies one amino acid. of protein (Figure 4. a bit more to a gene than just the amino acid sequence of a protein. In fact. In addition. which now contains a copy of the genetic code. the DNA template is used to make a complementary strand of RNA. This means that the gene encoding human insulin. That is.DNA. the DNA is unzipped and the information in the gene is transcribed into messenger RNA (mRNA). How can a code using only four “letters”—the four different bases in a DNA molecule—specify the assembly of at least 20 different amino acids into literally thousands of different proteins? DNA actually uses a three-letter. and humans. most amino acids are encoded by multiple codons. or triplet. For example. code based on a sequence of three nucleotides. so there are at least three times as many codons as are necessary to encode only 20 amino acids. As noted above. The RNA.

transfer RNA (tRNA) carry amino acids to the cell’s ribosomes.3 During translation.58 Plant Biotechnology Figure 4. on a strand of messenger RNA (mRNA). . known as a codon. Each tRNA molecule recognizes a sequence of three nucleotides. A protein is created when the amino acids are chained together.

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.DNA. one strand of the DNA is transcribed into messenger RNA (mRNA). The start codon. Messenger RNA is a single-stranded nucleic acid that carries the message of the gene into the cytoplasm. where the protein is actually synthesized. the codons in the mRNA will include U in place of T. lining up the tRNA in the proper position so that its amino acid can be added to the growing amino acid chain. transfer RNA (tRNA) binds to a specific amino acid and carries (or transfers) it to the mRNA. the complementary strand of RNA will incorporate uracil rather than thymine. which is complementary to the codon for the amino acid that it carries. there is enough DNA to code for over 1. the ribosome shifts one codon. is also the codon for the amino acid methionine. 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. and Protein 59 codes for human insulin is over 4. for tyrosine (Tyr) the codon is UAU. As shown in the illustration.000 bases long. . preparing it to receive the tRNA for the next amino acid in the sequence. A simplified version of how the code directs protein synthesis is illustrated in Figure 4. As a result. mRNA binds to a cytoplasmic protein unit called the ribosome. The anti-codon binds to the codon on the mRNA. 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.3. AUG. so the anti-codon on the tRNA is complementary. the codon for the amino acid alanine (Ala) is GCC.300 amino acids. When cellular conditions call for the synthesis of a particular protein. With a code of three bases for one amino acid. Genes. Then. so all proteins begin with a methionine unit. or three nucleotides. Each molecule of tRNA includes a sequence of nucleotides called an anti-codon. First. Similarly. but insulin is a small protein that contains just over 100 amino acids. downstream on the mRNA. so the anti-codon is AUA. or CGG. Each time a new amino acid is added to the chain.

introns. the host cell ruptures and the new generation of viruses is released into the environment. the Birth of moDerN BiotechNoloGy Bacteria have to contend with viruses just as humans do. Of course. It is difficult to say exactly when genetic engineering began. 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. . are little more than a piece of DNA surrounded by a coat of protein. but also the instructions for making the protein. It is because some of the bacterial DNA nucleotides have been methylated. the viral DNA takes over the synthetic machinery of the bacterium and directs the unlucky host to replicate more viral DNA and protein. called bacteriophages or simply phages. Once inside. The phage infects the bacterium by attaching to the surface of the host cell and injecting its DNA into the cell. etc. and so on. Because these enzymes restrict viral Restricting Restriction Enzymes You may wonder why a bacterium that produces restriction enzymes doesn’t digest its own DNA. Bacterial viruses. The addition of a few strategically placed methyl groups (–CH3) at the restriction site prevents the restriction enzymes from digesting the bacterial DNA.) contain the instructions that tell the cell when to make the protein. nature is a constant battle with one group trying to get ahead of the other. Some bacteria are protected from phages because they contain enzymes that can cut foreign DNA into shorter pieces that are unable to replicate.60 Plant Biotechnology It turns out that a gene is more like a recipe—it contains not only the list of ingredients (the amino acids). Eventually. Sectors of the gene (promoters. but certainly the discovery of restriction enzymes was an important first step. how much to make.

act as biological scissors. a researcher at Johns Hopkins University in 1970. Smith shared the Nobel Prize with two others for their discoveries of restriction enzymes. such as HindIII and EcoR1. and Protein 61 Figure 4. In 1978.DNA. In the early 1970s. He cut both .4). Genes. The first restriction enzyme (called HindIII) to be discovered was isolated and characterized by Hamilton Smith. Paul Berg at Stanford University isolated DNA from two sources: the bacterium Escherichia coli and a primate virus called SV40. they were called restriction enzymes. They allow scientists to cut DNA and recombine it into new configurations. for example. replication. Each one cuts DNA at a unique point identified by a particular sequence of base pairs. The enzyme EcoR1. There are now hundreds of restriction enzymes available from a diverse array of bacteria. while HindIII recognizes the sequence AAGCTT and cuts between the two A’s (Figure 4.4 Restriction enzymes. EcoR1 has a preeminent place in the history of bioengineering. recognizes the sequence GAATTC and cuts the DNA between G and A.

or “stick” together. coli may divide every 20 to 30 minutes. it was called recombinant DNA (rDNA). Berg’s work was recognized with the Nobel Prize in 1980. Cohen and Boyer found they could induce bacteria to take up hybrid plasmid DNA. coli DNA. Berg created the first recombinant DNA molecule. it leaves overhanging. or “sticky” ends. The result was a hybrid molecule of SV40 and E. the two plasmids would readily join to form a hybrid plasmid. With this experiment. Under optimal laboratory conditions. All pieces of DNA cut by EcoR1 will anneal with a similarly treated plasmid to form a hybrid plasmid (Figure 4. so that after . At the same time. also at Stanford. They are called “sticky” ends because when any two pieces of DNA cut by this enzyme are brought together. Stanley Cohen and Herbert Boyer. Plasmids are very small.62 Plant Biotechnology samples of DNA with EcoR1 and then mixed the two in a test tube. thus making genetic modification possible and providing the foundation for all of modern biotechnology. One of the principal functions of plasmids appears to be to carry genes that confer resistance to antibiotics. This is because when EcoR1 cuts the DNA. Cohen and Boyer then took advantage of another peculiar trait of bacteria—their capacity to take up bits of DNA from their environment and incorporate it into their own genome. The same thing will happen with a piece of “foreign” DNA—say from the chromosome of another organism—that has been cut out with the same enzyme. Because the new DNA was created by splicing together (or recombining) DNA from two different sources. and the bacterium that takes up the foreign DNA is said to be transformed. they naturally anneal. were studying bacterial plasmids.5). Once inside the cell. Cohen and Boyer found that if they cut plasmids from two different sources with the enzyme EcoR1. This process is called transformation. E. circular pieces of DNA that are separate from the normal chromosomal DNA of the bacterium. Plasmids are interesting little pieces of DNA that are found primarily in bacteria. the plasmid (including any newly introduced genes) is replicated normally as the bacteria divide.

In the illustration above.DNA. . or hybrid. and Protein 63 Figure 4. Genes. coli cell by a process known as transformation. The recombinant plasmid is incorporated into an E.5 Restriction enzymes are used to create recombinant. the restriction enzyme EcoR1 is used to insert the gene for insulin into a bacterial plasmid. plasmids.

zillions is not an official numerical unit. It is then easy enough to identify which colonies grew on ampicillin but not on tetracycline—those are the colonies that have been transformed with the recombinant DNA. so there has to be some method for selecting the cells that were successfully transformed and contain the desired DNA. After a period of incubation. only transformed cells having the ampicillin resistance gene will produce colonies. visible colony of cells but. For this. This technique spreads a dilute bacterial culture on the surface of an agar medium in a petri dish. (No. The DNA fragment containing the gene to be cloned was then inserted at a restriction site within the tet R gene. Using a technique called replica plating. but it is an easy way to express a really big number. This disrupts that gene and prevents the synthesis of the resistance protein. Cohen and Boyer developed another little trick. each live bacterium will give rise to a small. coli will give rise to over 16 million progeny. which is then pressed on the surface of a second plate containing tetracycline. Cells that contain the recombinant plasmid will form colonies on the ampicillin plate but will not grow and form colonies on the tetracycline plate. the cells that have been successfully transformed. a single E. Some of the cells from each colony will be picked up by the pad. . This transfers a few cells from each colony to the tetracycline medium in the exact image of the ampicillin plate. 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). Each cell contains an exact copy of the plasmid with the newly introduced gene.64 Plant Biotechnology 8 hours of laboratory culture. This replication of the genes to produce zillions of exact copies is called gene cloning. the culture is “plated out” on a medium that contains ampicillin. A sterile pad is then pressed against the colonies on the ampicillin plate. Genes such as amp R and tet R are called marker genes because they identify.) Transformation is not 100% efficient. or mark. because the medium contains ampicillin.

and it is made possible by the use of restriction enzymes. for inserting the new DNA into another organism. These experiments heralded the arrival of an entirely new way to modify cells. (continued on page 66) . • Plasmids that. and Protein 65 With these experiments. Genes. • Marker genes that enable the scientists to screen a culture and select for the cells that have been transformed with the rDNA. In the world of criminal investigation. that is. The new DNA inserted could be a specific sequence of nucleotides. First. provide a vehicle. a gene. Just what is this new technology? DNA fingerprinting is a way of looking at the unique signature found in an individual’s genetic makeup. We have: • Restriction enzymes that allow scientists to cut DNA in specific locations and insert new DNA to make recombinant DNA (rDNA). through the process of bacterial transformation. the investigator needs to obtain a sample of DNA. Detergents are used to extract and purify the DNA or it can be mechanically forced out of the cell through a syringe. but the polymerase chain reaction (PCR) can be used to amplify the quantity of DNA if the original sample is very small (for example. a hair follicle. DNA fingerprinting has helped to convict guilty parties and has led to the release of falsely convicted individuals. or a blood stain).DNA. DNA Fingerprinting: An Organism’s Bar Code DNA fingerprinting is one of the latest technologies to help forensic investigators bring the truth to light. saliva on an envelope. the stage was now set. or vector. The purified DNA is then cut with restriction enzymes. A milliliter of fresh whole blood is an ideal sample.

. just like the bar codes on manufactured products. The fragments are sorted according to size as they move through the gel because their movement is restricted by pores in 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 nitrocellulose membrane. and because different samples are run side by side on the same gel. The next step is to visualize the position of the different fragments on the gel. The image on the X-ray film may include 40 or more bands. unless they are identical twins. The fragments are permanently fixed to the surface of the membrane where they can be located with a probe. the mixture of DNA fragments of various sizes must be separated. When the film is developed. The “bar code” is the DNA fingerprint. The odds that DNA restriction fragments from two people will match all 40 bands. the positions of the various DNA fragments are indicated by a series of black lines that vary by position and intensity (amount). DNA fingerprinting is not limited to forensic situations or humans. single-stranded fragment of DNA that has been made radioactive and that contains the complementary code for a specific sequence of bases. so they move through the gel toward the positively charged electrode. are extremely low. A probe is a small. 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. Finally. Because the probes are radioactive they emit energy that exposes the film. a sheet of X-ray film is placed against the membrane. DNA fingerprinting can be used to establish the relatedness of virtually any organism. they may be compared directly. The probe will bind with the DNA on the membrane wherever the complementary base sequence is found. The DNA fragments are negatively charged. including plants. This is done using a technique known as electrophoresis.66 Plant Biotechnology (continued from page 65) After the restriction enzymes have done their job. Smaller fragments are less restricted by the pores and so will travel further through the gel than the larger fragments.

with each sequence of three nucleotides coding for a specific amino acid in the protein. The entire sequence of DNA nucleotides that specifies a complete protein is known as a gene. As the two strands separate.DNA. some containing a complete gene. free nucleotides naturally pair with each single strand to form a new double-stranded molecule. Limited sequences of DNA. Pieces of DNA can then be recombined in different combinations to form recombinant DNA (rDNA). The DNA code is based on the sequence of nucleotides. As the transformed bacterium reproduces. and Protein 67 summary DNA is a relatively uncomplicated. it produces many copies of that gene. Genes. double-stranded molecule. . A bacterium that has incorporated a foreign gene is said to have been transformed. can be isolated by cutting the DNA with restriction enzymes. In the same way. a process called gene cloning. The geometry of these pairings is responsible for maintaining the parallel spacing of the sugar-phosphate backbone of the molecule as it twists to form a helix. consisting of only four building blocks called deoxyribonucleotides. 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 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 complementarity of nucleotide pairing is also the key to both DNA replication and RNA synthesis. Bacteria have the capacity to take up pieces of DNA from their environment and incorporate it into their own genome.

