Applications of Transgenic Plants: 6 Applications

This article throws light upon the six applications of transgenic plants.

The six applications are: (1) Resistance to Biotic Stresses (2) Resistance to Abiotic Stresses (3) Improvement of Crop Yield and Quality (4) Transgenic Plants with Improved Nutrition(5) Commercial Transgenic Crop Plants and (6) Transgenic Plants as Bioreactors.

The genetic manipulations carried out in plants for the production of transgenic plants have been described .The ultimate goal of transgenic (involving introduction, integration, and expression of foreign genes) is to improve the crops, with the desired traits.

Some of the important ones are listed:

i. Resistance to biotic stresses i.e. resistance to diseases caused by insects, viruses, fungi and bacteria.

ii. Resistance to abiotic stresses-herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone, intense light.

iii. Improvement of crop yield, and quality e.g. storage, longer shelf life of fruits and flowers.

iv. Transgenic plants with improved nutrition.

v. Transgenic plants as bioreactors for the manufacture of commercial products e.g. proteins, vaccines, and biodegradable plastics.

Environmental stresses to plants:

The different types of external stresses that influence the plant growth and development are depicted in Fig. 50.1, These stresses are grouped based on their characters—biotic and abiotic stresses. The biotic stresses are caused by insects, pathogens (viruses, fungi, bacteria), and wounds. The abiotic stresses are due to herbicides, water deficiency (caused by drought, temperature, and salinity), ozone and intense light.

Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress to plants. This damages the cellular constituents of plants which is associated with a reduction in plant yield.

The major objective of plant biotechnology is to develop plants that are resistant to biotic and abiotic stresses.

Application # 1. Resistance to Biotic Stresses:

Genetic engineering of plants has led to the development of crops with increased resistance to biotic stresses which is described in three major categories:

1. Insect resistance.

2. Virus resistance.

3. Fungal and bacterial disease resistance

1. Insect (PEST) Resistance:

It is estimated that about 15% of the world’s crop yield is lost to insects or pests. A selected list of the common insects and the crops damaged is given in Table 50.1.

The damage to crops is mainly caused by insect larvae and to some extent adult insects.

The majority of the insects that damage crops belong to the following orders (with examples):

i. Lepidoptera (bollworms).

ii. Coleoptera (beetles).

iii. Orthoptera (grasshoppers).

iv. Homoptera (aphids).

Till some time ago, chemical pesticides are the only means of pest control.

Scientists have been looking for alternate methods of pest control for the following reasons (i.e. limitations of pesticide use):

i. About 95% of the pesticide sprayed is washed away from the plant surface and accumulates in the soil.

ii. It is difficult to deliver pesticides to vulnerable parts of plants such as roots, stems and fruits.

iii. Chemical pesticides are not efficiently degraded in the soil, causing environmental pollution.

iv. Pesticides, in general, are toxic to non- target organisms, particularly humans and animals.

It is fortunate that scientists have been able to discover new biotechnological alternatives to chemical pesticides thereby providing insect resistance to crop plants. Transgenic plants with insect resistance transgenes have been developed. About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants. Some of the approaches for the bio-control of insects are briefly described.

Resistance Genes from Microorganisms:

Bacillus thuringiensis (Bt) toxin:

Bacillus thuringiensis was first discovered by Ishiwaki in 1901, although its commercial importance was ignored until 1951. B. thuringiensis is a Gram negative, soil bacterium. This bacterium produces a parasporal crystalline proteinous toxin with insecticidal activity. The protein produced by B .thuringiensis is referred to as insecticidal crystalline protein (ICP). ICPs are among the endotoxins produced by sporulating bacteria, and were originally classified as δ-endotoxins (to distinguish them from other classes of α-, β- and ƴ-endotoxins).

Bt toxin genes:

Several strains of B. thuringiensis producing a wide range of crystal (cry) proteins have been identified. Further, the structure of cry genes and their corresponding toxin (δ-endotoxin) products have been characterized. The cry genes are classified (latest in 1998) into a large number of distinct families (about 40) designated as cry 1…… cry 40, based on their size and sequence similarities. And within each family, there may be sub-families. Thus, the total number of genes producing Bt toxins (Cry proteins) is more than 100.

There are differences in the structure of different Cry proteins, besides certain sequence similarities. The molecular weights of Cry proteins may be either large (~130 KDa) or small (~70KDa). Despite the differences in the Cry proteins, they share a common active core of three domains.

Mode of action of Cry proteins:

Most of the Bt toxins (Cry proteins) are active against Lepidopteran larvae, while some of them are specific against Dipteran and Coleopteran insects. The pro-toxin of Cry I toxin group has a molecular mass of 130 kilo Daltons (130 KDa).

When this parasporal crystal is ingested by the target insect, the pro-toxin gets activated within its gut by a combination of alkaline pH (7.5 to 8.5) and proteolytic enzymes. This results in the conversion of pro-toxin into an active toxin with a molecular weight of 68 KDa (Fig. 50.2).

The active form of toxin protein gets itself inserted into the membrane of the gut epithelial cells of the insect. This result in the formation of ion channels through which there occurs an excessive loss of cellular ATP. As a consequence, cellular metabolism ceases, insect stops feeding, and becomes dehydrated and finally dies.

Some workers i n the recent years suggest that the Bt toxin opens cation-selective pores in the membranes, leading to the inflow of cations into the cells that causes osmotic lysis and destruction of epithelial cells (and finally the death of insect larvae). The Bt toxin is not toxic to humans and animals since the conversion of pro-toxin to toxin requires alkaline pH and specific proteases (These are absent humans and animals).

Bt toxin as bio-pesticide:

Preparations of Bt spores or isolated crystals have been used as organic bio-pesticide for about 50 years.

This approach has not met with much success for the following reasons:

i. Low persistence and stability (sunlight degrades toxin) of the toxin on the surface of plants.

ii. The Bt toxin cannot effectively penetrate into various parts of plants, particularly roots.

iii. Cost of production is high.

Bt-based genetic transformation of plants:

It has been possible to genetically modify (CM) plants by inserting Bt genes and provide pest resistance to these transformed plants. For an effective pest resistance, the bacterial gene in transgenic plants must possess high level expression. This obviously means that the transgene transcription should be under the effective control of promoter and terminator sequences. The early attempts to express cry 1A and cry 3A proteins under the control of CaMV 35S or Agrobacterium T-DNA promoters resulted in a very low expression in tobacco, tomato and potato plants.

Modification of Bt cry 1A gene:

The wild type transgene Bt cry 1A(b) was found to express at a very low levels in transgenic plants. The nucleotide sequence of this gene was modified (G + C content altered, several polyadenylation signals removed, ATTTA sequence deleted etc). With appropriate sequence changes, an enormous increase (about 100 fold) in the Bt toxin product formation was observed.

The transgenic Bt crops that were found to provide effective protection against insect damage were given approval for commercial planting by USA in the mid-1990s (Table 50.2). Some biotechnological companies with their own trade names introduced several transgenic crops into the fields. Among these, only maize and cotton Bt crops are currently in use in USA. The other genetically modified plants met with failure for various reasons.

