• 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?

1. 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.
2. 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:

Physical gene transfer methods:

1. 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.
2. 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.
3. 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.
4. 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:

1. 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.
2. Calcium phosphate co-precipitation:
   1. The DNA is permitted to mix with calcium chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate.
   2. The cells are transformed when the actively dividing cells in the culture are exposed to this precipitate for several hours.
   3. The efficacy of this technique depends on the high DNA concentration and the protection of the complex precipitate.
   4. The addition of dimethyl sulfoxide (DMSO) enhances transformation efficiency.
3. DEAE-dextran mediated transfer:
   1. With a high molecular weight polymer diethyl amino ethyl(DEAE)dextran, the desirable DNA can be complexed and transferred.
   2. 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. 

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