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Technique
of
Transferring
Plant
Gene
Using
Ti
Plasmid - The simplest way to introduce T -DNA into plant cells is to infect them with A. tumefaciens containing the appropriate Ti plasmid and let nature do the rest. Therefore, we need to be able to insert desired genes into the T-regions of Ti plasmids.
Since the Ti plasmid is very large (up to 235 kbp), it is not feasible to modify it directly; hence it is useful to perform all manipulations on an excised piece of DNA including the T-DNA and then use in vivo recombination to swap the engineered T-DNA for its normal version in an intact Ti plasmid. The strategy developed is as follows.
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First, the T-region is cut out of a Ti plasmid with restriction enzymes and introduced into one of the standard cloning vector plasmids that are used with E. coli. Large amounts of the vector carrying the T -DNA can be grown in E. coli and then isolated.
The next step is to use restriction enzymes and recombinant DNA techniques to insert a particular gene into the T-DNA. This hybrid, containing the T-DNA and the gene inserted into it, can be grown in large amounts in E. coli and then introduced into A. tumefaciens cells containing the corresponding entire Ti plasmid.
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Homologous genetic recombination between the T-DNA segment of the native Ti plasmid and the cloned T-DNA segment carrying the foreign gene results in transfer of engineered T-DNA to the Ti plasmid and the displacement of its normal T-DNA. The outcome is A. tumefaciens with a Ti plasmid whose T region carries the desired foreign gene.
The last step is to infect plants with these engineered A. tumefaciens bacteria. The crown gall cells that result will be transformed by the T-DNA carrying the foreign gene and the goal of introducing a desired gene into plant cells thereby achieved.
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Three general methods are available for obtaining Agrobacterium transformed plant tissue. In one, plants are grown under aseptic conditions and the stem wounded. The wounded tissue is inoculated with the cells of A. tumefaciens by means of a syringe. The tumors that develop can be removed and cultured on hormone free agar on which the transformed cells can grow.
The second method is called cocultivation. Protoplasts lacking cell walls are first produced by dissolving the cell wall enzymatically. The protoplasts are then allowed to remain for about two days so that cell walls begin to form. At this stage (usually between 36-48 h) A. tumefaciens suspension is added. Some of the cells undergo transformation during the next few days of cocultivation.
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Then adding antibiotics, the bacterial cells are killed and the plant cells can be grown as callus on hormone free agar medium. The third method is called the leaf disk method in which large pieces are cut and incubated with Agrobacterium so that wounded cells at the cut edges become genetically transformed, as above Agrobacterium tumefaciens has also been shown to transform chloroplasts.
The usual method to transform plant cells with T -DNA is to paint agrobacteria that harbor Ti plasmids on a wound made in a plant shoot. However, with improvements in techniques for plant cells and protoplast culture, an often more convenient method that allows infection and transformation in vitro has been devised.
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Leaf cells are removed from plants, converted to protoplasts and put into culture. At this stage, when the protoplasts have just regenerated a cell wall and begun to divide, the culture is infected with Agrobacterium and left for several hours. Antibiotics are then added to kill the bacteria and the cells are grown in a medium containing plant hormones for a few weeks until they have formed small calli.
At this stage, the medium is changed to one lacking plant hormones. Only the transformed cells will continue to survive and multiply. The transformed cells can then be tested for the presence of T-DNA or its hallmark, the synthesis of opines. Sometimes cells from such cultures spontaneously regenerate into shoots or plants carrying T -DNA and making opines.
With a much lower efficiency, it is also possible to transform protoplasts directly with Ti plasmid DNA. Freshly prepared protoplasts are exposed to plasmid DNA in a medium containing polyethylene glycol and Ca++ ions-essentially the same medium used to induce protoplast fusion. T-DNA is taken up by the protoplasts, which are then cultured in a medium with plant hormones to allow regeneration of cell walls and cell division.
After a few weeks, when calli have developed, the medium is replaced with one lacking plant hormones. Only the transformed cells will survive and continue to multiply. The fact that protoplasts can be transformed by pure T-DNA proves that agrobacteria are not essential for transformation. Their role is solely that of a vector to bring T-DNA into plant cells.
The latest advance in the use of T-DNA as a vector for introducing genes into plants is the use of specific plant promoters to express the transferred genes. The Ti plasmid gene that codes for nopaline synthetase is isolated and sequenced and its promoter/ region is identified.
The structural gene for octopine synthetase is cloned downstream from this promoter and these hybrid genes are introduced into plant cells. These genes are found to be expressed in plant cells under the control of the nopaline synthetase promoter.
A glimmer of hope has been provided by the discovery that A. tumefaciens can transfer its T-DNA into certain monocots, resulting in expression of the opine gene within the plant cells but without inducing tumor formation.
If the T-DNA becomes integrated into the plant chromosomal DNA, and if similar results can be obtained using cereals, then the Ti plasmid will be even more suited to the transformation of monocots than dicots, since there seems to be no need to disarm the one gene when infecting monocots.
When we introduce novel DNA sequences into plants, the quantity of the novel protein produced will depend on the rates of transcription and translation and the stability of mRNA and the synthesized protein.
Studies on vector construction have identified strong promoters that control the function of certain genes. For gene transfer and expression, not only the genes but also their promoter sequences have to be identified, isolated, and transferred.
For tobacco, petunia and other members of Solanaceae, transformed calluses and transformed plants regenerated from such calluses can also be obtained by cocultivating protoplast-derived cells with A. tumefaciens.
Agrobacterium can be used to transfer foreign genes into the nuclei of maize cells. The bacterium inserts a part of its own DNA into the nuclei of cells it infects so as to force the cells to synthesize food materials on which the bacterium feeds. To achieve such gene transfer, the genes of the maize streak virus (which infects maize cells) are inserted into that part of Agrobacterium DNA which is naturally integrated into plant cell nuclei.
The maize plants are then inoculated with agrobacterial cells which have been so treated. Studies have shown that the plants become infected with the virus. The fact that the plants develop viral symptoms proves that the viral genes do integrate into the host cell nuclei with the bacterial DNA.
Of course the above experiment is of no direct commercial value but it nevertheless shows that a procedure has been standardized which may be used to insert useful genes such as those for resistance to insect pests or to herbicides into the viral DNA before inserting it into the bacterial DNA and inoculating the plants.
The idea in this approach is to use viral infection as a kind of tracer or marker, to show that foreign genes have become integrated into the maize cell nuclei.Although it is possible to readily isolate protoplasts from cereals and these protoplasts can also be induced to regenerate new cell walls and to divide repeatedly to form a callus, as yet there is no evidence that such cereal protoplast derived cells can produce transformed calluses after cocultivation with A. tumefaciens.
Failures to achieve Agrobacterium induced transformation in cereals have stimulated trials of other alternatives.Some alternative methods include direct uptake of DNA, fusion of bacterial protoplasts with plant cell protoplasts, liposome mediated DNA delivery, and microinjection of DNA into the cells.
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