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Introduction
4
Recent advances in biology have proceeded at an astonishing
rate, and biologists now have the means, by directly modifying genes,
to alter living organisms more quickly and more precisely than has
been done by nature and humans over millennia. There is general
agreement that this ability can yield far-reaching improvements in
our environment and in medical and agricultural practice. However,
field testing of prorn~s~g products of the new technology has been
slowed by the absence of a full scientific consensus on the relative
safety and risks of introducing modified organisms into the environ-
ment. Furthermore, the specific questions that are most important
to consider in making decisions have not been agreed on. Hence, this
NRC committee was formed to attempt to determine a reasoned con-
sensus about what scientific questions must be asked and how such
questions can aid In the development of a decision-making process
based soundly on the facts of science.
The history of efforts to reach a common ground about the
relative safety or hazard of genetic modification of organisms can
be traced directly to the early 1970s, when advances In biological
knowledge had given scientists the tools to recombine DNA ~ the
laboratory into new sequences (see Appendix).
7
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THE GENETIC MODIFICATION OF ORGANISMS:
MERGING CLASSICAL AND MOLECULAR TECHNIQUES
This report describes the properties of plants, rn~croorganisms,
and the environment that must be evaluated when the introduction
of a genetically modified organism into the environment ~ being
planned. IN this introductory section we explore the basic biolog-
ical principles that underlie both classical and molecular means of
altering the genetic makeup of organisms and explain how our inter-
pretation of these principles leads to the conclusion that the products
of classical and molecular methods are fundamentally sirn~lar. Both
methods of modifying DNA produce an organism (product) that
is genetically different from the starting organism regardless of the
method (process) used. The molecular techniques are often more
precise than classical techniques and can modify single nucleotides
of bacterial gnomes. Molecular mollifications surpass classical tech-
niques in their ability to introduce a great variety of traits from a
wide range of donor organ~srns into the recipient organisms. As a
coronary, the molecular techniques can generate a greater range of
phenotypes than the classical methods. These principles as they am
ply to plants and rriicroorganisms are discussed in greater detail in
the sections of this report dedicated to the two kinds of organisms.
Plants and microorganisms contain nucleotides ~ combinations
and arrangements that endow the organisms with genetic determi-
nants for many traits. Other regions of DNA may control the expres-
sion of the traits. The DNA provides the raw material upon which
genetic modifications depend. The evolution of new forms of crop
plants and microorganisms results from selecting organisms with
desirable traits from populations that possess heritable variation.
When genetic variants are selected to produce the next generation,
the population is changed with respect to the frequency of individuals
having the selected characteristic. In the terms used in population
genetics, selective breeding or propagation changes gene frequencies,
and the population differs in some aspect from its predecessor even
though the change may be small.
Modification of microorganisr~Ls and plants can be performed by
either classical or molecular methods. No hard line exists between
the two categories, especially with microorganisms. For this report,
we generally include as classical those means of genetically modifying
organisms that were used before recombinant DNA techniques were
developed. One major distinction of classical methods is that they
are relatively undirected modifications of the genome. Molecular
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methods provide more flexibility and control and thus are more
specific ~ directing the modifications toward a planned end product.
Methodological and biological distinctions exist In culturing mu-
croorgan~sms and plants, but one feature of the new genetic technolo-
gies is that they permit us to manipulate plants at the cellular level.
This technology provides new comrnonalities to plant and microbial
breeding.
Classical methods are those in which the genetic recombinations
occur essentially ~ a natural way; desirable offspring variants are
then selected in the laboratory or the field. Examples include spon-
taneously mutating m~croorganisrns and sexually cross-bred plants.
The term classical also includes some methods called that only be-
cause they predate the introduction of modern gene-splicing tech-
niques. The latter include such human-mediated techniques as ex-
posure of organisrrts to chemuca] mutagens or physical agents such as
x-rays and ultraviolet radiation. We also include as classical those
mechanisms of DNA transfer that occur without chemical treatment
of a cell's envelope, such as transformation, conjugation, and trance
auction In microorganisms.
