Experiments on Biotechnology | Biology

Are you researching experiments on biotechnology ? You are in the right place. The below mentioned article includes a collection of seven experiments on biotechnology: 1. Culture Media 2. Callus Tissue Culture 3. Tissue Culture Medium 4. Tissue Culture in Plants 5. Isolation of Single Cells 6. Cell Planting Technique 7. Isolation of Protoplasm.

Contents:

1. Experiment on Culture Media
2. Experiment on Callus Tissue Culture
3. Experiment on Tissue Culture Medium
4. Experiment on Tissue Culture in Plants
5. Experiment on Isolation of Single Cells
6. Experiment on Cell Planting Technique
7. Experiment on Isolation of Protoplasm

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1. Experiment on Culture Media:

Aim of the Experiment:

Prepare a list of nutritional requirements for root culture of Convolvulus arvensis.

Requirements:

Ca(NO₃)₂. 4H₂O, MgSO₄. 7H₂O, KNO₃, KCl, KH₂PO₄₎ H₃BO₃, ZnSO₄.7H₂O, MnSO₄.2H₂O, Na₂MoO₄.2H₂O, CuSO₄.5H₂O, FeCl₃.6H₂O, sucrose, thiamin, HCl, petri-dishes, autoclave.

Mix the above-mentioned macronutrient salts:

(A) micronutrient salts (B) and organic components (C). Adjust the pH of this medium to 4.5 and autoclave it for 15 minutes at 15 pounds per square inch.

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2. Experiment on Callus Tissue Culture:

Aim of the Experiment:

Prepare a list of nutritional requirements of callus tissue culture of roots of Convolvulus arvensis.

Requirements:

2,4-Dichlorophenoxyacetic acid (2, 4-D), nicotinic acid, pyridoxine HCl, adenine sulphate, myoinositol and 1-glutamine.

List of nutritional requirements:

All components mentioned above in Experiment No. 1 plus some additional organic components mentioned below:

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3. Experiment on Tissue Culture Medium:

Aim of the Experiment:

To prepare a tissue culture medium.

Tissue culture medium:

The simple method of preparing tissue culture medium is to use commercially available media of some good companies (e.g., SIGMA Chemical Company, St. Louis, USA; or Hi Media Laboratories Pvt. Ltd., 23 Vadhani Industrial Estate, Bombay-86) in the market.

These are dry powdered media containing the desired macronutrients, micronutrients, vitamins and amino acids. The powder is dissolved in distilled water. Agar, sugar and other constituents are added in it, and distilled water is again added to prepare the final volume. The medium is autoclaved after the adjustment of the desired pH .

Composition of murashige-skoog medium:

Murashige-Skoog’s (MS) medium is the most widely used medium in the tissue culture laboratories for culturing plant tissue and cell culture of both monocotyledons and dicotyledons.

The composition of the various constituents of this medium is under mentioned:

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4. Experiment on Tissue Culture in Plants:

Aim of the Experiment:

To work out the generalized steps used in the methodology of tissue culture in a plant material.

Requirements:

Plant material (e.g., mature carrot plant), water, scalpel or razor, cork borer, sterile petri-dishes, callus initiation medium (e.g., Murashige-Skoog’s medium) with 2,4-D, shoot development medium, pot with soil.

Method:

1. Take a mature carrot plant (Fig. 65 A) with its tap roots intact, remove its leaves and wash its tap roots thoroughly (Fig. 65 B).

2. Cut the tap root into 3 or 4 pieces (Fig. 65 C) with a sharp scalpel or razor.

3. Insert the cork borer into a tap root piece (Fig. 65 D) and take out the desired regions of root.

4. Put such a removed tap root piece in a sterile petri- dish and cut it transversely into small pieces as shown in Fig. 65 E.

Fig. 65. Various steps showing protocol for somatic embryogenesis in Carrot

5. Take some callus initiation medium (e.g., Murashige-Skoog’s medium or MS medium) with 2,4-D in a sterile petri-dish, place some discs or cut pieces of tap root on it and incubate for 6-8 weeks. Callus formation starts within 4-6 weeks (Fig. 65 F).

6. Transfer the callus to another petri-dish containing shoot development medium. Young plants with roots and shoots (Fig. 65G) start to develop within 4-8 weeks.

7. These young plants are transferred to pots containing soil (Fig. 65 H) where they develop into mature plants (Fig. 65 A).

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5. Experiment on Isolation of Single Cells:

Aim of the Experiment:

To demonstrate the isolation of single cells from intact plant organs.

Requirements:

Fresh leaves of plant, 95% ethyl alcohol, calcium hypochlorite (7% solution), sterile distilled water, blade, potter-Elvehjem glass homogenizer tube, Rossini culture medium, sterile metal Tyler filters, centrifuge, agar plates, and incubator.

