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OCR for page 40
Applying the Tools of
Biotechnology to
Agricultural Problems
Effort is now under way to apply these cellular and molecular tech-
nologies to specific agricultural problems. For instance, researchers are
attempting to clevelop herbicide-resistant plants and plants less suscep-
tible to environmental stresses, such as drought, salty soils, or climatic
extremes. They are working on increasing the nutritional quality of feed
crops. They are also trying to engineer soil microorganisms that can be
used to supply nitrogen to crop plants, or else mitigate or combat soil
diseases.
Herbicide Resistance
Some 420 million pounds of herbicides are used by farmers each year
in the United States. Herbicides are used to kill the weeds that compete
with crop plants. Unfortunately, they can also kill some crop plants as
well. This restricts each herbicide to use on a resistant crop. Neverthe-
less, crop Tosses from herbicides can still occur. A case in point is the
herbicide atrazine, which is commonly used in the culture of corn. In
Illinois, for instance, 90 percent of corn acreage is treated with atrazine
each year. Corn can tolerate atrazine because the plant naturally contains
an enzyme that rapidly breaks down and detoxifies the herbicide. Yet
in the Midwest, corn is often used in rotation with soybeans, which are
susceptible to atrazine. Sometimes winter climatic conditions cause res-
idues of atrazine to remain in the soil the following spring. Such residues
can dramatically reduce the yield of soybeans planted the following year.
To avoid such losses and to broaden the range of each herbicide, plant
breeders are interested in developing herbicide-resistant crops. An atra-
zine-resistant soybean, for example, would be ideal for the Corn Belt.
But before the trait can be transferred, it must be unclerstood. At
40
OCR for page 41
BIOTECHNOLOGY AND AGRICULTURE
41
Michigan State University, Charles Arntzen and colleagues have been
trying to elucidate the genetic mechanism of atrazine resistance in weeds.
Atrazine-resistant weeds began to appear spontaneously in the early
1970s, primarily in areas of prolongecl, steady use of the herbicide. Some
30 species of weeds have now become atrazine resistant. To find out
how, Arntzen, a plant biochemist, chew on the work of plant physiol-
ogists, other biochemists, and agroecologists, as well as molecular bi-
ologists.
Like the majority of herbicides, atrazine acts in the chIoroplasts where
it disrupts photosynthesis. Photosynthesis is the process by which plants
convert sunlight to chemical energy. Through photosynthesis, the sun's
energy is used to support all higher forms of life on earth.
In photosynthesis, sunlight is absorbed through chlorophyll, the green
pigment in the chIoroplast. The sun's energy is used to force electrons
to an excited state. In a complex series of reactions, the energy held by
the excited electrons is used to build carbohydrates.
One of the keys to photosynthesis is the transfer of solar energy from
the chlorophyll, where it is absorbed, to the place where it is used. The
excited electrons hold that energy. In bucket-brigade fashion, electron
carrier molecules transport those electrons from one molecule to another.
One of these carrier molecules is quinone.
It is now known that atrazine kills plants by disrupting this electron
transport, thereby blocking photosynthesis. It does this by competing
with quinone, one of the carrier molecules. When atrazine is taken into
the chIoroplast, it can take the place of the quinones on one type of
protein in the membrane that holds the electron transport chain together.
Without quinone to transport the electrons, photosynthesis is halted.
Atrazine resistance arises from a mutation that alters that membrane
protein so that it will no longer bind atrazine. The quinone, however,
still binds to the altered membrane protein; consequently, electron trans-
port remains undisturbed in the presence of atrazine.
Using traditional biochemical procedures, Arntzen's group spent three
years trying to purify that protein in orcler to study the nature of the
mutation. They did not succeed. The task of determining the sequence
of amino acids in a protein is difficult and time-consuming. It is easier
to sequence the nucleotide bases in the gene that codes for the protein.
From other studies, Arntzen knew that the protein was encoded by a
gene in the chioroplast. Arntzen's group began collaborating with Lee
McIntosh of Michigan State University and Lawrence Bogorad of Har-
vard to study the genes of the chIoroplast. One of the chIorop:tast genes
had already been isolated and cloned. It was the gene Arntzen was
seeking.
