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Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
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OCR for page 15
Gene Transfer
The Background
Most of the current excitement in agricultural research focuses on
gene-splicing or recombinant DNA technology. Lawrence Bogoract de-
scribed the development of this technology, its promise, and limitations.
This technique had its impetus in research in the 1940s and 1950s on
the molecular structure and function of genes. In the 1940s, Oswald
Avery, Colin MacLeod, and Maclyn McCarty presented evidence that
genes were made of deoxyribonucleic acid, or DNA. DNA is a molecule
consisting of sugar, phosphate, and four bases: adenine, guanine, thy-
mine, and cytosine (A, G. T. and C). At that time, no one could fathom
how such a simple molecule could contain and transmit hereditary in-
formation.
Watson and Crick provided the answer in 1953. They described DNA
as a two-stranded molecule, coiled in the now-famous double helix. The
backbone of the molecule is a string of sugar and phosphate. A nucleo-
tide base either an A, G. T. or C—sticks out from each of the sugars.
The two strands are held together by weak bonds between these bases;
A binds with T. and G binds with C. Thus, each strand is complementary
to the other.
The elucidation of that structure revealed how DNA passes on in-
structions from one generation to the next. Prior to cell division, the
two strands unwind. Each strand then serves as a template for the
faithful replication of another DNA molecule, which is then passed on
to progeny.
DNA contains hereditary information but the major question is how
is that information processed and turned into a trait? The answer lies
in the "central dogma" of molecular biology- that is, information con-
15
OCR for page 16
16
GENETIC ENGINEERING OF PLANTS
Old
~A.
{= T ~ /
.A~
Old New
,/
~T A~
New Old
Replication of DNA. The double-stranded DNA helix unwinds and each strand serves
as a template for the building of a complementary strand. The resulting daughter DNA
molecules are exact copies of the parent, with each double helix having one of the
parent strands. From U.S. Congress, Office of Technology Assessment, Impacts of Applied
Genetics: Micro-organisms, Plants, and Animals, U.S. Government Printing Office, Wash-
ington, D.C., 1981.
tained in DNA is copied into molecules known as ribonucleic acid, or
RNA, and RNA specifies the synthesis of all proteins. Thus, DNA carries
the instructions for proteins both for the structural proteins, such as
those in the framework of membranes, and for the enzymes, which
catalyze all the metabolic reactions of an organism.
Proteins are chainlike molecules composed of a sequence of amino
OCR for page 17
GENE TRANSFER
17
acids. That is where the genetic code comes in: a series of three nu-
cleotide bases in DNA codes for each amino acid in a protein. The
sequence TAC, for instance, cocles for the amino acid methionine; the
sequence TAT codes for the amino acid isoleucine. A single change in
a nucleotide means that one amino acid in the protein is replaced by
another. "That's a mutation in the gene and an alteration in the gene
product," Bogorad explained. "It came as a surprise 20 years ago when
it was discovered that a single amino acid change in a protein could
greatly affect the way it worked."
TRANSLATION
Messenger R N A
Cytoplasm
The "central dogma." DNA carries the instructions for the synthesis of proteins. A
series of three nucleotide bases in the DNA molecule code for a specific amino acid,
the building blocks of protein. Each gene, a relatively short segment of a long DNA
molecule, codes for a single protein. The genetic information is expressed in a two-step
process, described by the "central dogma" of molecular genetics: DNA is transcribed
to RNA, then RNA is translated to protein. During transcription, a strand of DNA serves
as a template for the formation of a complementary strand of messenger RNA. Next,
the messenger RNA moves from the cell nucleus to the cytoplasm. There ribosomes
attach to the messenger RNA and direct protein synthesis by reading the genetic code
and building a chain of amino acids.
OCR for page 18
18
GENETIC ENGINEERING OF PLANTS
The conversion of a gene to RNA and then to a protein product is
called expression. If a gene is present and its protein product appears,
the gene is said to be "on." If the gene is present but no product appears,
it is said to be "off." Gene expression is a two-step process: first, the
DNA is transcribed to RNA; then the RNA is translated to protein.
Transcription is similar to DNA replication. The DNA molecule un-
winds, but in this case the strands serve as a template for the formation
of an RNA molecule. RNA contains three of the same nucleotide bases
as does DNA A, G. and C. But in place of thymine, RNA has uracil,
U. Thus, during transcription, G binds with C, and now A bincTs with
U. Translation occurs when the RNA molecule, known as messenger
RNA, leaves the nucleus and travels to the ribosomes, the site of protein
synthesis in the cytoplasm. Here the RNA specifies the sequence of
amino acids in a protein according to the triplet codes mentioned earlier.
