Protoplast Isolation Protocol

Written by Super User. Posted in Botany

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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