BIOLOGY Volume 3 - A Guide to General Biology - 2004

25. APPLIED GENETICS

25.4. Transgenic Plants

Class="center">25.4.1. Introduction of new Genes into Plants

Using Agrobacterium

The most effective method for Gene transfer in plants is The Use of the soil bacterium Agrobacterium tumefaciens as a vector. This bacterium contains a plasmid into which the gene targeted for transfer can be inserted. Agrobacterium tumefaciens infects most dicotyledonous plants, causing The formation of large growths known as crown galls, which resemble cancerous tumors (Fig. 25.13). Normally, in response to injury, a plant secretes chemical compounds that stimulate Cell Division, resulting in the formation of a mass of Cells called a callus that rapidly seals the wound. Chemicals released by damaged plant cells also stimulate Agrobacterium cells, which infect the wound and induce gall formation. This process is controlled by a bacterial plasmid (the Ti plasmid, derived from tumor-inducing). It penetrates The plant cell and integrates into the plant's DNA, leading to unregulated growth. The bacterium itself does not enter the cells, but can live between them, utilizing nutrients produced by the plant Cells under the control of the plasmid DNA. Plant cells containing the Ti plasmid with a foreign gene integrated into their genome are termed transformed.

Unfortunately, for a long time this method was inapplicable to monocotyledonous plants, which include major agricultural crops such as maize and wheat. However, this problem has now been solved. Transformation techniques are currently used to improve varieties of tomatoes, potatoes, and many woody plants.

Using the cloning technique described in Section 21.3, an entire plant can be regenerated from a single transformed cell. To achieve this, cells are first grown in a liquid culture, and then the resulting undifferentiated mass, called a callus, is transferred to nutrient Agar. With the proper hormone balance, shoots and roots form, and a new plant develops. Another approach to obtaining Transgenic Plants also involves the use of Agrobacterium. Discs cut from leaves are infected with the bacterium and placed on nutrient agar. During growth, the transformed cells develop roots and shoots.

Fig. 25.13. A. Introduction of a new gene into a plant cell using Agrobacterium. B. Crown gall formed upon infection of a wound by the bacterium Agrobacterium.

Using Viruses

In bacterial Genetic Engineering, Bacteriophages (bacterial viruses) typically serve as vectors; similarly, plant viruses can potentially be used for plant transformation.

Using a "Gene Gun"

Surprisingly, introducing foreign DNA into plant cells using a specialized particle gun has proven highly effective. The desired DNA is coated onto microscopic gold or tungsten beads measuring 1 mm in diameter. These beads are placed at the tip of a plastic bullet inserted into the barrel of a specially designed gun. In the initial design, the bullet was propelled in the traditional manner, i.e., by an explosive charge; however, compressed gas is now used for firing. The bullet shatters within a chamber against a surface featuring microscopic holes. Some of the DNA-coated particles pass through these apertures into the target, which consists of plant cells or Tissues. Firing is conducted in a vacuum to prevent the DNA particles from settling. They are subsequently detected in the Cytoplasm of the transformed cells.

25.4.2. Pest Resistance — Insecticides

Insects inflict immense damage on agricultural crops and livestock. Since the mid-20th century, various chemicals, including DDT, have been employed to combat insect pests. The environmental impact of these agents was unknown at the time. Only after realizing the full scale of ecological destruction caused by insecticides did people begin to consider alternative strategies, one of which is biological pest control.

The soil bacterium Bacillus thuringiensis, which some biologists abbreviate as Bt, produces a protein toxin effective against many insect pests. It is 80,000 times more potent than organophosphate insecticides typically sprayed on crops, yet highly specific—it kills only certain insect species. Various strains of Bt act on different insects (primarily their larvae): Lepidoptera, Hemiptera, and Diptera. Some Bt strains even destroy nematodes, which are also agricultural pests. The toxin specifically binds to the midgut lining of insects and damages the epithelium, preventing the absorption of digested food and causing the larva to die of starvation (Fig. 25.14). The Bacteria themselves, their spores, or even the toxin can be sprayed onto plants; however, the toxin must first be isolated from spores and stabilized using Protein Engineering. A more ingenious approach, however, is to introduce the gene responsible for toxin production into plant cells via genetic engineering, thereby ensuring continuous protection. This method has been successfully tested on several plants, notably maize. In field trials, conventional plants and plants carrying the toxin gene were infested with corn borer larvae. Results were recorded after 6 weeks. The average length of tunnels bored by the pest was 6.3 cm in transgenic plants, whereas in conventional plants it reached 40.7 cm. Currently, the bacterial toxin gene has been successfully introduced into potatoes, tomatoes, cotton, rice, and other crops.

Fig. 25.14. A Pieris rapae larva six days after consuming a plant treated with Bt toxin. The larva is dead and undergoing decomposition.

