4.3 Quantification of gene expression
Expression of transgenes varies with the nucleotide
sequence of the gene, its promotor,
and the point of insertion of the gene in the DNA of the
transgenic variety, the internal
cell environment, as well as several external factors in the
environment. It is necessary to
know how a Bt gene is expressing in a transgenic
variety, in order to evaluate its
effectiveness against the targeted pest. Comparing the
density, morbidity and mortality
of pest populations, on the Bt and its isogenic non-Bt variety, is one way of doing this.
But a more direct way is to accurately quantify gene
expression in terms of the
protein/enzyme it helps to synthesize. There must be a
certain minimum quantity of the
Bt protein in the
plant parts, particularly during the more vulnerable phases of the crop, to
control the pest. The quantity of Bt protein present
in different parts of the plant during
the crucial phases of pest damage such as the boll
formation in cotton, would give an idea
of the effectiveness of the technology in a particular Bt variety.
Field kits have been developed to quantify Bt proteins
in transgenics. The Bt gene
construct is introduced into the experimental bacterium Escherichia
coli, so that the gene
product is more easily purified from the transgenic
bacterium, than from a transgenic
crop variety. Antibodies are raised against this purified
protein, and these antibodies are
used to quantify the Bt protein in the transgenic
variety, through an enzyme-linked
immuno-assay method. This procedure results in a colour
reaction whose intensity gives
the measure of the quantity of the protein involved.
Quantification of Bt proteins by this
procedure is relatively simple and with little instruction
and minimal facilities, a semiskilled worker can conduct the test. However, the
simplicity of the test itself is its
undoing. The test is expected to work with a little bit of
hand-crushed tissue of the Bt
transgenic plant. Unfortunately, quantification of
expression of the Bt gene is sensitive
to the following factors (Shantharam and Kameswara Rao,
2006):
a) Kits from different sources vary in their details, such
as whether the antibodies
used were monoclonal or polyclonal. Kits based on
polyclonal antibodies are
good enough to find out if any Bt protein is present
in the tissue, but are not very
exact to quantify the protein that occurs in microgram
quantities. Though
monoclonal antibodies provide for a more accurate
quantification, most kits are
based on polyclonal antibodies, as the production of
monoclonal antibodies is
more technically involved and so more expensive. There have
been complaints
on the accuracy and consistency of several of these kits,
but authentic data are
unavailable. Actually it is necessary that the kits
available on the market were
assessed for their reliability.
b) The tissue should be properly homogenized and the
protein extracted in an
appropriate buffer. Crushing a bit of a tissue in water is
not an exact scientific
way of extracting even most of, if not all of, the protein
in the tissue.
c) The excised plant part should be used immediately for
assay. Protein degradation
is quite rapid in excised and stored tissue.
d) There would be differences in the protein content
depending upon whether the
part used for assay was from a plant in the vegetative or
the reproductive phase.
Hence the results can be compared only between similar
parts of similar age taken
from plants that were in a comparable physiological state
of development.
e) Mature leaves, bolls and seeds are more fibrous and
harder, and contain several
chemical compounds such as resins, oils, phenolics, etc.,
which accumulate with
the age of the part and which may interfere with the
extraction of the protein in
the tissue.
Not observing these precautions would result in incomparable,
unreliable and misleading
data.
5. SUBSTANTIAL EQUIVALENCE OF TRANSGENICS AND THEIR
ISOGENICS
The US Food and Drug Administration (FDA) routinely and
stringently used the
Principle of Substantial Equivalence (PSE) for decades to
assure the public of the safety
of foods and drugs. This criterion refers only to the
product and not the process of its
production. On account of the high standards of FDA’s
regulatory oversight, most other
countries generally approve drugs and pharmaceuticals on
the basis of FDA’s approval.
PSE is now being applied to products from genetically
engineered organisms (GEOs), in
order to assure the consumer that the product is
'Substantially Equivalent' (SE) to its
conventional counterpart and so is safe for human consumption.
