NATURA MONTENEGRINA, PODGORICA, 6:137-149
INFLUENCE OF TRANSGENIC PLANTS ON ENVIRONMENT
Danko O B R A D O V I Ć 1
1
University of Montenegro, Faculty of Sciences, Department of Biology, P. O. Box 211, 81000 Podgorica,
Montenegro
Key words:
transgenic plants,
commercial use
Klučne riječi:
transgene biljke,
komercijalna
primena
Synopsis
In 1983, transgenic plants were introduced by four groups
of authors. However, commercial use of transgenic plants dates
from 1996. Since its first application, cultivation area of
transgenic crops around the world has been permanently
increasing. Transferring of a transgene from microorganisms,
animals, and distant plants, transgenic plants can obtain novel
treats that would probably never be introduced by conventional
breeding. New treats that plants can get in nature by
spontaneous mutations and breeding occur slowly. During the
evolution organisms have been accommodated to changes in
their environment, in contrast to revolutionary changes
introduced by molecular biology methods applied on production
of transgenic crops. This can make disbalance among organisms
and cause some ecological problems. Such problems can be
solved by regulations which forbid use of transgenic crops
without vigorous assays for safe application.
Sinopsis
UTICAJ TRANSGENIH BILJAKA NA ŽIVOTNU SREDINU
Četiri grupe autora su 1983. godine po prvi put dobile transgene
biljke. Ipak, komercijalna primena transgenih biljaka datira od 1996.
godine. Od svoje prve primene, obradiva površina pod transgenim
kulturama širom sveta je u stalnom porastu. Transferom nekog gena iz
mikroorganizama, životinja i nesrodnih biljaka, transgene biljke mogu
dobiti nove osobine koje verovatno ne bi nikada dobile konvencionalnim
ukrštanjem. Nova svojstva koja biljke mogu dobiti u prirodi spontanim
mutacijama i ukrštanjem događaju se sporo. U toku evolucije organizmi
su se prilagođavali na promene u njihovoj okolini za razliku od
revolucionarnih promena koje su uvele metode molekularne biologije
primenjene u proizvodnji transgenih kultura. Ovo može dovesti do
disbalansa među organizmima i uzrokovati neke ekološke probleme.
Takvi problemi mogu biti rešeni propisima koji zabranjuju primenu
transgenih biljaka bez prethodne temeljne provere bezbednosti primene.
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INTRODUCTION
Plant breeding has been a basic method for obtaining plants of particular traits
for several thousand years. People have used this method to improve some
characteristics of plants (sweeter and larger fruits, aridity and disease resistance,
faster growth) in spite of the fact that they did not understand breeding process and
its background. In 1865, Gregor Mendel showed that the inheritance of traits follows
particular regularities, but the significance of Mendel's work was not recognized until
1900. Traditional plant breeding is very difficult and time and labor consuming
process. By artificial plant crossing, plant breeders try to obtain plants of desirable
traits. To achieve this goal, they have to travel all around the world to find plants of
particular traits that are suitable for breeding. Variations of treats among plants arise
as result of mutations. However, these mutations, and as well as variations, are of
limited number. In 1926, Hermann Joseph Muller found that X-rays irradiation can
induce mutations (in 1946 he was awarded Nobel Prize for this discovery) (M u l l e r ,
1926). After this discovery, it was found out that irradiation of seed can greatly
increase amount of mutations in next generation. Since the end of World War II,
induced mutation have been widely applied in plant breeding. Such method was
named mutation breeding. Mutations, and thus, variations of traits, can be induced
with ionizing radiation (X-rays, gamma rays, alpha particles, beta particles, neutrons,
protons) or chemical agents (sodium azide, ethyl methanesulphonate). This is a way
to increase variability and to get plants of desirable traits that are not available in
nature. This method is named mutation breading. It became popular after World War
II, and it is popular at the present time as well.
