Thursday, May 21, 2009

Methods of Genetic Transformation

Among the several methods of plant transformation, four have yielded the best results: Agrobacterium species-mediated transformation, microprojectile bombardment, microinjection, and direct transformation. Each of these methods has merits and limitations and is used in specific situations. At this time there is no single technique that is suitable for all species.

Agrobacterium Mediated Transformation
Tumors and uncontrolled cellular growth in plants can occur due to genetic factors or bacterial and viral infections. An example is crown gall in plants, where tumors are caused by bacteria that causes uncontrolled growth on the stem of the infected plants. This problem is caused by Agrobacterium tumefaciens, a soil bacterium that infects some plants because of a wound on the plant. Plasmids present in the bacteria are responsible for tumor growth after infection by A. tumefaciens. The bacteria are able to recognize wounds on the plant, and this induces the transfer of the bacterial plasmid into the plant. The plasmids are capable of integrating into the DNA of the host plant, causing uncontrolled plant growth and the formation of tumors. The ability of A. tumefaciens to efficiently transfer plasmid DNA into the host has made it important in early studies in genetic transformation.

Agrobacterium tumefaciens was the first vector used for introduction of foreign DNA in plant cells. Although Agrobacterium has only been used to infect dicot plant species, such as soybean, tomato, pea, and cotton, the protocol has been modified to allow the bacteria to infect some monocot (grass) species as well. Many research groups working with plants have found this to be the preferred transformation approach. Another soil bacteria, Agrobacterium rhizogenes, causes the growth of secondary roots after infection. This bacterial species has also been used for plant transformation.

The basis of this transformation method is the bacterial plasmid, which contains the genetic sequence that is integrated into the host genome. One of the most important parts of a plasmid is the region responsible for the translocation of its DNA into the host plant genome. This is called transfer DNA (T-DNA), and this area of DNA is key to the tumor growth in infected plants. The region is located between the right border and left border (RB and LB) of the plasmid. Plasmids also contain other important DNA sequences; some of them control the production of auxin and cytokinin, two important plant hormones involved in tumor formation. With the use of the restriction enzymes, a transgene can be introduced between the right border and left border of the plasmid, allowing the bacteria to transfer novel genes into the recipient plant.

One of the techniques used for transformation mediated by A. tumefaciens uses leaf disks. Leaf disks of about 6 mm in diameter are cultured on a tissue-culture media containing A. tumefaciens with plasmids containing the transgene. After approximately a month of incubation in the tissue culture medium, seedlings start to develop on the leaf disks. Through selection methods, transgenic seedlings are identified for whole plant regeneration.

Microparticle Bombardment
This technique has also been called microprojectile acceleration or biolistics, but microparticle bombardment is the formal name for the machine called a gene gun. This method, developed at Cornell University, was designated biolistic (biologic + ballistics = biolistic), because high-speed microscopic projectiles (microprojectiles) are accelerated into the cells to be transformed.
This transformation method consists of the acceleration of a macroprojectile loaded with millions of tungsten or gold microspheres about 1 ┬Ám in diameter (microparticle). The microspheres are coated with the transgene, or DNA of the gene of interest. Microspheres have a high specific mass, allowing them to acquire the needed momentum to penetrate the target cells. The macroparticle is propelled in the direction of the cells at high speed, but it is retained, after a small distance, on a steel mesh so that the microparticles continue in the direction of the target cells. Helium gas at high pressure is used to propel the macroparticle, and the acceleration chamber operates under a partial vacuum, which allows for improved microsphere movement. Once inside the target cells, the DNA coating the microspheres is released and can be integrated into the plant's genome.

Many of the commercial transgenic crop varieties on the market today were developed using the gene gun. However, due to its cost and the complex integration patterns resulting from this method, several research groups are reducing its use.

This method was developed for animal transformation but has also been extended to plants. Although very difficult and laborious, DNA microinjection has yielded positive results and has been used in several laboratories.

In this technique, microcapillary needles are used to introduce DNA directly into cells. Each cell to be transformed must be manipulated individually. One of the advantages of this method is that the optimum amount of DNA can be injected into the target cells, which helps to ensure optimal integration. Positive results have already been obtained in several crop species such as corn, wheat, soybean, tobacco, and rice, and in animals like salmon, cattle, and swine.

Direct Transformation
Transformation using direct methods was accomplished soon after the first Agrobacterium-mediated transformation. These methods use protoplasts (cells after the removal of the cellular wall) as targets for transformation. This is a simple method that consists of adding great amounts of transgenic plasmids to a protoplast culture, which guarantees that a small proportion of the protoplasts will be taken up (assimilated) by the plasmids. The assimilation rate can be increased with the addition of polyethylene glycol (PEG) or the use of an electric discharge (electroporation). No barrier to direct transformation has been detected, indicating that this method can be used with virtually any species. The problem with this method lies in the difficulty of regenerating a whole plant starting from protoplasts. Therefore, it has not been used as widely as the other methods.

Tags: Bio Technology, Bio Genetics, Genetic Transformation

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