Another non-bacterial approach has also proven successful. Certain leguminous plants synthesize Polypeptides that inhibit proteinases in the guts of specific insect pests. Consequently, the insects lose their ability to digest protein and perish. The corresponding genes have been transferred into agricultural crops whose seeds are commonly attacked by these pests. Field trials have yielded successful results.

Currently, Genetic engineering Methods are also being explored to protect plants from other pathogens such as Fungi, bacteria, and viruses. The use of pest-resistant transgenic plants offers three principal advantages over other control methods:

1) pesticides are expensive and labor-intensive to apply; 2) pesticides kill both harmful and beneficial insects (such as pollinators); 3) certain pesticides accumulate in the environment and are capable of exerting mutagenic effects on multiple generations of animals.

25.4.3. Pest Resistance — Viruses

Plant viruses cause substantial damage to agriculture. The first attempts to generate virus-resistant varieties using genetic engineering were conducted on tobacco plants. Tobacco is susceptible to an RNA-containing virus known as tobacco mosaic virus (TMV — Fig. 2.18). This virus also threatens tomatoes, with annual losses in the USA exceeding $50 million. Using Agrobacterium, the gene encoding the TMV coat protein was introduced into tobacco plants. Trials demonstrated that transgenic plants exhibit significantly greater resistance to TMV compared to control plants. This phenomenon represents a form of vaccination (Fig. 25.15). Subsequent analogous experiments were performed on potatoes, tomatoes, and alfalfa to protect them against viral infections.

Fig. 25.15. Tobacco mosaic virus causes mosaic spotting on the leaves of infected plants.

25.4.4. Herbicide-Resistant Crops

Another promising direction in Plant Genetic Engineering is the introduction of herbicide-resistance alleles into their genomes. If fields planted with such crops are treated with herbicide, only the weeds will perish. Meanwhile, even in developed countries utilizing modern agricultural practices, weeds reduce crop yields by nearly 10%. It has been calculated that the application of herbicide-resistant varieties in certain regions of Africa—where weed problems are severe and people suffer from food shortages—could increase the yields of maize, wheat, sorghum, sunflower, and legumes by a factor of 2 to 4. Genetically modified varieties of maize, wheat, sugar beet, and oilseed rape resistant to herbicides are already in use in developed countries.

25.4.5. Nitrogen Fixation

A promising objective in agriculture is the introduction of genes controlling nitrogen fixation into crop plants. Nitrogen fixation is the process by which atmospheric nitrogen is reduced in cells to ammonium and subsequently utilized for the synthesis of Proteins and other Organic compounds. These chemical transformations are carried out by specialized nitrogen-fixing bacteria that inhabit the ROOT nodules of legumes such as peas, beans, alfalfa, and clover. These plants are in an advantageous position. Unlike other crops incapable of assimilating atmospheric nitrogen, they do not require nitrogen fertilizer supplementation. Globally, over 60 million tonnes of such fertilizers were used in 1987. If plants carried their own nitrogen fixation genes, it would save an immense amount of time, money, and energy expended on the production, transportation, and application of fertilizers. However, developing plants capable of assimilating atmospheric nitrogen is a formidable task, since nitrogen fixation is a complex process controlled by A large number (approximately 15) of genes (nif genes). Despite the tremendous amount of work already done by geneticists, proper functioning of nif genes in plant cells has not yet been achieved.

25.4.6. Transgenic Tomatoes

Soft fruits such as tomatoes, bananas, and bell peppers are typically harvested green and artificially ripened in storage using Ethylene gas (Section 16.2.9). The fruits are harvested while still firm to minimize damage during mechanized harvesting and container loading. While this technology ensures a maximally appealing product appearance, the taste and aroma are virtually absent. Using genetic engineering, American and British geneticists have developed tomatoes with a delayed ripening process. Such fruits can remain on the plant longer, thereby increasing yield and improving flavor characteristics; both farmers and consumers benefit. Such tomatoes first went on sale in the USA in 1995 and in the UK in 1996. They are more expensive, but significantly better-tasting. We will discuss food safety issues concerning genetically engineered foods in Section 25.6.

25.4.7. Other Applications of Plant Genetic Engineering

1. Creation of novel flower colors, patterns, and shapes. For instance, experiments are underway to breed blue roses.

2. Utilization of genetically modified plants for The production of pharmaceutical drugs. Such products will be less expensive than their analogs produced using animal cells. The human enkephalin gene has already been expressed in plant cells.

3. Utilization of plants for the production of mouse Monoclonal Antibodies.

4. Improvement of the baking properties of wheat through enhanced grain quality.

5. Enhancement of the Nutritional Value of plant products by increasing the content of Essential Amino Acids; for example, many legumes lack Sulfur-Containing Amino Acids. This deficiency can be corrected using genes from the Brazil nut.