Protoplast Isolation Protocol

Preparation of Metabolically Competent Protoplasts

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

(Table is listed below)

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.

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