How do plants have nucleic acids
Genetic engineering in plants
For a long time it was not possible to transfer foreign genetic information into plants. The basic requirement for the application of genetic engineering methods in plants - the availability of the plant cell in the test tube - had long been fulfilled by the development of biotechnical processes such as callus and protoplast cultures. However, since the plant cell differs from bacteria or animal cells in many ways, primarily due to the stable cell wall, specific processes for the transformation (for the transfer of foreign genes) of plant cells first had to be developed. Depending on the type of plant, two fundamentally different methods are used today: the indirect and the direct Gene transfer.
The indirect gene transfer into the plant cell via the agrobacteria system is an elegant method, but only works with dicotyledonous plants: Agrobacterium tumefaciens generally does not infect monocotyledonous plants; However, these include our most important types of grain. The bacteria enter the plant through injured parts of the plant and incorporate part of their DNA, a tumor-inducing plasmid, into the plant genome. Here, the information in the bacterial DNA is translated into products by the plant enzymes. These are genes for opines - substances that bacteria use as nutrients - and genes for plant hormones. The production of these plant hormones leads to uncontrolled growth of the plant cells.
One of the most frequently used methods of gene transfer is the use of vectors - only there were no suitable "gene ferries" in the plant kingdom. A solution to the problem: "How can foreign DNA be introduced into the plant cell?" accidentally emerged from the investigation of the formation of galls and tumors on dicotyledonous plants.
Plant galls and tumors are cell growths that are primarily triggered by certain soil bacteria, the agrobacteria. Agrobacterium tumefaciens is the most important type of these soil bacteria that penetrate the tissue through injuries to the plant and cause "root neck galls" in the area of the root neck. In addition to these growths triggered by the bacteria, the agrobacterium controls the metabolism in the plant cells in such a way that special protein molecules, the so-called Opine, are formed. These opines serve the agrobacteria as nutrients, with which they are supplied in abundance by the proliferating plant cells. The genetic information for the synthesis of opines and tumor formation is provided by the DNA of the agrobacterium.
The genes for this are not in the bacterial genome, but on the Ti plasmid, so-called because it has a tumor-inducing effect. After the plant has been infected by the bacterium, part of the Ti plasmid DNA, the T-DNA (T = transfer), is integrated into the plant genome. The T-DNA contains, among other things, the genes for opine synthesis and the genes that are responsible for tumor formation. These tumor-inducing genes encode plant hormones that trigger tumor growth.
The agrobacterial transport mechanism is now used for targeted gene transfer. The genes that you want to transfer are integrated into the natural vector, the Ti plasmid. Since tumor formation is naturally undesirable in the transgenic plants that develop, the genes responsible for tumor induction have been removed in the area of the transferable T-DNA region. A "defused" Ti plasmid is used in the recombination with a foreign gene. Restriction enzymes are used to cut out part of the T-DNA from the Ti plasmid that is already free of tumor DNA, and the foreign DNA is incorporated into this site with DNA ligase. Agrobacterium tumefaciens bacteria are transformed with the newly combined Ti plasmid and then used to infect the plants.
The natural "genetic engineering system" of the agrobacteria is used to smuggle DNA into the plant cell. Ti plasmids are used, from which the tumor-inducing genes have been removed. The piece of DNA intended for incorporation into the plant genome is integrated into these "disarmed" plasmids. After transformation of the agrobacteria with the recombinant plasmid and infection of the plant cells with the transformed agrobacteria, the foreign DNA is incorporated into the plant genome instead of the tumor DNA.
Also Protoplasts can be infected with agrobacteria in culture. The modified Ti plasmids reach the nucleus of the plant cell and are incorporated into the host cell's genome at various points. The protein can then be produced by a plant regenerated from the protoplasts. Since not all protoplasts are infected by agrobacteria, a selection must be carried out, similar to what we learned about bacteria. For this reason, a marker gene is also transferred here with the desired gene, the protein product of which enables the transformed cells to be found quickly. The marker genes used cause resistance or increased tolerance to substances that inhibit growth. Resistance to antibiotics or herbicides, for example, is used.
