Plant Biotech Blog

C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

ALLERGY
Allergy is an abnormal state of hypersensitivity of our body to normally innocuous substances in the food, medicine or the environment.  Allergy is neither new nor universal and it is not an infection that would spread.   Every one of us suffers from intolerance of, or allergic reactions to, one or the other element in our environment or certain foods or drugs, for a certain time in our lives.  Nevertheless, there is no single substance that causes allergy in every one of us.  Allergies affect millions of people and cause several thousand deaths globally every year.

Atmospheric pollen, dust mites, animal dander, insect stings, molds, latex, cosmetics, fragrances and several others in day to day contact may cause reactions in people.

Walnuts, pecans, Brazil nuts, cashews, peanuts, soybeans, some varieties of rice and wheat, cucumbers, mushrooms, fish, shellfish, eggs, milk, mother’s milk, some vegetables and fruits, etc., as also certain drugs like penicillin and aspirin, may cause intolerance or even true allergy in certain individuals.

Some people suddenly develop reactions to foods they have been eating for decades.  In some this happened when they shifted to a new place of residence, which did not revert when they moved back to the old place, showing that environmental influences are not necessarily the cause of the problem.  Most allergies disappear as mysteriously as they appear.

While there are no reliable data on food allergies in the developing countries, in the US five to eight per cent of the children and one to two per cent of the adults are prone to true allergy from some foods.   Children may overcome their sensitivities to certain food items as they grow up but the sensitivities acquired in adulthood would not easily go.  These people have to avoid the foods they are allergic to.

In a school class containing several children of diverse genetic backgrounds but of the same age group, some one or the other would be sensitive to one or the other food item, though sensitivity to eggs, fish and nuts is common.  As school children share their lunch boxes it is a hard task for parents to decide on what foods they can pack without risking sensitive reactions from any of their child’s friends in the class and the problem is worse when they arrange parties for their children’s friends.   The greater and unanticipated risk is from inadvertent or accidental servings of offending foods.

In highly sensitive individuals even 1/44,000 of a peanut kernel may threaten life.  Nevertheless, there never was even a simmer of protest against marketing any of the many conventional foods established as severely allergenic in certain individuals.

Since the scenario of food allergies is complex, it would be well to remember the following generalizations on food allergy: a) true food allergy is uncommon, but can be very serious, b) food intolerance, significantly different from food allergy, is quite common and confused with food allergy, c) the causes of true food allergies differ from individual to individual and from children to adults, d) the diagnosis of true food allergies is complex and time consuming and takes into consideration a detailed history of food habits and reactions, and elimination test to identify the causative food, and e) the best way to avoid food allergy is to avoid the offending food while emergency management involves drugs such as adrenaline.

RISK OF ALLERGY FROM GENETICALLY ENGINEERED CROPS
Risk of allergy from genetically engineered (GE) crops and foods is projected as a major biosafety issue, stemming from baseless allegations, rooted in two, but now irrelevant cases.

A gene for the Brazil nut protein was introduced into soybean to increase the content of methionine, an essential amino acid which the human body does not synthesize. The serum from people allergic to Brazil nuts cross-reacted with extracts of transgenic soybean and not with extracts of its isogenic, which links the problem to Brazil nut proteins, and not the soybean. Though no one actually developed allergy by eating the transgenic soybean that was never released for public consumption, 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, an example of self-regulation.

The Bt Cry 9c protein in the Aventis Starlink Bt corn controls the European corn borer.  Cry 9c protein binds to the pest’s gut at a site different from that of the Cry 1 proteins, and so would be effective even if the pest develops some resistance to Cry 1 proteins.  Cry 9c protein was found to be slightly more stable in simulated digestion than other Bt proteins, and so it was thought that it might be allergenic.  The United States Department of Agriculture (USDA) cleared the Cry 9c transgenic corn for use as both food and feed, but 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 Cry 9c protein was never demonstrated to be allergenic.  The US Centers for Disease Control (CDC) tested samples of blood from 17 people claimed to have developed allergenic reactions to Starlink and found that none of the blood samples showed cross-reactivity to Cry 9c protein.  The Cry 9c gene is not deployed in any commercial product now.   Since transgenic products approved as only feed may get into the food products, as has happened with Starlink Bt Cry 9c corn that appeared in Taco Bell taco shells, no transgenic is now approved exclusively for use as feed.    This shows that the regulatory regime is in fact vigilant.

Ignoring the scientific background of food allergies and the fact that the two questioned transgenes are neither allergenic nor deployed in any product, these two cases are repeated ad nauseam to make the world believe that all GE foods are allergenic and to repeatedly demand a blanket ban on GE foods.   In India, the charge was made against Bt cotton, though not a food crop.  Even while Bt brinjal is still in multilocation open field trials and not available for public consumption it is being projected as allergenic, trashing voluminous evidence that Cry 1Ac protein is not allergenic which was also confirmed during biosafety testing on Bt brinjal by Intox at Pune and Rallis at Bangalore.

IMMUNOLOGICAL BASIS OF ALLERGY
The term allergy is used very loosely and most people seem to have no idea of what it actually implies.   True allergy involves the immune system.   Often food allergy is not differentiated from other types of adverse non-immunological reactions to food.  Since the public fear allergy, it is being exploited to whip up fear against GE foods.

Mammalian systems produce four different classes of immunoglobulin proteins (Ig), the antibodies, in response to the presence of hazardous alien proteins (called antigens) that enter the body system through food or pathogens.  Vaccines contain antigens (of cholera or smallpox or other pathogens) and vaccination prepares the body into producing antibodies against specific pathogens.  The antibodies bind to the antigens when encountered in the body system affording the most valuable means of our body’s defense.

The IgM antibodies form first, but both the quantity and importance of the later formed IgG antibodies is far greater.   IgG antibodies are the most important body defense system.  They bind to the antigens neutralizing them.  While IgA antibodies are specifically involved in the defense of the oral cavity, the function of IgD antibodies is not very clear.

