Transgenic Crops: A Close View

Read this article to learn about the various types of transgenic crops:

(1) Insect pest control (2) Herbicide resistance (3) Virus resistance (4) Abiotic stress tolerance (5) Quality improvement (6) Pharmaceutical and industrial use (7) Environmental impact and (8) Edible vaccines.

1. Insect Pest Control:

The most talked insect pest control through use of trans-gene is Bt gene (from Bacillus thuringiensis) producing toxin. The adoption of insect resistant transgenic crops have been increasing annually since the commercial release of first generation maize and cotton expressing a single modified B. thuringiensis toxin (Bt) ten years ago.

Studies have shown that these Bt crops can be successfully deployed in agriculture, which has lead to a decrease in pesticide usage, and that they are environmental friendly. However, sustainability and durability of pest resistance continued to be discussed. Now, scientists are developing second and third generation insect resistant transgenic plants and examine the proposed models for longevity of such resistance.

First Generation Transgenic Plants:

Transgenic plants containing only marker genes, which are useful in the development of transformation systems.

Second Generation Transgenic Plants:

Transgenic plants containing, in addition to the selectable marker, one or two transgenic encoding simple agronomic traits (such as pest and herbicide resistance).

Third Generation Transgenic Plants:

Transgenic plants that contain multiple transgenes targeting multiple pests and disease, often in a temporal or spatial manner. These might also express additional value added or agronomic traits. By using a variety of technique, it has become possible to transform plants with foreign genes. Expression of foreign genes in plants makes it possible to produce a very wide range of new plants varieties. Transgenic plants have been developed to be resistant to a range of environmental stresses, including insects, viruses, herbicides, pathogens and salt stress to have flower with modified colour to a modified nutritional content including modifications in amino acids, lipids, discolouration and sweetness.

Bt Gene and Toxin (Bacillus Thuringiensis):

Several species of bacteria produce protein in abundance. When insect larvae ingest these bacteria with their food, protein present in bacteria kills larvae. The most widely studied of these bacteria is Bacillus thuringiensis or Bt in short.

This species lives all over the world. When these bacteria form spores, they also form a large crystal like structure in the bacterial cytoplasm, which is made out of protein. This bacterium comprises a number of different strains and subspecies, each of which produce’s different proteins (toxin) that can kill certain specific insects. Insecticidal toxins from some strains of Bt is shown in Table 17.1.

One of the proteins in the crystal like structure is called the Bt-protoxin. When insect larvae eat the bacterial cells along with leaves, the spores and the crystalline like structure containing the proteins are released in the larval gut, where the digestive enzymes cleave the protoxin producing an active toxin.

Protoxin activated within its gut by the combination of alkaline pH (7.5-8.0) and specific digestive proteases. The toxin binds to the membrane of the epithelium cells of the gut, inserts itself into that membrane and creates an ion channel through which other molecules (e.g., ATP) can freely pass. Punctured by many holes, the gut cell cannot survive long, so the insect larvae starve for lack of nutrition and ultimately die. Because conversion of the pro-toxin to the active toxin requires both alkaline pH and the presence of specific proteases, such conditions are not present in mammals and hence they are safe from the pro-toxin.

No significant role for the bacterium has been attributed to the parasporal crystal structure. The parasporal crystal usually consists mainly of protein (~95%) and small amount of carbohydrate (~5%). The crystal protein can generally be dissociated by mild alkali treatment into subunits. The insecticidal toxins of B. thuringiensis strains can be grouped into four major classes: Cry-I, Cry II, Cry III and Cry IV. This is based on insecticidal activity against various insect. These toxins are further classified in sub classes and sub groups according to DNA sequence of the toxin gene, e.g. Cry I gene has six sub classes (Cry IA to F) and Cry I has subgroup (Cry IA a to c).

As a result of co-evolution between insects and their pathogens, there is host specificity between Bt toxin and the membranes of the gut cells. The Bt toxin of a particular Bt strain will bind to the gut of Lepidoptera larvae, or only some species of Lepidoptera, but not to other. When toxin does not bind, there is no effect on the cells that line the gut, and the larvae do not die. Thus some Bt toxin will kill lepidoptera (butter flies and moths), other coleopteran (beetles and weevils) and others diptera (mosquitos).