Advantages of transgenic plants with Bt genes:

i. Bt genes could be expressed in all parts of the plants, including the roots and internal regions of stems and fruits. This is not possible by any chemical pesticide.

ii. Toxic proteins are produced within the plants; hence they are environmental-friendly.

iii. Bt toxins are rapidly degraded in the environment.

The problem of insect resistance to Bt crops:

The major limitation of Bf-gene possessing transgenic plants is the development of Bt-resistant insects. The Bf toxin is a protein, and the membrane receptor (of the gut) through which the toxin mediates its action is also a protein. It is possible that the appropriate mutations in the insect gene coding for receptor protein may reduce the toxin binding and render it ineffective. This may happen within a few generations by repeated growing of Bt crops.

Several approaches are made to avoid the development of resistance in insects:

i. Introduction of two different Bf toxin genes for the same target insect.

ii. Development of transgenic plants with two types of insect resistance genes e.g. Bt gene and proteinase inhibitor gene.

iii. Rotating Bt crops with non-Bf crops may also prevent the build-up of resistance in insect population.

The environmental impact of Bt crops:

The most serious impact of Bt crops on environment is the build-up of resistance in the pest population. In 1999, another issue was brought to light about Bt crops. It was reported that the pollen from Bt maize might be toxic to the larvae of Monarch butterfly. This generated considerable opposition to Bt crops by the public, since Monarch butterfly is one of the most colorful natives in USA.

It was later proved that the fears about the impact of Bt crops on the monarch butterfly were without the required scientific evidence. The lesson learnt from the monarch butterfly episode is that the risks of CM crops should be thoroughly assessed before they are reported.

Usage of Bt:

The usage Bt is commonly used for a transgenic crop with a cry gene e.g. Bt cotton. In the same way, Cry proteins are also referred to as Bt proteins. It may also be stated here that the authors use four different names for the same group of proteins-δ-endotoxin, insecticidal crystal protein (ICP), Cry and now Bt.

Resistance genes from other microorganisms:

There are certain other insect resistant genes from other microorganisms. Some of the important ones are listed.

1. Cholesterol oxidase of Strepotmyces culture filtrate was found to be toxic to boll weevil larvae. Cholesterol oxidase gene has been introduced into tobacco to develop a transgenic plant.

2. Isopentenyl transferase gene from Agro­bacterium tumefaciens has been introduced into tobacco and tomato. This gene codes for an important enzyme in the synthesis of cytokinin. The transgenic plants with this transgene were found to reduce the leaf consumption by tobacco hornworm and decrease the survival of peach potato aphid.

Resistance Genes from Higher Plants:

Certain genes from higher plants were also found to result in the synthesis of products possessing insecticidal activity. Some authors regard them as non-Bt insecticidal proteins. A selected list of plant insecticidal (non-Bt) genes used for developing transgenic plants with insect resistance is given Table 50.3. Some of them are briefly described.

Proteinase (Protease) Inhibitors:

Proteinase inhibitors are the proteins that inhibit the activity of proteinase enzymes. Certain plants naturally produce proteinase inhibitors to provide defence against herbivorous insects. This is possible since the inhibitors when ingested by insects interfere with the digestive enzymes of the insect. This results in the nutrient deprivation causing death of the insects. It is possible to control insects by introducing proteinase inhibitor genes into crop plants that normally do not produce these proteins.

Cowpea trypsin inhibitor gene:

It was observed that the wild species of cowpea plants growing in Africa were resistant to attack by a wide range of insects. Research findings revealed that insecticidal protein was a trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera (e.g. Heliothis virescans), Orthaptera (e.g. Locusta migratoria) and Coleoptera (e.g. Anthonous grandis).

Cowpea trypsin inhibitor (CpTi) has no affect on mammalian trypsins; hence it is non-toxic to mammals. CpTi gene was introduced into tobacco, potato and oilseed rape for developing transgenic plants. Survival of insects and damage to plants were much lower in plants possessing CpTi gene.

Advantages of proteinase inhibitors:

i. Many insects, not controlled by Bt, can be effectively controlled.

ii. Use of proteinase gene along with Bt gene will help to overcome Bt resistance development in plants.

Limitations of proteinase inhibitors:

i. Unlike Bt toxin, high levels of proteinase inhibitors are required to kill insects.

ii. It is necessary that the expression of proteinase inhibitors should be very low in the plant parts consumed by humans, while the expression should be high in the parts of plants utilized by insects.

α-Amylase inhibitors:

The insect larvae secrete a gut enzyme a- amylase to digest starch. By blocking the activity of this enzyme by α-amylase inhibitor, the larvae can be starved and killed. α-Amylase inhibitor gene (α-AI-Pv) isolated from bean has been successfully transferred and expressed in tobacco. It provides resistance against Coleoptera (e.g. Zabrotes sub-fasciatus).

Lectins:

Lectins are plant glycoproteins and they provide resistance to insects by acting as toxins. The lectin gene (CNA) from snowdrop (Calanthus nivalis) has been transferred and expressed in potato and tomato. The major limitations of lectin are that it acts only against piercing and sucking insect, and high doses are required.

Resistance Genes from Animals:

Proteinase inhibitor genes from mammals have also been transferred and expressed in plants to provide resistance against insects, although the success in this direction is very limited. Bovine pancreatic trypsin inhibitor (BPTI) and α₁– antitrypsin genes appear to be promising to offer insect resistance to transgenic plants.

Insect Resistance through Copy Nature Strategy:

Some of the limitations experienced by transferring the insecticidal genes (particularly Bt) and developing transgenic plants have prompted scientists to look for better alternatives. The copy nature strategy was introduced in 1993 (by Boulter) with the objective of insect pest control which is relatively sustainable and environmentally friendly.

The copy nature strategy for the development of insect -resistant transgenic plants has the following stages:

1. Identification of leads:

The first step in copy nature strategy is to identify the plants (from world over) that are naturally resistant to insect damage.

2. Isolation and purification of protein:

The protein with insecticidal properties (from the resistant plants) is isolated and purified. The sequence of the protein is determined, and the gene responsible for its production identified.

3. Bioassay of isolated protein:

The activity of the protein against the target insects is determined by performing a bioassay in the laboratory.

4. Testing for toxicity in mammals:

It is absolutely necessary to test the toxicity of the protein against mammals, particularly humans. If the protein is found to have any adverse effect, the copy nature strategy should be discontinued.

5. Gene transfer:

By the conventional techniques of genetic engineering, the isolated gene corresponding to the protein toxin is introduced into the crop plants.

6. Selection of transgenic plants:

After the gene transfer, the transgenic plants developed should be tested for the inheritance and appropriate expression of transgene. The efficiency of insecticidal protein to destroy insects is also evaluated.

7. Evaluation for biosafety:

Field trails have to be conducted to evaluate the crop yield, damage to insects, influence on the environment with respect to the transgenic plant. The copy nature strategy, though time consuming and many a times unsuccessful, takes into account the complex interplay in biological communities (between plants animals, microorganisms) and physical environment.