For some poorly understood reasons, our immune system also produces another class of antibodies, the IgE, in response to a few proteins, which through a complex sequence of cascading biochemical events lead to true allergic reactions, manifesting as skin rashes, intestinal inflammation, cramps and diarrhoea or respiratory disorders.   This process is anaphylaxis, on record since 2641 BCE, which varies in different individuals from mild and annoying to life threatening.  The active compounds, triggered by IgE involvement, such as histamine are mostly inflammatory agents that get into the blood stream making the problem systemic, when a number of different areas of the body are affected at the same time.  Some similar reactions do not involve the IgE antibodies (anaphylactoid reactions), but nonetheless are an important health hazard.

Some non-protein compounds, such as penicillin and aspirin, may also cause severe reactions, and these agents called haptens must bind to an endogenous carrier protein to cause the symptoms.

DIAGNOSIS OF ALLERGY
Identifying an allergen is a long drawn process.   For each individual a list of suspected sources allergens is drawn and through a dermal prick test the possibilities are narrowed down by eliminating those that do not cause any reaction at the test site on the skin.  Identification of the offending substance and demonstrating that it is true allergy involving IgE antibodies is done through an enzyme linked immunological assay procedure.

TREATMENT OF ALLERGY
The best way to avoid allergy is to avoid contact with the allergen, identified basing on each individual’s experience.

Repeated exposure to small quantities of an allergen over a long period of time results in higher and higher titres of IgG antibodies, which in course of time would be adequate to neutralize the allergen before it had a chance to elicit IgE antibodies. This is how we overcome allergies naturally or allergies are clinically treated (immunotherapy).   This slow process has worked well in treating environment based allergies and its success has just been demonstrated with peanuts on children who earlier developed strong reactions on eating even very small quantities.

While there is no assured treatment to cure allergies, anaphylactic reactions are treated using anti-histamine or steroid drugs.  Both children and adults prone to severe anaphylactic reactions carry a device to inject a measured quantity of the drug of choice, in case of an emergency.

PREDICTING ALLERGENIC POTENTIAL OF FOODS
A protein that is degraded by the gastric enzymes before reaching the intestine is very unlikely to cause allergy.     This has been the basis to investigate a protein further for its allergenic potential.

Basing on voluminous data on the biochemistry of over 200 known allergenic proteins, tests have been developed to identify potential food allergens.  It is now understood that only certain short stretches of amino acids (the components of proteins) constitute allergenic sites.  These identified sites, not the whole protein, trigger the production of IgE antibodies.  A consensus document on the biosafety of Bt in crops (Organization for Economic Cooperation and Development, July 2007), records that none of the Bt proteins deployed in crops, including Cry 1Ac, Cry 1Ab, Cry 2Ab and Cry 9c, share similar amino acid sequences with known proteinaceous food allergens.  So far, no allergenic reactions have been reported during extensive biosafety tests on GE crops in several countries or on consumption of foods from GE crops for more than a decade by over 350 million people in North America.

Transgenic crop varieties are substantially equivalent to their isogenics, except for the protein coded by the transgene.  The risk of allergy needs to be considered when a GE food or drug contains new protein(s), coded by the introduced genes, but not present in the isogenic variety.   For example, the Bt protein in the Bt potato tuber is new.   Now this protein is known to be safe for human consumption.   Similarly, the iron carrier protein ferritin, whose gene from bean or soybean is being introduced into rice to enhance its iron content, is not allergenic.

If a gene product in the non-transgenic (isogenic) variety were an allergen, it would be so in the transgenic as well.   Proteins that are normally not allergenic will not suddenly become allergens in a transgenic plant.   Whether a particular protein is allergenic or not depends more on the consuming individuals rather than on the protein itself.  This makes identification of allergenic proteins quite tricky.  The remote possibility that Bt crop foods might sporadically cause allergenic reactions in a few individuals, in spite of voluminous evidence to the contrary, cannot be the reason to dump the whole technology which is otherwise beneficial in a number of ways.

It is near impossible to test for all the antigens and haptens in a product for the potential of allergy.   Even so, scientists have not been complacent and every new protein in a transgenic food or feed is examined for allergenicity.   In fact, among all the foods we consume, the GE foods are the most thoroughly tested for allergenicity and toxicity.

Concern for public safety is very essential, but spreading fear on political compulsions, exploiting ignorance, is scare mongering.  What we need is a rational attitude with concern for the larger benefits for the larger sections of the society and not irrational blanket bans on whole technologies.  No one ever said that the production of any of the large number of conventional foods known to cause severe allergies in a few people should be stopped.

February 26, 2009

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Technologies come with some concomitant and some consequential benefits, both of which should be taken together in assessing the total benefits that accrue.   No technology is risk free.   Benefits of a technology should hence be weighed against minimal and acceptable risks and a favourable cost-benefit ratio.  

Risk assessment, mitigation and management are at the heart of regulatory processes.   Planting a non-Bt refugium along with Bt crops is a means of mitigating the risk of acquired resistance, and so is gene stacking (see the article ‘Transgenic Bt Technology: 2. Bt Crop varieties’ in this series). 

Stakeholder acceptance of a technology is rooted in a rational and balanced projection and not in hype.   Factual information will enhance the credibility of the establishment and help the consumer take educated and lasting decisions.

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 aubergine (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.  In view of the variation in the expression of Bt genes, due to various internal and external factors (see the article ‘Transgenic Bt  Technology: 4. Variation in Gene Expression, in this series), 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 costs and labour costs associated with pesticide application, provided the farmer does not resort to ill-advised or panic spraying.

A report from ISAAA (Brief No. 37, 2007) has reported diverse benefits from GE crops over a period of about 12 years of commercialization.  In India, from 2002 to 2007 there was an increase of farmer profits between 50 to 110 per cent, with yield increase between 30 to 60 per cent.  There was about 50 per cent reduction in pesticide usage.

Significantly, suppression of cotton bollworm in multiple crops in areas with Bt cotton was reported from China (Science, September 2008).

Optimal cultivation practices are mandatory:  Any crop should be grown under optimal conditions to obtain the best benefits from the new technology.   Although cotton is hardier than many other crops, it performs satisfactorily only under irrigation.   In India, cotton is often grown under near impossible conditions, as farmers are lured into growing a cash crop, irrespective of the inappropriate infrastructure, and suffer disastrous consequences.   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 largely unheeded.   In many developing countries, the record of both the advice given to the farmers and of farmers taking it seriously, is dismal.  

