Protoplast Isolation Protocol Protoplastfusion Dyrkning protokol
Preparation of Metabolically Competent Protoplasts Forberedelse af metabolisk Kompetente Protoplasts
Saji Menon and Vibha Tyagi Saji Menon og Vibha Tyagi
Birla Institute of Scientific Research, Statue circle, Jaipur (Rajasthan), India Birla Institute of Scientific Research, Statue cirkel, Horsens (Rajasthan), Indien
Materials Required for Protoplast Isolation Påkrævede materialer til protoplastfusion Dyrkning
- 70% Ethanol 70% ethanol
- 0.1% Mercuric Chloride 0,1% Mercuric Chlorid
- 0.5M Mannitol 0.5M Mannitol
- Enzyme solution : Enzyme løsning:
- 1% Cellulase 0.25% Macerozyme R-10 1% Cellulose 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 : Cell protoplastfusion Washing 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 Modified protoplastfusion Kultur medium
(Table is listed below) (Tabel er angivet nedenfor)
Protocol for the preparation of Metabolically Competent Protoplasts Protokol for forberedelse af metabolisk Kompetente Protoplasts
- Take fresh leaves from 4-5 week old aseptically growing plants. Tag friske blade fra 4-5 uger gamle aseptisk voksende planter.
- Surface sterilize in 70% Ethanol followed by treating with 0.1% Mercuric chloride for 1 minute. Overflade sterilisere i 70% ethanol efterfulgt af behandling med 0,1% Mercuric chloride for 1 minut.
- Wash the leaves thoroughly with sterile distilled water. Vask bladene grundigt med sterilt destilleret vand.
- Peel the lower epidermis of the leaf tissue and place it in 0.5M Mannitol solution for one hour in a petriplate. Skræl den nederste hud af blade væv og placere den i 0.5M Mannitol løsning for en time i en petriplate.
- Remove the Mannitol solution and replace it with filter sterilized enzyme solution. Fjern Mannitol løsning og erstatte det med filter steriliseret enzym løsning.
- Incubate the plates in dark for 16-20 hours at 25oC. Pladerne inkuberes i mørke i 16-20 timer ved 25oC.
- Separate the protoplast layer by filtration using 0.45µM nylon membrane. Adskil de protoplastfusion lag ved filtrering benytter 0,45 μ M nylon membran.
- Centrifuge the filtrate at 1000g for 10 minutes at 4 oC. Centrifuger filtratet på 1000g i 10 minutter ved 4 oC.
- Remove the supernatant and resuspend the pellet in cell protoplast washing medium. Fjern supernatanten og resuspender pillen i cellen protoplastfusion vask medium.
- Centrifuge at 1000g for 10 minutes. Der centrifugeres ved 1000g i 10 minutter.
- Repeat step 9 twice. Gentag trin 9 to gange.
- Finally suspend the protoplast pellet at a density of 1*105 ml/l in the modified protoplast culture medium. Endelig suspendere protoplastfusion pillen ved en densitet på 1 * 105 ml / l i den modificerede protoplastfusion dyrkningsmediet.
- Plate the protoplasts as fine thin layer in petriplates. Plade de protoplasts så fine tynde lag i petriplates.
- Incubate the plates at 25 oC in dark. Pladerne inkuberes ved 25 oC i mørke.
Modified Protoplast Culture Medium Modified protoplastfusion Kultur Medium
S.No. S.No. | Constituents Komponenter | Amount Mængde |
A En | Mineral Salts Mineralsalte | |
1 | Ammonium Nitrate Ammonium Nitrat | 82.5 gm/l 82,5 gm / l |
2 | Potassium Nitrate Kaliumnitrat | 95.0 gm/l 95,0 gm / l |
3 | Boric acid Borsyre | 1190 mg/l 1190 mg / l |
4 | Potassium Dihydrogen Phosphate Kaliumdihydrogenphosphat | 34 mg/l 34 mg / l |
5 | Potassium Iodide Kaliumjodid | 166 mg/l 166 mg / l |
6 | Sodium Molybdate Natrium Molybdat | 250 mg/l 250 mg / l |
7 | Cobalt Chloride Dihydrate Cobalt chlorid dihydrat | 25.0 mg/l 25,0 mg / l |
8 | Calcium Chloride Dihydrate Calcium Chloride Dihydrat | 88.0 gm/l 88,0 gm / l |
9 | Magnesium Sulphate Heptahydrate Magnesiumsulfat heptahydrat | 74.0 gm/l 74,0 gm / 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 Kobbersulfat pentahydrat | 25.0 mg/l 25,0 mg / l |
13 | Ethylene Diamino Tetra Acetic acid Disodium salt Ethylen Diamino Tetra Eddikesyre dinatriumsalt | 7.45 gm/l 7,45 g / l |
14 | Ferrous Sulphate heptahydrate Heptahydratiseret jernsulfat | 5.57 gm/l 5,57 g / l |
B | Sugars Sukker | |
1 | Glucose | 30.0 gm/l 30,0 gm / l |
2 | Ribose | 0.5 gm/l 0,5 g / l |
3 | Mannose | 0.5 gm/l 0,5 g / l |
4 | Sorbitol | 0.5 gm/l 0,5 g / l |
C | Organic Acids Organiske syrer | |
1 | Sodium Pyruvate Natrium pyruvat | 5.0 mg/l 5,0 mg / l |
2 | Citric acid Citronsyre | 10.0 mg/l 10,0 mg / l |
3 | Malic acid Æblesyre | 10.0 mg/l 10,0 mg / l |
D | Vitamins Vitaminer | |
1 | Thiamine Hydrochloride Thiamin hydrochlorid | 100 mg/l 100 mg / l |
2 | Nicotinamide Nikotinsyreamid | 500 mg/l 500 mg / l |
3 | Pyridoxine Hydrochloride Pyridoxinhydrochlorid | 500 mg/l 500 mg / l |
4 | Calcium pantothenate Calciumpantothenat | 250 mg/l 250 mg / l |
5 | Biotin | 100 mg/l 100 mg / l |
6 | Ascorbic acid Ascorbinsyre | 75.0 mg/l 75,0 mg / l |
7 | Riboflavin | 50.0 mg/l 50,0 mg / l |
8 | Inositol | 100 gm/l 100 gm / l |
E | Hormones Hormoner | |
1 | 2,4-Dichloro Phenoxy Acetic Acid (2,4-D) 2,4-Dichlor Phenoxy eddikesyre (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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