Engineering Plants 68 .

—Isaac Asimov (1920–1992) American author and biochemist .Science can amuse and fascinate us all. but it is engineering that changes the world.

then no fragments will contain an intact copy of the gene. In fact. Remember that making a copy of mRNA from DNA is called transcription. we are now faced with two questions. The enzyme reverse transcriptase is isolated from retroviruses. catch your hare. how do we insert that gene into a plant? In other words. When a virus invades a cell. So.” The recipe for genetic engineering is not a lot different. you have to first identify and isolate the gene of interest. cDNA is made by running RNA transcription in reverse. you have to isolate the messenger RNA (mRNA) from cells that contain the gene you are interested in. Before you can start moving genes around in order to create a transgenic plant. Random DNA fragments are of little value if you don’t know which fragment contains the gene you want to transfer. if the restriction enzymes you used to break up the DNA happened to cut the DNA in the middle of the gene you want. How do we find and clone a gene that we want to use to transform a plant? And once we have the gene.Engineering Plants A noted cookbook writer of the Victorian era began her recipe for jugged hare (rabbit stew) with the instructions: “First. it takes over the cell’s genetic machinery in order to make copies of its own DNA. 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. that is. When a 70 . First. Most animal viruses contain DNA as their genetic material. reverse transcription. It would be like looking for that needle in the haystack. But there are some viruses. called retroviruses. an enzyme called reverse transcriptase is mixed with this isolated mRNA. so making a copy of DNA from RNA is going the other way. that contain RNA instead of DNA. how do we actually go about genetically engineering a plant? GEnE HuntErs One approach to finding a particular gene is to create a complementary DNA (cDNA) library. Then.

The reverse transcriptase then catalyzes the reverse transcription of DNA. it ignores inactive genes and represents only genes that were actively being expressed in the cell at the time the mRNA was isolated. it is relatively easy to synthesize a DNA strand that encodes for those amino acids in that sequence. The viral DNA then instructs the host cell to produce the other bits and pieces necessary for virus multiplication.1). it is then replicated using the host cell machinery. The human immunodeficiency virus (HIV) is a familiar example of an animal retrovirus. If you don’t know anything about the sequence of a particular gene. After the cDNA strand has been synthesized. 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).Engineering Plants 71 retrovirus invades a host cell. so you must test each colony until you find those that contain the gene you want. and the method for determining the amino acid sequence of a protein is now a pretty routine laboratory exercise. it uses a portion of its RNA to direct the synthesis of the enzyme reverse transcriptase. a unique advantage to using cDNA for building this library. using the viral RNA as a template. however. you can often work back from the protein that it encodes. usually in a freezer. and this is the cDNA library. There is. The individual colonies are then stored. Because cDNA is made from mRNA. A mutation is nothing more than . Once you know the amino acid sequence. Once the DNA has been synthesized. Most proteins are easily isolated and purified. A third technique for coming up with new genes is to alter the DNA by inducing mutations. Most plant viruses are also RNA viruses (Figure 5. it must be cloned and the bacterial colonies grown as described in the previous chapter. Unfortunately. there is no card catalog for this library. 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.

Mutations occur naturally with a much greater frequency than you might imagine. although the truth is that most mutations are not . including tobacco.72 Plant Biotechnology Figure 5.1 The tobacco mosaic virus (TMV) infects the leaves. and fruit of many plants. a change—any change—in the DNA of an organism. TMV is an extremely persistent plant virus and has been known to survive for many years in dried plant parts. flowers.

a few seedlings will survive and can be screened for a new and desirable trait. however. 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. the seedlings will probably die from radiation or chemical poisoning.Engineering Plants 73 terribly beneficial. one transforms either cells in tissue culture (described in the micropropagation section in chapter 3) or protoplasts. Removing the cell wall. Protoplasts are naked plant cells that have been treated with a mixture of enzymes. This is one of the most controversial aspects of genetic engineering—creating transgenic plants. So how. At lower doses. plants do not spontaneously take up bits of DNA from their environment. a sugar alcohol. Occasionally. Of course. including cellulase. then. Instead. whole plants are not transformed. In order to get around this problem. however. One popular chemical mutagen is ethyl methane sulfonate (EMS)—a very nasty and highly toxic chemical. that strip away the cell walls and leave only the protoplasm surrounded by a cell membrane. if the level of radiation or EMS dose is too high. Mutations can easily be induced by exposing seeds or very young seedlings to ionizing radiation or chemical mutagens. It is not difficult to induce mutations in plants. however. creates certain problems for plant cells. Without the cell wall. because they depend on the strength and rigidity of the cell wall to keep intact. a mutation appears that gives the organism an edge and evolution takes a tiny step forward. does the genetic engineer transform plants? To begin with. There is one more method and that is to find another organism that has the gene you want. water will continue to diffuse into the protoplast by osmosis and the protoplast will swell until it ruptures. plant protoplasts must be maintained in a medium containing a high concentration of solute such as mannitol. The high solute concentration . Unlike bacteria.

but before the two strands can reunite. and so forth. some enzymes (DNA polymerase and ligase). or other microscopic sample. 20 cycles will produce approximately 220 or more than 1 million copies from a single starting molecule. which separates the double-stranded DNA into single strands. The basis for a PCR machine is a temperature-regulated block that can both heat and cool very rapidly. The sample is then cooled to about 60°C (140°F). A PCR machine is sort of like a photocopier for genes. The reaction is started by heating the sample to about 95°C (200°F). then 16. There are now two exact copies of the original DNA. the primers get in the way by binding to the DNA. Also in the mix are primers. . but the DNA polymerase used in PCR is a special form obtained from bacteria that are adapted to life in hot springs. Primers select the target gene to be amplified and help to avoid copying the entire genome. You may also have wondered how forensic scientists can do DNA fingerprinting with DNA from a single hair. The heating-cooling cycle is repeated to produce 4 copies.74 Plant Biotechnology 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. the number of copies is doubled. Bacteria can be used to clone or make multiple copies of genes. Most proteins are denatured or inactivated at temperatures near 95°C (200°F). 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. but now it is more common to use a method called the polymerase chain reaction (PCR). Every time the cycle is repeated. then 8. and a supply of each of the four nucleotides that make up DNA. spot of blood. The answer is that they use PCR to amplify the DNA. short pieces of DNA that have been constructed to bind with the DNA at either end of the gene. The sample chamber in the block is loaded with a sample of DNA that contains the gene.

the protoplasts are suspended in a liquid medium along . The term somatic hybrid refers to hybrids formed by fusion of vegetative cells rather than normal reproductive cells (sperm and eggs). 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. animal cells. protoplasts from two different sources can be induced to fuse. Somatic hybridization is an important tool for combining useful characteristics from different organisms. protoplasts will quickly regenerate cell walls. Protoplasts can be isolated from virtually any plant tissue. although leaf cells are most commonly used. eventually. Protoplasts are particularly useful to the bioengineer because the absence of a cell wall makes it easier to insert DNA directly into the cell. forming single hybrid cells called somatic hybrids. Under the right conditions. fertile plants. once the enzymes are removed from the medium. Incidentally. Protoplast fusion is one way to circumvent pollen sterility or other incompatibility barriers and introduce new characteristics into crop species. However.Engineering Plants 75 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. One technique is called electroporation. Most protoplasts are capable of continued cell division when cultured in an appropriate medium. avoid this problem because they have mechanisms for pumping water out of the cell to control cell volume. which do not have a cell wall. However. For example. many wild species of the genus Solanum have resistance to major diseases of the potato (Solanum tuberosum). In this technique. shoots and roots and intact. 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. Plant cells lack such pumping mechanisms. The cells can then be stimulated to form embryo-like cell clusters and.

fired by a discharge of high-pressure helium gas. Another way of transforming plant cells is literally a “shotgun” approach. known as biolistics. The protoplasts are then subjected to a brief pulse of electrical current (typically measured in milliseconds). The gene gun was originally developed in the early 1980s by a group of plant biologists and nanotechnologists (technologists who work with very small things) at Cornell University.000039 inch) in diameter. Believe it or not. In this technique. This growth. travel at about 400 meters per second. called crown gall. shows us that nature discovered genetic engineering long before we did. some laboratories actually used modified 0. Perhaps you have seen one in a local garden. in the early days of the gene gun technique.” which fires them at a suspension of cultured cells (Figure 5. . A curious pathological condition that affects many species of plants is a cancerous growth that commonly appears on stems (Figure 5.22-caliber rifles! The gene gun technique has been particularly useful for transforming corn or maize (Zea mays) cells. the DNA is first coated on gold or tungsten beads approximately 1 micrometer (0.76 Plant Biotechnology with microscopic gold beads that have been coated with pieces of DNA. The particles are then loaded into a “gene gun.2). Some of the DNA then becomes incorporated into the host cell’s genome and is reproduced along with the rest of the DNA when the cell divides. The particles. 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. which do not lend themselves to protoplast-based techniques.3). The electrical pulse opens up small holes in the cell membrane that allow the DNA-coated beads to enter the protoplast. naturE’s own GEnEtiC EnGinEEr The most widely used vector for introducing foreign genes into plants takes us back to bacteria and plasmids. The membrane quickly reseals itself and the DNA remains trapped within the protoplast.

Engineering Plants 77 Figure 5. The foreign DNA is coated on metal beads and then fired at a collection of plant cells. Once inside the cell.2 A gene gun can be used to inject foreign DNA into a plant cell. The genes that enable Agrobacter to cause crown gall are not particularly unusual—they are genes that encode enzymes for the synthesis of two naturally occurring plant hormones called auxin and cytokinin. The auxin and cytokinin genes are not expressed in the bacterium. Crown gall is caused by a common soil bacterium called Agrobacterium tumifaciens (we will call it Agrobacter. when the bacterium invades a plant. normally at the site of a wound. the DNA releases from the bead and becomes incorporated into the plant chromosome. it transforms the plant by integrating the hormone genes into the DNA of the infected plant cell. auxin normally causes cells to enlarge and cytokinin normally stimulates cells to divide. In the plant. However. The transformed plant cell then . for short).

Agrobacterium tumifaciens. The unique properties of this bacterium have made it a useful tool in genetic engineering of plants.3 A crown gall is the result of an infection by a common soil bacterium. .78 Plant Biotechnology Figure 5.

