I have discussed the Agrobacterium-mediated gene cloning process. In genetic engineering process transfer of genes from one organism to the other. Desired DNA is inserted into a vector and is transferred into the competent cell in a process is called transformation. All these steps are carried out in laboratories to engineer the desired organism genetically. The entire process of transformation is performed by scientists, known as genetic engineers. On the other hand, the bacterium Agrobacterium tumefaciens contains inside its a cell, a plasmid, i.e., an extrachromosomal self-replicating small DNA, known as Ti plasmid (tumor-inducing plasmid) that induce overproduction of growth factors in the host cells eventually leading to cell proliferation & tumor formation. This bacterium establishes contact with suitable root cells of plants by recognizing chemical signals and then prepares its plasmid ready to be delivered through t-pilus developed by type IV secretion system. Then, it transfers this DNA (also known as t-DNA) into the plant host cell. This delivery requires the assistance of several vir proteins. The t-DNA is transferred and stably integrated into the host chromosome which later wards lead in tumor development in the host. The whole process of successful DNA transfer and integration is performed & monitored by the bacteria itself. So, Agrobacterium tumefaciens is called the Natural genetic engineers of plants.

1. Dr. Divya Sharma
Assistant Professor

3.  The gene or other DNA fragment of interest is inserted
into a circular piece of DNA (Plasmid).
 The insertion is performed by using various enzymes like
Restriction enzymes (for cutting) and Ligase (for
connect) DNA, formed a Recombinant DNA.

4.  Multiple copies of a single gene
Step 1: Forming Recombinant DNA
Step 2: Transformation
(in-vivo amplification)
Step 3: Selection

5.  Disarmed Ti-Plasmid: Modified tumor inducing genes (Ti-
Plasmid) that lacks of transferable (T-DNA) genes.
 Gene of Interest: Addition or insert useful genes in place
of T-DNA genes -------- Transgenic Plants

6. Agropine – Type (strain EHA105:: pEHA105)
Carry genes for agropine synthesis and catabolism.
Tumors do not differentiate and die out.
Octopine- Type (strain LBA4404::pAL4404)
Carry genes (3 required) to synthesize octopine in the
plant and catabolism in the bacteria.
Nopaline-Type (strain GV3101::Pmp90 [pTiC58])
Carry gene for synthesizing nopaline in the plant and
for utilization (catabolism) in the bacteria.
Tumors can differentiate into shooty masses
(teratomas)

9.  Regeneration: shoot organogenesis, required Cytokinin (low amounts
of auxin)
 Selection: specifically 2 antibiotics (Kanamycin and Carbenicillin)
are required
First antibiotic, affect on to kill Agrobacterium cells, but neither effect on
the cell growth and division of plant’s; Another antibiotic, which allows
the transformed shoots growth; but inhibits the growth of untransformed
plant cells (simultaneously).
 Detection: detect Trait Gene
PCR method – detect presence of trait DNA
Protein detection method – gene products defines traits
Characterization – verification of trait genes on plant’s chromosomes; via
(a). southern hybridization; and (b). demonstration transfer of the trait to
the original transformant’s progeny

11. Advantages Disadvantages
 Less expensive  Time consuming
 High transformation efficiency  Not all kind of cells can be treated
by this method
 Transgenic crops obtained have
better fertility percentage
 Sometimes leads to false positive
result
 Protocols for both dicotyledons
and monocotyledon are available
 Relatively large length DNA
segment can be transferred.

12.  Golden rice
A recombinant variety of rice that has been engineered to
express the enzymes responsible for β-
carotene biosynthesis.
This variety of rice holds substantial promise for reducing
the incidence of vitamin A deficiency in the world's
population.
Golden rice is not currently
in use, pending the
resolution of regulatory and
intellectual property issues.

13.  Herbicide-resistant crops
Commercial varieties of important agricultural crops
(including soy, maize/corn, sorghum, canola, alfalfa and
cotton) have been developed that incorporate a recombinant
gene.
Results in resistance to the
herbicide Glyphosate (trade
name Roundup), and
simplifies weed control by
glyphosate application.
These crops are in common
commercial use in several
countries.

14.  Insect-resistant crops
Bacillus thuringeiensis is a bacterium that naturally produces
a protein (Bt toxin) with insecticidal properties.
The bacterium has been applied to crops as an insect-control
strategy for many years, and this practice has been widely
adopted in agriculture and gardening.
Recently, plants have been
developed that express a
recombinant form of the bacterial
protein, which may effectively
control some insect predators.
Environmental issues associated
with the use of
these transgenic crops have not
been fully resolved.