Transformation of plant by Agrobacterium Tumefaciens and rhizogenes mediated gene transfer ppt

Agrobacterium rhizogenes-mediated gene transfer in plants:Agrobacterium virulence and selection of transformation

Introduction-:

•         Rod shaped  gram negative bacterium Cause  diseases like crown gall and hairy roots disease in many dicotyledonous plants.
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•          The bacteria transfer a tumor inducing plasmid Agrobacterium mediated genetic transformation is the most widely used method of transferring genes into plants.
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•          The genomic integration of a set of genes is mediated through a specific transfer DNA from the Ri plasmid. 
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      What is a Agrobacterium rhizogenes ?

Agrobacterium rhizogenes is a gram-negative,rod shaped soil bacterium.Rhizogenes cause Hariy-root disease and formation and proliferate multi-branched adventitious roots at the site of infection  called hairy roots.

Agrobacterium the causative agent of hairy root syndrome is a common soil organism capable of entering a plant through a wound and causing a proliferation of secondary roots.

T-DNA -: 

The transfer DNA is the part of DNA, of the tumor-inducing plasmid of some species of Agrobacterium rhizogenes. The T-DNA is transferred from bacterium into the host plant nuclear DNA genome.The T-DNA is bordered by 25bp repeats on each end. Transfer is RB and LB require the vir genes of the Ti plasmid.

Ri plasmid-:

Ri plasmid are large 200 to greater than 800kb and contain one or more regions of T-DNA and virulence region. Ri plasmid is a circular and larger plasmid harbored by agrobacterium rhizogenes. The genes Ri plasmid T-DNA direct a variety of phenotopic traits in the transformed plant cells increased synthesis of two morphogenic auxins and cytokinin. The tumor synthesis specific compound called opines. Ri plasmid have been isolated from a variety of strains and appear to within two classes.

1.Mannopin 2.Agropine

Secondary metabolite: 

Hairy root culture are characterized by a high growth rate and are able to synthesize root derived secondary metabolite. Root culture exogenous phytohormone supply and grow very slowly.

Source of root derived phytochemicals use as pharmaceuticals , cosmetic and food additives.

Hairy root source of new cells are in the tips so proliferation occurs apical meristem and laterals from behind elongation zone. Growth pattern lead to accumulation of biomass in root culture.

Plant DNA Extraction protocol And Isolation Method

Plant DNA isolation

AIM -: Genomic DNA can be isolate from plant cells by disrupting the tissue and then exploiting the difference in properties between DNA, Protein, and other constituents.

Principle

•Methods for DNA Extraction from plant tissue can be vary depending on the 

material used. Essentially , any mechanical means of breaking down the cell wall and membranes to allow access to nuclear material, without it’s degradation is required.

For this usually an initial homogenization stage is employed to break down the cell wall and allow access to DNA while harmful cellular enzyme and chemical remain inactive.

•Phenol is employed to denature and dissolve protein leaving nucleic acids in the aqueous phase. Samples of DNA are then treated with phenol extracted with chloroform: Isoamyl alcohol to precipetate. Remaining protein and reduce the amount of dissolved phenol in aqueous phase.

• DNA must be then Precipitate from the aqueous phase and wash thoroughly to remove contaminating salt. The purified DNA is then resuspended and stored in T.E buffer or FSMQ(sterile D/W).

•This method has been shown to give intact genomic DNA from plant tissue. To check the quality of extrated DNA, sample is run on 0.8% agarose gel, stained with EtBr and visualized under UV-light

 Requirement for practical

§ 7.5 M Ammonium acetate,

§ PVP 4% (polyvinyl pyrrolidone) M.W 10k

§ Β-Mercaptoethanol

§ Equilibrate phenol

§ Chloroform: Isoamyl Alcohol: (24:1)v/v

§ Phenol: chloroform: Isomayl Alcohol- (25:24:1)v/v

§Ethidium Bromide Dye: 10 mg/ml (Stock) and working 0.5 µg/ml

§Extraction buffer: 1. 250mM Nacl

  2. 25mM  EDTA (ph-8.0)

  3. 0.5% SDS

  4. 200mM Tric HCl

Protocol

§ Collect fresh leaves from plant and weight 1 to 2 gm, then wash with tap water followed by D/W water.

§ transfer into mortar & pestle, crash homogenize thoroughly with the extraction buffer (5ml) and beta-mercaptoethanol (50µl) in mortar.

§Incubate the sample at 65°C for 60 minute. Measure the volume of homogenized sample, and add freshly prepaid 4% PVP.

§Add Ammonium acetate ½  of the final volume of sample and incubate in ice for 30 minutes.

§Centrifuge the homogenized sample at 10,000 RPM for 10 minute.

