Agrobacterium mediated Gene transfer # Gene of Interest # Gene Cloning # Transgenic Plants

1. Dr. Divya Sharma
Assistant Professor
A Biodiction (A Unit of Dr. Divya Sharma)

2. Formation of Recombinant
Plasmid

4. ➢ 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.

6. ➢ 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

7. 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 differentitate into shooty masses
(teratomas)

8. ➢ 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.

15. A Biodiction (A Unit of Dr. Divya Sharma)