For the biological control of insect pests, approximately 1.3 x 10⁸ to 2.6 x 10⁸ spores per sq foot of the target area are applied. Administration of the spores is timed to coincide with the peak of the larval population of the target organism.

Bt. Subspecies kurustaki contains a pro-toxin gene on one of seven different plasmids that approximately 2.0, 7.4, 7.8, 8.2, 14.4, 45 and 71kb in length. Pro-toxin is 130 kDa, therefore, not present on small plasmids. This gene has been transferred to other bacteria to kill mosquito larvae as well as gene has been modified to produce toxin during vegetative phase of bacterial growth rather than only during sporulation. Thus it is possible to produce toxin continuously in fermentor by growing bacteria. It has also been attempted to increase the host range.

This Bt toxin has been used in several ways to control the insects. A relatively simple way is to grow the Bt bacteria, dry them out, and prepare the heat killed and dried bacteria in such a way that they can be sprayed or dusted on crops. These preparations are initially highly effective, but the Bt pro-toxin is not stable after product is sprayed on plants. The Bt pro-toxin crystals are released from the bacteria and pro-toxin quickly disappears from the plants.

Scientists at Mycogen, a biotechnology company in San Diego, California (USA), introduced a Bt gene in a different bacterium (Pseudomonas fluorescents). These bacteria can readily be grown in large fermentors, killed and then formulated as a spray. With this bacterium the pro-toxin crystal remain in the bacterial cells, and as a result they are stable even after they have been sprayed on the plants.

The spraying Bt toxin works well with insect larvae that live on the surfaces of leaves, but would be less effective with insect larvae that live in soil or larvae living inside the plants. To control these insects, scientists have transferred Bt gene using particle gun transfer system in cotton, tomato, tobacco, potatoes, and other crop plants. Transgenic plants produced containing Bt gene are listed in Table 17.2.

Constitutive or Tissue Specific Expression:

Although constitutive expression of insecticidal trans-gene products has provided high levels of resistance in crop plants, tissue-specific or inducible expression might be desirable under some circumstances. Because the epidermal cells are first to be attacked by insects, defense genes expressed under epidermal cell-specific promoters (e.g., CEF6, an enzyme of curricular wax production) might be useful.

Phloem feeding insects can be targeted using the root phloem specific promoter AAP 3, the phloem specific pumpkin promoter PPZ and the rice sucrose synthase RSS promoter. Progress is being made with chemically inducible promoters, including those induced by ethanol, tetracycline, copper, glucocorticosteriod hormones and steroidal and non-steroidal ecdysone agonists.

2. Herbicide Resistant Plants:

Certain herbicides can be used as pre-emergence herbicides to kill weeds before the crops are planted. If the crop plants are resistant to these chemicals then they can be used with the crop plant (post emergency). By understanding the mechanism of action of these herbicides and development of resistance by certain bacteria to such chemicals can provide clone for developing herbicide tolerant plants. Some plants or bacteria are resistant because they have an enzyme that detoxifies the herbicides.

In other words they possess a gene for this action. Transfer of this gene to a crop plant should protect the crop plant by same action or mechanism. Some plants or bacteria become resistant to herbicide because of mutation in the target enzyme (or gene) and because of this change they are no more sensitive to herbicide or are not damaged by herbicides. The enzyme can work in presence of herbicide. Therefore the detoxifying mechanism or change in affected enzyme can make the organism herbicide tolerant.

Glyphosate (a herbicide) act by inhibiting one of the enzymes that is necessary for the synthesis of amino acids in the chloroplast. Glyphosate, initially produced and marketed by Monsanto under the trade name Roundup®, is widely used as non-selective herbicide. It effectively kills 76 of the world’s 78 worst weed species.

Scientist at Monsanto isolated a gene for an enzyme involved in amino acid biosynthesis enzyme EPSP-synthase (5 enol pyruvinyl shikimate 3-phosphate synthase) from resistant E. coli bacteria. They modified the gene in such a way that it could be expressed in plants, and then transferred it to plants e.g., tobacco, tomato and soybean.

