The following points highlight the five genetic engineering techniques for disease resistance. Some of the techniques are: 1. Developing Virus Resistance Food Crops 2. Developing Fungi Resistance Food Crops 3. Developing Bacteria Resistance Food Crops 4. Developing Insects Resistance Food Crops 5. Developing Nematode Resistance Food Crops 6. Improving Field-Crop Production and Soil Management and Others.
Techniques for Disease Resistance:
- Developing Virus Resistance Food Crops
- Developing Fungi Resistance Food Crops
- Developing Bacteria Resistance Food Crops
- Developing Insects Resistance Food Crops
- Developing Nematode Resistance Food Crops
- Improving Field-Crop Production and Soil Management
- Improved Nitrogen Utilization
- Stress Tolerance
- Regulation of Plant Hormones
Technique # 1. Developing Virus Resistance Food Crops:
Viruses are among the most ubiquitous pests in agriculture. Scientists are working to develop viral resistance in a variety of crops including squash, potato, sweet potato, wheat, papaya and raspberries.
Viruses are studied widely because they not only cause disease in humans, plants, animals and insects, but also are used as tools in the study of molecular biology and, in some cases, in the development of vaccines to fight the diseases they can cause.
Several techniques for virus resistance have been developed. These include viral coat protein technology and multiple gene transfers. A viral coat protein acts like a vaccine, causing the plant to develop resistance to the particular virus. Transferring the gene for a viral coat protein, a part of the outer shell of a virus that does not cause disease, into a plant acts like a vaccine for the plant.
The plant is then able to resist the virus, analogous to the way vaccines keep us from getting certain diseases like measles. The advantage of introducing only the coat protein is that it induces resistance without the introduction of the actual virus. The technique has been used successfully in many plants against several different viruses.
The first genetically engineered virus-resistant food crop in the marketplace was yellow crookneck squash. Using the viral coat protein approach, this squash was engineered to resist the watermelon mosaic virus and the zucchini yellow mosaic virus. Potatoes are highly susceptible to many viruses, including the potato mosaic virus and the potato leaf roll virus.
A leaf roll virus epidemic in 1996 was responsible for heavy potato crop losses in Idaho. The virus, spread by aphids, damaged the potatoes to the point that they were unmarketable. Scientists in Mexico, in collaboration with researchers at Monsanto, have developed potatoes resistant to several forms of this virus. Research on disease-resistant potatoes is continuing at other laboratories.
The feathery mottle virus has a damaging effect on sweet potatoes. In 1991, researchers began genetically engineering varieties of sweet potato grown in Africa, where it is an important subsistence crop. The sweet potato was engineered with coat protein from this virus and replicase genes. Replicase is an enzyme involved in the duplication of certain viral RNA molecules.
Current field-testing has demonstrated successful gene transformations and the desired development of resistance to sweet potato feathery mottle virus. Although wheat is an important food source, development of genetically engineered varieties has been slower than in corn, soy and cotton.
A major pest in wheat is barley yellow dwarf virus, which can cause damage in major wheat-growing regions such as North Dakota, because no resistant strains are known. Work is in progress to engineer resistance to this disease using the viral coat protein technique.
The wheat genome is highly complex-ten to twenty times larger than that of cotton or rice-and carries an exceptionally large amount of repetitive DNA sequences. Thus, targeting particular genes is challenging, and transgenic wheat biotechnology has advanced more slowly than that of other crops.
The papaya crop in Hawaii was nearly wiped out in the 1950s by the papaya ring-spot virus (PRSV). Transmitted by aphids, this virus causes one of the most serious diseases of papaya worldwide. Work to develop a transgenic virus-resistant variety began in the late 1980s. By 1992, resistant lines were field-tested; approvals for commercialization were granted in 1997.
The transgenic- resistant papaya is now in wide use in Hawaii, and similar work is in progress in the Philippines, Malaysia, Thailand, Vietnam and Indonesia to enhance resistance in local papaya varieties where ring-spot virus is a major pest. Researchers are also modifying other fruits for virus resistance.