Consequential benefits of Bt technology:  Bt technology’s consequential benefits are:

a) a healthy crop, more biomass and more yield;
b) reduced risk to farm labour involved in pesticide application; in the developing countries several thousand farm workers suffer or even die, due to unintended pesticide poisoning;
c) far lower concentrations of pesticide residues on the produce and in the environment;
d) reduced exposure of non-target organisms in the environment to pesticides, and so a better conservation of biodiversity; and
e) the Bt farmer experiences a far lower tension and is certainly better off with Bt technology than the earlier scenario of ‘spray and pray’.

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 yield due to prevention of loss of the crop produce caused by the pests; Bt farmers in India earned Rs. 6,000 (about US$ 135) more per acre, than the non-Bt farmers during the last season (ISAAA (Brief No. 37, 2007);
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 prevalent in northern India or the physiological disorder para-wilt 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 (October 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. 

Other articles in this series:
TRANSGENIC BT TECHNOLOGY: 1. BACILLUS THURINGIENSIS, BT PROTEINS AND TOXINS

TRANSGENIC BT TECHNOLOGY: 2. BT CROP VARIETIES

TRANSGENIC BT TECHNOLOGY: 3. EXPRESSION OF TRANSGENES

TRANSGENIC BT TECHNOLOGY: 4. VARIATION IN GENE EXPRESSION

TRANSGENIC BT TECHNOLOGY: 5. SUBSTANTIAL EQUIVALENCE OF TRANSGENICS AND THEIR ISOGENICS

TRANSGENIC BT TECHNOLOGY:  6. BIOSECURITY

January 1, 2009

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

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.  

Biosecurity issues raised to oppose GE crops by antitech activists are relevant to even to products of classical agricultural biotechnology, but were never made an issue in that context.  

It was the international scientific community, not the antitech activists, who have identified the possible biosecurity risks from the transgenic crops and devised testing and mitigation protocols.    Science has reasonable peer reviewed experimental evidence to answer biosecurity concerns.   The regulatory process in every country ensures that all questions are answered reasonably satisfactorily before commercialization is permitted. Most of those who raise biosecurity issues to voice their opposition to GE crops have no locus standi in terms of scientific knowledge and expertise to trash the combined global scientific wisdom.

Biosafety of Bt:  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 a variety of non-target organisms.   Cry proteins were shown to be harmless to vertebrates, including mammals and humans, even at high doses, by ingestion, inhalation or injection. For details see the other six articles in the series ‘Transgenic Bt Technology’ on this website.

Bt is one of the few pesticides recommended for widespread application in North America, 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).   Over 350 million people in North America have been eating Bt products for over a dozen years.  No greater testimony is needed for human safety of Bt than that no adverse effects on the human population have been reported so far.  

Toxicity and allergenicity:  Antitech activists raise issue after issue to brand GE crops as toxic.  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 have been effectively shown to be due to causes other than Bt protein toxicity. 

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. 

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.

Impact of Bt on non-target organisms:  Glare and O’Callaghan (‘Bacillus thuringiensis: Biology, Ecology and Safety’ 2000, John Wiley), and every country’s regulatory process provide extensive data demonstrating the safety of Bt proteins to non-target organisms.  

The much-brandished instance of toxicity of Bt proteins to non-target organisms was based on the study by Losey, et al.,  (Nature, 1999) who reported that transgenic Bt corn pollen harm monarch larvae, a conclusion immediately questioned by Hodgson (Nature Biotechnology, 1999).   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.

A February 2008 publication indicates that Cry 1Ab Bt proteins do not affect the performance of bumble bees in any manner.

In May 2008 Bt Cry1C proteins were shown to be safe to parasitoids that control pest populations in many crops, in contrast to the severe damage caused to the parasitoids by the traditional insecticides.

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, (Nature, 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., (PNAS, 2005) have analyzed 1,03620 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!

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 create the 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, a report in Transgenic Research (June, 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.

How safe are Bt transgenics?  All the evidence indicates that Bt transgenics are very safe and 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. 

A comprehensive report on the impact of agricultural biotechnology on biodiversity from the Bern University’s Botanic Garden (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 of March 2007 stated 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 (No. 42, 2007) on the safety of Bt proteins in transgenic plants did not identify any hazards caused by them.  

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. 

Other articles in this series:
TRANSGENIC BT TECHNOLOGY: 1. BACILLUS THURINGIENSIS, BT PROTEINS AND TOXINS

TRANSGENIC BT TECHNOLOGY: 2. BT CROP VARIETIES

TRANSGENIC BT TECHNOLOGY: 3. EXPRESSION OF TRANSGENES

TRANSGENIC BT TECHNOLOGY: 4. VARIATION IN GENE EXPRESSION

TRANSGENIC BT TECHNOLOGY: 5. SUBSTANTIAL EQUIVALENCE OF TRANSGENICS AND THEIR ISOGENICS

TRANSGENIC BT TECHNOLOGY: 7. BENEFITS

January 1, 2009

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

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 genetic engineering, 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.  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 to inform safety assessments. Codex Alimentarius sees it as a starting point in the regulatory process rather than an end point. 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, M.W. and Steinbrecher, R. (1998)). 

On account of such concerns, 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.

Other articles in this series:
TRANSGENIC BT TECHNOLOGY: 1. BACILLUS THURINGIENSIS, BT PROTEINS AND TOXINS

TRANSGENIC BT TECHNOLOGY: 2. BT CROP VARIETIES

TRANSGENIC BT TECHNOLOGY: 3. EXPRESSION OF TRANSGENES

TRANSGENIC BT TECHNOLOGY: 4. VARIATION IN GENE EXPRESSION

TRANSGENIC BT TECHNOLOGY:  6. BIOSECURITY

TRANSGENIC BT TECHNOLOGY: 7. BENEFITS

January 1, 2009

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Natural variation in gene expression: The tendency to vary is the only consistent feature of Nature.   All species of organisms, whether wild or cultivated, show naturally inherent variation in physical, chemical and physiological features, which is also the basis for distinguishing different species, varieties.   Each species or variety shows some variation in several features both between and within its populations.   Nevertheless, species and varieties have a set of discernible and invariable features characterizing their identity.   For example all transgenic Bt cotton varieties contain some quantity of Bt protein, though the actual quantities of the protein may vary from one variety to the other, as well as within each variety.   In addition, there is a) variation related to time (temporal), based in the age of the individual/population reflected in the growth phase such as vegetative, flowering, fruiting and other stages, and b) spatial variation within an individual specimen reflected in different parts of the plant such as the root, stem, leaf, floral parts, fruits and seeds. 

