BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Transgenic Plants

Plants Resistant to Insect Pests

To develop such plants, the expression of toxin genes is utilized directly within the plant Tissues. Transgenic Plants acquire pest resistance primarily through insecticidal toxins derived from the bacterium Bacillus thuringiensis. Forms of the protoxin Gene have been introduced into a wide range of crop species, including tomatoes, tobacco, potatoes, rice, corn, eggplants, apples, alfalfa, walnuts, poplars, spruces, cotton, and cranberries. Transgenic tomato and potato plants have proven resistant to the Colorado potato beetle, whereas transgenic cotton demonstrates resistance to the cotton bollworm. However, no single type of Bacillus thuringiensis protoxin is effective against all insect species.

Another effective mechanism for protecting plants from insect pests involves the Introduction of plant protein genes that encode amylase and proteinase inhibitors. Introducing the α-amylase inhibitor gene protects transgenic plants (such as cereals and legumes) against the cowpea weevil (Callosobruchus maculatus) and the adzuki bean weevil (C. chinensis). Likewise, incorporating the proteinase II inhibitor gene into rice plants protects them against the pink stem borer (Sesamia inferens), a major rice pest. The simultaneous use of the Bacillus thuringiensis toxin and a Serine proteinase inhibitor further enhances plant defense against insect pests.

An alternative approach for breeding pest-resistant plants relies on The Use of the bacterial Cholesterol oxidase gene. This enzyme, synthesized by various Bacteria, catalyzes The oxidation of 3-hydroxysteroids to yield ketosteroids and H₂O₂. The cholesterol oxidase gene was isolated from a Streptomyces strain and introduced into tobacco Cell protoplasts. The enzyme has been shown to exhibit insecticidal activity against the larvae of the boll weevil (Anthonomus grandis grandis). Utilizing this gene holds significant promise for protecting cotton plants from destructive insect pests.

The application of Genetic Engineering can reduce the use of chemical insecticides by 40 to 60%.

Virus-Resistant Plants

Viruses severely reduce crop yields. Natural Immunity to viral infections stems from various mechanisms, including blocking viral entry into the plant, preventing systemic spread, and suppressing the symptoms of viral infection. Ideally, transgenic plants should exhibit resistance to more than one virus.

Breeders have long attempted to transfer natural virus-resistance genes from one plant line to another. Viral genes encoding coat Proteins have been introduced into plant genomes. When a plant contains a viral coat protein gene, its susceptibility to viral penetration and systemic propagation is significantly reduced. This approach yields transgenic plants with high levels of viral resistance. Furthermore, research has revealed that the coat protein gene of one virus can confer resistance to other related viruses. The most effective strategy for protecting plants against viral pathogens involves introducing multiple genes that direct the synthesis of viral coat proteins.

Plants Resistant to Phytopathogenic Fungi and Bacteria

As a rule, in response to fungal or bacterial invasion, plants synthesize a group of specific Pathogenesis-related (PR) proteins, such as β-1,3-glucanases, chitinases, and proteinase inhibitors, which act directly against pathogens. Transgenic rice and tobacco plants have been successfully engineered to synthesize large amounts of chitinase—an enzyme that hydrolyzes β-1,4-linkages in molecules of N-acetyl-D-glucosamine, the primary component of fungal cell walls.

It is estimated that crop losses caused by the phytopathogenic soil bacterium Erwinia carotovora amount to approximately $100 million annually. Transgenic potato plants harboring the T4 bacteriophage Lysozyme gene have been developed and shown to be resistant to Erwinia carotovora. Because lysozyme targets various bacterial species (both Gram-positive and Gram-negative), this approach can be widely applied to protect crops against numerous bacterial pathogens.

Herbicide-Resistant Plants

Globally, more than $10 billion is spent annually on The production of over 100 chemical herbicides, yet crop losses due to weed infestation still exceed 10%. Moreover, many herbicides lack selectivity, affecting weeds and crops alike, and tend to accumulate in the environment.

Developing herbicide-resistant transgenic plants requires achieving the following objectives:

- ensuring the metabolic inactivation of the herbicide within the plant;

- reducing the binding affinity of the target protein for the herbicide;

- ensuring the overexpression of the target protein.

Some Examples of genetically engineered herbicide resistance in plants are summarized in Table 1.4.

Class="center">Table 1.4.

Genetically Engineered Herbicide Resistance in Plants

Salt-Stress-Resistant Plants. Under conditions of drought and soil salinity, plants synthesize low-molecular-weight compounds known as osmoprotectants, which facilitate Water uptake and retention, and prevent the degradation of cellular macromolecules under high salt concentrations. Osmoprotectants include CARBOHYDRATES, polyols, ammonium salts, Proline, and betaine. Betaine plays a major role in protecting plants against salt stress and is synthesized from Choline. Certain crops (such as potatoes, rice, and tomatoes) are naturally incapable of accumulating betaine. Introducing the E. coli betaine Biosynthesis gene into these plants enhances their tolerance to high salt concentrations (300 mM) by 80%.

Plants with extended fruit ripening periods

Premature ripening is a major challenge during fruit transport. As fruits ripen, specific genes encoding the Enzymes cellulase and polygalacturonase are activated in plants. Blocking the expression of these genes delays the ripening process.