Method:

1. Take the fresh leaves and immerse them in 95% ethyl alcohol.

2. Rinse these leaves for 15 minutes in calcium hypochlorite solution (7%) and then wash 2-3 times in sterile distilled water.

3. Cut these leaves into small pieces of about 1 sq. cm., and put 1.5 gm. of such pieces in a potter- Elvehjem glass homogenizer tube.

4. Add 10 ml of Rossini culture medium into this homogenizer tube and homogenize the leaves.

5. Filter the medium containing homogenized leaves through two layers of such sterile metal Tyler filters of which the mesh diameter of upper layer is 61 mm and of lower layer is 38 mm.

6. Centrifuge the filtrate and discard the supernatant.

7. The sediment consists of free mesophyll cells. Suspend this sediment in a volume of medium.

8. Inoculate the free mesophyll cells into an agar plate or into the liquid medium and incubate these plates or vials in dark or light at 26°C.

Observations and results:

Sediment in the centrifuge tube contains free mesophyll cells. On a suitable medium these cells can be cultured.

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6. Experiment on Cell Planting Technique:

Aim of the Experiment:

To demonstrate the Cell Planting Technique or process of Single Cell Culture and callus formation.

Requirements:

Free mesophyll cells (as obtained in Exercise No 5), beaker, Murashige-Skoog (MS) liquid medium (as prepared in Experiment No. 3), MS solid medium containing 0.6% agar, fine gauze, petri-dishes, sealing agent, inverted microscope, glass-marking pencil, incubator.

Method:

1. Take MS liquid medium in a beaker and suspend in it the free cells as obtained in Experiment No. 5.

2. Pass this cell suspension through a fine gauze.

3. In a separate beaker, dissolve MS solid medium and allow it to cool down to 35°C.

4. In a separate beaker, mix this molten MS agar medium and cell suspension in equal proportions (50: 50) and shake it well. By doing so the cells, would be evenly distributed throughout the medium.

5. Take some sterile petri-dishes and pour about 10 ml of this medium containing cells in each of them. Seal these petri-dishes with a sealing agent and incubate them in dark at about 25°C for 3-4 weeks.

Observations and results:

Prior to incubation, observe the single cells in the petri-dishes under an inverted microscope and mark the location of these cells on the outside of petri-dishes by a glass-marking pencil. This will make you sure about the isolation of pure single cells. After 3-4 weeks calli or colonies will develop on the agar surface in each petri-dish.

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7. Experiment on Isolation of Protoplasm:

Aim of the Experiment:

Isolation of protoplast from different tissues using commercially available enzymes.

Requirements:

Root tips of Allium sativum, alcohol, distilled water, sodium hypochlorite, autoclave, mannitol, driselase enzyme, Knop’s solution, incubator, small sterile tubes, centrifuge, slides, microscope, agar- based culture medium, ultraviolet microscope.

Method and Observations:

1. Dip some young root tips of Allium sativum in 80% alcohol for 30 seconds and rinse them thoroughly with some sterile distilled water.

2. Now dip the root tips in 1.5% sodium hypochlorite for about 10 minutes and again rinse them thoroughly with sterile distilled water.

3. Repeat the rinsing process with distilled water 2-3 times.

4. Now cut the tips into small pieces in freshly prepared and autoclaved 0.5 M mannitol.

5. Prepare 5% stock solution of enzyme driselase by adding 2 ml of stock driselase in 18 ml of 0.5 M mannitol.

6. Now put the cut tips in 0.5% driselase for about 30 minutes.

7. Transfer the tips into a solution of mannitol and Knop’s solution (1:1) and incubate them at 37°C for about 15 hours.

8. The incubated tips are now taken in small sterile tubes to release the protoplast. Centrifuge them in mannitol two times for about 15 minutes at 1500 rpm.

9. After centrifuge process, discard the supernatant. The settled residue contains protoplasts.

10.Put a drop of this residue on a clean slide and observe under microscope carefully to see that cell wall has been removed.

Result:

The protoplasts have now been isolated. These isolated protoplasts can now be transferred to the culture medium for regeneration, and this process is called protoplast culture.

Protoplast culture:

Now suspend the residue containing the isolated protoplasts in isotonic solution of mannitol. This will provide appropriate concentration of protoplast. This is now transferred to a suitable agar-based culture medium. Wait for a few hours.

The isolated protoplasts now begin to develop new cell wall, which can be detected by ultraviolet microscopy. The cells soon start to divide and form small callus colony. From the so-formed small colonies of callus, new intact plants can be regenerated.