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Genetic Engineering with Plants.

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Genetic Engineering with Plants
By Peter Eckes, Gunter Donn, and Friedrich Wengenmayer*
Dedicated to Professor Heinz Harnisch on the occasion of his 60th birthday
Today there are no longer any basic problems involved in isolating and characterizing plant
genes. Our knowledge of the structure and function of the genes so far sequenced provides
a beginning understanding of gene regulation in plants. In connection with the recently
developed methods of gene transfer to plant cells, the most important application is the
production of plants with new characteristics through genetic engineering. Since the genes
can be transferred from any kind of plant-indeed even from any organism-completely
new possibilities are opened u p for plant breeding.
1. Introduction
Ten years ago molecular biologists succeeded in isolating the first structural gene of a plant enzyme.“] Since then,
developments in the field of plant molecular biology have
opened u p possibilities for practical applications that were
previously inconceivable.
The first isolated gene, which is localized on the DNA of
chloroplasts, encodes the large subunit of the enzyme ribulose- 1,5-bisphosphate carboxylase (rubisco). This enzyme
enables plants to bind C 0 2 from the air. Indeed, the entire
chloroplast DNA of tobacco has already been sequenced12]-it consists of 155844 base pairs. Meanwhile,
methods have become available for the stable transfer of
foreign genes into plants; “transgenic plants” have already
been obtained in numerous laboratories. In the USA, official approval has recently been given for the first field
trials with plants that have been altered by genetic engineering.
The growing interest in the biochemistry and molecular
biology of plants is also indicated by the increasing number of publications in this field. Industry, mainly in the
United Kingdom, the United States, and Japan, has also
begun to establish research groups in this area (Table 1).
Table I . Number of companies involved in the application of biotechnology
to agriculture (taken from [3]).
Country
Number
Italy
Federal Republic of Germany
France
Japan
United Kingdom
USA
1
2
5
12
15
13
Several factors are responsible for the recent successes
and the growing interest in the biochemistry and molecular
biology of plants. One of the most important is scientific
interest in the gene structure and gene regulation of plants.
Knowledge in this area lags far behind what is known
about prokaryotic and animal cells.
[*] Dr. F. Wengenmayer, Dr. P. Eckes, Dr. G . Donn
Hoechst Aktiengesellschaft
Postfach 800320, D-6230 Frankfurt am Main 80 (FRG)
382
0 VCH Verlaysyesellrchaft mbH. D-6940 Weinheim. 1987
A further reason for the great interest in the molecular
biology of plants is the close relationship between the basic knowledge obtained and its possible applications in agriculture. In principle, genes that are responsible for the
expression of an important trait can be isolated from any
plant or other organism and transferred into crop plants.
In this way, characteristics that were not previously accessible to the classical methods of plant breeding become
transferable to plants. Thus, for example, resistance genes
could be isolated from exotic plants and transferred to
domestic crops. Previously, genes and the corresponding
characteristics could only be transferred naturally from
parent plants that already possessed them. These limitations are now being overcome with the help of genetic engineering.
Scientists are also becoming more actively engaged in
the molecular biology of plants because of the possibility
that the knowledge obtained might help in an important
way to solve the problem of worldwide famine. According
to the most recent calculations of the U. S . Bureau of Statistics, the world population by the year 2000 will have
risen from the present level of 4.9 billion to 6.2 billion.
The following factors have accounted for the impressive
increase in food production over the last few decades:
-the introduction of new, high-yield varieties of plants
-the use of fertilizers and crop protection chemicals
-the increasing mechanization of agriculture
Crop yields will continue to increase in the future, but
exponential increases such as occur in the world population will not appear. The large agricultural surpluses in the
EEC and the USA at present d o not represent a solution
for the 80% of the world population that will be living outside Europe, the United States, and the Soviet Union in the
year 2000. Especially in tropical and subtropical countries,
plants with genetically engineered resistance toward pests,
diseases, o r stress conditions could acquire great importance.
A further consideration is that, after genetically engineered plants have been produced in the laboratory, they
still have to be incorporated into cultivation programs and
tested. This process takes several years, so that such plants
will not be available to farmers in sufficient quantities un-
0570-0833/87/0805-0382$ 02.80/0
Angew. Chem. Int.
Ed. Engl. 26 (1987)382-402
ti1 the mid- 1990s. Consequently, an evaluation of the possibilities of genetic engineering for agriculture must be
based not on present food requirements but on the requirements anticipated for the year 2000. Of course, the problem of worldwide famine cannot be resolved solely by genetic engineering; political and sociological factors are
also of decisive importance. However, genetic engineering
could provide the basis for an adequate food supply in the
form of high-yield and resistant plants.
2.1. Vegetative Plant Reproduction
Plants are characterized by a large regenerative capacity.
If plant shoots or segments of shoots are cultivated under
suitable conditions (high humidity and absence of pathogens), these shoot segments develop roots. This asexual reproductive process, which is familiar in horticultural practice, can be carried out particularly efficiently on synthetic
culture media under sterile conditions. The simplest of
these culture media contain a balanced mixture of the salts
and trace elements essential to plant nutrition, plus several
required vitamins and suchrose."]
2. Plant Cell Biology
In a medium that is free of phytohormones, shoots or
shoot meristems grow to complete plants. If the culture
The development of procedures in cell biology to regenmedium contains phytohormones, however, the reaction of
erate plants from individual cells and to transfer genes to
the explant depends on the ratio of the auxin to the cytoplant cells provided the prerequisite for the practical use
kinin concentration in the medium.['] If both phytohorof genetic engineering. Specific objectives of plant breedmones are present in adequate concentrations, undifferening could be achieved, in many cases, through the methods
tiated tissue, named callus, typically grows at the cut surof genetic engineering as well as by suitable cell-biological
selection procedures. Hence, this section will examine a
face of the explant. This is true regardless of whether the
explant is a leaf fragment, a piece of stem, or a root. If
few aspects of cell biology that are important for obtaining
plants with new characteristics or for the application of gesuch a primary callus is transferred to fresh medium at regular intervals, it can be propagated for an indefinite time.
netic engineering. The techniques of plant cell cultivation
that have been the focus of interest for the past decade have
If cytokinin predominates in the medium, the formation
been described in several review articles and m o n ~ g r a p h s . l ~ - ~ ] of buds is favored, both on primary explants and on pre-
C
Fig. I . Cell culture techniques used for mutant selection and production of transgenic plants. Shoot meristems ( a ) , stem segments (b), leaf fragments ( e ) , and
mesophyll protoplasts (d) are used as explants both for the initiation of in vitro cultures and for gene-transfer methods. Through protoplast fusion, hybrid
protoplasts ( e ) can be obtained. Plant regeneration may occur via shoot formation (9) either immediately at the explants ( f - h ) or at the callus stage ( i . k , 0 ) .
Alternatively, somatic embryos may develop (r). A cell wall is regenerated from protoplasts and cell division ( I ) begins, leading to cell clusters (microcalli) (m).
They can be cultured as suspension cultures (p). In some species, the development of somatic embryos (u) can then be induced. On agar media, microcalli grow to
form callus ( o ) , which, as shown above, can undergo differentiation. If toxins, antibiotics, or herbicides are added to the media, resistant mutants or transformants
can be selected preferentially at stages f - p . Shoot cultures (n), shoot-forming calli (9). and embryo cultures ( t ) are used for clonal propagation of genotypes (s, v).
Under suitable conditions, somatic embryos may be convened into artificial seeds (w).
Angew. Chem Int. Ed Engl. 26 (1987) 382-402
383
viously formed c a l k However, the capability of a callus to
differentiate depends strongly on its genotype. In some
plant varieties (for example, tobacco) regeneration of a
shoot, and hence of the plant, succeeds with practically every explant and even with callus that has already been cultivated for many years.'"] In other varieties (for example,
soybeans), on the other hand, shoot regeneration from established callus cultures is not successful.
Regeneration from shoots is a prerequisite for the production of transgenic plants by the usual methods (Section
3). Alternative routes to shoot regeneration are shown in
Figure I . Shoots cultivated in vitro on culture media containing a cytokinin produce adventitious shoots, so that
within a short time thousands of individuals of the same
genotype can be produced from a single shoot. This
method of vegetative propagation has so far proved economical for only a few types of plants (ornamental plants,
potatoes'"]) because of the expensive manual labor required.
In a number of crop plants, including alfalfa,[121soybean,"31 and rape,['41the formation of somatic embryos can
be induced in cell cultures. In contrast to the zygotic (sexual) embryos of seeds, somatic embryos arise from somatic
cells (Figs. 1-3).
2.2. Protoplasts
Protoplasts are individual plant cells without cell walls
which can be obtained from plant tissue by the digestion
of the cell walls by hydrolytic enzymes (pectinases, hemicellulases, and cellulases). Several million mesophyll protoplasts can be obtained from 1 g of leaf tissue. In 1971,
the first successful regeneration of plants['61 from mesophyll protoplasts was achieved with tobacco. Meanwhile,
methods of plant regeneration have been worked out for a
number of useful plants, including rape,'"] potatoes,["] toma toe^,"'^ alfalfa,'*'] and rice.I2'l Figure 3 shows the devel-
Fig. 2. Alfalfd callus (white) developing somatic embryos (green).
Somatic embryos can be cultivated in any desired number in synthetic media. On a laboratory scale it has already
been shown that somatic embryos can be encapsulated together with a nutrient stock, stored for a short time, and
then induced to germinate.[''] Such biotechnologically produced plant embryos that are enclosed in polymer capsules
are called "artificial seeds." When this technology is further perfected it is conceivable that such artificial seeds
could be sown like natural seed. This would make possible
a productive and economical form of vegetative propagation of elite genotypes.
