Biotechnology - Yu.O. Sazykin 2006

Applied Biotechnology
Biotechnology of Medicinal Products Based on Plant Cell and Tissue Cultures
Transgenic Plants

Unlike Traditional Methods of genetics and breeding, modern biotechnology allows for combining Genes from different biological species to produce transgenic plants. Transgenic plants are those species in which genes (or a Gene) transferred from plants or animals of other species successfully function. This is done to grant the recipient plant new traits beneficial to humans, such as enhanced resistance to Viruses, herbicides, pests, and plant diseases. Food products derived from such genetically modified crops may boast improved flavor, better appearance, and a longer shelf life. Furthermore, these plants frequently yield richer and more stable harvests than their natural counterparts.

If we take disease-resistance genes from viruses, frost-resistance genes from fish, and pest-resistance genes from Bacteria, and introduce this "cocktail" into The Genome of a plant, such a combination will impart entirely new properties to the plant while preserving its biological species. A potato remains a potato, but it now becomes invulnerable to the Colorado potato beetle. Scientists have already developed frost-resistant beets, lawn grass that glows at twilight, and even a banana that, upon consumption, provides "vaccination against tropical diseases."

The search for ways to introduce foreign genes into higher plant Cells has been actively pursued worldwide since the 1970s. It was discovered that tumor-inducing Ti Plasmids of agrobacteria (Ti — tumor-inducing), which are mini-circular DNA molecules, serve as an excellent natural vector system currently used for gene transfer in plants. The plasmid contains T-DNA (transferred DNA), consisting of 12–22 thousand Base Pairs and encoding Enzymes for the synthesis of phytohormones and opines—Amino Acid Derivatives utilized by the bacterium as a source of carbon, nitrogen, and energy. In addition to T-DNA, the Ti plasmid contains the vir region responsible for T-DNA transfer into the plant, opine utilization genes, and loci controlling plasmid Replication within the bacterial Cell and its transfer via bacterial conjugation. The agrobacterium plasmid transfers a portion of its DNA into The plant cell's DNA, meaning the "target" gene becomes integrated into the latter's genome.

The entire process of excising and integrating T-DNA into the plant chromosome is carried out by the products of genes localized within the vir region. The induction of vir genes is reversible, which is vital for the pathogen: if the host is a diseased and unviable Organism, T-DNA transfer does not occur. The insertion of T-DNA into the plant genome is a multi-step process, during which multiple copies of T-DNA can be integrated into the plant genome.

Once integrated into the chromosome, the T-DNA becomes a regular part of the plant genome and is transcribed in plant cells by the host plant's RNA polymerase. The bacterium itself does not penetrate The Cell; instead, it remains in the intercellular space and uses the plant cells with the integrated T-DNA AS A factory producing opines.

The T-DNA of Ti plasmids possesses two properties that make it essentially an ideal vector for introducing foreign genes into plant cells. First, the host range of agrobacteria is very broad: they transform cells of virtually all dicotyledonous plants (and sometimes even monocots, including cereals). Second, T-DNA integrated into the plant genome is inherited as a simple dominant trait in accordance with Mendelian laws, and its genes feature their own promoters (the regulatory region of a gene that determines the timing and Location of its expression), under the control of which foreign genes inserted into the T-DNA can be expressed. Overall, an ideal Ti plasmid-based vector system must contain all signals required for transfer and stable integration into plant nuclear DNA, a system for expressing foreign genes in plants (a regulated promoter recognized by plant polymerases), and a marker (reporter gene) necessary for the Selection of transformed cells, while remaining free of oncogenes—that is, genes that suppress plant Cell Differentiation.

Today, a wide arsenal of methods is employed to produce transgenic plants. One approach for introducing T-DNA into plant cells involves The Use of binary vector systems, which are designed such that an agrobacterial cell must contain at least two distinct modified Ti plasmids. One of these must contain only the vir region, whose genes participate in T-DNA excision. Such plasmids are referred to as helper plasmids. The second Ti plasmid must contain the T-DNA region with the desired inserted gene. The products of vir genes are capable of excising T-DNA both on their own plasmid and on a neighboring one, meaning that vir genes can function regardless of their physical location.

