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THE IMPACT OF BIOTECHNOLOGY
ON FOOD PRODUCTION
Ernest G. Jaworski
The application of recombinant DNA techniques to
biological organisms, systems, and processes constitutes
an exciting new biology that is being used to increase
agricultural productivity and to improve the health of
humans and animals. These advances coupled with those
resulting from more traditional genetic and chemical
approaches are having and will continue to have an
enormous impact on the production of food throughout the
world.
These applications could each be described in some
depth, but this would require more pages than are
available in this volume. Therefore, this paper focuses
mainly on the most recent advances in the transformation
of plants, even though the creation of transgenic animals
is actively being explored at the fundamental and applied
levels.
Advances in plant cell and tissue culture have made it
possible in some cases to insert genetic information into
the chromosome of an organism and then to regenerate
whole plants from single cell cultures. Such techniques
have been used to produce protoplasts, i.e., plant cells
without a cell wall, which are useful in transformation.
This significant development, coupled with the ability to
clone pieces of functional DNA using bacterial systems
and restriction endonuclease-generated DNA fragments, has
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provided the basic tools for the creation of transgenic
plants. The key ingredient in the most recent advances
in plant transformation resulted from the use of a
naturally occurring bacterium (A~robacterium tumefaciens)
that has the capacity to insert its DNA stably into the
chromosome of plant cells (Fraley et al., 1986~. This
system can be used in conjunction with plant cell culture
to successfully produce whole plants containing foreign
gene inserts.
CELL TRANSFORMATION SYSTEM
A~robacterium tumefaciens contains a large Ti, or
tumor-inducing, plasmid that in its wild form is capable
of creating crown gall tumors in plants. The Ti plasmid
can transfer a small portion of its DNA (T-DNA) and
stably insert it into the nuclear DNA of the transformed
cell. Since the T-DNA contains genetic information
responsible for the synthesis of plant hormones as well
as novel metabolites called opines, its transfer and
insertion creates the crown gall tumor when these genes
are expressed. Although the mechanisms by which these
transfer and insertion processes take place are not well
understood, it has been possible to take advantage of
A~robacterium's properties to genetically engineer the Ti
plasmid into a useful transforming vector. Intermediate
vectors containing selectable antibiotic resistance
markers for the introduction of foreign genes into the Ti
plasmid have been constructed and the tumor-inducing
properties of A~robacterium deleted by removal of the
plant hormone genes.
The neomycin phosphotransferase (NPT) coding sequences
from a bacterial transposon (Tn5) were joined to the 5'
and 3' regulatory sequences of nopaline synthase--a gene
derived from the Ti plasmid, which is known to be
constitutively expressed in plants (Fraley et al., 1986~.
This chimeric gene confers resistance to kanamycin, an
aminoglycoside antibiotic that is lethal to plant cells.
Thus this chimeric gene construct provided a selectable
marker for the transformation vector.
Direct cloning approaches using the Ti plasmids were
not practical. It was therefore necessary to create
intermediate or shuttle vectors either to integrate with
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a resident Ti plasmid by recombination or to replicate
independently of the Ti plasmid as transvectors. The
integrated vector was then used for the transfer of a
number of foreign genes into A~robacterium cells. The
characteristics of these plasmids include a segment of
the pBR322 DNA for replication in Escherichia colt, a
portion of a Ti plasmid (pTiT37) containing the
functional nopaline synthase gene for ease in scoring
transformed plant cells, a streptomycin/spectinomycin
resistance determinant from Tn7 for selection in
A~robacterium, a portion of DNA from another Ti plasmid
(pTiA6) to provide homology for recombination with a
resident octopine-type plasmid in A. tumefaciens, a
synthetic multilinker containing unique sites for gene
insertion, and the chimeric kanamycin resistance gene
(NOS/NPT II/NOS). These plasmids and derivatives were
introduced into A. tumefaciens by conjugation procedures
and homologous recombination between the plasmid and the
wild-type octopine Ti plasmid to produce cointegrates
(Fraley et al., 1986~. Although this system was useful
for the study of gene expression and inheritance of
traits, it was not sufficiently efficient for routine
production of transformed plants. A subsequent series
of derivatives led to the formation of a variant and
selectable T-DNA system, which was highly efficient in
its transformation frequency (Fraley et al., 1986~.
