In-plant genetic engineering, plant genes can be modified for required characteristics. Desired plant genes can be transferred from one species to another, or introducing new genes can be done by genetic transformation technology. The transferred genes are known as a transgene, where the process is called transgenesis. This results in new genetically transformed new plants, also known as transgenic plants. These transgenic plants are developed on the basics of the Totipotency of the plant cell, where desired or genetic traits are introduced to the new plants for successive generations.
Gene transfer methods
Gene transfer technique is the primary requirement for genetic transformation technology. It avails transfer for genes to plant cells by various gene transfer methods.
Gene Transfer methods are classified as:
1. Vector mediated gene transfer method
2. Direct transfer or vector less gene transfer method
a) Physical methods
III. Liposome fusion
IV) Silicon carbide fibers
b) Chemical methods
I) Polyethylene glycol (PEG) mediated
II) Diethylaminoethyl (DEAE) Dextran mediated
III) Calcium phosphate precipitation
1.Vector mediated gene transfer method
Gene transfer is generally done by Agrobacterium-mediated transformation or the use of Plant viruses as vectors.
a) Agrobacterium-mediated gene transfer: a soil-borne, gram-negative bacterium Agrobacterium tumefaciens is used for gene transfer. It is a motile and a rod-shaped bacterium belongs to the bacteria family of Rhizobiaceae. It is a phytopathogen and hence, treated as nature’s effective plant genetic engineer. In this method, the agrobacterium carrying vector with the desired gene is developed. Then explant is co-cultured with Agrobacterium. Followed by, killing of agrobacterium with the use of antibiotics without harming explant cells. Finally, transformed cells are selected and regenerated into the whole plant. It is a natural method of gene transfer because Agrobacterium can infect any explant. Large fragments of DNA can be transferred by this method. The stability of the transferred DNA is good and hence whole plants can be regenerated effectively.
b) Virus mediated gene transfer: viruses are used as natural vectors for genetic engineering because they infect the plants and multiply the transferred genes by viral genome replication. Hence they are efficient for gene transfer methods. Three groups of viruses are used for gene transfer in plants e.g. Caulimoviruses, geminiviruses, and RNA viruses. By the use of virus vectors the, transmit of the desired genes to generations is not possible.
2. Direct transfer
a) Physical methods
I) Electroporation: In this technique uptake of DNA is done by, incubating plant material in a buffer solution containing DNA. This mixture solution is subjected to high voltage electrical impulses, which causes pores in the cell membrane. Reversible Uptake of DNA is facilitated by the pores on the cell membrane, this DNA then integrated into the host cell genome. This technique is simple and rapid to use. The physiological state of transformed cells is not altered. The efficiency of Transformation can be improved by electrical field strength optimization. Under normal conditions, DNA transformation in cells is very low. Plant regeneration is not easy by this method, especially protoplast is used.
II) Microinjection: In this method, mechanical transfer of gene to target cell is done by using a pipette of 0.5 to 10µm tip into the cytoplasm of a plant cell. Recipient cells are immobilized in Agarose embedding and then held by suction holding pipette. Later, this genetically modified cell is then cultured and grown into a transgenic plant. This process is very slow and is only performed by skilled or trained personnel.
III) Liposome mediated gene transfer: In this method liposomes carrying desired genes are fused with protoplasts and transfer the genes. Liposomes are first attached to the target protoplast, then these liposomes fuse with the protoplast at the site of attachment and release plasmid inside the cell. DNA is protected from the environment by encapsulation, and hence DNA is stable, can be stored prior to transfer. This method is used for a wide range of plants. Regeneration of plants from protoplasts is difficult using this method.
IV) Silicon carbide fibers mediated transformation: in this method silicon carbide fiber of 0.3 to 0.6 µm diameter and 10-100 µm length. The DNA coated silicon fibers are vortexed with suspension culture of plant material. These fibers penetrate the cell wall and plasma membrane hence, DNA adhering fibers enter the cell and get integrated with the host genome. This process is simple and cost-saving. These fibers are carcinogenic hence should be carefully handled. This technique is not applicable to embryonic plant cells, as they are hard and compact, so resistant to SCF penetration.
b) Chemical mediated gene transfer:
I) Polyethylene glycol mediated transformation: In this procedure plasmid, DNA is added to the protoplast suspension followed by slow addition of 40% PEG-4000 w/v dissolved in mannitol and calcium. This mixture is then incubated. These protoplasts get transformed, by destabilization of the plasma membrane by PEG in presence of divalent calcium ions. By this method, a large number of protoplasts can be transformed easily. There are chances of DNA degradation and rearrangement.
II) Dextran mediated transfer (DEAE): High molecular weight polymer diethyl aminoethyl (DEAE) dextran is complexed with desired DNA to be transferred. This method does not yield stable transformations.
III) Calcium Phosphate Co-Precipitation mediated transfer: The calcium chloride solution and isotonic phosphate buffer is mixed with DNA. This mixture allows precipitating DNA-Calcium phosphate precipitate. This precipitate is then exposed to the culture medium of actively dividing cells, by which the DNA is transformed into the cells. This method required a large amount of DNA samples.
Multiple Choice Questions
1. Plant gene transfer method is used in genetic engineering to __________
a) Transfer modified gene
b) Introduce new gene
d) Only A
2. In Genetic engineering the a modified or new gene transfer is done to ___________
a) Make diseases resistant plant
b) To introduce new traits
c) To increase the yield of secondary metabolites
3. Which of the following is used in vector-mediated gene transfer?
a) Agrobacterium tumefaciens
b) Agrobacterium lufifeciens
c) Agrobacterium mutafaciens
4. Agrobacterium tumefaciens is _________
5. ______________is the physical method for direct transfer of genes
a) PEG mediated
b) Dextran mediated
c) Calcium mediated
d) Liposome fusion
6. Large fragments of DNA can be transferred by ___________
a) Virus mediated transfer
b) Agrobacterium tumefaciens transfer
7. __________ method uses high voltage electrical impulses for gene transfer
a) Liposome fusion
d) Silicon carbide fibers
8. Silicon carbide fibers used in the direct method of gene transfer are carcinogenic
9. _____________method of gene transfer requires skilled personnel
a) Liposome fusion
d) Silicon carbide fibers
10. In liposome-mediated direct gene transfer method genes are __________
a) Gene is stable
b) Gene is protected
c) Both a) and b)
d) Nor protected nor stable
- Satyanarayana, biotechnology, 1st e.d. Kolkata, books & allied (p) ltd publishers, 2005 page:573-588