Plant Callus Initiation Assignment | Online Assignment

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PLANT CALLUS INITIATION

Carrot

1. Wash the outside of the carrot using antibacterial soap.
2. In the hood, cut the carrot into slices and dip in 70% ethanol.
3. Place in 15% v/v Clorox bleach with 2-3 drops of Tween 20 for 15 minutes.
4. Using aseptic technique, rinse in 3 separate distilled water rinses by placing slices in the water and leaving for 5 minutes each rinse.
5. Hold in forceps and place into sterile dish. Use a sterile cork borer to collect pieces, avoiding the central xylem. Also avoid tissue in direct contact with bleach.
6. Place five suitable pieces onto each of two B5, two B5+, and two MSI plates.
7. Parafilm plates
8. Incubate at room temperature in the dark until callus appears.

Plate Recipes

To prepare Gamborg’s B5:

1. To make 0.5 L, dissolve 1.6 g of Gamborg’s B5 powder in 450 ml of water.
2. Add 10 g of sucrose (final concentration is 2% w/v).
3. Add 0.5 ml of 2,4 D (1 mg/ml).
4. Raise pH to 5.6 using 2N KOH.
5. Top up to 500 ml.
6. Add 2.5 g of Agarose.
7. Autoclave 15 minutes.
8. Cool to 55’C.
9. Pour plates

To prepare Gamborg’s B5+

1. Add 1.6 g Gambourg’s B5 powder to 450 ml of water.
2. Add 10 g of sucrose.
3. pH to 5.7 (2 small drops of 1M KOH).
4. Top up to 499 ml.
5. Add 2.5 g Agarose.
6. Autoclave for 15 minutes.
7. When cool, add 0.5 ml of NAA (1mg/mL) and 0.5 ml of kinetin (1mg/mL).
8. Pour plates.

To prepare MCI medium:

1. Top up to 1 liter water.
2. Add 5 g Agarose.
3. Autoclave for 15 minutes.

Note:  I usually store my media at 4C, and it we can store it up top 3 months without problems with contamination.

Note: 2,4 D not dissolve in cold water, you have to bring the solution to boil.

To prepare 2,4 D (1mg/ml):

• Take a little bit powder from the bottle
• Weight, for example, if it is 35.5 mg
• Add 30.5ml water to make it at the dose of 1 mg/ml
• Bring to the boil until the powder is totally dissolved in the water
• Add 0.5ml to 0.5L media, Discard the rest.