The Technique
In theory, gene-splicing is relatively straightforward. In practice, it is
far from routine. Bogorad outlined the basic procedure. The first step
is to locate the desired gene among the 5 million or so in the cell nucleus.
Each gene has three regions, all essential for successful functioning. The
beginning is the promoter region, the series of nucleotides that is rec-
ognized by the enzyme that triggers the transcription of DNA to RNA.
The middle sequence of nucleotides contains the cocle, the instructions
for producing a specific protein. The end series of nucIeotides, or ter-
minator, is a signal to stop the transcription process.
Next, the gene must be isolated from the others on the chromosome.
For this task, the genetic engineer uses a restriction enzyme. These
enzymes recognize specific nucleotide sequences and cut the DNA at
precisely those points. It is these enzymes that allow researchers to snip
a gene out of the DNA sequence from one organism and splice it into
the DNA of another. In fact, the advent of recombinant DNA technology
can be traced to the discovery of these restriction enzymes in the early
1970s. Now, a decade later, biological suppliers offer hundreds of re-
striction enzymes for sale, each one recognizing a different sequence of
nucleotides.
Once the gene is isolated, it must be cloned, or duplicated, and in-
serted into the host cell. Both steps are accomplished by inserting the
gene into a plasmid. A plasmid is a tiny, circular piece of bacterial DNA.
Plasmicis reside as separate units of DNA inside the cytoplasm of a
bacterial cell. With the same restriction enzyme that was used to excise
the gene from the donor cell, the genetic engineer cuts open the plasmid.
OCR for page 19
GENE TRANSFER
LEA
~T...A~//
/~
A...T~7
C .,/
19
Eco Rl
Hpa 1
—C G—
—T. A—
—T. A—
—A, T—
—A, T—
~ A
—G C—
—G C—
—T A—
T—
—C G—
Restriction enzymes. Restriction endonucleoses are enzymes that cleave DNA at specific
sites. The illustration shows examples of cleavages made by two such enzymes, Eco RI
and Hpa 1. Eco RI recognizes the DNA sequence CLANG and cleaves each strand
between the G and A yielding single strand ends. Such "sticky ends" can readily join
on to other DNA fragments created by the same enzyme. Hpa 1 does not leave "sticky
ends"; it recognizes the sequence CGATT^=c and cleaves each strand between the A and
T. Restriction enzymes can be used to isolate single genes.
OCR for page 20
20
GENETIC ENGINEERING OF PLANTS
This leaves the plasmid with two "sticky" ends, which will now accept
the foreign gene. After the foreign gene is inserted, another enzyme,
called a ligase, is used to sew the plasmid together. Plasmids are ideal
vectors for carrying the new gene into a host cell, because, in nature,
plasmids are routinely passed from one bacterium to another, where
they are readily accepted. When the recombinant molecule part bac-
terial plasmid, part plant gene—is taken up by the bacterial cell, that
cell is said to be transformed. As the plasmid replicates inside the host
cell, it copies the foreign gene along with its standard gene allotment.
The goal is not just to clone the gene, but to have that gene ex-
pressed—to have the DNA transcribed to RNA, and the RNA translated
into the desired protein in the host cell. Most work to date has in-
volved the insertion of a gene from a higher organism—usually an
animal—into a bacterial host, where the animal gene produces proteins
such as insulin, interferon, and human growth hormone. The genes of
higher organisms (eukaryotes) have different control signals—signals
that turn genes on and off than do the genes of primitive organisms
(prokaryotes) like bacteria, which lack a nucleus. These signals must be
read correctly for the gene to be expressed. Gene expression has been
achieved by removing the specific control signals from genes of higher
organisms, in essence tricking the bacterium into accepting the foreign
gene as a bacterial gene.
Up to this point, the techniques for plant genetic engineering are
similar to those used to design bacteria to produce insulin or other
pharmaceuticals. In short, a gene is isolated, spliced into a vector, in-
serted into a host cell, and expressed. Pharmaceutical applications de-
pend upon a method of culturing large batches of these recombinant
bacteria. By contrast, plant genetic engineering depends upon a means
of regenerating a whole plant from the cells in culture.
