What is Plant transformation?
- Genetic transformation: The term genetic transformation is defined as the directed desirable transfer of gene from one organism to another along with the subsequent stable integration and expression of a foreign gene in the genome.
- The gene that is transferred is termed as ‘transgene’ and the organisms that are formed after a successful gene transfer are termed as ‘transgenics’.
- Plant transformation: It is defined as the method of insertion of the DNA from other organism, usually a plant into the genome of the plant of interest.
Why do we use plant transformation technology?
- To develop variety that are resistance to pests:
- It is now possible to introduce foreign genes that impart resistance to insects into the plant genome with the advent of genetic engineering techniques based on recombinant DNA technology.
- Combined with plant tissue culture, recombinant DNA technology has helped to develop innovative methods for the economic management of various types of biotic stresses, including insect pests.
- In reducing the losses incurred by biotic stresses, including insect pests, such innovations will be of tremendous benefit.
- Transgenic plants that display insecticidal Bt proteins alone or in combination with herbicide resistance proteins are revolutionizing agriculture.
- For Quality enhancement of the plant variety:
- In improving plant productivity and improving the quality of plant products, plant transformations/transgenics have tremendous utility.
- Improvements may be related to improving the nutritional value of the plant or to improving the functional properties of the production or consumption process.
- In order to block the development of certain metabolites, transgenic plants can be used by regulating the over-expression or inhibition (antisense expression) of some of the essential enzymes as shown.
What are the biological requirements for plant transformation?
- For the gene transfer to produce transgenic plant, the biological requirements are listed below:
- There must be a target tissue that consists of competent cells for plant regeneration.
- A technique for the introduction of DNA into these regenerable cells.
- A procedure for the selection and regeneration of transformed plants at an optimum frequency.
What are the physical requirements for plant transformation?
- Highly efficient, economic, reproducible, and should readily produce several transformants for testing.
- Should be safe for operators.
- Should be simple technically including a minimum of demanding or inherently variable manipulations, like protoplast production and regeneration.
- Tissue culture should be completed in minimum time as to reduce the related costs and to avoid unnecessary somaclonal variation.
- Stable, if vegetatively propagated then uniform transformants and if sexually propagated species then, fertile germline transformants.
- Integration patterns should be simple and the introduced genes should have low copy number, so as to reduce the probability of undesired gene disruption at insertion sites.
Plant transformation methods:
| Direct method of gene transfer (Non-biological based transformation) | Indirect method of gene transfer |
| Physical Microinjection Biolistics-gene gun/particle bombardment Electroporation Silica/carbon fibers Laser mediated SAT Chemical PEGDEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers | Biological Agrobacterium mediated Basically done by two methods: Co-cultivation with the explants tissue In planta transformation Virus mediated |
Physical gene transfer methods:
- Electroporation:
- In this method, electric pulse of high field strength is used inorder to form pores in the cell membrane.
- If DNA is present at an appropriate concentration in the buffer solution, it will be taken up via these pores.
- Plant materials is incubated in a buffer solution that contains DNA and exposed to high voltage electric pulse.
- Plant materials is incubated in a buffer solution that contains DNA and exposed to intense electric pulse.
- Plant materials is incubated in a buffer solution that contains DNA and exposed to intense electric pulse.
- Advantages:
- It is possible to transform both intact cells and tissue.
- The transformation efficiency depends on the materials of the plant.
- Disadvantages:
- DNA is obtained by 40 to 50 percent of incubated cells.
- Near to 50 percent of the cells transformed will survive.
- Biolistic gene gun / particle bombardment:
- It is also termed as microprojectile bombardment.
- To deliver DNA into cells, foreign DNA is coated with high-velocity gold or tungsten particles.
- This method is popularly being used for its ability to transfer foreign DNA into the mammalian cells and microorganisms.
- Advantages:
- All plant species can be transformed using this process.
- All plant species can be transformed using this process.
- The protocol for transformation is relatively simple.
- Disadvantages:
- Difficulty in acquiring single copy transgenic events.
- High costs for microcarriers and supplies.
- Intracellular target is irregular or random (cytoplasm, vacuole, nucleus, plasmid etc.)
- Transfer DNA is not protected.
- Microinjection method:
- A direct physical approach involving the mechanical introduction of the desirable DNA into a target cell is microinjection.
- The microinjection technique involves transferring the gene into the cytoplasm or nucleus of a plant cell or protoplast through a micropipette.
- The most important application of this is the introduction of DNA into animal oocytes and embryos, either in the study of transient expression or in the generation of transgenic animals.
