Plant Physiology - Musienko M.M. 2001

Plant Physiology and Biotechnology: Achievements and Prospects for Development
Transgenic Crop Plants

The first Transgenic Plants were created in the 1980s–1990s, and since then, they have been obtained for numerous plant species of practical value. This achievement has allowed scientists to delve deep into the fundamental mechanisms of plant developmental physiology. These studies tested the specific ability of Agrobacterium tumefaciens to transfer a portion of its DNA into plant Cells, using higher plants as a model. The Introduction of a foreign Gene into a plant Cell via agrobacteria was first accomplished in the early 1980s at the Max Planck Institute for Plant Breeding Research in Germany. As a result, the experimental plant cells acquired resistance to the antibiotic kanamycin, which inhibits neomycin phosphotransferase II and halts growth. This served as proof that foreign genes could be successfully expressed in plants. Furthermore, kanamycin resistance can serve as a marker because only a very small fraction of cells incorporate the introduced DNA into their genome, thereby allowing marker genes to identify such cells.

Thanks to the totipotency of plant cells, fully functional, fertile plants can be regenerated from these transformed cells. Various types of explants, such as leaf discs and stem segments, are used for genetic transformation. Today, this method of genetic material transfer has become standard in laboratories worldwide. However, despite the method's simplicity, many plant species—most notably major cereal crops (corn, rice, wheat)—are not natural hosts for agrobacteria, making transformation via this pathway quite problematic.

Consequently, researchers began searching for alternative approaches. One of the first steps toward solving this problem was the introduction of naked DNA into protoplasts—plant cells stripped of their cell walls. The pores in Plant Cell Walls are simply too small for DNA molecules to pass through, whereas in protoplasts, the Plasmalemma is the sole barrier to DNA delivery. Polyethylene glycol (PEG), an organic polymer capable of penetrating The Plasma Membrane, is frequently used to facilitate DNA delivery. Alternatively, transfer is achieved through short, high-voltage electrical pulses that transiently open pores through which the DNA passes—hence the name of the method, electroporation. Yet, once again, the regeneration of plants from isolated protoplasts presents significant difficulties in many species, particularly wheat and corn.

To improve the efficiency of gene delivery into intact cells, J. Sanford (1987) proposed a method of bombarding A large number of plant cells with genetic material. In this technique, microscopic metal particles (1–2 µm) made of tungsten or gold are coated with DNA and fired into intact cells using a specialized device known as a gene gun (Fig. 215).

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Fig. 215. A gene gun used for bombarding plant cells with DNA-coated tungsten microprojectiles

The microscopic punctures created in The Cell membranes and walls during this process quickly reseal. Such devices are successfully used to produce transgenic plants, including transformed corn and wheat. Thus, these two primary Methods for creating transgenic plants are now widely employed in Genetic Engineering (Fig. 216).

In both cases, the introduced DNA integrates into the Chromosomes, after which the cell divides and regenerates into a whole plant (Gasser & Fraley, 1992). Other noteworthy genetic transformation techniques include DNA microinjection into plant cells, as well as treating protoplasts with ultrasound or a UV laser beam.

However, delivering DNA into a cell is only the first step in plant transformation. The next phase involves the meticulous work of engineering plants with predetermined traits, a task somewhat facilitated by the inherently modular nature of genes. Protein-coding genes share a fundamental tripartite Structure consisting of three distinct regions: a promoter, which is a nucleotide sequence that dictates when and where the gene is expressed; a coding region, which contains the genetic blueprint for the protein product; and finally, a poly(A) region (a tail of adenylic NUCLEOTIDES) responsible for the timely termination of METABOLISM/31.html">Transcription—the synthesis of Messenger RNA complementary to the coding sequence.

Fig. 216. Two main METHODS FOR PRODUCING transgenic plants: 1 — DNA introduction into Agrobacterium cells; 2 — DNA delivery via microscopic metal particles using a gene gun; 3 — DNA; 4 — metal particles; 5 — tumor-inducing plasmid; 6 — chromosome; 7 — DNA transfer; 8 — nucleus; 9 — chromosome; 10 — Cell Division

Using genetic engineering techniques, these three regions can be recombined, assembling components from different genes to create so-called chimeric genes (Fig. 217) in virtually any given Organism.