2. Virus Resistance:

Virus infections of crops may result in retarded cell division (hypoplasia), excessive cell division (hyperplasia), and cell death (necrosis). The overall effects of virus infections are growth retardation, lowered product yield and sometimes complete crop failure. The chemical methods used to control various plant pathogens will be ineffective with respect to plant viruses since the viruses are intracellular obligate parasites.

There are however, certain safe agricultural practices to control/reduce viral infections to plants:

i. Use of seeds that are virus – free.

ii. Control insects that spread plant viruses.

iii. Control weeds that serve as alternate hosts for viruses.

iv. Use cultivars that possess virus resistance.

It is possible to immunize plants against viral damages by expressing viral proteins in the plant cells. With the advances made in genetic engineering, it has become a reality to develop transgenic plants with virus resistance.

This is mostly done by employing virus-encoded genes – virus coat proteins, movement proteins, transmission proteins, satellite RNA, antisense RNAs and ribozymes. In recent years, some attempts are also made to provide virus resistance to plants by using animal genes. Some of the developments are briefly described.

Virus coat proteins:

The virus coat protein-mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV. Excited by this remarkable success, scientists have worked with many more viruses (around 30 or so) and developed crops with virus coat protein-mediated protection.

A selected list of the virus resistant transgenic plants with sources of virus coat protein genes is given in Table 50.4. The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, wheat, tobacco, peanut, sugar beet, alfalfa etc. The viruses that have been used include alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), potato virus X (PVX), potato virus Y (PVY), citrus tristeza virus (CTV) and R rice stripe virus (RSV).

Advantages of virus coat proteins:

The coat protein gene from one virus sometimes provides resistance (cross protection) to some other viruses, which may be unrelated e.g. TMV of tobacco plant provides resistance to potato virus X, alfalfa mosaic virus and cucumber mosaic virus.

Limitation of virus coat proteins:

The virus coat protein-mediated protection is successful for viruses with single-stranded RNA genomes. However, this approach is of not much use for viruses with genomes containing double- stranded RNA and single-stranded DNA.

Mechanism of action of virus coat proteins:

As the transgenic plant expresses the gene for coat protein of a given virus, the ability of the same virus to infect the plants again is drastically reduced. Despite a remarkable success in the virus coat protein-mediated protection, the molecular mechanism of the protection is not clearly known.

Movement proteins:

As the virus infects the plant cells, its rapid spread through intercellular junctions (plasmadesmata) of vascular tissue occurs through the participation of movement proteins produced by the viruses. Good examples of movement proteins are 30KDa protein of tobacco mosaic virus (TMV) and 32KDa protein of brome mosaic virus (BMV).

Transgenic tobacco plants that express a mutated 30KDa movement protein have been developed. The TMV infection to these plants is much less. It is believed that the mutated movement protein competes with the wild-type TMV-coded protein thereby reducing the spread of the virus (TMV).

In recent years, a recombinant movement protein having the components of golden mosaic virus and African cassava mosaic virus have been developed. This protein effectively interferes with the spread of both the viruses. The advantage with movement protein strategy is that it is applicable to single-stranded DNA viruses (Gemini viruses) also.

Transmission proteins:

There is a good coordination and interaction between plant viruses and insect vectors for the spread of viruses from one plant to another. Certain viral-encoded transmission proteins do this job effectively. It is possible to produce mutated transmission proteins and block the spread of viruses. Thus, the spread of insect-transmitted viruses can be prevented by engineering crops to express a defective virus-transmission protein.

Satellite RNAs:

Plant viral satellite RNAs are small RNA molecules that multiply in the host cells with the help of specific helper viruses. These satellite RNAs are encapsulated together with the respective helper viruses. In general, the presence of satellite RNAs reduces the severity of the viral disease and the symptoms, and thus reduces the effect of the virus.

Transgenic plants containing satellite sequences have been developed to provide resistance to virus diseases. One example is given here. When cucumber mosaic virus (CMV) infects pepper plants, severe symptoms appear. These symptoms can be minimized with higher plant yield when CMV is co-inoculated with a satellite RNA.

Satellite RNA approach is not widely used due to several limitations:

i. Some of the satellite RNA may increase the severity of disease symptoms in some plants.

ii. Satellite RNAs mutate very rapidly which may sometimes result in a highly virulent agent.

iii. Re-combinations between satellite RNAs have been detected. This may lead to serious consequences.

Antisense RNAs:

The antisense RNA approach is designed to specifically interfere with virus replication. By use of genetic engineering, a complementary DNA Strand of a gene (DNA sequence) can be inserted in reverse orientation (3′ → 5′ as opposed to 5′ → 3′) and this is referred to as antisense gene.

The mRNA produced by antisense gene is complementary to the mRNA synthesized by normal gene. As a result, both these mRNAs hybridize and thus the normal translation of mRNA is blocked. The net effect of employing an antisense gene into a cell is that it blocks a specific gene expression.

It is possible to introduce viral antisense genes into plants and produce mRNAs complementary to viral sequences involved in virus replication. The antisense mRNAs can block the replication of viruses (Fig 50.3). Initially, antisense RNA approach was carried out in single-stranded RNA viruses. The success of this approach however, was limited probably due to the following reasons.

i. High concentration of antisense mRNA may be required.

ii. Protein association with mRNA interferes with hybridization (between sense mRNA and antisense mRNA).

Antisense RNA approach may be more useful for DNA viruses. In fact, tomato golden mosaic virus (TGMV) replicase coding sequence was cloned in antisense orientation and introduced into tobacco plants. The transgenic tobacco plants expressed antisense RNA of TGMV replicase. These plants were resistant to TGMV infection.

Ribozymes:

Ribozymes are small RNA molecules which promote the catalytic cleavage of RNA. For providing virus resistance, ribozymes in the form of antisense RNAs capable of cleaving the target viral (sense) RNAs have been developed. The ribozyme (antisense RNA) binds to a small sequence of viral RNA and splits (Fig. 50.4).

In this way, it is possible to block the replication of viral RNA. However, the ribozymes approach has not been very successful in plants.

3. Fungal and Bacterial Diseases Resistance:

The plants do posses general defence systems against invading pathogens. This is however, not truly comparable with the immune system of the animals. Whenever there is a cellular damage caused by pathogens (fungi, bacteria) and plant pests, the general defence system of plants get geared up to provide some amount of protection to the plant. This natural disease resistance of plants is inadequate.

However, knowledge on the natural systems of plant resistance is useful for the biotechnological approaches to develop disease resistance. Some of the defenses of plants and the biotechnological approaches are briefly described.

Pathogenesis-Related (Pr) Proteins:

To defend themselves against the invading pathogens (fungi and bacteria), plants accumulate low molecular weight proteins which are collectively regarded as pathogenesis-related (PR) proteins. The different types of PR proteins and their properties are given in Table 50.5. Some of the most important ones are described.

Chitinase:

Chitin is a constituent of fungal cell walls which can be hydrolysed by the enzyme chitinase. Certain chitinase genes from plants have been isolated and characterized. A bacterial chitinase gene obtained from a soil bacterium (Serratia marcescens) was introduced and expressed in tobacco leaves. Some other workers isolated a chitinase gene from bean (Phaseolus vulgaris) and developed transgenic plants of tobacco and Brassica napus with this gene. The transformed tobacco plants were found to be resistant to infection of the pathogen Rhizoctonia solani. In case of B. napus, the protection however, was comparatively less.

