Protocol for the preparation of metabolically competent protoplasts

[Saji Menon]

Protoplast Isolation Method has been attached

Preparation of Metabolically Competent Protoplasts

Saji Menon and Vibha Tyagi

Birla Institute of Scientific Research, Statue circle, Jaipur (Rajasthan), India

Materials Required

·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

1.Take fresh leaves from 4-5 week old aseptically growing plants.
2.Surface sterilize in 70% Ethanol followed by treating with 0.1% Mercuric chloride for 1 minute.
3.Wash the leaves thoroughly with sterile distilled water.
4.Peel the lower epidermis of the leaf tissue and place it in 0.5M Mannitol solution for one hour in a petriplate.
5.Remove the Mannitol solution and replace it with filter sterilized enzyme solution.
6.Incubate the plates in dark for 16-20 hours at 25oC.
7.Separate the protoplast layer by filtration using 0.45µM nylon membrane.
8.Centrifuge the filtrate at 1000g for 10 minutes at 4 oC.
9.Remove the supernatant and resuspend the pellet in cell protoplast washing medium.
10.Centrifuge at 1000g for 10 minutes.
11.Repeat step 9 twice.
12.Finally suspend the protoplast pellet at a density of 1*105 ml/l in the modified protoplast culture medium.
13.Plate the protoplasts as fine thin layer in petriplates.
14.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

[moleculardude]

Dear Saji,
hello and welcome to the forums!

please try reposting the protocol as the attachment is missing. if you need help posting the protocol or anything else do not hesitate to post here...

thanks!

[admin]

Thank you very much. We have added the protocol at the following location

Protoplast Isolation Protocol