This document provides an overview of genetic engineering techniques used to create transgenic plants, specifically focusing on Agrobacterium-mediated transformation. It discusses what transgenic plants are, genetic engineering methods like microprojectile bombardment and electroporation, and how Agrobacterium tumefaciens is used. A. tumefaciens causes crown gall disease by transferring T-DNA from its Ti plasmid into plant cells. The Ti plasmid and vir genes control this process. Researchers have developed Ti plasmid vectors to insert foreign genes between the T-DNA borders and transfer them to plant genomes, creating transgenic plants. Selectable marker genes like NPTII allow identification of transformed cells.

1. Genetic Engineering
Kuldeep Gauliya
Research Scholar,
Dept. of Biotechnology
DHSGU, Sagar, M.P.
Altering God’s Creation….
Lecture I – Introduction to Agrobacterium
Nov. 18, 2022

2. What are TRANSGENIC PLANTS ?
 “A transgenic plant is a modified organism where genes are
transferred from one organism to another through genetic
engineering techniques”.
 This may involve changing a single base pair (A-T or C-G),
deleting a region of DNA or adding a new segment of DNA
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3. What is Genetic Engineering ?
Genetic engineering (also called genetic modification) is a
process that uses laboratory-based technologies to alter the
DNA makeup of an organism.
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4. Methods of production of Transgenic plants
 Microprojectile bombardment: shooting DNA-coated
tungsten or gold particles into plant cells.
 Electroporation: use of a short burst of electricity to put
the DNA into cells.
 Agrobacterium tumefaciens-mediated transformation.
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5. What is Agrobacterium?
 Agrobacterium is a soil pathogenic bacterium. This
plant pathogen causes crown-gall disease or hairy
root disease in infected dicotyledonous plants.
 The galls or tumors are formed at the junction
between the root and the stem of infected plants.
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6. How does Crown Gall Disease looks like ?
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7. What is Agrobacterium-mediated
transformation ?
Agrobacterium-mediated transformation is a process of
using Agrobacterium to transfer a gene of interest into the
plant cells, generating transgenic plants.
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8. Why is Agrobacterium used to make transgenic plants?
• Agrobacterium is a useful tool bcz it can carry, transfer, and
integrate a gene of interest into the plant genome.
• In the development of transgenic plants, this system allows
plants to stably harbor and pass a particular gene of
interest to the next generations relatively quicker than by
using the more traditional plant breeding method.
• This method is relatively inexpensive and easy to perform. In
addition, it provides convenient way to screen and select the
transformed plant tissues.
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9. 1. The ability of A. tumefaciens to induce crown galls in
plants is controlled by genetic information carried on Ti
plasmid (tumor-inducing plasmid).
2. Ti plasmid has two components the T-DNA (Transferred
DNA) and
3. the vir region, which are essential for the transformation
of plant cells.
4. During the transformation process, the T-DNA is excised
from the Ti plasmid, transferred to a plant cell, and
integrated into the plant cell genome.
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How does Transformation occurs…?

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Fig. shows the Plant -Agrobacterium
Interaction.
It depicts how does the T-DNA
gets incorporated into the plant
cell that ultimately causes Crown
Gall Disease.

11. Structure of Ti Plasmid
1. T-DNA: In nopaline-type Ti plasmids the T-DNA is a 23,000-
nucleotide-pair segment.
2. It carries 13 known genes including genes encoding enzymes that
catalyze the synthesis of phytohormones (the auxin indoleacetic acid
and the cytokinin isopentenyl adenosine).
3. These phytohormones are responsible for the tumorous growth of
cells in crown galls.
4. The T-DNA region is bordered by 25-nucleotide-pair imperfect
repeats.
5. One of this must be present in cis for T-DNA excision and transfer.
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12. Fig. Structure of the nopaline Ti plasmid
pTi C58, showing ori, origin of replication.
1. Tum, genes responsible for tumor
formation;
2. Nos, genes involved in nopaline
biosynthesis;
3. Noc, genes involved in the catabolism of
nopaline;
4. vir, virulence genes required for T-DNA
transfer.
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13. Structure of Ti Plasmid: The vir (virulence) region
• It contains the genes required for the T-DNA transfer process.
• These genes encode the DNA processing enzymes required for excision,
transfer, and integration of the T-DNA segment.
• They are expressed at very low levels in A. tumefaciens cells growing
in soil.
• Exposure of the bacteria to wounded plant cells or exudates from plant
cells induces enhanced levels of expression of the vir genes.
• This induction process is very slow for bacteria, taking 10 to 15 hours to
reach maximum levels of expression.
• Phenolic compounds such as acetosyringone act as inducers of the vir
genes.
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Fig. Structure of Ti Plasmid Vector.

