Procedure
The tobacco mesophyll protoplasts used in this study are isolated according to Wu’s method [1]. The plant protoplasts are
prepared as follows:
1. Take fully expanded leaves of tobacco plants grown for 40–60 days (pick thick leaves in a similar growth state) to prepare
the protoplasts.
2. Excise fully expanded leaves and sterilize by rinsing with 75% alcohol for 10 s and 0.1% mercuric chloride for 4 min,
respectively, then wash three to five times with distilled water. Dry off leaves with sterilized filter paper.
3. Remove large vein tissues, pile two to four leaves, and cut only the middle part of the leaves into 0.5–1.0 mm strips using a
sharp razor blade without bruising the leaves.
4. Transfer leaf strips quickly and gently into the prepared enzyme (four to six tobacco leaves in 5–10 ml) by dipping both