Thus, if agrobacterium cells contain a Ti plasmid with the vir segment and another plasmid with T-DNA carrying the inserted gene, these bacteria can successfully transform plant cells. Other methods for introducing foreign DNA into plant cells also exist. For instance, Enzymes can be used to dissolve the thick Cell wall of the plant cell, which otherwise hinders the direct penetration of foreign DNA; such purified cells are then placed in a solution containing DNA and a chemical agent that facilitates DNA entry (polyethylene glycol is most commonly used).

Sometimes, micro-pores are created in The cell membrane using short, high-voltage pulses, allowing DNA fragments to pass into the cell. In some cases, direct "microinjection" of DNA into the cell using a micro-syringe under a Microscope is employed. A few years ago, a method was proposed to coat ultra-small metallic "bullets"—such as tungsten beads 1–2 µm in diameter—with DNA molecules, and then "SHOOT" them into plant cells using a specialized device called a "gene gun" (Shotgun). The holes created in The cell wall heal rapidly, and the "bullets" trapped within the protoplasm are so small that they do not interfere with cell function. A fraction of the "volley" yields success, with some "bullets" delivering their DNA to the target site, thereby achieving plant cell transformation.

In recent years, scientists have adopted a novel approach for generating transgenic plants using "antisense RNA," which allows for The regulation of a target gene's activity. In this method, during vector construction, the complementary DNA (cDNA) copy of the inserted gene is inverted by 180 degrees. As a result, the transgenic plant produces both a normal mRNA molecule and an inverted one; due to complementarity with the normal mRNA, the inverted molecule forms a complex with it, preventing the Synthesis of the encoded protein.

This approach was utilized to produce transgenic tomato plants with enhanced fruit quality. The vector incorporated the cDNA of the PG gene, which controls the synthesis of polygalacturonase—an enzyme involved in The breakdown of pectin, the primary component of the intercellular matrix in plant Tissues. The PG gene product is synthesized during tomato fruit ripening, and an increase in its concentration leads to fruit softening, which in turn significantly reduces shelf life. Silencing this gene yielded tomato plants with novel fruit characteristics that not only kept fresh much longer but also exhibited enhanced resistance to fungal diseases.

The most pressing issue is the Development of Plants resistant to agricultural pests (primarily phytopathogenic Fungi and insects), as plant diseases have become the primary limiting factor in crop yields. It was discovered that plant pest resistance can be genetically programmed by introducing foreign genes into the plant genome whose products cause pest mortality. Traditionally, the bt gene is employed for this purpose, encoding a bacterial toxin produced by the bacterium Bacillus thuringiensis. This large protein (protoxin), controlled by the bt gene, enters the gut of insect larvae where it is cleaved by enzymes, releasing a fragment (endotoxin) that leads to insect death. Transgenic potato, cotton, and maize plants carrying the bt gene are already produced by companies such as Monsanto and Ciba Seeds, and are successfully marketed globally.

It is well known that plants, much like animals, are capable of developing Immunity. However, this remarkable property is possessed exclusively by resistant plants, whose METABOLISM changes dramatically upon pathogen attack. As a result, these plants accumulate chemical compounds such as hydrogen peroxide, salicylic acid, and phytoalexins. Elevated levels of these substances help the plant fend off pathogens. For instance, transgenic tobacco plants containing a bacterial gene that controls the synthesis of salicylate hydroxylase (an enzyme that degrades salicylic acid) were found incapable of mounting an Immune Response. Therefore, engineering the levels of salicylic acid or The production of hydrogen peroxide in plants in response to pathogens holds great promise for developing resistant transgenic plants.

A highly promising direction in Plant Introduction/32.html">Genetic Engineering is the creation of transgenic plants harboring a combination of specific bacterial hormonal genes. It turns out that the fruits of some such transgenic plants are parthenocarpic—that is, formed without pollination. These fruits feature either a complete absence of seeds or a very low seed count, which resolves Structure/149.html">The problem of "unwanted seeds" in crops such as watermelons and citrus fruits. Researchers have already successfully obtained transgenic zucchini plants that are phenotypically indistinguishable from control plants yet are virtually seedless.

Efforts are actively underway worldwide to develop so-called "edible Vaccines" based on transgenic plants, which could subsequently be used to prevent the most dangerous human diseases. For instance, scientists at the Siberian Branch of the Russian Academy of Sciences (SB RAS) are successfully developing a tuberculosis vaccine. In creating this vaccine, researchers utilize human genes encoding the Synthesis of specific Antibodies against Proteins of the disease causative agent, Mycobacterium tuberculosis. These antibodies provide immunity against the given disease. Genes encoding antituberculosis antibodies were integrated into the plant cell genome. From cells where the protective genes were successfully incorporated, fully developed plants capable of synthesizing antituberculosis antibodies were regenerated.