In the context of
modern agricultural biotechnology, PSE is frequently an
issue for serious discussion
The FDA has long considered GE crops to be substantially
equivalent to conventional
varieties and required no other regulatory review. However,
using the ‘provision for
voluntary consultation’, biotech companies in the
US
seek independent SE certification
by FDA, of all GE varieties and their products that are
marketed in the
US
.
The policy of the FDA did not result in any health concerns
but invited criticism on
account of, a) the FDA itself has a mandatory process for
approving transgenic animals,
b) the
US
Environment Protection Agency (EPA) and the
US
States Department of
Agriculture (USDA) have a mandatory and open process for
evaluating the biosafety of
transgenic plants, and c) the data are provided by the
product developers (and so are
suspect).
Products from transgenics of such crops as soybean, tomato,
corn, cotton, etc., on the
US
markets have been tested extensively and judged
substantially equivalent to their
conventional counterparts. Some products may contain
miniscule quantities of one or
two additional proteins, which are usually broken down
during processing or digestion, or
some others may contain some compounds not occurring in the
counterparts but at below
threshold levels. Such products are categorized as
'Generally Recognized As Safe'
(GRAS).
The presence in the GEOs, of new genes that would code for
fats, proteins or
carbohydrates, that may be toxic or may cause allergies or
may adversely affect the
nutritional value of the product, prevents certification as
SE or GRAS, without
appropriate and adequate testing.
While in the
US
no labeling as SE or GRAS is
mandatory, it is not so in several other
parts of the world. This leads to considerable confusion
and controversies. Suggestions
were made for the application of PSE to all products of GE,
including livestock feed and
GE crops, which raises certain questions.
In the application of PSE, the comparison should be between
the GE variety and its
isogenic, which is the basic variety into which a transgene
was inserted. The
certification is to the effect that the GE crop variety is
substantially equivalent to its
isogenic, in genotype, marked characteristics and
performance, but for the transgenes and
their anticipated characteristics. If the isogenic were
safe, the transgenic would be
equally safe, provided that the newly introduced transgenes
do not exercise any adverse
effects by themselves or through altering the expression of
any other genes of the
isogenic, in the transgenic environment. Such an assurance
requires scientific evaluation
of the crop variety first, and then of its products. This
involves additional efforts, time
and expense, raising consumer costs.
All
US
agricultural biotechnology companies submit to the FDA, voluminous dossiers on
the safety and risk analysis of the GEOs and their products
developed by them, before the
products are on the
US
markets. Such a voluntary
mechanism should be global, although
antitech activists look down upon data provided by the
product developers themselves,
even when gathered by different recognized laboratories
outside the companies. If
testing standards and procedures in different countries
were uniform, what is considered
safe in one country should also be considered so in other
the countries. This will
eliminate the need for repeating the same and every test in
every country.
At no time, transgenics can be substantially equivalent to
their isogenics in their entire
genotypes and this is not related to transgenic technology.
Even to start with, members
of the same population are not entirely genetically
identical. In addition, mutations
occur naturally and randomly, involving different genes.
Lethal mutations are naturally
eliminated. Mutations of the genes of the desired
characteristics are eliminated in the
process of selection, but those that do not affect the
desired characteristics escape
attention and accumulate. After a certain number of
generations, a critical genetic
analysis will contravene SE, although SE can be established
for the genes of the desired
characteristics. Such a situation would cause problems in
some countries, where the
regulatory authorities apply the principle of SE more in
letter than in spirit, and a lot
more strictly than in other countries.
The official European consensus is that SE should only be
used to guide and inform
safety assessments. Codex Alimentarius, the
international set of guidelines for food
standards and safety, sees it as a starting point in the
regulatory process rather than an end
point (Codex Alimentarius Commission, 2008).
However, in the
US
,
SE still plays a
significant role in the regulation and commercialization of
GE foods.
Notwithstanding the importance given to PSE, it has been criticized
as vague, ill defined,
flexible, malleable, open to interpretation, unscientific
and arbitrary (Ho and
Steinbrecher, 1998).
