TRANSFORMATION OF INDICA RICE

-srinath

AGROBACTERIUM MEDIATED TRANSFORMATION IN RICE

-srinath

GM RICE IN CHINA

TRANSGENIC PLANTSDNA 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…

Methods Employed for TransformationDirect gene Transfer-Electroporation,Particle

Bombardment,Micro injection,PEG mediated.In-direct gene Transfer-Virus

mediated,Agrobacterium mediatedAgrobacterium mediated transformation is

the only high Efficient Transformation Till date than,Remaining Transformation Techniques.

Agrobacterium mediated Transformation.

RICE

RICERice,wheat,maize are worlds 3 leading food

cropsRice is staple food of more than half of the

worlds populationMore than 3.5 billion people depend on rice

Challenges in Rice Disease,many disease such as

bacterial,fungal,viral diseases,pestsCreate huge loss to farmersSome fungal disease like Blast created

Famines.like the great bengal famineRice requires huge water for cultivationRice is not a salt tolerant plant.Rice is not a Drought Resistant crop.

Rice plant affected with Blast disease.

TRANSGENIC RICEPid2-Rice plants

resistant to Rice blastCry1la5-rice resistant

to stemborrerAm sod-rice plant

showing high level salt tolerance

AtDREBIA-plants showing improved drought tolerance

Agb,mediatedTransformation in RiceFirst 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 .

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.

Immature rice for transformationImmature embryos

are used as Explants in rice transformation.

Rice is grown in a green house for obtaining good immature embryos at the right stage.

Sterilization and isolation of embryosImmature 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

TransformationAll 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.

Infection and co-cultivationEmbryos 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.

RESTING AND SELECTIONThe 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.

REGENERATIONAfter 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.

GUS ASSAYGUS 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

Geno typing of Transformed PlantsComplete 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