Expression of bacterial gene in plants required a control region that would direct the expression at the gene in the plant (because bacterial control regions do not work in plants).

In addition to this the gene had to be modified in such a way that the enzyme, which is synthesized in cytoplasm, would be transported to chloroplast. This is important that when gene of prokaryotic origin is used, the product should be transported to right cellular compartment in the plant. This should not affect the quantity or quality of yield. The gene has been successfully transferred in soybean where the plants showed resistance without change in yield.

Phosphinothricin is a herbicide that acts by inhibiting another enzyme necessary for amino acid biosynthesis (glutamine synthetase) and nitrogen metabolism. This enzyme converts ammonia to glutamate. Inhibiting the activity of this enzyme leads to rapid accumulation of ammonia within the plant cell. Higher concentrations of ammonia are toxic to the cell. Phosphinothricin, produced and marketed by Hoechst AG under the trade name Basta®, is also a very effective non selective herbicide. This product is related to an antibiotic that is also a herbicide, ‘produced by the fungus Streptomyces hygroscopicus.

Scientists at plant Genetic systems, Belgium obtained a gene from this fungus that encodes an enzyme that converts phosphinotricin to a non-herbicidal derivative by combining it with a cell metabolite. This gene, known as bar-gene, has been transferred in tobacco and potato, where it is expressed showing herbicide tolerance in these plants. The yield performance of the plants remained unchanged.

Herbicides are simply chemical compounds that kill or inhibit the growth of plants without deleterious effects on animals. Herbicides usually inhibit processes that are unique to plants, e.g. photosynthesis. Mostly herbicides act as inhibitors of essential enzyme reactions. Any change which can reduce the inhibitory effect of herbicide will provide increased herbicide tolerance.

Glyphosate acts by inhibiting the enzyme 5 enol pyruvinyl shikimate 3 phosphate synthase (EPSP synthase), an essential enzyme in the biosynthesis of the aromatic amino acid, tysosine, phenylalanine and tryptophan. These are essential components in the diets of higher animals. Therefore higher animals do not contain EPSP synthase, and are not affected by glyphosate.

Glyphosate does inhibit the EPSP synthase of microorganisms as well as those of plants. Selection of organisms is made on inhibitory concentration of herbicides by growing them in presence of herbicide. This way researches isolated glyphosate tolerant mutant of Salmonella typhimurion, Aerobacter acrogens, and Escherichia coli.

In bacteria, EPSP synthase is encoded by the aero A gene. When aeroA genes (with plant promoter and adenylation signals) were transferred in plants, the transgenic plants showed increased tolerance to glyphosate. In plants, aromatic amino acids are synthesized in chloroplasts, but gene for EPSP is localized in nucleus. Therefore, a protein is attached to EPSP synthase, which translocate the EPSP synthase into chloroplast, where the protein is removed by cleavage. It has been shown that the petunia transit peptide will target the E. coli aeroA gene product into tobacco chloroplasts and will impart glyphosate tolerance.

In another method, glyphosate-tolerant plants have also been produced by using an EPSP synthesis cDNA isolated from a glyphosate tolerant petunia cell culture line. Such lines can be selected by growing cells on medium containing increasing concentration of selection factor, e.g. glyphosate. In the cell line, tolerance resulted from amplification (an increase in copy number) of the EPSP synthesis gene, resulting in over production of EPSP synthase in these cells.

The EPSP synthase cDNA isolated from the cell line was joined to the CaMV 35s promoter and to the Ti nos. polyadenylation signal. The strong CaMV 35s promoter (35s +EPSP synthase + nos) gene was introduced into petunia plants on a Ti vectors, the transgenic developed were tolerant to the four times higher concentration which kills control plants.

The other examples of herbicides resistance plants are given in the table 26.4. It is evident from these examples that a resistant factor is developed based on mode of action of herbicide, by modifying or over producing the target product. Canola (Brassica napus) cultivars engineered to tolerate the application of broad-spectrum herbicides have been developed both via transgenic and mutagenesis. This type of canola has been adopted rapidly by the Canadian farmers. The proportion of farmers growing transgenic herbicide- resistant canola has increased from 7% in 1995 to 80% in 2000.

