Agrobacterium tumefaciens is a soil bacterium that naturally transfers DNA to plant cells and causes crown gall disease. It contains a tumor-inducing plasmid (Ti plasmid) that can be engineered to transfer foreign genes of interest into plant genomes. This process, called Agrobacterium-mediated transformation, is commonly used in genetic engineering due to its high efficiency. The Ti plasmid contains T-DNA that is transferred to plant cells, along with virulence genes that facilitate transfer without being transferred themselves. Transformed plants are regenerated from cultured plant cells or tissues containing the novel genes. Agrobacterium-mediated transformation is useful for genetic engineering but has limitations such as a narrow host range and time-consuming

1. Agrobacterium tumefaciens in genetic
engineering
PRESENTED BY
SURESH KUMAR SAHU
M.Sc. PLANT PATHOLOGY

2. Contents
• Scientific Classification
• Introduction
• Ti-Plasmid
• Plant Transformation Using Agrobacterium tumefaciens
• Production of transgenic plants
• Uses of Agrobacterium tumefaciens
• Advantages of using Agrobacterium in Genetic
Engineering
• Disadvantages of Agrobacterium mediated Genetic
Engineering

3. Agrobacterium tumefaciens
Scientific Classification
Kingdom : Bacteria
Phyllum : Proteobacteria
Class : Alpha proteobacteria
Order : Rhizobials
Family : Rhizobiaceae
Genus : Agrobacterium
Species : A. tumefacience

4. Introduction
 It is a rod-shaped, Gram-negative, Peritrichous flagella, Soil
bacterium.
 Agrobacterium is well known for its ability to transfer DNA
between itself become an important tool for genetic
engineering.
 Agrobacterium tumefaciens is a bacterium which naturally
causes tumors in plants which is caused by the transfer of DNA
from the bacterium to the plant it infects.
 Economically, A. tumefaciens is a serious pathogen of
of walnuts, grape vines, stone fruits, nut trees, sugar
beets, horse radish, and rhubarb.

5.  The crown gall bacterium Agrobacterium tumefaciens was
discovered by Erwin Smith in the 1890s.
Agrobacterium tumefaciens, a natural pathogen of many dicotyledon
species, has been successfully used in transformation experiment.
Bacteria carrying a tumor inducing (Ti) plasmid induce crown gall.
 The plasmid is known as a tumor-inducing (Ti) plasmid, and the
segment of the plasmid DNA that is inserted by the bacterium into the
plant cell is known as T-DNA.
 The T-DNA carries genes Oncogene: auxin and cytokinin; and
Opine( octapine and nopaline) respectively, that the bacterium can
use as food, and sequences forming the left and the right border of T-
DNA.

8. Agrobacterium tumefacience - The Natural
genetic Engineer
 Agrobacterium tumefacience is a gram negative, rod shaped,
soil bacterium which is non sporing, and closely related to the
N-fixing rhizobium bacteria which form root nodule on
leguminous plants. The bacterium is surrounded by a small
number of peritrichous flagella.
 Virulent bacteria contain one or more large plasmid, one of
which carries the genes for tumor induction and is known as the
Ti plasmid. The Ti plasmid also contain the genes that
determine the host range and the symptoms, which the
infection will produce. Without this Ti plasmid, the bacterium is
described as being non virulent and will not be able to cause
disease on the plant.

9. Why is Agrobacterium tumefaciens known as
natural genetic engineer?
This bacterium establishes contact with suitable root cells
of plants by recognising chemical signals and then prepares
it's plasmid ready to be delivered through t-pilus developed
by type IV secretion system . ... So, Agrobacterium
tumefaciens is called the Natural genetic engineers of
plants .

10. Ti Plasmid-: The large-sized tumor inducing plasmid found in
Agrobacterium tumefaciens. It directs crown gall formation in
certain plant species.
Ti-Plasmid

11. Ti-Plasmid
 The Ti plasmid (approx. size 200 kb each) exist as independent
replicating circular DNA molecules within the Agrobacterium cells.
The T-DNA is variable in length in the range of 12 to 24 kb.
The Ti plasmid has three important region:-
1. T-DNA region(between right and left T-DNA border)
2. Virulence region
3. Opine Catabolism region

13. 1. T-DNA region: This region has the genes for the biosynthesis
of auxin (aux), cytokinin (cyt) and opine (ocs), and is flanked by left
and right borders.
 T-DNA borders- A set of 24 kb sequences present on either side
(right & left) of T-DNA are also transferred to the plant cells.
 It is clearly established that the right border is more critical for T-
DNA transfer.
2. Virulence region: The genes responsible for the transfer of T-
DNA into host plant are located outside T-DNA and the region is
referred to as vir or virulence region.

