Introduction
Gene transfer/Uptake of DNA referred to as a transfer of a specific piece of DNA into a cell.
Genetic Transformation stands for the directed desirable gene transfer from one organism to another organism and after that the subsequent stable integration and expression of the foreign gene into the genome.
The transferred gene is known as transgene and the organism that develops after a successful gene transfer is known as transgenic plants.
Transgenic plants are the plants that carry the stable integrated foreign genes.
These plants may also be called transformed plants.
Benefits of Gene Transfer
Provide resistance against viruses.
Develop drought resistance plants.
Herbicide resistance plants can be created.
Acquire insecticidal resistance property.
To improve the nutritional quality of plants.
To make the plants such that they can grow in any season.
Delayed ripening can be done.
Steps In Transformation
Identification of useful genes :
Desirable genes located in wild species, unrelated plant species, unrelated organisms and animals.
Designing gene for insertion:
The gene of interest is isolated from the donor source and cloned in the laboratory. The cloning is done generally using plasmid.
Insertion of gene into target plant:
The cloned gene i.e multiple copies of the gene of interest are inserted into the host plant or the recipient plant.
Identification of transgenic cells:
Transformed cells are identified using selectable markers and are regenerated into whole plants in nutrient medium. Regenerate plant compared with plant variety. It should look like parent variety except the gene of interest.
Gene Transfer Methods in Plants
Electroporation
Electroporation is the process where the electrical impulses of high field strength are used to reversibly permeabilize cell membranes to facilitate uptake of large molecules, including DNA.
In this procedure, a sample of protoplasts is pulsed with high/low voltage pulses in the chamber of an electroporator. The electric pulse disrupts the phospholipid membrane and forms temporary pores making it permeable. The electric pulse also causes change in electric potential across the membrane allowing the charged molecule namely DNA or RNA to move across the membrane.
It uses relatively high initial field strength (1-1.5Kv) with a low capacitance and therefore a short decay time. It has been reported that linear DNA is transfected more efficiently than circular DNA.
How does Electroporation work?
Pulse Application: A brief, high-voltage electrical pulse is applied to the cells.
Membrane Disruption: The pulse creates temporary pores in the cell membrane.
Molecular Uptake: The pores allow molecules in the surrounding solution to enter the cell.
Membrane Resealing: Once the pulse is removed, the pores quickly close, and the cell membrane returns to its normal state.
Microprojectile
The technique of particle bombardment also known as biolistics, microprojectile bombardment, particle acceleration etc, is the most versatile and effective way for the creation of many transgenic organisms. The procedure in which high velocity microprojectiles were utilised to deliver nucleic acids into living cells was described by KLEIN and SANFORD in 1987.
The desired gene is coated on tungsten or gold particles (3 micrometres in diameter) called microprojectiles. These are accelerated with pressure on the plant cells or tissues.
The microprojectile is stopped by a perforated plate while allowing the microprojectile to pass through it. When microprojectiles enter the plant cells, the desired genes are released from microprojectiles and may get inserted into the plant chromosomal DNA and get transformed.
DNA markers are used to distinguish the transformed and non-transformed cells. The transformed plant cells with desired characteristics are further developed to whole plants.
Microinjection
It is the direct mechanical method for gene transfer in the target. A target can be a defined cell within a multicellular structure such as embryo, ovule, and meristem or protoplasts, cells or a defined compartment of a single cell. Microinjection is able to penetrate intact cell walls. Glass micropipettes with 0.5-10.0 micrometre diameter tips (needle) are used to transfer the micromolecules into the cytoplasm or the nucleus of a recipient cell or protoplast. Once injection has been achieved the injected cells must be cultured properly to ensure its continued growth and development.
Microinjection is a technique that uses a fine glass needle (micropipette) to inject a liquid substance at a microscopic level. The target is often a living cell, but it can also be intercellular space.
How does Microinjection work?
Micropipette Preparation: A fine glass needle is pulled to a very sharp tip using a micropipette puller.
Sample Loading: The substance to be injected is loaded into the micropipette.