Agrobacter is most commonly used to transform disks of tissue cut from leaves.Engineering Plants 79 synthesizes higher-than-normal amounts of the two hormones. herbicide tolerance usually involves only one gene. The first step is to disarm the plasmid by removing the hormone genes. such as corn and wheat. where the bacteria infect the wound cells at the edge of the disk. 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. are most commonly transformed by using the gene gun technique. Once transformation is complete. First. The bacterium Agrobacter is thus a natural genetic engineer. Plants engineered to tolerate (or resist) this herbicide have received a lot of publicity and are probably the most widely known example of GMOs worldwide. . This means that plants with only one cotyledon (monocotyledonous plants). 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. Scientists have learned to use the engineering skills of Agrobacter as an efficient way to introduce new genes into plants. the new gene becomes incorporated into the host cell DNA and is expressed along with all of the other host cell genes. A bacterium carrying the disarmed plasmid can still invade plant cells. When the bacterium carrying the engineered plasmid infects cells of the target plant. The bacterium carries the hormone genes on a Ti (tumor-inducing) plasmid. Plants engineered for herbicide tolerance were the first genetically engineered plants to be released for two reasons. Agrobacter infects only dicotyledenous plants (plants with two cotyledons or seed leaves in the seed) such as beans and peas. the bacteria are killed off with an antibiotic and the leaf disks can be manipulated in culture to produce shoots and roots. HErbiCidE tolEranCE: How GEnEtiC EnGinEErinG works You may have heard of the herbicide Roundup™. Unfortunately.

as we will see in chapter 6. they coupled this enzyme to a particularly active promoter (the piece of DNA that activates or turns on the gene). the plant is unable to synthesize proteins and it dies of protein starvation. Here. Another example is the herbicide glufosinate. is incorporated immediately into amino acids.4). leaving the ammonium ion to accumulate in the cells. and tyrosine (Figure 5. 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.80 Plant Biotechnology so the engineering is relatively simple. there appeared to be both an obvious benefit and a ready market for the product. Without those amino acids. called bialaphos. enolpyruvyl-shikimate-3-phosphate synthase (EPSPS). Using rDNA technology. Cells with more of the resistant enzyme will naturally tolerate higher doses of the enzyme inhibitor. phenylalanine. The term overexpression means that the cells produce higher-than-normal amounts of the messenger RNA for the resistant enzyme. Plants that are transformed with this new combination of gene and promoter will overexpress the gene for the more tolerant enzyme. that catalyzes a critical step in the synthesis of those three amino acids. Roundup™ is a trade name for a chemical compound called glyphosate. Glyphosate acts by inhibiting the activity of an enzyme. free ammonium ion is highly toxic and it doesn’t take too much to kill the cells. but this enzyme is involved in a complex set of reactions that incorporates nitrogen into amino acids. Second. a fungus comes to the rescue: Streptomyces hygroscopicus is a common soil fungus that secretes a chemical inhibitor. Glufosinate also inhibits an enzyme. Glufosinate blocks this last step. marketed under the trademark Liberty™. under normal circumstances. an herbicide that kills plants by blocking the synthesis of the so-called aromatic amino acids: tryptophan. Plants take up nitrogen from the soil in the form of nitrate (NO3-) and convert it to ammonium ion (NH4+) which. But how do . or fungicide. Unfortunately.

.4 Two test plots of soybeans are pictured side by side.Engineering Plants 81 Figure 5. The soybeans treated with Roundup™ herbicide (right) show less weed growth than the soybeans that have not been treated with Roundup™ (left).

Fortunately. The question of human toxicity naturally arises when discussing pesticides in general and extensive use of herbicides such as glyphosate and glufosinate in particular. this enzyme also deactivates other chemicals. Nor do we get our organic nitrogen from nitrate. Glyphosate and glufosinate are not generally toxic to animals and humans because they target specific enzymes that are not present in humans and other animals. as high nitrate levels are toxic to humans. 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. common sense tells us to always treat all synthetic chemicals with caution. construct a . While humans and other animals make some amino acids. The first is to isolate messenger RNA and. is a chlorinated hydrocarbon. 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. Keep in mind. especially infants. hygroscopicus resolves this problem by also producing an enzyme that deactivates bialaphos. We get our nitrogen from organic compounds in our diet.82 Plant Biotechnology organisms protect themselves against toxic substances that they themselves secrete? S. using the enzyme reverse transcriptase. that have a chemical structure similar to bialaphos.4-D. at least for genetic engineers. these so-called essential amino acids must be present in our diet. that the same is not necessarily true of other herbicides. As a result. even though glyphosate and similar chemicals are considered relatively safe. There are several ways to obtain a gene. they do not have the enzyme EPSPS and. The common lawn weed killer 2. On the other hand. consequently. for example. do not make the aromatic amino acids. such as glufosinate. a family of highly reactive chemicals that are known to have nasty effects on human genes and metabolism. however.

by “shooting” the gene into protoplasts with a gene gun. A third method is to induce mutations by treating seeds with ionizing radiation or chemical mutagens. based on the amino acid sequence of the desired protein. Inserting the cloned gene—transforming plant cells—is accomplished by treating protoplasts (naked plant cells) with a brief pulse of electricity. Another method is to reverse engineer DNA. . or by enlisting the aid of nature’s own genetic engineer. 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.Engineering Plants 83 complementary DNA (cDNA) library. the crown gall bacterium (Agrobacterium tumifasciens).

Genetically Modified Plants From Herbicides to Vaccines 84 .

…whoever could make two ears of corn. —Jonathan Swift (1667–1745) Anglo-Irish author of Gulliver’s Travels . would do … more essential service to his country. than the whole race of politicians put together. to grow upon a spot of ground where only one grew before. or two blades of grass.

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

Rich Jorgensen. In the original experiment. When the transgene produced double-stranded RNA. Unexpectedly. had the idea of using recombinant DNA to develop new colors in petunia flowers. extra copies of the gene for purple pigment were inserted into petunia plants. But remember that plant viruses are RNA viruses and their RNA happens to be double-stranded. because the cell’s own gene and the transgene produce RNA with the same base sequence. The expectation was that the extra genes would cause the petals to make more pigment than normal and thus produce flowers with a more intense purple color. However.Genetically Modified Plants: From Herbicides to Vaccines 87 to resist herbicides. the plant’s defense system could not discriminate between the two and the RNA produced by the normal gene was destroyed as well. genetically modified crops have saved farmers 475 million gallons of fuel over the last nine years and reduced pesticide use by 14%. a plant biologist at the University of Arizona. petunia plants have a mechanism that allows them to recognize viral RNA and destroy it. Apparently. A recent study by a British firm. The Mystery of White Petunias Dr. the cell apparently mistook it for virus RNA and set about destroying it. by reducing the number of trips across the field for spraying. fewer herbicides are sprayed on the fields. but (continued on page 88) . without any lasting damage to the crop plants. In the end. The gene for purple color remained intact. The result is that less diesel fuel is consumed. and the farmer’s costs go down. consumers benefit as well because of the reduction in greenhouse gas emissions and lower pesticide load on the environment. for example. the weeds can be killed with a single pass after the crop has emerged. 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. surveyed 18 countries and found that.

RNAi therapy has been used successfully to treat diseases such as Huntington’s disease. Jorgenson called this phenomenon RNA interference (RNAi). With no intact messenger RNA left in the cytoplasm. amounting to economic losses of more than $100 billion and significant losses in food production (Figure 6. beekeeping is a growth industry as beekeepers follow the pollinating seasons from one crop to the next. Crop losses due to insects and plant diseases far exceed those of weeds. It did not take long for scientists to recognize that RNAi was not a property solely of plant cells but could be used to explain similar phenomena in organisms from nematodes (roundworms) to fruit flies to humans. Traditional insecticides are not the answer because they kill beneficial insects and crop pests equally well. Most orchard growers now find it necessary to import hives of bees during the pollinating season because natural bee populations have been decimated by years of widespread insecticide use. the cell was unable to make any of the purple pigment. Nowhere is this more obvious than in orchards across North America. In fact. These phenomena are now referred to more broadly as RNA silencing because they prevent the gene from being expressed. And it all started with transgenic petunias. hepatitis. Crops . resisTance To insecTs and disease Weeds aren’t the only problem faced by farmers—they must constantly fight insects and diseases caused by bacteria and fungi as well. Lou Gehrig’s disease.88 Plant Biotechnology (continued from page 87) the messenger RNA that it normally produces failed to accumulate. 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.1). and breast cancer in mice and age-related macular degeneration causing loss of vision in humans.

a potato leaf.Genetically Modified Plants: From Herbicides to Vaccines 89 Figure 6. Enterotoxins are toxic chemicals that act in the gut. They could possibly even stimulate a resurgence in natural bee populations.1 A Colorado potato beetle feeds on its favorite food. engineered to control insects eating on plants could be beneficial because they would target only the insects that feed on the crop plants themselves. One of the most common enterotoxins is the protein produced by Bacillus thuringiensis (Bt). such as the Colorado potato beetle. the protein binds to recep- . When the protein is ingested by the larvae of susceptible insects. A major focus of the biotechnology industry is the creation of insecticides that will reduce the impact of destructive insects. Genetic modifications to control insects exploit naturally occurring bacterial poisons called enterotoxins (from the Greek word enteron meaning “intestine”). a common soil bacterium.

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tors in the insect’s 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 group’s 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.

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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 monarch 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 available 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 modification and how one should not jump to conclusions until all the facts are in.

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neered crops don’t 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 don’t 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, produce 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 modifications 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 increasing 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 photosynthesis and the amount of carbon lost through respiration. Both photosynthesis and respiration are governed by a large

and sunflower is a large component of the North American agricultural economy. but by traditional plant breeding. These oils are used extensively as cooking and salad oils and in manufactured food products. this rapeseed was developed in Canada not by genetic modification. One popular source of oil is rapeseed. is to simply increase the yield of oil. contain high amounts of glucosinolates and erucic acid. Almost all the world’s 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. If that is not enough to discourage the use of rapeseed oil. however. unsaturated fatty acids are considered healthier than saturated fatty acids. Scientists are now looking to recombinant DNA techniques in order to further improve canola and other oilseed crops. Another objective is to change the fatty acid composition of plant oils to better match different food or industrial applications (Table 6. broccoli. For example. Known commercially as canola. The production of oilseed crops such as canola. The bottom line is that any improvements to plant productivity through biotechnology are not likely to be achieved until scientists understand much more about which genes are involved and how these complex pathways interact. of course. such as cabbage. Glucosinolates are organic sulfur compounds that are easily converted to mustard oils. however. The oils of native rapeseed. and cauliflower. so engineering plants to produce oils with .1). One objective. the erucic acid content adds its own distinctively unpleasant taste. Changing the oils in seeds. safflower. This might be accomplished by engineering overexpression of certain enzymes involved in the biosynthesis of oils. a collection of Brassica species in the mustard family.Genetically Modified Plants: From Herbicides to Vaccines 93 number of genes that interact with each other in complex ways. is another story. flavor constituents that give the pungent taste to mustard and horseradish as well as the distinctive flavors of other members of the same family.