OCR for page 42
42
GENETIC ENGINEERING OF PLANTS
Since then, the genes in both atrazine-susceptible and atrazine-re-
sistant weeds have been sequenced. The only difference between the
two is one nucleotide base: in the resistant weect, an adenine is replaced
by a guanine. That one change spells one different amino acid, creating
a slightly different protein but different enough to cause a "glitch"
that prevents atrazine bincting. In short, one nucleoticle substitution in
a single gene determines whether a plant is resistant or susceptible to
this herbicide.
Engineering an Atrazine-Resistant Crop
Because resistance to atrazine is conferred by a single gene, this trait
seems amenable to molecular gene transfer. But there are other, simpler
approaches to develop herbicide-resistant crops.
To date, the greatest success has come from classical plant breeding
aided by knowledge garnered from molecular-level investigations. At
the University of Guelph, Ontario, a research team including W. Bev-
ersdorf and Vince Souza-Machado have bred atrazine-resistant strains
of oiTseed rape (Brassica napus) and summer turnip rape (Brassica cam-
pestris). This was accomplished through repeated backcrosses between
rape and a closely related resistant weed, wild turnip (Brassica campestris).
The key to this success was the knowledge that the trait is carried in
the chIoroplast. In sexual crosses, the chIoroplast is transmitted by the
maternal line alone; the mate contributes only nuclear DNA. That means
the only way to generate a resistant crop is to use the resistant weed
as the female parent and a crop plant as the source of pollen. The
resulting resistant progeny are then fertilized with pollen from the crop
plant, and the process is repeated for five to seven generations. "Finally
what you get is a crop a new plant in which the cytoplasm, including
the chIoroplast, is essentially donated by the weed, and the nucleus is
donated by the crop plant," Arntzen described.
Although backcrossing is laborious, it works. "Current estimates sug-
gest that by 1985, with increased seed stocks, there will be close to one
million acres of atrazine-resistant oilseed rape grown in Canada. It's a
dramatic success story, and it didn't take one iota of genetic engineer-
ing."
Unfortunately, an identical approach is not feasible with many crops,
as few are cross-fertile with weeds. There are, however, many herbicide-
resistant weeds that are closely related to—but not cross-fertile with
major crop plants. For example, the atrazine-resistant weed black night-
shade (Solanum nigrum) is in the same genus as potato (SoZanum tuber-
osum) and the same family as tobacco and tomato. That is where the
OCR for page 43
BIOTECHNOLOGY AND AGRICULTURE
Atrazine-Resistant Weeds That Are in the Same Botanical Family as Crop
Plants
43
Atrazine-Resistant Weed
Crop Plant
CHENOPODIACEAE
Chenopodium album (common lambsquarters)
Artiplex patula
COMPOSITAE
Senecio vulgaris (common groundsel)
Ambrosia artemisifolia (ragweed)
Brassica campestris (wild turnip)
Solanum nigrum (black nightshade)
Beta vulgaris (sugar beet)
Beta vulgaris (red beet)
Helianthus annuus (sunflower)
Carthamus tinctorius (safflower)
CRUCIFERAE
SOLANACEAE
Brassica campestris (turnip rape)
Brassica napus (oilseed rape)
Brassica oleraceae (cabbage)
Solanum tuberosum (potato)
Lycoperiscon esculentum (tomato)
Nicotiana tabacum (tobacco)
SOURCE: Charles T. Arntzen, Plant Research Laboratory, Michigan State University, East Lansing.
new genetic technologies come in. Because the species are closely re-
lated, their protoplasts can be fused in culture to create a hybric3. The
first of these experiments was reported) in 1982 by Horst Binding in West
Germany and lonathan Gresse} in Israe} and their colleagues, who fused
protoplasts of black nightshade and potato. The goal was a resistant
potato; unfortunately, the atrazine-resistant hybrid was more like the
weed than the potato. A solution may be in sight, according to Arntzen.
