Protoplast Isolation Protocol Protoplast Izolacija Protocol
Preparation of Metabolically Competent Protoplasts Priprema Metabolically kompetentan Protoplasts
Saji Menon and Vibha Tyagi Saji Menon i Vibha Tyagi
Birla Institute of Scientific Research, Statue circle, Jaipur (Rajasthan), India Birla Institute of Scientific Research, Statue krug, Jaipur (Rađasthanu), Indija
Materials Required for Protoplast Isolation Materials Required za Protoplast Izolacija
- 70% Ethanol Etanol 70%
- 0.1% Mercuric Chloride 0,1% Mercuric Chloride
- 0.5M Mannitol 0.5M Mannitol
- Enzyme solution : Enzim rješenje:
- 1% Cellulase 0.25% Macerozyme R-10 1% celulaza 0,25% Macerozyme R-10
- 27.2 mg/l KH2PO4 27,2 mg / l KH2PO4
- 101 mg/l KNO3 101 mg / l KNO3
- 1480 mg/l CaCl2.2H2O 1480 mg / l CaCl2.2H2O
- 246 mg/l MgSO4.7H2O 246 mg / l MgSO4.7H2O
- 0.5M Mannitol (pH 5.6) 0.5M Mannitol (pH 5,6)
- Cell Protoplast Washing Medium : Stanica Protoplast pranje Medium:
- 0.5M Mannitol (pH 5.6) 0.5M Mannitol (pH 5,6)
- 27.2 mg/l KH2PO4 27,2 mg / l KH2PO4
- 101 mg/l KNO3 101 mg / l KNO3
- 1480 mg/l CaCl2.2H2O 1480 mg / l CaCl2.2H2O
- 246 mg/l MgSO4.7H2O 246 mg / l MgSO4.7H2O
- Modified Protoplast Culture medium Promijenio Protoplast kulture srednje
(Table is listed below) (Tablica je navedena u nastavku)
Protocol for the preparation of Metabolically Competent Protoplasts Protokol za pripremu Metabolically kompetentan Protoplasts
- Take fresh leaves from 4-5 week old aseptically growing plants. Uzmi svježeg lišća od 4-5 tjedna stare aseptically uzgoj biljaka.
- Surface sterilize in 70% Ethanol followed by treating with 0.1% Mercuric chloride for 1 minute. Površina sterilize u 70% Etanol slijedi liječenje s Mercuric klorid 0,1% za 1 minutu.
- Wash the leaves thoroughly with sterile distilled water. Temeljito oprati lišće sa sterilnim destilirana voda.
- Peel the lower epidermis of the leaf tissue and place it in 0.5M Mannitol solution for one hour in a petriplate. Skinuti pokožica je niža od leaf tkiva i postavite je na 0.5M Mannitol rješenje za jedan sat u petriplate.
- Remove the Mannitol solution and replace it with filter sterilized enzyme solution. Uklonite Mannitol rješenje i opet staviti Internet sa filterom sterilizirano enzim rješenje.
- Incubate the plates in dark for 16-20 hours at 25oC. Izleći piliće na ploče u tamno za 16-20 sati na 25oC.
- Separate the protoplast layer by filtration using 0.45µM nylon membrane. Odvojite se protoplast sloj po filtracija pomoću 0,45 μ m najlona membrana.
- Centrifuge the filtrate at 1000g for 10 minutes at 4 oC. Centrifuga, procijediti u 1000g za 10 minuta na 4 oC.
- Remove the supernatant and resuspend the pellet in cell protoplast washing medium. Uklonite supernatant i resuspend je metak u ćeliji protoplast pranje medij.
- Centrifuge at 1000g for 10 minutes. Centrifuga u 1000g za 10 minuta.
- Repeat step 9 twice. Ponovite korak 9 puta.
- Finally suspend the protoplast pellet at a density of 1*105 ml/l in the modified protoplast culture medium. Konačno stanje mirovanja, protoplast metak u gustoći 1 * 105 ml / l u izmijenjeni protoplast culture medium.