By centuries of experience, biologists in general and agricultural scientists in particular, fully understand that the expression of the same gene or set of genes is influenced by several factors, some inherent in the organism and some in the environment. Some of this variation, called genotypic variation, is based in the differences in the genetic constitution (genotype) between the varieties.   The other kind, called phenotypic variation, is the result of an interaction between the genotype and the environment, so much so the same genotype behaves differently in different areas and seasons.     Cultivation and management practices also influence gene expression and so the crop’s performance.  Consequently, no crop variety, either conventional or genetically engineered, can be expected to perform uniformly throughout the entire area, or history of its cultivation.  

The full expression of the transgenes in a transgenic crop variety is crucial, but transgenic varieties may behave differently depending upon the genotype of the recipient variety and on where and how it is being cultivated, as has happened also in conventional agriculture all through.   Most of the factors that affect gene expression are beyond the control of the plant breeders and biotechnologists, once a variety is chosen for transgenic development.  

Agro-climatic zones and crop varieties: The physical and chemical characteristics of a) the soil, b) the quantity, periodicity and distribution of rainfall and/or irrigation facilities, and c) the range of temperature, are factors important for a healthy crop life.   These factors, which vary from country to country and even within a country from region to region, are very critical to successful agriculture.  Taking all such relevant factors together, several agro-climatic zones, each characterized by a set of soil, rainfall (or irrigation facilities), and temperature parameters, are identified in countries with diverse geographical features.   The Planning Commission of India has recognized 15 agro-climatic zones in India, and these are further divided into about 120 sub-zones.   Each agro-climatic zone or sub-zone requires varieties of crops particularly suitable to be grown there.   Consequently, a very large number of varieties of different crops was developed by farmers and agricultural scientists in different parts of the world, over centuries, either to suit a particular agro-climatic zone and for certain beneficial traits in them.   As a result, there are over 1,00,000 varieties of cultivated rice, some 80,000 varieties of wheat, and about 15,000 varieties each of potato and the bean in the world today.  

Transgsenic Bt crop varieties: Transgenic Bt cotton containing Cry1Ac was originally developed using the American cotton variety Cocker 312, and this variety is not suitable for cultivation outside America.   Different local varieties of cotton are chosen for developing transgenic Bt cotton varieties, incorporating the same Cry1Ac event, for cultivation in different agro-climatic zones in different countries.    There are now over about 140 approved varieties of transgenic Bt cotton in India, containing the same Cry1Ac transgenic event, and most of them marketed under the Monsanto’s trade name Bollgard I.    The situation is similar with all transgenic events.   The Golden Rice event was first inserted into the genome of a temperate japonica variety and the event had to be transferred to the indica varieties for cultivation in different rice growing agro-climatic zones in tropical countries.   The costs of developing so many varieties with the same transgenic event and the costs of the associated regulatory processing of all these varieties escalate steeply by the time the transgenic products reach the consumer.

Variation in the expression of Bt genes: Even when Bt crop varieties are cultivated in the recommended agro-climatic sub-zone, there would be significant differences in the expression of Cry1Ac gene in them.  

The general health of the crop is an important factor in realizing the full genetic potential of a crop variety.   The expression levels of a gene may decrease as the age of the crop advances.   There may be differences in expression levels between young and older parts such as the leaves or between comparable parts in vegetative and reproductive phases.   Such variation in the expression of Bt event in cotton was observed in Australia and India.

Soil characteristics, rain fall, the severity of pests and diseases, adequate, appropriate and timely farming inputs such as irrigation, weeding, fertilizer, supportive pesticide application, all have a direct or indirect influence on the performance of the crop and may affect the expression of the transgenes and so the benefits to be derived from transgenic technology.   All these factors, inherent in the varieties and/or the environment vary from crop season to season, make the difference between supraoptimal, optimal or suboptimal performance of a crop or even its failure.
 
Transgenic Bt technology produces crop varieties that are only tolerant of the targeted pests and not fully resistant to them.   The farmer has to be advised on the varieties suitable for cultivation in an area and the appropriate practices and precautions needed in every crop season, in order to derive the maximum possible benefit during each season.  The objective of transgenic technology is to derive cost effective benefits of the technology over a considerable period of time and not in a particular season or in a particular region in a season.   No crop variety has ever performed uniformly season after season in all regions of its cultivation.  

Ignoring the factors that control crop performance is poor crop husbandry.   Technology should not be blamed for ills befalling for reasons of poor management that lie beyond the realm of a particular technology.

Other articles in this series:
TRANSGENIC BT TECHNOLOGY: 1. BACILLUS THURINGIENSIS, BT PROTEINS AND TOXINS

TRANSGENIC BT TECHNOLOGY: 2. BT CROP VARIETIES

TRANSGENIC BT TECHNOLOGY: 3. EXPRESSION OF TRANSGENES

TRANSGENIC BT TECHNOLOGY: 5. SUBSTANTIAL EQUIVALENCE OF TRANSGENICS AND THEIR ISOGENICS

TRANSGENIC BT TECHNOLOGY:  6. BIOSECURITY

TRANSGENIC BT TECHNOLOGY: 7. BENEFITS

 January 1, 2009

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Genes and gene expression:  A gene, the basic unit of inheritance and diversity, is a segment of DNA containing a specific sequence of nucleotides (the building blocks of DNA). Genes determine the characteristics and life processes of organisms.   Each species contains several thousand genes.  Most genes occur in one of three combinations of two variant forms (for example, AA, Aa or aa).  The genotype of an organism is the specific and characteristic combination of variants of all genes the organism carries.  