Classical plant breeding uses deliberate interbreeding of closely or distantly
related species to produce new crops with desirable properties. Breeding is possible
between plants within the same species, genus and, less commonly, between plants
of different genera. Plants that are evolutionally distant have larger amount of different
properties. Thus, amount of plant property variations that are available for breeding
and obtaining of new crops is limited, because breeding between plants that are
evolutionally distant is not possible. Even application of mutation breading produces
new crops with limited variations in treats. Additionally, induced mutations which are
obtained by mutation breeding produce new treats that cannot be predicted, because
mutations are random events. All these features of classical and mutation breeding
limit their application.
In 1983, four groups of authors, almost simultaneously, introduced a novel
method for production of new crops. They worked independently, and three of them
announced their work at a conference in Miami, USA in January 1983 (Framond et al.,
1983; Schell et al., 1983; Fraley et al., 1983). Their researches enable gene transfer
from bacteria into plants. These works were published in scientific journals as well
(B e v a n , et al. 1983; H e r r e r a - E s t r e l l a et al., 1983; F r a l e y et al., 1983). The
fourth had transferred a plant gene from one species into another species. They
announced their research at a conference in Los Angeles, USA, in April 1983, and
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later it was published in a journal (M u r a i et al., 1983). Plants obtained this way are
transgenic plants. This method enables gene insertion of one or more genes from a
plant which can be evolutionally very distant into another plant. The genes can be
transferred from any organism (animals, bacteria, viruses etc.) into a plant. This
makes possible production of novel crops with new treats which in practice can never
be obtained by breeding. Very important feature of this method is possibility to predict
novel treats of transgenic crops. It enables to skip millions of years of evolution that
would be necessary for nature to produce plants with so different treats. This skip
made impossible accommodation between transgenic crops and environment, and
develops concerns regarding the application of transgenic plants. However, transgenic
plants can provide substantial benefits: better nutritional characteristics, increased
productivity, longer shelf life, environmental tolerance, pest and disease resistance,
etc.
Production Process of Transgenic Plants
Molecular biology methods enable gene transfer and production of transgenic
plants. These methods are commonly named genetic engineering. Actually, all
molecular biology methods that can be involved as tools for changing of genetic
constitution are named genetic engineering. Production of transgenic plants consists
of: isolation of a gene from donor organism, insertion of the gene into plant cell,
obtaining of whole plants from transformed cells grown in tissue culture, plant
breeding and testing.
Isolation of a Gene from a Donor Organism. This is the most difficult step in
production of transgenic plants. This step is mostly based on fundamental research in
determination of structure and function of donor organism genes. It is very important
to locate and determine the role of a gene, its function and treats that the gene is
responsible for. Identification of expression mechanisms of the gene and its influence
on other genes, metabolic pathways of gene products is very important. Procedure of
this step depends very much on results of such researches. In contrast to this step, all
other steps are processed under well determined procedures. Isolated gene that is to
be inserted into a plant is termed transgene.
Insertion of the Gene into Plant Cells. Currently, there are two types of
vehicles which can be used for gene insertion into plants. The most often used vehicle
is Agrobacterium tumefaciens (Figure 1). A. tumefaciens is a gram negative, rod
shaped, bacteria, which is the causal agent of Crown Gall disease (the formation of
tumors) of dicots. It has been used as gene transfer vehicle into dicots, and just
recently into monocots. A. tumefaciens contains chromosomal DNA and plasmid
(known as the Ti-plasmid for tumor-inducing plasmid). In order to be virulent the
bacterial Ti-plasmid has to contain a small segment of DNA termed T-DNA
(transferred DNA), and vir (virulence) genes that direct the infection process. A.
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tumefaciens are attracted to wound sites of a plant by chemotaxis. This is a response
to the release of some common root components (sugars and particularly phenolic
compounds such as acetosyringone). Acetosyringone activates vir genes on the Tiplasmid. Activity of this gene leads to the production of opine permease, that is
inserted in the bacterial cell membrane for uptake of compounds (opines) that will be
produced by the tumors, and production of an enzyme restriction endonuclease that
excises part of the Ti-plasmid termed the T-DNA. Excised T-DNA released by the
bacterium enters the plant cells and in an unknown way integrates into the
chromosome. T-DNA dictates change in the functioning of those cells which cause
formation of tumors.