- The key disadvantages of microinjection are that it is long, costly and needs to be carried out by trained and certified workers.
- Advantages:
- This method does not need protoplast.
- The device is easy and inexpensive.
- Methods may be helpful for the transfer of genes into cereals that do not easily regenerate from cultured cells.
- Technically straightforward.
- Limitations:
- Less specific
- Less efficient
- The transformation frequency is low.
- Liposome mediated transformation:
- Liposome-mediated transformation includes liposome adhesion to the surface of the protoplast, its fusion at the attachment site, and the release of plasmids inside the cell.
- Liposomes are lipid spheres that are used to bring molecules into cells.
- These are artificial vesicles that can behave as delivery vehicles for exogenous materials including transgenes.
- They are regarded as sphere of lipid bilayers encircling the molecule to be transported and encourage transport after fusing with the cell membrane.
- Cationic lipids are those with a positive charge that are used for nucleic acid transfer.
- Liposomes can interact more readily with the negatively charged cell membrane than uncharged liposomes.
- Fusion between the cationic liposome and the cell surface results in the transmission of DNA directly through the plasma membrane.
- Advantages:
- High reproducibility degree.
- Stability for the long term.
- Low toxicity level.
- Nucleic acid protection from degradation
Chemical mediated gene transfer methods:
- PEG mediated gene transfer:
- Polyethylene glycol (PEG) disrupts the plasma membrane of protoplasts in the presence of divalent cations (using Ca2+) and makes it permeable to naked DNA.
- Polyethylene glycol (PEG) disrupts the plasma membrane of protoplasts in the presence of divalent cations (using Ca2+) and makes it permeable to naked DNA.
- The protoplastic culture is taken into a tube and 40 percent PEG 4000 (w/v) dissolved in mannitol is added to this tube and calcium nitrate is added slowly and incubated for a few minutes.
- Advantages:
- A large number of protoplasts can be transformed concurrently.
- A wide variety of plant species can be used successfully.
- Limitations:
- The DNA is prone to rearrangement and degradation.
- Random integration of foreign DNA into genome may lead to unfavorable characteristics.
- A challenging task is the regeneration of plants from converted protoplasts.
- Calcium phosphate co-precipitation:
- The DNA is permitted to mix with calcium chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate.
- The cells are transformed when the actively dividing cells in the culture are exposed to this precipitate for several hours.
- The efficacy of this technique depends on the high DNA concentration and the protection of the complex precipitate.
- The addition of dimethyl sulfoxide (DMSO) enhances transformation efficiency.
- DEAE-dextran mediated transfer:
- With a high molecular weight polymer diethyl amino ethyl(DEAE)dextran, the desirable DNA can be complexed and transferred.
- This approach’s main drawback is that it does not generate stable transformants.
Biological method of gene transfer:
Agrobacterium-mediated gene transfer in Plants:
- Agrobacterium tumefaciens is a widely occurring soil bacterium that is responsible for causing crown gall, and has the capability to integrate new genetic material into the plant cell.
- T DNA is a term given for the genetic material that is situated on a Ti plasmid.
- A Ti plasmid is a common circular fragment of DNA found in almost all bacteria.
- Agrobacterium-mediated transformation is highly efficient and hence is most usually used method for plant genetic engineering.
- In the course of transformation, various components of Ti plasmid plays a role in effective transfer of the gene of interest into the plant cells. They are:
- T-DNA border sequences that are responsible for demarcating the T-DNA to be transferred to the plant genome.
- vir genes that are necessary for the transfer of T-DNA region to the plant but they cannot be transferred by themselves.
- Modified T-DNA region where the genes that are responsible for the formation of crown gall are eliminated and replaced with genes of interest.
Steps for Agrobacterium-mediated Plant Transformation Process:
- The Agrobacterium-mediated transformation process comprises of several steps. They are:
- Isolation of the gene of interest from the source organism.
- Development of a functional transgenic hybrid involving the gene of interest, promoters to drive expression; codon modification, if required to enhance successful protein production; and marker genes to favor tracking of the introduced genes in the host plant.
- Insertion of the transgene into the Ti-plasmid.
- Integration of the T-DNA-containing-plasmid into Agrobacterium.
- The transformed Agrobacterium is mixed with plant cells to permit transfer of T-DNA into plant chromosome.
- Regeneration of the new plantlets that are genetically modified.
- Examination for characteristic performance or expression of transgene at lab, greenhouse and field level.