Plant Biotech Blog

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

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

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