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Table of Content
- Introduction:................................................................................................................................................ s
- What is genetic engineering?.......................................................................................................................
- History of genetic engineering:...................................................................................................................
- Techniques of genetic engineering:.............................................................................................................
- Genome editing:......................................................................................................................................
- Homologous recombination:...................................................................................................................
- Meganucleases and ZNF.........................................................................................................................
- TALEN and CRISPER............................................................................................................................
- Genetic transformation techniques:.............................................................................................................
- Indirect method:......................................................................................................................................
- Agrobacterium mediated gene transfer:...............................................................................................
- Direct gene transfer method:.....................................................................................................................
- Physical gene transfer method:..............................................................................................................
- Electroporation:.................................................................................................................................
- Particle bombardment:.......................................................................................................................
- Microinjection:..................................................................................................................................
- Liposome mediated transformation:..................................................................................................
- Silicon carbide mediated transformation:..........................................................................................
- Chemical gene transfer method:............................................................................................................
- Polyethylene Glycol mediated transfer:.............................................................................................
- DEAE dextran mediated transfer:......................................................................................................
- Calcium phosphate co-operation mediated transfer:..........................................................................
- Conclusion:...............................................................................................................................................
- References:................................................................................................................................................
- Figure 1: Genetic engineering in plants....................................................................................................... Table of figures:
- Figure 2: Gene transfer method in plant......................................................................................................
- Figure 3: Indirect gene transfer...................................................................................................................
- Figure 4: Electroporation...........................................................................................................................
- Figure 5: particle bombardment gun.........................................................................................................
- Figure 6: microinjection.............................................................................................................................
- Figure 7: Liposomes mediated transformation..........................................................................................
Figure 1 : Genetic engineering in plants discoveries that were made in 1900s paved the way for genetic engineering. In 1922, Robbin and Kolte cultured root and stem tips. In 1950, Barbara McClinton explains about the transposons. Transposons are the DNA sections that move from one place or location of chromosome to another and this process is known as jumping gene. She said that, this all jumping led to alteration of DNA. When the DNA was discovered by Watson and Crick in 1953 then , a scientist with the name of Kornberg made the DNA in test tube for the very first time in 1958 by extracting the DNA polymerase from bacteria and then assembled the nucleotides of the DNA. 1n 1962, Osamu isolated the GFP (green florescence protein) from the jelly fish and introduced it into a plasmid. In 1967, discovery of the DNA ligase was made. In 1968, restriction endonucleases were discovered and purified in 1970 [4]. In 1973, genetic modification was done by Herbert and Stanley. A mouse is the organism that was first genetically modified by Rudolf Jaensch, in 1974 [5]. In 1971, Takebe developed the first plant from the protoplast. In 1982, Mary Dell created the first genetic engineered tobacco plat. In this process, a gene resistant to bacterium was introduced into the tobacco plant to make it resistant to bacteria. This achievement paved the way for the scientists to manipulate the genetic makeup of organisms [6]. In 1994, first genetically modified food i.e. tomato was brought into the market. This was done to lower the ripening of tomato but the scientists didn’t get the results that they wanted. In 1996, a sheep was cloned and named as dolly. In 2003, human genome was sequenced [7].
Techniques of genetic engineering:
The process of genetic engineering is usually taking place with the help of other techniques or in simple words, other techniques are also used in the genetic engineering. If its method is discussed in simple word, then according to this method first of all, a gene is selected whose characteristics have to be show in another organism. Then isolate the gene
and then insert it into plant and then grows that seeds. Then it will turn out into a genetic engineered plant having desired phenotype or characteristics and as a result, it will become novel plant. In genetic engineering, a foreign gene is inserted by gene targeting through different techniques and these techniques provide safe transgenic crops and gene integration.