Cell Positioning: The cell to be injected is positioned under a microscope.
Membrane Penetration: The micropipette is carefully inserted into the cell membrane.
Substance Injection: The desired substance is injected into the cell.
Micropipette Withdrawal: The micropipette is slowly withdrawn from the cell.
Liposome Mediated Gene Transfer
Liposomes are synthetically created lipid vesicles enclosed by a phospholipid membrane.
They exhibit amphipathic properties, possessing both hydrophilic (water-loving) and hydrophobic (water-repelling) components.
This fluid-like nature allows them to fuse with protoplasts when induced by polyethylene glycol (PEG). This property has been harnessed for gene transfer.
In this method, DNA is introduced into protoplasts through a process involving liposome endocytosis. The steps include:
Adhesion: Liposomes attach to the protoplast surface.
Fusion: Liposomes merge with the protoplast membrane at the adhesion site.
Plasmid Release: The plasmids encapsulated within the liposomes are released into the cell.
Indirect or Vector mediated Gene transfer
For Transfer of DNA two bacterias are used
Agarobacterium Tumefaciens
Agarobacterium rhizogenes
Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil-borne, Gram-negative bacteria.
These are phytopathogens (that cause infection in plants) and are treated as nature's most effective plant genetic engineer.
A. tumefaciens induces crown gall disease and A. rhizogenes that induces hairy root disease in plants.
Crown Gall Disease
Crown gall formation occurs when the bacterium releases its Ti plasmid (Tumour- inducing plasmid) into the plant cell cytoplasm.
A fragment of Ti plasmid, referred to as T-DNA, is actually transferred from the bacterium into the host where it gets integrated into the plant cell chromosome (i.e. host genome). Thus, crown gall disease is a naturally evolved genetic engineering process.
The T-DNA carries genes that code for proteins involved in the biosynthesis of growth hormones (auxin and cytokinin) and novel plant metabolites namely opines-amino acid derivatives and agropines-sugar derivatives.
Structure of Ti plasmid
Ti plasmids are large, approximately 200 kb in size.
They are circular DNA molecules that replicate independently within Agrobacterium cells.
T-DNA (Transferred DNA)
T-DNA is a specific DNA segment within Ti plasmids that gets transferred to plant cells.
T-DNA length varies between 12 to 24 kb, depending on the bacterial strain.
Types of Ti Plasmids
Nopaline Strain
Contains one T-DNA region.
T-DNA length in this strain is about 20 kb.
Octopine Strain
Contains two distinct T-DNA regions, named TL and TR.
TL region: 14 kb in length.
TR region: 7 kb in length.
Ti Plasmid Size
It is a large plasmid, approximately 200 kb in size, known as a megaplasmid.
T-DNA (Transferred DNA) Region
The T-DNA region is about 15-40 kb in length.
It contains genes for the synthesis of plant hormones Auxins and Cytokinins, which promote disease symptoms in plant tissue.
It also has genes for Opine production, produced by infected cells, which are unusual amino acids used by Agrobacterium as nutrients.
T-DNA Border Sequences
T-DNA is flanked by 25 base pair (bp) repeat sequences on both sides.
These repeats help facilitate the transfer of T-DNA into the plant genome.
Virulence (Vir) Region
This region consists of around 8 operons with approximately 24-25 genes.
The Vir genes aid in the transfer process of T-DNA into the plant cells.
Host Specificity Region
Contains genes for conjugative transfer (movement between bacteria) and opine catabolism (breakdown of opines).
Origin of Replication
Ti plasmid also includes an origin of replication which allows it to replicate independently within Agrobacterium.
The Ti plasmid has three important regions
T DNA Region
Genes in T-DNA
The T-DNA region contains genes for the biosynthesis of:
Auxins (aux)
Cytokinins (cyt)
Opines (ocs)
These genes are referred to as oncogenes as they determine the tumour phenotype in plants.
T-DNA Borders
The T-DNA is flanked by left and right borders, which are 24 kb sequences.
Both borders are transferred to plant cells, but the right border is more critical for T-DNA transfer and tumour formation.