In a half dozen laboratories around the worId, researchers are now trying
to inactivate the nuc:teus in the protoplast from the donor weed, so that
just the weed's cytop:Lasm which contains the resistant chIoroplast—
will be introduced into the potato protoplast. "If somebody doesn't have
a herbicide-resistant potato plant within the next year or two, 1'd be
very surprised," Arntzen said.
An alternative approach is to find a mutant in nature yet this par-
ticular mutation rarely occurs. Arntzen and others are investigating
methods to induce this mutation.
The most powerful technique, if it can be mastered, will be to transfer
the resistant gene from a weed into a crop plant using recombinant
DNA technology. "There has been a Tot of progress along these lines,"
Arntzen said, "but we still have a great deal of work left to do." So far,
the gene for herbicide resistance has been isolated and cloned inside a
OCR for page 44
44
GENETIC ENGINEERING OF PLANTS
bacterium. Now the Michigan State and Harvard University groups are
trying to achieve gene expression. The biggest hurdle will be finding a
vector to carry the herbicide resistance into a crop, since the gene must
be inserted into the chIoroplast. The only successful plant vector that
has been developed to date the Ti plasmid—does not work for chIo-
roplasts.
Bioengineered Microorganisms to Combat Plant Diseases
One of the most promising, and relatively unexplored, applications
of the new genetic technologies is in combatting soil-borne plant dis-
eases, according to Milton N. Schroth, a plant pathologist at the Uni-
versity of California at Berkeley. Some of the most destructive plant
diseases, such as Fusarium wilts and Phytophthora root rots, are caused
by microorganisms that inhabit the soil. Other, less virulent microor-
ganisms also exact their toll: the presence in the soil of low-grade disease
agents can lower yield significantly. For example, if strawberries are
grown on fumigated soil, in which all the soil microorganisms have been
destroyed, the yield is approximately 20 tons per acre, or four times
higher than if they are grown on unfumigated soil. Because the effects
of soil pathogens are sometimes insidious, it is difficult to estimate
exactly the economic costs of soil-borne diseases, but they undoubtedly
can be considerable.
The increasing adoption of minimum or no-till farming practices pro-
vides an extra incentive for developing effective controls. Both of these
practices leave organic debris on the soil surface, which makes the soil
both wetter and colder creating a more favorable environment for
pathogenic microorganisms.
There are several possible strategies. Fumigation of the soil effectively
controls disease, but it is costly and impractical for large areas. Moreover,
its benefits are transitory, as clisease-causing microorganisms can be
easily reintroduced by wind or animals.
Another approach is to breed resistant cultivars. Many resistant va-
rieties have been developed, yet for unknown reasons the introduction
of disease resistance often results in a loss in yield. Tobacco plants bred
for resistance to Fusarium wilt fungus, for example, commonly show a
nearly 6 percent reduction in yield as compared to susceptible plants.
Resistant plants could also be developed through gene-splicing tech-
niques. "it would be ideal if genes conferring resistance to pests could
be introduced to the plant, expressed, remain stable, and not result in
a cost to the plant," Schroth said. Unfortunately, demonstrated tech-
niques for cloing this are not yet available. Moreover, it is not clear
OCR for page 45
BIOTECHNOLOGY AND AGRICULTURE
Reductions in Yield and Quality in Disease-Resistant Tobacco Lines in
Comparison to the Susceptible Linea
45
Resistance
Percent Yield Percent Price
Reductionlha Reduction
Tobacco mosaic 5.9 1.5
Fusarium wilt 6.9 1.9
Mosaic + Fusarium wilt 9.9 1.9
Mosaic + bacterial wilt 7.1 2.7
Mosaic + root knot nematode 5.0 1.8
Fusarium wilt + bacterial wilt 10.3 4.0
Fusarium wilt + black shank 6.6 3.2
a Based on data given in Chaplin, 1970. Agron. J. 62:87-91.
SOURCE: Milton N. Schroth, Department of Plant Pathology, University of California at Berkeley
whether the introduction of resistance through gene-splicing would re-
sult in the same Toss in yield as does conventional plant breeding.