3 4.9 7.2 1.1 Fatty Acid Composition of Common Plant and Animal Fats and Oils Fatty Acid (% of total lipid) Oil Saturated Unsaturated Ratio (Unsaturated/ Saturated Safflower oil Corn oil Olive oil Soybean oil Peanut oil Margarine Fish Beef fat Butter 8 11 11 15 18 26 15 48 55 87 85 84 79 76 70 78 47 39 10. canola.7 7.7 5. The development of golden rice is one of the greatest biotech success stories. This would bypass the need for hydrogenation and the associated generation of unhealthy trans fats.0 0. On the other hand.7 . and flax have all been engineered to produce more polyunsaturated and fewer saturated fatty acids.6 5.2 2. While North Americans and Europeans tend to favor potatoes. white rice is the staple food throughout much of Table 6.94 Plant Biotechnology a higher proportion of unsaturated fatty acids for use in cooking and salad oils would benefit health-conscious consumers. Soybean. oils with a higher proportion of stearic acid (a natural saturated fatty acid) could be used to solidify margarine and shortenings at room temperature.

both fats. a triglyceride made up of two or three saturated fatty acids will be solid at room temperature—a fat—while oils have a high proportion of unsaturated fatty acids. . hydrogenation also converts some of the cis double bonds to trans double bonds. Thus. the oils are cooked in the presence of hydrogen at high temperature and under pressure. are manufactured from plant oils. or trans fats. an extra bond (a carbon-carbon double bond) forms between the two carbon atoms. the double bond is always in the cis configuration. In the fatty acids that occur naturally in plants. Another interesting feature of the double bond is that it can occur in two configurations called cis and trans. A fatty acid with more than one double bond is often referred to as polyunsaturated. for example. Fats and oils have a similar chemical composition. 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. Unfortunately. A fatty acid is referred to as either saturated or unsaturated. This may be a confusing bit of circular reasoning. the only difference is that fats are solid at room temperature and oils are liquid. Lipids are molecules that are soluble in lipid solvents. “saturates” some of the double bonds in order to give the product the required melting properties. in which three long-chain fatty acids are attached to a three-carbon alcohol. Trans fats have lately been linked to heart disease and other human health problems. depending on the number of hydrogen atoms it contains. When margarine and shortening. Saturated fatty acids.Genetically Modified Plants: From Herbicides to Vaccines 95 Fatty Acids and the Trans-fat Problem Plant fats and oils belong to a large and diverse group of molecules called lipids. The key to the composition of fats and oils lies in the fatty acid mix that makes up the triglyceride. Where a pair of hydrogen atoms is missing. called hydrogenation. glycerol. have a higher melting point than unsaturated fatty acids. The principal components of fats and oils in plants are triglycerides. but it does emphasize that lipids share an aversion to water (hydrophobic). This process. 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.

Unfortunately. 8 of them are synthesized only in microorganisms and plants. potentially reducing the incidence of iron deficiency that affects an estimated 3. Lysine is expensive to produce. High-lysine corn varieties have been available through conventional breeding for more than 30 years. and it is estimated that the livestock feed industry purchases $1 billion worth of lysine every year to supplement animal feeds. especially in young children. polished white rice does not contain any of the orange pigment beta-carotene. which leads to blindness. A team of Swiss and German scientists successfully modified rice. to convert the precursor to beta-carotene. Vitamin A deficiency. however. but corn protein contains exceptionally low amounts of the essential amino acid lysine. using genes from daffodils. that must be included in the diet of all vertebrates. is a chronic problem throughout much of Southeast Asia and other parts of the developing world. . but yields were too low to be profitable. hogs. Corn is a popular feed for livestock such as cattle. The team was also able to add additional genes that increased the iron content of rice. 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.7 billion people worldwide. Rice does. a pro-vitamin that the body converts to vitamin A. Nutritional deficiencies can affect farm animals as well. The transgenic rice is known as golden rice because of the color imparted by the accumulated beta-carotene. mentioned earlier in chapter 5. These are the essential amino acids. One biotech company has genetically modified high-yielding varieties of corn for high lysine content and is expected to bring the new varieties into commercial production by 2007 or 2008.96 Plant Biotechnology the world. Of the 20 amino acids that go into making protein. and poultry. produce a precursor to vitamin A.

however. Many of these molecules have found use in antiquity as folk remedies. Among plants that are being used for molecular pharming are alfalfa. Why engineer plants to produce these products? The answer is that traditional methods are very costly. bananas. resins. on the other hand.” Most of the therapeutic products produced in transgenic plants are proteins. Even though the recent trend has been toward chemical synthesis of drugs designed to target specific illnesses more effectively. it produces a large leaf mass. and tomatoes.Genetically Modified Plants: From Herbicides to Vaccines 97 Molecular FarMinG Plants make a lot of strange chemicals. is tobacco. And the . Using transgenic plants as bioreactors to produce useful molecules is called molecular farming (Figure 6. it is a non-food crop. the technique is often referred to as “molecular pharming. In most plants. With the advent of rDNA technology. carrots. Plants. are easily transformed and they can produce huge quantities of soluble protein at relatively low cost. Tobacco is one of the easiest plants to transform. however. a significant proportion of their assimilated carbon and energy is diverted to the synthesis of molecules that have no obvious role in their growth and development. The significance of this last attribute will be addressed in chapter 7. rubber. and. potatoes. and others) and even more have found use as therapeutic drugs. cultured in chicken eggs. One of the favored crops for molecular pharming. and essences. plants traditionally have been the principal source of therapeutic drugs. 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. soaps. Others have found use as dyestuffs and feedstocks for chemical industries (gums. or simply not produced at all. perhaps most important. such as antibodies and antigens or vaccines—products that have previously been extracted directly from other animals.2) Because many of these molecules are intended to have use as therapeutic drugs (or pharmaceuticals).

Pioneering work in molecular pharming has been carried out in the laboratory of C. also known as molecular pharming.98 Plant Biotechnology Figure 6. purified pharmaceuticals are not contaminated with nicotine or any other nasty tobacco chemicals. An antigen is a protein that stimulates the body’s . 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. Arntzen. This technique is often used in molecular farming. a plant biologist at Arizona State University.2 A researcher inoculates a plant with an engineered virus that produces proteins for human therapeutic use. J.

trained medical personnel. 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 only successful oral vaccine has been the oral polio vaccine. In 2001. The estimated cost for delivery of an edible vaccine was as low as $0. The transgenic potatoes were also successful in invoking immune responses in mice. . and refrigeration can thwart conventional vaccination programs.02. 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. the cost of injecting a patient in remote areas with a conventional vaccine was estimated to be about $120.Genetically Modified Plants: From Herbicides to Vaccines 99 immune cells to produce antibodies that fight off invading organisms. Arntzen believed that a more cost-effective method would be an oral vaccine that could be delivered in a locally grown food product. The results of this trial clearly illustrated the potential for transgenic plants to be used as production vehicles for edible vaccines. so. coli) and the coat protein for the Norwalk virus. and polio has been effectively eradicated worldwide. These are countries where high cost and logistical problems such as lack of transportation. But Arntzen was primarily interested in developing a plantbased system for producing low-cost vaccines that could be used to immunize people in developing countries. So. Arntzen’s group showed that the antigens isolated from the transgenic plants did indeed invoke an immune response when injected into mice. Eleven volunteers were fed potatoes transformed with the E. the first human trials were conducted. in 1998. Both E. They had successfully produced an oral vaccine! Prior to this. coli and the Norwalk virus are common agents for severe diarrhea in humans and are a serious problem in developing countries. Arntzen and his group also successfully cloned into potatoes the genes for the toxin produced by the bacterium Escherichia coli (E.

Acetyl-CoA is also the primary building block for fats and oils. more palatable plant sources. molecular farming is in no way limited to pharmaceuticals. In fact. so plants produce it in fairly large . 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. that is found in virtually all cells. It is likely that other.100 Plant Biotechnology There is. Cooking. They must be eaten raw. In the meantime. apparently as a means for storing carbon. most humans do. called acetyl-CoA. by 2002. unfortunately. there are many species of bacteria that naturally produce fully biodegradable plastics known by the somewhat daunting name of polyhydroxyalkanoates (PHAs). While mice may not object to eating raw potatoes. In bacterial cells. denatures the protein and renders it inactive. such as bananas or tomatoes. the plastic accumulates as large bodies or inclusions. the product is no more biodegradable than those made from petroleum. especially those in countries where potatoes are not a normal part of the diet. PHAs are synthesized from a metabolic intermediate. however. scientists around the world were getting into molecular pharming. one problem with using potatoes as a vaccine delivery vehicle. One example is an Australian group that has cloned the gene for measles vaccine into tobacco. Although production of therapeutic proteins is an important step forward and captures the imagination. Plants could also be used to produce these plastics. On the other hand. there were an estimated 400 plant-derived genetically engineered pharmaceutical products (drugs and vaccines) in clinical development in the United States and Canada alone. They are now trying to clone the gene into rice. One of the more interesting applications is the production of biodegradable plastics. in much the same way that plant cells store carbon as starch and animals store carbon as glycogen. would be more acceptable. remote communities. Although corn oil has long been used as a substitute for petroleum in the manufacture of traditional plastics.

3). In genetically engineered cotton plants. By altering the genes of the cotton plant. or PHB) produced was not large. Recently. the genes for biodegradable plastics were expressed in the seed hair (fiber) cells. polyhydroxybutarate. One plant that has interesting possibilities is cotton (Gossypium hirsutum) (Figure 6.3 The cotton plant is a promising candidate for future genetic engineering. amounts. scientists may be able to produce a cotton fiber that has qualities superior to non-genetically engineered cotton fiber. The amount of PHA (in this case.Genetically Modified Plants: From Herbicides to Vaccines 101 Figure 6. 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. but it was enough .

The plastics made from petroleum do not break down very readily. This would be a real benefit for the environment. It may be possible to change the oil composition to . PHA-based consumer products discarded into landfills would be 100% biodegradable. suggesting that transgenic fibers could have enhanced insulation properties. might also be adapted to produce PHAs on a commercial scale. Consumers benefit indirectly by the reduction of greenhouse gas emissions from farming operations and lowered pesticide load in the environment. summary The first generation of genetically modified plants has focused on herbicide tolerance and resistance to insects and disease. but fermentation requires large industrial facilities and consumes substantial amounts of energy. 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. who benefit by reduced production costs. Some biodegradable PHAs are currently being produced by bacterial fermentation.102 Plant Biotechnology to alter the thermal properties of the cotton fiber. Producing biodegradable plastics by fermentation is currently about five times more expensive than the cost of conventional plastics made from petroleum. plastics are a mainstay of our modern consumer economy and because of that they also represent about one-fifth of the municipal waste across North America. The improvement of quantity and composition of oils from oilseed crops is one area under development. Since there are many bacteria capable of using PHAs as a carbon source. which are known for producing large quantities of products such as starch and oils. on the other hand. The primary beneficiaries of these products are the farmers. Plants. Another objective of using rDNA in plant breeding is to produce plants with enhanced nutrition. The transgenic fibers conducted less heat than normal fibers.

Producing edible vaccines in plants would drastically lower the cost of delivery and improve access to immunizations in the developing world. Molecular farming is the use of plants as bioreactors to produce a variety of useful molecules. It may also be possible to engineer plants to produce biodegradable plastics and other useful products. 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. . This would avoid the need to hydrogenate oils. increasing the proportion of stearic acid in plant oils could help to solidify shortenings at room temperature. a process that creates unhealthy trans fats. The primary focus is presently on using plants to produce vaccines and other protein-based pharmaceutical products. For example.Genetically Modified Plants: From Herbicides to Vaccines 103 serve particular needs.

Putting Genetically Modified Organisms in Perspective 104 .

You can’t get around it. —Lee Iacocca (1924–) American business executive .Every business and every product has risks.