Technique # 2. Developing Fungi Resistance Food Crops:
The search for genetic engineering tactics to combat fungi has intensified with the need to find adequate substitutes for fungicides such as methyl bromide, widely used on fruit and vegetables but being phased out due to its links to ozone depletion.
One emerging area is directed at a plant’s production of defensins, a family of naturally occurring antimicrobial proteins which enhance the plant’s tolerance to pathogens, especially bacteria. Certain defensins also demonstrate an ability to fight fungal infections. Defensins are found throughout nature in insects, mammals (including humans), crustaceans, fish and plants.
Defensins from moths and butterflies, the fruit fly, pea seeds and alfalfa seeds all show potent antifungal activity. The first transgenic application of defensins was the incorporation into potatoes of the antifungal defensin from alfalfa. Laboratory and field trials showed that the transgenic potatoes were as resistant to the fungal pathogen Verticillium dahliae as non-transgenic potatoes treated with fungicide.
Although studies are continuing, the chance that fungi will build resistance to defensins is thought unlikely. No known resistant strains of bacteria or fungi have yet evolved that can overcome these highly protective, pesticidal proteins.
On-going research involving banana and cassava is directed to cloning resistance genes for major tropical diseases such as black sigatoka, a leaf fungus that widely infects bananas, cassava mosaic disease and cassava bacterial blight.
In bananas, transgenic lines combining several antifungal genes have been generated. Selected lines are currently being tested for resistance to black sigatoka and Panama disease under greenhouse and field conditions.
Scientists are devising protection against the plant fungus Botrytis cinerea, a serious pathogen in wheat and barley. The strategy uses the gene for a natural plant defence compound named resveratrol. Scientists have also introduced a gene from a wine grape into barley to confer resistance to Botrytis cinerea. Field trials are underway.
Resistance to potato late blight, a disease caused by Phytophthora infestans, receives high priority in potato research. Plant disease from this fungus can be destructive to crop production, as was dramatically illustrated in the Irish potato famine.
In 1995, a U.S. late blight epidemic (caused by new aggressive strains of Phytophthora infestans) affected nearly 160,000 acres of potatoes, or about 20 per cent of domestic production.
Research is underway to genetically engineer potatoes that express the enzyme glucose oxidase and develop resistance to Phytophthora blights (Douches undated). At present, however, no products are close to commercialisation. Potatoes are also being transformed using a soybean gene for a protein (beta-1, 3-endoglucanase) that confers resistance to infection by Phytophthora.
Other studies report that transgenic potatoes expressing a protein called osmotin showed reduced damage from lesion growth in leaves inoculated with the Phytophthora infestans pathogen.
Still other research is attempting to boost fungal resistance in potatoes by transferring resistance genes from peas. Infection of these transgenic potatoes with the fungus triggers hormone-like signals in the potatoes that turn on the pea resistance genes.
One substance that is produced, chitosan, stops fungal growth and activates the potato’s own natural defence systems. In rice, blast and sheath blight are major fungal diseases. Scientists created transgenic strains resistant to sheath blight that are currently being field-tested.
Technique # 3. Developing Bacteria Resistance Food Crops:
Most food crops are susceptible to bacterial diseases, but bacteria rarely attack certain plants, such as mosses, ferns and conifers. Bacterial infections in plants may cause leaf and fruit spots (lesions), soft rots, yellowing, wilting, stunting, tumours, scabs or blossom blights.
When tissue damage occurs on the blossoms, fruit or roots of food crops, yields may be reduced. Potatoes are susceptible to blackleg and soft rot diseases caused by the bacterial pathogen Erwinia carotovora.
To combat these bacteria, scientists have exploited the family of enzymes known as lysozymes that catalyze the breakdown of bacterial cell walls. Using cloned lysozyme genes and a promoter, transgenic potatoes were created that produced lysozyme.
In laboratory tests, the transformed potatoes exhibited substantially enhanced resistance to Erwinia carotovora. Field tests and further development of resistant lines are in progress. A different transgenic strategy to combat Erwinia carotovora was demonstrated in tobacco engineered to overexpress a peptide that kills bacteria.