A gene expresses through the synthesis of a protein or an enzyme (most proteins are enzymes), which is the means of functioning of a gene.   Gene expression 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.

Quantification of gene expression:  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 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 transgenic varieties.  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 semi-skilled worker can conduct the test.   However, the simplicity of the test itself is its Achilles’ heel.   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:

a) Kits from different sources vary in their details, such as whether the antibodies used were monoclonal or polyclonal (see the article on Immunology and Immunotechnology.  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 a proper solvent, an appropriate buffer.   Crushing a bit of a tissue 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.   The rate of 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., all of which may interfere with the extraction of all the protein in the tissue.     

Not observing these precautions would result in incomparable, unreliable and misleading data.

December 19, 2008

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com  

Specific Bt protein-encoding genes were isolated from Bacillus thuringiensis and incorporated into the genetic complements of several crop plants such as cotton, corn, rice, tomato, potato, soybean, and others, to develop transgenic Bt varieties, using complex yet elegant procedures of genetic engineering.  This results in a crop variety with a single systemic insecticide that kills specific caterpillars feeding on the respective crop.   For each crop the most damaging pest has been targeted, as for example, the bollworms of cotton, the stem borers of rice and corn and the stem and fruit borers of aubergine (brinjal, egg plant).   The objective is that, while the Bt proteins take care of the major pests, the rest can be controlled by conventional pest management practices.  

The choice of Bt genes depends upon the crop and the targeted pest, as most of the Bt toxins are insect group specific.   For example, the proteins encoded by the genes Cry1Ac and Cry2Ab control the cotton bollworms, Cry1Ab controls corn borer, Cry3Ab controls Colorado potato beetle and Cry3Bb controls corn rootworm.    

Transgenic Bt varieties:  A gene construct (or a cassette) consisting of the chosen Bt gene is made, along with other molecular components needed for its expression in the transgenic crop variety.  The construct consists of sequences of nucleotides (the building blocks of DNA, the genetic material) a) to initiate the expression of the selected gene, b) to promote such expression, c) the actual sequence for the gene and d) a nucleotide sequence to signal the completion of the process of expression.   This construct is then incorporated into the tissue of a (chosen primary) variety of the crop, and this is called an event.   A large number of plants are developed from the event, through micropropagation (tissue culture) for agronomic and biosecurity evaluation.   Since this primary variety may not be suitable for cultivation in all countries or even in different regions in the same country, the event has to be transferred into the genetic component of other varieties suitable for cultivation in different parts of the world.   For example, the event MON 531, containing the Cry1Ac gene, was used to develop the Bt cotton variety Coker 312, which is not suitable for cultivation in India.   The chosen Indian regional varieties were repeatedly backcrossed with Coker 312 to develop different Bt cotton varieties.   All Bt cotton varieties containing Cry1Ac gene and developed from MON 531 are marketed under the trade name Bollgard I.   In India there are over 135 Bt cotton varieties permitted for commercial cultivation in different parts of the country and most of them are Bollgard I varieties as they were developed from MON 531 and contain Cry1Ac gene, marketed by several seed companies under license from Monsanto and its partner Maharashtra Hybrid Seed Company (Mahyco).

Acquired resistance and refugium: A prolonged exposure to a toxin at sub-lethal doses may result in the development of gene-based resistance in organisms, called acquired resistance.   Famous examples of such acquired resistance are mosquitoes resistant to DDT and human pathogenic bacteria resistant to antibiotics, which are being so casually used, particularly in the developing countries.   There is a possibility of crop pests acquiring genetic resistance to Bt proteins in Bt crop varieties, due to natural variation in susceptibility to a particular toxin, in the caterpillar populations.   Nevertheless, over a decade of cultivation of various Bt transgenics in different countries, has not thrown up even a single instance of acquired resistance of the concerned pests to Bt toxins.
 
In order to de-accelerate the development of acquired resistance, the regulatory frame work in all countries has stipulated that a certain number of rows of the isogenic non-Bt plants should be planted along with the Bt crop and this is called the refugium (border or barrier).   A certain number of the caterpillars feeding on Bt plants may escape death and if there was mating among these worms, the resulting progeny are likely to be resistant to Bt toxins to various degrees.   Acquired resistance is a very slow process but may build up to significant levels if such mating continues for several generations.  The caterpillars feeding on the non-Bt refugium are not exposed to the Bt toxin and so would be susceptible to it.   In the presence of a refugium, a certain proportion of the progeny would be from the mating of Bt-exposed and Bt-unexposed worms, and this progeny would be far less resistant to the Bt toxin than the progeny from Bt-exposed worms.  The refugium is thus expected to retard the pace of acquired resistance.  

Cotton farmers are reluctant to lose the product form the non-Bt refugium and often no refugium is planted.   Cotton bollworms also feed on several other crops (polyphagous) and do not seriously affect the commercial product in them.   A non-cotton refugium in a cotton field will function as well as a cotton refugium and should be a viable alternative.      

Gene stacking: Most transgenics contain only one gene, such as for pest tolerance or herbicide tolerance.   In order to compound the benefits, more than one gene is used in the development of a transgenic, by gene stacking or pyramiding.  Transgenic cotton containing both Cry1Ac and Cry2Ab (Bollgard II) has been developed.  Possibilities are being explored to incorporate both pest and herbicide tolerance in the same variety.  In future, there would be transgenic varieties with three or even four different genes stacked.

Gene stacking can also occur in nature.   If two transgenic varieties of the same crop are tolerant of a different herbicide each, intercrossing of these two varieties may result in a hybrid tolerant of both the herbicides.  Similarly, the progeny of a cross between a pest tolerant and a herbicide tolerant variety may be tolerant to of both the pest and the herbicide.

December 19, 2008

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Transgenic technology, involving a wide range of pesticidal genes from the bacterium Bacillus thuringinesis (Bt), dominates the scenario of agricultural biotechnology.   At the same time, Bt technology is also the most focused target of vehement anti-tech activism.  