Traditional treatments for tuberculosis are not always harmless or effective, and scientists hope that utilizing transgenic plants offers a chance not only to cure the disease but also to avoid side effects. Furthermore, the Institute of Plant Physiology and Biochemistry of the SB RAS is developing vaccines against AIDS and hepatitis based on transgenic tomatoes and cucumbers. A group of German geneticists from Giessen University in Frankfurt successfully cultivated genetically modified carrots containing a hepatitis B vaccine, which significantly reduces the costs associated with preventing this disease (according to official WHO statistics, approximately 350 million people worldwide are infected with the hepatitis B virus, which causes severe Liver damage, chronic progression, and fatalities, claiming 1 million lives annually). Transgenic carrots can grow in various climate zones and on diverse soils, store and transport well, and can be consumed raw. By ingesting this product, an individual continuously receives micro-doses of the vaccine, thereby sustaining active immunity against the hepatitis B virus.

The United States is the world's largest producer and consumer of transgenic plants, leading both in cultivated acreage and societal acceptance of transgenic food. The widespread adoption of genetically modified crops there (expressed as a percentage of the total baseline) is as follows: 40% of the country's corn, 81% of soybeans, 65% of canola (rapeseed), and 73% of cotton, and these figures continue to rise. Hundreds of commercial firms with a combined capital exceeding $100 billion are engaged in developing and testing such plants.

Currently, the leading cultivator of insect-resistant transgenic cotton is China. In Argentina, where the government Supports the cultivation of genetically modified crops, the share of transgenic soybeans reaches 90%, while corn and cotton account for 50%.

The Republic of South Africa is the only African nation with large-scale plantings of transgenic crops, with 80% of cotton, 20% of corn, and 11% of soybeans being genetically modified. The rest of Africa is viewed by agricultural biotechnology firms primarily as a testing ground for future trials. Japan has prioritized agricultural biotechnology within its scientific budget, despite the fact that the application of genetically modified products faces strong public resistance there.

In Europe, following the EU's adoption of regulations on the use of genetically modified products in 1998, France, Italy, Denmark, Greece, and Luxembourg banned these products. Currently, the EU has adopted new certification and labeling rules for such products and significantly softened its stance. Nevertheless, transgenic corn is cultivated only in Spain in negligible quantities. In Russia, not a single transgenic plant is authorized for open-field cultivation.

The sole exception is transgenic soybeans, and even then, only as a food raw material or product component (it may be consumed, but not cultivated).

Despite the remarkable successes of biotechnology in producing transgenic plants alongside all necessary toxicity, allergenicity, and mutagenicity testing, a cautious attitude toward genetically modified foods persists in society. There are very real concerns that the pollen and seeds of transgenic plants and weeds might cross-pollinate, ultimately giving rise to a "superweed" that no herbicide can eradicate.

At the same time, the uncontrolled spread ("Horizontal Gene Transfer") of foreign genes into populations of cultivated crops, traditional varieties, and wild plant forms can disrupt the equilibrium of biocenoses, contaminate traditional varieties with transgenic insertions, and eventually lead to their complete eradication. Enhancing the nutritional profile of potatoes, corn, and soybeans could trigger severe allergic reactions in humans. Several scientists have expressed serious concern regarding the potential emergence of new virulent strains resulting from genetic recombination between transgenes and natural viral genes.

Review Questions

1. What pharmaceutical production challenges are addressed through the use of plant cell cultures?

2. What is the specific nature of plant cells that determines their cultivation conditions in the production of Pharmaceuticals?

3. What are the growth Characteristics of plant cells in culture and how do they affect the final product yield?

4. What are the specific requirements for nutrient media used in plant cell cultures?

5. What is The Role of biotransformation (bioconversion) in the production of pharmaceuticals based on plant cell cultures?

6. What are the advantages of plant cell immobilization in the production of medicinal substances?

7. What forms and Methods of Plant cell immobilization exist, and what makes plant cell immobilization more challenging compared to microbial cells?

8. What are the Prospects for The Development of biotechnology in producing pharmaceuticals based on plant cell cultures?

9. What are the main METHODS FOR PRODUCING transgenic plants?

10. Can transgenic plants be utilized for the production of pharmaceutical agents?