Glucanase:

Glucanase is another enzyme that degrades the cell wall of many fungi. The most widely used glucanase is β-1, 4-glucanase. The gene encoding for β-1, 4-glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani.

The resistance to fungal pathogens is much higher if both chitinase and glucanase producing genes are present in transgenic plants. By this approach, fungal resistant tobacco, tomato and carrot have been developed.

Ribosome-In Activating Proteins (RIPs):

Ribosome-inactivating proteins offer protection against fungal infections. They act on the large rRNA of eukaryote and prokaryote ribosomes (remove an adenine residue from a specific site), and thus inhibit protein biosynthesis.

Certain RIPs that do not inhibit plant ribosomes were identified and the corresponding genes have been used to develop transgenic plants e.g. Type-I barley RIP is used to provide resistance to fungal infections. Some authors use the term antimicrobial proteins to RIPs. The other examples of antimicrobial proteins are lectins, defensins, lysozyme, thionins etc.

Lysozyme:

Lysozyme degrades chitin and peptidoglycan of cell wall and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed.

Defensins:

Defensins are antimicrobial peptides (26-50 amino acid residues) found in all the plant cells. They attack the microbial plasma membrane; however this is not adequate to provide resistance to pathogens. In recent years, an artificial defensin gene has been developed and introduced into potatoes. These potatoes developed resistance to the bacterium Eswinia carotovora.

Thionins:

Thionin proteins also offer protection against bacteria. Thionin coding genes have been introduced into tobacco and the transgenic plant so developed showed resistance to Pseudomonas syringae.

Phytoalexins:

Phytoalexins are secondary metabolites produced in plants in response to infection. They are low- molecular weight and antimicrobial in nature. The phytoalexins usually present in specialized cells or organelles are mobilized when infection occurs. Further, during infection there occurs induction of genes for increased production of phytoalexins.

Stilbene synthase is a key enzyme for the synthesis of a common phytoalexin. The gene coding stilbene synthase has been isolated from peanut and introduced into tobacco, rice and Brassica napus. The transgenic plants carrying stilbene synthase gene were resistant against some fungi. A selected list of transgenic plants developed, along with the genes transferred and the controlled pathogens is given in Table 50.6.

Nematode Resistance:

Nematodes are simple worms found in the soil. They possess a complete digestive tract. The annual crop loss of the world due to nematode (roundworm) infestation is very high.Some workers have identified and cloned a nematode resistance gene from wild beet plants.

It is proposed that this gene encodes a protein that detects the pests (nematodes) and triggers a defensive reactions in the plant. It is believed that some chemical compounds that destroy the gut of the nematode are produced. Attempts were also made to transfer the nematode resistance gene to sugar beet. Not much success was reported, the major limitation being the difficulty in cultivating the gene-altered cells of sugar beet.

Application # 2. Resistance to Abiotic Stresses:

Plants are constantly being subjected to environmental stresses that may result in deterioration of crop plants, and a very low or even no yield. Plants are dependent on the subtle internal mechanisms for tolerance of various stresses.

The in situ tolerance of crop plants, whenever present, is inadequate and therefore cannot give protection against the stresses. A wide range of strategies are required to engineer plants against a particular type of stress tolerance. Some of the abiotic stresses and the recombinant strategies developed to overcome them are described.

Herbicide Resistance:

Weeds (wild herbs) are unwanted and useless plants that grow along with the crop plants. Weeds compete with traps for light and nutrients, besides harbouring various pathogens. It is estimated that the world’s crop yield is reduced by 10-15% due to the presence of weeds.

To tackle the problem of weeds, modern agriculture has developed a wide range of weed killers which are collectively referred to as herbicides. In general, majority of the herbicides are broad-spectrum as they can kill a wide range of weeds.

A good or an ideal herbicide is expected to possess the following characteristics:

i. Capable of killing weeds without affecting crop plants.

ii. Not toxic to animals and microorganisms.

iii. Rapidly trans-located within the target plant.

iv. Rapidly degraded in the soil.

None of the commercially available herbicides fulfills all the above criteria. The major limitation of the herbicides is that they cannot discriminate weeds from crop plants. For this reason, the crops are also affected by herbicides, hence the need to develop herbicide-resistant plants. Thus, these plants provide an opportunity to effectively kill the weeds (by herbicides) without damaging the crop plants.

Strategies for engineering herbicide resistance:

A number of biological manipulations particularly involving genetic engineering are in use to develop herbicide-resistant plants.

1. Overexpression of the target protein:

The target protein, being acted by the herbicide can be produced in large quantities so that the affect of the herbicide becomes insignificant. Overexpression can be achieved by integrating multiple copies of the genes and/or by using a strong promoter.

2. Improved plant detoxification:

The plants do possess natural defense systems against toxic compounds (herbicides). Detoxification involves the conversion of toxic herbicide to non-toxic or less toxic compound. By enhancing the plant detoxification system, the impact of the herbicide can be reduced.

3. Detoxification of herbicide by using a foreign gene:

By introducing a foreign gene into the crop plant, the herbicide can be effectively detoxified.

4. Mutation of the target protein:

The target protein which is being affected by the herbicide can be suitably modified. The changed protein should be capable of discharging the functions of the native protein but is resistant to inhibition by the herbicide.

Once the resistant target protein gene is identified, it can be introduced into the plant genomes, and thus herbicide-resistant plants can be developed. For success in the development of herbicide resistant plants, good knowledge of the target protein and the action of herbicides is required.

Some of the developments made in the herbicide resistance of plant are briefly described:

Glyphosate Resistance:

Glyphosate, is a glycine derivative. It acts as a broad-spectrum herbicide and is effective against 76 of the world’s worst 78 weeds. Glyphosate is less toxic to animals and is rapidly degraded by microorganisms. In addition, it has a short half-life. The American chemical company Monsanto markets glyphosate as Round up.

Mechanism of action of glyphosate:

Glyphosate is rapidly transported to the growing points of plants. It is capable of killing the plants even at a low concentration. Glyphosate acts as a competitive inhibitor of the enzyme 5-enoylpyruvylshikimate 3-phosphate synthase (EPSPS). This is a key enzyme in shikimic acid pathway that results in the formation of aromatic amino acids (tryptophan, phenylalanine and tyrosine), phenols and certain secondary metabolites (Fig. 50.5).

The enzyme EPSPS catalyses the synthesis of 5-enoylpyruvylshikimate 3-phosphate from shikimate 3-phosphate and phosphoenoylpyruvate. Glyphosate has some structural similarly with the substrate phosphoenol pyruvate (Fig 50.6). Consequently, glyphosate binds more tightly with EPSPS and blocks the normal shikimic acid pathway. Thus, the herbicide glyphosate inhibits the biosynthesis of aromatic amino acids and other important products.

This results in inhibition of protein biosynthesis (due to lack of aromatic amino acids). As a consequence, cell division and plant growth are blocked. Further, the plant growth regulator indole acetic acid (an auxin) is also produced from tryptophan. The net result of glyphosate is the death of the plants. Glyphosate is toxic to microorganisms as they also possess shikimate pathway.

