Protocol for Isolation of Protoplasts from Root Nodules
Protocol
for
Isolation
of
Protoplasts
from
Root
Nodules -
1. Wash the root nodules (1-5 mm in length) excised from aseptically grown plants inoculated with Rhizobium trifolii in protoplast dilution buffer (PDB). (PDB consists of: 250 mmoll-1 sorbitol, 250 mmoll-1 mannitol,10 mmoll-1 K2HPO4, 2 m1T1011-1 CaCl2, pH 5.8).
2. Cut the nodules into four pieces and again wash in PDB.
3. Incubate the nodule pieces in the enzyme solution (4% cellulysin, 2% macerase, 1% driselase in PDB, pH 5.8) in darkness at 23°C.
4. After 3-4 h dissociate the partially digested nodules by passing through the orifice of a Pasteur pipette and continue incubation for a further 90 minutes.
5. Sieve through a 50 I-Lm nylon mesh and wash the digested tissue twice in PDB by spinning at 200 g for 10 min.
6. Suspend the washed protoplasts in 30% sucrose solution and spin at 100 g for 10 min.
7. Collect the intact protoplasts from the top of the sucrose pad and plate them in a nutrient medium following usual procedures.