PLANT TRANSFORMATION SYSTEM
Initially, an In vitro transformation was developed by
incubating plant protoplasts directly in A. tumefaciens
cell suspension (Fraley et al., 1986~. Protoplasts were
prepared from a variety of leaf tissues by conventional
enzyme digestion. The bacteria attached to the proto-
plasts during cell wall regeneration and subsequently
transferred the T-DNA into the plant cells by an unknown
mechanism. Such cells were easily identified within 3
weeks by selection for kanamycin resistance.
Since the protoplast system had a number of techni-
cal drawbacks, an improved alternative procedure was
developed to obviate problems in the isolation and
regeneration of protoplasts. The modification involved
cutting disks from leaves and infecting them with A~ro-
bacterium. These disks were then placed on nutrient
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agar. Subsequently, callus formation was observed around
the circumference of the disks. Within 3 to 4 weeks,
plant regeneration occurred under appropriate conditions.
Stable maintenance and expression of foreign genes
(kanamycin resistance) were demonstrated in cells and
plants derived from either the protoplast cocultivation
or leaf disk systems. Subsequent progeny seed derived
from the transformed plants inherited the kanamycin
resistance in a simple Mendelian manner.
GENE EXPRESSION
The development of a transformation system for the
stable and heritable introduction and expression of a
foreign gene provided a tool for the analysis of gene
expression in general. The first study in this effort
involved light-regulated, tissue-specific gene coding for
the small subunit of ribulose-1,5-bisphosphatecarboxylase
(RuBPss) from peas (Fraley et al., 1986~. The investi-
gators demonstrated that the genomic clone for pea RuBPss
could be introduced into petunia cells by cocultivation
with _. tumefaciens. Molecular analyses of transformed
cells revealed that the small subunit gene of the pea was
indeed expressed in the petunia under the control of its
own promoter and was regulated by light in a manner
identical to that seen for the endogenous gene in peas.
The pea RuBPss retained its tissue-specific pattern of
expression in leaves derived from regenerated transformed
petunia plants. Subsequent studies using In vivo radio-
labeling, followed by immunoprecipitation of ribulose-l,
5-bisphosphatecarboxylase, demonstrated that the heter-
ologous RuBPss protein of the peas could be separated
from the endogenous petunia RuBPss, thereby indicating
that the pea RuBPss protein was correctly processed In
vivo by petunia chloroplast (Fraley et al., 1986~. In
vivo pea RuBPss was also recovered from the holoenzyme,
which was immunoselected with the petunia antilarge
subunit antibody, indicating that the small subunit of
the pea could form a hybrid holoenzyme assembly with
large subunits of the petunia.
Two mammalian genes were also demonstrated to be
expressed in plant cells. A cDNA clone encoding
a-human chorionogonadotropin (a-hCG) under the
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control of the cauliflower mosaic virus 35s promoter and
a mouse cDNA clone encoding a methotrexate-insensitive
dihydrofolate reductase (DHFR) gene also under the
control of the cauliflower mosaic 35s promoter expressed
their gene products in transformed petunia cells. The
results with ~x-hCG and the mouse DHFR indicate the
broad utility of the A~robacterium system for the study
of gene expression and regulation.
Finally, the transfer of a legume storage protein gene
into the petunia resulted in the tissue-specific accumu-
lation of the storage protein in the seeds of the trans-
formed plants. A cDNA clone encoding the soybean 7s
c''-conglycinin protein was engineered into an
appropriate A~robacterium plasmid and transferred into
petunia cells (Fraley et al., 1986~. Analysis of sub-
sequent petunia seeds indicated that there was regulated
expression of the soybean storage protein gene in the
petunia seed. This exciting model system for storage
protein expression should permit the determination of the
structural sequences required for protein translocation,
glycosylation, and processing as well as the study of the
regulatory sequences essential for seed-specific expres-
sion.
TECHNOLOGY APPLICATIONS
Plants
In the past 3 years, the gene transfer systems
described above have led to important new insights into
gene regulation and protein transport in plants. The
basic applications of this technology should provide a
means for developing deeper understandings of the speci-
fic promoter/enhancer DNA sequences involved in gene
expression and fundamental information on cats and bans
gene regulation.