Figure 1. Simplified organization of a sequence constructed for insertion into a plant genome.
Insertion of a transgene into plant cell genome does not mean that the gene will
be expressed. Unexpressed gene will not code any product (protein), and thus, will
not provide any new treat to the plant. In order to provide gene expression a promoter
DNA sequence has to be inserted together with the gene, and as well as a termination
sequence (terminator). Terminator signals that gene coding sequences ends, and that
transcription of the DNA has to be ended. A given promoter and terminator can be
combined with various genes (Figure 2). The degree of gene expression, the region of
plant body where the gene will be expressed, and the plant life cycle stage depend on
which promoter is applied.
A marker gene has to be inserted together with the transgene. It is necessary for
selection of cells which received and expressed a transgene. It encodes a protein
which provides resistance to an agent (usually antibiotics or herbicides) which is toxic
to plant cells. Thus, cells that received transgene will be resistant to the toxic agent
(they will survive), and all other cells will die.
Figure 2. Bacterium Agrobacterium tumefaciens.
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Ti-plasmid used in gene transfer process is not a wild type plasmid, but plasmid
especially constructed for this purpose. T-DNA sequence which causes tumor growth
is deleted and just its border sequence is retained. A. tumefaciens with such Tiplasmid is not a pathogen. It retains ability to insert the plasmid into the cell, but the
plasmid cannot cause tumor growth. Transgene is to be inserted into the Ti-plasmid,
instead of removed T-DNA sequence and transferred into a plant cell. The cell is
maintained in tissue culture and transgene is incorporated into the plant chromosome.
The cells in tissue culture are grown in media containing nutrients and hormones
necessary for cell growth.
Obtaining of Whole Plants from Transformed Cells Grown in Tissue
Culture. After the treatment with A. tumefaciens, it is necessary to select transformed
cells. The selection is carried out by replacement of standard media with selectable
media, which contain an agent (usually antibiotic or herbicide) toxic for the cells.
Marker gene inserted into plant chromosome together with the transgene provides
resistance to the toxic agent. Thus, the cells that received marker gene will survive in
the tissue culture, and all other will die. Survived cells will be maintained to produce
an embryo and eventually whole plant.
Plant Breeding and Testing. A limited number of plant lines is possible to use
in effective gene transfer. They are usually not elite lines. To obtain an elite line with a
transgene it is necessary to breed transgenic plant with an elite line. After the
breeding process, a transgenic plant containing at least 98% of elite genes is
produced. The next step is testing of the plant (expression of transgene, stability of
inheritance of the treats, unexpected features of the plant etc.).
Another method for insertion of a transgene is the gene gun. All steps in
production of transgenic plants applying gene gun are pretty much the same as with
A. tumefaciens, except gene insertion. Gene gun method applies microscopic gold
particles to deliver a transgene into the plant cell nucleus. Golden particles coated
with a large amount of transgenes together with a marker gene are accelerated with
air pressure and shoot at tissue culture cells. The golden particles will pass into the
cell nucleus. Coated DNA will be dissolved and inserted into the chromosome. The
method with A. tumefaciens is more effective than gene gun method, but it cannot be
applied to every plant species. A. tumefaciens is ussualy applied with dicots, and
gene gun with monocots.
Transgenic Plants Benefits
New treats introduced by insertion of transgenes into plants can provide a
number of benefits: pest and disease resistance, herbicide tolerance, better nutritional
characteristics, increased productivity, longer shelf life, environmental tolerance, etc.