By employing appropriate promoters, researchers can direct the expression of a given gene exclusively to a desired target organ—such as a leaf, ROOT, tuber, or grain—or even to specific cell types within those Organs.

Gene transfer holds immense promise, particularly for cellular Selection. Initial breakthroughs have been achieved in developing organisms resistant to viral diseases. The Genetic Methods behind these plants rely on a profound understanding of the physiology of infected plants. Through this approach, numerous crops have acquired The ability to effectively resist more than ten different Viruses.

Fig. 217. A chimeric kanamycin-resistance gene assembled from multiple sources: the promoter from a plant virus, the coding region from the bacterium Escherichia coli, and the poly(A) region from the T-DNA of Agrobacterium

Efforts are also underway to engineer insect-resistant plants. In the mid-1980s, researchers in the U.S. successfully isolated bacterial genes encoding insecticidal Proteins. For cotton, these technologies promise to reduce insecticide use by 40–60% in the near future. A modified gene has been developed that confers resistance to the Colorado potato beetle. Field trials conducted across various Regions of the United States in 1991 using plants expressing this "insecticidal" gene demonstrated that they are exceptionally resistant to Colorado potato beetle damage.

Furthermore, genes targeting nematodes and mosquitoes have been identified. Unlike conventional pesticides with prolonged persistence, these proteins do not accumulate in the environment; they degrade just like other proteins, are non-toxic, and therefore represent the most environmentally friendly class of pest control agents. In a similar vein, research aimed at creating herbicide-resistant transgenic plants serves as an alternative to traditional weedkillers and addresses a wide array of other agricultural challenges.

For instance, recent years have seen the identification and isolation of several genes responsible for synthesizing Ethylene, the hormone that triggers fruit ripening. It is also possible to enhance the nutritional quality of various foods and direct plant organisms to synthesize specific compounds, such as starch, industrial oils, Enzymes, and BIOLOGICALLY ACTIVE SUBSTANCES.

The early 1990s marked the launch of field trials with genetically modified plants across the globe, encompassing apple, raspberry, walnut, wheat, soybean, pea, sugar beet, corn, rice, rye, potato, tomato, grapevine, and many others. While the global acreage of transgenic crops was 3 million hectares in 1996, it expanded rapidly to 12 million hectares in 1997, 26 million hectares in 1998, and nearly 40 million hectares in 1999 (James, 2000). In Ukraine, pioneering work in this field was initiated by Academicians K.M. Sytnyk and Yu.Yu. Gleba, and is currently ongoing at various research institutions of the National Academy of Sciences of Ukraine and specialized branch institutes.

One of the primary challenges faced by genetic engineering at the dawn of the new millennium is public apprehension—particularly in certain developed European countries—regarding the appearance of genetically modified crops in fields worldwide and

the consumption of food products derived from them. Scientific opinions regarding the risks associated with transgenic plants remain divided. Some researchers maintain that transgenic crops, as well as the foods derived from them, are safe and require only minimal oversight. Proponents of this view argue that there are no fundamental differences between Genetic Engineering and traditional selective breeding practiced by humans for centuries; the only difference lies in the speed with which the end goal is attained. Supporting this stance on safety is the fact that since 1985, over 25,000 field trials involving at least 60 different genetically transformed plant species have been conducted across 45 countries with no adverse environmental consequences observed.

Conversely, another perspective holds that the release of transgenic plants into the natural environment increases the potential risks of disrupting ecological equilibrium, causing unpredictable gene flow to other organisms, and reducing agricultural biodiversity.

Finally, there is an extreme viewpoint suggesting that this branch of biotechnology should be banned altogether, arguing that our current knowledge is still insufficient to guarantee absolute safety.

The majority of scientists agree that research involving transgenic organisms requires rigorous oversight, yet there is no scientific basis to anticipate catastrophic outcomes. There is a pressing need for internationally harmonized principles to assess risks and manage all aspects of emerging biotechnologies. Appropriate legislation and regulatory mechanisms must be established to address any potential risks arising from the introduction of genetically transformed organisms.