Protoplast Isolation Protocol Protoplastfusion Isolering protokoll
Preparation of Metabolically Competent Protoplasts Beredning av metaboliskt Behöriga Protoplasts
Saji Menon and Vibha Tyagi Saji Menon och Vibha Tyagi
Birla Institute of Scientific Research, Statue circle, Jaipur (Rajasthan), India Birla Institute of Scientific Research, Statue cirkel, Jaipur (Rajasthan), Indien
Materials Required for Protoplast Isolation Nödvändigt för protoplastfusion Isolering
- 70% Ethanol 70% etanol
- 0.1% Mercuric Chloride 0,1% merkuriklorid
- 0.5M Mannitol 0.5 miljoner euro Mannitol
- Enzyme solution : Enzyme lösning:
- 1% Cellulase 0.25% Macerozyme R-10 1% Cellulase 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) 0.5 miljoner euro Mannitol (pH 5,6)
- Cell Protoplast Washing Medium : Cell protoplastfusion Tvätt Medium:
- 0.5M Mannitol (pH 5.6) 0.5 miljoner euro 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 Ändrad protoplastfusion odlingssubstratet
(Table is listed below) (Tabell visas nedan)
Protocol for the preparation of Metabolically Competent Protoplasts Protokoll för beredning av metaboliskt Behöriga Protoplasts
- Take fresh leaves from 4-5 week old aseptically growing plants. Ta färska blad från 4-5 veckor gamla aseptiskt växande växter.
- Surface sterilize in 70% Ethanol followed by treating with 0.1% Mercuric chloride for 1 minute. Surface sterilize i 70% etanol följt av behandling med 0,1% merkuriklorid i 1 minut.
- Wash the leaves thoroughly with sterile distilled water. Skölj bladen noggrant med sterilt destillerat vatten.
- Peel the lower epidermis of the leaf tissue and place it in 0.5M Mannitol solution for one hour in a petriplate. Peel nedre epidermis av löv vävnad och placera den i 0.5 miljoner euro Mannitol lösning för en timme i en petriplate.
- Remove the Mannitol solution and replace it with filter sterilized enzyme solution. Ta bort Mannitol lösning och ersätta det med filter steriliserad enzym lösning.
- Incubate the plates in dark for 16-20 hours at 25oC. Inkubera plattorna i mörker i 16-20 timmar vid 25oC.
- Separate the protoplast layer by filtration using 0.45µM nylon membrane. Separera de protoplastfusion skikt genom filtrering använder 0,45 μ M nylon membrane.
- Centrifuge the filtrate at 1000g for 10 minutes at 4 oC. Centrifugera filtratet vid 1000g i 10 minuter vid 4 oC.
- Remove the supernatant and resuspend the pellet in cell protoplast washing medium. Avlägsna supernatanten och återsuspendera pelleten i cell protoplastfusion tvätt medium.
- Centrifuge at 1000g for 10 minutes. Centrifugera vid 1000g i 10 minuter.
- Repeat step 9 twice. Upprepa steg 9 två gånger.
- Finally suspend the protoplast pellet at a density of 1*105 ml/l in the modified protoplast culture medium. Slutligen avbryta protoplastfusion pellet vid en densitet på 1 * 105 ml / l på den modifierade protoplastfusion odlingssubstrat.
- Plate the protoplasts as fine thin layer in petriplates. Plåt de protoplasts så fina tunt lager i petriplates.
- Incubate the plates at 25 oC in dark. Inkubera plattorna vid 25 ° C i mörker.
Modified Protoplast Culture Medium Ändrad protoplastfusion odlingssubstratet
S.No. S.No. | Constituents Beståndsdelar | Amount Belopp |
A En | Mineral Salts Mineralsalter | |
1 | Ammonium Nitrate Ammoniumnitrathaltiga | 82.5 gm/l 82,5 g / l |
2 | Potassium Nitrate Kaliumnitrat | 95.0 gm/l 95,0 g / l |
3 | Boric acid Borsyra | 1190 mg/l 1190 mg / l |
4 | Potassium Dihydrogen Phosphate Kaliumdivätefosfat | 34 mg/l 34 mg / l |
5 | Potassium Iodide Kaliumjodid | 166 mg/l 166 mg / l |
6 | Sodium Molybdate Sodium Molybdate | 250 mg/l 250 mg / l |
7 | Cobalt Chloride Dihydrate Cobalt Chloride Dihydrat | 25.0 mg/l 25,0 mg / l |
8 | Calcium Chloride Dihydrate Kalciumkloriddihydrat | 88.0 gm/l 88,0 g / l |
9 | Magnesium Sulphate Heptahydrate Magnesium Sulphate heptahydrat | 74.0 gm/l 74,0 g / l |
10 | Manganese Sulphate tetrahydrate Mangansulfat tetrahydrat | 4460 mg/l 4460 mg / l |
11 | Zinc Sulphate Heptahydrate Zinksulfat heptahydrat | 1720 mg/l 1720 mg / l |
12 | Copper Sulphate Pentahydrate Kopparsulfat pentahydrat | 25.0 mg/l 25,0 mg / l |
13 | Ethylene Diamino Tetra Acetic acid Disodium salt Eten Diamino Tetra Ättiksyra Disodium salt | 7.45 gm/l 7,45 g / l |
14 | Ferrous Sulphate heptahydrate Järnsulfat heptahydrat | 5.57 gm/l 5,57 g / l |
B | Sugars Sockerarter | |
1 | Glucose Glukos | 30.0 gm/l 30,0 g / l |
2 | Ribose Ribos | 0.5 gm/l 0,5 g / l |
3 | Mannose Mannos | 0.5 gm/l 0,5 g / l |
4 | Sorbitol | 0.5 gm/l 0,5 g / l |
C | Organic Acids Organiska syror | |
1 | Sodium Pyruvate Natrium pyruvate | 5.0 mg/l 5,0 mg / l |
2 | Citric acid Citronsyra | 10.0 mg/l 10,0 mg / l |
3 | Malic acid Äppelsyra | 10.0 mg/l 10,0 mg / l |
D | Vitamins Vitaminer | |
1 | Thiamine Hydrochloride Tiaminhydroklorid | 100 mg/l 100 mg / l |
2 | Nicotinamide Nikotinamid | 500 mg/l 500 mg / l |
3 | Pyridoxine Hydrochloride Pyridoxinhydroklorid | 500 mg/l 500 mg / l |
4 | Calcium pantothenate Kalciumpantothenat | 250 mg/l 250 mg / l |
5 | Biotin | 100 mg/l 100 mg / l |
6 | Ascorbic acid Askorbinsyra | 75.0 mg/l 75,0 mg / l |
7 | Riboflavin | 50.0 mg/l 50,0 mg / l |
8 | Inositol | 100 gm/l 100 g / l |
E | Hormones Hormoner | |
1 | 2,4-Dichloro Phenoxy Acetic Acid (2,4-D) 2,4-Diklor Phenoxy ättiksyra (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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