CaMV 35 S Promoter

cropped-picture1.jpgCaMV 35 S Promoter

The CaMV 35 S promoter, most commonly used for constitutive expression of foreign proteins in plants is a sequence of 528 base pairs. In the viral genome, the nucleotide sequence of CaMV P6 protein precedes the 35 S promoter sequence. However, the third part of P6 gene sequence overlaps the promoter sequence.

  1. The 35 S promoter sequence used in transgenic plants does not have ATG at 5’ end nor a translation start site which excludes the possibility of plant cells making the third domain of P6 protein.
  2. Expression of P6 sequence needs a plant active promoter of its own.
  3. No scientific literature has been reported on any allergenic properties of CaMV and no similarities have been shown to know allergens.
  4. Most likely the P6 protein is not an allergen as concluded by Podevin 2012.
  5. P35S variants do not contain ORFs that encode for proteins that have allergenic or toxic properties.
  6. These sequences/proteins (like P6) are ubiquitous in nature and have been part of mammalian diets even before human beings evolved.
  7. We find plant viral genomes in most crops and plant species.  This is because plants and viruses have been exchanging genes since millions of years.
  8. The cauliflower, broccoli, knoll-khol and cabbage we buy in local markets carry CaMV virus due to natural infestation and conatain P6 protein.
  9. There is a history of safe use of CaMV by humans both in rDNA form and in natural forms.
  10. Fifty four commercialized GM events (carrying CaMV 35 S Promoter) have passed biosafety tests.

Directions:

  1. The possibility of transgenic plants expressing P6 protein (third fragment)  can easily be checked by using monoclonal antibodies to P6 protein.
  2. The quantitative analysis to measure the amount of third fragment of P6 can be carried out.
  3. Even if the third domain/fragment of P6 is produced in plant cells, it needs to be checked if it is functional.
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GM Events-GUS

COMMERCIAL GM CROP EVENTS CARRYING UidA gene (Gus)

beta-D-glucuronidase  (Escherichia coli)

Approved/deregulated GM Crop events at global level

Crop

Event(s)

Trait

Developer

Countries

Papaya 55-1/63-1 Ring spot virus resistance Cornell University USA, Canada
Sugar beet GTSB77 Glyphosate tolerance Novartis, Monsanto USA, Australia, Japan, Philippines
Soybean G94-1, G94-19 and G168 Modified oleic acid Dupont USA, Australia, Japan, Canda
Soybean W62 and W98 Basta tolerance Bayer USA
Plum C5 Pox virus resistance USDA USA
Cotton 15985 (BG II) Insect resistance Monsanto USA, EU, India and 10 others
Cotton LL Cotton25 x MON15985 Basta tolerance +

Insect resistance

Bayer Japan, Korea, Mexico
Cotton MON15985 x MON88913 Insect resistance + Glyphosate tolerance Monsanto Australia, Colombia, South Africa and 3 others

Biosafety of E. coli beta-glucuronidase (GUS) in plants.

Transgenic Res. 1998 May;7(3):157-63.

Gilissen LJ, Metz PL, Stiekema WJ, Nap JP.

Department of Molecular Biology, CPRO-DLO, Wageningen, The Netherlands.

Abstract

The beta-glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers.

 

Relevance of Bt Crops

Relevance of Bt-Crops

Introduction

Insect pest management in agriculture is important to safeguard crop yields and productivity. Chemical insecticides that effectively control insect pests have been proven to be harmful to human health and environment. There is a need to reduce the dependence on pesticides by using safer alternatives to manage insect pests. Many insecticidal proteins and molecules are available in nature, which are effective against agriculturally important pests but innocuous to mammals, beneficial insects and other organisms. Insecticidal proteins present in the soil borne bacterium, Bacillus thuringiensis (Bt), which has demonstrated its efficacy as a spray formulation in agriculture over the past five decades, have been expressed in many crop species with positive results. Three such transgenic crop species (cotton, corn and potato) have been commercialized with substantial benefits to the farmers. Bt crops occupy an area of 42 million hectares out of the global transgenic area of >150 million hectares in 2011. In India, Bt cotton was cultivated in more than 10.0 million hectares.
Bacillus thuringiensis
Bt is a gram-positive, aerobic, endospore-forming bacterium belonging to morphological group I along with Bacillus cereus, Bacillus anthracis and Bacillus laterosporus. All these bacteria have endospores. Bt, however, is recognized by its parasporal body (known as the crystal) that is proteinaceous in nature and which possesses insecticidal properties. The parasporal body comprises of crystals varying in size, shape and morphology. The crystals are tightly packed with proteins called protoxins or d-endotoxins. The first record on Bt goes back to 1901, when a Japanese microbiologist Ishiwata discovered a bacterium from diseased silkworm larvae, which he named Bacillus sotto. Between 1909 and 1912, Berliner, working at a research station for grain processing in Germany, investigated an infectious disease of the Mediterranean flour moth and described a spore-forming bacterium as the causative agent and designated it as B. thuringiensis.

