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Plant Transformation Methods
- Transformation is defined as the process in which the genetic material of an organism is altered by the integration of new genes into its genome.
- The transformation is done by using vectors such as plasmids.
- The importance to produce transgenic plants is to:
- Improve crop yields.
- Improvement of varietal traits.
- transgenic plants have protection against their parasites, pests and harsh weather conditions.
- The transformation was the first time applied by British Bacteriologist Frederick Griffith in 1928 in diplococcus pneumonia.
- Transformation using electroporation was developed in the late 1980s.
- Particle bombardment discovered (gene gun) by John Sanford in the 1980s.
Methods of plant transformation
Agrobacterium-mediated gene transfer.
- Particle gun/biolistic/ballistic method of DNA delivery.
- Chemical method; Polyethylene glycol (PEG)/protoplast fusion.
- Laser-induced DNA delivery.
Agrobacterium tumefaciens mediated gene transfer
- Agrobacterium tumefaciens is a natural genetic engineer.
- It is achieved in two ways:
- Co-culture with tissue explants.
- In-plant transformation.
Agrobacterium tumefaciens Characteristics
- Soil-borne, gram – ve, motile, rod-shaped bacteria that are present in the rhizosphere.
- Encodes by a tumor-inducing plasmid that is a large (250kbp) plasmid, which is a vector that transfer T-DNA region into the genome of plants host.
- Causes Crown gall disease.
- Have able to transferred bacterial genes into the plant genome.
- Attracted to wound site via chemotaxis in response to chemicals (Sugar and Phenolic molecules: like Acetosyringone) released from damaged plant cells.
Vectors, the DNA carriers must have:
- Origin of replication.
- Antibiotic-resistant genes.
- Give permission to host to grow on selective media.
- Increase the volume of a vector in the host cell.
- Multiple cloning sites.
- Allow insertion of foreign DNA.
- Contains one or more T-DNA region that is integrated into the plant host genome of size~12to24kb.
- Right and left border sequence(24bp) which will be transferred into the host plant genome.
- Contain a virregion~40kb, at least 8~11virgenes.
Process of T-DNA transfer and integration
1. Signal recognition by Agrobacterium:
- Agrobacterium perceives signals such as sugar and phenolic compounds(Acetosyrinzon) which are released from plants when got wounded.
2. Attachment to cells of the plant
- Have two processes:
- Initial attachment via polysaccharide.
- Bacteria produce cellulose fiber mesh.
- Vir genes involved in the attachment of bacterial cells to plant cells and this attachment is stable.
3. Induction of Vir gene
- Vir-A senses are phenolic and phosphorylating due to activated VirG.
- Vir genes expression are induces by VirG.
4. T-strand production
- The VirD1 (having topoisomerase activity) that is bound to RB.
- After binding it relaxes the supercoiling that induce the action of VirD2 (as endonuclease activity) and binds to 5’ end.
- Similarly, at the site of nick 3’ end produced and serves as a primer.
- As a result, virE is displaced by a single strand of T-DNA.
- The T-DNA is again nicked at LB to generate an ssT-DNA copy.
- The protection of VirE2 is done by nucleases.
- VirB is necessary for virulence which has ATPase activity.
- It also helps in T_DNA delivery into the plant cell through a nuclear pore complex.
5. Transfer of Vir proteins and T-DNA into the plant nuclear localization
- The single-stranded T-DNA is converted to double-stranded by replication in the nucleus.
- The dsT- DNA integrates from multiple sites into the host genome by unauthorized recombination.
- Practical application of Agrobacterium-mediated plant transformation
- Agrobacterium-mediated transformation is considering to induce less re-arrangement of the transgene.
1. Particle gun method
- A biolistic particle or gene gun system are designed for the transformation of the plant.
- It is a device that injects cells with genetic information.
2. Polyethylene (glycol mediated transformation) the chemical method
- The transformation of plant protoplast done with naked DNA through treatment with PEG, the treatment also takes place in the presence of divalent cations.
- The divalent cations and PEG de-stabilize the plant protoplast plasma membrane and provide it permeable to naked DNA.
- DNA then enters into the nucleus and integrates into the genome of the host.
- Protoplast are isolated and transformed into a number of plant species.
- Regeneration of fertile plants from protoplasts is problematic for some species because of secondary metabolites accumulation or any compounds.
- The transformation of DNA is susceptible to rearrangement and degradation.
- The delivery of DNA into the plant cells and protoplasts is done through electroporation.
- Plant regulatory sequence is necessary for the gene of interest.
- After that incubate the plant material in a buffer solution that contains DNA and subjected to high-voltage electric current.
- The DNA than transfer through high voltage and induced opening in the plasma membrane and enter into the plant genome.
- It may be used to transform all the major cereals particularly rice, wheat, maize.
- Both intact cells and tissue can be transformed.
- The efficiency of transformation depends upon the plant materials, electroporation and tissue treatment conditions used for transformation.
- Only 40 to 50% of incubated cells receive DNA.
- Around 50% of the transformed cells can survive.
4. Laser-induced DNA delivery
- LASERs produce holes that are transient in the cell membrane in which DNA enters into the cell cytoplasm.
- But, no information about gene expression and stable integration.
- The cereal crops that are transformed are widely used which is difficult to transform with agrobacterium.
- They have a tendency to guide better frequency of transgene rearrangement and better copy range.
- This can result in a high frequency of gene silencing.
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