The use of transgenic herbicide – tolerant canola varieties had increased net return by 32%, had reduced pesticide use by 6000 tones and fuel consumption by 31 million litres. There are undoubtedly very real benefits both for the farmers and for the environment.

3. Virus Resistant Plants:

Plants viruses often cause considerable damage and significantly reduce yield. Breeding for disease resistance is the best method to protect plants from viral and other infections. Recently scientists have used the techniques of genetic engineering to develop virus resistant transgenic plants. These methods used immunization with viral coat protein genes, other viral genes, or viral gene antisense sequence to confer resistance.

Potato is one of the most important food crops after cereals and pulses. It is very difficult to improve potato through breeding techniques as it is a tetraploid. Most of the cultivars are susceptible to various diseases caused by fungi, nematodes, and virus. Potatoes are vegetatively propagated. Therefore, seed material (tubers) for planting must be virus free. Potatoes suffer from three important virus diseases called Photo virus X, (PVX, PVY) and potato leaf-roll virus.

The phenomenon of cross protection or immunization of plant is not clearly understood. It is similar to immunization of human being for bacterial disease. When a plant is inoculated with a form of the virus, when virus infects, plant cell start synthesizing coat proteins instead of its own proteins. This cross protection is in some way related to the synthesis of the coat protein by the plant cell.

1986, Roger Beachy and colleagues at Washington University introduced the gene that encodes that coat protein of TMV into tobacco plants, resultantly each and every cell of the transgenic plant start producing coat protein. These plants showed considerable resistance to infection by TMV.

The virus was unable to multiply in the cells already containing some coat proteins. Therefore, the number of virus particles per cell remained low in transgenic plants as compared to normal control plants. Scientists at Mogen International in the Netherlands used the same approach to make potatoes resistant to PVX. The gene encoding the coat protein of PVX was introduced into two cultivars. The transgenic showed 100 times less virus particles as compared to control plants after two weeks of inoculation. The yield performance of most cultivars was same but potatoes produced were elongated.

The viral coast protein gene approach has been used to transfer tolerance to a number of transgenic plants for a number of different crops. Although complete protection is not usually achieved high levels of virus resistance have been reported. Moreover, a coat protein gene from one virus sometimes provides tolerance to a number of unrelated viruses.

In both eukaryotes and prokaryotes, an RNA molecule that is complementary to a normal gene transcript (that is mRNA) is called antisense RNA. The mRNA, being translatable, is considered to be a sense RNA. The presence of antisense RNA can decrease the synthesis of the gene product by forming a duplex molecules with the normal sense mRNA. Thereby, preventing it from being translated.

The antisense RNA-mRNA duplex is also rapidly degraded, a response that diminishes the amount of that particular mRNA in the cell. Therefore, in principle it should be possible to prevent plant viruses from replicating and subsequently damaging plant tissues by creating transgenic plants that synthesize antisense RNA that is complementary to virus coat protein mRNA.

The Ti binary vector system was used to transfer both protein producing sense and antisense RNA producing cDNA sequence to separate tobacco cells, from which transgenic plants were regenerated. The transgenic tobacco plants that expressed the cucumber mosaic virus (CuMV) coat protein were produced from viral particle accumulation and did not show symptoms of viral infection, irrespectively of whether the inoculum of the challenge virus was high or low.

However, the transgenic tobacco plants expressing the CuMV coat protein antisense RNA were protected only when the concentration of the challenge virus in the inoculums was low. Therefore, this approach is not successful when virus infection is high.

4. Abiotic Stress Tolerance:

Abiotic stresses such as drought, salinity and extreme temperatures cause significant losses of crop productivity and quality. Development of crops with an inherent capacity to withstand abiotic stress would help stabilize the crop production and significantly contribute to food and nutritional security in developing countries.

Transcriptome engineering or over expression of a master switch gene (such as stress sensors, protein kinases or transcription factors) that regulate several target genes coding for osmolyte biosynthesis enzymes, antioxidant enzymes and stress protein (such as late embryogenesis abundant proteins) is emerging as an important tool to combat abiotic stress. Stress-induced transcription factors such as c-repeat binding protein (CBF) or dehydration responsive element binding proteins regulate the expression of many genes for compatible osmolyte biosynthesis and oxidative stress management.