What this statement usually implies is that they do not want some scientist 106 . Such foods are considered natural. GMOs should be vigorously debated. safe. no doubt. as the product of any new technology should be. worse yet. Informed debate on the GMO issue can only come about when the public has a broader understanding of the underlying scientific foundation. The debate should not be distorted by scientific misinformation and misinterpretation or. however. Many feel that GMOs. GMO supporters might be tempted to laugh at such antics. but it is important to remember that genetic modification (GM) is a new technology.Putting Genetically Modified Organisms in Perspective Perhaps you have seen them in the news—anti-GMO protesters dressed like a cob of corn or a tomato with a fish head. Most consumers accept without question foods produced through what is now called conventional plant breeding. but the debate must be based on an informed understanding of the facts. personal invective or political objectives. or acceptable.1). 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. safe. and acceptable. but for others there are legitimate 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. There are. are not natural. You should now be able to appreciate that the scientific concepts behind genetically modified foods are not all that difficult to comprehend. some protesters who are simply “anti” new technology.

But how natural is “natural”? The truth is that humans have been “messing around” with their food for a very long time.1 A Greenpeace activist wears a mask and shows a hand full of peas in front of the European Council building in Brussels. and the hybrid . Belgium. We have. 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. been practicing genetic modification since the dawn of agriculture some 10.000 years ago.Putting Genetically Modified Organisms in Perspective 107 Figure 7. The wheat that goes into your bread and breakfast cereal is the product of three different species. or large multinational company “messing” with their food. in fact.

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corn of today bears little resemblance to its ancestor (a somewhat 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 transforming plants on a daily basis. We have all occasionally, if unknowingly, 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 year’s 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 plant—yields 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 farmers (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. Marquis 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

Putting Genetically Modified Organisms in Perspective 109

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 actually 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 openpollinated 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, numerous 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

110 Plant Biotechnology

Figure 7.2 Double cross seed is produced by the careful breeding of four different inbred lines. Two of the inbred lines must be detasseled in order to prevent them from pollinating themselves.

It is impractical. One advantage cited by proponents of GM crops is that producing a transgenic “hybrid” is far more precise. A large amount of the conventional breeder’s effort is spent simply getting rid of this genetic garbage. 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. Many of these genes may be undesirable—they may cause the stem to grow long and weak. It may take 10–12 generations—meaning 10–12 years—to eliminate the undesirable genes. reduce the yield. If you don’t want to resort to rDNA technology. but you are unable to find a compatible wild type or other plant with that particular trait. what are your options? A conventional plant breeder actually has a variety of methods for creating the gene for a trait of . to remove them all. Recombinant DNA technology allows the breeder to select a single gene and insert only that gene into the genome of the commercial cultivar.000 or more genes that come along for the ride. The resulting progeny are then identical to the parent with the exception of that one additional gene. This means that all of the progeny. develOPinG new traits fOr the COnventiOnal Breeder Suppose you are a plant breeder and would like to introduce a new trait. or lower the quality of the flour. Even then.Putting Genetically Modified Organisms in Perspective 111 25. a process called backcrossing. if not impossible. must be carefully screened and those with undesirable traits discarded. There are no undesirable or unknown genes carried along. the new commercial strain will still be contaminated with unknown foreign genes contributed by the wild relative. which may number in the thousands. The remaining progeny are again backcrossed to the original cultivar. and the new cultivar with the new trait can be available to growers within two to three years. This is usually accomplished by crossing the hybrid back to the original commercial cultivar.

Earlier. and bacteria. What is the solution to this problem? Well. Lodged plants are a real problem for the farmer. in chapter 5. 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 pencil of the same diameter. including several short-stemmed wheat and rice cultivars. This is known as lodging. Those seedlings are then introduced into a conventional breeding program. a condition commonly precipitated by heavy rains or winds (Figure 7. for example. The same is true of plant stems. Thus. genetic dwarfs with reduced gibberellin levels are quite common in nature. Great idea. stem length is regulated by a natural plant hormone called gibberellin. are shorter than their “tall” vinelike climbing relatives simply because they make less gibberellin. They are more difficult to harvest.3). we mentioned the use of mutations to generate new traits. Over the last half of the 1900s. the plants developed a tendency to fall over and lie on the ground. Mutation breeding is a common technique in conventional breeding. so one solution to the lodging problem would be a wheat plant with a shorter stem. one way to create a dwarf plant is to induce a mutation that reduces gibberellin levels. Let’s use wheat as an example and see how mutations can be used to advantage. more than 1. As breeders succeeded in producing plants with larger numbers of heavier grains. but how do you go about it? In most cases. These varieties are accepted as “natural” foods solely because they are not GMOs. In fact. Mutated seedlings can be screened for a desired trait such as shortened stem length. Embryo rescue is a . now in contact with the ground. Bush beans or peas. have been developed through induced mutations. A variety of other techniques for manipulating genes are also available to the conventional breeder. and the grains. are more susceptible to losses from soil-dwelling insects. Traditional wheat varieties carry their grain at the top of long stems.400 cultivars. fungi.112 Plant Biotechnology interest. Plants that make less of the hormone generally have shorter stems.

Finally. but can be removed (or “rescued”) from the ovary and encouraged to reproductive maturity by culturing them artificially.3 The toppling of plants before harvest is known as lodging. which now have two exact copies of every gene. To the . These corn plants became lodged after the roots of the plants were damaged by insects. The cells. that would not normally mate in nature. often from different species. by mutation) in the vegetative cells. are treated with chemicals that force them to make a copy of their own chromosomes.Putting Genetically Modified Organisms in Perspective 113 Figure 7. The resulting embryos do not often survive naturally. there are often odd plantlets that arise apparently by spontaneous genetic changes (that is. Haploid breeding is a method in which pollen or egg cells. technique used to mate two plants. when plants are cloned by micropropagation. which normally have only one set of chromosomes. can be induced to form plantlets in tissue culture and grown to sexual maturity.

As intrusive as mutational breeding and these other techniques may sound. have recognized that these genetic changes. maturity date. but even those might contain traces. these “sports” are simply defective plants to be discarded. can we evaluate the safety of conventional foods? First. the popular Russet Burbank variety of potato arose as a somaclonal variant of the original Burbank variety. But in what way could rDNA techniques compromise food safety? Would it be the “new” or “foreign” DNA that is introduced or would it be the “new” protein encoded by that DNA? And. So. might be sources of new and useful traits. they illustrate the extent to which conventional plant breeding involves genetic modification but without involving rDNA techniques. for comparison. our diets contain a fair amount of bacterial . and similar products do not contain significant amounts of DNA.” A recent survey indicated that a surprising percentage of the public believe that natural foods do not contain DNA but that GMOs do. now called somoclonal variation (SCV). and tuber characteristics in potatoes. You can’t avoid bacteria—they are everywhere in our environment. SCV has been exploited to improve growth habit. We take steps such as pasteurizing milk to destroy harmful bacteria in the food we eat. All of the foods we eat contain DNA because DNA is a constituent of all living cells. 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. let’s address the question of “foreign” DNA and proteins. refined cooking and salad oils. In his book Pandora’s Picnic Basket. Alan McHughen relates an anecdote about an anti-GMO activist who stormed out of a meeting declaring that “You’ll never convince me to eat DNA.114 Plant Biotechnology micropropagationist. In fact. Others. Only highly processed foods such as soy protein. Many natural foods such as buttermilk and yogurt also contain cultures of harmless bacteria. but the bacteria are still there. they are just dead. however.

it has persisted with a life of its own. was engineered to delay softening and prolong shelf life while continuing to develop normal color and flavor. They live in very different worlds and what works in one will not necessarily work in the other. Although the story has been thoroughly discredited. called the Flavr-Savr™.Putting Genetically Modified Organisms in Perspective 115 Fish Genes and GMO Myths At the beginning of this chapter. This should not be too surprising since fish and plants are very different organisms. The tomato. The inverted gene could not be read by the cell’s genetic machinery any better than you could read this sentence if it were printed backwards. In writing the story. which encodes an anti-freeze protein that helps the fish to survive in icy waters. This is one of the myths of GMOs that arose probably out of a misinterpretation by an overzealous and non-critical reporter. would also help crop plants survive unseasonable frosts. not tomatoes—and it didn’t work. The flounder gene idea was eventually tried—with strawberries. but they were not modified with fish genes. we mentioned anti-GMO activists who dress as tomatoes with fish heads. The idea was that this gene. 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. recent research has shown that winter strains of rye and other plants make their own anti-freeze proteins. This costume is based on the belief that tomatoes were engineered with genes from a fish. the reporter apparently combined the Flavr-Savr™ project with the anti-freeze concept. GM tomatoes were the first GMO vegetable to be released for human consumption in the early 1980s. (!ees dna flesruoy ti yrT) The engineered tomato made less of the softening enzyme and the shelf life of the tomato was prolonged. so it is not necessary to look to fish genes for frost protection in plants. specifically arctic flounder. It was accomplished by removing a tomato gene—one that codes for the enzyme that causes the tissue to soften—and reinserting it backwards. Moreover. .

Bt protein does not affect humans. which are absorbed into the bloodstream. Potatoes and tomatoes are in the same family as belladonna. The potato alkaloid solanine accumulates in the green plants but not . We also eat lots of protein in the form of enzymes and structural proteins in our food. animals. cherries. DNA and protein are further denatured by the acids in our stomachs. and intestinal enzymes break down these molecules into their basic building blocks. The protein is then digested as any other protein is. and Datura (jimson weed). and the strongly acidic environment of the human gut immediately denatures the protein. contain cyanogenic glycosides. For example. These are chemical compounds that release cyanide when the cells are disrupted. but a half cup of dried seeds releases enough cyanide to kill an adult. The odd apple seed eaten with a core will probably cause little harm. It kills the larva by interfering with the absorption of nutrients in the gut. These DNA and protein building blocks are the same regardless of whether they came from plants. But cooking denatures both DNA and protein. however. plants have had billions of years to develop toxins that discourage creatures such as us from eating them. Remember that Bt protein is toxic only to the larval stage of insects in the family Lepidoptera. as well as some strains of lima beans. After all. Keep in mind as well that Bt is an approved insecticide for organic gardening. The human gut does not have the receptors that are attacked by Bt protein. the seeds (but thankfully not the fruit) of apples. because the human gut is fundamentally different from the insect gut. but neither are conventional foods. 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 earthworms and more than 95% with mice! GM foods are not immune to safety and security problems. and apricots. Some people express concern about eating a “pesticide” or “toxin” when corn or potatoes are transformed with the gene that encodes Bt protein. all of which contain deadly alkaloids. or bacteria. deadly nightshade.116 Plant Biotechnology DNA as well. Finally.

• Coffee contains caffeine. Glucosinolates are also called goitrogens because they can interfere with iodine uptake by the thyroid gland. brussels sprouts. cauliflower.Putting Genetically Modified Organisms in Perspective 117 in the potato tubers themselves. the concentrations of toxins in common food plants are so low that they can be tolerated without ill effects. potatoes that have turned green following exposure to strong light will contain elevated levels of solanine. • Kidney beans. cabbage. which is why you should avoid eating green potatoes. contain serotonin and other chemicals that raise blood pressure in animals. molecules that cause red blood cells to clump and in large doses can interfere with the body’s immune system. Numerous other foods contain various toxins as well. • Aside from allergenic protein that can lead to anaphylactic shock in sensitive individuals. We have . Here are just a few examples of toxins that are natural constituents of common foods: • Banana peels and. • Papaya and pineapple contain proteolytic enzymes (enzymes that break down proteins) that will cause irritation of the mouth’s mucous membranes. except in rare circumstances. and others) contain glucosinolates (mustard oils) that are responsible for the pungent taste of these vegetables. peanuts can become contaminated with molds that produce deadly aflatoxins. This is not intended to put you off from eating fruits and vegetables. However. the pulp. What you have to remember is that. and peas contain lectins. to a lesser extent. soybeans. resulting in goiters (an enlargement of the thyroid gland). • Brassicas (broccoli. an alkaloid that is toxic to the central nervous system. This is not a concern if the diet contains sufficient iodine.