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Pest and Disease Resistance. Insertion of a transgene (isolated from a soil
bacterium Bacillus thuringiensis) coding a crystalline (Cry) protein can introduce pest
resistance to transgenic plants. In the intestine gut, the protein is broken down to
release delta-endotoxin that creates pores in the intestinal lining of pets. This creates
ion imbalance, dysfunction of digestive system, and death of the insect in a few days.
Product of Cry gene is considered safe for human and birds, and less harmful for nontarget insects than other insecticides. Insecticides based on Cry gene have been used
for long time, but in this case they are produced by plant in the plant body. Thus,
insects eating such plant will eventually die. Plants containing this transgene are
named Bt (Bacillus thuringiensis) plants. Bt plants available by now are: Bt cotton (to
control European corn borer, Southwestern corn borer, and corn earworm), Bt cotton
(to control cotton bollworm and tobacco budworm), and Bt potato (to control Colorado
potato beetle). Bt plants can decrease application of insecticides, and so, the
surrounding environment is no longer exposed to large amounts of harmful insecticide.
Time and labor saving is achieved too.
Transgenic papaya and squash carrying virus coat protein gene are resistant
to virus. This gene produces virus coat protein before an infection. After the infection,
plant cells will not produce this protein because of co-suppression (plant cell
mechanism which suppresses production of the viral protein), and virus cannot
replicate.
Herbicide Tolerance. Weeds can significantly decrease crop yield, and so
herbicides are widely applied for weed control. Sometimes farmers apply more than
one herbicide because they are specific for a particular weed, and they are applied at
particular growth stage. Herbicides are long lasting and can persist in the soil for
years. Transgenic herbicide tolerant plants have a transgene providing resistance to
new herbicide that can kill all kinds of plants (including weeds). These herbicides
break down in the soil quickly. So far, there are two herbicides that are used with
herbicide tolerant plants: Liberty (glufosinate) and Roundup (glyphosate). These
herbicides break down quickly in contrast to conventional herbicides that can remain
in the soil for more than a year, and thus prevent farmers to plant crops sensitive to
them in future. Fast degradation of these new herbicides protects environment of large
scale contamination, which can be common in the case of conventional herbicides. An
example of transgenic herbicide tolerant plant is Roundup Ready soybean.
Long-Lasting Products. Some plants (strawberries, tomatoes, pineapples,
sweet peppers and bananas) are genetically modified to produce less enzyme that
cause products to rot. These plants can remain firm and fresh for long time. The first
long-lasting plant modified plants was tomato, which came on the market in 1994.
Such tomato can tolerate a longer transport time and it can be allowed to ripen in the
sun before picking - resulting in a better tasting tomato.
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Improvements of nutritious characteristics of the plants. Nutritious
characteristics of a plant can be improved by inserting a transgene. An example is
golden rice. It contains beta-carotene which is converted into vitamin A in the body.
For the golden rice to make beta-carotene three new genes are implanted: two from
daffodils and the third from a bacterium.
Transgenic Plants Risks and Concerns
Transgenic plants can provide many benefits, but in the same time some risks
and concerns. Insertion of transgenes into plants, which introduces novel treats to the
plants, can change interactions among plants and animals. This can cause ecological
and other kinds of problems.
Gene Transfer from Transgenic Crops to Their Wild Relatives. Many crops
have wild relatives which can be crossed by pollination. Thus, there is real danger of
gene transfer from crops to wild population including weeds (K a i s e r , 2001; P o p p y
and W i l k i n s o n , 2005). This possibility is supported by researches. Wild population
would receive novel treats that can make them resistant to many factors. This is
especially critical for weeds. Weeds resistant for herbicides, plant diseases, climatic
factors etc. would be a nightmare for every farmer. Transgene transfer to wild
relatives that are not weeds is not desirable either, because this could change natural
balance in the wild nature and cause ecological problems.
Crop to Crop Gene Flow. Gene transfer is possible from transgenic to
conventional crops (H a l l et al., 2000; R i t a l a e t al., 2002).The transfer is processed
by pollination, and pollen can be carried by wind and insects. After this gene flow,
farmers that plant conventional crops will have transgenic even if they dislike them.