Over expression or stress responsive promoter driven expression of CBF3 gene in transgenic Arabidopsis provided protection against multiple environmental stresses such as cold, salt and drought. Components of the Arabidopsis CBF pathway are conserved in B. napus, wheat, rye, and tomato. Transgenic tomato with CBF1 gene and a CAM35s promoter showed significant chilling tolerance.

Transgenic tomato plants expressing Arabidopsis thaliana CBF1 gene, showed enhanced tolerance to oxidative stress, as CBF1 over expression induced a high level of expression of a catalase gene in these transgenic tomato plants.

5. Quality Improvement:

The goal of plant biotechnology is not confined to improvements of crop plants for agronomic traits and significant efforts are also being made to improve the nutritional content and organoleptic qualities such as taste and aroma in fruits and vegetables.

Nutritional Improvement:

Plant produces various compounds such as storage proteins, vitamin, flavonoids, carotenoids that perform vital functions for plants and also have nutritional importance for human beings. Vegetables are sources of minerals, proteins, micronutrients, vitamins, antioxidants, phytosterols and dietary fibre. However, some of the vegetables are deficient in essential amino acids such as methionine and lysine.

The amino acid content can be modified or enhanced by expression of synthetic protein, over expression of homologous or heterogeneous proteins, modifying the amino acid sequence of the protein or through metabolic engineering.

Potato is an important food crop; the nutritive value of potato protein is diminished due to deficiency in essential amino acids lysine, tyrosine and the sulphur containing amino acids methionine and cysteine. To improve the nutritive value of potato an Amaranthus seed albumin gene AmAl has been expressed in transgenic potato tubers.

This protein is non-allergenic and rich in all essential amino acids corresponding with WHO standards for human diet requirements. Similarly, a 292 bp artificial gene (asp-1) encoding a storage protein composed of essential amino acids was introduced in sweet potato. One of the transgenic lines showed a fourfold increase in protein as compared to that of storage roots of control plants.

Carotenoids, such as B-carotene and lycopene, give the fruit its characteristic colour. Carotenoids are good antioxidants and are precursors of vitamin A. These are synthesized through the isoprenoid biosynthetic pathway. Provitamin content of tomato was increased by transferring a bacterial gene encoding for the phytoene – desaturase enzyme that converts phytoene to lycopene into transgenic tomato.

These transgenic plants produced three-fold more B-carotene content than that of control plants. Similarly, a six-fold increase in carotenoid content and two to three fold increase in tocopherol content was achieved in transgenic potato plants by antisense technology.

Another group of metabolites exploited for its antioxidant property are the flavonoids. These are a diverse group of polyphenolic secondary metabolites, which impart colour to the fruits. Flavonoids are present only in tomato peel. A transgenic approach has been used to increase the flavonoid content by over-expression of either the enzymes involved in flavonoids biosynthesis or transcription factors that regulate the genes of this pathway.

Transgenic tomato plants expressing petunia CHI-A gene encoding chalcone isomerase showed significant increase in flavonoids content. Similarly, a 10-fold increase in flavonoid content has been achieved by ectopic expression of the maize transcription factors LC and CI in transgenic tomato.

Golden Rice-with Pro-Vitamin A:

According to the World Health Organization (WHO), vitamin A deficiency (VAD) is the leading causes of preventable blindness in children. For children, a lack of vitamin A causes severe visual impairments and blindness and significantly increases the risk of severe illness and even death from common infections such as diarrhea and measles.

The genes from daffodil and one from the bacterium Erwinia uredovora were inserted in the rice genome. These three genes produce the enzymes necessary to convert GGDP to pro vitamin- A. The inserted genes are controlled by specific promoters such that the enzymes and the provitamin-A are only produced in the rice endosperm.

Provitamin-A is not produced by traditional rice varieties. However, geranylgeranyl diphosphate (GGDP), a compound naturally present in immature rice endosperm, with the help of several enzymes not normally found in rice can be used to produce provitamin-A.