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Fig. Showing the Agrobacterium mediated gene Transformation

16. Ti plasmid vector for creating transgenic
plants
• Foreign genes could be inserted into the T-DNA and then
transferred to the plant.
• In the modified Ti plasmid the genes responsible for tumor
formation are deleted.
• Selectable markers added along with appropriate regulatory
elements.
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17. Ti plasmid vector for creating transgenic
plants
• The kanr gene from the E. coli transposon Tn5 has been used
extensively as a selectable marker.
• It encodes an enzyme called neomycin phosphotransferase
type II (NPTII), it detoxify the kanamycin.
• The NPTII coding sequence are provided with a plant
promoter and plant termination and polyadenylation signals.
• Such constructions with prokaryotic coding sequences flanked
by eukaryotic regulatory sequences are called chimeric
selectable marker genes.
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18. 1. One widely used Chimeric selectable marker gene
contains the cauliflower mosaic virus (CaMV) 35S
promoter, the NPTII coding sequence, and the Ti
nopaline synthase (nos) termination sequence; this
chimeric gene is usually symbolized 35S/NPTII/nos.
2. The Ti vectors used to transfer genes into plants have the
of the plasmid replaced with a chimeric selectable tumor-
inducing genes marker gene such as 35S/NPTII/nos.
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Ti plasmid vector for creating transgenic
plants

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21. Ti plasmid-based vectors: Components
1. A selectable marker gene, such as neomycin phosphotransferase, put under the
control of plant (eukaryotic) transcriptional regulation signals, including both a
promoter and a termination–polyadenylation sequence.
2. An origin of DNA replication that allows the plasmid to replicate in E. coli .
3. A polylinker to facilitate insertion of the cloned gene into the region between T-
DNA border sequences.
4. A “killer” gene encoding a toxin downstream from the left border to prevent
unwanted vector DNA past the left border from being incorporated into transgenic
plants.
5. If this incorporation occurs, and the killer gene is present, the transformed cells
will not survive.
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22. Binary vector system
1. The binary cloning vector contains either the E. coli and A. tumefaciens origins of
DNA replication or a single broad host range origin of DNA replication.
2. All the cloning steps are carried out in E. coli before the vector is introduced into A.
tumefaciens.
3. The recipient A. tumefaciens strain carries a modified (defective or disarmed) Ti
plasmid that contains a complete set of vir genes but lacks the T-DNA region, so that
this T-DNA cannot be transferred.
4. With this system, the defective Ti plasmid synthesizes the vir gene products and acts
as a helper plasmid.
5. This enables the T-DNA from the binary cloning vector to be inserted into the plant
chromosomal DNA.
6. Since transfer of the T-DNA is initiated from the right border, the selectable
marker, is usually placed next to the left border. 22

23. Binary vector system
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24. Limitations as routine Ti plasmid vectors
1. The production of phytohormones by transformed cells prevents
them from being regenerated into mature plants.
2. A gene encoding opine synthesis is not useful to a transgenic plant
and may lower the final plant yield.
3. Ti plasmids are large (approximately 200 to 800 kb).
4. Ti plasmid does not replicate in Escherichia coli, therefore it cannot be
cloned in E. coli.
5. Transfer of the T-DNA, which begins from the right border, does not
always end at the left border. Rather, vector DNA sequences past the
left border are often transferred.
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