Department of Agriculture (USDA) and the Food and Drug Administration (FDA) in the United States and the Canadian Food Inspection Agency (CFIA) in Canada. it must be genetically stable. regardless of its origin. There are two factors that help mitigate any concerns about food safety. into the marketplace. Is this a reasonable approach? Can you .S. any new crop variety. First. especially with respect to allergenic and toxic properties. Some opponents of GMOs insist that no transgenic plant should be released until it is proven safe. This is called the precautionary principle. The second factor is that. for example. at least in North America. new crop varieties are subjected to the scrutiny of several government agencies—the U. We assume a certain level of risk when we eat these foods. The attendant publicity would likely be the death knell for the company and that is not what investors in those companies want. The risk is manageable. In general. must meet three criteria before it can be registered and approved for release: it must be genetically distinct from other approved varieties. so we do not use it as an excuse to stop eating fruits and vegetables. but the same is true of conventionally bred foods. not only for GMOs but for conventional foods as well. than the food they replace. both GMOs and conventionally bred foods are carefully scrutinized to ensure that they are no more dangerous. A while back. a new strain of potato produced by conventional breeding was found to have unacceptably high levels of solanine and had to be pulled from the market. The point is that some plants do make things that are not necessarily good for you. Is it possible to create a toxic food by genetic modification? Of course it is. and the new variety must produce a uniform population of plants. In addition. it is unlikely that any company would knowingly release an unsafe food. but we have learned that the nutritional value of fruits and vegetables far outweighs the risk of death by starvation.118 Plant Biotechnology also learned to avoid eating plants or parts of plants that produce unacceptably high levels of toxins. whether produced by GM or conventional plant breeding.

These include traits such as cold tolerance or fungal resistance—traits that have yet to be developed. Even if it were necessary to use herbicide. . However. the USDA has reported that in 2002 American farmers planted nearly 80 million acres of genetically modified corn and soybeans. for example. a glyphosate-resistant plant could be killed with any one of several other herbicides. Why? A plant can succeed in the wild only when it enjoys some advantage over its competitors. would enjoy no competitive advantage because it would not be challenged with herbicide. That acreage is without doubt much higher at this time and there have been no ill effects reported. 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 secondgeneration GMOs. 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. there have been no unpredicted results from the release of any genetically modified product. however.Putting Genetically Modified Organisms in Perspective 119 anticipate or test for every possible eventuality? Probably not. An herbicide-resistant plant. In addition. Finally. 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. what aBOut the envirOnMent? Beyond food safety. Moreover. but no one seems unduly concerned about their release. after more than 25 years of experimentation. it is not likely to be a major problem. If pesticide resistance is a problem. pesticide-resistant plants have been produced by conventional breeding. we know that resistance to herbicides arises naturally in wild and weed populations based on selection pressure. creating a race of super weeds.

and farmers alike are conspicuous by their silence. fearing the loss of European and other markets where the import of GMOs has been banned. Consumer groups were concerned about the release of yet another GMO and corporate control over a major crop. and farmers who wanted to continue growing conventional wheat feared contamination of their crops with wind-spread pollen. The announcement was met with vocal resistance from consumer groups. and farmers themselves. and it would be tolerant to Monsanto’s very popular herbicide. they will arise because of the nature of the A Tale of Two Wheats In 2004. killed off the competition. Environmentalists were concerned that the “foreign” gene would spread into native grasses. Another concern is molecular pharming: Plants that are used as bioreactors to make pharmaceuticals 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. Even the Canadian wheat marketing board expressed opposition. was created by chemical mutagenesis . escaped plants could suddenly acquire an adaptive advantage that could be exploited if a sudden cold snap. environmentalists.000 acres in 2005—without a sound of protest. Fearing a public relations disaster. Consumers.-based chemical company Monsanto announced plans to seek permission for the release of a genetically modified. environmentalists. If there is a final lesson when it comes to using this new technology. called CDC Imagine. herbicide-tolerant wheat variety into the Canadian market. the U. glyphosate. Meanwhile. The wheat was modified by inserting a gene from a soil bacterium. for example.120 Plant Biotechnology In this case. Why the difference? Only that this wheat. thousands of acres of herbicide-tolerant wheat are already being grown across the Canadian prairies—more than 200. Monsanto wisely decided to cancel its plans.S. it is this: if there are going to be problems with food safety or the environment.

Some would say that our concern should be focused on the product. regardless of whether it was generated by conventional breeding or genetic engineering. So. In Monsanto’s case. Either variety has the same potential for cross-pollinating with wild grasses or contaminating the wheat growing in a neighbor’s field. rather than how that tolerance was bred into the plant. not the method. This does not mean. that there are not potential problems with GMOs. The question is whether the product—in this case. the wheat was transformed with a gene for herbicide tolerance. not because of the method that was used to generate that product. There are. 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. herbicidetolerance in wheat—presents a measurable risk. Both methods end up with the same result. 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 BASF’s proprietary brand of imidazolinone herbicides. It is more important that we be able to trust our government agencies and other organizations to carefully scrutinize the safety and potential environmental impact of any new crop variety. in fact. than it is to worry about the breeding method that was used to arrive at that product. we have two cases involving genetic modification at the level of a single gene. Both problems rather than by insertion of a bacterial gene. however.Putting Genetically Modified Organisms in Perspective 121 product itself. while in the BASF case an existing wheat gene was mutated. 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. two problems that are of particular concern to a growing number of farmers. What do you think? .

based on patented genes showing up in saved seed. The genes used to modify crop plants are patented. commonly referred to as “terminator technology” or “suicide seeds. Organic farmers must meet a number of conditions in order to have their crops certified as organic.” This genetic technology causes plants to produce sterile seeds—the crop is normal in all respects . Many farmers still exercise the traditional practice of saving seed from each year’s crop to use as seed for the following season. There are a number of ongoing court cases between seed companies and farmers. The farmers claim the seed was contaminated through no doing of their own. One of those conditions prohibits the use of genetically modified crops (Figure 7.122 Plant Biotechnology involve the potential for gene spread through cross-pollination.4). Organic farmers are also concerned about the spread of modified genes. If a genetically modified crop is planted in a neighboring field. while the seed companies claim the farmer is breaking patent law by saving seed that contains their patented gene. Organic farmers risk loss of their certification if their crops are contaminated with modified genes. This is called bin run seed because the seed is taken from the farmer’s own storage bin. Most of these court cases have involved herbicide resistance in oilseed rape (or canola). Saving seed reduces costs for the farmer who does not have to purchase new seed each year and often results in the selection of seed that performs especially well within the conditions of a particular farm. They sign agreements with the seed company and are legally prohibited from saving seed. Both seed savers and organic growers are also concerned about the possibilities of a new technology called genetic use restriction technology (GURT). and farmers who wish to plant the modified crops must purchase new seed each year. there is the danger that pollen from the genetically modified crop will contaminate the non-modified plants. Contamination can cause problems for the farmer who saves seed. Oilseed rape is a particularly precocious plant that readily crosspollinates over long distances.

some manufacturers have chosen to label their foods as being free of genetically engineered ingredients.Putting Genetically Modified Organisms in Perspective 123 Figure 7.4 Due to the public’s concern over the safety of GM foods. .

On the one hand. or eat food in a restaurant. which can be substantial. and further research on the technology was banned under a moratorium issued by the United Nations convention of biological diversity in 2000. there is concern that research continues in the United States. Terminator technology could seriously erode the farmer’s ability to save seed and increase their dependence on multinational corporations. consumers.4 billion people depend on seed saving to feed themselves. This would be particularly serious in Third World countries. climb into an automobile or airplane. terminator technology would help seed companies protect their investment in modified crops. Although several large U.” We subject ourselves to risk in hundreds of ways every day of our lives. where an estimated 1. Our recreation—Rollerblading. 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. every time we step off the curb to cross a street. skiing. It will likely be very difficult to balance the rights of seed companies against those of farmers and consumers. But in each . virtually everything we do involves some level of risk. noted biochemist Daniel Koshland wrote in Science that “to be alive is to be at risk.124 Plant Biotechnology except that the seeds produced by that crop will not germinate.S. Terminator technology generated substantial protest when it was first brought to the public’s attention in 1998. Terminator technology may help to focus farmers. it offers no agronomic benefit of any kind for farmers. governments. the technology was banned outright in several countries. swimming in shark-infested waters—and. whose rights must also be protected. in fact. which did not sign on to the moratorium. a QuestiOn Of risk Back in 1987. On the other hand. seed companies indicated they would not use the technology in their products. As a result. and seed companies on the real risks and benefits of genetic modification. bicycling.

allowing the insertion of one specific gene expressing a single trait. Both as individuals and as a society. against the risks associated with conventional food production. Conventional breeding. for example. simply by selecting the best seeds and crossing their best plants to improve the quality of grains and other food crops. In the end. we have to weigh the risks associated with biotechnology in all its forms. however. we should approach biotechnology with the same caution. we also take steps to minimize risk. A strong argument can be made that concern over food safety and protection of the environment should not be focused on the method. mutations. That means we should continue the necessary oversight to ensure that any new crop varieties. yet this method is unacceptable to many. we wear knee pads and helmets when we Rollerblade. to minimize any risk. And we have to take appropriate steps. meet acceptable guidelines for food safety and environmental protection. we add more safety features to our automobiles and airplanes. haploid breeding. farmers began what we now know as conventional plant breeding. We install traffic lights at pedestrian crossings. By contrast. and somaclonal variation. and the products of these methods are considered acceptable by the public. Herbicide tolerance. a whole new arsenal of methods was developed by plant breeders. including education. always carries unknown genes into the new variety. embryo rescue. regardless of the method of production. Each of these new methods has allowed the introduction of new traits into our food plants.Putting Genetically Modified Organisms in Perspective 125 case. however. summary Very early in the development of agriculture. In the twentieth century. can be introduced . we attempt to educate our children and others about these risks and how to minimize our exposure. and especially GMOs. Moreover. including double-crossed hybrids. genetic modification (GM) is a far more precise technique.

The question of food and environmental safety of any new crop really comes down to a question of risk management. continue to meet acceptable guidelines for food safety and environmental protection.126 Plant Biotechnology by both genetic modification and conventional breeding. rather than the method. The real focus should be that all new crop varieties. If herbicide-resistance trait is a potential problem. and nutritious. healthy. We know that many foods contain potentially toxic substances and that genes naturally spread through the environment. regardless of how they are produced. should it matter how that trait was introduced into the crop? It would be more productive to focus on the safety of the product. but over the millennia we have learned to manage any potential risk and produce foods that are high-yielding. .

also called simply “a phage. 127 . Bin run seed Seed saved from a farmer’s current crop to be used to plant the fields the following year. Biogas Methane gas that is generated by the microbial decomposition of organic matter in the absence of oxygen. and uracil. Backcross A cross between a hybrid and one of its parents (or a geneti- cally equivalent organism). Batch culture A bioreactor that is emptied and set up anew each time the reaction has run to completion. Bioreactor Any system that uses living organisms or enzymes to effect chemical changes. and morphine are examples of alkaloids. Nicotine. Anti-parallel The condition in double-stranded DNA in which the two strands with polarity run in opposite directions. such as decomposition. to decon- taminate polluted soils.Accumulator species Plants that take up large amounts of heavy metal ions from the soil without injury. Aseptic Free of contamination with microorganisms. such as at the base of stem cuttings or from a clump of callus tissue in culture. Bioremediation The use of living organisms. Allele One of the two or more possible variants for a gene. caffeine. cytosine. guanine. as opposed to buying new seed each season. Adventitious shoot (root) A shoot (or root) that arises where it is not normally expected. decontamination. Bioengineering The application of engineering techniques to biological processes. usually bacteria. thymine. A diploid organism contains two alleles. such as one of the five bases that make up nucleic acids: adenine. such as large-scale cultures of fungi to produce drugs. Bacteriophage A virus that infects bacteria. Apical meristem The growing point at the tip of a shoot or root. or production of chemical products. Alkaloid A nitrogen-containing chemical produced by plants that is physi- ologically active in vertebrates. one contributed by each parent.” Base A type of molecule.