There are three tissue-culture techniques for regenerating plants from
culture (see Cell Culture, p. 34~. For gene-transfer experiments, the pre-
ferred route is the culture of protoplasts single cells from which the
cellulose wall has been removed. That is because it is easier to insert
genes into protoplasts than into cells containing the tough outer wall,
which animal cells lack.
Thus, before a foreign gene is introduced into a plant cell, that cell
is treated with enzymes that dissolve the outer wall. The protoplast,
containing its new gene, is then placed in a broth of plant hormones
and nutrients that induce it to regenerate. It first re-forms a cell wall.
By changing the nutrient mixture, the cells can be induced to multiply
and form embryolike structures. Known as somatic embryos, these give
rise to tiny plants, which then can be transferred to the soil.
OCR for page 21
GENE TRANSFER
Current Constraints
21
Though major strides have been made in the past few years, only the
barest beginnings have been made in the transfer of genes among higher
plants. As Bogorad explained, the major limitation is the lack of knowI-
edge about basic plant biology necessary to exploit this new technology.
Each step of the process presents its own difficulties. For instance,
just finding the desired gene is a monumental task—"one of the most
difficult and challenging operations in molecular biology," Bogorad said.
The plant genome is large and exceedingly complex. Some genes are
located on chromosomes in the cell nucleus. Others are contained in
two organelles the chIoroplasts and mitrochondria. Similarly, there are
difficulties in identifying all the important parts of the gene, including
any DNA sequences necessary to regulate expression of the gene; in
developing an appropriate vector to carry the foreign gene into the plant
cell; and, finally, in regenerating plants from the transformed cells in
culture.
Vectors
One of the major challenges is the development of vectors to ferry
foreign DNA into the plant genome. Only a few bacterial plasmids will
work in plants. One of these is the Ti plasmid from the soil-borne
bacterium Agrobacterium tumefaciens. It is the most promising vector to
date for plant genetic engineering. Agrobacterium causes crown gall dis-
ease: it infects the plant stem tissues, inducing tumors. The disease-
causin~ agent is the bacterium's Ti (for tomor-ind~rin~N nln~mi~ Thin
. . . . . . . . . . ~
plasmas does its damage by inserting itself into the plant cell's genome,
where it is replicated and expressed along with the plant's DNA. The
expression of the bacterial Ti plasmid genes causes the abnormal cell
growth characteristic of crown gall disease. Actually, only a small piece
of the Ti plasmid is inserted into the plant genome this piece is called
T DNA (for transferred DNA).
The Ti plasmid is a natural vector that routinely inserts new DNA
into plant cells. Moreover, it comes equipped with a trait molecular
biologists were seeking: its genes can be expressed in the environment
of the plant genome; the regulatory signals of the bacterial genes can
be read by the plant cell. Several scientists reasoned that the Ti plasmid
could be tricked into carrying additional genes into the plant genome
as well.
For the past several years, there has been an intensive research effort
to develop the Ti plasmid as a genetic engineering vector. Much of it
OCR for page 22
22
GENETIC ENGINEERING OF PLANTS
Bacterial ~'
e T P ~ ~ If
Agrobacter/um
tumefac/ens T-D NA Jon
Chromosomes ii7
Transformed Plant Cell
The Ti plasmid vector. In plant genetic engineering, the Ti plasmid can be used to carry
foreign genes into plant cells. The Ti plasmid is the disease-causing agent of the soil-
borne bacteria Agrobacterium tumefaciens. When the bacteria infect a plant, a part of the
Ti plasmid called the T DNA is transferred to a plant chromosome. When the T DNA
is expressed as part of that chromosome, it causes the plant cell to divide and grow
abnormally. Researchers have recently developed procedures for removing the tumor-
causing genes from the T DNA and replacing them with desirable genes. The Ti plasmid
containing the altered T TUNA region can then he llCt°~ to insert the H~cirPH ~PnPc into
plant chromosomes.
~ _ _ _ _ _ ~ ~ ~ ~ ~ ~ _ ~ ~ ~ ~ ~ _ _ _ v ^^ _ ~ ~t~ _ ~ ~ ~ v ~
has been performed by two research groups: one led by Mary Dell
Chilton at Washington University and the other a European group led
by Jeff Schell of the Max Planck Plant Breeding Institute in Cologne,
Germany, and Mark Van Montagu of State University in Ghent, Bel-
gium. They have addressed a number of questions, such as how to
insert new genes into the T DNA region without disrupting the se-
quences that control its insertion into the plant genome and how to
remove the disease-causing part of the plasmid so that the vector could
be used in practical as well as experimental gene transfer.
