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Protocol for Isolation of Protoplasts from Leaf Cells by Simaltaneous Method -
1. Select fully expanded leaves from 7- to 8-week-old plants growing in a greenhouse.
2. Surface sterilize the leaves by first immersing in 70% ethanol for 30 s, followed by rinsing in 0.4-0.5% sodium hypochlorite solution for about 30 min.
3. Peel the lower epidermis with fine forceps and cut out the peeled areas with a fine scalpel.
4. Place the peeled leaf pieces on a thin layer of 600 mmol 1-1 mannitol CPW solution in such a way that the peeled surface is in contact with the solution. CPW (cell protoplast washing medium mg 1-1 contains: KH2PO4(27.2), KNO3(101), CaCl2,2H2O(1480. MgSO4.7H2O(246). KI (0.16), CuSO4.5H2O(0.025; pH 5.8).

5. After about 30 min replace the mannitol-CPW solution by a filter sterilized solution of enzyme containing 4% cellulase, 0.4% cellulase, 0.4% macerozyme, 600 mmoll-1 mannitol and CPW salts.
6. Incubate the petri plate with enzyme solution and leaf pieces in darkness at 24-26°C for 16-18 h.
7. Gently squeeze the leaf pieces with a Pasteur pipette to liberate the protoplasts. Remove the large debris by passing through a 60-80 μm mesh sieve.

8. Transfer the sieved protoplast solution to a screw cap centrifuge tube and spin at 100 g for 10 min.
9. Remove the supernatant and resuspend the sediment in 860 mmoll-1 sucrose solution (prepared in CPW) in a screw cap centrifuge tube and spin at 100 g for 10 min.
10. Green protoplasts form a band at the top of the sucrose solution; transfer them with a pipette to another tube. Add the protoplast culture medium to the protoplasts and centrifuge at 100 g for 3 min. Repeat such washing three times.
11. After the final wash add enough culture medium to achieve a protoplast density of 0.5 x 105 to 1 X 105 ml-1.
12. Plate the protoplasts following any accepted plating procedure.

 
 

 

 
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