Glyphosate is non-toxic to animals (including humans), since they do not possess shikimate pathway. Of the three aromatic amino acids (synthesized in this pathway), tryptophan and phenylalanine are essential and they have to be supplied in the diet, while tyrosine can be formed from phenylalanine.

Strategies for glyphosate resistance:

There are three distinct strategies to provide glyphosphate resistance to plants:

1. Overexpression of crop plant EPSPS gene:

An overexpressing gene of EPSPS was detected in Petunia. This expression was found to be due to gene amplification rather than an increased expression of the gene. EPSPS gene from Petunia was isolated and introduced into other plants. The increased synthesis of EPSPS (by about 40 fold) in transgenic plants provides resistance to glyphosate. These plants can tolerate glyphosate at a dose of 2-4 times higher than that required to kill wild-type plants.

2. Use of mutant EPSPS genes:

An EPSPS mutant gene that conferred resistance to glyphosate was first detected in the bacterium Salmonella typhimurium. It was found that a single base substitution (C to 7) resulted in the change of an amino acid from proline to serine in EPSPS. This modified enzyme cannot bind to glyphosate, and thus provides resistance.

The mutant EPSPS gene was introduced into tobacco plants using Agrobacterium Ti plasmid vectors. The transgene produced high quantities of the enzyme EPSPS. However, the transformed tobacco plants provided only marginal resistance to glyphosate. The reason for this was not immediately identified.

It was later known that the shikimate pathway occurs in the chloroplasts while the glyphosate resistant EPSPS was produced only in the cytoplasm. This enzyme was not transported to the chloroplasts, hence the problem to provide resistance. This episode made scientists to realize the importance of chloroplasts in genetic engineering.

In later years, the mutant EPSPS gene was tagged with a chloroplast-specific transit peptide sequence. By this approach, the glyphosate-resistant EPSPS enzyme was directed to freely enter chloroplast and confer resistance against the herbicide.

3. Detoxification of glyphosate:

The soil microorganisms possess the enzyme glyphosate oxidase that converts glyphosate to glyoxylate and aminomethylphosponic acid. The gene encoding glyphosate oxidase has been isolated from a soil organism Ochrobactrum anthropi. With suitable modifications, this gene was introduced into crop plants e.g. oilseed rape. The transgenic plants were found to exhibit very good glyphosate resistance in the field.

Use of a combined strategy:

More efficient resistance of plants against glyphosate can be provided by employing a combined strategy. Thus, resistant (i.e. mutant) EPSPS gene in combination with glyphosate oxidase gene are used. By this approach, there occurs glyphosate resistance (due to mutant EPSPS gene) as well as its detoxification (due to glyphosate oxidase gene).

Phosphinothricin Resistance:

Phosphinothricin (or glufosinate) is also a broad spectrum herbicide like glyphosate. Phosphino­thricin is more effective against broad-leafed weeds but least effective against perennials. (Note: Phosphinothricin and glufosinate are two names for the same herbicide. However, to avoid confusion between glyphosate and glufosinate, phosphino­thricin is more commonly used. Basta Aventis and Liberty are the trade names for phosphinothricin).

Phosphinothricin-a natural herbicide:

Phosphinothricin is an unusual herbicide, being a derivative of a natural product namely bialaphos. Certain species of Streptomyces produce bialaphos which is a combination of phosphinothricin bound to two alanine residues, forming a tripeptide. By the action of a peptidase, bialaphos is converted to active phosphinothricin (Fig 50.7).

Mechanism of action of phosphinothricin:

Phosphinothricin acts as a competitive inhibitor of the enzyme glutamine synthase (Fig 50.7). This is possible since phosphinothricin has some structural similarity with the substrate glutamate. As a consequence of the inhibition of glutamine synthase, ammonia accumulates and kills the plant cells. Further, disturbance in glutamine synthesis also inhibits photosynthesis. Thus, the herbicidal activity of phosphinothricin is due to the combined effects of ammonia toxicity and inhibition of photosynthesis.

Strategy for phosphinothricin resistance:

The natural detoxifying mechanism of phosphinothricin observed in Streptomyces sp has prompted scientists to develop resistant plants against this herbicide. The enzyme phosphinothricin acetyl transferase (of Streptomyces sp) acetylates phosphinothricin, and thus inactivates the herbicide.

The gene responsible for coding phosphinothricin acetyl transferase (bar gene) has been identified in Streptomyces hygroscopicus. Some success has been reported in developing transgenic maize and oilseed rape by introducing bar gene. These plants were found to provide resistance to phosphinothricin.

Sulfonylureas and Imidazolinones Resistance:

The herbicides namely sulfonylureas and imidazolinones inhibit the enzyme acetolactate synthase (ALS), a key enzyme in the synthesis of branched chain amino acids namely isoleucine, leucine and valine. Mutant forms of this enzyme and the corresponding genes have been isolated, identified and characterized. Transgenic plants with the mutant genes of ALS were found to be resistant to sulfonylureas and imidazolinones e.g. maize, tomato, sugar beet.

Resistance to other herbicides:

Besides the above, some other herbicide resistant plants have also been developed e.g. bromoxynil, atrazine, phenocarboxylic acids, cyanamide. A list of selected examples of gene transferred herbicide resistant plants is given in Table 50.7.

It may however, be noted that some of the herbicide-resistant transgenic plants are at field-trial stage. Due to environmental concern, a few of these plants are withdrawn e.g. atrazine- resistant crops.

Environmental Impact of Herbicide-Resistant Crops:

The development genetically modified (GM) herbicide-resistant crops has undoubtedly contributed to increase in the yield of crops. For this reason, farmers particularly in the developed countries (e.g. USA) have started using these GM crops. Thus, the proportion of herbicide resistant soybean plants grown in USA increased from 17% in 1997 to 68% in 2001.

The farmer is immensely benefited as there is a reduction in the cost of herbicide usage. It is believed that the impact of herbicide-resistant plants on the environment is much lower than the direct use of the herbicides in huge quantities.

There are however, other environmental concerns:

i. Disturbance in biodiversity due to elimination of weeds.

ii. Rapid development of herbicide-resistance weeds that may finally lead to the production of super weeds.

Tolerance to Water Deficit Stresses:

The environmental conditions such as temperature (heat, freezing, chilling), water availability (shortage due to drought), and salinity influence the plant growth, development and yield. The abiotic stresses due to temperature, drought and salinity are collectively regarded as water deficit stresses (See Fig 50.1).

Causes of water deficit:

Water deficit may occur due to the following causes:

i. Reduced soil water potential.

ii. Increased water evaporation (in dry, hot and windy conditions).

iii. High salt concentration in the soil (decrease soil water potential).

iv. Low temperature resulting in the formation of ice crystals.

Effects of water deficit:

i. Results in osmotic stress.

ii. Inhibits photosynthesis.

iii. Increases the concentration of toxic ions (reactive oxygen species) within the cells.

iv. Loss of water from the cell causing plasmolysis and finally cell death.