While the terms Bt cotton, Bt corn, Bt potato, etc., are familiar, the level of understanding of what the technology actually means, what it can and what it cannot do, is very poor.   A variety of issues such as the biology of Bacillus thuringiensis, its proteins, use of Bt as a biopesticide, transgenic Bt crops, benefits and limitations of the technology and biosecurity, are important facets of public awareness.  

Bt proteins are per se not toxic.  To function as toxins Bt proteins require a specific set of biochemical and biological parameters which are available for different Bt proteins only in specific insect groups. 

Bacillus thuringiensis:  Bt is a rod shaped, Gram-positive, soil bacterium, discovered in 1901.  Bt is among the most thoroughly studied bacterial species of agricultural importance, its diverse aspects having been researched for over a century.   The book ‘Bacillus thuringiensis: Biology, Ecology and Safety’ (T.R. Glare and M. O’Callaghan, 2000, John Wiley) refers to over 8,000 research publications by over 10,000 biologists, in over 60 years, and deals with most of the issues raised against the use of Bt.  Ignorance of this and other subsequent publications on Bt or a deliberate indifference to them, have resulted in a much exploited misunderstanding of Bt technology.

Concept of Bt:  The term Bt now refers to not a single simple species entity, but to a large group of subspecies and varieties, based on over 60,000 isolates, collected from all over the world.   There are more than 80 serologically characterized (using specific antibodies) types of Bt.

The question of difficulty in distinguishing Bacillus thuringiensis from the related pathogenic Bacillus cereus and Bacillus anthracis was adequately addressed (Ruud A de Maagd, Alejandra Bravo & Neil Crickmore, www.agbioworld.org : July 11, 2005).   When types of Bt can be identified serologically, a microbiologist can certainly distinguish the three species.  

Bt in nature: Bt is a universally occurring soil bacterium, isolated from several thousand soil samples from 80 different countries.   It commonly occurs also on the aerial parts of plants such as leaves and on even washed fruits and vegetables we consume.   It may be present in water, possibly as a wash off from the soil and plant surfaces.   Bt may be transported in the atmosphere, as inferred from its presence deep in the polar ice cap.

Bt grows and competes, but poorly in soil.   Bt or its proteins may persist for about 100 days in soils, for 24 hr in running water and for 12 days in stagnant water bodies.   Bt seems to require an association with plants and insects to perpetuate for longer periods in nature.

Bt as a biopesticide: Bt produces a wide range of insecticidal proteins that have been in use in pest control since 1938.   There are about a 100 biopesticides exclusively based on Bt and over 90 per cent of commercial biopesticides, used even in organic farming, contain Bt.  

Bt proteins and their encoding genes:  Bt produces a large number of proteins that are toxic to specific insect groups under specific conditions.   Bt also produces a) several enzymes, b) some compounds that lyse erythrocytes, and c) some that are enterotoxic to vertebrates.   Bt toxins are produced either within the bacterial cell (endotoxins), or on the cell surface (exotoxins).  

More than 170 toxin-encoding genes have been isolated form Bt collections.     Among the endotoxins, the insecticidal crystalline proteins, called the delta-endotoxins, are significant in Bt technology.  The crystalline proteins are described para-sporal, as they are co-produced and co-exist along with spores (the means of bacterial propagation), in the bacterial cells.   When the bacterial cell lyses to release the spores, the crystalline proteins are also routinely released into the soil.  

The names of the genes that encode the crystalline proteins are prefixed with ‘Cry’, as for example Cry1Ab, Cry1Ac, Cry9c, etc., and the proteins that are encoded by these genes are ‘Cry’ proteins.   The non-crystalline endotoxins are prefixed with ‘Cyt’.

Pest specificity of Bt  toxins:
Most of the Bt toxins are insect group specific.   Cry1Ac and Cry2Ab control the cotton bollworms, Cry1Ab controls corn borer, Cry3Ab controls Colarado potato beetle and Cry3Bb controls corn rootworm.   The Bt genes that are incorporated into different crops are species specific to moths and butterflies (Lepidoptera, having wings covered by scales).  

Pre-requisites for pesticidal activity of Bt proteins: 
The following conditions are essential for an effective insecticidal activity of the Bt proteins:

a) The pest must take a few bites of the plant tissue; Bt transgenics are not effective against sucking pests (Homoptera, with wings without scales), as they do not ingest plant tissue.

b) An alkaline environment (pH 9.5 and above) in the gut of the insect pest is essential for the Cry proteins to dissolve in the gut fluids and to be converted into an active molecule to function as an insecticidal compound.

c) The lining of the mid-gut (brush border) of the pest must have an appropriate receptor for a particular toxin to bind to.   The pest specificity of different Bt toxins depends upon the presence of appropriate receptors, which are absent form some pests, as evidenced by different Bt proteins being toxic to specific pest species.  The receptor bound toxin causes disturbance in the integrity of the gut wall, leading to leakage of the contents, followed by starvation and death of the pest.  

Fundamentally, the alkaline gut environment and the presence of an appropriate toxin binding receptor are crucial for insecticidal activity of Bt proteins. Basing on such requirements, the genes that encode pest specific toxins are chosen for developing different transgenic crops.

December 19, 2008

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Modern Biotechnology

Modern Biotechnology provides novel goods and services using organisms or their products, applying innovative scientific and engineering principles.  Modern biotechnology found several new applications in agriculture, medicine, industrial production and environmental management, using one or more of the following procedures:

a) recombinant DNA (rDNA) technology to incorporate new genes from an organism into the genome of another organism cutting across genetic relationships, resulting in transgenic organisms (with transgenes from the bacterium Bacillus thuringiensis into crop plants, or human insulin genes into bacteria and yeasts),

b) the control of gene expression through several different means (coffee seeds without caffeine through gene silencing or enhanced production of hepatitis B antigens through somatic hybridization).

Cloning of cells, tissues, organs and organisms through culture techniques is a very important tool in biotechnological procedures, but do not constitute biotechnology per se.