T-DNA Size and Structure
The T-DNA is a 23 kb segment (varies between 15-40 kb).
It is bordered by 25 bp direct repeat sequences on both sides, which aid in its transfer to plant cells.
Genes for Tumour Induction
T-DNA contains genes such as:
IAAM and IAAH: For auxin production.
IPT: For cytokinin production.
These genes lead to tumour formation by producing auxins, cytokinins, and opines.
Regulatory Sequences
All T-DNA genes have eukaryotic regulatory sequences and are expressed only in plant cells.
Virulence (Vir) Region
Location:
Found outside the T-DNA region.
Function:
Contains genes responsible for transferring T-DNA into the host plant.
Components:
At least nine vir-gene operons have been identified:
Vir A, Vir G,
Vir B1,
Vir C1,
Vir D1, D2, D4,
Vir E1, and Vir E2.
Opines Catabolism Region
Function:
Codes for proteins involved in the uptake and metabolism of opines, which are nutrient sources for Agrobacterium.
Origin of Replication (ori) Region
Function: This region contains the origin of DNA replication.
Importance: It allows the Ti plasmid to replicate and be stably maintained in Agrobacterium tumefaciens.
Mechanisms Of Transfer of T DNA
Wounding of Plant Tissue
Wounded dicot plant tissue releases phenolic compounds (e.g., Acetosyringone and o-Hydroxyacetosyringone).
Activation of Vir A Protein
Acetosyringone binds with Vir A protein on the inner membrane of Agrobacterium.
Vir A is activated and begins to function as an autokinase.
Phosphorylation of Vir A
Vir A phosphorylates itself using ATP.
Phosphorylation of Vir G Protein
Phosphorylated Vir A transfers the phosphate to Vir G protein.
Vir G becomes active, dimerizes, and gains DNA-binding ability.
Induction of Other Vir Operons
Phosphorylated Vir G binds to DNA, initiating the expression of other Vir operons needed for T-DNA transfer.
Action of Vir D1 and Vir D2 Proteins
Vir D1 protein binds to the right border of T-DNA.
Functions as a topoisomerase and endonuclease.
Vir D2 protein nicks the right border of T-DNA.
Function as an endonuclease.
Remains bound to the 5' end of T-DNA, aiding in T-DNA transfer.
It shows the path.
DNA Synthesis and Strand Displacement
The 3' end of the nick at the right border acts as a primer.
DNA synthesis occurs in the 5' to 3' direction.
This process displaces one strand of T-DNA from the DNA duplex.
Nicking at the Left Border
The displaced T-DNA strand is nicked at the left border.
A single-strand copy of the T-DNA is produced.
Protection of Single-Stranded T-DNA
Vir E2 protein binds to the single-strand T-DNA.
Vir E2 protects the T-DNA from exonucleases.
Helps in crossing the cell wall.
Conjugal Tube Formation
Vir B operon encodes membrane-bound Vir B proteins (11 genes).
Vir B proteins, along with Vir D4 proteins, form a conjugal tube.
The conjugal tube facilitates T-DNA transfer between Agrobacterium and plant cells.
Transport into the Plant Cell Nucleus
Vir D2 protein, bound to the 5' end of the T-DNA, has a signal sequence.
This signal sequence directs T-DNA into the plant cell nucleus.
T-DNA integrates into the plant genome.
Integration of T-DNA into Plant Genome
T-DNA Entry and Conversion
T-DNA enters the plant cell as a single-stranded DNA (ssDNA).
Inside the plant cell, it is quickly converted into a double-stranded DNA (dsDNA).
Role of Vir E2 Protein
Vir E2 has a nuclear localization sequence that assists in transporting the T-DNA into the plant cell nucleus.
Integration into Plant Genome
The double-stranded T-DNA integrates into the host plant genome at random sites.
During the integration process, 23-79 base pairs are deleted at the integration or target site.
Expression of T-DNA Genes
Once integrated, the T-DNA genes for auxins, cytokinins, and opines are expressed.
This expression causes uncontrolled cell growth, resulting in the formation of a tumour (crown gall) on the plant.