There are many subspecies and serotypes of Bt with a range of well-characterized insecticidal proteins or Bt toxins (d-endotoxins). At present it has been estimated that over 60,000 isolates of Bt are being maintained in culture collections worldwide. Known Bt toxins kill subsets of insects among the Lepidoptera, Coleoptera, Diptera and nematodes. The host range of Bt has expanded considerably in recent years due to extensive screening programs. Currently more than 160 different genes encoding Bt toxins have been cloned. Recent information about Bt toxins/genes can be obtained from http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/.

Mode of action

The Bt toxins exert their toxicity by forming pores in the larval midgut epithelial membranes. Initially the protoxins are activated in the midgut by trypsin-like proteases to toxins. The active toxins bind to specific receptors located on the apical brush border membrane of the columnar cells. Binding is followed by the insertion of the toxin into the apical membrane leading to pore formation. The formation of toxin-induced pores in the columnar cell apical membrane allows rapid fluxes of ions. Different studies revealed that the pores are K+ selective, permeable to cations, anions or permeable to small solutes like sucrose, irrespective of the charge. It appears that the toxin forms or activates a relatively large aqueous channel in the membrane. The disruption of gut integrity results in the death of the insect from starvation or septicemia.

Applications of Bt

The first practical application of Bt dates back to 1938 when it was sold as ‘Sporeine’ in France for the control of European corn borer. The growing realization that organic insecticides are deleterious to the environment and human health spurred a renewed interest in Bt in the 1960s, which led to the introduction of viable Bt biopesticides like Thuricide and Dipel. Bt is the most popular biological control agent with worldwide sales of about $100 million. Bt spray formulations comprise 5% of total global pesticide market. The use of conventional Bt biopesticides, however, was found to have limitations like narrow specificity, short shelf life, low potency, lack of systemic activity, and the presence of viable spores.

An elegant and the most effective delivery system for Bt toxins is the transgenic plant. The major benefits of this system are economic, environmental, and qualitative. In addition to the reduced input costs to the farmer, the transgenic plants provide season-long protection independent of weather conditions, effective control of burrowing insects difficult to reach with sprays and control at all of the stages of insect development. The important feature of such a system is that only insects eating the crop are exposed to the toxin. Genetic transformation of almost all the major crop species is now feasible with the development of an array of techniques ranging from the Agrobacterium approach to electric discharge-mediated particle acceleration procedure (Pattanayak et al., 2000).

The initial attempts to introduce and express native Bt genes encoding protoxins or truncated toxins in plants were not very successful because the levels of toxin expression were very low. In 1990, researchers at Monsanto made a significant advancement in the expression of Bt genes in plants. They noticed that Bt genes were excessively AT rich in comparison with normal plant genes. This bias in nucleotide composition of the DNA could have a number of deleterious consequences to gene expression because AT-rich regions in plants are often found in introns or have a regulatory role in determining polyadenylation. Introduction and expression of codon-modified genes in crop plants conferred significant protection against target pests. The first transgenic Bt-crops viz., cotton, corn and potato were commercialized in USA in 1995 and 1996. Currently more than a dozen countries cultivate Bt-crops. In 2009, China’s Ministry of Agriculture released biosafety certificates for Bt rice Huahui No. 1 and Bt Shanyou 63 with possible planting in 2012. Bt-cotton was permitted for commercial cultivation in India in 2002, which has brought about a revolution in cotton production.

Benefits of Bt-crops

In the past fifteen years, all the countries that have introduced Bt cotton and maize have derived significant and multiple benefits.  These include increases in yield, decreased production costs, a reduction of at least 50% in insecticide applications, resulting in substantial environmental and health benefits to small producers, and significant economic and social benefits.

Bt maize: Globally, the farm level benefit of using Bt maize cumulatively since 1996 has been $6.34 billions. In terms of the total value of maize production from the countries growing Bt maize in 2008, the additional farm income generated by the technology is equal to a value added equivalent of 2.2%.

Bt cotton: Globally, the farm level benefit of using Bt cotton cumulatively since 1996 has been $15.61 billions.  In terms of the total value of cotton production from the countries growing Bt in 2008, the additional farm income generated by the technology is equal to a value added equivalent of 19.3%. The economic benefits of Bt cotton in India have been enormous and well documented (http://www.apcoab.org/publications.html). Millions of farmers benefited from Bt cotton in developing countries such as China, India and South Africa where Bt cotton contributed to the reduction in poverty by increasing incomes of small farmers.