Medical biotechnology aims at a) safe, effective and inexpensive medicine, b) the rapid and low cost synthesis of purer industrial products, c) restoring damaged environmental components such as soil and water, d) better crops in terms of crop health, yield, quality and reduced labor and financial inputs, to help the farmer whose focus is lower cultivation inputs, higher quality and yield. Ingenious techniques have led to innovative crops that produce nutritionally important compounds (Golden Rice with β-carotene) or therapeutically active agents (vaccines, antibodies).   

Biodiversity

Biodiversity, the short form of Biological Diversity, refers to the variety of life in all its forms, levels and combinations and includes the diversity of ecosystems, species and genes, focusing on bioresources and gene pools. 

The composition of biodiversity is not static and it has changed and will continue to change gradually, responding to numerous factors inherent in the organisms or the environment, and their interactions.  

Biodiversity Loss

Some component or the other of biodiversity was always lost as a natural process, and was replaced by new diversity components over time. 

Sudden and catastrophic changes in the habitat due to natural causes (fires, earth quakes, cyclones and floods) or human intervention (excessive use of forest resources like timber, development projects such as townships, roads, dams) cause often irreplaceable loss of biodiversity.   

Agriculture has been the single largest cause of habitat destruction and biodiversity loss.  Nevertheless, agriculture has also created new and different kinds of diversity in the same environment. 

Impact of Genetically Engineered Products on Biodiversity

The impact of medical and environmental biotech products on biodiversity and environment is largely confined to the management of effluents and pollutants from the production sites.  Environmental biotechnology actually restores degraded environmental components like soil and water. 

Agricultural biotechnology caused very contentious arguments regarding its ‘impact’ on biodiversity and the environment.  A particular Bt gene (like Cry 1 Ac) and its product have no impact if the target pest (like cotton bollworm) of the gene is not present on a plant.  Herbicide tolerance genes have no impact if an appropriate herbicide is not sprayed.  Drought, flood and salinity tolerance genes have no impact in the absence of the respective stress factors.

Activists persistently projected GE technology as a serious threat to biodiversity and the environment.  Three issues are commonly raised in this context: a) Bt corn pollen are harmful to Monarch butterflies, b) corn transgenes have introgressed into native corn varieties in Mexico and c) Bt pollen are responsible for bee Colony Collapse Disaster (CCD) in North America and Europe.  All these claims have been challenged and disproved by the scientific community.  A February 2008 publication indicates that Cry 1Ab Bt proteins do not affect the performance of bumble bees in any manner.

Crop Variety Diversity and Genetically Engineered crops

The dimensions of crop diversity are reflected in the vast numbers of varieties of globally important crops such as rice (about 1,000,000 varieties), wheat (70,000), corn (45,000), and many others.  As crops are bred to improve over the existing varieties, the new varieties of a crop, preferred by the farmers and the consumers, displaced the older ones, resulting in a continuous reduction in the number of older varieties under cultivation.  The present day farmers prefer genetically engineered (GE) varieties of crops, which replace the conventional varieties.

Anti-tech activists argue that GE crops will destroy the existing gene pools of crops, since discontinued cultivation results in the disappearance of genetic traits that may prove to be valuable in future.  There are hundreds of thousands of crop seed collections in different institutions which may contain genes for some valuable traits.  Even though these seeds may not germinate now to produce plants needed for research, modern techniques of molecular biology could be used to rescue any useful genes from them. 

A comprehensive report on the impact of agricultural biotechnology on biodiversity from the Bern University’s Botanic Garden (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. 

Crop Field Diversity

Another aspect related to crop diversity is crop field diversity, constituted by a) the insects colonizing on the crop plants, b) other insects, birds and small animals that feed on the former, c) the pathogens of the crop and d) the weeds in the crop fields. 

Pest or pathogen tolerant GE crops affect only the target pest or pathogen.  They reduce application of chemical pesticides that harm non-target organisms and enhance populations of beneficial insects, facilitating visits by birds preying on insects, thus restoring crop field biodiversity that was restrained by excessive inputs of agricultural chemicals.  Reduced handling of chemical pesticides reduces the risk farm workers face from exposure to them. 

The composition of weeds in a given crop field depends upon the crop and agricultural practices, as for example the weeds in a rice field are not necessarily the same as those in sugarcane or corn or pulse crop fields in the same area.  Weeds cause over 30 per cent crop yield losses and so need to be removed effectively and scientifically.    

Activists insist that the weeds in crop fields are valuable components of diverse diets and that they are important as medicinal plants and so herbicides should not be used.  The crop field weeds are common species that are found easily in the same area and there is no special merit in them to be protected at the expense of crop productivity.

Anti-tech activists have also contended that the genes from GE crops would get incorporated into the wild and weedy varieties of the crops, through ‘gene flow’ and result in ‘super weeds’.  Nothing, in centuries of agricultural experience, or about 15 years’ of research and regulatory processing of several GE crops in different countries, has indicated such a possibility.  A ten year long experiment in the UK has shown that pest tolerant and herbicide tolerant GE crops have actually perished earlier than their non-GE counter parts.  None escaped into the environment to become a super weed.  Crops are heavily pampered with inputs and care, which are unavailable outside a crop field. 

A peer reviewed report of March 2007 stated 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. 

November 28, 2008

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C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

There are two kinds of ‘gene flow’, the natural processes of transfer of genes from one species/variety to another.  Gene flow through natural interspecific or intervarietal hybridization mediated by pollen is ‘vertical gene flow’, which results in the transfer of entire genomes.  The genes are transferred from the parent to the offspring and express only in the next generation, the offspring. 

The other kind is ‘horizontal or lateral gene flow’, which is a rare natural biological event that transfers a few genes at a time from one species to an unrelated species, without involving sexual reproduction. 

Crossability Barriers and Reproductive Isolation

Species and even their varieties do not cross with each other freely.  They are reproductively isolated, with their identity being maintained through several genetically controlled physical and/or physiological crossability (reproductive) barriers.  Species and varieties differ in chromosome number and structure, and genetic constitution, which are often serious impediments to the fertility of the offspring of hybrid crosses.  Reproductive barriers may also operate at one or more stages of pollen germination, viability, fertilization, embryo development and seed germination.  When reproductive isolation from reproductive barriers is strong, even persistent attempts at artificial hybridization have failed.  When such restrictions are weak, natural intervarietal or interspecific hybridization and gene transfer may occur. Even intergeneric gene transfer such as known from wheat (Triticum aestivum) to a distant wild relative (Aegilops peregrina) is a possibility, though extremely rare.  Repeated (back) crossing with the donor parent results in introgression, the stable incorporation of genes from one differentiated gene pool into another.