Cyanogenic glycoside A chemical. Chemical mutagen A substance that induces mutations. Effervescence The property of producing large numbers of bubbles that rise to the surface with a fizzing sound. Continuous culture A bioreactor that is fed by a stream of nutrients and produces a continuous stream of effluent to be processed. that releases toxic hydrogen cyanide when the cells are disrupted. such as in beer or soda pop. a second round of copying restores the sequence of the original strand. in the DNA of a living organism. Electroporation The momentary induction of small pores in the membrane of a cell or protoplast by a brief pulse of electric current. Crown gall A cancerous growth on plants due to transformation of the host cells by the bacterium Agrobacterium tumifaciens. in which the nucleotide sequence in the original strand is preserved in the newly formed complementary strand. or genetic changes. Butanol A type of alcohol consisting of four carbon atoms per molecule. Conventional plant breeding Any method for producing new plant variet- ies that does not involve recombinant DNA. Cellular respiration A sequence of metabolic reactions that converts sugars and other substrates to carbon dioxide and water in the presence of oxygen. Codon A sequence of three adjacent nucleotides in DNA or RNA that com- prises the genetic code for one amino acid in a protein. It enables plant protoplasts to take up small pieces of DNA from the surrounding medium. The principal energy-generating metabolism in a cell. found in some plants.Biotechnology The use of living organisms to generate products for the use and benefit of humans. DNA ligase An enzyme that catalyses the formation of strong covalent bonds along the sugar-phosphate backbone of DNA. Complementarity The property of base pairing in nucleic acid synthesis. Edible vaccine A vaccine produced in a common food (such as bananas) that can be administered simply by eating the food. Embryo rescue A technique for culturing the hybrid embryo formed when two plants that do not normally mate are crossed. 128 .

129 .Enterotoxin A toxin that has its effect in the gastrointestinal tract. amino acids in a protein. Fermentation The metabolic breakdown of sugars and other sub- strates in the absence of oxygen. The principal component of fats and oils. or a similar chemical. A sequence of nucleotides in DNA that encodes the sequence of RNA. The difference between respiration and fermentation is in the fate of the pyruvic acid. Fatty acid A long-chain hydrocarbon (composed of only carbon and hydrogen) with an oxygen-containing carboxylic acid group at one end. Genome The sum of all the genes in an organism. Genetic modification (GM) Any change to the genetic constitution of an organism. or carries other instructions for the synthesis of proteins. crude oil is the feedstock for the production of gasoline and diesel fuel in a refinery. The technique is known as biolistics. It is common to both cellular respiration and fermentation. Fructose A six-carbon sugar molecule with a structure similar to glucose. 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. For example. The product of fermentation is usually ethanol. lactic acid. Glufosinate An herbicide that kills plants by blocking the incorpora- tion of inorganic nitrogen into organic molecules. although in common usage it refers specifically to changes involving recombinant DNA technology. Common table sugar (sucrose) is made up of one molecule each of glucose and fructose. Feedstock The raw or starting material for an industrial manufac- turing process. Gene The basic unit of heredity. Gene gun A laboratory tool for shooting foreign DNA into plant cells. Glycolysis A sequence of enzymatic reactions in a cell that convert one molecule of glucose to two molecules of pyruvic acid.

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 molecular 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 generation 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

Molecular Biology of the Cell. Inc. Eating in the Dark.. Szczawinski. W. Food.. New York: Garland Science. P. 1999. The Uses of Life: A History of Biotechnology. 1993. A. 2001. Hopkins. Tagliaferro.Alberts. Lewis. P. Portland. New York: Simon & Schuster. N. New York: Atheneum. E. Genetically Engineered Food: A Self-Defense Guide for Consumers. Hart. J. Introduction to Biotechnology. Plasterk. F. K. J. 1997. Pandora’s Picnic Basket: The Potential and Hazards of Genetically Modified Foods. Biotechnology Unzipped. DC: ASM Press. New York: Marlowe & Co. B. 1991. 2002. R. W. Toronto: Trifolium Books. Massey. A.. G. Evert.. Kreuzer. 2002. H. J. Eric R. R. R. J. Eichhorn. England: Cambridge University Press. Grace. L. and A. OR: Timber Press. H. A.” Science 296 (2002): 1263–1265. 2003. 2004. Oxford: Oxford University Press. Genetic Engineering: Progress or Peril? New York: Lerner Publications. 1997. 133 . A. Turner. 3rd ed. Common Poisonous Plants and Mushrooms of North America.. Johnson. Raven. Bud. Thieman. “RNA Silencing: The Genome’s Immune System. 2004. F. Watson. Lilliston.: Mendel to Monsanto—The Promises and Perils of the Biotech Harvest. and M. Cummins. A. et al. P. San Francisco: Benjamin Cummings. Washington. and B. H. Pringle. New York: Worth Publishers. R. New York: John Wiley & Sons. and N. S. McHughen. Cambridge.. The Double Helix. Hüner. New York: Vintage Books. and A. 1968.. 2000. 2000.. Recombinant DNA and Biotechnology. Biology of Plants. D. Palladino. Introduction to Plant Physiology.

International Survey of Herbicide-Resistant Weeds http://www. Tagliaferro. J. 2003.Hart. D.nysaes. Inc.org This site has many links to both Canadian and American renewable fuels sites.greenfuels. New York: Atheneum.: Mendel to Monsanto—The Promises and Perils of the Biotech Harvest. Watson. Eating in the Dark. 2002.asp With extensive use of herbicides and herbicide-resistant crops. Genetic Engineering: Progress or Peril? New York: Lerner Publications. The Double Helix. Food. National Center for Biotechnology Information http://www. This interesting Website tracks the origin and distribution of herbicide-resistant weeds.html This site describes the origins of the helium-powered gene gun. 1968.nih.molecularfarming. Nobel Prize in Physiology or Medicine http://www.dnalc.ncbi. Cold Spring Harbor DNA Learning Center http://www. 1997.org/in.cornell.org A student-friendly resource with links to current topics in gene cloning and recombinant DNA techniques. the development of herbicide-resistant weeds is an increasing problem. 134 . L. Gene Gun http://www. New York: Simon & Schuster.com Well-constructed site with extensive links to other sites both for and against genetic engineering. Pringle. P.nobel. K.nlm.edu/pubs/press/1999/genegun. Molecular Farming http://www. Web Sites Canadian Renewable Fuels Association http://www.weedscience. New York: Vintage Books.se/medicine Information on past recipients of the Nobel Prize.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 emphasis on human and animal applications).

html Provides animated video and links to questions and answers. which provides access to other useful information about transgenic crops.“RNAi. Includes a link to the Transgenic Crops Homepage (http://filebox.pbs.edu/cals/cses/chagedor/crops.html A brief description of how the genetic restriction technology works. html).org/wgbh/nova/sciencenow/3210/02. Terminator Technology and Transgenic Crops http://filebox.vt.vt.” Nova (Public Broadcasting System) http://www. 135 .edu/cals/cses/chagedor/terminator.

Bt and. 77 Bioreactors. 5. 80–82 Bin run seed. 29 Biotechnology. PHAs and. 16 Caffeine. 20 Astralgus. 17–19 Cellulose. 23. 77–79 Axillary buds. 32 Alkaloids. 90. 41. 22 Antibiotics. 32 Auxins. 21. 122 Catalysts. fermentation and. 100–102 136 Biodiesel. toxicity of. defined. 27 Amylase. defined. 50–52 Cheese. 122 Biodegradable plastics. 120–121 cDNA. 4–5. Paul. C. 115 Antiparallel. 62. 26–28. 41. 20–22. 73. 96 Bialaphos. 52–55 Batch culture. defined. 121 Cane sugar. 54 Apical meristems. 114 Bacteriophages. 116 Backcrossing. 36 Aspergillus niger. 120–121 Chemicals.J. 111 Bacteria. Erwin. 40 CDC Imagine wheat. 44–46 Bioengineering. 97–102 Chitinase. 36 Aflatoxins. 44 Biolistics. 26. 116–117 Amino acids. 22. 56. 94. peanuts and. gasohol and. 18. 117 Agrobacterium tumefaciens. bioremediation in. 122 Chargaff. 22 Aspergillus oryzae. 98–99 Aseptic technique. 19 Clones. 32–35 Acetic acid. 62 Colorado potato beetles. 8 Biofuels. 32. 22 Citric acid cycle. 27. 76. viral resistance and. 70–71 Cellular respiration. 7 Alkali poisoning. 28 Alcohols. 32 Boyer. 35 Codons.Accumulator species. 5. 60–62 Base pairing. 61–62. 20 Adaptive advantages. 33 Chemical mutagens. 119–121 Advantages of GMO crops. 10. 17. bioremediation and. 91 . enzymes as. crown gall and. 23. 61–62 Beta-carotene. 40–44. 89–92. 101 Arntzen. 118. fermentation and. 97–102 Bioremediation. 90–91 Butyl rubber. 19 Acetyl-CoA. 34. 8. 25. 62 Brassica species. Stanley. 16 Butterflies. 98–99 Antifreeze genes. 93 Brazil. 57 Cohen. 41–43. 7 Butanol. 4–5 Berg. 5–7 Chelation. 108 Alaska. 43–44 Canola. 64 Antibodies. 21. 38–39 Bacillus thuringiensis (Bt) protein. 24 Cattails. 24. 111 Adventitious shoots.. 92 Citric acid. 47 Biogas. 24. in foods. 89. 43–44 Certification of organic crops. 100–102 Actinomucor elegans. Herbert. protein synthesis and. 43–44 Breadmaking. defined. 10 Blind staggers disease. 117 Canadian Food Inspection Agency (CFIA). 21 Beer. 38 Arabidopsis thaliana. 93. 26 Cattle. defined. 77–79. 80–82 Ammonium perchlorate.