Long-term application of transgenic crops and gene flow can lead even to complete
loss of conventional crops. This is the most likely to occur in the case of highly
outcrossing plants in contrast to highly self-pollinating plants.
Antibiotic resistance. All transgenic crops in the process of their production
have to contain a marker gene which is inserted to insure that a transgene has been
inserted into the plant. It is usually an antibiotic resistance gene. There is a concern
that bacteria, which inhabit intestine of human and animals, could be transformed by
transfer of a DNA fragment carrying the antibiotic resistance gene from the intestine
into the bacterial cell. After such transformation, bacteria would become resistant to a
given antibiotic. This could lead to increase of amount of antibiotic resistant bacterial
strains in the nature, and would cause problems in human and animal medical
treatment. However, application of new markers that do not represent antibiotic
resistance genes would resolve this problem. An example of such markers is green
fluorescent protein and mannose (Joersbo et al., 1998). Another way to resolve this
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problem is to remove the marker after the development of transgenic plant, when its
presence is not required any more (Zuo, 2001). Transgenic plants with green
fluorescent protein as a marker and with removed markers are already submitted for
authorization and their large scale production can be expected soon.
Allergenicity. Insertion of a transgene into a plant can cause production of a
product which is an allergen. This allergen will cause allergic reaction, which can be
fatal to persons who consume such plants and are allergic. Some plants do not
contain any allergen, but insertion of a transgene which produces them can cause
health problem and even death of people who are allergic. Allergic people who do not
know that a transgenic plant contains a given allergen can be in great danger. An
example is transgenic soybean with inserted gene from Brazil nut (Nordlee et al.,
1996). The gene was inserted to improve nutritional characteristics of soybean, but
people who were allergic to Brazil nut were allergic to mentioned transgenic soybean
too. This soybean has never been approved for market application. Now, all
transgenic plants are tested for allergens to solve this problem.
Terminator Technology. In 1990s U.S. Department of Agriculture and Delta
and Pine Land Company developed a method for protection of technology. The goal of
this method is to force farmers to pay intellectual property for development of
transgenic plants. Actually, many farmers all over the world save some seeds to plant
next year. If they buy transgenic seeds from a company developer of the transgenic
plant once, they do not have to buy it anymore because they can produce transgenic
seeds by themselves, saving some of the seeds in next generation. In 1998,
developers of mentioned method were awarded a joint patent. The method is named
Technology Protection System (TPS). TPS makes possible production of seeds which
is sterile in the second generation. Seeds obtained after first generation is good for
consumption, but plant embryo is killed by TPS, making seed unsuitable for planting.
Thus, if farmers want to plant transgenic plants, they have to buy seeds from company
developer of the seed.
Advantage of this technology is that plant pollinated with transgenic plants will
be sterile as well, and it will prevent gene flow from transgenic plant to the other
plants. Another advantage is that this will encourage biotech companies for further
development of transgenic plants with novel treats, especially those that are not so
profitable for them.
Disadvantage is that some farmers, especially small and farmers in developing
countries, will not be able to buy seeds every season. That would increase cost of the
production, and it can lead to their bankruptcy in competition with large
manufacturers. Another disadvantage is that neighboring non-transgenic crops and
wild plants would be affected, because after possible pollination their seeds would be
sterile and it could cause lower yield. This impact would be higher on non-transgenic
crops than on wild plants.
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There is another system for protection of technology named Trait-specific
Genetic Use Restriction Technology or T-Gurt. It was developed by agro-chemical
companies including Monsanto and AstraZeneca. T-Gurt does not cause production of
sterile seeds. The seeds in next generation would not express transgenes. However, if
seeds are sprayed with a chemical, sold solely by the manufacturer of seeds, the
transgene would be expressed.