384
Fig. 3 . Plant regeneration from protoplasts of alfalfa (Medicago .satiua). a)
Freshly isolated protoplasts (day I); b) four-cell stage (day 4); c) microcallus
(day I I):d) formation of embryogenic clusters (day 14); e) germinating somatic embryos (5 weeks after protoplast isolation); 0 regenerated plant (8
weeks after protoplast isolation).
opment of alfalfa protoplasts to complete plants. Protoplasts are especially interesting because they are able to
take u p macromolecules such as DNA(221and even cell org a n e l l e ~ [ and
~~]
On the basis of these
early observations, a method of direct D N A transfer to
protoplasts has been developed in the last few years (see
Section 3).
Angew Chem. Inr. Ed. Engl. 26 (1987) 382-402
2.2.1. Protoplast Fusion
Table 2. Pathogen-resistant crop plants obtained through tissue culture
(taken in part from 1351).
Plant protoplasts can fuse under various conditions, including the presence of C a 2 + ions at pH values over 9.5,[251
polyethylene glycol,[2b1or dimethyl sulfoxide. Brief electric
pulses[ZX1
will also trigger protoplast fusion. The protoplasts of any plants can be fused, but, in general, the fusion products of protoplasts from different plant genera
are difficult to regenerate. In only a few cases has it been
possible so far to regenerate intergeneric hybrids, which,
moreover, proved to be infertile.[29,301
A familiar example is
the somatic hybrid from tomato and potato. This somatic
hybrid is of theoretical importance, showing that, by
means of protoplast fusion, hybrids can be produced that
are not accessible via the sexual route. Fertile hybrid
plants can be obtained by fusing the protoplasts of closely
related plant species, even when these plants cannot be hybridized
If different selectable marker genes
are present in the genomes of the two fusing protoplasts,
the somatic hybrids can be sorted out easily in a selection
medium. One method of protoplast fusion, which was already described in principle in 1980,[321has excited much
interest recently. Donor protoplasts whose genome has
been fragmented by exposure to intense X-irradiation are
fused with nonirradiated acceptor protoplasts. In some of
the hybrid protoplasts, the fragments of the donor genome
are integrated into the acceptor genome. If the donor genome contains genes that are responsible for the expression of resistances, it is possible to select out these recombinants. In this way, a chromosome fragment containing a
gene for resistance against the antibiotic methotrexate
could be transferred from carrots to tobacco protoplasts.
The plants regenerated from these hybrid protoplasts resemble tobacco plants and, in addition to the transferred
gene for methotrexate resistance, they possess some further carrot genes.f331The plants are fertile (i.e., they produce seed) and transmit the methotrexate resistance to
their progeny. This asymmetric protoplast fusion method
could be a way of transferring several, even unknown,
genes between species.
Crop plant
2.3. Mutant Selection in vitro
The isolation of protoplasts has enabled large populations of homogeneous, diploid, and totipotent cells to be
obtained for many varieties of plants and to be regenerated
to plants. Early in the development of protoplast techniques, attempts were made to use protoplast populations
for mutant selection.[341A single petri dish may contain 1-5
million protoplasts, all of which are potential plants. If
mutations can be induced in this cell population and the
mutants selected, it should be possible to regenerate plants
with new characteristics.
Protoplasts are not a necessary condition for mutant selection, however; any readily regenerating plant cell culture may be used for this purpose. There are now a number
of reports describing the successful selection of mutants
from plant cell cultures, including pathogen-resistant (Table 2) and herbicide-resistant plants (cf. Table 5 ) as well as
plants that overproduce specific amino acids. Most mutants and variants found in this way could already be seAngew Chem. In1 Ed. Engl. 26 (19871 382-402
Pathogen
Ref.
a ) After
in vitro selection /or toxin resistance
Tobacco
Pseudomonas tabaci
Alternaria solani
(Nicotiana tabacurn)
Potato
Phytophtora infestans
(Solanurn tuberosum)
Fusarium oxysporurn
Erwinia carotouora
Tomato
Fusariurn oxysporum
(Lycopersicon esculenturn)
Rape
Phoma Iingom
(Brassica napus)
Alfalfa
Fusarium oxysporum
(Medicago sativa)
Verticilliumalboatrum
Maize
Helminthosporium rnaydis
(Zea mays)
b) Resistance observed without prior in vitro selection
Potato
Alternaria solani
Phytophtora rnfestans
Potato Y virus
Potato curling virus
Tobacco
Tomato
Tobacco mosaic virus
Tobacco mosaic virus
lected and isolated at the cell level by means of a selection
pressure-e.g., the addition to the culture medium of enzyme inhibitors, amino-acid analogues, pathogen toxins,
or herbicides.‘501 Strikingly, these variants were also found
in experiments in which the mutation rate was not accelerated by chemical or physical mutagens.
2.3. I . Somaclonal Variation
The high variability described above seems to arise
spontaneously through the process of in vitro cultivation,
especially when the cell cultures have been cultivated for a
prolonged period as undifferentiated cells. Under such
conditions, chromosome mutations can accumulate easi l ~ . [ ~Only
’ ] part of the observed variability, however, can
be explained by the loss of individual chromosomes or by
the presence of supernumerary chromosomes. The variability induced by cell cultivation is named “somaclonal
variation.”[s21An obvious application would be to use this
source of variability for breeding programs.*531
Meanwhile,
increasing evidence has appeared, indicating that, under
the stress of in vitro cultivation, mechanisms are activated
in plant cells that lead to reorganization of the genome.1541
In maize, for example, “jumping genes” can be activated
by in vitro cultivation.[551
2.4. Importance of Cell Biology for Genetic Engineering
The contribution of plant cell biology to genetic engineering consists in making competent (Le., .transformable)
plant cells available, effecting DNA transfer, and regenerating plants from the transformed cells. These prerequisites have not been met to the required extent for important crop plants such as soybeans, cotton, and the cereals,
with the exception of rice. The cell culture systems of species that are difficult to regenerate have to be improved
further. It would be ideal to be able to regenerate proto385
plasts of these plants, too, since protoplasts are capable of
taking up foreign DNA directly and integrating it into
their genomes.
Hope that progress is also possible with the cereals has
been inspired by the recent reports of three research
groups that rice plants have been successfully regenerated
from protoplasts.[”I This breakthrough was achieved by
the use of embryogenic suspension cultures as starting material for the isolation of protoplasts.
3. Transfer of DNA
3.1. The Agrobacterium tumefaciens System
3.1. I . Tumor Formation
Until recently it was not possible to transfer foreign
genes to plant cells. Although DNA could be isolated from
plants and characterized, the function of certain D N A sequences was difficult to analyze because the possibility of
transfer was lacking. Surprisingly, this dilemma was solved
through studies of the formation of certain plant tumors,
the so-called crown galls. These tumors are undifferentiated cell growths caused by the infection of a plant with
gram-negative soil bacteria of the species Agrobacterium
tumefaciens (Fig. 4). The formation of these tumors is
caused by the introduction of bacterial D N A into plant
ceIIs.[561
Agrobacterium tumefaciens is a soil bacterium that is
capable of infecting a large number of dicotyledonous
plants, such as tobacco, tomatoes, carrots, and potatoes,
after an injury.[”] At the site of infection, strong undifferentiated cell growth begins, leading to the crown gall tumors. Once a tumor has formed, it can continue to grow
even in the absence of agrobacteria.[sxlTwo properties of
the tumor tissue should be especially emphasized: the tumor proliferates on sterile culture media of a defined composition without the addition of plant hormones (auxins
Fig. 4. Undifferentiated, growing tumor tissue (crown gall) on the stem of a
tomato plant after infection of the wounded area with Agrobacteriurn tumefaciens.
and cytokinins), although these hormones are essential for
the growth of normal cell tissue.[’’] Furthermore, the tumor
synthesizes specific metabolites, the so-called opines, that
are not present in other plant tissues.[591Opines are derivatives of amino acids and sugars; they can be used by agrobacteria but not by plants as sources of nitrogen and carbon.[s9.601The opines found in plants belong to four different classes: nopalines, octopines, agropines, and agrocinopines (Fig. 5).@’’
A given strain of agrobacteria can, within certain limits,
utilize only the class of opines whose synthesis it induces
in the plant. Nopaline-inducing bacteria, for example, cannot metabolize ~ c t o p i n e . [ ~Corresponding
~I
to the opines,
the agrobacteria have been divided into different classes,
HZN,
octopine family
C-NH-(CH~),-CH-COOH
H,N-(CH,).+-CH-COOH
I
I
I
I
HN+
H,N-(CH,)~-CH-COOH
I
NH
NH
NH
H3C-CH-COOH
H3C-CH-COOH
HgC- CH -COOH
octopine
Lysopine
I
octopinic acid
HN~CH,-CH-COOH
I
\=N
NH
I
H3C-CH-COOH
histopine
HZN,
nopaline family
C-NH-(CHZ),-CH-COOH
I
HN+
I
NH
I
HOOC-(CH,),-CH-COOH
nopaline
HOH,C-(CHOH),
agropine family
H,N-(CH,)S-CH-COOH
NH
I
HOOC-(CH,),-CH-COOH
nopalinic acid
HOH,C-(CHOH),-CHZ
Y N H
o+(CH,),-CONH,
0
agropine
I
NH
I
HOOC-CH-(CH,),-CONH,
mannopine
HOH,C-(CHOH),-CH,
I
NH
I
HOOC-CH-(CH,),-COOH
mannopinic acid
HOH2C-(CHOH)4-CH2
I
HOOC e
agropinic acid
Fig. 5. Chemical structure of the opines (data from IS,]). Agrocinopines are phosphorylated sugars of unknown structure.
386
Angew. Chem. Int. Ed. Engl. 26 (1987) 382-402
0
of which the nopaline and octopine classes have been most
thoroughly characterized.
3.1.2. The Ti Plasmid as the Tumor-inducing Principle
The hormone-independent growth of gall tumors in the
absence of agrobacteria and the biosynthesis of opines led
to the conclusion that agrobacteria must transmit a “tumor-inducing principle” to the plants.[621 Indeed, infectious bacteria were found to contain a megaplasmid, the Ti
plasmid (Ti = tumor-inducing), which is approximately 200
kilobases (kb) long.‘h31When the plasmid was removed
from Agrobacterium tumefaciens, the bacterium lost its
ability to induce plant tumors. Reintroduction of the plasmid restored the tumor-inducing capability. The plasmid
was thus identified as the tumor-inducing principle being
This Ti plasmid contains the genes that are responsible for hormone-independent growth of the tumor
and those involved in opine formation and utilization (Fig.
