Genetic engineering for crop improvement

It illustrates about the importance of Genetic engineering for Crop improvement

1. PLANT TRANSFORMATION WITH
A. TUMIFACIENS

BY
T.NIVETHITHA
À TII-BIOTECHNOLOGY

= BON SECOURS COLLEGE
FOR WOMEN.

2. Agrobacterium tumefaciens are gram-negative
organisms responsible for transfer of target DNA(T-
DNA).

These phytopathogens, transforms plant cells

(trasngenic plants), which genetic transformation
leads to the formation of Crown gall tumors.

As it helps in the transformation of plants,
Agrobacterium sp. are known as Natural Genetic
engineers.

The involvement of Agrobacterium sp. was discovered
by Smith and Townsend in 1907.

3. Ti-plasmid carries about
approximately 10-30kbp Ai
of T-DNA. 4
The virulent strains of
A.tumifaciens harbor large ‘tty
plasmids of about 140-

235 kbp, known as Ti- |
Plasmids. |
It consists of vir region, 1
origin of replication, region
enabling conjugative Viuance
transfer, o-cat region for region
catabolism of opines and

most importantly T-DNA.

Cytokinin

Ti plasmid

Ine

sa Bigin of
= replication (ORI)

4. STRUCTURE OF T-DNA

+ T-DNA is a part of the “tumor-inducing” (Ti) plasmid, that is
carried by most strains of A. tumefaciens.
+ T-DNA is defined by its left and right borders and includes

genes for the biosynthesis of auxin,cytokinin and opine.

Structure of T-DNA

ONC REGION

SHOOTY LOCUS ROOTY Locus

GGCAGGATATATTCAATTGTAA GGCAG GATATATACCGTTGTAAT

5. The initial step in the infection is the attachment of
A.tumefaciens to a plant cell at the site of an open
wound, often at the crown(base), which produces a
network of cellulose fibrils that bind the bacterium
tightly to the plant cell surface.

These bacteria respond to certain plant phenolic
compounds, acetosyringone and

hydroxyacetosyringone, excreted by wounded plants.

These small molecules act to induce the virulence (vir)

genes located on 35kb region of Ti-plasmid.

6. After Ti-plasmid carrying
A.tumifaciens attaches to a
host plant, the vir genes
are induced and becomes
integrated into the
chromosomal DNA. The T-
DNA also contains iaaM and
iaaH genes.

The iaaM codes for the
enzyme tryptophan 2-
monooxygenase which
converts tryptophan to
indole 3-acetamide
hydrolase, which coverts it
to indoleacetic acid.

7. Agrobacterium tumefaciens

Tumor inducing (T})-plasmid

Bacterial
chromosome

3. T-DNA synthesis
and vir tracweription À

Se
= a

=
S qu

'Opine catabolisn

ME Origin of replication
(ORI)

=

6. Mmipulation aloe ot

TNA VIDA complex

el

Crown gall

EX

8. The T-DNA also carries ‘tmr’ gene(ipt), that encodes
isopentenyltransferase responsible for the synthesis of
cytokinin isopentenyladenine.

The opines are synthesized within the crown gall and
then secreted, which can be used as a carbon source,
and sometimes as nitrogen source by A. tumifaciens.

The binary cloning vector system and cointegrated
cloning vector system are being used in this transfer
technique.

Finally, the transformed cells are being tested by
Kanamycin treatment, and if they grew the
transformation is confirmed and those transgenic
plants or crops are cultivated in a large scale.

9. Genetic Engineering using

10. Physical Methods of Transferring
Genes to Plants

Q MICROPROJECTILE BOMBARDMENT. (Biolistics) Used
with a wide range of plants and tissues; easy and
inexpensive.

OVIRALVECTORS- Not an effective way to deliver DNA
to plant cells.

Q DIRECT GENE TRANSFER INTO PLANT
PROTOPLASTS- Can be used only with the plant cell

protoplasts that can be regenerated into viable plants.

11. Q ELECTROPORATION- Generally limited to plant cell
protoplasts that can be regenerated into viable plants.
Q LIPOSOME FUSION- Can be used only with plant cell
protoplasts that can be regenerated into viable plants.

Q MICROINJECTION- Has limited usefulness because

only one cell can be injected at a time; requires the

services of a highly skilled individual.

12. APPLICATIONS:

- Crop Improvement
- Genetically Engineered Traits:
The Big Six

Herbicide resistance

Insect resistance
Virus resistance
Altered oil content
Delayed fruit ripening
Pollen control

13. - Biotech Revolution: Cold and Drought
Tolerance and Weather-guard genes

1. Genetically Engineered foods
2. Soybeans

3. Corn
4. Cotton
5. Other crops

14. Reference:

Molecular Biotechnology: Principles
and Applications of Recombinant
DNA(2010) — Bernard R. Glick, Jack
J. Pasternak, and Cheryl L. Patten -
4th Edition.