“UNIVERSITY OF THE PUNJAB”

INSTITUTE OF BIOCHEMISTRY AND BIOTECHNOLOGY

Submitted by: Ayesha, Mehwish Rafiq, Aiman Abdul Sittar

Roll: no: 27, 17, 18
Major: BS. Biochemistry
Semester: 6th
Course: Plant Biochemistry
Submitted to: Dr. Mahjbeen Saleem

TOPIC OF ASSIGNMENT

PLANT TRANSFORMATION FACTORS

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TABLE OF CONTENTS:

Transformation:.....................................................................................................................................3
Introduction:..........................................................................................................................................3
Production of transgenic plants:.............................................................................................................3
1. Isolate and clone the gene of interest:............................................................................................4
2. DNA Insertion into the plasmid:.....................................................................................................4
3. Insertion of the plasmid into the bacteria:......................................................................................4
4. The flowering plant is dipped in a large number of bacteria:..........................................................4
5. DNA inserted into the plant cells:...................................................................................................4
Agrobacterium Mediated Plant Transformation:....................................................................................5
Factors affecting Plant Transformation:.................................................................................................5
I. Host Factors Involved in Plant Transformation:..........................................................................5
II. Vir Inducers:................................................................................................................................6
III. Bacterial Strains:.....................................................................................................................6
IV. Co-cultivation Factors:............................................................................................................6
Methods of Plant Transformation:.........................................................................................................7
1) Indirect Method:.........................................................................................................................7
2) Direct Method:...........................................................................................................................7
Transformation Vector Requirements:...................................................................................................8
Advantages............................................................................................................................................8
Disadvantages........................................................................................................................................9
References:............................................................................................................................................9

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 PLANT TRANSFORMATION FACTORS

Transformation:
When we incorporate or express the foreign DNA (exogenous DNA) into the
specific cell, there occur genetic changes in the cell this is called the term
transformation. Similarly, when we express DNA in the plant genome called
plant transformation.

Introduction:
The process of Genetic modification has been widely used in plants, yeast, and
mammalian cells for the betterment of certain traits, production of certain
nutrients and to develop disease resistance. In plants, scientists and
researchers use plant transformation techniques to see the influence of
certain genes, and also the concern about the improvement of a gene. Plants
often suffer from many stresses such as high salt level or drought etc. so the
main aim of plant transformation is to improve stress tolerance. Also, it gives
us desire traits. Because of this technology, breeding of plants occurs and we
get the crops and plant product with genetic diversity with the desirable
characteristics. We can also remove the undesirable characters from the
plants with the help of conventional breeding so it is used to create diverse
genotypes. This technology depends upon the method of delivery, expression,
and incorporation of foreign DNA in a plant cell. We have to need the
regulatory sequence for the successful incorporation of the desired gene.
Those regulatory sequences are a promoter, gene of interest, and terminator.

Production of transgenic plants:

There are several chemical, biological and physical methods for the transfer of
gene plants and the production of transgenic plants. In the case of the
biological method, agrobacterium is more influenced by nature’s plant genetic
engineer. Agrobacterium is one of the main two methods for delivery of genes
in the case of stable transformation. Agrobacterium is a naturally occurring

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soil bacterium. It is the most widely used bacteria for transformation because
of its ability to transfer its DNA in the plant cell. This bacterium is used
because it can cope with whole-plant tissue and easy to handle and use.
Following are the steps for the production of transgenic plants;

1. Isolate and clone the gene of interest:

First of all the gene of interest is isolated from the normal plant that has the
desire characteristic gene.

2. DNA Insertion into the plasmid:

The DNA containing a gene of desire trait is inserted into the plasmid

3. Insertion of the plasmid into the bacteria:

The plasmid is inserted into the bacteria which scientists or researchers used
for producing transgenic plant mostly used bacteria is agrobacterium. Then
provide a culture medium to the bacteria so that bacteria having plasmid that
containing gene of interest grow in large number. For insertion, suitable
markers are used.