6),[60.65 -681
Only a relatively small region of the overall Ti plasmid is
integrated stably into the plant
Hence, the
transfer of D N A to plant cells is a natural process that has
been practiced by Agrobacterium tumefaciens for ages. The
integrated region of DNA, the so-called T-DNA, comprises about 20 kb. The genes responsible for opine bio-
octopine TL - DNA
1
A
b
1
synthesis and those causing tumor growth are localized on
the T_DNA.[67.72.731
Besides their striking similarities, the T-DNA regions of
octopine and nopaline bacteria also exhibit several significant differences. Whereas nopaline T-DNA represents an
approximately 23-kb-long D N A fragment on the Ti plasmid and is transmitted as such, octopine T-DNA is divided
into two adjoining sections of about 14 kb (left T-DNA,
TL) and about 7 kb (right T-DNA, TR).174.751
TL and TR
are integrated independently of each other into different
sites o n the plant g e n ~ m e . ’ ~ ’TL-DNA
.~~]
and nopaline TD N A possess a very homologous, approximately 9-kb-long
nucleotide region, on which genes 5, 2, 1, 4, 6a, and 6b are
localized (Fig. 6).[691The functions of the known genes are
compiled in Table 3. At least three of these genes (genes 1,
2, and 4) are responsible for tumor growth in infected
plants. Since all three genes are active in wild-type agrobacteria, both auxins and cytokinins are formed, which
leads to tumors with fully undifferentiated growth (cf. Fig.
4). Agrobacteria in which gene 4 is inactivated (much auxin, no cytokinin) induce root growth in the tumor, so that
gene 4 (roi gene: root inhibition gene) prevents the differentiation of roots. Shoots are induced by inactivation of
genes I and 2 (much cytokinin, no auxin). Thus, genes 1
and 2 (shi genes: shoot inhibition genes) prevent the formation of shoots.~83~85~
TR - DNA
9
tumor formation
52 L l L W ! ?gcs
LE
- - ___-___
c
-____
d
e
z?y
1
L-665
-8
LX.2
T . 2
xRB
tumor formation
nopaline T- DNA
Vir
nopaline
Ti
plasmid
Ica. 200kbl
Rep
Inc
Angew. Chem. In!. Ed. Engl. 26 (1987) 382-402
Noc
Fig. 6. Genetic organization of a Ti plasmid.
Lower part: Functionally characterized regions
of a nopaline Ti plasmid. Tra, functions for plasmid transfer between bacteria; Noc, nopaline catabolism; Rep, functions for plasmid replication
in agrobacteria; Inc, functions that confer incompatibility of two different Ti plasmids in a
single cell of Agrobacterium tumefaciens (i.e., nopaline and octopine Ti plasmids can’t exist together in the same cell); Vir, virulence region; TDNA, DNA region that becomes integrated in
the plant genome. Upper part: Comparison of
the T-DNAs of nopaline and octopine Ti plasmids. Polyadenylated transcripts are shown as
either half-arrows or bars according to whether
the direction of transcription is known or not.
Nomenclature of transcripts is according to 1691.
Black areas mark the regions of homology between the two T-DNAs. Nos, nopaline synthase
gene; ocs, octopine synthase gene; acs, agrocinopine synthase gene; Aux, genes involved in auxin
biosynthesis; Cyt, Gene involved in cytokinin
biosynthesis; RB, right border sequence: LB, left
border sequence.
387
Table 3. Functions of T-DNA genes. Functions of other genes included in
Figure 6 are still unknown.
Gene
Function
Protein
Ref.
I
2
Auxin biosynthesis
Auxin biosynthesis
Tryptophan 2-monooxygenase
lndoleacetamide hydrolase
[77]
nos
ocs
Nopaline biosynthesis
Octopine biosynthesis
Cytokinin biosynthesis
Tumor growth
Opine secretion
Nopaline synthase
Octopine synthase
lsopentenyltransferase
1791
4
5
6a
1781
1801
PI1
[781
184
6b
0’
Tumor growth
Conversion of
mannopine to agropine
1831
I841
I’
Mannopine biosynthesis
Mannopine biosynthesis
1841
2‘
1841
Accordingly, T-DNA genes are responsible for tumor
growth. They are not required, however, for the actual
plant infection or for the integration of T-DNA into the
plant genome.‘861Deletion of various regions of the Ti plasmid has shown that only two regions of the plasmid are
essential for infection and integration. One of these is the
virulence region (“vir”) and the other the “border” regions
of the T-DNA. These borders consist of two nearly identical nucleotide seq~ences,[~’.’~’
which are 25 base pairs (bp)
long and exhibit very strong conservation in all types of
T-DNA (Fig. 7). Any D N A sequences up to 50 kb long that
are inserted between these borders can be integrated into
the plant genome.6891The mechanism of this integration
process has not yet been clarified. It has been shown, however, that during an infection the T-DNA is cut out of the
Ti plasmid and circularized beyond these 25-bp-long
border sequences to form unstable intermediate products.[901
Fig. 7. Comparison of the conserved border sequences of nopaline and octopine T-DNAs. Homologous areas are boxed. RB, right border sequences;
LB, left border sequences.
The DNA of the approximately 40-kb-long virulence region is not itself integrated into the plant genome,[751but
the virulence genes are responsible for the infectiousness
of the agr~bacteria.~”’
The vir region consists of at least six
different complementation g r o u p ~ . [ ~ ’ . ~To
’ ’ determine a
complementation group, a mutation that leads to avirulence was inserted into the vir region. An attempt was then
made to restore virulence by introducing into the bacteria
a second vir region with a mutation at a different site. If
both mutations are in the same complementation group,
the bacteria remain avirulent, but if they lie in different
groups, the two groups complement each other and the
bacteria become virulent again. Each complementation
group corresponds to a single transcription unit. Two of
these transcripts are expressed constitutively; the remain388
ing four transcripts are induced only in the presence of
plant
The exact mechanism of virulence induction
is not yet fully understood. Since in nature agrobacteria
infect only injured plant cells, it was suspected that certain
substances are synthesized by these plant cells to which the
bacteria “react.” Two of these compounds, acetosyringone
1 and a-hydroxyacetosyringone 2, have so far been isolated (Fig. 8). These phenolic compounds interact with the
2
1
Fig. 8. The two compounds synthesized by wounded plant cells which activate the virulence region of Agrobacferium tumefaciens: acetosyringone 1
and a-hydroxyacetosyringone 2.
products of the two constitutively expressed virulence
genes and thus stimulate the activity of the other four vir
l ~ c i . [ ~ ’In
. ~ addition
~l
to the borders and the vir region of
the Ti plasmid, agrobacteria contain at least two chromosomal loci that are responsible for the attachment of the
bacteria to the plant cell W ~ I I S . [ ~ ~ . ~ ~ ’
3. I . 3. Agrobacterium tumefaciens as a Gene Vector
Knowledge of the basic processes involved in the infection of plant cells by Agrobacteriurn tumefaciens led to the
development of two basically different methods of introducing foreign genetic material into a plant: the use of “integrative vectors” and the use of “binary vectors.” In constructing the “integrative vectors,” advantage is taken of
the fact that the tumor-inducing genes are not required for
the actual infection and that any DNA sequence that is inserted between the two borders will also be transmitted to
the plant. In these integrative vectors, the tumor genes of
T-DNA, for example, are replaced by sequences of the E.
coli cloning vector pBR 322.[Xh1
The foreign DNA that is to
be integrated into the plant genome is simply cloned in a
pBR vector in E. coli and then inserted by homologous recombination, via these pBR sequences, into the Ti plasmid
(Fig. 9).
In the construction of the “binary vectors,” the fact is
utilized that the vir region and the “borders” can be localized on two separate plasmids without affecting the infectiousness of the a g r ~ b a c t e r i aAccordingly,
.~~~~
the T-DNA
borders are transferred to a plasmid that can be replicated
in E. coli as well as in agrobacteria. These plasmids can be
very much smaller than the Ti plasmid (about 10-20 kb),”8J
and the different cloning steps can readily be carried out in
E. coli. If the DNA to be transferred to plants has been
cloned on such a plasmid, this plasmid is transferred to a
strain of agrobacteria that already has a Ti plasmid, but
which, although it still has a vir region, contains no further
T-DNA. Agrobacteria with these two plasmids are also
capable of stably integrating the DNA lying between the
borders into the plant genome.
Angew. Chem. Int. Ed. Engl.
26 (1987) 382-402
X
t
single crossover
selection for R i f R ; CarbR : SmR
,
SpR
Fig. 9. Schematic drawing of the mobilization of a pBR vector of E . coli into
a modified Ti plasmid of Agrobacreriurn tumefaciens (modified according to
1861). Antibiotics: Carb =carbenicillin: Rif= rifampicin; Srn=streptomycin;
Sp=spectinomycin. nmmIspBR322 sequences; -=transferred
gene X ;
= border sequences.
3.1.4. Methods of Gene Transfer with Agrobacterium
tumefaciens
The prerequisite for the transfer of D N A by Agrobacterium tumefaciens in plants is the infection of plant cells by
the bacterium. The techniques for infecting plant tissue
with Agrobacterium tumefaciens are relatively easy to perform. In the simplest case, plants are injured mechanically,
e.g., with a scalpel, and a suspension of agrobacteria is applied to the site of the wound.
Better transformation efficiencies are achieved by incubating parts of the tissue with agrobacteria and then cultivating the tissue on synthetic culture media. Thus, with the
"leaf disc" method, small parts of leaves are dipped into a
bacterial suspension and then cultivated.'991 If a selectable
marker gene such as the resistance gene to the phytotoxic
antibiotic kanamycin (see Sections 3.3 and 4.1), is transferred by means of the agrobacteria, new plants can b e regenerated only from transformed cells of the leaf parts on
kanamycin-containing media (Fig. 10).
