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There is big temporal scale difference between traditional plant improvement technique and genetic engineering in experimental time. For example, it may need to use about ten years for selecting genes for cereal improvement, but only two to three years time for the practice of genetic engineering. (Tourte, chapter 2)
In the sexual reproduction of plants, the offsprings contain half genetic information from each of the two parental genetic blocks. The process of the genetic transferring can be by conjugation, like the bacteria, or transduction with the help of viruses. The development of genetic engineering began when the biologists first discovered the happening of transportation of genetic materials from bacteria to plants.
Natural Gene Transfer in Plants
Crown gall disease has been common in Dicotyledons, especially in cabbages (Brassica), for a long time. Farmers knew the causal agent is one of the soil bacteria class called Agrobacterium, which cause tumors in the crown. It is the area between the root and the stem in plants. The disease cause plants death from necrosis in the above-ground part of the plants. Biologists found the tumor cells produce opines to support the agrobacteria and initiate the multiplication of agrobacteria. The normal metabolism of the plant was then affected. Opines also support the aggressive reproduction of all tissue cells at the crown which is the formation of tumors.
The most commonly studied species of agrobacteria are Agrobacterium tumefaciens and A. rhizogenes, which inlet tumor-inducing plasmid (Ti plasmid), and root-inducing plasmid (Ri plasmid) respectively. The figure below shows the simplified map of Ti plasmids with octopine.
The large circular plasmids of double-stranded DNA contain three major parts. They are region of origin of replication, transfer DNA (T-DNA) region and virulence genes region (vir region). T-DNA region containing carcinogens for tumor formation and is limited by left and right borders (LB and RB). Vir region consists of different virulence factors, such as the capacity to infect plant cells, leading the T-DNA insert into plant cells and the integration of T-DNA into the plant cell genome. T-DNA contains important genes for the production of plant hormones. The gene iaaM and iaaH for the integration of auxin, while the gene iptZ for that of cytokinin. Both hormones are important for the plant growth. Before transporting, T-DNA is split into single DNA strands with the help of the borders and the origin of the replication. After plant cells contacting the Agrobacterium tumefaciens directly, the single-stranded T-DNA is inserted into the plant cells. Then the T-DNA acts as a template to replicate the missing strand. Eventually plants became infected when the genome of the plant cell is altering by integration of T-DNA. The secretion of opines and tumor formation are also the outcomes.
Gene Transferring Technique
Molecular biologists have developed several techniques in genetic engineering. Indirect transformation and direct transformation are two main pathways in gene transformation into plant cells. Indirect pathways make use of biological vector, such as agrobacteria and E. coli. And direct methods use particle cannon, microinjection and electroporation. The figure below shows the brief outline of the both pathways.
Indirect transformation via Agrobacterium
Inserting gene indirectly into plant cells is invented after studying the crown gall disease and the action of agrobacteria inside. Agrobacterium tumefaciens can infect and transform protoplasts. Protoplasts are plant cells without a cell wall and are usually derived from leaf tissue (Halford, p.21). Enzymes, which contain cellulases, pectinases and hemicellulases, are incubated in the cells and break down the cell wall. The protoplasts can then be cultured and callus, is a clump of undifferentiated cells, are formed with the help of plant hormones, such as auxins and cytokinins. Eventually, GM plants can be produced from the callus and the process is called
Agrobacterium tumefaciens mediated transformation of explants material (Halford, p.22).
The Co-integration method was the eldest method practiced in transformation of plant cells. The drawback was the need of suppressing tumor formation in plants. The condition can be solved by inserting modified T-DNA into Agrobacterium by the vector E. coli. It resulted in production of disarmed Ti plasmid, which the onc part of the Ti plasmid was deleted. Another limitation was the size of plasmids needed to be reduces in relation to the initial Ti plasmid.
Binary vector method
The binary vector method is the more prefer technique, which consists of two types of vectors in the cell. They are disarmed Ti plasmid and a vector carrying target DNA. The T-DNA of the autonomous vector can enter the plant cells because the vir region of disarmed Ti allows the agrobacterium to express its virulence against the plant cell. The figure below shows the following condition.
The technique is successful in lots of Dicotyledons and is useful in transferring DNA with several genes in a series. Biotechnologists make use of the space on the disarmed Ti plasmid after the removing of oncogenes. Foreign genes can then be inserted into the space. Two genes are commonly transferred together which are target gene and following by marker gene or reporter gene. Marker gene act as an indicator for the target gene since it is easily distinguished by their properties. The property expressions include antibiotic resistance and fluorescence emission. Antibiotic-tolerant and herbicide-tolerant gene are useful in agriculture. They are located between the border sequences and then are inserted into the plant cells.
As indirect transformation of gene is not suitable in Monocotyledons, direct methods of genetic transformation are then developed. It was significant to invent other methods as major food resource crops, such as wheat, maize and rice are Monocotyledons. They are not able to regenerate to whole plant from protoplast as Agrobacterium tumefaciens does not infect monocotyledonous plants, although some are available nowne. Direct transformations include microinjection, electroporation and biolistics.
Microinjection must be conducted on protoplasts in culture and one time can transform one protoplast only. The setup of the transformation is shown in the figure below. It includes a micromanipulator accompanied an inverse light microscope. They are kept under a laminar flow hood in order to maintain aseptic conditions during the manipulation. During the process, a protoplast is detached from a culture and held at the end of a tiny glass cannula, whose diameter is much smaller than that of the protoplast. In another side, a glass microneedle acts as a microelectrode holding the target DNA. The protoplast is then brought into contact with the top of the microneedle and the DNA is released into the nucleus of the protoplast with the help of air pressure. Then, the protoplast is cultured and regenerate into a whole plant finally. The efficiency of this method is low and the technical skill required is high, even the inventor, Croshaw, had 60% successful rate.
Protoplasts are transformed with the help of electroporator in this method. The protoplast culture is put into a chamber of concentrated solution of plasmidic DNA with electrodes. An electric field of 200 to nearly 1000V per cm is created and the process carries out for a few microseconds to milliseconds. This causes the pores formation in the plant cell membrane and allows protoplasts to take up DNA. Many types of protoplasts are found to be able for transformation by this method, although the product yield is low. GM maize and rice can be produced under this process. The equipment for electroportation is shown in the following figure.
In this method, originally, plant cells are bombarded with tungsten microbullets, with about one micrometer diameter, coated with DNA and then projected by a particle gun. It is a 22LR calinbre gun and with the help of small explosive charge to bombarded the plant cells. After shooting, some of the DNA on the microbullet is washed off and then integrated into the plant genome. This method is then modified which the gun is replaced by a cylinder of pressured helium gas and gold particles are used instead of tungsten. This method is useful in studying the function and transient activity of genes which are inserted into plant cell and stay for a short period of time. It is because the transgene only penetrates the cell but does not integrate into the host plant DNA. After bombardment, the isolated explants plant tissues are induced to become embryogenic cells and regenerated. The embryonic cells which have received the DNA will then grow into a whole plant. Production of genetic modified cereals is very successful by this method. For example, modified maize, wheat, rice and barley are commonly produced by biolistics.