The document discusses the development of transgenic rice plants through genetic engineering techniques, primarily using Agrobacterium-mediated transformation. It highlights the advantages of these modifications, such as resistance to diseases, pests, and improved environmental tolerance. The process includes various steps from embryo sterilization and transformation to regeneration and analysis of genetically modified plants.

1. -srinath

2. -srinath

3. GM RICE IN CHINA

4. TRANSGENIC PLANTS
 DNA is modified using Genetic Engineering
techniques.
 Transgenic Plant contains Genes inserted Artificially
called Transgenes.
 Provides advantages in producing plants wich are
Drought Resistant,cold tolerance, pest resistant, etc…

5. Methods Employed for
Transformation
 Direct gene Transfer-Electroporation,Particle
Bombardment,Micro injection,PEG mediated.
 In-direct gene Transfer-Virus mediated,Agrobacterium
mediated
 Agrobacterium mediated transformation is the only
high Efficient Transformation Till date
than,Remaining Transformation Techniques.

6. Agrobacterium mediated
Transformation.

7. RICE

8. RICE
 Rice,wheat,maize are worlds 3 leading food crops
 Rice is staple food of more than half of the worlds
population
 More than 3.5 billion people depend on rice

9. Challenges in Rice
 Disease,many disease such as bacterial,fungal,viral
diseases,pests
 Create huge loss to farmers
 Some fungal disease like Blast created Famines.like the
great bengal famine
 Rice requires huge water for cultivation
 Rice is not a salt tolerant plant.
 Rice is not a Drought Resistant crop.

10. Rice plant affected
with Blast disease.

11. TRANSGENIC RICE
 Pid2-Rice plants
resistant to Rice blast
 Cry1la5-rice resistant to
stemborrer
 Am sod-rice plant
showing high level salt
tolerance
 AtDREBIA-plants
showing improved
drought tolerance

12. Agb,mediatedTransformation in
Rice
 First step is to Transform
the Agrobacterium by
using molecular biology
tools
 Inserting our GOI with
Marker,into the bacteria.
 Once Agrobacterium is
engineered it can be
multiplied easily.
 Agrobacterium with
GOI,is streaked on LB
plates.
 Agb,is selected with
R,K,T,S
 LB plates are stored at 4
0 c .

13. EXPLANT
 Rice is a monocot plant.
 Monocot plants are
complicated compared
to dicots
 Unlike other
Dicots,Explants used in
monocots are Immature
Embryos or Calli.

14. Immature rice for transformation
 Immature embryos are
used as Explants in rice
transformation.
 Rice is grown in a green
house for obtaining good
immature embryos at the
right stage.

15. Sterilization and isolation of
embryos
 Immature rice is de-
husked,surface sterilized
with 2% Naocl,for 20
mins.
 Using a
microscope,immature
embryos are dissected.
 Embryos are collected in
liquid media (infection
media)in ependorf tubes

16. Transformation
 All the Embryos are
collected in liquid
media.
 Collected embryos are
pre treated,that means
they are kept in water
bath at 430Cfor 30
mins,and ice shock for 1
min.
 Agrobacterium culture is
freashly streaked on LB
plates,one day before
Transformation.
 O.D for infection should
be set as desired.
 Desired OD is adjusted
and culture is
resuspended in liquid
infection media.

17. Infection and co-cultivation
 Embryos after pre
treatment are infected with
agrobacterium suspended
media for 15 mins
 Agrobacterium suspension
is removed,and embryos
are kept in co-cultivation
media,in dark for 7 days.
 This step is called co-
cultivation.

18. RESTING AND SELECTION
 The embryos are kept in resting
media,ie..media can kill
agrobacterium but withou any
selection pressure.so embryos
can grow(both transformed and
non-transformed).
 After Resting,embryos are
moved to Selection
Media.ie..media with
hygromycin or kanamycin…
 Selection media is that in wich
only Transformed cells survive
and remaining cells die,so
Transformed embryo forming
calli survive and Remaining Die.

19. REGENERATION
 After 3 rounds of
selection,calli formed is
kept for Regeneration of
transformed plant.
 After 3 regeneration
subcultures on suitable
media,Plants are
Regenerated on media.
 Regenerated plants are
Transferred to Rooting
media.
 After rooting plants are
transffered to pots for
whole plant Regeneration.

20. GUS ASSAY
 GUS gene is used as Reporter
gene in Transformation
studies
 The enzyme β-glucuronidase
cleaves the substrate X-gluc
into glucuronic acid and
indoxyl derivative. The
indoxyl derivative dimerizes
and is then oxidized to form
insoluble, dark indigo dye the
presence of which can
indicate the site of expression
of the gene

21. Geno typing of Transformed Plants
 Complete regenerated
plants in a green house
can be analyzed for
GOI(gene of interest),via
pcr, southern blot or
other molecular
techniques.
 PCR,GUS(if used in gene
construct) can
demonstrate if Plant is
Transformed