87–88 Food and Drug Administration (FDA). 63 Edible vaccines. 115 Fleming. 102 butanol production and. 75–79 modern biotechnology and. 115 Flower coloration. Alexander. 61–62. 76. 56–60 DNA fingerprinting. 19. 59. See also specific enzymes Ereky. 76–79 CryIAb. 118. 80. citric acid and. 122 Continuous culture.. 55.S. 116 Cytokinins. 41. 74 DNA polymerase. 100 Corn. 89–92 Environmental Protection Agency (EPA). 44. micropropagation and. 52–54 EcoRI enzyme. 119–121 Complementarity. 4–5 Double helix model. 73 Explants. 67 Complementary DNA (cDNA). defined. 26 Deoxyribonucleic acid (DNA). 70–71 Contamination. crown gall and. 23–24. 118 137 . DNA. 97–102. 46. 5. 110 Corn steep liquor. 122 Crown gall. 66 Electroporation. 8 gasohol and. 112–113 Enolpyruvyl-shikimate-3-phosphate synthase (EPSPS). 93–95 Feedstocks. 65–66. 60–66 overview of. 82 Enterotoxins. 108–110 Cross-pollination. U. 101–102 Crick. 23 Flounder genes. 24–25. 111–114 Cooking oils. 77–79 Denitrifying bacteria. 108. 23 Cotton. tomatoes and. 93 Escherichia coli (E. 43–44 Ethyl methane sulfonate (EMS). 109–111 herbicide tolerance and. 74 Domestication. 8 Fermentation biodegradable plastics and. 106 disadvantages of. 22. 44 glycerol production and. 77 Embryo rescue. 14 overview of. 74 DNA ligase. 14 Erucic acid. 38 Exxon Valdez. 28 Farming. 54–56 insertion into plant cells. 108–109 trait development by. 114–117. 120–121 history of. 52–53 Crossing. 75–76. 53–55. 96. 99–100 Effervescence. 116 proteins and. 119 Disease resistance. 21. 99 Ethanol. Karl. 115 Flavr-Savr tomatoes. 22 Electrophoresis. 14–15 industrial production and. Francis. coli). 17–19 Fingerprinting. seeds and. 10 Environmental safety. 21 Conventional plant breeding acceptance of. molecular. 54. 89–92 DNA genes and. 16 feedstock production and. 28. 65–66. 118 Food safety. 42. 107–108 pesticide tolerance and. 50–54 presence of in all foods. 61–62. 119 process overview.Competitive advantages. 74 Fish genes. 119–124 Enzymes. 20–25 Louis Pasteur and. 91 Cyanogenic glycosides. 46. 120 Fatty acids. See DNA Department of Agriculture. 114.

42 Glycine max. 123 . 76. 61 Human immunodeficiency virus (HIV). 7–8. 8 Genetic modification (GM). 82 Hybridization. 66 Gene guns. 89–92 Insulin. 93. GMOs and. 98–100 Inbred lines. Rosalind. 14–15. defined. 112 Glucose. 46 Glycerol. 116 precautionary principle. 18. fermentation and. 56–57 public resistance to. 44–46. 63 Invertase. 76–79 Gasohol. 9. 22 Ionizing radiation. 8 Genetic engineering. 80–82 Glycerin. 94–96 Greenpeace. defined. 5 Glucose isomerase. 71 Human toxicity. Rich. 73 Jorgensen. 117 Golden rice. 53 Fungi. 75. 93–95 Hydrogen bonds. 32 Helicase. 114. 109 pesticide consumption. 43–44 Gels. 41. 19. 16 Haploid breeding. 28 Hydrogenation. 119–122 Genetically modified organisms (GMOs). 81 Glycolysis. 116 gene spread. See also specific species Fungicides. 79–82 protein structure and. 87–88 Krebs cycle. overview of. 21. 20. 21 Groundwater. 88–89. 120–121 GM crops. 26. 120–121 tobacco.Forensics. 109 High-fructose syrups. 55 Helices. 108–109 Indicator species. 113 Heavy metals. 35 Genetic use restriction technology (GURT). 86–87. 52–54 Hemophilus influenzae. 61 Hepatitis B antigen. 118–119 risk assessment. 22. 56 Gibberellin. 122–124 toxin presence as. 52–55 Immunizations. 109 Genomes. 65–66 Franklin. 119–122 myths. 98–99 Herbicide tolerance criticisms of GMOs and. 17–18 Glyphosate. 119 genetic engineering and. 80–81 Fusarium infections. 115 patenting. 77 Genes. 107 Griseofulvin. 79–82. biofuels and. 124–125 terminator technology. 114 DNA consumption. 24 Glucosinolates. 9 Fusion of protoplasts. 122–124 Genetic variability. 75 Gall. 8 Genetic reassortment. 116–118 138 Goitrogens. 32–33 Insecticides. 108–109 Hydrocarbons. 10 value of. 86–87 Heritage varieties. 27 Gun cotton. 24–25 Hind III enzyme. 117 Glufosinate. 19 Labeling. 54. criticisms of bacterial consumption. 54–56 Gene spread.

26–27 Oils. 100 Oilseed rape. 15 Pasteurization. 40 Nature. 109 Orchids. 16 Nobel. 39 Organic farming. cDNA. 119 Petroleum products. 22 Penicillin. 73–75 Marker genes. 62–64. 23 Perchlorate. 60 Microbiology. protoplasts and. 95 Locoweed. 51. 73–75 Overexpression. 32 Lodging. 34–35 Phytochelatins. 113–114 Molecular farming. 17. aflatoxins and. 14–15 Nickel. pigmentation and. 14 Osmosis. 60–62 Pharming. 28 Petunias. methane and. Linus. edible vaccines and. 34 Nitroglycerin. 120 Monarch butterflies. 23 Penicillium chrysogenum. 80 Oxygen. 62 Norwalk virus. 87–88 Metabolites. 24 Paper mills. chemical. 90–91. 65 Marquis wheat. biofuels and. 32–33 Phytoremediation. 4–7. 40–41 Methylation. 114 Papain. Louis. 112. 28 Landfills. 93–95. 14 Micropropagation.. 122 Open-pollination. 116. biodegradable. 50. 57. Alfred. 93–96 Oil refineries. molecular. 73 Mutation breeding.A. 44 Pandora’s Picnic Basket. 97–102. 76 Mannitol. feedstock and. 20 Lactobacillus. 116. 120 Phytoaccumulation. 16–17 Nobel Prize. 73. 87–88 Mutagens. 64. 59. 46. 14. 113 Lysine. Bt and. Bt and.Lactic acid. 19. 14. 87–88 Phages. 100–102 139 . 112 Nanotechnology. 33 Phytoprospecting. 36–40. 10 Natural gas. 18–19. Pasteur and. 90–91 mRNA. 79 Plastics. 90 Neuberg. 39–40 Pauling. 34 Pasteur. 65. protoplasts and. 70–71 Ligase. production of. 58. 27 Pesticides. 94. transformation of. 57. 41–44. 117 Pectinase. 93. improving. 40–41 Landraces. 32 Methane. 97–102. See Polymerase chain reaction Peanuts. 54. defined. 100 Messenger RNA (mRNA). 20 Lake Ontario. C. 80–82 Libraries. 122 Origins of biotechnology. 74 Lipids. 61. 40–41 Measles. 71–73. 114 Pathogens. 21. 70–71. biofuels and. methane and. 20–25 Metals. 41–44 Plasmids. phytomining and. 52–54. 109 Lepidopterans. 76 National Center for Biotechnology Information. 59 Nutrition. 108 Marsh gas. 112 Mutations. 116 Liberty. 59. 96 Maize. 75–76 Plant oils. 26. 52–54 PCR. 58. 70–71. 99 Nucleotides. 32–35 Plant cell transformation.

75 Soybeans. 28 Probes. bioremediation and. 22. 36 Sterility. 108 Selenium. 43–44 Sugar-phosphate backbone. 74 Resistance. 116–117. 74 Polymerases. 65–66. 24. 22 Rennin. 61–62 Solanine. 111 Prince William Sound. 40 molecular farming and. 119–124 Salix species.Pollination. 35 Saturation. 118. 88–92. 20 140 Riboflavin. 40. 89–92 Protoplasts. 19 Safety. synthetic. 99 somaclonal variation (SCV) and. 9. 44 micropropagation and. 118–119 Precision. 64 Replication. 93–95 Sedges. 93 Reassortment. fingerprinting and. 41 Smith. 62–64. 59 Stearic acid. 65–66. 86–87. 17–19 Restriction enzymes. 60–62. 118 disease resistance and. 24 Replica plating. 28 Pyruvic acid. 114 Saccharomyces cerevisiae. 114–117. 114 Precautionary principle. 50. 35 Recombinant DNA (rDNA). 38 Shotgun biolistics. 94 Sterile technique. 70–71 Rhizopus. 93–94 Selection. 81 Soy sauce. 75 Somaclonal variations. 22 Ribonucleic acid (RNA). 67 Rennet. 120–121 Rubber. 70–71 Reverse engineering. 8. 56–60. assessment of. 43–44 Start codons. 62–64. 18–19 Radiation. 94–96 Risk. 20. 117 Sewage treatment plants. 87–88 RNA silencing. 76. 92. 7. 100–102 Polymerase chain reaction (PCR). 33 Serotonin. 20 Sugar cane. defined. 73 Rapeseed. 67 Retroviruses. 16 Russet Burbank potatoes. 26 Seed oils. 122 Polyhydroxyalkanoates (PHAs). 75 gasohol and. Golden. bananas and. 54. 109. 74 Potatoes alkaloids in. 73–75 Pseudomonas. 44–46. 8. 55. See also Herbicide tolerance Respiration. Hamilton. 5. 124–125 RNA interference (RNAi). 87–88 Roundup. 79–82. 66 Productivity. 59 Rice. 80–82 Sufu. 41. 20 Starches. methane and. 122–124 Sticky ends. 88–90. 57 Ribosomes. 62 Streptomyces hygroscopicus. 87–88 Silos. industrial production of. 92–94 Proteases. 17. gasohol and. 6. 71 Reverse transcriptase. 40 Shoot-tip culture. 32. 61–62. 113–114 Somatic hybridization. ionizing. 118 Solanum tuberosum. 64. 117 Proteins. 34 Sequestration. 52–55 . 116–117. 77 Silencing. as advantage.

41. 16 Weizmann process. 92 Visualization. 73. 92–94 Zea mays. RNA and. 26 Watson. James. 82 Toxins. 34 Thermal properties. 34 Transcription. 36 Vetch. 40 Vegetative reproduction. 39–40. 14 141 . 108. 122–124 Thallium. 5 Yeasts. 16. 97–98 Tobacco mosaic virus (TMV). 41 Suppression. 42. 52–53 Weeds. 56–57 Triglycerides. 26 Wheat. 44 Tempeh. 112. fermentation and. 9. 40–41 Sweet potatoes. 14 Zymotechnology. 62–63. genetic. 98–100 Variability. clones and. Chaim. 5 Yields. 96 Waste management. Phillip. 35 Wines. 58 Triazine resistance. 32 Vinegar. 76–79 Translation. 38 Toxicity. 72 Tomatoes. 74 Teosinte. 60–62. 35–36 Vaccines. 108 Terminator technology. 75–76. defined. 57 Transfer RNA (tRNA).Suicide seeds. 19 Viruses. 20 Temperature. 76 Zymology. 17 Wetlands. biofuels and. 36 Wilkins. 53 Willows. 33 Ti plasmids. 70–72. 22. 101–102 Thlaspi species. 58. human. somaclonal. 115 Totipotency. 59 Transformation of bacteria. 79 Tissue culture. 64 of plant cells. Maurice. 109 Variations. 116–118 Trace elements. 87–88 Swamp gas. 120–121 White. in all foods. 66 Vitamins. PCR and. 36 Tobacco. 122–124 Sunflower oil. control of. 10. 86–87 Weizmann. phytomining and. 95 Uniformity.

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the organization of chlorophyll-protein complexes. and energy transformations in chloroplasts. Hopkins has taught primarily in the areas of plant physiology and cell biology. Hopkins has been a contributing author to two high school biology textbooks and is the senior author of Introduction to Plant Physiology. His research and publications have focused on the role of light and temperature in plant development. was responsible for design and implementation of an honors program in cell biology. in biology from Wesleyan University and a Ph. where he is now professor emeritus (Biology).William G.A. a textbook published by John Wiley & Sons and now in its third edition. In 1988. 143 . Dr. Dr. Hopkins received a B. and served many years as an undergraduate counselor.D. Hopkins was awarded the university’s Gold Medal for Excellence in Teaching. Dr. His post-doctoral training was conducted at Brookhaven National Laboratories. in botany from Indiana University. He has taught at Bryn Mawr College and the University of Western Ontario.

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