4. The flowering plant is dipped in a large number of bacteria:

For the production of transgenic plants it is necessary to dip them and wait
until plants are flowering.

5. DNA inserted into the plant cells:

Naturally, agrobacterium can insert new DNA into the plant genome.
Agrobacterium inserts DNA in many types of cell including those cells that are
involved in forming seeds so when these seeds with inserted DNA are grown
produce plant having desirable characteristics. Then regenerate the whole
plant.

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Agrobacterium Mediated Plant Transformation:
Agrobacterium is a soil-dwelling bacterium that affects the plant cells in the
roots of plants. This bacterium infects plants through plant wounds.
Agrobacterium-mediated plant transformation refers to the regeneration of
transgenic plants by altering the plant chromosome through the plasmid of
agrobacterium.

Agrobacterium tumefaciens comprises a bacterial chromosome and an

additional Ti-plasmid. The Ti-plasmid has a stretched region of DNA that is
known as T-DNA. The remaining region of the plasmid contains various Vir
genes which direct the bacteria to cause infection in plants. When
agrobacterium infects plants, it sends signals to plasmid and these signals
direct the plants to activate Vir genes. The activation of these genes directs a
series of events to transfer the T-DNA from Ti-plasmid into the plant’s
chromosome.

 Agrobacterium-mediated plant transformation is done by cleaving the

T-DNA from Ti-plasmid at restriction sites.
 The T-DNA is removed from the T-plasmid and desired gene of interest
is inserted by restriction enzyme in its place.
 Recombinant Ti plasmid is formed when DNA ligase joins the gene of
interest with the plasmid.
 The recombinant Ti plasmid is introduced into the plant cells through
culture media.
 The introduction of the recombinant plasmid into plant cells causes the
alteration of the plant’s DNA by entering into the plant chromosome.
 The plant regenerates with new desired traits, thus we called it
transgenic plants, and the process is referred to as agrobacterium-
mediated plant transformation.

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 Factors affecting Plant Transformation:
I. Host Factors Involved in Plant Transformation:
The insertion of a certain vector into the plant genome has serious effects on
plant growth. The Ti-plasmid of agrobacteria infects the plants due to Vir
genes. But the certain types of proteins of the bacterium are essential for
plants to develop the new desired traits. So, the selection of hosts is a major
factor that contributes a lot to plant transformation.

II. Vir Inducers:

Specific destructive effector proteins move from the phone divider and the
plasma layer of Agrobacterium into the host plant cells. Agrobacterium has a
few sensors that perceive signals discharged by the host tissue and destruct in
light of these signs. At first, acetosyringone was recognized as one of the plant
cell exudates that appeared to go about as a Vir inducer with changing
efficiencies relying upon plant species. For example, Trycirtis hirta is a
decorative plant in which change recurrences expanded when acetosyringone
was added to the co-development medium.

III. Bacterial Strains:

The kind of strain utilized can influence change recurrence. In Iris Germania,
LBA4404 gave astoundingly higher change rates than EHA105. In Agapanthus
praecox, a similar LBA4404 was discovered to be more compelling than
EHA101. The action of LBA4404 is credited to the very parallel vector
pTOK233, which has VirG, VirB, and VirC qualities got from the Ti-plasmid.

IV. Co-cultivation Factors:

Numerous geophytes are monocotyledonous plants that are non-hosts of A.
tumefaciens. Monocotyledonous cells deliver troublesome phenolic
compounds in light of injuring. Nonetheless, Danilova et al. tracked down that
a concentrate of sterile tobacco leaves and stems expanded maize change
more adequately than acetosyringone. The stimulatory impacts of tobacco are
due to its wide scope of ideal phenolic mixtures, sugars, and amino acids
which prompt Vir qualities answerable for T-DNA move. Tobacco concentrate
is helpful in the change of ornamentals.