The plant growth regulator Ethylene initiates the expression of numerous genes responsible for fruit ripening. Ethylene is synthesized from S-adenosylmethionine. Treating plants with chemical agents that block ethylene synthesis delays fruit ripening. Transgenic plants have been developed whose fruits exhibit an extended shelf life due to the suppression of ethylene synthesis.

German scientists at the Cologne Institute for Plant Sciences have developed a tomato incorporating thermoregulation-related genes from the winter flounder (flatfish). This tomato can be stored unripe at 12°C for several months (and ripens within a few hours in warm conditions).

Modification of Flower coloration

Flower color modification plays an exceptionally crucial role in floriculture. Roses, carnations, tulips, and chrysanthemums account for over 70% of the floriculture industry's volume. Genetic engineering techniques are employed to create new flower varieties with unusual colors. This is achieved by manipulating the genes encoding enzymes for anthocyanin pigment biosynthesis. Phenylalanine serves as the initial precursor for anthocyanin synthesis.

In the future, manipulating The genes of scent-determining proteins could enable the creation of plants with virtually any desired fragrance.

Enhancing the Nutritional Value of plants

Genetic engineering has made it possible to improve the nutritional value of plants by altering their protein and lipid profiles. Rice and corn varieties with increased PROTEIN AND AMINO acid content have been successfully developed. Plant varieties containing significant amounts of Essential Amino Acids are particularly valuable. Transgenic soybean varieties have been created in which the content of the essential amino acid Lysine is 5 times higher than that of conventional plants.

Soybean, oil palm, rapeseed, and sunflower account for 75% of total oilseed crop production. Transgenic rapeseed varieties that synthesize Lipids with altered fatty acid compositions have been developed. Fats with a high content of polyunsaturated Higher Fatty acids are especially valuable, as they cannot be synthesized by The Human Body.

The synthesis of vitamin A in "golden rice" is the result of a collaborative research effort between the research groups of Ingo Potrykus (Switzerland) and Peter Beyer (Germany). The rice contains 2 daffodil genes and 1 bacterial gene, giving the grains a golden-yellow color.

Genetic engineering enables the improvement of fruit flavor profiles. The protein monellin, which is 100,000 times sweeter than sucrose, was isolated from the fruits of the African plant *Dioscorephyllum cumminsii* Diels. Monellin is a two-chain dimer: the A-chain contains 45 amino acid residues, and the B-chain contains 50. To create transgenic plants capable of synthesizing monellin, both genes had to be cloned. To solve this problem, the gene encoding both the A- and B-chains was chemically synthesized. Transgenic tomatoes and lettuce synthesizing monellin have been successfully produced. The synthetic monellin gene was introduced into plants using Ti Plasmids of *Agrobacterium tumefaciens*.

Plants as Bioreactors

Using plants to synthesize valuable compounds offers several advantages over microbial Fermentation systems:

1) plant protein Processing and folding closely mimic those of animals;

2) plant transformation is stable, with foreign DNA integrating directly into the plant genome;

3) plant cultivation is cost-effective and not constrained by the operational limits of fermentation processes;

4) products derived from transgenic plants are safe to use (eliminating the risk of protein contamination by viral or prion pathogens);

5) conditions can be established where foreign proteins are synthesized within seeds, ensuring their structural integrity over long periods.

Ideally, a biopharmaceutical-producing plant should be non-edible and incapable of surviving in natural ecosystems if its seeds are dispersed by wind or animals. Transgenic plants must be cultivated in regions devoid of closely related wild species.

Experimental systems have been established for the production of enzymes, Monoclonal Antibodies, functional antibody fragments, and polymers using plant platforms.

It is known that the bacterium *Alcaligenes eutrophus* synthesizes the polymer Poly-β-hydroxybutyrate. A key characteristic of this polymer is its biodegradability. In bacteria, poly-β-hydroxybutyrate is synthesized from acetyl-CoA via three enzymatic steps. The genes for these enzymes were integrated into the chloroplast DNA of *Arabidopsis thaliana* using Ti-plasmid-derived vectors. Leaves of transgenic plants containing all three bacterial genes synthesize more than 1 mg of poly-β-hydroxybutyrate per gram of fresh leaf tissue.

Technologies for producing pharmaceutical and diagnostic agents using plant systems have been established. Transgenic corn is utilized to produce monoclonal antibodies against respiratory syncytial virus, rheumatoid Arthritis, and lymphoma. Transgenic bananas containing inactivated forms of cholera, hepatitis B, and diarrhea viruses have been developed. Furthermore, the Moscow Potato Research Institute has bred a potato variety that synthesizes human Blood interferon.

Plant METABOLISM/14.html">Chloroplasts execute the synthesis of human proteins more efficiently than microorganisms or plants with nuclear-integrated genes. Tobacco is exceptionally well-suited for producing human proteins. Research and development of technology for yielding human somatotropin in tobacco chloroplasts are currently underway.

An important objective of biotechnology is to prevent the contamination of food and feed crops with biopharmaceutical products, as well as to minimize their environmental impact.