Cloning Vectors For Higher Plants

Cloning Vectors
for Higher Plants

DR. DARSHAN MARJADI
SRKI, SURAT
Can be occur at two place
1. Nucleus
2. Chloroplast
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.
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.
First described as a method of gene transfer into plants John
Sanford at Cornell University in 1987.
The micro projectile bombardment method was initially named
as biolistics by its inventor Sanford (1988). Biolistics is a
combination of biological and ballistics. There are other names
for this technique- particle gun, gene gun, bio blaster.
This device is able to transform almost any type of cell, including
plants, and is not limited to genetic material of the nucleus: it can
also transform organelles, including plastids.
Advantages of particle bombardment:
i. Gene transfer can be efficiently done in organized

tissues.
ii. Different species of plants can be used to develop

transgenic plants.
Limitations of particle bombardment:
i. The major complication is the production of high

transgene copy number. This may result in instability
of transgene expression due to gene silencing.
ii. The target tissue may often get damaged due to

lack of control of bombardment velocity.
iii. Sometimes, undesirable chimeric plants may be

regenerated.
Microinjection:
Microinjection is a direct physical method involving the mechanical

insertion of the desirable DNA into a target cell. The target cell may be
the one identified from intact cells, protoplasts, callus, embryos,
meristems etc. Microinjection is used for the transfer of cellular
organelles and for the manipulation of chromosomes.
The technique of microinjection involves the transfer of the gene

through a micropipette (0.5-10.0 pm tip) into the cytoplasm/nucleus of
a plant cell or protoplast. While the gene transfer is done, the recipient
cells are kept immobilized in agarose embedding, and held by a suction
holding pipette.
As the process of microinjection is complete, the transformed cell is cultured
and grown to develop into a transgenic plant.
In fact, transgenic tobacco and Brassica napus have been developed by this
approach. The major limitations of microinjection are that it is slow,
expensive, and has to be performed by trained and skilled personnel.
Liposome-Mediated Transformation:
Liposomes are artificially created lipid vesicles containing a

phospholipid membrane. They are successfully used in
mammalian cells for the delivery of proteins, drugs etc.
Liposomes carrying genes can be employed to fuse with
protoplasts and transfer the genes.
The efficiency of transformation increases when the process is

carried out in conjunction with polyethylene glycol (PEG).
Liposome-mediated transformation involves adhesion of
liposomes to the protoplast surface, its fusion at the site of
attachment and release of plasmids inside the cell
Advantages of liposome fusion:
i. Being present in an encapsulated form of liposomes, DNA is protected from

environmental insults and damage.
ii. DNA is stable and can be stored for some time in liposomes prior to transfer.
iii. Applicable to a wide range of plant cells.
iv. There is good reproducibility in the technique.
Limitations of liposome fusion:
The major problem with liposome-mediated transformation is the difficulty

associated with the regeneration of plants from transformed protoplasts.
Silicon Carbide Fibre-Mediated Transformation:
The silicon carbide fibres (SCF) are about 0.3-0.6 pm in

diameter and 10-100 pm in length.
These fibres are capable of penetrating the cell wall and
plasma membrane, and thus can deliver DNA into the
cells.
The DNA coated silicon carbide fibres are vortexed with
‘plant material (suspension culture, calluses).
During the mixing, DNA adhering to the fibres enters the
cells and gets stably integrated with the host genome.
The silicon carbide fibres with the trade name Whiskers
are available in the market.
Advantages of SCF-mediated transformation:
i. Direct delivery of DNA into intact walled cells. This avoids the protoplast isolation.
ii. Procedure is simple and does not involve costly equipment.
Disadvantages of SCF-mediated transformation:
i. Silicon carbide fibres are carcinogenic and therefore have to be carefully handled.
ii. The embryonic plant cells are hard and compact and are resistant to SCF penetration.
In recent years, some improvements have been made in SCF-mediated transformation.

This has helped in the transformation of rice, wheat, maize and barley by using this
technique.
Chemicalbased Method:
1. Polyethylene glycol (PEG)-mediated transfer:
Polyethylene glycol (PEG), in the presence of divalent cations (using Ca2+), destabilizes
the plasma membrane of protoplasts and renders it permeable to naked DNA. In this
way, the DNA enters nucleus of the protoplasts and gets integrated with the genome.
The procedure involves the isolation of protoplasts and their suspension, addition of
plasmid DNA, followed by a slow addition of 40% PEG-4000 (w/v) dissolved in mannitol
and calcium nitrate solution.
As this mixture is incubated, protoplasts get transformed.
Advantages of PEG-mediated transformation:
i. A large number of protoplasts can be simultaneously transformed.
ii. This technique can be successfully used for a wide range of plant species.
Limitations of PEG-mediated transformation:
i. The DNA is susceptible for degradation and rearrangement.
ii. Random integration of foreign DNA into genome may result in undesirable traits.
iii. Regeneration of plants from transformed protoplasts is a difficult task
Calcium Phosphate Co- Precipitation-Mediated Transfer:
The DNA is allowed to mix with calcium chloride solution and isotonic
phosphate buffer to form DNA-calcium phosphate precipitate.
When the actively dividing cells in culture are exposed to this
precipitate for several hours, the cells get transformed.
The success of this method is dependent on the high concentration of
DNA and the protection of the complex precipitate.
Addition of dimethyl sulfoxide (DMSO) increases the efficiency of
transformation.

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Cloning Vectors

for Higher Plants

Both intact cells and tissue can be transformed.

Only 40 to 50% of incubated cells receive DNA.

i. Gene transfer can be efficiently done in organized

ii. Different species of plants can be used to develop

Limitations of particle bombardment:

i. The major complication is the production of high

ii. The target tissue may often get damaged due to

iii. Sometimes, undesirable chimeric plants may be

Microinjection is a direct physical method involving the mechanical

The technique of microinjection involves the transfer of the gene

Liposomes are artificially created lipid vesicles containing a

The efficiency of transformation increases when the process is

i. Being present in an encapsulated form of liposomes, DNA is protected from

iii. Applicable to a wide range of plant cells.

iv. There is good reproducibility in the technique.

Limitations of liposome fusion:

The major problem with liposome-mediated transformation is the difficulty

The silicon carbide fibres (SCF) are about 0.3-0.6 pm in

ii. Procedure is simple and does not involve costly equipment.

Disadvantages of SCF-mediated transformation:

In recent years, some improvements have been made in SCF-mediated transformation.

1. Polyethylene glycol (PEG)-mediated transfer:

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