Tolerance to osmotic stress:

The plant cells are subjected to severe osmotic stress due to water deficit. They however, produce certain compounds, collectively referred to as osmoprotectants or osmolytes, to overcome the osmotic stress. Osmoprotectants are non-toxic compatible solutes and are divided into two groups.

1. Sugar and sugar alcohols e.g. mannitol, sorbitol, pinitol, ononitol, trehalose, fructans.

2. Zwitterionic compounds: These osmo­protectants carry positive and negative charges e.g. proline, glycine betaine.

The production of a given osmoprotectant is species dependent. The formation of mannitol, proline and glycine betaine are more closely linked to osmotic tolerance.

Strategies to develop water deficit tolerance plants:

As explained above osmoprotectants offer good protection to plants against osmotic stress and therefore water deficit. It is therefore, logical to think of genetic engineering strategies for the increased production of osmoprotectants.

Some progress has been made in this direction. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding key enzymes isolated. In fact, some progress has been made in the development of transgenic plants with high production of osmoprotectants.

Transgenic plants with glycine toetaine production:

Glycine betaine is a quaternary ammonium compound and is electrically neutral. Besides functioning as a cellular osmolytes, glycine betaine stabilizes proteins and membrane structures. Some of the key enzymes for the production of glycine betaine have been identified e.g. choline mono-oxygenase, choline dehydrogenase, betaine aldehyde dehydrogenase.

The genes coding these enzymes were transferred to develop transgenic plants. By using choline oxidase gene from Arthrobacter sp, transgenic rice that produces higher glycine betaine (which offers tolerance against water deficit stress) has been developed.

Resistance against Ice-Nucleating Bacteria:

Formation of ice on the plant cells (outer membrane) is a complex chemical process. The importance of ice-nucleating bacteria is recognized in recent years. The occurrence of these bacteria has been reported in most of the plants — cereals, fruits and vegetable crops. The ice-nucleating bacteria synthesize proteins, which coalesce with water molecules to form ice crystals at temperature around 32°F. As the ice crystals grow, they can pierce the plant cells and severely damage the plants.

Chemical treatment of plants to protect from ice formation:

Plants can be treated with copper containing compounds to kill the bacteria. Another approach is to use urea solution so that the ice formation is minimized.

Ice-minus bacteria to resist plants from cold temperatures:

The bacterium Pseudomonas syringae is one of the highly prevalent ice-forming organisms in nature. With genetic manipulations, the gene that directs the synthesis of ice-related bacterial proteins in P. syringae was removed. These newly developed bacteria are referred to as ice-minus bacteria.

The researchers proposed to spray the transgenic ice-minus bacteria on to young plants. The intention was that these bacteria would give frost tolerance to the plants; and thus increase the crop yield. The opponents of DNA technology were against this approach—the main fear being that the bacterial mutants may create some health complication in humans.

The researchers argued and justified that no new genetic information is introduced into P. syringae and it is closely related in all aspect to the parent one which is already in the environment. After prolonged court proceedings in USA, clearance was given for spraying the ice-minus bacteria in the fields.

It was in 1987, ice-minus bacteria were sprayed on to the field of potato plants and strawberry plants. Another strain of P. syringae commercially labeled, as Frostban was later developed and used in crop fields. It may be noted here that ice-minus bacteria of P. syringae were the first transgenic bacteria that were used outside the laboratory. Fortunately, the experiments yielded encouraging results, since crop damage due to frost formation was found to be reduced.

Arabidopsis with cold- tolerant genes:

Scientists were successful in developing cold- tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperatures. They also identified a coordinating gene that encodes a protein, which acts as a transcription factor for regulating the expression of cold-tolerant genes. By introducing the coordinating gene, expression of cold-tolerant genes was triggered, and this protected the plants against cold temperatures. More work is in progress in this direction.

Application # 3. Improvement of Crop Yield and Quality:

With the advances made in plant genetic engineering, improvement in crop yield and quality have become a reality. The crop yield is primarily dependent on the photosynthetic efficiency and the harvest index (the fraction of the dry matter allocated to the harvested part of the crop). The quality of the crop is dependent on a wide range of desirable characters-nutritional composition of edible parts, flavour, processing quality, shelf-life etc.

Green Revolution:

The ‘Green Revolution’ led by Borlaug, Swaminathan and Khus enabled the world’s food supply to be tripled during the last three decades of 20th century. This was made possible by adopting genetically improved varieties of crops, coupled with advances in crop management.

The development of high-yielding varieties of wheat and rice has enabled several developing countries (a good example being India) to move from a position of food scarcity to become net exporter of these cereals.

The Green Revolution became a reality as the farmers adopted to new cereal seeds, besides employing high-input methods of agriculture — use of nitrogen fertilizers, herbicides, pesticides, modern equipment of agriculture etc.

Selected examples of crops for quality and yield:

There are a wide range of crops that have been manipulated by scientists for improved yield and quality. Only selected examples are briefly described.

Genetic Engineering for Extended Shelf-Life of Fruits:

The genetic manipulation of fruit ripening has become an important commercial aspect in plant genetic engineering.

Delay in fruit ripening has many advantages:

i. It extends the shelf-life, keeping the quality of the fruit intact.

ii. Long distance transport becomes easy without damage to fruit.

iii. Slow ripening improves the flavour.

Genetic engineering work has been extensively carried out in tomatoes, and some of the development are described.

Biochemical Changes during Tomato Ripening:

Fruit ripening is an active process. It is characterized by increased respiration accompanied by a rapid increase in ethylene synthesis. As the chlorophyll gets degraded, the green colour of the fruit disappears, and a red pigment, lycopene is synthesized (Fig. 50.8).

The fruit gets softened as a result of the activity of cell wall degrading enzymes namely polygalacturonase (PG) and pectin methyl esterase. The phytohormone ethylene production is intimately linked to fruit ripening as it triggers the ripening process of fruit. Addition of exogenous ethylene promotes fruit ripening, while inhibition of ethylene biosynthesis drastically reduces ripening.

The breakdown of starch to sugars, and accumulation of a large number of secondary products improves the flavour, taste and smell of the fruit. Three distinct genes involved in tomato ripening have been isolated and cloned. The enzymes encoded by these genes and their respective role in fruit ripening are given in Table 50.8.

Genetic Manipulations of Fruit Ripening:

Scientists have been trying to genetically manipulate and delay the fruit ripening process. Almost all the attempts involve antisense RNA approach.

Manipulation of the enzyme polygalacturonase (development of Flavr Savr tomato):

As already stated, softening of the fruit is largely due to degradation of the cell wall (pectin) by the enzyme polygalacturonase (PG). The gene responsible for PG, the rotting enzyme, has been cloned (pTOM 6). The genetic manipulation of polygalacturonase by antisense RNA approach for the development of Flavr Savr tomato (by Calgene Company in USA) is depicted in Fig. 50.9, and mainly involves the following stages.

