Transfer of desired genes and expression of these genes into plant cells is a modern application of plant tissue culture. In vectormediated gene transfer, some biological agent is employed for transferring desirable genes into the plant cell. This vector-mediated transfer is also known as indirect gene transfer.
(1) Vector Mediated or indirect Gene Transfer: In this procedure, the DNA is transferred with the help of some vector. There are three methods of this –
1.Agrobacterium-mediated Gene Transfer: Among the various vectors used in plant transformation, the Tiplasmid of Agrobacterium tumefaciens has been used extensively. This bacterium contains large-sized plasmid, known as Ti-plasmid (tumour-inducing plasmid) and the portion of this plasmid referred as T-DNA (transferred DNA) when is transferred to plant genome, in the infected cells, it causes plant tumours (crown galls).
This means that A. thermogenesis has natural ability to transfer TDNA of its plasmid into plant genome at the site of infection, and therefore this bacterium is known as “natural genetic engineer of plants”. Because of this unique property. Ti-plasmid can be used as vectors for inserting useful foreign genes into plant cells and tissues.
The foreign genes (transgenes), i.e., the gene of interest(e.g. Bt gene for insect resistance) and plant selection marker gene, usually an antibiotic gene like no-till which confer resistance to kanamycin are cloned in place of the T-DNA region of Ti plasmid.
There is a possibility of tumour formation in plants by Ti plasmid. Hence for developing transgenic plants the tumour inducing gene (T-DNA) is separated from the plasmid DNA of Agrobacterium and in place of this desired gene is incorporated. Now the desired gene containing Agrobacterium is cultured with the tissues of the plant in which the desired gene is to be transferred.
Normally the rings or discs of leaves of tomato, tobacco, Petunia, rose etc. are used for composite culture because the acetosyringone produced by the rings or discs of leaves activate the operons of Ti plasmid. As a result of activation of these operons the desired gene containing Ti plasmid enters in several cells and gets incorporated in the genome of the plant cells. After 2 to 3 days of composite culture, the transformed cells are cultured in a suitable medium. This technique can be used for dicotyledon plants only.
Up to the beginning of 20th century Agrobacterium tumefaciens and associated species were considered as causative of plant diseases. But after knowing its ability to transfer foreign DNA, it is now used in Genetic engineering and due to extensive use in genetic engineering, it is known as Natural Genetic engineer.
Transformation of the gene in plants can be detected on the basis of production of amino acids by the genes present in the plasmid of this bacterium. The genetically transformed cells produce opines. These opines are of different types which depend on the strain of the Agrobacterium. The strains of A. thermogenesis produce octopine and nopaline whereas strains of A. rhizogens produce atropine and man opine opines.
In various research institutes, scientists are using Ti plasmid as a vector. By inserting desired and important genes in T DNA we can produce many important characteristics in plants such as Herbicide tolerance, Pathogen tolerance, Stress tolerance, increase in nutritive value (enrichment of rice with vitamin A, golden rice), improvement in nitrogen fixation.
Agrobacterium does not infect monocot plants normally but in 1994, Japanese scientists were able to do transformation with the help of Ti plasmid in rice.
2. Virus Mediated Gene Transfer: Both DNA and RNA viruses act as an ideal vector for transfer of desired genes. Two virus groups Caulimo viruses and Gemini viruses, which have DNA genome are the most widely used DNA viruses for gene transfer. Retroviruses, Lentivirus and Adenovirus are also used widely in genetic engineering for gene transfer.
3. In-planta method: This technique of gene transfer was developed by Failzadman and Markakes (1987). They kept Arabidopsis seed with genetically modified Agrobacterium and then raised the plants. Seeds obtained from these plants were germinated on antibiotic-free medium and identified the transformed plants.
Similarly, the apical meristematic part of embryo of germinated seeds can also be infected with Agrobacterium to produce genetically modified plants. In this method of gene transfer, the genes are directly transferred into a plant and for this reason, this technique is called In planta technique.
(2) Direct gene transfer: In this method, the DNA of the desired gene(s) is transferred directly with some technique without involving any vector. Normally gene transfer by Agrobacterium is possible only in dicotyledons. In monocotyledons, which are chief cereals, gene transfer by Agroinfection is normally not possible.
For improvement in these plants and to incorporate desired features, new techniques for gene transfer have been developed, in which biological vectors are not required. Hence the techniques in which biological vectors such as Agrobacterium or viruses are not required are called direct gene transfer.
Direct gene transfer in plants can be done by the following methods:
1. Chemical mediated gene transfer: Certain chemicals like polyethene glycol (PEG), polyvinyl alcohol, calcium phosphate etc. induce DNA uptake into plant protoplasts Among chemical methods PEG is more commonly used chemical.
In this method, first plasmid DNA is mixed with protoplasts and after some time 15-25% PEG is added. This amount of PEG promotes DNA uptake in the protoplasts. The transformed protoplasts are cultured on selected medium and with the help of marker genes, the transformed protoplasts are selected.
Liposomes, diethyl aminoethyl (DEAE) and dextran proteins are also used for gene transfer in plants and animals. PEG directed gene transfer does not cause any harm to protoplasts.
2. Physical methods of Gene Transfer: Direct gene transfer in plants is done effectively by several physical methods also. Some main physical methods are as follows
1. Gene gun: Gene gun is also known as particle gun, shotgun or microprojectile etc. By this technique, gene transfer is possible in the intact plant cells. (cells having intact cell wall). This technique of gene transfer was first used by Klein and Co-workers (1987) in onion cells for transferring DNA and viral RNA.
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In this process, the minute gold or tungsten particles of 1-3 mm diameter (microparticles) coated with desired DNA are shot (Bombarded) in the target cells with the help of microprojectile. These desired DNA coated gold or tungsten particles penetrate the cell wall and enter the cell where desired DNA incorporates with the host cell DNA and forms transgenic DNA. By the use of this method, gene transfer has been successfully achieved in wheat, rice, maize, tobacco and soybean etc. This technique is being used now in all type of plants worldwide.
3. Liposome mediated gene transfer: In this method of gene transfer, spherical lipid molecules filled with desired DNA and water are used for transferring genes. These DNA containing lipid capsules first stick to the plasmalemma (plasma membrane) and then fuse with it. The DNA present in these first enter into the cell and then enter the nucleus where it gets incorporated in the genome of the host cell. The liposome directed gene transferring technique also called as lipofection technique is a highly effective technique for transferring genes in bacteria, animal & plant cells
4. Microinjection: In this method, genes are injected in plant protoplasts or cells with the help of glass needle of 0.5 – 1.0 mm diameter or micropipette directly in the cytoplasm or nucleus of the protoplasts or cells of plants. This is the suitable method of transferring genes in the isolated protoplasts.
Note: Besides the above methods of gene transfer other methods are also employed for transferring genes. These are Ladder dependent gene transfer and Silicon carbide fibre dependent gene transfer and Silicon carbide fibre dependent gene transfer.