(Plant Cells Without Walls)
1993 Woodrow Wilson Biology Institute
This activity allows students to strip away the cell walls of plant cells (using enzymes) and then observe the resulting spherical protoplasts (plant cells minus the cell wall). Students see that plant cells indeed have a plasma membrane in addition to a cell wall.
The activity can also be used to introduce students to the concept of genetic transformation of cells (insertion of foreign DNA into cells). Bacterial, yeast, animal and plant cells have all been transformed. When transforming any type of cell, plasma membranes and/or cell walls must be penetrated without permanently damaging the cell. Different techniques are used to transform different types of cells, and the mechanisms of most of these techniques, while useful, are incompletely understood. Some types of plants can be transformed via infection with Agrobacterium tumefaciens. However, not all types of plants are susceptible to such infection (Walden, p.3) and direct DNA transfer is the usual alternative.
In the case of plants, removal of the cell wall is a first and crucial step required before introduction of DNA directly through the plasma membrane. Molecular biologists produce protoplasts when preparing to transform a variety of plant types, especially members of Solanacea (the potato and tomato family). Following protoplast production, the cell membrane can be made permeable to DNA by using electroporation (using an electric field) or polyethylene glycol (PEG). Microinjection of DNA is used less often. Protoplasts have also been used (with very limited success) to do "protoplast fusion," in which protoplasts from different species of plant are treated so that they fuse together, resulting in cross-species hybrid cells.
The following simple activity allows students to prepare protoplasts for observation. However, with modifications (also included), these protoplasts can also be used to do actual transformations or callus-induction and tissue-induction projects. Protocols for those projects are not included in this publication.
Introductory biology (9th or 10th grade) and beyond.
CLASS TIME REQUIRED:
Two days, 30 minutes each day (for actual protoplast production and observation). Additional activities, discussions, applications will require more time.
TEACHER GUIDE FOR PREPARATION OF MATERIALS
Preparation Time: 1 hour
A. Buffer Solution
(0.625 M mannitol, 500 ml)
- Dissolve 56.94 g of mannitol in 200 ml of
distilled water. Add distilled water to bring
the final volume to 500 ml.
- Measure 10 ml of the solution into a test tube
or beaker for each group OR have student-
groups measure their own 10 ml volumes from
a central supply during the lab activity.
- Mannitol is a disaccharide which helps to
maintain an osmolarity similar to that of the
protoplasts. A concentration of 12-14% is
needed. (Dodds, p. 5)
- The mannitol solution described above has a pH
of 4-5. Greater numbers of protoplasts have
been obtained by adding 0.1 N NaOH to the
mannitol solution to obtain pH 6-7. One drop of
NaOH should be sufficient to adjust the pH of 50
ml of solution.
- A 0.625 M sucrose solution (56.25 g
sucrose/500 ml water) has worked quite well
with tobacco leaves, and has been marginally
successful with Elodea and spinach leaves.
Sucrose (from the grocery store!) can be
metabolized by the cells, so concentration may
decrease over the incubation period, affecting
the osmotic buffering ability. (Dodds, p.6)
- For protoplast production with the intent to
do electroporation or other transformation
techniques, use a 0.6 M mannitol, 25 mM MES
buffer solution. Add 54.66 g mannitol and 2.44
g MES (Sigma catalog #M8652) to 200 ml
distilled water. Adjust pH to 5.7 using 1.0 N
NaOH (approximately 15 drops per 50 ml), then
add water to 500 ml total. Autoclave or
otherwise sterilize the solution in 100 ml
- MES is a pH buffer.
B. Enzymes: macerase and cellulysin
- Students add enzymes to buffer solution
immediately before use. These enzymes are not
stable at high temperatures or for long periods
of time. If working under sterile conditions, use
a syringe to push the freshly-made enzyme
solution through a filter (0.45u).
- Macerase is Sigma's proprietary name for a
crude pectinase obtained from the fungus
Rhizopus; pectinase separates cells from one
- Cellulysin is Sigma's proprietary name for a
cellulase isolated from Trichoderma uride. This
enzyme degrades cell walls.