The genetically engineered tobacco plants were resistant to both Erwinia carotovora and Pseudomonas syringae pv tabaci, the pathogen responsible for wild fire disease in rice. Scientists have also successfully transferred a bacterial resistance gene from wild rice to cultivated rice.
4. Developing Insects Resistance Food Crops:
There are several different combat tactics, including engineering for the expression of toxins in plants that kill insects when they consume the plant material, but are nontoxic to other species that eat the plant. Other alterations focus on inducing sterility in the pest organism or affecting the digestion or metabolism of the pests.
In addition, attempts to enhance a plant’s natural ability to produce leaf wax could make the plant more difficult for insects to consume. The best known and most widely used transgenic pest-protected crops are those that express insecticidal proteins derived from genes cloned from the soil bacterium Bacillus thuringiensis, more commonly known as Bt. Crystal (Cry) proteins or delta-endotoxins formed by this bacterium are toxic to many insect species.
Delta-endotoxins bind specifically in the insect gut to receptor proteins, destroying cells and killing the insect in several days (shown below). There are several different Bt strains containing many different toxins. Scientists have identified and isolated the genes for several toxin proteins from different Bt strains.
In recent years, these genes have been introduced into several crop plants in an effort to protect them from insect attack and eliminate the need for spraying synthetic chemical pesticides. There are more than 100 patents for Bt Cry genes. Bt field corn, sweet corn, soy, potato and cotton are commercialized in the U.S., and one or more of these are commercialized in at least 11 other countries.
Bt controls the larvae of butterflies and moths (Lepidopteran insects) that eat the plants. It is especially effective against the larvae of the European corn borer (shown left), a significant corn pest in the U.S., as well as the Southwestern corn borer and the lesser cornstalk borer. In sweet corn, Bt toxins effectively deter corn earworm and fall armyworm.
Recently, a different strain of Bt, Bacillus thuringiensis tenebrionis, was used as a gene source to confer resistance to corn rootworm, another major pest in cornfields. The resistant corn is currently in field trials. Bt hybrid rice is also undergoing field-testing and is showing considerable effectiveness in resisting major pests in Asia such as the leaf folder, yellow stem borer and striped stem borer.
Bt canola is also under development. Borers also create a good environment for fungi to grow. Where fusarium fungi grow, they reduce plant quality and generate fumonisins-toxins that can be fatal to farm animals and have been linked to liver and esophageal cancer in African farmers. Thus, one way to reduce fungal contamination is to control pests.
Scientists have measured reductions in fumonisin levels in Bt corn of 90 percent or greater. Bt works against insects that eat plant tissue. However, those pests that do not eat the leaves, but rather pierce and suck nutrients from the plant, require different defence strategies. These insects include aphids, white flies and stink bugs.
White flies are a major pest in poinsettias, sweet potatoes and cotton. Because these insects do not consume large amounts of plant material, a leading way to combat them is the genetic expression of toxic proteins that are strong enough to kill the pest, yet safe for the plant and non- target organisms.
Avidin in transgenic corn demonstrates a different approach. Avidin is a glycoprotein, an organic compound composed of both a protein and a carbohydrate, and is usually found in egg whites. Avidin is known for chemically tying up the vitamin biotin, making it unavailable as a nutrient. Insects eating transgenic corn modified to produce avidin die from biotin deficiency.
Although this corn was not toxic to mice, further evaluation of its potential for insect toxicity and safety for human consumption is awaited. Plants produce wax as a natural protective coating. Genetic modification can increase the expression of this inherent trait.
Experiments to increase leaf wax are in the early stages, but scientists have already raised wax content by as much as 15-fold. This strategy is aimed at increasing the plant’s resistance to both pests and fungal pathogens.
Technique # 5. Developing Nematode Resistance Food Crops:
The most common of nematode plant parasites found worldwide is the root-knot nematode. Probably every form of plant life, including field crops, ornamentals and trees, is attacked by at least one species of nematode. They are responsible for 10 per cent of global crop losses worth an estimated $80 billion a year.