On account of the concerns raised, PSE should be
re-examined, and for re-defining its
applicability to GE crop plants and their products, laying
emphasis on a reasonable
application of the principle, addressing only those genes
and their products that are
relevant to the objectives of developing a particular
transgenic variety or product. There
is also a dire need for a uniform and harmonized
international policy. At the moment,
there is no evidence that SE is an issue that adversely
affects the safety of Bt transgenics
or their products.
6. BIOSECURITY
In the context of modern agricultural biotechnology the
term Biosecurity has two
components: a) Biosafety, the safety of genetically
engineered (GE) organisms and/or
their products to humans and animals as food, feed and
medicine, and b) Environmental
safety, the safety of non-target organisms, soil and water.
The terms biosecurity and
biosafety are often used incorrectly as synonyms.
There is no risk-free technology. It was the international
scientific community, not the
activists, who have identified the possible biosecurity
risks from the transgenic crops and
devised protocols for the identification, assessment,
quantification and mitigation of risk.
Science has reasonable peer reviewed experimental evidence
to answer biosecurity
concerns.
Biosecurity issues are unfortunately often mixed up with
political, economic,
management, societal and ethical issues, emotionalizing and
sensationalizing the
concerns, to spread fear and suspicion of GE technology.
Biosecurity issues raised to
oppose GE crops by antitech activists are relevant to even
products of classical
agricultural biotechnology, but were never made an issue in
that context.
Every country that commercializes GE products has a strict
regulatory regime to ensure
biosecurity of GE products and that all questions are
answered reasonably satisfactorily
before commercialization is permitted.
India
has a
regulatory regime that is actually
more stringent than that of most other countries. Powered
by several Acts of the
Parliament, managed by the Department of Biotechnology and
the Ministry of
Environment and Forests, and supported a large number of
public sector research
institutions and scientists, the Indian regulatory regime
functions satisfactorily.
7. BIOSAFETY OF BT TRANSGENICS
Bt being a universally
occurring soil bacterium, all species of plants and animals in
agricultural and other situations, and those that use
plants as food have been exposed to
Bt and Bt proteins
for centuries. Bt proteins are transient in the environment. The
toxicity of Bt proteins is pest specific, dependent
upon a set of biological pre-requisites.
The use of Bt as a conventional pesticide for over
60 years has demonstrated that it is safe
to the consumers and a variety of non-target organisms.
Bt is one of the few
pesticides recommended for widespread application in North
America
(Glare and O’Callaghan, 2000), and was broadcast or
sprayed on crops and air
sprayed to control forest pests in
Utah
(US, 1990-1995) and
Ontario
(
Canada
, 1985-
1994). Water borne Bt was air sprayed to control the
Asian gypsy moth in
Vancouver
(
Canada
,1988), and
North Carolina
(US, 1993) and the white-spotted tussock moth in
Auckland
(New Zealand, 1996) and no adverse effects on the human
health have been
reported so far from these urban locations.
7.1 Toxicity
Cry proteins were shown to be harmless to vertebrates, including
mammals and humans,
even at high doses, by ingestion, inhalation or injection.
Nevertheless, antitech activists
raise issue after issue to brand GE crops as toxic in spite
of massive evidence on their
safety as food and feed. Over 350 million people in
North America
have been eating Bt
products for over a dozen years and no greater testimony is
needed for human safety of Bt
transgenic products than this.
7.2 Allergenicity
Several claims have been made of allergenicity of
transgenic crops, including Bt cotton in
some places in
India
, but there has never been any
scientific evidence, as discussed
elsewhere in detail (Kameswara Rao, 2009).
A transgenic soybean with a gene for the Brazil nut protein
developed to increase the
content of methionine, an essential amino acid, was one of
the targets. Though no one
actually developed allergy by eating the transgenic
soybean, since the transgenic is likely
to affect people who are allergenic to Brazil nuts, Pioneer
Hi-Bred International, the
developer of the product, did not proceed with it, setting
an example of self-regulation.