1. Isolation of the DNA from tomato plant that encodes the enzyme polygalacturonase (PG).

2. Transfer of PG gene to a vector bacteria and production of complementary DNA molecules.

3. Introduction of complementary DNA into a fresh tomato plant to produce a transgenic plant.

Mechanism of PG antisense RNA approach:

In the normal tomato plant, PG gene encodes a normal (sense) mRNA that produces the enzyme polygalacturonase that is actively involved in fruit ripening. The complementary DNA of PG encodes for antisense mRNA, which is complementary to normal (sense) mRNA. The hybridization between the sense and antisense mRNAs renders the sense mRNA ineffective. Consequently, no poly­galacturonase is produced, hence fruit ripening is delayed.

The rise and fall of Flavr Savr Tomato:

The genetically engineered tomato, known as Flavr Savr (pronounced flavour saver) by employing PC antisense RNA was approved by U.S. Food and Drug Administration on 18^th May 1994. The FDA ruled that Flavr Savr tomatoes are as safe as tomatoes that are bred by conventional means, and therefore no special labeling is required. The new tomato could be shipped without refrigeration too far off places, as it was capable of resisting rot for more than three weeks (double the time of a conventional tomato).

Although Flavr Savr was launched with a great fanfare in 1995, it did not fulfill the expectation for the following reasons:

i. Transgenic tomatoes could not be grown properly in different parts of U.S.A.

ii. The yield of tomatoes was low.

iii. The cost of Flavr Savr was high.

It is argued that the company that developed Flavr Savr, in its overenthusiasm to become the first Biotech Company to market a bioengineered food had not taken adequate care in developing the transgenic plant. And unfortunately, within a year after its entry, Flavr Savr was withdrawn, and it is now almost forgotten!

Manipulation of ethylene biosynthesis:

It has been clearly established that ethylene plays a key role in the ripening of fruits. The biosynthetic pathway of ethylene is depicted in Fig 50.10. Ethylene is synthesized from S-adenosyl methonine via the formation of an intermediate, namely 1 -aminocyclopropane-1 carboxylic acid (ACC), catalysed by the enzyme ACC synthase. The next step is the conversion of ACC to ethylene by ACC oxidase.

Three different strategies have been developed to block ethylene biosynthesis, and thereby reduce fruit ripening.

1. Antisense gene of ACC oxidase:

Transgenic plants with antisense gene of ACC oxidase have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in fruit ripening.

2. Antisense gene of ACC synthase:

Ethylene biosynthesis was inhibited to an extent of 99.5% by inserting antisense gene of ACC synthase, and the tomato ripening was markedly delayed.

3. Insertion of ACC deaminase gene:

ACC deaminase is a bacterial enzyme. It acts on ACC (removes amino group), and consequently the substrate availability for ethylene biosynthesis is reduced. The bacterial gene encoding ACC deaminase has been transferred and expressed in tomato plants. These transgenic plants inhibited about 90% of ethylene biosynthesis. The fruit ripening was delayed by about six weeks. The strategies 1 and 2 may be referred to as antisense ethylene technology.

Longer Shelf-Life of Fruits and Vegetables:

The spoilage of fruits, vegetables and senescence of picked flowers, collectively referred to as post- harvest spoilage is major concern in agriculture. This hampers the distribution system particularly when the transport is done to far off places. The successful manipulations to delay ripening, senescence and spoilage of various foods will significantly contribute to the appropriate food distribution and thus good economic practices in agriculture.

Suppressing the biosynthesis of ethylene appears to be a promising area to reduce the spoilage of fruits, vegetables and senescence of flowers. The three different strategies to block ethylene synthesis in tomato have been described. The same approaches in fact can be successfully used for other fruits, vegetables etc., to achieve longer shelf- life.

Genetic Engineering for Preventing Discoloration:

Discoloration of fruits and vegetables is a major postharvest problem encountered in food industry. Certain food additives are added to prevent discoloration. However, these additives may cause health complications in humans.

Biochemically, discoloration of fruits and vegetables is mainly due to the oxidation of phenols (mono- and diphenols) to quinones, catalysed by a group of enzymes namely polyphenol oxidases. These enzymes are localized in the membranes of mitochondria and chloroplasts. Genetic manipulations using antisense approach to inhibit the synthesis of polyphenol oxidase has been carried. Some success has been reported in preventing the discoloration of potatoes by this strategy.

Genetic Engineering for Flower Pigmentation:

There are continuous attempts in flower industry to make the ornamental flowers more attractive (by improving or creating new colours), besides prolonging post-harvest lifetime. The cut flower industry is mostly (about 70%) dominated by four plants—roses, tulips, chrysanthemums and carnations.

The most common type of flower pigments are anthocyanin’s, a group of flavonoids. They are synthesized by a series of reactions, starting from the amino acid phenylalanine (Fig. 50.11). The colour of the flower is dependent on the chemical nature of the anthocyanin produced.

i. Pelagonidin 3-glucoside — brick red/orange.

ii. Cyanidin 3-glucoside — red.

iii. Delphinidin 3-glucoside — blue to purple.

Manipulation of anthocyanin pathway enzymes:

The enzymes responsible for different reactions, in the anthocyanin pathway have been identified. By genetic manipulations and mutations, it is possible to develop flowers with the desired colours. Most of the flowers (roses, carnations chrysanthemums) lack blue colour due to the absence of the key enzyme flavonoids 3′, 5′- hydroxylase (F 3′ 5′ H) that produces delphinidine 3 glucoside. One company, by the name Florigene, has genetically manipulated and introduced the gene encoding the enzyme F 3′ 5′ H (from Petunia hybrida) into the following plants.

The world’s first genetically modified (GM) flower was introduced in 1996. It was a mauve (bluish) coloured carnation with a trade name Moondust™. Subsequently, many other flowers have been produced and marketed.

Can GM-flowers be eaten?

In majority of countries, flowers are used for ornamental purposes and not usually eaten. However, in some countries like Japan, flower petals are used for decoration of foods, and frequently eaten also. This raises an important question about the safety of GM flowers, since they are not thoroughly screened for human consumption. But the present belief is that the anthocyanin’s (the colouring chemical molecules) are natural plant materials, and their consumption may be in fact beneficial to health.

Genetic Engineering for Male Sterility:

The plants may inherit male sterility either from the nucleus or cytoplasm, cytoplasmic male sterility (cms) is due to the defects in the mitochondrial genome. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility.

In tobacco plants, male sterility was introduced by using a mitochondrial mutated gene encoding the enzyme ribonuclease. The gene encoding ribonuclease namely barnase gene from Bacillus amyloliquefaciens was transferred to tobacco plants.

The ribonuclease is toxic to tapetal cells, and thus prevents the development of pollen, ultimately leading to male sterility. By this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce etc., with male sterility have been developed. It is possible to restore male sterility in the above plants by crossing them with a second set of transgenic plants containing ribonuclease inhibitor gene.

Application # 4. Transgenic Plants with Improved Nutrition:

Genetic manipulations for improving the nutritional quality of plant products are of great importance in plant biotechnology. Some success has been achieved in this direction through conventional cross-breeding of plants. However, this approach is very slow and difficult, and many a times will not give the traits with the desired improvements in the nutritional quality. Selected examples of genetic engineering with improved nutritional contents are described.