The incubation of plant protoplasts with agrobacteria
and the subsequent regeneration of the treated protoplasts
to plant tissue or intact plants, which is known as coculture, also leads to good transformation results."001
However, the advantages of the agrobacteria system, for
example, the simple transformation technique and good
transformation frequency, must be weighed against a large
disadvantage, namely, the limited host range of the agrobacterium. A large number of dicotyledonous and also
some monocotyledonous plants of the Liliaceae family, for
example, Asparagus officinalis
and Chiorophyfum capense (spider plant),~'O1hl
are susceptible to infecAngew. C'hem. Inr. Ed. Engl. 26 (1987) 382-402
Fig. 10. Production of a transgenic plant by transferring a gene, imparting
resistance against the phytotoxic antibiotic kanamycin. a) Leaf pieces after
transformation with Agrohacreriurn turnefuciens Transformed (right) and
nontransformed leaf fragments (left) are cultivated on media containing kanamycin. Only the transformed leaf pieces form shoots. b) TrdnSgenlC tobacco plant regenerated from a shoot (right) and nontransformed tobacco
plant (left) on media containing kanamycin. Only transformed plants survive.
tion. With the exception of maize,''('*] however, no susceptibility to infection by Agrobacteriurn tumefaciens has yet
been observed for the economically important Gramineae
to which all other cereal species belong.
3.2. Direct Transformation of Plant Cells with Isolated
DNA
Owing to the limited host range of the agrobacteria,
other methods were also sought at the same time for introducing genetic material into plants. The most successful
method so far has proved to be the transformation of plant
protoplasts with isolated DNA/'03.
as had already been
carried out with bacteria""51 and animal cells.l'ohl In this
approach, the transfer of the foreign D N A into the plant
cells is carried out without using a natural plant-infectious
system such as Agrobacterium tumefaciens. The cells to be
transformed are simply incubated together with the D N A
in a suitable solution and subjected to certain procedures
which have already been used in microbiological research
to transfer foreign genetic material. These procedures include, in particular, treatment with polyethylene glycol,
3 89
heat shocks, and electrical shocks (electroporation). In
electroporation, short electrical pulses are used to produce
small pores in the cell membrane through which the D N A
is able to pass into the ce11.~'04~'071
Although this method
may appear to be easy, the detailed procedure presents difficulties since the individual experimental parameters have
to be optimized for each plant species.
Direct transformation has the undisputed advantage that
it can be used with all plants from which protoplasts can
be obtained. These include some of the Gramineae as well
as many dicotyledonous plants. So far it has been possible
to introduce foreign D N A into protoplasts from rice
(Oryza satiua),"081maize (Zea mays),['091
and wheat (Triticum monococum),I"olamong others.
One disadvantage of the direct transformation of protoplasts compared with transformation with Agrobacterium
tumefaciens is the fact that the regeneration of intact plants
from protoplasts takes much longer than regeneration
from tissue parts. However, the greatest disadvantage of
direct transformation is that so far, despite extremely intensive research with cereals, only rice protoplasts could
be regenerated to intact plants.'"]
3.3. Other Methods of Gene Transfer
Since there has been only limited success in transforming and regenerating the most important crop plants by using either the direct transformation of protoplasts or by
means of agrobacteria, other transformation methods have
also been investigated, including the introduction of genetic material via phytopathogenic viruses. Although most
plant viruses belong to the group of RNA viruses, experiments have mostly been carried out with D N A viruses,
since work with RNA on the molecular biological level is
vastly more difficult than with DNA. Only two classes of
phytopathogenic DNA viruses that can be used as D N A
vectors are currently known, namely, the gemini viruses
and the caulimoviruses.[' "I By far the best characterized
DNA virus, namely, cauliflower mosaic virus (CaMV),
which belongs to the class of caulimoviruses, has already
been used for transferring genetic material into plants." "I
However, with CaMV, owing to the special genome structure of the virus, only very small pieces of DNA u p to 500
nucleotides long can be integrated into the virus genome.
In addition, the narrow host range of CaMV (only plants
of the Brassicaceae family) and the fact that virus DNA is
not integrated stably into the plant genome prevent cauliflower mosaic virus from being suitable as a universal
transformation system.
In order to avoid the difficulties in regenerating intact
plants from the protoplasts of many species, attempts have
been made to transfer D N A directly into certain tissues.
Several authors have described how, after incubation of
pollen with foreign D N A and subsequent dusting of plants
with this pollen, transformed progeny were obtained.["3.'141However, it has not yet been possible to reproduce these experiments.[' '']
Microinjection of DNA into cell nuclei using a very fine
cannula is a proven method for the genetic manipulation
of animal cells.1"61 Microinjection has also been used for
390
transforming plant ce11s.1"71 Owing to the very large plant
cell vacuole and the small cell nucleus into which the
DNA has to be injected, this method requires special
equipment and is not very efficient.
The injection of DNA into reproductive plant organs is
also carried out. A breakthrough seems to have been
achieved here with the injection of D N A into young inflorescences of rye
After the injection of a gene for
resistance to the phytotoxic antibiotic kanamycin, it was
possible, for the first time, to regenerate resistant, transformed rye plants.
4. Gene Expression in Plants
4.1. Regulatory Regions of Plant Genes
The first successful attempts to introduce foreign DNA
into plants by means of the Ti plasmid were carried out
with bacterial genes for resistance to the antibiotics streptomycin and kanamycin." '9.831 These genes were inserted
into the T-DNA and introduced in this way into the plant
genome. However, n o transcription could be detected. The
same result was obtained for an alcohol dehydrogenase
gene obtained from yeast"'"' and a 8-globin gene from
mammalian
These genes were integrated into the
genome but were not active in the plant. It was therefore
assumed that certain transcription signals specific to plants
are necessary in order to activate the genes. From the study
of animal systems, the general structure of functional eukaryotic genes was already known.["" In principle, plant
genes show the same structure.["'] A typical plant gene is
illustrated in Figure 11. The promoter is located in the
nontranscribed 5' region of eukaryotic genes. The enzyme
RNA polymerase 11, which catalyzes the synthesis of messenger RNA, binds to the promoter sequences, in particular to a region approximately 10 b p long which is rich in
adenine and thymine, the so-called TATA
Promoter sequences are therefore responsible for the correct
initiation of the transcription process. The transcription is
terminated at D N A sequences in the 3' region, namely at
the end of the gene. These terminator sequences are involved, in a way that is not yet exactly clear, in the termination of transcription and the attachment of the poly-A
sequences to the RNA chains produced (polyadenylation).11261I n order to express foreign genes in plants, attempts were therefore made to use promoter and terminator sequences of genes known to be actively expressed in
all plant tissues. The best gene for this purpose was the
nopaline synthase gene from Agrobacterium tumefaciens,
which was first isolated at that time.'791 It possessed eukaryotic promoter and terminator sequences and, as was
known from transformation experiments with wild-type
agrobacteria, it was also expressed in callus and other
plant tissues. The fusion of the promoter and terminator
regions of this gene to the bacterial gene for chloramphenicol acetyltransferase (resistance to chloramphenicol) resulted in the first chimeric gene that could be expressed in
plant cell^.^'^^' Thus, certain regulatory regions are necessary to express genes in plants (Fig. 11).
Angew. Chem. Int. Ed. Enql. 26 (1987) 382-402
5' region
translation
s t a r t codon
start
translation
stop codon
(TAA)
polyadenyiation
end mRNA
I
.
Intl
Ex1
Ex2
Int2
structural gene
(transcribed region)
enhancer
element
(organ specifity,
transcription
enhancement)
promoter
ibinding O f
RNA Po'Ymerase
t o T A T A box,light
regulation in callus)
'
3' reg'on
Ex3
L
terminator region
(termination o f
:ranscrip t ion)
Fig. I I . General btructure of a plant gene (ss-rubisco gene). Enhancer, promoter, and terminator elements regulate the expression of
the structural gene. T h e whole structural gene is transcribed. After removal of introns (Int), the exons (Ex) are spliced to build up the
regions of the m-RNA. Technical terms are exmature m-RNA. UItlID=nontranslated regions of the m-RNA: -=translated
plained in 11241.
Most of the following research focused on achieving the
expression of resistance genes to phytotoxic antibiotics in
order to use these genes as selectable markers to differentiate transformed and nontransformed plant cells. Resistance to kanamycin 3, hygromycin, and methotrexate could
be expressed in plant cells.['281Resistance to kanamycin,
for example, is based on expression of the transferred gene
for neomycin-kanamycin phosphotransferase, which catalyzes phosphorylation of the antibiotic and consequent
inactivation.
H, CNH
HO 4
0
,
e"'"".-"
OH
HO
3
NH2
Fig. 12. Chemical htructure of the phytotoxic antibiotic kanamycin 3 11291.
The hydroxyl group marked by an arrow is phosphorylated by the enzyme
neomycin-kanamycin phosphotransferase, resulting in inactivation of the antibiotic.
4.2. Differential Gene Expression
Once evidence had been supplied that foreign genes can
be introduced into the plant genome and expressed, more
complex gene regulatory mechanisms in plants were investigated on the molecular level. The development of integrative and binary vector systems (see Section 3.1.3) was
essential for this purpose. Using these systems it was possible for the first time to regenerate morphologically normal
plants after the transformation.
Two phenomena of developmental biology are currently
being investigated intensively, since their parameters can
be defined quite easily: the light-specific and the organspecific activation of certain genes.
The light-induced gene expression is being examined
primarily in genes coding for the small subunit of ribulose1,5-bisphosphate carboxylase (ss-rubisco) or for the chlorophyll a / b binding protein (CAB). Rubisco and CAB are
key enzymes in C 0 2 fixation and in plant photosynthesis.li3"'
A n y r w Cliuni I n / Ed Engl 26 (1987) 382-402
The organ-specific gene expression is also being investigated in the genes mentioned above, since, apart from their
light-dependent expression, they are only activated in tissues containing chloroplasts (e.g., leaves and stems). Another important class of differentially expressed genes
codes for the storage proteins, which are important from a
nutritional and physiological point of
Thus, the
zein genes of maize (Zea mays) or the legumin genes of pea
(Pisum satiuum), for example, are active in seeds but not in
other organs such as leaves or roots.