- Plate the protoplasts as fine thin layer in petriplates. Plate, protoplasts kao fini tanki sloj u petriplates.
- Incubate the plates at 25 oC in dark. Izleći piliće na ploče na 25 oC u tamno.
Modified Protoplast Culture Medium Promijenio Protoplast Kultura Medium
S.No. S.No. | Constituents Komponente | Amount Količina |
A Neki | Mineral Salts Mineralnih soli | |
1 | Ammonium Nitrate Amonij nitrat | 82.5 gm/l 82,5 gm / l |
2 | Potassium Nitrate Kalij nitrat | 95.0 gm/l 95,0 gm / l |
3 | Boric acid Borić kiselina | 1190 mg/l 1190 mg / l |
4 | Potassium Dihydrogen Phosphate Kalij Dihydrogen Fosftna | 34 mg/l 34 mg / l |
5 | Potassium Iodide Kalij Iodide | 166 mg/l 166 mg / l |
6 | Sodium Molybdate Natrij Molybdate | 250 mg/l 250 mg / l |
7 | Cobalt Chloride Dihydrate Kobalt Chloride Dihydrate | 25.0 mg/l 25,0 mg / l |
8 | Calcium Chloride Dihydrate Calcium Chloride Dihydrate | 88.0 gm/l 88,0 gm / l |
9 | Magnesium Sulphate Heptahydrate Magnesium Sulphate Heptahydrate | 74.0 gm/l 74,0 gm / l |
10 | Manganese Sulphate tetrahydrate Mangan Sulphate tetrahydrate | 4460 mg/l 4460 mg / l |
11 | Zinc Sulphate Heptahydrate Cink Sulphate Heptahydrate | 1720 mg/l 1720 mg / l |
12 | Copper Sulphate Pentahydrate Bakrenim Sulphate Pentahydrate | 25.0 mg/l 25,0 mg / l |
13 | Ethylene Diamino Tetra Acetic acid Disodium salt Etilen Diamino Tetra octena kiselina Disodium sol | 7.45 gm/l 7,45 gm / l |
14 | Ferrous Sulphate heptahydrate Željezni Sulphate heptahydrate | 5.57 gm/l 5,57 gm / l |
B | Sugars Šećeri | |
1 | Glucose Glukoza | 30.0 gm/l 30,0 gm / l |
2 | Ribose | 0.5 gm/l 0,5 gm / l |
3 | Mannose | 0.5 gm/l 0,5 gm / l |
4 | Sorbitol | 0.5 gm/l 0,5 gm / l |
C | Organic Acids Organske kiseline | |
1 | Sodium Pyruvate Natrij Pyruvate | 5.0 mg/l 5,0 mg / l |
2 | Citric acid Limunska kiselina | 10.0 mg/l 10,0 mg / l |
3 | Malic acid Jabučna kiselina | 10.0 mg/l 10,0 mg / l |
D | Vitamins Vitamini | |
1 | Thiamine Hydrochloride Thiamine hidroklorid | 100 mg/l 100 mg / l |
2 | Nicotinamide | 500 mg/l 500 mg / l |
3 | Pyridoxine Hydrochloride Pyridoxin hidroklorid | 500 mg/l 500 mg / l |
4 | Calcium pantothenate Calcium pantothenát | 250 mg/l 250 mg / l |
5 | Biotin | 100 mg/l 100 mg / l |
6 | Ascorbic acid Askorbinska kiselina | 75.0 mg/l 75,0 mg / l |
7 | Riboflavin | 50.0 mg/l 50,0 mg / l |
8 | Inositol Inozitol | 100 gm/l 100 gm / l |
E | Hormones Hormoni | |
1 | 2,4-Dichloro Phenoxy Acetic Acid (2,4-D) 2,4-Dichloro Phenoxy octena kiselina (2,4-D) | 0.5 mg/l 0,5 mg / l |
2 | Benzyl Amino Purine (BAP) Benzyl Amino Purine (BAP) | 1.0 mg/l 1,0 mg / l |
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