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 cm
3 stock to 100 cm
3 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
- Place 12.5 cm3 of 13% sorbitol solution into the Petri dish.
- Place the squares of lettuce leaf bottom side in the Petri dish.
- Cut up the lettuce leaf into pieces approximately 5 mm × 5 mm.
- Place the lid on the dish and incubate in the oven at 35°C for 5 minutes.
- Add the viscozyme and cellulase enzymes to the dish. Swirl gently.
- Incubate for a further 20 minutes; swirl gently at regular intervals.
- Place the 60-mm mesh into the funnel and secure with tape.
- Hold the funnel over the lid of the Petri dish.
- Pour the digested lettuce into the filter funnel. Try to minimize the distance
the protoplasts have to fall!
- Place the funnel over a centrifuge tube and gently pour the contents of the
Petri dish lid through the funnel again.
- Balance your tube with another and centrifuge for 5–10 minutes at 2000
rpm.
- 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.
- Place a drop of the suspension on a slide, cover with a cover slip, and
examine under high power.
- 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.
- 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
- Place a drop of the protoplast suspension on the glass slide.
- Observe under the medium power of the microscope.
- Add 1 drop of the fusion mix.
- Observe over a period of ~3 minutes.