§Collect the supernatant in fresh sterilized tubes and add equal volume of isopropyl alcohol or ethanol followed by incubate at -20°C for 30 minutes.

§Centrifuge at 10,000 rpm for 10 minute at 4°C

§Discard the supernatant & air dry pellet .

§ Dissolve the pellet in TE buffer (add depending on pellet size).

§Add 20µl/ml of Rnase and incubate at 37 degree for 30 minutes.

§Add equal volume of phenol : chloroform : isoamylalcohol (25:24:1) and gently mix during Incubation for 2 minutes,

§Centrifuge it 1000 rpm 4 degree 10 minute.

§Collect supernatant and add equal volume  of chloroform : isoamylalcohol (24:1) followed by centrifugation 1000 rpm 4 degree 10 minute.

§Collect aqueous phase (upper layer) and add equal volume of ethanol followed by incubation in -20 degree for 15 minutes.

§Finally load the aliquot on 0.8% Agarose gel, To check the purity of DNA

§Again centrifuge 1000 rpm 4 degree 15 minute. Discard supernatant and air dry pellet, afterward dissolve the pellet in TE buffer/TAE/FSFM.

§Measure the concentration and purity of DNA by obtaining the OD at A260/280  ratio with UV spectrophotometer. Pure DNA A260/280  ratio is 1.8, and pure RNA A260/280  ratio is 2.

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DNA isolation Protocol, DNA From Bacteria

  

DNA isolation Protocol from bacterial cell

Principle:

§The isolation and purification of DNA from bacterial cells is the most common procedure in genetic engineering (molecular biology ). The isolation of DNA from bacteria is simple process.

§The organism we use in DNA isolation should be grown in a favorable medium at an optimum temperature.

§Should be harvested in late log phase to early stationary phase for maximum yield of desirable DNA.

§The genomic DNA isolation needs to total separate from RNA and proteins, lipid, etc..

§Initially the cell disrupted in order to release DNA in the extraction buffer, SDS is use to disrupt the cell membrane. Once cell is disrupted the endogenous nuclease And nuclease also present on human fingertips tend to cause extensive hydrolysis of nucleic acid (DNA).

§ DNA can be protected from endogenous nuclease by chelating Mg+2 ions using  EDTA.

§Mg+2 is consider as a cofactor for most of the nuclease enzymes. And nucleoprotein interaction are disrupted by SDS action, and phenol act as proteinase K enzyme.

§Proteinase K degrade the proteins in the disrupted cell soup.

§chloroform(protein denaturant) are used to denature and separate protein from DNA by making boundary between aqueous phase and organic phase.

§The denaturant protein form a layer at interface between the aqueous phase and  organic phase which remove by centrifugation.

§DNA release from disrupted cells and precipitated by child ethanol or isopropanol.

Reagents for DNA isolation

1.SET Buffer-  (PH-7.5)

                        Sodium chloride : 75mM

                                         EDTA : 25mM

                                             Tris : 20mM

2.Lysozyme: 1mg/ml

3.Protinase K : 0.5mg/ml

4.Nacl: 5M

5.Rnase: 2mg/ml

6.Ethanol: 80% child

7.Chloroform: child

DNA isolation Protocol:-

1.Harvest the bacterial culture (18hrs old) in 2ml Eppendrop tube and centrifuge at 10000 rpm, 4°C for 15 minutes.

2.Discard the supernatant and re-suspend pellet in SET Buffer(according to pellet density) then vertex till the pellet dissolve in SET Buffer, afterword add lysozyme enzyme (10 µl), mix gently by inverting the tube 2 to 4 time. Then give Rnase treatment (10 µl) and gently mix.

3.Incubate at 37°C for 60 to 90 minutes, meanwhile mix the suspension every 20 minutes.

4. Add 1/10th volume of SDS, mix uniformly and incubate for 5 minutes, then add proteinaseK(10 µl) mix gently.

5. Incubate 55°C in water bath up to 120 minutes.

6. Add 1/3th volume of 5M Nacl, gently mix then add equal volume of chloroform, mix gently by inverting micro-centrifuge tube.

7.Incubate at room temperature for 30 minutes.

8.Centrifuge at 10000 rpm, 4°C for 10 minutes.

9.Collect the supernatant in 1.5 ml tube

10. Add the equal volume of 80% child ethanol in tube. Mix gently,

11.Centrifuge 10000 rpm, 4°C for 15 to 20 minute.

12.Discard the supernatant and allow pellet to air dry.

13.Then dissolve the pellet in TAE buffer or FSMQ

14.Then run it on Agarose gel electrophoresis,(0.8%)

15.Also you can check purity of DNA by taking 260/280 ratio

Result and Discussion:

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