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The co-development period likewise achieves pros and cons about alterations
of a given plant. This period should be pre-resolved to keep away from the
excessive inconsistency of change or Agrobacterium excess because of
delayed co-development time. A co-development time of 2–3 days gave the
best outcomes in Agapanthus praecox, while in Typha latifolia, a multiday co-
development brought about the most elevated level of GUS articulation. Since
T-DNA move from Agrobacterium into the plant genome during the S-period
of the phone cycle, it is fundamental to build up ideal co-culture states of
explants and the Agrobacterium at the earliest reference point of the
hereditary change convention.

Methods of Plant Transformation:

1. Indirect Method
2. Direct

1) Indirect Method:
 Agrobacterium mediated gene transfer
 Rhizogenic Virus mediated method
 Tumefaciens

2) Direct Method:
Direct Physical Method
 Pressure
 Ballistics gene
 Gun Bombardment
 Particle bombardment
 Microinjection
 SAT
 Silica or carbon fibers
 Electroporation
 Microinjection

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 Direct Chemical Method:
 PEG
 DEAD dextran
 Dendrimers
 Calcium phosphate
 Artificial lipids
 Proteins

Transformation Vector Requirements:

Transformation vector is DNA. Vector should have following
characteristics:

 Multiple Cloning sites

 Increase the volume of a vector in the host cell
 Origin of replication
 Allow insertion of foreign DNA
 Antibiotic resistant genes
 Bacterial selectable marker
 Compatible with helper plasmid if Agrobacterium is used.
 T DNA borders and other Agrobacterium gene if Agrobacterium is used.

Advantages
 Agrobacterium mediated transformation is natural mean of transfer and
considered as most acceptable.
 By this transformation plant can be regenerated more rapidly.
 Without any subsequent arrangements agrobacterium cell mediated
transformation method has ability of transferring large fragment of DNA
more effectively.
 In the precise process named as integration of T-DNA , it serve as
insertional mutagenesis vehicle.
 As agrobacterium is used for stable transformation, the stability of gene
transfer is excellent.

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Disadvantages
 It has limitation of host range.
 Complex and time consuming
 Most of the important food crops is not infected by this technology
 Mostly the problem occurs is that the cell that are capable of
regenerating the whole plant face difficulty to transform, the reason is
that it may have layers too deep so that agrobacterium not reached
there.
 Sterile conditions are required foe regeneration
 Dependent to plant genotype
 Vulnerable to damage from vacuum treatment
 Low seed production with less efficiency of floral dip

References:
1. https://www.jic.ac.uk/blog/what-is-plant-transformation/
2. https://www.sciencedirect.com/science/article/pii/
S0254629915000228
3. https://www.sciencedirect.com/science/article/abs/pii/
S1878818113001175
4. Aitken CJ, Kozai T, Smith MAL. 1995. Automation and Environmental
Control in Plant Tissue Culture. Dordrecht: Kluwer
5. Albert H, Dale EC, Lee E, Ow DW. 1995. Site-specific integration of DNA
into wildtype and mutant lox sites placed in the plant genome. Plant J.
7:649–59
6. An GH, Mitra A, Choi HK, Costa MA, AnKS, et al. 1989. Functional
analysis of the 3′ control region of the potato wound-in- ducible
proteinase inhibitor II gene. Plant Cell 1:115–22
7. Ardley J, Hoptroff CGM. 1996. Protecting plant ‘invention’: the role of
plant variety rights and patents. Trends Biotechnol. 14: 67–69
8. Baker BF. 1993. The 5′ cap of mRNA: biosynthesis, function and
structure as related to antisense drugs. In Antisense Re- search and
Applications, ed. ST Crooke, B Lebleu, pp. 37–53. Boca Raton, FL: CRC
Press
9. Barks AH. 1994. Patent information in bio technology. Trends
Biotechnol. 12:352– 64