Genome editing:
It is a type of genetic engineering, in which insertion, deletion or replacement of DNA into the genome of organisms take place. In the genome, double stranded breaks are produced by using following techniques: Homologous recombination MEGANUCLEASES Zinc Finger Nucleases CRISPR Transcription Activator Like Effector Nucleases Among all of these, most commonly used techniques are two; one is TALEN and second is CRISPR. Homologous recombination: It is a technique that plays important role in maintenance of chromosome. It protects the DNA from damage. Damage can be double stranded break or inter stranded crosslinks. There are three ways to insert the foreign gene that include; single strand annealing, end joining and illegitimate recombination. Breaks are made at the target site to introduce the gene. Meganucleases and ZNF Meganucleases: In 1998, this technique was performed on mammalian cells. Another name of them is endo deoxyribonucleases , because they perform their function as restriction enzymes. They are highly specific and act at specific sites and are highly specific then restriction enzymes. They are specific because of their acting on specific site rather then disturbing the whole genome. Zinc-finger nucleases:
Genetic engineering techniques are broadly classified into two: Indirect gene transfer Direct gene transfer Figure 2 : Gene transfer method in plant
Indirect method:
As the name indicating, there is not direct transfer of gene into plant. In this method a vector act like a messenger that transfer the gene to plant after inoculating the required gene to it. In this method, agrobacteria cat as a second messenger. That’s why it is known as agrobacterium- mediated transformation. Another name for it, is plant gene vector. According to this term, vectors that are having potential to transfer the gene from an organism to the plant. Agrobacterium mediated gene transfer: Agrobacterium is the bacteria that is present naturally in environment or surrounding. It is a gram-negative bacterium that is naturally found in soil. In 1983, there was successful plant
transformation by the use of the agrobacteria. The two main species of this bacterium are following: A. Rhizogenes that have the ability to cause hairy root disease in plant. A. Tumiefacines that have the ability to cause gall disease in plant. Crown gall disease: It is the disease that is due to entry of A. Tumiefacines into the plant cell. If the plant is injured or wounded, then the plant will secret or release the phenolic and these compounds would be recognized by this bacterium and the bacterium will attack or enter into the plant. This bacterium is having Tumor inducing plasmid. That T-DNA will be transferred to the host cell that is plant. As the plant divide, then the division of bacterium will also occur and a result it will cause disease. Figure 3 : Formation of gall tumor in plant affected by A. tumefaciens
Figure 4 : Indirect gene transfer
Direct gene transfer method:
In this method, there is not any need of the vector or plasmid to transfer the gene into plant. Rather, the gene is directly transferred into plant without using second messenger. In this method, direct transfer of naked DNA occurs. This method is effective and simple. Because of this method, many transgenic plants have been produced. This method is categorized into following:
Chemical gene transfer method Physical gene transfer method
Physical gene transfer method:
It is divided into sub categories that are following: Particle bombardment Silicon carbide fibers Microinjection Electroporation Liposome fusion Electroporation: It is a method in which electrical impulses are used in order to create hole to transfer the DNA into plant. In this method, the plant material in buffer solution that is also containing the foreign DNA is incubated and then subjected the electrical impulse to create holes in plasma membrane. Through this plasma membrane, DNA will enter and become the part of host cell genome. In past, protoplast was the only one that is used for gene transfer. Now, callus culture, premature embryo has been using to produce transgenic plant. Figure 5 : Electroporation Advantages: This method is simple, cheap, and less time consuming. By changing the electrical field, efficiency of this method can be changed or improved. The physiological state will remain same after this method. Limitations:
Figure 8 : Liposomes mediated transformation In this method, plasmid containing liposomes will introduced and they will bind at attachment site. After that, they will fuse and as a result, plasmid will enter. Advantages: Can be applied on wide variety of plant. DNA can be stored in them for long time. It protects DNA from damage. Limitation: The problem is only in case protoplast regeneration into whole plant. Silicon carbide mediated transformation: Silicon carbide fiber is used of 0.3-0.6pm width and of 10-100pm length. This fiber has ability to enter cell wall and cell membrane and then inject the material. The DNA does not bind properly to the fiber so, can easily transfer into call and bind with genetic material. Advantages: This one is easy and cheap method. Disadvantages: Carefully handling is required because silicon carbide is carcinogenic. In embryonic cell, SCF faces resistance.
Chemical gene transfer method:
It is sub categorized into following: DEAE dextran mediated transfer Calcium phosphate co-operation mediated transfer Polyethylene glycol mediated transfer Polyethylene Glycol mediated transfer: In this method, PEG with the help of cations (e.g. Ca++) disturbs the plasma membrane and as a result DNA enters to nucleus and then integrates with genome. Advantages: Can be used for large variety of plant species. Disadvantages: Degradation of DNA can be occurred in this process. Undesirable traits can be obtained due to random insertion of DNA in genome. DEAE dextran mediated transfer: DNA is combined with diethyl amino ethyl (DEAE) that is polymer containing high molecular weight and then shift it. Stable transformation is not obtained through this method. Calcium phosphate co-operation mediated transfer: In this process, precipitates of DNA-calcium phosphate forms, when DNA is mixed with calcium chloride solution in phosphate buffer. Continuously dividing cells then placed on these precipitates, as a result, cell will be transferred after few hours. DMSO has ability to increase efficiency [10] [11].
Conclusion:
It is a technique that is used for transfer of gene or genetic makeup from one organism to another organism. There are different techniques for the editing of genome but the best one is CRISPR. There are two methods for the transfer of gene; one is direct while another is indirect method. So we can use both of these method in genetic engineering of plant and as a result variety of transgenic plant can be produced.