The environmental benefits of cultivating Bt crops are:

  1. Reduction in use of pesticides: The cumulative reduction of pesticide applications due to Bt crops from 1996-2008 is estimated to be 356 million kilograms of active ingredient.
  2. Less insecticides in aquifers and the environment: The substantial decrease in insecticides associated with the cultivation of Bt cotton has lead to significant decrease in insecticide run off into watersheds, aquifers, soils and generally into the environment. More widespread global cultivation of Bt-cotton will further improve the water quality.
  3. Reduced farmer exposure to insecticides and improvement of human health: Chemical insecticides used in cotton have high toxicity to humans and animals. Substitution of the chemical insecticides with Bt cotton has clearly reduced the risks to farm workers and to others in the farm community who may be exposed. These effects are particularly important in developing countries where modern application techniques are neither always adopted nor available for use.
  4. Increased populations of beneficial insects: The global use of broad spectrum insecticides on cotton has adversely affected and decreased the populations of non-target species including the arthropod natural enemies that can provide effective control of non-lepidopteran pests. Various studies confirmed that the arthropod natural enemy populations in Bt cotton are greater than in non-Bt cotton. In addition to reducing the number of sprays for the bollworm/budworm complex, Bt cotton has also reduced the number of sprays for other insects such as thrips and aphids. This effect has been attributed to higher populations of beneficial predators and parasitic insects that are eliminated by insecticide sprays.
  5. Reduced risk for wildlife: Reduction in the use of insecticides, many of which are highly toxic to wildlife will reduce the risks to mammals, birds, bees, fish and other organisms. Many birds are dependent on insects for food and their elimination through the use of insecticides deprives birds of their food source.
  6. Reduced fuel and raw material consumption and decreased pollution: Lowering the demand for insecticides, through the use of Bt cotton reduces tractor fuel usage as a result of reduction in number of sprays, which in turn reduces air pollution.

Biosafety

Safety of Bt toxins in terms of toxicity and allergenicity towards mammals and other non-target organisms is well documented. Lack of receptors that bind to Bt toxins and instant degradation of Bt toxins in human digestive system makes them innocuous to human beings. Community exposure to Bt toxins/spray formulations over a period of six decades has not resulted in any adverse effects. Human volunteers consumed Bt toxins at very high concentration without any undesirable effects. Lack of homology to any allergenic protein/epitope sequences makes Bt toxins non-allergenic. Extensive testing of Bt cotton and Bt maize has proven that the crops and the products derived thereof are totally safe.

Insect resistance management

One of the important considerations of introduction of insect resistant transgenic plants into environment is to prevent the development of resistance in insects towards Bt toxins. Instances where resistance has developed in laboratory and field populations of Diamondback moth and Indian meal moth point towards great caution to be applied in the implementation of this technology. Various resistance management strategies have been suggested to prevent or delay the development of resistance to Bt. Proposed strategies include the use of multiple toxins (stacking or pyramiding), crop rotation, high or ultra-high dosages, and spatial or temporal refugia. Gene stacked Bt cotton is currently available which will ensure the longevity of the technology. The most promising and currently practical strategy is that of using refugia. This strategy calls for reducing the possibility of long-term impact by preventing the creation of a resistant population. This is achieved by ensuring that there are always plenty of susceptible insects nearby for the few resistant ones to mate with.

Future perspectives:

Introduction of various Bt crops is extremely important towards achieving the goals of eco-friendly and sustainable pest management in agriculture. Deployment of Bt vegetables will go a long way to protect human health by reducing the pesticide use in vegetable crops, which are often consumed raw. There is an urgent need to develop pod borer-resistant pulses, which would usher in a revolution in pulse production in India. Caution is needed, however, while introducing a particular Bt gene in multiple crops. Large-scale bioprospecting of Bt strains will provide us novel Bt genes, which can widen the spectrum of insecticidal activity as well as ensure resistance management.

Bt Crops

Bt crops

There have been several misconceptions about Bt cotton, Bt brinjal and other genetically modified  (GM) crops.  Some of these reports emanated because of the basic misunderstanding of the scientific facts related to a bacterium called Bacillus thuringiensis (Bt). As a result the whole gamut of biotechnology applications in agriculture has been under cloud in public perception.

In this article, I seek to dispel some of the misconceptions about Bt crops and emphasize how beneficial these crops would be for the mankind and the environment. Ever since Man domesticated crops ten millennia ago the major problem encountered by the crops is that of insect pests. The crop varieties selected by Man over several centuries do not have natural capability to resist insect attack. Discovery of insecticides in 1940s has brought about a sea change in the way we protected crops from insect damage. However, the adverse effects of many of the pesticides were realized in 1960s as highlighted in the most well known book “Silent Spring” written by Rachel Carson who poignantly described the disappearance of birds because of widespread use of pesticides such as DDT. Protection of environment has got increased attention and research into safe biological pesticides has got a boost.