Biosecurity of Genetically Engineered Products

Biosecurity, the safety of genetically engineered (GE) products as food and feed and the safety of the environment, is a matter of concern both for the biotechnologists and the critics.  It was the scientists, not the anti-GE activists, who first thought of biosecurity issues, investigated the extent of risk and devised means to mitigate any possible risk.  Before commercialization, each GE product is subjected to a very lengthy and stringent biosecurity regime and only when the product is found to be safe and has a favorable risk-benefit ratio; it is released for public use. 

The opponents of GE (transgenic) crops and products made gene flow a contentious issue and raised several biosecurity concerns in their anti-GE arguments. 

Vertical gene flow

The apprehension is that pollen from the transgenic crops pollinate non-GE varieties (gene escape) causing genetic contamination or pollution.   According to the anti-tech activists such an event would cause untold horrors to the crops and the environment.  The activists argue that the non-target organisms would be affected and the transgenics would become super weeds.

Issues of gene flow should take into consideration several factors related to the reproductive biology of the species involved.  Vertical gene flow requires a) populations of two different varieties/species that grow together in the same geographical area (sympatric), b) they should be synchronous in flowering time, pollen viability and stigma receptivity, and c) be genetically compatible allowing random mating (panmictic) between the two populations, which means that there are no reproductive barriers preventing hybridization or at least that they are weak. 

The means of pollination, extent of natural cross-pollination, duration of pollen viability and stigma receptivity, extent of cross-fertilization, embryo survival and seed and fruit set are the critical events in gene flow.   The degree of fertility of the hybrids depends upon the degree of genetic compatibility.  Analysis of the hybrids would indicate the extent of gene transfer.  If the transferred genes add to the competence of the species/variety (adaptive value), and if there is repeated back crossing with the donor parent, the genes will be fixed in the population (introgressed) which in course of time differentiates as a new variety or even a new species.

Gene flow can occur between transgenics and the isogenic varieties of a crop, to the same extent as was happening earlier between non-transgenic populations of that crop.  Transgenes do not promote promiscuity.  Unintended transgenic hybrids resulting from gene flow from GE crops to wild or weedy relatives is possible, but cannot easily or routinely occur.   

Hybridization experiments have shown that intercrossing among the indica varieties of rice was 3.9 per cent and among japonica varieties it was 3.6 per cent, while practically there was no intercrossing between indica and japonica varieties.  Gene flow of an herbicide resistant gene from a cultivated rice variety to a weedy rice variety was 0.011 to 0.046 per cent and from a cultivated variety to a wild variety was 1.21 to 2.19 per cent. 

A comparative analytical study of GE and non-GE corn has shown that the transgenic content of a non-GE population was 0.9 per cent at 10 m from the GE population.   The claim that corn transgenes have introgressed into native corn varieties in Mexico was disproved.

In Canola, the presence of an herbicide resistant gene in a non-GE population was 0.1 per cent at 5 m, 0.02 per cent at 10 m and zero at more than 15 m distance, from the GE variety.

Since the conditions under which these experiments were conducted do not exist in nature, the possibility of gene flow and of the impact of such an event would be even less. 

The transgene, such as herbicide resistance in rape seed, may introgress and the gene may even persist for some time in a very small fraction of the population for a while.  A recent report in Molecular Ecology (October 2007) on transgenic herbicide tolerance genes in two species of Brassica indicates that such individuals have intermediate genome structure and their frequency rapidly decreases from about 44 per cent to 2.5 per cent in three years.  They have reduced pollen viability and reduced male fertility. 

The mere presence of a transgene in a plant is of no consequence. The question is what is the impact of few miniscule weedy populations with rapidly deteriorating vigor in agronomic terms? A Bt gene has no impact if the intended pest is not present and an herbicide tolerant gene is of no consequence if an appropriate herbicide is not sprayed.  The gene should introgress and should impart an advantage to the recipient population over the others which do not have the transgene. 

Horizontal gene flow

The other set of anti-GE arguments relate to the horizontal or lateral gene flow, raised in the context of antibiotic resistance genes used as selectable markers in GE technology.  It is argued that these genes would be taken up by soil pathogens which would become resistant to the antibiotics, and people infected by them cannot be cured by that antibiotic, leading to incurable diseases and death.

In horizontal gene flow which does not involve sexual reproduction and bypasses reproductive isolation, the few genes transferred can express in the same cell generation.  In nature, horizontal gene flow is a fundamentally prokaryotic (organisms with no nucleus or cell organelles such as bacteria) phenomenon.   Other natural examples of horizontal gene flow relate to endoparasites and their hosts, which share several genes as a result of millions of years of co-habitation and co-evolution. 

Horizontal gene flow occurs through one of five different biological processes known as transformation, conjugation, transfection, lysogenization and transduction.  Recombinant DNA (rDNA) technology that involves transformation is a good example of artificial horizontal gene transfer that makes transgenics possible irrespective of genetic relationship. 

The issue of horizontal transfer of antibiotic resistance marker genes from transgenic plants to bacteria was discussed in depth at an international symposium on the Biosafety of GE organisms in 2000.  The consensus was that such an event is very unlikely and even if it happens it would not contribute significantly to the horizontal spread of the genes in question, for three reasons: a) the antibiotic resistance genes are already widespread in bacterial populations, b) there was no experimental evidence for horizontal gene transfer from plants to bacteria, and c) horizontal gene transfer events from transgenic plants to bacteria have not been detected.  There is no change in this position till to date.

A recent paper in Transgenic Research (June, 2007) concluded, supported by numerous studies, most of which are commissioned by some of the very parties that have taken a position against the use of antibiotic selectable marker gene systems, is that there is no scientific basis to argue against the use and presence of selectable marker genes in transgenic plants.

November 28, 2008

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