Molecular methods of genetic modification include the newer
methods for modifying DNA In which one nucleotide can be substi-
tuted for another at a predetermined site In a DNA molecule (site-
directed mutagenesis). Molecular gene transfer methods are used
for transfer of genetic material between donor and recipient cells
that have diverged widely through evolution and probably do not
exchange DNA without laboratory manipulation. However, it ~ im-
portant to recognize that certain gene transfers thought impossible in
nature a few years ago because of the phylogenetic distance between
donor and recipient have now been shown to occur in the laboratory,
and they may occur in nature. For example, there is evidence that
a gene or genes for erythromycin resistance was transferred between
the gram-negative bacterium Campylobacter and unrelated gram-
positive bacteria (Brisson-Noe} et al., 19883. Recent laboratory ex-
per~rnents have accomplished gene transfer between Eschertchia cold
and streptomyces (Mazodier et al., 1989) or yeast tHeinemann and
Sprague, 1989~. Another example relates to the natural transfer of
DNA from the bacterial species Agrobacterium to plant cells (Nester
et al., 1984~. Plasmid genes from this bacterium probably were trans-
ferred into a species of tobacco early in the evolution of the genus
Nicotiana, and they became integrated into the plant chromosome.
These genes, or their remnants, have beers detected in a variety of
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different species of Nicotiana, which presumably evolved from the
original infected plant (Furner et al., 1986~.
PLANT MODIFICATION~CIASSICAL TECHNIQUES
Spontaneous and mutagen-induced variation in plants has pro-
duced a great variety of genetic traits that may be used in plant
breeding. The crop plants of today had their origins ~ the fields of
early farmers who selected plants with desirable traits and perpetu-
ated plants to meet agricultural needs.
Controlled matings (hybridization) of plants through the sexual
process is the cornerstone of classical plant breeding. Hybridization
and selection of plants with new combinations of traits have been
used to increase genetic diversity. By repeated hybridization and
selection, new traits could be introduced into varieties already proven
successful In agriculture.
Hybridization is often possible between species, usually within
the same genus. However, many interspecific hybridizations require
human-mediated intervention to facilitate the sexual process. For
example, developing embryos me excised and cultured on nutrient
media before being grown as plants In the field. The male or female
fertility of such hybrids ~ often reduced so that they themselves must
be hybridized with one of the parents or with a closely related species.
Alternatively, fertility can be restored by doubling the chromosome
number. With sexual hybridization, the resulting progeny contain
fuB complements of genes from each parent. The challenge for plant
breeders is to select for the genes which result in a plant's exhibiting
the desired combination of traits. Because interspecific hybrids, and
even many intraspecific hybrids, have a parent that may be poorly
adapted to survive and grow in an agriculturally useful way, consid-
erable effort Is required to examine large numbers of plants to find
the desired combinations of traits.
Two major limitations exist with classical plant breeding. The
first is an extraordinarily large degree of variability from which a
low frequency of desired plants must be identified. Second, the gene
pool-the source of genes accessible to the breeder is Tented to
those species which can be sexually hybridized.
PLANT MODIFICATIONS MOLECULAR TECHNIQUES
TO principle, any gene can now be introduced into any plant
by one of several possible molecular modification techniques. At
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present, the most frequently used agent for DNA transfer is the
common soil bacterium Agrobacterium (Nester et al., 1984~. This
organism evolved a mechanism for transferring part of its plasmas
into plant ceils, where it is integrated randomly into the chromosome
(Peerbolte et al., 1986~. The introduced DNA is inserted within this
plasmas DNA as a "hitchhiker. Once integrated into the plant's
chromosome, the DNA ~ transmitted from parent to offspring and
follows the pattern of Mendelian inheritance. Virtually all dicotyI~
donous plants are amenable to transformation by Agrobacterium, but
most monocotyTedonous plants appear to be resistant.
A technique frequently used to transform monocotyledonous
plants, such as maize and rice, is electroporation; this technique
requires removal of the plant cell wails before the DNA is added.