Below, we shall briefly describe a few attempts to elucidate gene regulatory mechanisms by the application of
D N A transfer methods, as exemplified by ss-rubisco.
The enzyme ribulose-l,5-bisphosphate carboxylase is
the most frequently occurring protein in the world, accounting for approximately 50% of all soluble plant protein. It is composed of eight identical large subunits ( M ,
56000 daltons, LS-rubisco) and eight identical small subunits ( M , 14000 daltons, ss-rubisco). Whereas the gene of
the large subunit is localized on the chloroplast genome,
the small subunit is encoded by the nuclear DNA. The ssrubisco subunits are formed in the cytosol and then transported to the stroma of the chloroplasts after proteolytic
cleavage of a transit peptide of approximately 6000 daltons. Here, they combine with the LS-rubisco subunits
formed in the chloroplasts to form the functional holoenzyme.
The enzyme rubisco can only be detected in the tissue of
illuminated plants. In the dark, the synthesis of the enzyme
stops after a short while.['321 Since no rubisco-specific
mRNA can be detected once the plants have been put in
the dark, the regulation of gene activity is effected on the
level of transcription."331 In order to transcribe a gene,
RNA polymerase 11 has to bind to the corresponding promoter sequence. It was therefore assumed that a specific
sequence exists in this promoter region which is somehow
linked to light-dependent expression. Factors that enhance
or inhibit polymerase binding to the DNA could bind to
this sequence.['341A possible way to investigate this hypothesis would be to isolate these DNA regions and fuse
them with structural regions of suitable marker genes. The
differential expression of these chimeric genes could then
be investigated in transgenic plants. In order to achieve
this, a region approximately 1000 nucleotides long that
391
was situated directly in front of the start site for transcription of an ss-rubisco gene was inserted into the corresponding position of the gene for the bacterial chloramphenicol acetyltransferase. This construction was light-regulated in transformed tobacco cells and therefore confirmed the above-mentioned hypothesis."3s1 in the meantime, similar experiments showed that a 33-bp-long DNA
region is sufficient for light-specific regulation of an ssrubisco gene in callus tissue."361 However, it was not possible to obtain strong expression of the gene with this region
alone. A further "enhancer" (see Fig. 11) with a length of
approximately 250 b p was discovered, which, independent
of its orientation but dependent on light, enhanced the activity of an otherwise constitutively acting pr0moter.['~~1
The plant-specific promoters that have been isolated to
date can be divided into three categories: (1) the light-dependent promoters, (2) the tissue-specific regulated promoters, such as those from the seed storage protein genes
in maize and wheat, and ( 3 ) the promoters of constitutively
expressed genes. The degree of expression of transferred
genes is usually lower in the transgenic (i.e., transformed)
plants than in the plants from which the genes were isolated."371 Therefore, promoters of highly expressed genes
are of special interest. One of the strongest promoters so
far used in plant transformation experiments is of viral origin. It is the constitutively acting promoter of the 35s transcripts of the phytopathogenic cauliflower mosaic virus
and seems to be about 30 times as strong as the nopaline
synthase p r ~ m o t e r . ~ 'In
~ *the
~ search for other inducible
genes, gene sequences that are inducible by heat,"391 darkn e ~ s , ~and
'~~]
have been isolated.
Promoters of dicotyledonous plants appear to regulate
the expression of genes in other dicotyledonous plants correctly. Thus, in addition to many other examples, an organ-specifically expressed seed-storage-protein gene from
soybeans, after transfer into tobacco plants, was expressed
in exactly the same time and tissue-specific pattern as in
the soybean."421
There are contradictory reports, however, on the function of promoters of monocotyledonous plants in dicotyledonous species. For example, a CAB gene from wheat was
correctly transcribed in tobacco
whereas an ssrubisco gene from wheat was not active in
In addition to the strength of a promoter, other factors
play an important role in the level of expression of a transferred gene. Thus, often not just one gene copy is integrated into the plant genome when a gene is transferred.
Indeed, more than five copies of a transferred gene have
been detected in the genomes of transgenic plants.['37' But
even when the different number of gene copies is taken
into account, a large variability in expression is observed
with different transgenic plants in a transformation experiment. This phenomenon is usually explained by what is
called the position effect.['451The integration loci of transferred DNA in the plant genome have so far shown no obvious similarities as regards the nucleotide sequence. The
integration sites seem to be entirely coincidental.['461Since
the genes are presumably integrated into DNA regions that
are transcribed to different extents, they are expressed to
varying degrees. Owing to this variability in the degree of
expression of a transferred gene, those plants that show
392
the desired characteristics to the required extent have to be
selected from a number of transgenic plants. However,
once a gene is integrated in the plant genome, it will be
stably maintained and transmitted to the progenies according to the Mendelian laws.['471
5. Aims of Applied Genetic Engineering with Plants
Some of the most important aims of genetic engineering
with plants are shown in Table 4. The aims mentioned only
represent examples, which were selected to illustrate the
many possibilities of genetic transfer to plants. This is
therefore not a complete list.
Table 4. Genetic engineering with plants-some
possible applications.
1. Generation of resistance to
2.
3.
4.
5.
herbicides
viral diseases
fungal diseases
pests
microorganisms
stress conditions
Changes in the compositions of plant substances
Modification of amino-acid composition
Modification in the composition of vegetable oils, carbohydrates, or plant
metabolites
Modification in the content and activity of enzymes important for processing, transportation, or storage of agricultural products
Changes in physioiogical processes
Photosynthesis/photorespiration
Improvement of nitrogen fixation
Genetic engineering as an aid to plant breeding
Diagnosis of plant diseases
Restriction fragment length polymorphism (RFLP)
5.1. The Production of Resistant Plants
5. I . 1. Resistance to Herbicides
The cultivation of plants that show resistance to herbicides has long been part of agricultural practice, "selective
herbicides" being in widespread use. These herbicides are
metabolically transformed in certain cultivated plants (e.g.,
by hydroxylation, conjugation with carbohydrates or glutathione, demethylation) in such a way that herbicidal action
is lost. Examples are maize with resistance to atra~ine,['~"]
wheat with resistance to diclofop-methyl,['4y1sugar beet
with resistance to p h e ~ ~ r n e d i p h a mand
) ' ~ ~cereal
~
species
with resistance to chlorsulphuron. Thus, these plants have
a physiologically based, natural herbicide resistance,
which is exploited by the farmer.
Often, several different herbicides have to be used, because this is the only way that all weeds that occur can be
controlled. By introducing a resistance to a nonselective
herbicide, the use of several herbicides may no longer be
necessary.
Plants with resistance to nonselective herbicides will be
one of the first results of genetic engineering with plants to
have a n application in agriculture. The site of action of
most modern herbicides is known and biochemically characterized. This knowledge is the prerequisite for the development of strategies for producing herbicide-resistant
Angew. Chem. Inr. Ed. Engl. 26 (1987) 382-402
plants with the aid of genetic engineering. This aim seems
to be achievable in several cases by transferring only one
or a few genes. Further reasons for intensive work on herbicide resistance are the advantages that resistant plants
offer to the farmer.
The cultivation of resistant cultures makes the preventative use of herbicides superfluous. The farmer now has to
use the herbicide only when it is needed, that is, only if the
weeds exceed a tolerable level. This is contrary to usual
practice today, where a preventative application of herbicides is commonly made.
Soil erosion can also be reduced by using herbicideresistant plants, since the later application of herbicides
makes it possible to avoid the occurrence of exposed areas.
Herbicide-resistant cultures are also particularly well
suited to the no-till (or "slot" planting) m e t h ~ d . " ~ ' ]
The possible use of herbicides according to the damage
threshold concept and the replacement of several different
herbicides by a single nonselective herbicide should result
in decreased overall use of herbicides as herbicide-resistant cultures are introduced. In addition to simplified use,
the economic advantage for the farmer is obvious.
The methods of classical cell biology (see Section 2.3)
can also be used to obtain herbicide-resistant plants. The
addition of a herbicide to the plant-cell culture medium
enables the selection of resistant calli from which herbicide-resistant plants can later be obtained.["'.
The various possibilities for obtaining herbicide-resistant plants
by means of cell biology and genetic engineering will be
exemplified here for several newer herbicides (Table 5).
Relatively new herbicides have been obtained from the
compound classes of sulfonylureas and dihydroimidazolones. These herbicides inhibit the enzyme acetolactate
synthase, which is involved in the biosynthesis of the
Herbicide
Class
Mode of action
Ref.
4
Sulfometuronmethyl
Sulfonyl
urea
Inhibition of acetolaclate synthase, thereby
blocking biosynthesis of
branched amino acids
1154, 1561
5
lmazapyr
Dihydroimidazolone
Same as for 4
(155, 1571
6
Glyphosate
Amino-acid
derivative
Inhibition of 5-enolpyruvylshikimate 3-phosphate synthase, thereby
blocking biosynthesis of
aromatic amino acids
1153, 159,
161)
7
Phosphinothricine
(glufosinate)
Amino-acid
derivative
Inhibition of glutamine
synthetase, thereby increasing the intracellular
concentration of ammonia and blocking biosynthesis of glutamine
[160, 1621
Angen. Chem i n f Ed Engl. 26 (1987) 382-402
branched amino acids leucine, isoleucine, and valine in
p]ants.l'54. '551 W'ith the sulfonylureas chlorsulfuron and
sulfometuron-methyl, resistant plants were obtained by using selection against these herbicides.""] The selection of
plants resistant to dihydroimidazolones was also successf ~ l . ~ 'These
' ~ ' plants show cross-resistance to sulfonylureas
owing to the common site of action.
I n both cases, the cause of the resistance seems to lie
either in excess production of the enzyme or in a mutation
of the gene. Both possibilities can easily be checked and
reproduced with the aid of genetic engineering after isolation of the gene for acetolactate synthase.