Amino Acids of Seed Storage Proteins:

Of the 20 amino acids present in the humans, 10 are essential while the other 10 can be synthesized by the body. The 10 essential amino acids (EAAs) have to be supplied through the diet. Cereals (rice, wheat, maize, corn) are the predominant suppliers of EAAs. However, cereals do not contain adequate quantity of the essential amino acid lysine.

On the other hand, pulses (Bengal gram, red gram, soybean) are rich in lysine and limited in sulfur-containing amino acids (the essential one being methionine). Transgenic routes have been developed to improve the essential amino acid contents in the seed storage proteins of various crop plants.

Overproduction of lysine by deregulation:

The four essential amino acids namely lysine, methionine, threonine and isoleucine are produced from a non-essential amino acid aspartic acid (Fig. 50.12). The formation of lysine is regulated by feedback inhibition of the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DHDPS). Theoretically, it is possible to overproduce lysine by abolishing the feedback regulation. This is what has been accomplished.

The lysine feedback-insensitive genes encoding the enzymes AK and DHPDS have been respectively isolated from E.coli and Cornynebacterium with appropriate genetic manipulations, these genes were introduced into soybean and canola plants. The transgenic plants so developed produced high quantities of lysine.

Transfer of genes encoding methionine-rich proteins:

Several genes encoding methonine-rich proteins have been identified:

i. In maize, 21 KDa zein with 28% methionine.

ii. In rice, 10 KDa prolamin with 20% methionine.

iii. In sunflower, seed albumin with 16% methionine.

These genes have been introduced into some crops such as soybean, maize and canola.

The transgenic plants produced proteins with high contents of sulfur-containing amino acids.

Production of lysine-rich glycinin in rice:

Glycinin is a lysine-rich protein of soybean. The gene encoding glycinin has been introduced into rice and successfully expressed. The transgenic rice plants produced glycinin with high contents of lysine. Another added advantage of glycinin is that its consumption in humans is associated with a reduction in serum cholesterol (hypo- cholesterolaemic effect).

Construction of artificial genes to produce proteins rich in EAAs:

Attempts are being made to construct artificial genes that code for proteins containing the essential amino acids in the desired proportion. Some success has been reported in the production of one synthetic protein containing 13% methionine residues.

Genetic Engineering for Improving Palatability of Foods:

More than the nutritive value, taste of the food is important for attracting humans. It is customary to make food palatable by adding salt, sugar, flavors and many other ingredients. It would be nice if a food has an intrinsically appetizing character.

A protein monellin isolated from an African plant (Dioscorephyllum cumminsii) is about 100,000 sweeter than sucrose on molar basis. Monellin gene has been introduced into tomato and lettuce plants. Some success has been reported in the production of monellin in these plants, improving the palatability.

Golden Rice —The Provitamin A Enriched Rice:

About one-third of the world’s population is dependent on rice as staple food. The milled rice that is usually consumed is almost deficit in P-carotene, the pro-vitamin A. As such, vitamin A deficiency (causing night blindness) is major nutritional disorder world over, particularly in people subsisting on rice.

To overcome vitamin A deficiency, it was proposed to genetically manipulate rice to produce β-carotene, in the rice endosperm. The presence of β-carotene in the rice gives a characteristic yellow/orange colour, hence the pro-vitamin A-enriched rice are appropriately considered as Golden Rice.

In Fig. 50.13 an outline of the biosynthetic pathway for the formation of P-carotene is given. The genetic manipulation to produce Golden Rice required the introduction of three genes encoding the enzymes phytoene synthase, carotene desaturase and lycopene β-cyclase. It took about 7 years to insert three genes for developing Golden Rice.

Golden Rice has met almost all the objections raised by the opponents of GM foods. However, many people are still against the large scale production of Golden Rice, as this will open door to the entry of many other GM foods.

Another argument put forth against the consumption of Golden Rice is that it can supply only about 20% of daily requirement of vitamin A. But the proponents justify that since rice is a part of a mixed diet consumed (along with many other foods), the contribution of pro-vitamin A through Golden Rice is quite substantial.

Recently (in 2004), a group of British scientists have developed an improved version of Golden Rice. The new strain, Golden Rice 2 contains more than 20 times the amount of pro-vitamin A than its predecessor. It is claimed that a daily consumption of 70 g rice can meet the recommended dietary allowance for vitamin A.

Genetic Engineering to Increase Vitamins and Minerals:

The transgenic rice (Golden Rice) developed with high pro-vitamin A content is described above. Transgenic crop plants are also being developed for increased production of other vitamins and minerals. A transgenic Arabidopsis thaliana that can produce ten-fold higher vitamin E (α-tocopherol) than the native plant has been developed. This was done by a novel approach. A. thaliana possesses the biochemical machinery to produce a compound close in structure to α-tocopherol.

A gene that can finally produce α-tocopherol is also present, but is not expressed. This dormant gene was activated by inserting a regulatory gene from a bacterium. This resulted in an efficient production of vitamin E. Some workers are trying to increase the mineral contents of edible plants by enhancing their ability to absorb from the soil. Some success has been reported with regard to increased concentration of iron.

Application # 5. Commercial Transgenic Crop Plants:

The very purpose of production of transgenic plants is for their commercial importance with high productivity. It was in 1995-96; transgenic plants (potato and cotton) were, for the first time, made available to farmers in USA. By the year 1998-99, five major transgenic crops (cotton, maize, soybean, canola and potato) were in widespread use. They accounted for about 75% of the total area planted by crops in USA.

A selected list of transgenic crop plants (approved in USA) for the commercial use is given in Table 50.9. Some examples of transgenic crop plants, which are at the developmental stages are given in Table 50.10. These plants are carefully designed to give rise to products, which will improve human health and increase of crop yield.

Goals of biotechnological improvements in crops:

There are about 30-40 crops that have been genetically modified, and many more are being added. However, very few of them have got the clearance for commercial use. A selected list is already given in Table 50.9.

The ultimate goals of genetically modified (CM) crop plants are listed below:

i. Resistance to diseases (insect, microorganisms).

ii. Improved nitrogen fixing ability.

iii. Higher yielding capacity.

iv. Resistance to drought and soil salinity.

v. Better nutritional properties.

vi. Improved storage qualities.

vii. Production of pharmaceutically important compounds.

viii. Absence of allergens.

ix. Modified sensory attributes e.g. increased sweetness as in thaumatin.

Concerns about transgenic plants:

The fears about the harmful environmental and hazardous health effects of transgenic plants still exist, despite the fact that there have been no reports so far in this regard. The transfer of almost all the transgenic plants from the laboratory to the crop fields is invariably associated with legal and regulatory hurdles, besides the social and economic concerns.

The major concern expressed by public (also acknowledged by biotechnologists) is the development of resistance genes in insects, generation of super weeds etc. Several remedial measures are advocated to overcome these problems.

The farmers in developing countries are much worried about the seed terminator technology which forces them to buy seeds for every new crop. These farmers are traditionally habituated to use the seeds from the previous crop which is now not possible due to seed terminator technology.

Application # 6. Transgenic Plants as Bioreactors:

Another important application of genetically transformed plants is their utility as bioreactors to produce a wide range of metabolic and industrial products.