These naked cells, or protoplasts, often do not synthesize new cell
walls readily. Thus, regeneration of whole, fertile plants from pro-
toplasts has limited use for molecular gene transfer, especially in
cereal grasses. More recently, DNA-coated gold or tungsten parti-
cles have been upshots into plant ceils, and stable, genetically trans-
formed plants have been regenerated from the cells or organized tissue
(Klein et al., 1987~. This technique may be suitable for introducing
DNA into plant chIoroplasts (Boynton et al., 1988) and niitochondria
(Johnston et al., 1988), as wed as into the nucleus. Current research
is directed toward introducing DNA into specific plant tissues that
have the greatest probability of regenerating genetically modified
plants.
COMPARISON OF CLASSICAL AND MOLECULAR
TECHNIQUES IN PLANTS
The major difference between classical and molecular techniques
is the greater diversity of genes that can be introduced by molecular
techniques and the greater precision of these introductions. From
a single gene to more then 50 genes can be introduced with the
Agrobacterium system, although the site in the plant chromosome at
which the foreign DNA has been integrated appears to be random.
The donor DNA can be derived from the same or different plant
species, or even from m~croorganisrns or anneal cells. For example,
the DNA from fireflies (Ow et al., 1986) and bacteria (Koncz et
al., 1987) that cocles for luminescence has been inserted into plants.
Thus, no species barrier exists, because the chern~cal nature of DNA
is universal in its structure, irrespective of the organism of its origin.
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After being integrated, the gene, to be useful, must be expressed in
the host plant. Genes have regions at one end of their nucleotide
chain that control when and under what conditions the gene wiD
be expressed. These regions determine specific conditions for gene
expression, for example, in the light, In specific tissues, or at cer-
ta~n stages of development (Goldberg et al., 1989~. On the basis of
this knowledge and recombinant DNA technology, one can attach
the desired region of a gene to a bacterial gene and introduce the
combination into a plant cell, where it will be expressed In a spe-
cific tissue. Particular conditions, such as wounding, may be needed
for expression of the added gene or genes, and knowledge of these
conditions can be used to precisely control expression (Ryan, 1988~.
GENOME MODIFICATION OF
MICROORGANISMS CLASSICAL TECHNIQUES
The classical methods of genome modification in microorganisms
faD into two classes, selection of spontaneous and induced mutations
and the exchange of DNA between (usually) closely related organ-
isms. Spontaneous mutations result In a variety of heritable changes
In the DNA, including the substitution of one nucleotide for another,
the deletion or addition of one or more nucleotides, and other types
of DNA rearrangements. Many spontaneous mut~ts appear to re-
sult from the movement of transposable elements to new locations
in the ceD's DNA. Transposable elements, first discovered in maize,
also occur In other plants (McClintock, 1950), bacteria, and anunals.
Another mechanism for generating variability in niicroorganisuts
is through the introduction of new genetic information from either
chromosomal or plasniid DNA. DNA from a donor organism's chro-
mosome is integrated into the recipient genome. Plasrn~ds, being
selfreplicating, do not have to integrate their DNA into the genome
of the recipient. Consequently, plasmid DNA can be transferred to
more widely divergent organisms than DNA from the chromosome
of a donor organism. Plasmas movement can be monitored because
the DNA often provides the genetic code for readily distinguishable
traits, such as antibiotic resistance.
In bacteria, gene transfer can occur by three different classical
means: DNA-mediated transformation, in which the DNA Is trans-
ferred as Naked DNA; transduction, In which the DNA is enclosed
in a virus coat and the virus mediates the transfer; and conjugation,
in which the DNA is transferred during cell-t~ceD contact between
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donor and recipient cells. Presumably, all these mechanisms operate
in nature (Freifelder, 1987~.
GENOME MODIFICATION OF
MICROORGANISMS MOLECULAR TECHNIQUES
The range of techniques to mutate bacteria has expanded and
become sophisticated in recent years. It now is routine practice to
mutate specific genes (insertion mutagenesis) (Ruvken and Ausubel,
1981) as well as to alter specific nucleotides within a gene (site-
directed mutagenesis) (Kunkel, 1985~. These techniques are possible
not only for microbial genes, but, In principle, for genes from any
organism.
The range of microorganisms among which DNA can be trans-
ferred has also been expanded through the use of new technologies.