The alternative genetic engineering approach is to harness and im-
prove upon the beneficial microorganisms that inhabit some soils and
use them to combat plant disease. In nature some soils, known as
disease-suppressive soils, contain beneficial microorganisms that help
to protect plant roots from pathogens. The mechanism of disease
suppression in these soils is poorly understood, and it does not come
from the beneficial microorganisms alone. Instead, it seems to be con-
trolled by complex interractions among both biotic and abiotic factors.
it has long been known, for instance, that physical conditions such as
the salinity, acidity, temperature, and moisture levels of soils can render
plants less or more susceptible to a disease. Nonetheless, beneficial
microorganisms seem to play a major role. If a disease-suppressive soil
is fumigated to destroy all microorganisms, the soil loses its capacity to
suppress plant pathogens. But when some of the unfumigated sup-
pressive soil is reintroduced, the disease-suppressive quality of the soil
is restored.
These naturally suppressive soils provide a substantial boost to crop
yield. In Provence in southeastern France, the suppressiveness of the
soil varies greatly from region to region. This has been determined by
infesting the soil with pathogens and then comparing the severity of
the ensuing disease. For centuries, muskmelons have been grown in
the Chateaurenard area with little trouble from Fusarium wilt, even though
the fungus is present in the soil. Yet in the neighboring two regions of
Cavaillon and Carpentras, the disease can be so severe that the musk-
melon crop sometimes has to be abandoned.
OCR for page 46
46
GENETIC ENGINEERING OF PLANTS
Little is known about the microorganisms that inhabit the rhizosphere,
or soil-root zone. Most research to date has focused on nitrogen-fixing
bacteria because of their potential to reduce the need for chemical fer-
tilizer (see Nitrogen Fixation, p. 48~. In just a few years, molecular bi-
ologists have made great strides in understanding the genetic control
of this trait. The same tools can be used for studying, and ultimately
improving, these other beneficial microorganisms.
Some of the most promising candidates for biocontrol agents are the
root-colonizing bacteria, generically known as rhizobacteria. Some of
these have the beneficial effect of promoting plant growth; others have
deleterious or neutral effects. For use as biocontrol agents, the bacteria
must be able to colonize the roots aggressively and have the potential
to dominate the ecological niche. Finding such bacteria will be difficult,
Pseudomonas colonizing the surface of a sugar beet root. This scanning electron micro-
graph shows chainlike colonies of bacteria against the ribbed background of a sugar
beet root (x 3000~. These beneficial bacteria suppress the growth of plant pathogens
that could otherwise attack sugar beets. Courtesy of Milton N. Schroth, Department of
Plant Pathology, University of California at Berkeley.
OCR for page 47
BIOTECHNOLOGY AND AGRICULTURE
47
as less than 5 percent of bacteria isolated to date from plant roots are
able to colonize the roots effectively and promote plant growth. Several
strains of Pseudomonas can.
Rhizobacteria seem to work through two generic mechanisms. One
is antagonism the bacteria compete with and displace the deleterious
organisms on the plant root. The second is inhibition some rhizobac-
teria produce antibiotics that inhibit a variety of pathogens.
It seems likely that disease protection in soils is conferred through a
variety of microorganisms and favorable environmental conditions. The
hope is to identify some of the key beneficial microorganisms and adapt
them to use as biocontrol agents in conducive soils, either by manipu-
lating the soil environment or modifying these microorganisms to im-
prove their efficiency. That, of course, depends on an understanding of
their normal mode of action.
Research to date has been promising. Though relatively little is known
about rhizobacteria, their application to seeds and roots at planting time
can increase plant growth and yield. In Idaho, California, and Penn-
sylvania, potato yields increased 5 to 33 percent following application
of Pseudomonas. It has also been effective on sugar beets and radishes.
It might be simpler to introduce the microorganisms directly into the
soil, but that approach is "unreachable" at this time and may remain
but that approach is ''unreachable''
impractical for commercial agriculture, Schroth said. The biocontro} agents
would have to compete with the other organisms already present in the
soil. Those long-term residents are "well entrenched, and not easily
displacecI by intruders." In such a scheme, vast amounts of inocuTum
would be required on a regular basis; the cost might be prohibitive.