Genetic engineering may be used to obtain herbicide-resistant plants via the following paths (the site of action refers to enzymes in these cases):
-change in the site of action through mutation (e.g., glypho~ate['~~])
-increase in the concentration of the site of action (e.g.,
g l y p h ~ s a t e , [ l phosphinotricine"'"])
~~]
-introduction of metabolizing enzymes
I n order to choose the most favorable strategy, exact
knowledge of the site of action and the degradation pathway of the herbicide is necessary. Usually, several of the
paths indicated are successful for producing herbicide resistance.
The herbicide glyphosate prevents the synthesis of aromatic amino acids in plant cells by inhibiting the enzyme
5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase).["'l Glyphosate is one of the most widespread nonselective herbicides. Resistant plant cell cultures were obtained by selection in the presence of glyphosate. The resistance was found to be due to an increase in the concentration of EPSP synthase, which, in turn, was caused by an
increase in the number of genes (gene amplification)."5y1
For the production of tolerant plants by means of genetic
engineering, a similar strategy was followed. The gene for
EPSP synthase was first isolated from cell cultures of Perunia hybrzda. Overproduction of the enzyme in plants was
then achieved by exchanging the promoter of the EPSP
synthase gene for the constitutively acting 35s promoter of
the cauliflower mosaic virus (Section 4.2). After reintroducing this hybrid gene into petunia cells, a 40-fold increase in enzyme activity was detected in calli and is presumably responsible for the observed tolerance.['"31 Tolerant plants were obtained from these calli.
Tolerant plants may also be obtained by means of mutants that have an altered site of action for the herbicide
glyphosate. After mutagenesis of Salmonella typhimurium,
a strain was obtained whose EPSP synthase was insensitive to glyphosate. The resistance was based on replacement of one of the 421 amino acids of the Sulmone/lu enzyme." '] The mutated structural gene was isolated, fused
with regulatory elements that are recognized in plants, and
then integrated into the genome of tobacco plants. With
this bacterial resistance gene, it was thus possible to obtain
plants that are tolerant to g l y p h ~ s a t e . ~ " ~ ~
A final example is provided by the herbicide phosphinotricine,""' which, by inhibiting the enzyme glutamine synthetase, prevents ammonium ions from being metabolized
393
in plant cells. This enzyme catalyzes the following reaction:
glutamate
+ NHf + ATP
-t
glutamine + H 2 0 + ADP + P,
Resistant cell cultures were also obtained in this case.
Gene amplification and a correspondingly increased concentration of the enzyme glutamine synthetase was determined as the cause of this resistance.[’601Owing to the increased number of copies of the glutamine synthetase
gene, this cell line proved suitable for the isolation of the
Recently, the gene of a plant glutamine synthetase was
expressed in an E. coli mutant that no longer had any glutamine synthetase of its own.L1661
5.1.2. Resistance to Viruses
genes may lead to numerous scientific and practical applications, since these genes are induced by infection and the
genes for the PR proteins (about ten have been identified
to date) may have different promoters. The overexpression
of some of these proteins could activate specific protective
mechanisms.
5.1.3. Resistance to Fungal Diseases and Microorganisms
In spite of the use of crop protection agents, damage
caused by plant diseases continues to be very high. For
1976, worldwide crop losses due to disease, mainly of fungal origin, were estimated at 50 billion U.S. dollars.”721
Similarly to most of the previous examples mentioned, the
genetic expression of resistances to diseases can be based
on the utilization of natural resistance mechanisms, which
are either intensified or transferred by genetic engineering
between plants that were previously not capable of being
crossed. As is the case with virus infections, an induced
resistance also occurs in fungal and bacterial infections. It
has not yet been possible, however, to explain its cause.[’731
Plants possess a number of different defense mechanisms
against infections:
In the production of virus-resistant plants by genetic engineering, considerable successes have recently been
achieved. It was known for a long time, that, after an infection with a weakly pathogenic virus, plants do not develop
any more symptoms when they are subsequently infected
with a virulent strain. This property is used in the cultivation of tomatoes and potatoes. The molecular mechanism
of this type of “cross resistance” has not yet been clarified.
--Increased lignin formation at the cell wall and thus
greater protection against invaders
Different theories, some of them conflicting, are still waiting for experimental confirmation. Based on the consider-Formation of hydrolases, whose function may be to deation that cross-resistance could possibly be produced by
grade polysaccharides in the cell wall of invading fungi
-Formation of low-molecular-weight, secondary metabolthe expression of specific genes of a virus, Beachy et al.Ii671
transferred the gene for the coat protein of the tobacco
ites, which can have a toxic effect on infectious organmosaic virus (TMV) by means of Agrobacterium tumefaisms
ciens to tobacco plants. Indeed, a high percentage of the
-Formation of “hydroxyproline-rich proteins” of untransformed plants, which now form the coat protein of
known function, which occur in the cell wall of plants
the virus, showed no symptoms after artificial infection
and whose concentration rises appreciably following an
infection
with TMV. By contrast, a clear infection was observed in
-Formation of infection-specific proteins whose function
all the control plants after three to four days.
This very important success in producing virus-resistant
has not yet been clarified, but which probably are an implants enables specific investigations to be carried out into
portant factor in the defense against infections[i741
the phenomenon of cross-resistance, since only the expression of a single gene is required. This approach thus opens
There are a number of possible approaches to producing
up the possibility of producing virus-resistant seed.
resistance mechanisms against fungi or microorganisms by
Induced resistance is possibly also linked to the formagenetic engineering methods:
tion of “pathogen-related proteins” (PR proteins). Several
plants, after infection with viruses, viroids, bacteria, or
I. Clarification of the inducers and the mechanisms necesfungi, form specific proteins whose occurrence can be corsary for the expression of defense reactions in plants is
related with resistance to further
The connecessary. Several triggers of defense mechanisms (elicitors) have already been identified. In addition, the genes of
centration of PR proteins increases at the beginning of an
infection to u p to 100 times the initial v a l ~ e . [ ‘ ~They
~ . ’ ~ ~ ~ several important proteins whose expression rises sharply
when infection occurs have been c h a r a ~ t e r i z e d . ” 1761
~~,
usually occur extracellularly and are frequently resistant to
proteases. By crossing tobacco cultivars that form the same
2. Numerous plants contain the enzyme chitinase, whose
PR protein after induction with a pathogen, hybrids were
activity increases considerably after fungal attack.[i771
Chiobtained which form this PR protein constitutively and
tin (poly-N-acetyl-D-glucosamine)
is not found in plant
showed much greater resistance to virus infection^."^']
cells, but is an important constituent of the cell wall of fungi. Therefore, the function of the chitinase formed during
Further investigations into the still unknown functions of
an infection is presumably to hydrolyze the fungal cell
these proteins promise to provide a better understanding
walls and thus defend the plant against fungal attack. Sevof the resistance mechanism of plants.
Since gene sequences of some of these proteins have aleral chitinase genes have already been
The
ready been cloned,“701the practical use of these resistance
next step would be to use genetic engineering methods to
achieve an increase in the formation of chitinase when
mechanisms may become possible in the foreseeable fufungal attack occurs.
ture. Investigation of the promoters of the PR protein
394
Angew Chem In!. Ed. Engl. 26 (1987) 382-402
3 . Undomesticated relatives of our crops often possess important resistance genes. It has not in all cases been possible, however, to transfer these resistance genes to crops by
means of crossing experiments. Here, genetic engineering
could develop into an important tool for the breeder. One
example is the occurrence of rust in soybeans (Glycine
max). which is caused by Phakospora pachyrhizi and can
result in heavy crop losses. Although resistance genes have
not been found in corresponding breeding plants, they are
present in species of the genus Gfycine. Their transfer to
soybeans by genetic engineering methods would yield considerable economic benefits.['791
4. Strains closely related to some plant-pathogenic organisms are often no longer able to cause infection in plants.
Since it is often easier to carry out molecular biology studies in microorganisms than in plants, an attempt is frequently made to characterize the genes responsible for "recognizing" the susceptible host plants and for initiating the
plant-pathogenic reaction."x".'x'l Once these mechanisms
are understood, genetic engineering strategies can be devised to produce a resistance to these pathogens.
5. I . 4. Resistance to Pests
As in the case of the examples discussed above, plants
have presumably also developed defense mechanisms
against insects. Fragments of the cell wall (oligosaccharides) are probably the inducing factors in some
case^!''^. lXZ1
One possible defense mechanism against animal pests is the formation of protease inhibitors as a reaction to plant
These are proteins that act as potent inhibitors of serine proteases, which are involved in
the digestive process of insects."s31 The inhibitors can
therefore act as feeding suppressants. Since the substances
in question are proteins, genetic engineering should make
it possible through overexpression of the corresponding
genes to obtain increased formation of these inhibitors,
perhaps specifically in leaves or fruits. The gene structure
and sequence of an inhibitor of this type was published
re~ently.['~~l
The transfer of genes for proteins toxic to insects represents a further possibility for the creation of insect-resistant plants by genetic engineering methods. One example
of such a protein is the toxin of Bacillus thuringiensis. The
bacteria producing this toxin have already been used for
decades in the biological control of certain species of caterpillars. However, because of their slow action compared
with chemical crop protection agents, they have not been
employed on a wide scale. Recently, the corresponding
gene could be isolated and incorporated into tobacco
plants."x51 Information is not yet available, however, on
the insect resistance of a plant changed by this means. Another effective defense may consist in the formation of
low-molecular-weight substances whose concentration
rises considerably in the event of mechanical damage or
insect attack and which at least suppress feeding.['75.'".'x71
These compounds can belong to a wide range of different
chemical classes and are often secondary metabolites: phytoalexins, tannins, and even substances that function as
hormones or signals (ecdysone, pheromones, and anaAnqew Cliem. In! Ed. Engl. 26 11987) 382-402
logues of the juvenile hormone of insects). The utilization
of this defense strategy for genetic engineering purposes
appears problematic at present, since, on the one hand, numerous enzymes (and genes) are involved in the biosynthesis of the compounds mentioned, and on the other, these
compounds frequently have unpleasant side effects on humans.