Benefits of this technology are that transgenes would not be expressed in the
wild plants, and farmers can plant seeds obtained in the next generation (but without
expression of transgenes if they do not buy the chemicals). Disadvantages are that
transgenes would be transferred by pollination to the neighboring population (crops
and wild plants) in spite the fact that it will not be expressed and spraying of the seed
with the chemicals can make negative impact on environment.
A new sophisticated system for protection of technology which produces sterile
seeds is under development. This seed becomes fertile if it is sprayed with a chemical
sold by company developer of the transgenic plant.
None of terminator technologies has been applied yet. A great resistance to its
application given by farmers all around the world is one of the reasons for delaying of
the application. However, it is hard to believe that large agro-chemical companies will
quit these projects which can enable them to make additional large scale profit.
Production of Transgenic Crops around the World
In 1996, the first transgenic crops were planted in the USA for commercial use.
Cultivation of transgenic crops has shown permanent increase (Figure 3) according to
ISAAA (ISAAA, 2006). In 2006, they were grown on 102 million hectares worldwide.
The increase has been observed in both, industrial and developing countries. The
USA is the country with the largest area planted with transgenic crops. European
countries do not plant large areas with transgenic plants, and their amounts are
modest in comparison to some other countries (table 1). In 2006, the most often
planted transgenic crops around the world were: soybean (58.6 million hectares),
maize (25.2 million hectares), cotton (13.4 million hectares), canola (4.8 million
hectares).
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Figure 3. Global area of transgenic plants in
million hectares (1996-2006).
COUNTRY
CULTIVATION
AREAS
TRANSGENIC PLANTS
USA
54,6
S,M,C,R, Squash, Papaya,
Alfalfa
Argentina
18,0
S,M,C
Brazil
11,5
S, C
Canada
6,1
R,M,S
India
3,8
C
China
3,5
C
Paraguay
2,0
S
South Africa
1,4
M,S,C
Uruguay
0,4
S,M
Philippines
0,2
M
Australia
0,2
C
Romania
0,1
S
Mexico
0,1
C,S
Spain
0,1
M
Colombia
<0,1
C
France
<0,1
M
Iran
<0,1
Rice
Honduras
<0,1
M
Czech Republic
<0,1
M
Germany
<0,1
M
Portugal
<0,1
M
Slovakia
<0,1
M
Table 1. Cultivation areas for GM crops in 2006: by Country (million hectares)
S = Soybeans, M= Maize, R = Rapeseed, C = Cotton
Source: ISAAA Briefs No 35-2006 (executive summary)
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CONCLUSION
There are many contradictory opinions in connection with transgenic plants.
Even experts in that field do not have common opinion. Some experts have opinion
that application of transgenic plants can be very dangerous for environment and
human health. The others have opinion that risks are overestimated, and that future is
in transgenic plants. Actually, probably both groups of experts are right. Application of
transgenic plants without appropriate regulations can be very risky. The new
transgenes inserted into them, and novel treats, can disturb harmony among
organisms that has been built by nature for many millions of years. This
disorganization of an ecosystem is not predictable and cannot be always prevented.
Restoring of the consequences could be very difficult and probably in majority of the
cases impossible. Transgenes that are accidentally transferred to the other plants,
including wild environment, will stay there forever. However, application of transgenic
plants can increase agricultural production, decrease cost of products and save
environment by decreasing amount of applied chemicals (or by application of safer
chemicals). Transgenic plant can be resistant to various plant diseases and
environmental conditions which can make them very suitable for agricultural
production. The problems caused by introduction of transgenes cannot be solved by
nature, or to be more accurate, nature would need very long time to do that with
dangerous consequences. Thus, only solution is to prevent all known problems and to
begin with application of transgenic plants with precautions. Regulations, which would
prevent large scale application without preliminary assays and approval, are
necessary at national and as well as international levels.
Contemporary obtained improvements obtained by transgenic technology are
y just
very modest in comparison to benefits that will be in the future. Now, we enjoy
novice movements in this field. Benefits in the future will be so huge that agricultural
production without transgenic plants can be compared to transportation with animaldrawn vehicles.
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