Genetic transformation is a key research tool with regard to the study of molecular biology relating to plants. In this connection more than a hundred plant species have been successfully transformed.
The two commonly used gene delivery systems are agrobacterium and biolistics. However host-range specificity is a biological barrier when agrobacterium is used for transformation purposes. This can be overcome by using particle bombardment techniques. An example is the ease of transformation of papaya tree using biolistics.
Biolistics is a wholesome word for particle bombardment. In other words, it is an acronym for biological ballistics. It is the method of physically impregnating the cells with nucleic acids or with other biological molecules.
How biolistics originated?
John Stanford is credited with developing the biolistic particle delivery system or the gene gun. The experiments conducted by Sanford in relation to biolistics originally used onion epidermal cells. Subsequent research by Klein and others confirmed viral replication of cells when genomic RNA of tobacco virus was transferred. This confirmed the workability of the biolistic particle delivery system.
In biolistics the impregnation of the cells with nucleic acids or with other biological molecules is achieved by coating the molecules that are intended to be introduced into the cells onto micro-carriers. These micro-carriers could be:
High density gold microparticles
High density tungsten microparticles
Then a gene gun is used to carry out the procedure. The gene gun with the help of helium pulse accelerates these particles to extremely high velocities. The result is they are able to penetrate the cell walls and membranes and get into the cell.
A gene gun can also be used for:
In vivo and in vitro assay of gene expression and regulation
For eliciting immune response in animals
How a typical gene gun works?
This is how a typically modern gene gun works. Firstly the cartridges are loaded with DNA or RNA coated micro-carriers. Then simply point the nozzle at the target and activate the device.
The coating given to microparticles could be either of DNA or RNA. Usually for DNA prior to coating it is first precipitated with the following:
As a result, microprojectiles of dimension of 1 to 4 microns are created. These are targeted into cells at high velocities using a carrier nylon micro-projectile.
The stopping plate of the barrel of the gene gun stops the micropiles while the microprojectile with the DNA coating penetrates the cell wall to deposit the DNA.
While on the context of gene guns the way the genes are prepared for it is quite different for plants and animals. The gene must have a promoter sequence which is appropriate for the organism it is being put into. For example, a gene with a plant promoter wouldn't be appropriate for a mammal.
In biolistics it is not necessary to use binary vectors with T-DNA borders as for instance in transformation using Agrobacterium.
Uses of biolistics
Biolistics can be used to transform a plethora of targets. These could be:
• Animal tissues
• Cell cultures
• Apical meristems
• Floral tissues
Some animal cells have specific growth requirements. That apart, biolistics is especially suitable for sensitive animal tissue cells because no manipulation is required either before or after. It can be used for successful transformation of the following type of animal tissues:
• Intact tissues
• Cells in culture
Biolistics has been successfully tried in several acquatic mammals like fishes and even oysters. And transgenic embryos have resulted subsequently. But in acquatic mammals microinjection remains the popular method of transformation.
Biolistics can be used to transform other targets like
Biolistics can be used for transforming whole cells in culture media or for transforming chloroplast and mitochondria which are essentially components of cells.
Why is biolitics important?
Some monocotyledonous plants especially agricultural varieties are not amenable to other transformation methods. For such cells and tissues biolistics is important. For example the following targets could be transformed through biolistics: apical meristems, seedlings, embryos, cultured cells, leaves, and floral tissues.
The cell walls of plants are pretty tough. To get a DNA to penetrate the cell walls of plants requires force. Biolistics can make this happen when gold or tungsten microscopic particles coated with DNA construct are fired at extremely high velocities through the cell walls of plants.
• Biolistic method of transformation can be applied to a wide range of cells/tissues.
• In mammalian cells biolistics helps retain cell morphology as there is no need to tryptinize the cells.
• Biolistics can be used to transform even intracellular organelles unlike other methods of transformation.
• In comparison with microinjection of nucleic acids into plant cells, biolistics offers an easier method that is less time consuming.
• Both transient and stable transformation can be carried out using biolistics
• Biolistics needs less number of cells to work with and also less DNA
• There is no need for carrier DNA
• Biolistics does not deliver extraneous genes or proteins. The emergence of toxic possibilities such as viruses or chemicals can be avoided.
• Biolistics makes it possible to co-deliver multiple plasmids.
• Using biolistics a high level of co-transformation is possible.
• With biolistics it is possible to transfect non-dividing cells and plants
• With biolistics it is possible to transfer large DNA fragments as well as siRNA's.
• In biolistics the particles used for bombardment can carry nucleic acids, DNA or RNA. Other DNA delivery systems have some limitations.
• With the technique of biolistics multiple genes can be put into a single plant. Example: the incorporation of fifteen foreign genes into rice.
Biolistics is undoubtedly a potent method of introducing nucleic acids into plant cells aided by helium pressure that enables the micro carriers to penetrate the cell walls effectively. But transformation using Agrobacterium is easier to accomplish as there is no need for specialized equipment.
On the negative side biolistics has not produced transgenic varieties in cereals so far. But using this method transgenic plants have been produced in tobacco and soybeans and may find use in different cassava genotypes as well.
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