The United States Department of Agriculture (USDA) cleared
Aventis Starlink Bt corn
for use as both food and feed. Since the Bt Cry9
protein in this transgenic corn was
projected to be allergenic, the US Environment Protection
Agency (EPA) took a
precautionary measure and approved this corn only for
animal feed, as animals do not
generally suffer from food allergies. Bt Cry9
protein was never demonstrated to be
allergenic. The US Centers for Disease Control (CDC) tested
17 samples of blood from
people claimed to have developed allergenic reactions to
Starlink and found that none of
the blood samples showed cross-reactivity to Cry9 Bt
protein. The Cry9 gene is not
deployed in any commercial product now. Since transgenic
products approved as only
feed may accidentally get into the food products, no
transgenic is now approved
exclusively for use as feed. This shows that the regulatory
regime is in fact functioning
effectively.
Among the commonly consumed food items, several such as
walnuts, pecans, Brazil nuts,
cashews, peanuts, soybeans, some varieties of rice and
wheat, cucumbers, mushrooms,
fish, shellfish, eggs, milk, mother’s milk, etc., and
certain drugs like penicillin, cause
clinically well known anaphylactic reactions in certain
individuals. Even 1/44,000 of a
peanut kernel may cause severe anaphylaxis in some. Food
and drug based allergies
cause several deaths every year. Yet, there was not even a
simmer of protest against
marketing such products.
8. ENVIRONMENTAL SAFETY
All the evidence indicates that Bt transgenics are
very safe to all components of the
environment. Over a decade’s cultivation of Bt transgenics
has neither confirmed the
scary scenarios aired by the critics nor has thrown up any
new threats to the environment.
8.1 Super weeds
A serious negative factor projected by the antitech
activists from transgenics is that they
would escape cultivation and become super weeds placing
other vegetation at risk.
Crawley et al., (2001), basing on a 10-year study of
pest and herbicide tolerant transgenic
crops demonstrated that the transgenics do not become more
competitive to invade the
environment as super weeds, but that in fact they perished
earlier than their isogenic
counterparts.
8.2 Impact of Bt on non-target organisms
Glare and O’Callaghan (2000) and every country’s regulatory
process provide extensive
data demonstrating the safety of Bt proteins to
non-target organisms.
The much publicized instance of toxicity of Bt proteins
to non-target organisms was
based on the study by Losey et al., (1999), who
reported that transgenic Bt corn pollen
harm monarch larvae, a conclusion immediately questioned by
the scientific community.
Subsequently, Sears et al., (2001) re-examined the
issue, avoiding the flaws in the
experimental design in the study of Losey et al., and
concluded that impact of Bt corn
pollen on monarch butterfly populations was not
significant. The performance of bumble
bees was not affected in any manner by Cry 1Ab Bt proteins
(Babendreier et al., 2008).
Chen et al., (2008) showed that Cry1C proteins were
safe to parasitoids that control pest
populations in many crops, in contrast to the severe damage
caused to the parasitoids by
the traditional insecticides.
Reports of the death of peacocks and the death of farm
animals in Andhra Pradesh and
honey bee Colony Collapse Disaster in Europe and
North America
, were attributed to the
presumed toxicity of Bt proteins in GE crops. These
incidents, projected as major issues,
were shown to be due to causes other than Bt protein
toxicity (Kameswara Rao, 2008
a,b).
8.3 Gene flow from transgenics
The possibility of gene flow from transgenics and the
negative impact of this on other
crops, biodiversity and the environment occupy the centre
stage in discussions that
denigrate modern agricultural biotechnology, although the
experience gained from the
regulatory processes of transgenic crops and their
cultivation for over two decades have
not indicated any serious possibilities of gene flow or its
negative consequences. Gene
flow depends upon the reproductive biology and breeding
behaviour of the crop in
question (Kameswara Rao, 2008 c,d), which the activists
have not taken into
consideration.
8.4 Vertical gene flow
The essential pre-requisite for vertical gene flow is
sexual reproduction between the
transgenics and related plants. The transferred genes
express only in the next generation.
The ease of vertical gene flow depends upon the genetic
relationships between the
varieties and whether the crop is self or open pollinated,
which Bt technology cannot
change. Transgenics are no more promiscuous than their
isogenics. If vertical gene
flow were possible between isogenics and any related
varieties or species, it would be so
between transgenics and related plants too. However,
centuries of agricultural
experience does not indicate any alarming possibilities.
