This document discusses methods for plant genetic engineering and transformation. It describes three main methods: electroporation, biolistics/particle bombardment, and Agrobacterium-mediated transformation. Agrobacterium transformation uses Agrobacterium tumefaciens bacteria to insert foreign DNA into plant cells. It involves removing crown gall genes from the Ti plasmid and replacing them with genes of interest. The process then involves inserting the modified Ti plasmid into Agrobacterium, mixing it with plant cells, and regenerating genetically modified plantlets.

1. Plant Genetic Engineering delivery System
M.PHOOL BADSHAH
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2. 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 ‘transgenic’.
• 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.

3. Plant transformation methods
Direct method of gene transfer (Non-biological based
transformation)
Indirect method of gene transfer
Chemical
Calcium phosphate
Artificial lipids
Proteins
Dendrimers
Physical
Microinjection
Biolistics-gene gun/particle bombardment
Electroporation
Silica/carbon fibers
Laser mediated
Biological(biologicalbasedtransformation)
Agrobacterium mediated Basically
done by two methods: Co-cultivation
with the explants tissue
In plant transformation
Virus mediated

4. 1-Electroporation Method
• In this method, electric pulse of high field strength is used in
order 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.

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• Advantages:
i. It is possible to transform both intact cells and tissue.
ii. The transformation efficiency depends on the materials of
the plant.
• Disadvantages:
i. DNA is obtained by 40 to 50 percent of incubated cells.
ii. Near to 50 percent of the cells transformed will survive.

6. 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.

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• Advantages:
i. All plant species can be transformed using this process.
ii. The protocol for transformation is relatively simple.
• Disadvantages:
i. Difficulty in acquiring single copy transgenic events.
ii. High costs for micro carriers and supplies.
iii. Intracellular target is irregular or random (cytoplasm, vacuole,
nucleus, plasmid etc.)
iv. Transfer DNA is not protected.

8. 3-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.

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• 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.

10. 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.

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• 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.