Transgenic strategies to combat nematodes are emerging. Nematodes are particularly destructive in bananas, soybeans, rice and potatoes. Scientists are fighting these parasitic worms in potato and banana crops using the genes for cystatins, defence proteins that occur naturally in rice and sunflowers. Incorporation of the genes in potatoes produced as much as 70 per cent nematode resistance in field trials.
Nematodes are particularly fond of soybeans. In the U.S., the soybean cyst nematode is considered the most devastating pest. Standard plant breeding led to a highly resistant variety of soybeans from a wild strain, but it did not cross well with modern soybean lines.
Using genetic markers, a means of identifying cells with particular traits, scientists bred plants containing the resistance gene with domesticated varieties, circumventing the poor performance characteristics of the wild variety. While the new varieties are not transgenic, they resulted from combining the use of modern genetic markers with conventional breeding techniques.
Technique # 6. Improving Field-Crop Production and Soil Management:
Improving field-crop production and soil management is another central aim of genetic engineering technology in commodity crops. Applications include crop resistance to herbicides; improved nitrogen utilisation, reducing need for fertiliser; increased tolerance to stresses such as drought and frost; regulation of plant hormones, which are key to plant growth and development; attempts to increase yield, and a multitude of other, less widespread applications.
There are many negative effects when weeds grow with crop plants, the most common being competition for sunlight, water, space and soil nutrients. If weeds grow with crops, they too use these growth factors, and may cause losses great enough to justify control measures.
In addition to economic yield loss, other concerns may determine when weed control is justified. For example, eastern black nightshade in soybeans or late-emerging grasses in corn may not reduce yield, but these weeds can clog equipment, causing harvest delays. The most common method currently employed to manage weeds is the use of herbicides.
The use of genetic modification techniques has created crops that are both tolerant and resistant to herbicides, or weed killers. This technology allows herbicides to be sprayed over resistant crops from emergence through flowering, thus making the applications more effective.
To date, six categories of these crops have been engineered to be resistant to the herbicides glyphosate, glufosinate ammonium, imidazolinone, sulfonylurea, sethoxydim and bromoxynil.
Probably the best-known herbicide for which tolerance has been genetically engineered into crops is glyphosate, known commercially by brand names such as Roundup, Rodeo and Accord. Resistance to glyphosate is the transgenic trait most common in agriculture worldwide. To date soy, corn, cotton, canola, sugar beets and, most recently, wheat, have been genetically transformed for glyphosate tolerance.
Although glyphosate has been used as an herbicide for 26 years, transgenic glyphosate-resistant crops are a more recent development and are widely deployed on acres devoted to soy and cotton. Research is underway to create other glyphosate tolerant crops. To date, two weed species, annual rigid ryegrass and goose grass, have built resistance to glyphosate.
Corn, soy, rice, sugar beet, sweet corn and canola have also been genetically modified to tolerate the herbicide glufosinate ammonium. The seeds for these crops are sold commercially under brand names such as Liberty Link. Transgenic soybeans, cotton and flax with a tolerance to the herbicide sulfonylurea are also on the market.
Other strains of engineered soybeans and corn are resistant to sethoxydim, the active ingredient in the commercial herbicides Poast, Poast Plus, and Headline, used to control undesirable grass species.
The herbicide bromoxynil, sold under the commercial name Buctril, is normally toxic to cotton, a broadleaf crop, and is primarily used on grass-like crops, such as corn, sorghum and small grains, to kill invading broadleaf weeds. Scientists have genetically modified cotton plants for resistance to this herbicide, allowing its use to control broadleaf weeds in cotton fields.
Technique # 7. Improved Nitrogen Utilization:
There appear to be relatively few biotechnology applications specifically designed to enhance the characteristics of farm crops, such as size, yield, branching, seed size and number. Scientists have, however, created some enhancements. A recent example is the discovery of a gene in the alga Chlorella sorokiniana that has a unique enzyme not found in conventional crop plants.