Of specific interest, however, has been the recent
demonstration of agronomically significant transforma-
tions involving the generation of herbicide-tolerant,
insect-resistant, and viral disease-resistant plants.
The herbicide N-(phosphonomethyl~glycine, or glyphosate,
is the active ingredient in Roundups . It inhibits the
aromatic biosynthetic pathway at its sixth step; namely,
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enolpyruvylshikimate-3-phosphate (EPSP) synthase (Fraley
et al., 1986~. This pathway is involved in the biosyn-
thesis of phenylalanine, tyrosine, and tryptophan, and
when the pathway is inhibited at the EPSP synthase level,
the formation of these essential amino acids ceases.
Early studies indicated that overproduction of a bac-
terial EPSP synthase in bacteria results in herbicide
tolerance (Fraley et al., 1986~. Subsequent efforts led
to the development of a chimeric gene construct consist-
ing of a petunia EPSP synthase cDNA flanked by the
cauliflower mosaic 35s 57 promoter and the nopaline syn-
thase, 3' regulatory regions. Transformation of petunia
cells with this construct resulted in herbicide resis-
tance and the overproduction of EPSP synthase 30- to
60-fold. Plants regenerated from these cell lines were
found to be tolerant to Roundup ~ when sprayed at
concentrations of 0.9 kg/hectare. Control plants were
killed when sprayed with the herbicide at 0.22 kg/hec-
tare. Similar strategies are currently being used to
create plants tolerant to a number of other herbicides
such as atrazine, the imidazolidinone series, sulfonyl-
ureas, and phosphonotricine.
Within the past few years, numerous crop plants,
including canola, tomato, potato, tobacco, lettuce, sugar
beets, and poplar, have become amenable to the transfor-
mation technologies involving A~robacterium. On the
basis of these rapid advances, it can be expected that
practical demonstrations of the system are forthcoming.
Within the past year, field tests were initiated with
genetically engineered tobacco that is resistant to
atrazine.
The creation of a transformation vector for the
production of viral disease resistance in plants was
recently reported by the R. Beachy Group at Washington
University (Powell-Abel et al., 1986~. The transfor-
mation system described above was used to insert and
express the tobacco mosaic virus (TMV) coat protein gene
in both tobacco and tomato plants. The coat protein
gene was engineered into a plasmid similar to the one
described for herbicide tolerance. Both tobacco and
tomato plants transformed with the coat protein gene
were found either to be resistant to TMV infection or to
demonstrate significant delays (weeks) in symptomology
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following infection. Control plants always demonstrated
severe symptoms within days following inoculation with
the virus. This is one of the first demonstrations of
the genetic engineering of disease resistance in plants.
Studies by AgroCetus have recently been carried into the
field assessment stage with tobacco genetically engi-
neered to be tolerant of soil-borne A~robacterium
infections.
Finally, a Belgian company, Plant Genetic Sciences, has
recently demonstrated that the Bacillus thurin~iensis
(B.t.) toxin gene could be inserted into the Ti plasmid
system anti expressed in tobacco at sufficient concen-
trations to protect the tobacco plants from attack by the
tobacco hornworm. This was the first demonstration of
engineering to protect a plant against insect damage by
using biotechnology.
These results demonstrate that single gene traits can
be successfully introduced into plants for expression and
that they can function at potentially economic levels.
On the basis of these findings, it can be anticipated
that useful single gene traits will also be inserted into
plants in combination with other functionally important
single gene traits. Furthermore, the stage has now been
set for the examination of other genetic traits.