5.1.5. Stress Resistance
Many environmental factors produce stress conditions
for plants: heat, aridness, excess moisture, high salt content of the soil, etc. As defense against these stress conditions, various counterreactions are initiated by the plant,
the importance of which is not yet understood. In response
to heat shock, for example, various proteins are synthesized whose precise function is not yet known. They are,
however, certainly involved in a protective mechanism and
are partly transported to the chloroplasts['Xx1
and incorporated there.
Under other stress conditions, the formation of the same
heat shock proteins frequently occurs, but o n a smaller
scale.[1x91
Stress-related fluctuations in the amino-acid content also occur; for example, a sharp increase in the proline content of plants is often
The genetic engineering approach to increasing stress resistance could lie in the identification and isolation of the
most important genes involved in protection against stress
conditions and the subsequent transfer of them from resistant to sensitive plants.
5.2. Changes in the Composition of Plant Substances
Several of the examples discussed in Section 5.1 in relation to the creation of resistance in plants already entailed
the increased formation of specific plant substances. Further possibilities, which result in an "improvement" in the
characteristics of a specific crop, will now be mentioned.
This approach can be used, for instance, to influence both
the production and processing of foodstuffs as well as
plant raw materials used in industry.
Because of their high protein and amino-acid content,
the seeds of cereals and vegetables constitute an important
nutritional factor for humans and animals. However, the
composition of the substances contained in this important
food source does not always conform to requirements,
since essential amino acids are often not present in the
necessary amounts. Maize, for example, only has a low lysine and tryptophan content. For this reason, soybean
flour is generally added when using maize as animal feed.
Soya flour, however, is deficient in the amino acid methionine. A long-term goal of genetic engineering might be
to change the amino-acid content of the storage proteins of
important plants in such a way that a balanced composition is obtained. Using conventional breeding methods it
has already been possible to develop maize varieties with a
higher lysine content. However, this also resulted in undesirable features such as lower crop yields, lower protein
content, and susceptibility to disease.""' The genetic engineering approach to such a project is not simple, since
large gene families are generally responsible for the forma39 5
tion of storage
The increased expression of a sin- proteins.
.
gle gene therefore does not appreciably affect the aminoacid content. Genetic engineering can be used, however,
either to influence the metabolism in the seed in such a
way that the content of a specific free amino acid is greatly
increased or to introduce several, suitably altered genes.
Other possibilities of improving the quality of plant seeds
have been
Apart from genetic changes in plant products used as
food, the increased formation of substances of medicinal
or industrial importance is doubtless also important.[’y31
An example is the genetic engineering of oil plants (e.g.,
soybean, rape, sunflower) with the aim of achieving a
change in the oil composition. In principle, by transferring
specific genes, it should be possible to obtain, within certain limits, uniform oil fractions which could be tailormade for industrial application. For this purpose, however,
much intensive basic research in the fields of biochemistry
and molecular biology is still required. The new possibilities opened u p for both agriculture and industry certainly
appear to justify this kind of commitment.
5.3. Influencing Physiological Processes in Plants
Ribulose-l,5-bisphosphatecarboxylase (rubisco) is a key
enzyme in photosynthesis, since it is responsible for the
fixation of carbon dioxide from the air (see Section 4.2). In
addition to carbon dioxide, this enzyme also transfers oxygen to ribulose-l,5-bisphosphateand thus gives rise to the
formation of undesirable side products. This reaction,
known as photorespiration, diminishes the efficiency of
photosynthesis.
Because of the fundamental importance of photosynthesis and photorespiration to all plants, there have been numerous attempts to use mutagenesis to create enzymes that
exhibit a lower degree of photorespiration. Up to now,
however, no commercially utilizable results have been obtained. The chances of reducing photorespiration have recently improved considerably, since the genes for the two
subunits of rubisco have been isolated from photosynthetic
bacteria and transferred to E. coli. An active enzyme was
then formed in E. C O I ~ . ~ ’Furthermore,
~~]
the three-dimensional structure of the enzymes from the photosynthetic
bacteria Rhodospirillum rubrumilg5.”and Alcaligenes eutropublished.
p h ~ s ~ were
” ~ ~recently
]
Moreover, gene transfer in E. coli has been used to
create a hybrid, active enzyme of rubisco consisting of the
large subunit from cyano bacteria and the small subunit
from wheat.[’yh1It should now be possible, by means of
site-directed mutagenesis with synthetic genes, to change
almost any amino acid and subsequently study the effect
on enzymatic activity. The great importance of an enzyme
that, by means of an increased rate of fixation of carbon
dioxide, gives rise to more efficient photosynthesis doubtless does not need to be emphasized.
5.4. Influencing Nitrogen Fixation
It should first be noted that nitrogen-fixing plants are
unlikely to be produced before the turn of the millennium.
396
Table 6. Some applications of genetic engineering to nitrogen fixation
a) Fixationby free-livingmicroorganisms
Improvement of fixation reactions
b) Fixation by symbiotic microorganisms
Increase of efficiency (number of nodules, regulation)
Host-range extension of rhizobia among leguminous plants
Transfer o f the capability to undergo symbiosis with other plants
c) Nitrogen fixation in piants
However, this represents the “maximum requirement.” A
number of smaller steps could certainly be achieved in the
next few years. Various possibilities for influencing nitrogen fixation are listed in Table 6. Numerous microorganisms in the soil, such as Azotobacter, Azospirillurn, Klebsiella, and Rhizobiurn, are capable of fixing nitrogen from
the air and forming ammonia. As a result of their metabolism, these bacteria contribute to nitrogen fertilization. The
quantity of nitrogen bound by this biological fixation process is considerable. Worldwide, it amounts to many times
the quantity of industrially produced a m m ~ n i a . ~ ” ”
Nitrogen-fixing microorganisms can be divided into two
large groups: free-living fixers and symbiontic fixers. Only
in partnership with plants, frequently a leguminous crop,
are the latter able to fix nitrogen. In this partnership, the
microorganisms supply the product of their nitrogen fixation to the plant and in exchange receive from the plant
assimilation products of photosynthesis. The proportion of
symbiotically fixed nitrogen is far higher than the nitrogen
combined by free-living microorganisms.
Structure and function of the genes involved in nitrogen
fixation have up to now been investigated most thoroughly
for Klebsiellu pneurnoniue. a free-living fixer, and Rhizobiurn meMoti, which forms a symbiosis with alfalfa.[""^ l”yl
In K . pneurnoniae, the enzymes specifically involved in
nitrogen fixation are coded by a block of 17 adjacent
genes. The large number of genes involved in this process
is one of the difficulties that has to be overcome for their
transfer to plants. The control regions (promoters) of all
these genes have to be changed and adapted to the conditions in the plant. Additional difficulties arise owing to the
oxygen sensitivity of nitrogenase, the enzyme that reduces
nitrogen, and the high energy requirement of the whole
process of nitrogen fixation and assimilation.
When sufficient ammonium ions are present in the surrounding environment, the energy-consuming process of
nitrogen fixation by free-living fixers is discontinued, since
the requirement can now be met by uptake of ammonium
ions. This regulation is achieved by specific genes, which,
in the presence of ammonium ion, prevent the formation
of the nitrogen-fixing enzymes. For agricultural application, strains in which this regulation is no longer operative
can represent an important contribution toward improving
the nitrogen supply.
The symbiotic nitrogen fixation of rhizobia takes place
after formation of typical root nodules (Fig. 13). Development of a symbiosis depends on numerous complex steps:
mutual recognition between microorganism and plant, infection of the root, formation of nodules, and finally the
actual nitrogen fixation and exchange of molecules between plant and microorganism. About 40 genes of the
plant are involved in the formation and functioning of the
nodules.[2001I n principle, each of the steps mentioned opAngew. Chem. Inr. Ed. Engl. 26 (1987) 382-402
fewer than ten plant cells.[2n31There are numerous possible
applications of this technique in plant breeding: rapid examination of the progeny of crosses and of unknown genotypes for the presence of specific genes, characterization of
certain varieties by identifying characteristic DNA restriction patterns (RFLP: restriction fragment length polymorphi~m),[’’~~
information on the relationship between specific varieties, and tests for the presence of viruses in seed
or breeding material. A comparison between such tests and
the corresponding serological tests revealed that the results
obtained by hybridization with D N A are more reliable and
informative in determining the presence of viruses.[20s1
6. Problems and Risks
Fig. 13. Symbiotic nodules on the roots of a broad bean plant as a consequence of infection with rhizobia. Fixation of nitrogen takes place in these
nodules.
ens up a number of possibilities for influencing the overall
process by means of genetic engineering. In the mutual recognition and formation of nodules, for instance, low-molecular-weight substances (flavones), which are excreted by
the plant, are involved.[2n” By influencing the type and
quantity of the flavones that are biosynthesized and distributed, it may be possible to influence the number of nodules formed and thereby to achieve an increase in nitrogen fixation. The discovery of soybean plants that in
comparison to the wild variety, possess a higher number of
nodules, supports this possibility.[2n21
The various symbiotic rhizobium strains exhibit a pronounced specificity for certain host plants. Marked differences arise in this regard in the growth behavior and fixation rates of the various bacterial strains. A realistic goal
would be to widen the host spectrum of the rhizobia involved. This could result in a marked improvement in the
supply of nitrogen to leguminous plants that u p t o now
have only been capable of forming a symbiosis with slowgrowing rhizobia.
Several of the plant gene products that are involved in
the formation and functioning of nodules also occur in
plants not able to form a symbiosis. It may be easier to use
genetic engineering methods to enable these plants to form
a symbiosis with bacteria than to transfer to them the ability to fix the nitrogen themselves.