A study, much quoted by the critics as a risk of vertical
gene flow, relates to Bt maize in
Mexico
. Quist and Chapela (2001) reported the presence CaMV 35S
promoter and a Bt
gene, ‘traced’ to Bt maize, in native maize
populations in
Oaxaca
,
Mexico
. They
claimed that the genes got incorporated into the native
land race and that the promoter
was out of control and may activate any other genes. The
scientific community
challenged the methodology and the conclusions, which lead
Nature to announce that it
should never have published the paper. Ortiz-Garcia et
al., (2005) have analyzed
1,03,620 corn seeds collected during 2003-04, from 125
fields at 18 locations, in the State
Oaxaca
,
Mexico
, the same area as of Quist and Chapela’s study, and found
no evidence
of the transgenes in native maize populations. The defense
was that the genes were there
in 2001 and vanished subsequently!
8.5 Lateral/horizontal gene flow
Lateral/horizontal gene flow involves exchange of genes
between genetically unrelated
organisms, a fact of evolution, but not of day-to-day
occurrence. It does not involve
sexual reproduction and the transferred genes can express
in the same generation.
Transgenic technology itself is an example of lateral gene
transfer. All known examples
of lateral gene transfer relate to endoparasites and their
hosts, as for example, the
commonality of about 30 per cent of genes between mammalian
intestinal parasites and
their hosts.
The use of antibiotic markers in transgenic technology, to
confirm genetic transformation
was used to promote fear of GE technology. The argument,
not supported by any
tangible evidence, is that if there were lateral transfer
of antibiotic resistance genes to
pathogenic organisms, it would result in pathogens
resistant to the antibiotics used as
markers and endanger our prospects in the fight against the
new pathogens using the
antibiotics to which they are resistant. Supported by
numerous studies, Ramessar et al.,
(2007) concluded that there is no scientific basis to argue
against the use and presence of
selectable antibiotic resistant marker genes in transgenic
plants. However, to assuage the
fears expressed, the use of antibiotic resistance marker
genes is now minimized, as
alternatives are found. The antibiotic marker genes can
also be removed, after
confirming genetic transformation.
8.6 Impact on biodiversity
A comprehensive report on the impact of agricultural
biotechnology on biodiversity
(
Amman
,
2004) reiterated that the introduction of GE crop varieties does not represent
any greater risk to crop genetic diversity than the
varieties of conventional agriculture.
GE actually increases crop diversity by adding new
varieties.
A peer reviewed report (Sanvido et al., 2007)
concluded that no aspect of credible
science based on ten years of field research and commercial
cultivation has indicated that
GE crops have harmed biodiversity or the environment.
The Consensus Document from the Organization for Economic
Cooperation and
Development (OECD, 2007) on the safety of Bt proteins
in transgenic plants did not
identify any hazards caused by them.
9. BENEFITS FROM BT TRANSGENICS
Technologies come with some concomitant and some
consequential benefits, both of
which should be taken together in assessing the total
benefits that accrue. Benefits of a
technology should hence be weighed against minimal and
acceptable risks and a
favourable cost-benefit ratio.
9.1 Optimal cultivation practices are mandatory
In order to realize the benefits from the full potential of
any crop variety, it should be
grown under optimal conditions. Although cotton is hardier
than many other crops, it
performs satisfactorily only under irrigation and on a
right soil type. In
India
,
cotton is
often grown under near impossible conditions, as farmers
are lured into growing a cash
crop, irrespective of the inadequate and/or inappropriate
infrastructure, and suffer
disastrous consequences. A few years ago, the Government of
Andhra Pradesh, India,
rather unsuccessfully advised the farmers to avoid growing
cotton on red soils,
particularly as a rain fed crop. A long time advice to grow
cotton only in areas with the
average rainfall of more than 60 cm per year, uniformly
distributed throughout the crop
season, is also largely unheeded. In many developing
countries, the record of both the
advice given to the farmers and of farmers taking it
seriously when given, is dismal.