Thus, it is now possible to transform cells by physically altering
their cell envelopes so that they become permeable to most DNA
molecules. One such technique Is electroporation, in which recipient
cells and the genetic material to be transferred are subjected to an
electric current (Eromm et al., 1987~. The successful use of these
techniques for genome modification requires that the entering DNA
be able to replicate inside its new host. In principle, the techniques
for performing these manipulations are straightforward. With such
techniques, plasm~ds have been constructed that can replicate in
both the bacterium E. cold and the yeast Saccharomyces cerev~siae
(Ereifelder, 1987~.
COMPARISON OF CLASSICAL AND MOLECULAR
TECHNIQUES ~ MICROORGANISMS
Recent molecular technological advances in mutagenesis and
gene-transfer methods have opened new possibilities for expanding
the range of rn~croorganisrns into which DNA from unrelated organ-
isms can be introduced. The genus barrier and, indeed, the kingdom
barrier are no longer complete obstacles.
Recombinant DNA methodology makes it possible to introduce
pieces of DNA, consisting of either single or multiple genes, that
can be defined in function and even in nucleotide sequence. With
-classical techniques of gene transfer, a v~iable number of genes can
be transferred, the number depending on the mechanism of trans-
fer; but predicting the precise number or the traits that have been
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transferred ~ difficult, and we cannot always predict the phenotypic
expression that will result. With organisms modified by molecular
methods; we are in a better, if not perfect, position to predict the
phenotypic expression.
With classical methods of mutagenesis, chemical mutagens such
as alkylating agents modify DNA In essentially random ways; it is
not possible to direct a mutation to specific genes, much less to sped
cific sites within a gene. ~deed, one common alkylat~g agent alters
a number of different genes simultaneously. These mutations can go
unnoticed unless they produce phenotypic changes that make them
detectable in their environments. Many mutations go undetected un-
ti! the organisms are grown under conditions that support expression
of the mutation.
SUGARY
We have reviewed briefly the various means by which plants
and microorganisms can be genetically modified by methods termed
Classical or "molecular. Genetic variability in microorganisms and
plants is enhanced by classical modifications such as spontaneous or
mutagen-~duced variation, by hybridization, and by gene transfer.
These methods are relatively ~rnprecise and undirected and less pow-
erful than molecular techniques for modifying genes. However, no
conceptual distinction exists between genetic modification of plants
and microorganisms by classical methods or by molecular techniques
that modify DNA and transfer genes.
Figure 2-l graphically depicts this view. The difference in the
modes of genetic modification are not deemed critical, and both
methods are included in one box. This figure also illustrates that no
distinction exists between so-called classical and molecular breeding
methods at the steps of evaluation In laboratory, field, or large-scale
environmental introduction.
This understanding of the biological nrincinles has the f~ll~nn~
implications for the report:
To r ~r^~~"~ ~
1. The deliberations of the committees were guided by the
conclusion (NAS, 1987) that the product of genetic modification and
selection should be the primary focus for making decisions about the
environmental introduction of a plant or microorganism and not the
process by which the products were obtained.
2. Information about the process used to produce a genetically
modified organism is unporta~t in understanding the characteristics
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BIOLOGICAL SOURCE MATERIAL
GENETIC MODIFICATION
BY CLASSICAL OR
CELLULAR/MOLECULAR METHODS
g
SELECTION OF DESIRED FORM
-
l
EVALUATION IN LABORATORY
AND/OR FIELD TESTS
LARGE SCALE INTRODUCTION
l
FIGURE 2.1 Genetic modification of an organism and its introduction into
the environment.
Of the product. However, the nature of the process is not a useful
criterion for determining whether the product requires less or more
oversight.
3. The same physical and biological laws govern the response
of organisms modified by modern molecular and cellular methods
and those procluced by classical methods. Scientists have vast expe-
rience with the products of classical modification, and the knowledge
gained thereby is directly applicable to understanding, evaluation,
and decision-mak~g about the relative safety or risk of field tests on
products of molecular modification techniques.
Representative terms from entire chapter:
genetic modification
