Direct gene transfer method : Direct gene transfer method Presented by
Birendra kumar contents : contents Introduction
Why direct gene transfer????????
Conclusion Introduction : Introduction In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector
It is the best method for the production of transgenic plant.
They high importance both in agriculture and medical.
Production of diseases resistance, high yield, drought resistance and many more….
In medical, production of edible vaccine, plantibodies etc…. protocol : protocol Physical methods : Physical methods Mainly there are three as follows
Particle bombardment – gene gun Microinjection : Microinjection Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette
This method of gene transfer is used to introduce DNA into large cells
normally performed under a specialized optical microscope setup called a micromanipulator. The process is frequently used as a vector in genetic engineering and transgenetics to insert genetic material into a single cell Electroporation : Electroporation Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus
Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5 V to 1 V) Particle Bombarment : Particle Bombarment In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).
The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues Slide 12: A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with geneticinformation. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to asbioballistics or biolistics.
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated tungsten particle, whose DNA eventually migrates to and integrates into a plant chromosome.
Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots. Gene Gun : Gene Gun Slide 14: Selected single cells from the callus can be treated with a series of plant hormones, suchas auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant.
The new plant that originated from a successfully shot cell may have new genetic (heritable) traits. Chemical method : Chemical method Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) induce DNA uptake into plant protoplasts. Calcium phosphate is also used to transfer DNA into cultured cells.
Liposome are mainly used for transferring GI.
Liposome are sac made of phospholipid. liposome : liposome Liposome are sac made up of phospholipid.
This sac are filled up with GI and markers.
Liposome are able to activate Na/K pump. Then the DNA are inserted into the nucleus.
They get hybridised there and tansformation takes place in the host.
The transformed cell are screened in the selective media with the helps of markers. Calcium Phosphate method : Calcium Phosphate method Foreign DNA can also be carried with the Ca ++ ions, to be released inside the cell due to the precipitation of calcium in the form of calcium phosphate. In the past, this method was considered to be very important for gene transfer in plants.
Calcium phosphate transfection method is a very efficient means of introducing DNA into cells in many cell systems. This method works best in cell lines that are 1) highly transformed and 2) adherent. conclusion : conclusion Transgenic plant are produced for our convienence
They have high value both in agriculture and medicinal…
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