9.2 Concomitant benefits of Bt technology
The most direct and the most important benefit of Bt technology
is the control of the most
damaging pest of particular crop, such as the American
bollworm of cotton, stem borers
of rice and corn, rootworm of corn,
Colorado
beetle of potato or stem and fruit
borers of
brinjal. As systemic pesticides, Bt proteins take
care of these pests. The other pests, on
which Bt proteins have little or no effect, need to
be controlled by pesticide application,
preferably as a part of Integrated Pest Management (IPM)
practices.
Bt technology imparts
only tolerance of the targeted pest of a particular crop and not total
resistance to it (GEAC, 52nd Meeting, 2005). In view
of the variation in the expression of
Bt genes, due to
various internal and external factors, two or three pesticide applications
are needed, against even the targeted pest, such as the
bollworms of cotton, instead of the
usual 10 to 20. Even so, in a country like
India
, where
over 50 per cent of pesticide
application is on cotton, Bt technology results in a
very substantial savings on pesticide
and labour costs associated with pesticide application
(James, 2008), provided the farmer
does not resort to ill-advised or panic spraying.
9.3 Consequential benefits of Bt technology
Bt technology’s consequential
benefits are:
a) drastic reduction of pest pressure; suppression of
cotton bollworm on multiple
non-Bt non-cotton crops in areas with Bt cotton
was reported from
China
(Wu et
al., 2008);
b) healthy crop, more biomass and more yield from saving
losses;
c) reduced risk to farm labour involved in pesticide
application; in the developing
countries several thousand farm workers suffer any many of
them die, due to
unintended pesticide poisoning;
d) far lower concentrations of pesticide residues on the
produce and in the
environment;
e) reduced exposure of non-target organisms in the
environment to pesticides, and so
a better conservation of biodiversity; and
f) the Bt farmer experiences a far lower tension and
is certainly better off with Bt
technology than the earlier scenario of ‘spray and pray’.
9.4 What is not to be expected of Bt technology
Bt technology has no
role to play in the following areas:
a) Yield: Bt technology has no gene based influence
on crop yield; nevertheless,
there is a substantial increase in crop product recovery
due to prevention of loss of
the crop produce caused by the pests; Bt farmers in
India
experienced sizeable
increase in yield per acre, compared to non-Bt farmers
(James, 2008);
b) Seed germination: failure of seed to germinate is often
mischievously attributed to
Bt technology; causes
for the failure of seed germination lie in the varieties or
cultivation practices or environmental factors; the
percentage of germination of
the seed of a Bt variety would be about the same as
that of its isogenic;
c) Non-target pests: Bt technology is specific pest
targeted and has little or no effect
on other pests;
d) Diseases: Bt technology does not cause or control
any viral, bacterial or fungal
diseases; such diseases as the viral leaf curl of cotton
prevalent in northern
India
or the physiological disorder para-wilt of cotton that
occurs after a heavy rain fall
preceded by drought conditions, are erroneously or
deliberately attributed to Bt
technology.
It is a compulsive habit of the antitech activists to
repeatedly attribute farmer suicides in
India
to the failure of Bt cotton crop. A comprehensive
review on the issue (Gruere et al.,
2008) found no evidence in support of the allegation and it
even pointed out that the
number of suicides has actually come down after the
introduction of Bt cotton cultivation.
10. ANTITECH ACTIVISM
The antitech activists endlessly criticize the whole
technology and the biosecurity
regulatory regime. They use junk science to pursue their
vested interest and cause fear
psychosis on the public mind against the technology. They
have used diverse media,
filed petitions in the Supreme Court demanding moratorium
on the technology and even
vandalized GE crops in field testing. Most of those who
raise biosecurity issues to voice
their opposition to GE crops have no locus standi in
terms of knowledge and expertise to
trash the combined global scientific wisdom. Unfortunately,
the scientists, the agribiotech
industry and the Governmental agencies have failed to stand
up against the
onslaught and in support of what they obviously believe as
sound and safe technology.
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