Even much earlier in 1938, a biological pesticide by name ‘Sporeine’ was used by corn farmers in France to control Corn Borer with good results. This spray pesticide was a crude preparation of a naturally occurring soil bacterium called Bacillus thuringiensis, in short Bt.

Bt is a bacterium that exists all over the globe. It has been in existence for millions of years. A Japanese scientist Ishiwata discovered it in 1901. It was re-discovered by a German scientist Berliner in 1911 who found that the bacterium has insecticidal properties. Extensive research on Bt in 1960s and 1970s revealed that a class of proteins called delta endotoxins are responsible for the insecticidal property and that these proteins do not cause any harm to mammals including human beings. In addition, the endotoxins are harmless to beneficial insects such as honeybees, ladybird beetles, spiders, mites etc. A multi million dollar industry based on Bt spray formulations has come into being in western countries. Since 1970s, Bt spray formulations have been in extensive use for pest management in fruit and vegetable crops, forestry and animal health (control of lice). Certain Bt formulations are used in controlling mosquitoes too.

Extensive research on various aspects of Bt including search for novel Bt strains, safety of Bt proteins, biochemistry and molecular biology of Bt insecticidal proteins has been carried out in 1980s. Bt has been found to be safe for human beings, other mammals, soil dwelling organisms etc. The biochemical nature of Bt proteins makes them safe towards non-target organisms because a counterpart protein called Bt protein-receptor is absent in the guts of such organisms. Receptor protein is essential for Bt protein to act. In addition, the human digestive system is highly acidic in which Bt proteins are instantly degraded. Human volunteers in USA consumed enormous quantities of pure Bt proteins without any adverse effects.

Early 1990s witnessed a revolution in plant biology research. Powerful techniques were discovered by which foreign genes can be introduced and expressed in plant cells. This opened up a plethora of opportunities to make crop plants resistant to pests, diseases etc. The genes responsible for the production of pesticidal proteins were isolated from Bt and expressed in important crops such as cotton, maize and potato. These genetically modified (GM) crops were approved by USA in 1995 after rigorous testing for bisafety and environmental safety. Currently, Bt crops are cultivated in thirteen countries including India. Bt crops occupy about 40% of the global area of GM crops (160 million hectares). Several studies have documented the social, economic and environmental benefits of Bt crops. Protection of environment in terms of reduced pesticide use is the major benefit. Similarly, conservation of biodiversity that includes beneficial insects, non-target organisms such as birds and other wild life has been ensured. There have been several reports of the benefits of Bt cotton in India in the past eight years. No scientifically proven adverse effects of Bt crops have been reported in the past sixteen years around the world.

Bt crops such as Bt cotton are only protected from insect attack. One cannot expect an increase in the yield because of Bt. The Bt crop varieties are as susceptible as other crop varieties to drought, diseases and other environmental factors. The benefits are basically economical and environmental because the pesticide consumption is reduced. Indirectly, Bt crops help protect human health, animal health, food and water quality etc.,

There is an urgent need to develop Bt varieties in vegetables, pulses and staple food crops such as rice primarily to safeguard our health and protect biodiversity and the environment in general. It is all the more important in vegetables because several of them are consumed raw and vegetables laced with pesticides or their residues are highly deleterious to our health. The efforts being made in various public research laboratories should not be jeopardized because of misconceptions about Bt crops. A lot is at stake!

It is generally agreed that GM organisms (GMOs) including Bt crops should be thoroughly tested for their biosafety, environmental safety and protection of biodiversity before their release into the environment. In addition, the GMO regulatory system should be robust, transparent and amenable to public probity.

References:

  1. OECD 2007. Consensus document on safety information on transgenic plants expressing Bacillus thuringiensis derived insect control proteins. ENV/JM/MONO (2007) 14.
  2. WHO 1999. Microbial Pest Control Agent BACILLUS THURINGIENSIS. WHO, Geneva. ISBN 92 4 157217 5
  3. Karihaloo J. L. and Kumar P. A. 2009. Bt-Cotton in India: A Status Report. Asia-Pacific Consortium on Agricultural Biotechnology, New Delhi.
  4. Kumar P A, Sharma R P and Malik V S. 1996 Insecticidal proteins of Bacillus thuringieensis. Advances in Applied Microbiology. 42: 1-43.
  5. Glare, T. R. and O’Callaghan, M. 2000. Bacillus thuringiensis: Biology, Ecology and Safety. John Wiley, Chichester.
  6. NAAS, 2011. Biosafety Assurance for GM Food crops in India. Policy Paper 52. NAAS, New Delhi.