14. Virulence genes present in the Ti plasmid of Agrobacterium
tumefaciens and their function -
A- receptor of wound signal
B- protein form membrane pore
C- enhances transfer of T- DNA
3. Opine catabolism region: This region codes for proteins
involved in the uptake and metabolisms of opines.
 Besides the above three there is ori. region that responsible for
origin of DNA replication which permit the Ti plasmid to be stably
maintain in A. tumefaciens.

15. • If A. tumefaciens bacteria that
contain an engineered Ti plasmid
are introduced into a plant in the
natural way, by infection of a
wound in the stem, then only the
cells in the resulting crown gall will
possess the cloned gene. This is of
little value.
• Instead a way of introducing the
new gene into every cell in the
plant is needed.
• There are several solutions, the
simplest being to infect not the
mature plant but a culture of plant
cells or protoplasts in liquid
medium.
Production of transformed plants with the Ti plasmid

16. Plant Transformation Using Agrobacterium
tumefaciens
Agrobacterium tumefaciens is a widespread naturally
occurring soil bacterium that causes crown gall, and has the
ability to introduce new genetic material into the plant cell.
The genetic material that is introduced is called T-DNA
(transferred DNA) which is located on a Ti plasmid. A Ti
plasmid is a circular piece of DNA found in almost all bacteria.
This natural ability to alter the plant’s genetic makeup was the
foundation of plant transformation using Agrobacterium.
Currently, Agrobacterium-mediated transformation is the
most commonly used method for plant genetic engineering
because of relatively high efficiency. Initially it was believed
that this Agrobacterium only infects dicotyledonous plants,
but it was later established that it can also be used for
transformation of monocotyledonous plants such as rice.

17. During transformation, several components of the Ti plasmid
enable effective transfer of the genes of interest into the plant
cells. These include:
• T-DNA border sequences, which demarcate the DNA
segment (T-DNA) to be transferred into the plant genome
• vir genes (virulence genes), which are required for
transferring the T-DNA region to the plant but are not
themselves transferred, and
• modified T-DNA region where the genes that cause crown
gall formation are removed and replaced with the genes of
interest.

18. Agrobacterium-mediated Plant Transformation Process

19. Production of transgenic Plants
Isolate and clone gene of interest
Add DNA segments to initiate or enhance gene expression
Add selectable markers
Introduce gene construct into plant cells(transformation)
Select transformed cells or tissues
Regenerate whole plants

20. Approved Transgenic Plants
• Soybean • Corn
• Cotton • Oil Seed rape
• Sugar beet • Squash
• Tomato • Tobacco
• Carnations • Potato
• Flax • Papaya
• Melon • Rice
Approved Traits
• Glufosinater • Sethoxydimr
• Bromoxynilr • Glyphosater
• Sulfonylurear • Male Sterility
• Modified Fatty Acid
• Flower colour • Flower life
• Delayed Fruit ripening • Virus resistance

21. Uses of Agrobacterium tumefaciens
 Agrobacterium tumefaciens, a natural pathogen of many
dicotyledon species, has been successfully used in
transformation experiments. It accomplishes genetic
engineering of the host plants naturally by transferring its
tumor-inducing plasmid (Ti plasmid) to the compatible host
cells.
 Agrobacterium tumefaciens is a soil-born pathogen with the
unique ability to genetically transform plants. Agrobacterium
infects plant wound sites, causing crown gall disease.
 Agrobacterium tumefaciens is a soil bacterium, which
is used to transfer a small segment of DNA into plant genome
by the process known as transformation. The whole plant is
regenerated from individual plant.

22. Advantages of using Agrobacterium in Genetic
Engineering
• It is relatively inexpensive than other gene transfer methods.
• It is reliable and precise.
• It is relatively less laborious.
Disadvantages of Agrobacterium mediated
Genetic Engineering
• Though Agrobacterium has a wide host range, it cannot be
used to transfer genes to almost all the plants.
• It is not used to transfer genes to organisms other than plants.
• It is a relatively time consuming method.
• Antibiotic resistance genes can be transferred to bacteria in
the human/ animal digestive system.

23. Conclusion
Agrobacterium tumefaciens is more than the causative
agent of crown gall disease affecting dicotyledonous plants. It
is also the natural instance for the introduction of foreign
genes in plants allowing its genetic manipulation.

24. References
Agrios, G.N. 2005, Plant Pathology, Elsevier Academic Press.
pp 664-665.
Das A (1998) DNA transfer from Agrobacterium to plant cells
in crown gall tumor disease. Subcellular Biochemistry 29:
343–363.
Agrobacterium mediated gene transformation. Google
scholars article.
Chamikara Pasindu, 2018, Agrobacterium in Genetic
Engineering, 10.13140/RG.2.2.20898.94402.
S.S. Purohit, The Cell and Molecular Biology: Fundmentals
and Applications. AGROBIOS (INDIA) pp 1038-103.