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Plant Cell Laboratory Experiment-Hairy Root Induction
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Objective: To study the hairy roots induction in A. indica by leaf disc method.

 

Introduction: Infection of wounded plants by a soil bacterium Agrobacterium rhizogenes results in hairy root induction, which is characterized by rapidly growing and highly branched roots at the host’s, wound sites. T-DNA of the bacterial Ri (root inducing) plasmid, which contains genes encoding endogenous hormones, is integrated in the plant genome and allows the proliferation of these adventitious roots. These transformed tissues are comparatively more advantageous for secondary metabolite production.

 

Requirements:

1.   A. indica leaves

2.   Overnight grown Agrobacterium rhizogenes culture

3.   Rectified spirit, 90% (v/v) alcohol, 70%(v/v) alcohol

4.   Aqueous solution of 1.0% sodium hypochlorite (NaOCl) Sterilized distilled water (SDW)

5.   Forceps

6.   Scalpel and blades

7.   Cork borer

8.   Beakers, Erlenmeyer flasks

9.   Sterilized Petri dishes with and without blotting paper

10.  MS Medium with and without antibiotic

 

Protocol:

Bacterial strain used - Agrobacterium rhizogenes NRRL B193

 

Sterilization of explants: As leaf explants are collected from the plants growing under natural environment, surface sterilization of explant is necessary to prevent microbial contamination.

 

1.  Wash the leaves with 1 % Savlon approximately for 10 minutes.

2.  Rinse with sterile distilled water (SDW) two times.

3.  Dip the explants in 70% ethanol for 30 seconds and wash with SDW two times.

4.  Soak the explants in 0.1% sodium hypochlorite or HgCl2 for 3 minutes followed by several rinses of SDW.

5.  Put the leaves on sterile petridish and cut into discs using cork borer.

6.  Put the discs on the culture medium.

 

Preparation of Agrobacterium rhizogenes culture:

 

1.  Grow the bacterial culture overnight at 280C on yeast mannitol broth.

2.  Reinoculate this overnight grown culture and incubate in a gyratory shaker for 24 h at 28˚C and 220rpm.

3.  These two times activated culture is used for infection of explant materials.

4.  Measure the optical density of the culture broth at 600 nm.

5.  Centrifuge the suspension at 6000 rpm for 10 minutes.

6.  Discard the supernatant and resuspend the pellet in the fresh media to obtain an OD value equivalent to 1.0 (108cells/ml).

 

Inoculation:

 

1.  400 ml of the bacterial culture was dispersed on the upper surface of the discs.

2.  Discs were incubated at 25°C in complete darkness initially for two days.

3.  After two days all the discs were kept in 16/8 h light/dark regime.

4.  Discs incubated on MS media were transferred to antibiotic (cefotaxime, 1.0 g/l) containing medium to check the overgrowth of bacteria.

5.  The control was run along with each set in which the fresh medium was dispersed on the discs and the incubation was done under similar conditions.

 

Observations:

 

Observe the root disc at routine interval for the development of hairy root induction.

 

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