The enzyme, ammonium-inducible glutamate dehydrogenase, increases the efficiency of ammonium incorporation into proteins. In some plants, it increases the efficiency of nitrogen use. The practical implication is that less fertilizer would be necessary to grow these plants. When the gene was incorporated into wheat, biomass production, growth rate and kernel weight all increased, as did the number of spikes in the plant.
Technique # 8. Stress Tolerance:
Stress tolerance involves a family of genes, rather than a single one. They are rapidly activated in response to cold, inducing the expression of “cold-regulated” genes, and resulting in enhanced freezing tolerance.
Over-expression of these genes in Arabidopsis-small plants of the mustard family that are commonly used to study plant genetics-increases freezing tolerance and leads to elevated levels of proline and total soluble sugars, substances that protect against cold. Common stress responses in plants involve water retention at the cellular level.
As a result, researchers have given special attention to osmoprotectant molecules, or molecules that hold water, such as sugars, sugar alcohols, certain amino acids (proline) and quaternary amines like glycinebetaine.
Various plants genetically engineered for increased levels of protectant sugar have shown increased drought tolerance. For instance, Arabidopsis and tobacco plants engineered to produce mannitol, a sugar alcohol, withstood high saline conditions and had enhanced germination rates and increased biomass. Other strategies have addressed different stress factors.
Improved cold tolerance and normal germination under high salt was reported in Arabidopsis engineered to express the enzyme choline oxidase. Transgenic rice, engineered to express the late embryogenesis abundant protein gene transferred from barley, was significantly more tolerant to drought and salinity than conventional varieties of rice.
Another transgenic rice engineered in the laboratory for enhanced expression of the enzyme glutamine synthetase had increased photorespiration capacity and increased tolerance to salt. Preliminary results suggested enhanced tolerance to chilling as well.
Technique # 9. Regulation of Plant Hormones:
Plant hormones such as auxin, cytokinins, gibberellins, abscisic acid, ethylene, etc. have been targeted for genetic modification to influence plant growth and development- fruit development and ripening; stem elongation and leaf development; germination, dormancy and tolerance of adverse conditions.
These hormone classes are highly interactive; the concentration of one affects the activity of another. For example, the ratio of the hormone abscisic acid to gibberellin in a plant determines whether a seed will remain dormant or germinate.
Recent discovery of an enzyme involved in the production of the hormone auxin enabled researchers to investigate the effects of moderating auxin production in determining plant characteristics. When auxin is overproduced, branching is inhibited and leaves curl down as the plant elongates, a reaction typically related to reduced light exposure. The same gene that produces this enzyme is apparently related to a gene in mammals that governs enzymes that detoxify certain chemicals.
In wheat, the hormone abscisic acid slows seed germination and improves the tolerance to cold and drought. Extending or enhancing the production of abscisic acid may also delay germination, a useful characteristic in climates where spring rain is sparse or falls late in the season.
Production of abscisic acid is increased in response to environmental stress, and a family of enzymes called protein kinases stimulates its production. Selecting plant varieties high in abscisic acid, or engineering plants to produce more of the hormone, may confer greater drought and cold tolerance.
Introduction of dwarfed, high-yielding wheat contributed to the ‘Green Revolution’ of the 1960s and 1970s, during which world wheat yields almost doubled. Shorter varieties of wheat grains, with a greater resistance to damage by wind, resulted from a reduced response to the hormone gibberellin.
Scientists have since shown that the gene called Rht can cause “dwarfing” in a range of plants, opening up the possibility of quickly developing higher-yielding varieties in several crops. Researchers believe that this strategy could be applied to a still wider range of crops through genetic engineering.
The plant hormone ethylene regulates ripening in fruits and vegetables. Controlling the amount and timing of ethylene production can initiate or delay ripening, which might reduce spoilage that can occur between the time produce is picked and brought to market.
Transgenic techniques aim to regulate the enzyme that breaks down a precursor of ethylene production. By regulating the timing and rate of this degradation, ripening can be controlled. This technology has been applied and field- tested in tomatoes, raspberries, melons, strawberries, cauliflower and broccoli, but has not yet been commercialized.