Through the work of two European scientist, Dr. Ingo Potrykus of the Swiss Federal Institute of Technology in Zurich and Dr. Peter Beyer of the University of Freiburg in Germany, rice plants were developed containing two daffodil genes and one bacterial gene that carry out the four steps required for the production of beta-carotene in rice endosperm.

Endosperm is the nutritive tissue surrounding the embryo of a seed and makes up the majority of the rice grain that we eat. The resulting plants appear normal expect the after milling (to remove the brown bran), their grain is golden yellow in colour due to the presence of pro vitamin-A.

When golden rice is ingested, the human body splits the pro-vitamin-A to make vitamin A. Detailed information can only be obtained once the golden rice trait is transferred to local varieties and produced in quantities sufficient to support necessary field experiments.

According to Swiss scientist Potrykus, “The intent of golden rice is to supplement to diet with vitamin A, not provide 100% of the Recommended Daily Allowance (RDA)”.

Potrykus maintains the goal of golden rice having a beneficial effect on vitamin A-deficient people is realistic with experimental golden rice lines ready in the 20-40% RDA range.

Golden rice is the result of an effort to develop rice verities that produce pro-vitamin-A (beta- carotene) as a means of alleviating vitamin A (retinol) deficiencies in the diets of poor and disadvantaged people in developing countries. Because traditional rice verities do not produce vitamin-A, transgenic technologies were required.

Improvement of Aroma:

The aroma of fruits, vegetables and flowers are mixtures of volatile metabolites such as alcohols, phenols, ethers, adehydes, ketones etc. Some of the short-chain adehydes and alcohols are derived from lipid components by the action of lipases, hydro-peroxide lipases and alcohol dehydrogenases. When yeast ∆-9 desaturase gene was transferred in tomato plants, changes in certain flavour compounds such as cw-3-hexenol, 1-hexanol, hexanal and cis-3-hexenal was recorded.

Linalool, an acyclic monoterpene alcohol, markedly influences the flavour of tomatoes. Linalool imparts a sweet, floral alcoholic note to fresh tomatoes. Hence linalool levels were altered by engineering the S-linalool synthase (LIS) gene from Clarkia breweri in tomato plants. The expression of S-linalool synthase enzyme, which catalyses the formation of linalool, resulted in elevated levels of linalool in the transgenic fruit.

Seedless Vegetables:

Browning and loss of flavour are two problems associated with potato. Transgenic potato have been generated in which browning is overcome by antisense inhibition of polyphenol oxidase. Cystathionine gamma synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants.

To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosiac virus 35s promoter into potato. Increase in 2.4 – to 4.4 fold increase in methional level in transgenic potato tubers was recorded.

The seedless nature of parthenocarpic (development of fruit without fertilization) fruits increases consumer acceptance, makes processing of vegetables easier, and also improves the quality of vegetables, e.g., brinjal (where seeds are associated with bitter substances).

Parthenocarpy has been shown to be regulated by auxins. Hence, efforts have been made to increase the auxin production or the sensitivity of ovary to auxins, towards inducing parthenocarpy. Expression of iaaM gene driven by the ovule specific promoter defH9 has been shown to confer parthenocarpy to transgenic tomato and eggplant.

In another approach, the Agrobacterium rhizogenes derived gene rol B has been used for the induction of parthenocarpy in tomato. Transgenic tomato plants transformed with the rol B under the control of ovary and young fruit specific promoter TPRP-F1 developed parthenocarpic fruits.

6. Pharmaceutical and Industrial Use:

The ability to transfer gene across different plant species and kingdoms through genetic engineering is being exploited in term of bio-farming. Bio-farming refers to production of proteins and bio-molecules in transgenic plants at agricultural scale. The proteins mainly include antigens, antibodies, enzymes that are of immense importance in therapeutics, pharmaceutical and industrial applications. Though many of these proteins are being made in bacterial, fungal or animal systems, plants are now being preferred for manufacturing these proteins.

The use of plants as bio-factories is attributed to many factors;

(i) Plants offer cost effective and environmentally safe production of proteins as they use low cost inputs such as light, water and minerals,

(ii) Plants allow mass production,

(iii) Suitable for production of eukaryotic proteins which many require post- translational modifications, oligomerization etc., and

(iv) Plants are not pathogenic to human beings.