Their efforts have paid off. In January 1983 the European researchers
and another group at Monsanto Co. announced that they had used a
Ti plasmid to carry a functioning bacterial gene into a plant cell. This
was the first demonstration that a foreign gene could be inserted into
OCR for page 23
GENE TRANSFER
r - - -- - -A
23
a plant cell and be expressed. The Monsanto group included Robert
Horsch, Stephen G. Rogers, and Robert T. Fraley. Both teams, working
indenendentlv, inserted a bacterial gene for antibiotic resistance into the
T DNA portion of a Ti plasmid. The Ti plasmid was then used to
transform petunia cells in culture. The foreign gene was expressed: the
cells in culture were resistant to the antibiotic. When a plant was re-
generated from these cells, it retained the antibiotic resistance.
Commenting on the widely heralded gene transfer, Robert M. Good-
man of Calgene, Inc., interjected a note of caution. "Notwithstanding
the excitement generated by the recent demonstration that the expected
is possible, we are only at the beginning of a long period of research
and development" on vectors. At this stage, biologists do not under-
stand how the Ti plasmid works specifically, they clo not understand
the signals that control the insertion anct expression of T DNA.
Even without that knowledge, the Ti plasmid can be effectively used
as a vector, as shown by the recent petunia experiment. But Goodman
cautioned against becoming too intent on applications, on getting "too
carried away with the short-term excitement that we overlook the need
to invest in work that leads to an understanding of the underlying
principles . . . that will allow the design of sophisticated genetic engi-
neering vectors." For example, he said, "we must understand how the
T DNA inserts if we ever hope to control the location and perhaps the
multiplicity of the insertion."
According to Goodman and others, adclitional work is necessary to
improve the efficiency of Ti plasmid as a vector. In most work to date,
only a small percentage of plant cells inoculated with Agrobacterium
carrying the Ti plasmid are transformed. if the Ti plasmid is to be used
in a practical gene-transfer system, then much higher rates of transfor-
mation must be achieved. Such research has already begun.
Other vectors will also be necessary. A major limitation of the Ti
plasmicl is that it works only in those plants that the Agrobacterium
normally infects. It does not infect plants in the grass family, which
include the important cereal crops like corn, rice, and wheat. Conse-
quently, there is now no vector available for their genetic engineering.
It may be possible to modify the Ti plasmid so that it can infect grasses,
yet other vectors having different host ranges should be explored.
Another limitation is that the Ti plasmid can be used only to ferry
genes into the nuclear DNA. Several agronomically useful traits, how-
ever, are controlled by genes locater! outside the nucleus in either the
chloroplasts or mitochondria. For example, male sterility in several cases
is affected by genes in the mitochondria. Male sterility is a desired trait
in plant breeding because it allows the inexpensive production of hybrid
OCR for page 24
24
GENETIC ENGINEERING OF PLANTS
seecT. Yet at present, no vector is available to manipulate genes in the
organelles.
Plant viruses are also being studied as possible vectors. Viruses are
tiny bundles of either DNA or RNA encased in a protein coat. Viruses
are of interest because they somehow command the plant cell's ma-
chinery to replicate the virus and express the viral genes. As with a
plasmid, the idea is to insert foreign DNA into the virus and use it to
transform a plant cell. Again, the virus would have to be "disarmed"
so that it would not cause disease before it could be used as a practical
vector. One advantage viral vectors offer is that almost all plants are
susceptible to one virus or another. By contrast, the host range of the
Ti plasmid is quite limited.
Most work has been performed on the cauliflower mosaic virus, a
small, double-stranded DNA virus. Researchers have inserted short pieces
of foreign DNA into the virus. This virus has then been used to transform
plant cells. In these experiments, the foreign DNA has been replicated
inside the plant cell as part of the viral genome.
But much work needs to be done to develop the cauliflower mosaic
virus as a vector. Its major drawback is the size of the foreign DNA it
can accept. So far, only pieces of DNA smaller than a gene have been
stably inserted into the virus genome. It seems that the protein coat on
the virus could be the limiting factor. But there are other plant viruses.
RNA viruses, for example, can function without their protein coats and
thus conceivably could accept larger pieces containing foreign genes.