MICROORGANISMS
Transformations of microorganisms that colonize plants
are also expected to be useful in enhancing the produc-
tivity of plants. For example, transformed Pseudomonas
florescens, which is a natural colonizer of roots in such
major crops as corn and soybeans, has been engineered to
carry and express the Bacillus thurin~iensis toxin gene
mentioned earlier. Greenhouse tests have indicated that
such microorganisms are capable of protecting the root
systems of corn plants from attack by certain soil-borne
insects. Similar advances can be expected in the
development of both root-colonizing and leaf-colonizing
organisms, which will protect plants from diseases,
pests, and environmental stresses. Laboratory and
greenhouse studies certainly promise that such organisms
will effectively aid in plant productivity. Much further
work will be needed to develop performance character-
istics for these microorganisms under normal field
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conditions and soil types, and a special need exists
to generate more basic data on the ecology of these
microbes. In this regard, the E. cold lacZY genes
(coding for p;-galactosidase and lactose permease)
have been engineered into Pseudomonas florescens to
create a well-marked microorganism for microbial ecology
research. The microorganism contains four marker char-
acteristics that make it possible to isolate it from soil
samples and detect it at levels of one bacterium per gram
of soil. The characteristics of the engineered pseudo-
monad include its fluorescent properties, natural
rifampicin resistance, ability to grow on a simple
lactose media, and detection by the X-gal chromogenic
dye. Thus this microorganism provides a very sensitive,
selectable tracking system that should be extremely
useful in ecological studies under natural environmental
conditions.
FUTURE NEEDS AND EXPECTATIONS
The applications of genetic engineering and modern
molecular biology have provided us with the ability to
insert novel genetic traits into plants and into
microorganisms that interact with plants. It can be
predicted that this technology will have an impact on
the production of food and on the efficiency of crop
production throughout the world, since weeds, insects,
and diseases create enormous losses in food production.
Advances in the more qualitative traits in our food
supply can also be anticipated and are in fact receiving
attention at present. For example, extensive efforts are
being devoted to the development of higher levels of
solids in tomatoes by enhancing the concentration of
natural polymers normally present in tomatoes. Protein
engineering may well see its first application in simple
amino acid codon shifts in seed storage proteins to
enhance their content of lysine (e.g., in corn) or
methionine (e.g., in soybeans). Gene engineering
techniques may also be useful in improving the yield ot
protein levels in a variety of crops, especially forage
crops. Site-directed mutagenesis may be applied to the
creation of more stable proteins for storage purposes.
The loss of food products and gains in storage may be
reduced by subsequent genetic engineering of the crops to
create disease- and insect-resistant products. We now
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have the technologies and tools to approach some of these
important food supply issues, but we should consider some
of the future needs that could help advance progress
more rapidly.
Technical hurdles that still remain include cell
culture procedures to broaden the base of crop species
that can be regenerated, especially from protoplasts.
Interestingly, a recent announcement by Japanese and
French scientists indicates that it is now possible to
regenerate rice plants from protoplast cultures. New
transformation systems are needed, especially for monocot
transformation. Other methods for the transfer of DNA
into plant cells are being investigated, and there are
indications that methods such as electroporation,
microinjection, laser treatment, and the use of gemini
virus vectors will soon have an impact on this field.
Gene structure and function will continue to receive
attention, especially with reference to the regulatory
sequences affecting gene expression at the developmental
and tissue-specific level. Among the most practical
needs for the modification of our food supply will be
the identification of additional agronomically important
genes that can be used to create stress- and pest-
tolerant plants.
At present, the lack of knowledge about the basic
biochemistry of plant systems remains one of the major
limiting factors in the advancement and exploitation of
the technology described. This knowledge is vital for
our understanding of the genetic components involved in
achieving such traits as frost tolerance, heat tolerance,
drought tolerance, metal tolerance, disease resistance,
and insect tolerance. Incentives have been provided to
improve this situation, but additional resources must be
directed toward improving plant tissue culture and
regeneration; novel transformation systems, understanding
of gene structure organization and function; the selec-
tion, isolation, and characterization of agronomically
important genes; and the development of unique plant-
breeding techniques. Only then will the sociological and
economic impacts of these exciting technologies and tools
be fully realized.
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REFERENCES
Fraley, R.T., S.G. Rogers, and R.B. Horsch. 1986.
Genetic transformation in higher plants. CRC Crit.
Rev. Plant Sci. 4~1~: 1-46.
Powell-Abel, P., R.S. Nelson, B. De, N. Hoffman, S.G.
Rogers, R.T. Fraley, and R.N. Beachy. 1986. Delay
of disease development in transgenic plants that
express the tobacco mosaic virus coat protein gene.
Science 232:738-743.
18
Representative terms from entire chapter:
protein gene
