Protoplast Isolation

Technical & Teaching Notes

Apparatus

 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 m gauze square (approximately 12 cm X 12 cm)
 tape
 centrifuge tube
 slide and coverslip

A centrifuge, high power microscope and incubator set at approximately 35 oC must be

available in the lab.

Preparation of Materials

It is best to use limp green lettuce which has been left in an incubator at about 35 oC for
an hour. This causes the cells to plasmolyse slightly.

Viscozyme, pectinase and cellulase enzymes can be bought from NCBE. Do not dilute
the enzymes. The enzyme solution should be stored in a fridge until use.

Sorbitol solution (70%) can be bought from Boots the Chemist. To prepare a 13%
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.

Making the fusion mix

PEG (12 ml. of 50% solution)

HEPES buffer 0.9g
10 ml. water
pH 8.0

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.

Suppliers

Viscozyme, pectinase and cellulase enzymes can be bought from NCBE.

Sorbitol solution (70%) can be bought from Boots the Chemist.

60 m gauze can be ordered from Clarcor, www.clarcoruk.com, (0)1925 654321

PEG 6000 (50% solution) and HEPES buffer can be ordered from Sigma-Aldrich (was
Fluka Chemicals) www.sigmaaldrich.com

A microcentrifuge can be obtained from the NCBE, http://www.ncbe.reading.ac.uk/

Teaching Notes

Protoplasts are cells which 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 which causes the cells to plasmolyse). 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 which 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 for 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.

Teachers’ Queries

In your document about Protoplast Isolation there is a line that reads "Fusion
products can be selected for on media containing antibiotics or herbicides. These
can then be induced to form shoots which grow into roots and hybrid plants that
can be tested for desirable characteristics." I was wondering how this would be
carried out?

Protoplasts from plant A with herbicide resistance could be fused directly with
protoplasts from plant B which has other desirable qualities (eg potatoes which make
good chips). In that case when you grow the protoplasts into calli, and then shoots, i)
some calli will be from unfused protoplasts from the parent plants, ii) some from fusion
products which result from protoplasts from plant A fusing with other plant A
protoplasts, iii) some from fusion products resulting from protoplasts from plant B fusing
with other plant B protoplasts, and iv) a few which are the result of fusion between plant
A and B protoplasts - THESE are the ones you want. You have to have some way of
selecting the calli/shoots which are of the latter type. If all the calli/shoots are placed on a
medium containing the herbicide, then only calli/shoots which have the gene for
herbicide resistance from plant A can grow ( these could have been regenerated from
either fused or unfused protoplasats of plant A). You can carry out other tests (eg DNA
analysis) to find out which ones are the desired fusion products, with the qualities of both
parents. This is hugely simplified but the basic idea is correct.
 
Alternatively if protoplasts have been transformed (eg by incubation with Agrobacterium
containing a geneticallly modified plasmid which contains a marker gene for antibiotic
resistance along with a desirable gene), then when the calli initially formed start to
produce shoots (usually on a medium containing a high cytokinin:auxin ratio) some of
them will produce shoots with the desired gene AND with antibiotic resistance. If grown
on medium containing that antibiotic, only shoots with the antibiotic resistance gene (and
the desired gene) will grow (ie it is a selective medium). This is the cause of a lot of
debate, as many people feel that antibiotic resistance will with the antibiotic resistance
gene are grown.

What varieties of lettuce could be used for protoplast fusion?

We advise that you use the 'normal' green lettuce as a source of green protoplasts and try
to isolate protoplasts from a red lettuce, preferably the least crispy variety you can find.
That way you can count not only the percentage of protoplasts fusing but also calculate
the percentage of heterofusions (red-green) and the percentage of homo fusions (green-
green or red-red) which obviously would not be desirable in a fusion experiment where
one is trying to combine the characteristics of two different varieties/species. 
 
The problem you may encounter is that the osmoticum (sorbitol) concentration may need
to be altered for the red lettuce, and that is an investigation in itself!  But it should be
possible and would yield lovely quantitative results.

Acknowledgements

Dr Lucy Payne, Dollar Academy

This experiment is modified from an original by NCBE

http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PRACBIOTECH/protoplasts.html