5.5. Genetic Engineering as an Aid to Plant Breeding
Molecular biology methods can be used to supply precise information on the presence of specific genes in
plants. The sensitivity of these techniques has been increased to the extent that they can be carried out with
Angen Chem Inr Ed Engl. 26 (19871 382-402
The objectives of genetic engineering with plants, described in Section 5, are unlikely to cause any risk. Serious
objections can hardly be raised against the development of
resistant or high-yield varieties, since this has been the aim
of conventional plant breeding methods for many decades.
In the case of herbicide resistance, however, a discussion
would appear to be useful in order to prevent misunderstandings. In addition to the hypothetical risks that might
arise as a result of modifying plants by genetic engineering
methods, herbicide resistance will also be discussed here.
6.1. Risks Resulting from Plants Modified by Genetic
Engineering
Genetic engineering permits the isolation of genes from
widely differing organisms and their incorporation in
plants. With present methods it is only possible to transfer
specific genes whose function is generally known. The following hypothetical sources of risk exist:
1. Development of rampant-growing plants with uncontrolled propagation (weeds);
2. Transmission of the transferred genes from crops to
weeds by cross-fertilization;
3. Formation of toxic substances in plants modified by genetic engineering.
First, the release of plants modified by genetic engineering is far less problematic than the release of modified microorganisms or animals. Undesired plant growth can after
all be controlled by various means (herbicides, mechanical
weed control), which belong to the daily activities of the
farmer.
Second, prior to a field experiment, plants modified by
genetic engineering are first tested under laboratory conditions and in greenhouses. During this phase, any undesirable or harmful characteristics in a plant can be identified.
Third, plant breeders have for centuries achieved good
results with widely differing crosses, including some that
would never have come about without human assistance.
In such experiments, tens of thousands of different genes
are recombined. This has not u p to now resulted in any
serious problems. The methods of cell biology have also
been used to create gene combinations in new plants,
391
which would never have resulted under natural conditions.
Here one may call to mind the protoplast fusions between
tomato and potato (cf. Section 2.2.
Even a critical examination gave no indication here of any possible risks. It
is therefore inconceivable that the transfer of a few genes
by genetic engineering methods might give rise to a dangerous situation.
Fourth, characteristics such as rapid, uncontrolled
growth (weed formation) can be obtained only by means of
a large number of genes which must be suitably adapted to
one another. For this reason, it is also inconceivable that
new weeds could be created unintentionally through genetic engineering with plants.
Fifth, characteristics unintentionally transferred to other
plants (e.g., weeds) can only be preserved and dispersed if
the necessary selection pressure is present. If the customary rules of good agricultural practice are observed (e.g.,
rotation of crops and herbicides), this is unlikely to occur
when an unintentional transfer of individual genes takes
place. Moreover, only in a few instances can crops cross
with closely related weeds.
Sixth, in conventional breeding methods the possibility
cannot be entirely ruled out that the concentrations of
toxic substances peculiar to plants will be greater after a
crossing experiment. One example of this is the development of varieties of potato possessing an unpleasant, burning taste due to an increased concentration of glycoalkaloids.[2"h1This was not a very surprising result, since many
wild varieties of potato, which are frequently used in the
development of new varieties, already contain high concentrations of these glycoalkaloids. Here, too, similar effects are unlikely to be obtained by selective transfer of a
few genes. Moreover, toxic side effects can be easily identified by appropriate analyses and test methods.
In summary, it is difficult to recognize any specific or
potential risk due to plants modified by genetic engineering.
6.2. Release of Plants Modified by Genetic Engineering
In the Federal Republic of Germany, organisms modified by genetic engineering may not be tested or released
under field conditions. On request and after a hearing of
the "Central Committee for Biological Safety," the Federal
Health Department can, in agreement with the responsible
federal biological institutes, allow
As far as
we know, n o such experiments have hitherto been carried
out.
It was recently reported in the press that in the USA two
field experiments with tobacco plants modified by genetic
engineering were approved by the responsible authorities.
In one case, the plants contain an additional gene from
yeast (the alcohol dehydrogenase gene) and, in the other
case, they contain the toxin gene from Bacillus thuringienxis. The aim of the experiments now being conducted is to
show that under field conditions an additional gene does
not cause a change in growth behavior. In the latter case,
resistance to caterpillars is also being tested under field
conditions.
Within Europe, the preconditions and rules for carrying
out field experiments on organisms modified by genetic
398
engineering still vary considerably. From the viewpoint of
both safety and competitiveness of the companies and research institutes involved, a uniform ruling, at least within
Europe, would be extremely desirable.
6.3. Herbicide Resistance
The most important advantages resulting from the cultivation of herbicide-resistant crops are again summarized
below:
-Use of herbicides only when required, and thus no further need for preventive application
-Increased economic efficiency for the farmer
-Increased use of ecologically favorable (more rapidly
degradable) herbicides in place of older products
-Reduced soil erosion by preventing the appearance of
exposed areas
-Replacement of herbicide mixtures by a single nonselective herbicide
These advantages are opposed by a number of potential
disadvantages and apprehensions:
--Creation of ecological problems owing to increased use
of herbicides when herbicide-resistant crops are grown
- Emergence of herbicide-resistant weeds
-Further decrease in the number of different varieties
The discussion on herbicide-resistant plants[2('x1
should
not overlook the fact that many years of experience have
already been acquired in the use of selective herbicides in
agriculture. The resistant crops possess metabolic pathways that render individual herbicides ineffective. A natural herbicide resistance thus exists, which applies only to
the individual crop. The use of these selective herbicides
on crops with natural resistance does not differ from the
application of nonselective herbicides and the corresponding herbicide-resistant crops created by genetic engineering.
Because of the possibility of using herbicides only when
required, i.e., not until weed infestation has gone beyond
an acceptable level, and because of the replacement of several selective herbicides by a single nonselective one, the
use of herbicide-resistant plants should result in less use of
herbicides.
In the development of resistances to herbicides with
long-lasting action, it cannot be ruled out that over a period of years the herbicide will accumulate in the soil and
thus cause ecological problems. The trend observed at all
research institutes toward the development of resistances
only against ecologically favorable herbicides is therefore
to be welcomed. This shows that the specific properties of
the respective herbicide are the decisive factors in the discussion on herbicide resistance (degradation, environmental behavior, toxicity, possibility of accumulation, etc.). A
separate discussion on each herbicide is therefore necessary.
The possible formation of herbicide-resistant weeds is
probably not a major problem in the use of nonselective
herbicides. For example, in spite of the fact that the herbiAngew. Chem. I n f . Ed. Engl 26 (1987) 382-402
cide glyphosate has been used for over twelve years, no
problems have yet arisen as a result of the development of
resistances. In the event that herbicide-resistant weeds d o
indeed emerge, these can be controlled at any time by herbicides with other sites of action.
Herbicide resistance in a specific plant variety will probably be as important a characteristic, as, for example, resistance to disease, hardiness, or high yield. None of these
characteristics alone, however, is a decisive factor in seriously eliminating other varieties of crops. Therefore, herbicide resistance is unlikely to reduce the existing number of
varieties to a greater extent than the other breeding objectives mentioned. In this connection, it should also be emphasized that, as regards the modification of plants by genetic engineering, the preservation of as many different varieties as possible is of very considerable importance, since
only in this way is a wide range of natural genes accessible.
Herbicide resistance is probably one of the most easily
attainable objectives in genetic engineering with plants.
The experience gained here can subsequently be utilized in
solving more complex problems, such as the creation of
resistances to virus infections, animal pests, or fungal diseases.
The further development of the biochemistry and molecular biology of plants will result in the elucidation of the
molecular mechanisms of previously unexplained plant
diseases, for example, fungal and viroid diseases. AIthough the complete sequence of the infectious RNA of a
number of viroids has been determined,[2091
there is still no
explanation for the mechanism and causes of plant damage.
The efforts being made in the genetic engineering field
will result in more resistant and productive plants. These
plants will be available to the farmer in the 1990s. On the
one hand, this will enable an important contribution to be
made toward solving the problem of world famine; on the
other hand, the production of tailor-made raw materials
for industrial application will open u p additional markets
for agricultural products in industrialized countries. It may
even be possible to use wild plants to create new types of
crops with advantageous characteristics (e.g., salt tolerance, high biomass production, good protein quality in the
harvested crop) by employing breeding and genetic engineering
Close cooperation between biochemists, cell biologists,
molecular biologists, plant pathologists, and plant breeders is urgently necessary in order to obtain more productive crops with high adaptability and less susceptibility to
disease.
7. Summary and Prospects
Our thanks are due to H . Drager for preparation of the
light-optical micrographs. We wish to thank B. Diehls and R .
Sfehrfor typing the manuscript and F. Sponemann f o r drawing the figures.
A number of crop plants can be regenerated from single
cells or protoplasts in any desired quantity by means of
cell culture techniques. Such regenerating cell cultures are
used both for selection of mutants and for D N A transformation experiments. D N A transfer by means of engineered Ti plasmids and Agrobacterium tumefaciens has become an established technique for a rapidly growing number of dicotyledonous plants.
Proof of general transformability by agrobacteria is still
lacking in cereals. Alternative methods of DNA transfer
are thus being developed. Direct gene transfer in protoplasts has proved to have universal applicability. Transformed cereal cell cultures are accessible from DNAtreated protoplasts by routine methods. The dogma that
cereal protoplasts are incapable of being regenerated has
been refuted by the successful plant regeneration from rice
protoplasts.
Major efforts are being undertaken to render gene transfer independent of cell culture methods. For example, a n
attempt is being made to transfer foreign D N A into reproductive cells both by pollen transformation and by direct
DNA injection into young inflorescences.
The number of isolated plant genes is rapidly increasing.
By analysis of the regulation sequences of these genes,
DNA segments are becoming accessible that are responsible for tissue-specific or environmentally stimulated expression of genes in plants.
The first genes that may be of economic importance to
plant breeding and crop protection have already been
transferred to plants. These include genes that impart to
the transformants resistance against viruses, insects, or herbicides.
A n y r r . Cbem. lnt Ed Engl. 26 (1987) 382-402
Received: January 26, 1987 [A 618 IE]
German version: Angew Cliem. YY (1987) 392
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