Because strains of the same species are best adapted to occupying the
same ecological niche, the ultimate approach may be to engineer the
pathogen to control itself. This would entail inactivating the disease-
causing agent from the microorganisms and then using this disarmed
bug to displace its pathogenic relative. Eventually, when more is known
about complex interactions in the rhizosphere, it may even be possible
to manipulate the soil ecosystem to favor beneficial microorganisms.
1 , . ~
ldentitying and improving rhizo bacteria will require the combined
efforts of bacterial ecologists, plant pathologists, biochemists, and ge-
netic engineers. Specifically, they need to determine the genetic factors
that govern root colonization. They need to identify the key factors that
enable a microorganism to compete successfully in an ecological niche.
One means of increasing their competitive ability might be to bioengineer
them to tolerate greater moisture stress.
Schroth cautioned against underestimating the complexity of the ag-
roecosystem. It may be possible to design a strain of bacteria that gives
OCR for page 48
48
GENETIC ENGINEERING OF PLANTS
dramatic results in the laboratory. But in the field, competing with
microorganisms that have evolved for hundreds of thousands of years,
a successful laboratory strain might not perform well, or even survive.
Nitrogen Fixation
Nitrogen is an essential plant nutrient and a key determinant of crop
productivity. Unfortunately, the nitrogen content of agricultural soils is
quickly depleted. Farmers worldwide supplement the available nitrogen
with some 60 million metric tons of nitrogen fertilizer annually. By the
year 2000, an estimated 160 million metric tons of nitrogen fertilizer may
be used each year. Producing that fertilizer is both expensive and energy
. .
Intensive.
As farmers face the prospect of rising bills for nitrogen fertilizer, their
crops are literally being bathed in nitrogen gas, as roughly 80 percent
of the air is nitrogen. Yet plants are unable to use nitrogen from the
air. Soybeans and other legumes are an exception; they have a symbiotic
relationship with nitrogen-fixing bacteria, Rhizobium. In some soils, where
Rhizobium are indigenous, the farmer need only plant the legume. In
areas where the bacteria are not present, the farmer adds or inoculates
the soil with Rhizobium. In either case, no nitrogen fertilizer is necessary.
The Rhizobium infect the roots of the plants, causing nodules to form.
Insicle the nodules, millions of bacteria convert the nitrogen that is in
the air to ammonia, which the legume, like other plants, needs for
protein synthesis.
Agricultural yields could be sustained at tremendous savings if bio-
Togical nitrogen fixation can be improved and extencled to major crops,
such as corn and wheat, that now depenct on costly nitrogen fertilizer.
The cluster of genes that control nitrogen fixation in microorganisms
has been isolated and analyzed. In numerous academic and industrial
laboratories, researchers are trying to understand how those genes are
regulated and how they can be usecT in practical crop improvement
schemes. Winston Brill of the University of Wisconsin and Cetus Mad-
ison, Corp., described that work.
The earliest payoff may come from attempts to improve the efficiency
of nitrogen fixation in legumes. The approach is to engineer either the
Plant or the Rhizobium or both to improve the symbiotic relation~hin
. . . . ~ - .. . . . . ~ ~ .
According to Frill, genetic manipulation of the bacterium is tar simpler
than manipulation of the plant. He has used both standard mutagenesis
and recombinant DNA techniques to develop improved strains of Rhi-
zobium. When inoculated with the mutant strains, plants show increased
vigor and growth.
OCR for page 49
OCR for page 50
50
GENETIC ENGINEERING OF PLANTS
The most efficient means of supplying nitrogen would be to transfer
the nitrogen-fixing genes from the bacteria into the plant. It is also one
of the toughest tasks imaginable. A number of laboratories have isolated
the nitrogen-fixing genes from Klebsiella, bacteria similar to Rhizobium
but easier to work with in the laboratory. Nitrogen fixation is a complex,
multigene trait controlled by a cluster of 17 genes. These genes are
broken clown into smaller units, each of which is regulated separately.
To endow a plant with the ability to fix its own nitrogen would mean
transferring all 17 genes, along with the complete collection of regulatory
signals.
