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Protocol for protoplast isolation.

protoplast

Preparation of Metabolically Competent Protoplasts

 

Saji Menon and Vibha Tyagi
Birla Institute of Scientific Research, Statue circle, Jaipur (Rajasthan), India

 

Materials Required for Protoplast Isolation

  • 70% Ethanol
  • 0.1% Mercuric Chloride
  • 0.5M Mannitol

 

  • Enzyme solution :
  • 1% Cellulase 0.25% Macerozyme R-10
  • 27.2 mg/l KH2PO4
  • 101 mg/l KNO3
  • 1480 mg/l CaCl2.2H2O
  • 246 mg/l MgSO4.7H2O
  • 0.5M Mannitol (pH 5.6)

 

  • Cell Protoplast Washing Medium :
  • 0.5M Mannitol (pH 5.6)
  • 27.2 mg/l KH2PO4
  • 101 mg/l KNO3
  • 1480 mg/l CaCl2.2H2O
  • 246 mg/l MgSO4.7H2O
  • Modified Protoplast Culture medium       

(Table is listed below)

 

Protocol for the preparation of Metabolically Competent Protoplasts

  • Take fresh leaves from 4-5 week old aseptically growing plants.
  • Surface sterilize in 70% Ethanol followed by treating with 0.1% Mercuric chloride for 1 minute.
  • Wash the leaves thoroughly with sterile distilled water.
  • Peel the lower epidermis of the leaf tissue and place it in 0.5M Mannitol solution for one hour in a petriplate.
  • Remove the Mannitol solution and replace it with filter sterilized enzyme solution.
  • Incubate the plates in dark for 16-20 hours at 25oC.
  • Separate the protoplast layer by filtration using 0.45µM nylon membrane.
  • Centrifuge the filtrate at 1000g for 10 minutes at 4 oC.
  • Remove the supernatant and resuspend the pellet in cell protoplast washing medium.
  • Centrifuge at 1000g for 10 minutes.
  • Repeat step 9 twice.
  • Finally suspend the protoplast pellet at a density of 1*105 ml/l in the modified protoplast culture medium.
  • Plate the protoplasts as fine thin layer in petriplates.
  • Incubate the plates at 25 oC in dark.

 

Modified Protoplast Culture Medium

S.No.

Constituents

Amount

A

Mineral Salts

 

1

Ammonium Nitrate

82.5 gm/l

2

Potassium Nitrate

95.0 gm/l

3

Boric acid

1190 mg/l

4

Potassium Dihydrogen Phosphate

34 mg/l

5

Potassium Iodide

166 mg/l

6

Sodium Molybdate

250 mg/l

7

Cobalt Chloride Dihydrate

25.0 mg/l

8

Calcium Chloride Dihydrate

88.0 gm/l

9

Magnesium Sulphate Heptahydrate

74.0 gm/l

10

Manganese Sulphate tetrahydrate

4460 mg/l

11

Zinc Sulphate Heptahydrate

1720 mg/l

12

Copper Sulphate Pentahydrate

25.0 mg/l

13

Ethylene Diamino Tetra Acetic acid Disodium salt

7.45 gm/l

14

Ferrous Sulphate heptahydrate

5.57 gm/l

B

Sugars

 

1

Glucose

30.0 gm/l

2

Ribose

0.5 gm/l

3

Mannose

0.5 gm/l

4

Sorbitol

0.5 gm/l

C

Organic Acids

 

1

Sodium Pyruvate

5.0 mg/l

2

Citric acid

10.0 mg/l

3

Malic acid

10.0 mg/l

D

Vitamins

 

1

Thiamine Hydrochloride

100 mg/l

2

Nicotinamide

500 mg/l

3

Pyridoxine Hydrochloride

500 mg/l

4

Calcium pantothenate

250 mg/l

5

Biotin

100 mg/l

6

Ascorbic acid

75.0 mg/l

7

Riboflavin

50.0 mg/l

8

Inositol

100 gm/l

E

Hormones

 

1

2,4-Dichloro Phenoxy Acetic Acid (2,4-D)

0.5 mg/l

2

Benzyl Amino Purine (BAP)

1.0 mg/l

 

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