Generating Genetically Modified Plants

HAMID M. SADEGHI
Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran

Transformation

Two mostly used transformation pathways for making transgenic plants are:
•Bacterial carriers
•Biolistics

Bacterial Carriers
Agrobacterium tumefaciens contains a plasmid, a small circular piece of DNA that has its own origin of replication and is replicated independently of other nuclear material, that is key to its use in genetically modifying plants. This plasmid, called a tumor-inducing (Ti) plasmid, interacts with compounds released by fractured plant cells. When a wounded plant is exposed to A. tumefaciens, it integrates a stretch of its DNA, called transferred DNA (T-DNA), to the plant's genome.Normally, the bacterium transfers its own T-DNA, but if the T-DNA is removed and replaced with another gene, A. tumefaciens can be used to introduce that gene into the plant genome, thereby providing a vector for scientists to engineer beneficial genes into plants. Studies have shown that native T-DNA genes are not necessary for this process. Inserted genes get transferred to plants as long as two repeated border sequences of 25 base pairs flanking the genes are present in the vector.


Biolistics
The gene gun is part of the gene transfer method called the biolistic (also known as biobalistic or particle bombardment) method. In this method, DNA or RNA adhere to biological inert particles (such as gold or tungsten). By this method, DNA-particle complex is put on the top location of target tissue in a vacuum condition and accelerated by powerful shot to the tissue, then DNA will be effectively introduce into the target cells. Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus picking up the genetic material and proceeding into the living cells. The efficiency of the gene gun transfer could be depended on the following factors: cell type, cell growth condition, culture medium, gene gun ammunition type, gene gun settings and the experimental experiences, etc.


Agrobacterium versus biolistics (particle bombardment)
In general Agrobacterium is considered the method of choice for transformation. Advantages include:
•Low copy number of the transgene
•Higher proportion of stable transformants
•Large DNA segments can be transferred
•More time efficient


Other techniques that can be used:

Calcium phosphate precipitation
Electroporation
Gene silencing
Gene splicing
Lipofection
Microinjection
Viral carriers


Protein Based Testing Methods

•Transgenes encode for novel proteins
•Immunoassay techniques using antibodies
•Analyte must be known
•Interference from non-specific interactions of proteins, surfactants (saponins), phenolics, fatty acids and phosphatases
•Polyclonal antibodies
•Monoclonal antibodies

Key words: Western Blot ,ELISA Microwell Plate , Antibody-coated Tube ,ELISA Considerations , DNA-Based Methods , Southern Blot , PCR Fragment Confirmation

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