Viroids are another possibility, though their use is even more spec-
ulative, both because of their size and because so little is known about
them. ViroicTs consist of RNA without the protein coat. They are the
smallest pathogenic agents known, smaller than the average gene. The
potato spindle viroicT, for example, contains only 359 nucleotide base
pairs, as opposed to about 8,000 in the cauliflower mosaic virus.
Gene Expression
Until recently, one of the key uncertainties of genetic engineering was
whether a foreign gene would be correctly expressed in a higher or-
ganism. The recent successful transfer and expression of a foreign gene
into a plant cell answered that question. But, as Bogorad pointed out,
achieving expression is only the first step. The next step is to control
that expression so that the gene is switched on in the right place at the
right time. That selective expression is what occurs in nature: although
all cells contain the genes for photosynthesis, for instance, they are
turned on only in the leaf, not the root. Molecular biologists have yet
OCR for page 25
GENE TRANSFER
25
to master those controls. Until they do, a transferred gene could simply
be on all the time in all the cells.
In numerous laboratories, biologists are searching for the control ele-
ments that regulate gene expression. If these controls can be identified
and transferred to a plant along with the desired gene, they will permit
gene expression to be "targeted" to specific organs and developmental
stages. The regulation of gene expression is also of enormous theoretical
interest, for the switching on and off of genes determines how a single
cell differentiates into a plant.
Another question is whether the introduction of a foreign gene will
affect the expression of the other genes. The introduction of new genes
through conventional plant breeding can have deleterious effects, sug-
gesting that gene interaction is quite complex. For instance, in 1964 a
strain of high-lysine corn was identified. Lysine is an essential amino
acid in the diet of nonruminant animals. Though the strain has improved
nutritional value for swine and poultry, it is not grown commercially
because the yield is reduced 10 percent over other strains. Another
drawback is that the kernels of the high-lysine strain do not have good
storage quality. It is too soon to say whether molecular genetic engi-
neering will involve similar trade-offs.
Single and Mulligene Traits
Based on the recent advances in identifying and isolating genes as
well as advances with vectors, many molecular biologists are confident
that they will be able to engineer traits controlled by a single gene or a
small cluster of genes. Yet many commercially valuable traits, such as
yield and stress resistance of various kinds, are controlled by numerous
genes somehow acting in concert. These are called multigene traits. Just
finding the genes will be difficult, as evidence suggests that they are
scattered throughout the chromosome. Determining how the expression
of these genes is regulated, and how their gene products interact, is an
even more formidable task. Consequently, it is not clear that techniques
can be developed to engineer multigene traits.
Plant Regeneration
Successful gene transfer ultimately depends on the ability to regen-
erate plants from cells in culture. Yet protoplast culture is far from a
proven technology. Although it works well in some species, such as
carrots, tomatoes, tobacco, and petunia, some of the major crop species
are notoriously difficult to regenerate from protoplasts. Potatoes and
OCR for page 26
26
GENETIC ENGINEERING OF PLANTS
alfalfa have been successfully regenerated from protoplasts, but the
technique does not work reliably for corn, wheat, or soybeans. Com-
pounding the difficulty, no one knows exactly why.
The secret lies in the signals that turn genes on and off during de-
velopment. Cell culture is an attempt to mimic that process in the lab-
oratory. As the ability of plants to regenerate reveals, cells are totipo-
tent that is, each cell, such as a leaf cell, contains the instructions for
the whole plant. In the differentiatec! state in the leaf, for example
most of those genes are shut off. The trick is to induce the cell in culture
to regress to an undifferentiated state in which the genes can be switched
on ancT off again in proper sequence.
The development of culture methods has been handicapped by the
lack of knowledge about the regulation of gene expression during de-
velopment. Research to understand the genetic mechanisms involved
in regeneration is proceeding in tandem with efforts to develop practical
culture techniques. Although an increasing number of plants are yielding
to protoplast and the other culture techniques, these advances have
stemmed as much from guesswork as from science. Whether or not a
plant will respond in culture is influenced by several factors, including
the composition of the nutrient broth, the specific genotype of the donor
plant, and the site from which the explant is taken. In working with
unresponsive species, biologists are often confined to juggling these
factors perhaps screening hundreds of genotypes in search of one that
will work. To a lesser degree, similar uncertainties surround the other
two in vitro regeneration techniques: callus and suspension culture (see
Cell Culture, p. 34~.
Representative terms from entire chapter:
foreign gene