The feasibility of vegetables as plant factories is very well illustrated in the form of edible vaccines, plant-bodies (plant derived antibodies) and plant derived recombinant enzymes.

Terminator Gene:

One potential use of transgenic technology is to allow seed producers to realize profits from their investments in new product development. Seed companies have preferred to invest heavily in developing new varieties of crops such as corn for which the farmers typically purchases new seeds each year. Two biotech protection methods, dubbed ‘Terminator’ and ‘Traitor’ by opponents, may allow companies to increase profits on their cultivars. Terminator, officially named as “Technology protection system” (TPS), incorporates a trait that kills developing plant embryos, so seed cannot be saved and replanted in subsequent years.

Traitor, officially known as “Trait-specific genetic use restriction technology” or T-Gurt, incorporates a control mechanism that requires yearly application of a preparatory chemical to activate desirable traits in the crop. The farmer can save and replant seeds, but cannot gain the benefits of the controlled traits unless he pays for the activating chemical each year.

Both methods avoid the difficulties associated with enforcing ‘no replanting’ agreements. Because TPS and T- Gurt plants would be transgenic, their commercial use will require approval by the government. Scientist from agricultural research service (USDA) and Delta and Pine Land Company jointly developed this technology in 1998.

The technology protection system (TPS) inserts half a dozen sequences into the DNA of the parent plant that is slated for protection. These DNA sequences are arranged into a system that kills seeds at a prearranged time in their development. The system can be left inactive while the seed company grows several generations of seeds for sale.

The system is switched on by soaking the seeds in a special chemical before the seeds are delivered to the farmer for planting.

The special chemical triggers a slow cascade of events that lead eventually to the death of progeny seeds developed on the protected plant. For the purpose of preventing replanting, the progeny seeds should be killed only after they have completed production of all commercially valuable products such as oil. Therefore, the system is designed to take effect only after the crop has grown to maturity in the field and the progeny seeds are nearly ripe.

7. Environmental Impact:

The use of Bt gene containing crops has been the most hotly debated issues regarding GM crops. Two different concerns have been broadly raised regarding such engineered insecticide resistance. The first concerns the broader impact of the presence of such insecticidal proteins on other organisms coming in contact with the transgenic crop. The second centres on the possibility of the target insets developing resistance to the insecticidal protein.

The possibility of detrimental environmental impacts of Bt corn become headline news in 1999. A paper was published suggesting that the presence of this insecticidal protein in the pollen of transgenic corn was detrimental to the larvae of the Monarch butterfly (Danaus plexippus). This was a laboratory based study and not a field study, even though sparked a controversy about use of transgenic crops and its impact on ecosystem.

Later on, based on field studies by American Universities, the issue was settled in 2001. This episode illustrates the fact that the first generation of transgenic crops is largely lacking mechanisms to target gene expression to precise cell organ or cell types. Rather, the introduced trans-genes are typically expressed constitutively in all the cells of the plant.

In case of Bt corn, the presence of insecticidal cry protein in the pollen, where it serves no useful purpose as the insect attacks the stem of the plant, raised environmental concerns without any reason. If the expression is controlled, particularly in open-pollinated crop like corn, the incidental damages to the environment can be minimize.

The second concern expressed regarding Bt gene was that insect would develop resistance to the insecticidal protein. This would not only make the transgenic crop worthless but might also the usefulness of Bt spray. One of the few tools available to organic farmers in the fight against insect pests. The seed industry is encouraging farmers to keep some area for non-GM crops, where insect can multiply, and also cross with resistant insect, if any, generating susceptible progeny. This will delay the true breeding resistant strain of insect.

8. Edible Vaccines:

Edible fruits and vegetables are good choice to develop transgenic oral vaccines. Potato, tomato, banana, grapes are the examples of plant species grown all over the world and particularly in developing countries, where cheap vaccines are required the most. This reduces the cost of purification and downstream processing and transportation. Transgenic plants have been produced in the following other edible plants and species can be suitably modified to desired vaccine production: – apple, asparagus, cabbage, carrot, cauliflower, cucumber, egg plant, papaya, pea etc.

