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Protocol for Isolation of Protoplasts from Mesophyll Cells of Cereals -

1. Select leaves from 5-6-day-old seedlings by cutting at the base of the leaf. Discard the apical (0.5 cm) region.
2. Surface sterilize the leaves in 0.1 % Zephiran-10% ethanol for 5 min.
3. Wash twice in a solution containing 600 mmoll-1 sorbitol and 10 mmol 1-1 CaCl2
4. Cut leaves into transverse (1-2 mm wide) strips and transfer them to conical flasks containing enzyme solution (0.5% macerozyme, 1 % hemicellulase, 2% cellulysin, 600 mmol-1 sorbitol, pH 5.4). The proportion of enzyme solution for each gram of leaf tissue is 10:1.

5. Infiltrate the leaves under partial vacuum for 3-5 min.
6. After 2 h filter the leaf digest first through a 0.7 mm mesh nylon sieve and then through a 0.05 mm sieve.
7. Transfer the material that has passed through the sieves to centrifuge tubes and spin at 50 g for 90 s.
8. Remove the supernatant and wash the pellet thrice with washing medium.
9. Suspend the protoplasts in the nutrient medium and plate them in the culture medium.
10. Seal the culture plates with parafilm. Place them in large glass plates lined with filter paper moistened with 0.001 % CUSO4 Finally, store in darkness at 23°C.

 

  

 
 

 
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