Protoplast Isolation, Culture, and Fusion

Background information
Protoplasts are cells that have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed, the resulting protoplast is spherical in shape.

Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents that causes the cells to plasmolyze). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension, the protoplasts can be cultured on media that induce cell division and differentiation. A large number of plants can be regenerated from a single experiment—a gram of potato leaf tissue can produce more than a million protoplasts, for example.

Protoplasts can be isolated from a range of plant tissues: leaves, stems, roots, flowers, anthers, and even pollen. The isolation and culture media used vary with the species and with the tissue from which the protoplasts were isolated.

Protoplasts are used in a number of ways for research and plant improvement. They can be treated in a variety of ways (electroporation, incubation with bacteria, heat shock, high pH treatment) to induce them to take up DNA. The protoplasts can then be cultured and plants regenerated. In this way, genetically engineered plants can be produced more easily than is possible using intact cells/plants.

Plants from distantly related or unrelated species are unable to reproduce sexually, as their genomes/modes of reproduction, etc. are incompatible. Protoplasts from unrelated species can be fused to produce plants combining desirable characteristics such as disease resistance, good flavour and cold tolerance. Fusion is carried out by application of an electric current or by treatment with chemicals such as polyethylene glycol (PEG). Fusion products can be selected for on media containing antibiotics or herbicides. These can then be induced to form shoots and roots and hybrid plants can be tested for desirable characteristics.

Materials

• Lettuce (round, green lettuce, not iceberg)
• Scalpel or sharp knife
• Forceps
• Tile
• Glass Petri dish
• 10 cm³ syringe
• 1 cm³ syringe
• 13% sorbitol solution
• Viscozyme enzyme
• Cellulase enzyme
• Dropper
• Small filter funnel
• 60-mm gauze square (approximately 12 cm × 12 cm)
• Tape
• Centrifuge tube
• Slide and cover slip
• A centrifuge, high-power microscope, and incubator set at approximately 35°C must be available in the lab.

Preparation of Materials
It is best to use limp lettuce that has been left in an incubator at about 35°C for an hour. This causes the cells to plasmolyze slightly.

Do not dilute the enzymes. The enzyme solution should be stored in a fridge until use.

To prepare a 13% sorbitol solution, add 18.5 cm³ stock to 100 cm³ distilled water.

Waterproof sticky tape can be used to secure the gauze mesh in a small filter funnel. This can be washed and reused.

For Protoplast Fusion
A microcentrifuge can be used to spin the protoplasts. A spin of 3 minutes at the lowest voltage is sufficient. Care should be taken when resuspending the pellet, as the protoplasts are very fragile.

Making the Fusion Mix
- PEG (12 mL of 50% solution)
- HEPES buffer 0.9 g
- 10 mL water
- pH 8.0

Materials

• Lettuce leaf (2 squares, each approximately 3 cm × 3 cm)
• Scalpel or sharp knife
• Forceps
• Tile
• Glass Petri dish
• 10 cm³ syringe
• 1 cm³ syringe
• 13% sorbitol solution
• 1.2 cm³ viscozyme enzyme
• 0.6 cm³ cellulase enzyme
• Dropper
• Small-filter funnel
• 60-mm gauze
• Tape
• Centrifuge tube
• Slide and cover slip
• Centrifuge
• Incubator
• Microscope

Procedure

1. Place 12.5 cm³ of 13% sorbitol solution into the Petri dish.
2. Place the squares of lettuce leaf bottom side in the Petri dish.
3. Cut up the lettuce leaf into pieces approximately 5 mm × 5 mm.
4. Place the lid on the dish and incubate in the oven at 35°C for 5 minutes.
5. Add the viscozyme and cellulase enzymes to the dish. Swirl gently.
6. Incubate for a further 20 minutes; swirl gently at regular intervals.
7. Place the 60-mm mesh into the funnel and secure with tape.
8. Hold the funnel over the lid of the Petri dish.
9. Pour the digested lettuce into the filter funnel. Try to minimize the distance the protoplasts have to fall!
10. Place the funnel over a centrifuge tube and gently pour the contents of the Petri dish lid through the funnel again.
11. Balance your tube with another and centrifuge for 5–10 minutes at 2000 rpm.
12. The protoplasts will have formed a pellet at the bottom of the tube. Use the dropper to remove the liquid supernatant from the tube, without disturbing the pellet. Remove all but 0.5 cm³ of the liquid and then resuspend the pellet by gently tapping the tube.
13. Place a drop of the suspension on a slide, cover with a cover slip, and examine under high power.
14. If your suspension is too concentrated to see the protoplasts, clearly you can add several drops of the sucrose solution to the suspension in the centrifuge tube.
15. You should be able to try to fuse your protoplasts together if you have time.

Protoplast Fusion (Somatic Hybridization)

• Glass slide
• Cover slip
• Glass dropper
• Fusion mix
• Protoplast suspension
• Microscope

Instructions

1. Place a drop of the protoplast suspension on the glass slide.
2. Observe under the medium power of the microscope.
3. Add 1 drop of the fusion mix.
4. Observe over a period of ~3 minutes.