There are currently two methods of protein production from plants:

1. Stable integration of foreign DNA into plant genome introduced either by genetic transformation: Agro-bacterium mediated or directly by using micro projectile bombardment and,

2. Transient expression of candidate DNA using viral vectors. The stable integration is advantageous because it passes in subsequent generations of large number of transgenic plants, either by vegetative or sexual means and also the possibility to introduce more than one gene for possible multi-component vaccine production.

To produce sufficient amount of vaccines by recombinant cell culture technology, fermentation and purification are required which are very expensive. If the antigens are expressed in edible tissues of transgenic plants, it will become a cost effective production and delivery system. This is referred to as ‘edible plant vaccine technology’. In addition tissue or organ specific expression of foreign antigens is possible by using tissue specific promoters.

Vaccines are the most important and cost effective sources for fighting infection diseases. Every year, an estimated 17 million people die of infectious diseases which include 7 million children. Although there have been opportunities for the production of cell cultures and recombinant vaccines, there is an increasing demand and the current production facilities are inadequate to supply the vaccines on a large-scale at an affordable price for the people living in developing countries.

In recent years, considerable progress has been made in producing functionally active proteins, peptide of medical importance in transgenic plants. The expression of subunit antigens of infectious microorganisms in transgenic plants and their subsequent immunogenic properties led to the production of edible vaccines.

The modern biotechnological tools demonstrated the feasibility of using a genetically engineered food as an inexpensive oral vaccine production and delivery system for diarrhea disease. Recently clinical trials have been conducted for heat labile enterotoxin (LT-B vaccine against cholera) from E. coli in the form of edible vaccine.

In the developing countries, diarrhea disease is a leading cause of death, especially among children and travelers. Travelers who visit these tropical areas are victim of diarrheal diseases because they are frequently exposed to bacterial contaminations in food, water and common places. Enter toxigenic E. coli, which produce a heat Labile (LT) and heat stable (ST) enterotoxin, are the most common causes of travellers diarrhea throughout the world.

LT is comprised of six sub-units. LT-A is an enzymatically active protein which enters the epithelial cells of the gut and initiates cellular metabolic changes that lead to loss of water from cells. LT-B has five identical enzymatically inactive proteins, which form a pentamer that binds to GM1 gangliosides in the membranes epithelial cells. Binding of LT-B initiates transport of the active subunit inside the cells resulting in diarrhoea and any interference with binding will block the action of toxin. LT-B elicits oral immune response when given orally without any symptoms of disease.

Tobacco plants containing LT-B bacterial gene accumulated LT-B toxin and this LT-B was similar to that produced by bacteria. When mice were orally inoculated were with tobacco derived LT-B, both serum and mucosal antibodies were induced. In another experiment; potato plants were transformed to produce LT-B and upon feeding the transgenic tubers directly to mice, serum antibodies were induced. Production of serum and mucosal antibodies was confirmed on feeding transformed potatoes.

Norwalk Virus:

Norwalk virus is the causative agent of acute epidemic gastroenteritis in humans. Recent advances in cloning the Norwalk virus genome and expression of the capsid protein in insect cell cultures have facilitated the study of the virus and the development of candidate vaccines for oral immunization. Norwalk virus capsid protein (NVCP) gene has been transferred and expressed in tobacco leaves and potato tubers. Partially purified antigen from tobacco or potato tubers is used for vaccination.

Hepatitis-B Surface Antigen (HbsAg):

Hepatitis is the single most important cause of viremia in humans and currently there are about 300 million carriers all over the world. The worldwide problem of infection and its association with chronic liver disease has necessitated the development of an effective vaccine. In many parts of the developing world, the expense of immunization programme limits the usage of the currently available serum or yeast could offer as a relatively low-cost method.

The transfer of hepatitis-B surface antigen gene in tobacco, expression of recombinant gene in tobacco followed by partial purification of protein from the plant. When this protein was injected into mice, it elicited antibody response similar to that obtained with yeast derived commercially available vaccine. This is clear that gene product obtained from two different organisms has same property and transgenic plants can be used as source of antibodies.