PROBING THE MECHANISMS OF NITROGEN FIXATION
Soybeans, alfalfa, and other legumes have a symbiotic relationship
with the bacteria Rhizobium that enables these plants to obtain nitrogen
from the soil. The increasing interest in extending the ability to fix
nitrogen to other crops has spurred efforts to understand the unusual
v ,
. . . . .
relationship between Rhizobium and legumes.
When Rhizobium intact plant roots they cause the cells to proliferate,
giving rise to nodules on the roots. Rhizobium induce another, appar-
ently unique, change in the plant cells: at the spot where the bacteria
first come in contact with the root, the plant cells form a tubular~ike
structure, known as an infection thread. These infection threads wind
throughout the cells in the root nodule, providing a conduit through
which bacteria migrate from one cell to another. Once inside the cells,
the bacteria convert nitrogen to a chemical form the plant can use.
The top photograph, a scanning electron micrograph (magnification
x 2000), shows an infection thread traversing a ceil in an alfalfa root
nodule. At the end of the thread (upper lefty bacteria are being released
into the cell.
Bacteria other than Rhizobium are not known to induce the formation
. . .. ~ . . . . . ~ . .
Of infection threads. Research is under way, using gene-splicing tech-
niques, to transfer the genes that control nodule formation (the nod
genes) and nitrogen fixation (the nif genes) from Rhizobium to other
bacteria. If bacteria that infect nonieguminous plants can be endowed
OCR for page 51
BIOTECHNOLOGY AND AGRICULTURE
with the properties of Rhizobium, it might offer a means of extending
nitrogen fixation to other crops.
The nod and nif genes have recently been transferred from Rhizobium
to Agrobacterium tumefaciens. When the genetically engineered Agro-
bacterium infected an alfalfa plant, it induced root nodules and infection
threads to form (bottom photograph, magnification x 7601. The genes
for nitrogen fixation, however, were not expressed.
51
OCR for page 52
52
GENETIC ENGINEERING OF PLANTS
Brill predicted that this cluster of genes will soon be moved into dicots
such as potatoes and tomatoes and later into monocots like corn and
other cereals. "Is that the end of the story? Do we now have nitrogen-
fixing corn?" he asked. The answer is no. The entire cluster of genes
has already been transferred into yeast, but the genes were not ex-
pressed.
Not only must gene expression be achieved, but the genes must be
inserted into the proper place in corn and turned on at the appropriate
time. Nitrogen fixation requires an energy-rich, oxygen-depleted mi-
croenvironment the corn's mitochondria or chioroplasts might be a
suitable site for the genes. In addition, the chemical reactions involved
in nitrogen fixation require far more iron and molybdenum than are
normally found in most plant cells. It is too early to tell if the hurdles
are simply formidable or if they are insurmountable.
The Bottom Line
The final test of these new agricultural products the improved va-
rieties and bioeng~neered microorganisms, for instance will be their
performance in the marketplace. The new products must offer an ad-
vantage over existing ones if the farmer is to adopt them. As Schroth
explained, the bioengineerecl products must improve the profit margin
per hectare either by increasing yield or reducing production costs.
That, in turn, depends on how well the new products can be integrated
into existing agricultural techniques.
Though disease-resistant plants can be bred, they often have reduced
yield. It may be more economical for the farmer to use a susceptible
variety and a fungicide to control the disease. Similarly, the benefit of
a biocontroT agent will have to be weighed against a fungicide. Key
factors influencing the commercial success of a biocontro! agent might
be its shelf life and the ease with which it can be applied to the soil.
So far, many researchers have been so caught up in what is scientif-
ically possible that they have neglected the practical considerations, such
as market analysis, Schroth added. He suggested that molecular biolo-
gists work with plant breeders, agronomists, and pomologists in iden-
tifying scientifically and economically attractive projects for genetic en-
gineering. "It will not be a simple task to improve productivity per
hectare," he said. "And it certainly will not be done by the unilateral
efforts of one discipline."
Bogorad concurred. "It is clear that we need molecular biologists plus
plant pathologists and agronomists—people who know about real plant
problems. One of the difficulties we have today is that there are very,
very few people who understand both sides of the problem."
Representative terms from entire chapter:
genetic engineering