In addition to marker genes in plants, Reporter genes (Indicator genes) used. They are not used for selection, but to prove that the corresponding gene is actually present in the plant and is translated into a protein, i.e. that the promoter in front of the gene is also functioning properly. The reporter genes code for enzymes whose activity can be detected easily and quickly, e.g. by simple color reactions.
The decidedly "elegant" agrobacterial system works well on dicotyledonous plants such as soybeans. Our most important crops, however, including all types of grain, are monocotyledons and cannot be infected or only poorly infected by agrobacteria. Other vector systems (e.g. geminiviruses) now exist for these crops. However, these still have disadvantages. On the one hand, the viral genetic material can only rarely be integrated into the plant genome, i.e. the next generation already loses the new property, on the other hand, the plants become sick from the virus infection, i.e. the damage to the plant affects the formation of the desired protein product. However, viral promoters are often transferred together with a desired gene and a marker gene / reporter gene as a "gene construct", since they very effectively ensure that as much of the desired protein as possible can be produced.
Other effective systems have also been developed for monocot plants because of the lack of suitable vectors. In these direct gene transfer systems, the DNA is transferred using membrane-destabilizing processes. The Electroporation is carried out in a liquid-filled chamber in which plant protoplasts and the DNA to be transferred "swim". The chamber is connected to electrodes, high-voltage pulses of up to 1500 volts temporarily increase the permeability of the plasma membrane and thus enable the DNA to penetrate the protoplasts. In about one percent of the cells used, it actually works that the DNA is incorporated into the genome of the cell. In the Microinjection DNA is introduced into the cell through a very fine glass capillary that is inserted into a protoplast. With this technology, transformation rates of up to 15 percent can be achieved. The membrane of protoplasts can also be chemically destabilized in a medium containing polyethylene glycol. This method is particularly suitable for monocot plants.
The Particle bombardment (Particle bombardment; magic bullets) is the most successful method for direct gene transfer. Gold or tungsten particles are coated with the DNA to be introduced and shot into the plant cells at high speeds using a "gene cannon" (particle gun). The diameter of these microprojectiles is only 1 to 3 µm, the necessary high speeds are achieved by shock or pressure waves. The pressure waves are generated, for example, by applying a microprojectile to a larger projectile, a macroprojectile. The macro projectile is fired and braked abruptly on impact with a locking plate. As a result, the microprojectile is torn away and penetrates the target cell with force: however, the gold or tungsten particles themselves do not remain in the target cells, only the DNA is dumped; the particles are "stripped". With this technique, e.g. meristems, calluses and spread cell suspensions can be bombarded; protoplasts are not necessarily needed here. For crops such as maize, whose attempts at regeneration from protoplasts have been unsuccessful for years, particle bombardment is therefore the method of choice.
Bombardment with gold or tungsten particles is a physical method of transferring genes. Particles coated with DNA are accelerated and shot into the cells. The DNA is dumped in the cells and - if successful - also incorporated into the plant genome. With this method, which has now been used very successfully, not only cells, but also tissues and even entire plants can be transformed.
The particles (microprojectiles) are applied to a macroprojectile. The acceleration takes place e.g. by means of a pressure wave triggered by an explosion of cordite. When the macro projectile accelerated by the pressure wave hits a blocking plate, the micro projectiles are torn away. The steel sieve that the microprojectiles pass is used for greater dispersion.
As with the other methods mentioned, the incorporation of the foreign DNA is random and not targeted. Whether the integrated foreign gene is actually translated into an intact protein depends on many factors. Among other things, the integrity of the integrated DNA plays a very important role. In the case of the methods described, however, the DNA is very often damaged, especially by mechanical stress. Therefore, such experiments often have to be repeated up to a thousand times before success is achieved. Then whole plants have to be regenerated from the transgenic cells. This is nowhere near as easy as it might sound here.
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