Plant Transformation Methods

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.

History

• 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

Indirect method:

Agrobacterium-mediated gene transfer.

Direct methods:

• Particle gun/biolistic/ballistic method of DNA delivery.
• Chemical method; Polyethylene glycol (PEG)/protoplast fusion.
• Laser-induced DNA delivery.
• Electroporation

Image Sources: Gerhard Obermeyer and IntechOpen

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

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.

Ti plasmid

• 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.

Direct Methods

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.

Advantages

• Protoplast are isolated and transformed into a number of plant species.

Disadvantages

• 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.

3. Electroporation

•  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.

Advantages

• 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.

Disadvantages

• 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.

Advantage

• The cereal crops that are transformed are widely used which is difficult to transform with agrobacterium.

Disadvantage

• 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.

References

• https://www.ncbi.nlm.nih.gov/pubmed/12378268
• https://bioone.org/journals/the-arabidopsis-book/volume-2017/issue-15/tab.0186/Agrobacterium-Mediated-Plant-Transformation-Biology-and-Applications/10.1199/tab.0186.full
• https://www.intechopen.com/books/transgenic-plants-advances-and-limitations/methods-to-transfer-foreign-genes-to-plants.
• https://onlinelibrary.wiley.com/doi/abs/10.1002/0470869143.kc014
• http://nepad-abne.net/biotechnology/process-of-developing-genetically-modified-gm-crops/plant-transformation-using-particle-bombardment/
• https://www.ncbi.nlm.nih.gov/pubmed/15310913
• https://en.wikipedia.org/wiki/Electroporation
• https://link.springer.com/chapter/10.1007/978-1-4613-1159-1_10
• https://www.sciencedirect.com/science/article/pii/S0091679X08610432
• https://scialert.net/abstractmobile/?doi=biotech.2008.385.402
• https://www.slideshare.net/gnsk143/gene-transformation-methods

Internet Sources

• 4% – https://www.slideshare.net/gnsk143/gene-transformation-methods
• 2% – https://www.slideshare.net/sushantasarma/agrobacterium-mediated-gene-transfer-42137395
• 2% – https://chantikaturi.blogspot.com/2008/05/bio-technolgy-and-its-applications.html
• 1% – https://www.slideshare.net/purakichha/agrobacterium-mediated-gene-transfer-in-plantsdoc-for-wiziqcom
• 1% – https://www.researchgate.net/publication/257470932_Current_Status_of_Sugarcane_Transgenic_an_Overview
• 1% – https://www.powershow.com/view/407dc3-MTE0M/3b_Techniques_for_plant_transformation_powerpoint_ppt_presentation?varnishcache=1
• 1% – https://mafiadoc.com/naveen-kumar-arora-editor_599951711723dd96bd931735.html
• <1% – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824253/
• <1% – https://en.wikipedia.org/wiki/T-DNA
• <1% – https://en.wikibooks.org/wiki/Structural_Biochemistry/Nucleic_Acid/DNA/Supercoiling_and_Nucleosomes

Plant Transformation Methods