Lab Notes and Procedures-08 Biology 1004
North Arkansas College
Topic: Cloning African Violets
Sterilization of Plant Tissue
- Cut one fully expanded healthy African Violet leaf. Avoid using bruised or damaged tissue.
- In the hood, use sterile forceps to dip the leaf in 70% ethanol and swirl for 10 seconds.
- Transfer to beaker with 0.5% bleach and swirl for 20 seconds. From this point on, the tissue should be treated as sterile. Take precautions not to touch or contaminate the plant tissue.
- Using sterile forceps (flame off the alcohol) transfer the tissue to the 1% bleach and swirl for 30 seconds.
- Use the sterile forceps to transfer (dip) the leaf to (in) 4 beakers of sterile water consecutively.
- After the final rinse, transfer the leaf to a sterile petri dish.
Explanting
- Use sterile forceps and scalpel to excise 4-6 pieces (0.5-1.0 cm) of sterile leaf tissue. Cut pieces from different areas of the leaf. Include some tissue from near the main vein. Return forceps to alcohol.
- Use sterile forceps to transfer the pieces to 2 plates of explant media (shoot initiation). Place the piece upright with about ¼ of the tissue inserted in the media. Replace the lids and label with preparer’ initials.
- Wrap the plates with a double strip of parafilm. Hold the strip with your thumbs and stretch it with your other hand to pull it tightly around the plates. Overlap at ends and seal.
- Incubate plates upright under a grow light for 3-5 weeks.