TEACHER OUTLINE FOR PRESENTATION OF ACTIVITY
The procedure and analysis questions found on the student page can stand alone or can be embedded within any context provided by the teacher. The following outline uses the problem of transformation as the context for the activity.
- Introduce the concept of transformation
(introduction of foreign DNA into cells). Discuss
reasons why a person would transform cells (to
introduce new genes and therefore new traits
into a cell/organism).
- Have students describe cellular barriers (plasma
membranes, cell walls). Discuss substances that
are kept within the cell by these barriers (water,
solutes, organelles, DNA) and consider the fate of
a cell if these substances aren't kept within the
cell. Brainstorm and discuss reasons why cells
would evolve to keep DNA out of their cells (a
membrane that keeps DNA inside will probably
keep it out as well; if viral DNA/RNA enters, it
usually destroys the cell).
- Challenge groups of students to develop a
method for transforming plant cells without
killing the cells. Students develop a general
method with justifications for each step.
- Students produce protoplasts and answer
- Students review their proposed transformation
methods, determining which might have
ADDITIONAL TEACHER INFORMATION
Osmotic issues: A protoplast is a plant cell having a plasma membrane but no cell wall . Having no cell wall, protoplasts are very sensitive to osmotic differences and must be stored in an isotonic solution to prevent rupture. A hypertonic solution will result in very tiny protoplasts.
Appearance of protoplasts: Protoplasts are spherical, clearly 3-dimensional and float freely within the solution. Chloroplasts may be pushed against a small portion of the membrane by the vacuole (the vacuole membrane cannot be seen). The sizes of the protoplasts are generally consistent within a particular preparation, but different tissue sources and different osmotic solutions may result in varying sizes of protoplasts. Compare the size of the protoplasts with the size of cells in the untreated leaf squares.
Tissue sources: Various plant tissues can provide the cells for protoplast production. This procedure works well for leaf mesophyll of solanaceous plants (potato and tomato family). Protoplasts have also been isolated from suspension cultures (single cells floating and growing in solution), callus cultures (a callus is undifferentiated tissue), embryos, shoots and seedlings. Tissue with a thin cuticle works best.
Possible Extensions: Place protoplasts in different (known) osmotic solutions. Students can do dilutions of the buffer solution (0.625M) to obtain a range of solutions.
What do the results of these experiments suggest about the ability of plants to tolerate varying osmotic and dehydration conditions? Compare plants to animals in terms of tolerance to changing osmostic conditions and dehydration. Compare the different adaptations for cellular water-regulation found in land plants and land animals.
For further sterile work with protoplasts: Do all steps using sterile technique. For instructions on sterile technique and surface sterilization of leaves see SURFACE STERILIZATION
OF BIOLOGICAL MATERIALS. Use student procedure #1-7 and continue with the following steps.
8. Filter the protoplast/leaf suspension through
sterile cheese cloth into a sterile 50 ml conical
centrifuge tube. Rinse the Petri plate with 5-10
ml of sterile buffered mannitol without enzymes
and pass it through the cheese cloth filter as
well, washing protoplasts through the filter and
into the centrifuge tube.
9. Gently centrifuge at 75xg for 2 minutes in a
swing-out rotor (if available). Use a sterile
pipette to remove and discard the supernatent.
10.Treat the protoplasts according to your protocol.
Note: No protocols for transformation or
regeneration of protoplasts have been included
in this lesson.
Cell walls compared among four kingdoms: (All cells have plasma membranes)
animals: no cell wall; bacteria: cell wall of interlinked proteins and polysaccharides; yeast (fungi): cell wall of polysaccharides and chitin; plants: cell wall of cellulose (a polysaccharide) "embedded in a matrix of other polysaccharides and protein." (Raven and Johnson, 3rd edition, p. 87); cell walls can be of varying thicknesses in different tissues and ages of plant cells.
- If you intend to make protoplasts for observations
only, then use of careful sterile technique is not
necessary; the procedures can be performed
without any use of ethanol.
- Preparation of protoplasts for transformation
requires use of sterile technique, which requires
the use of ethanol and flame.
- Ethanol is FLAMMABLE!
MAKING PROTOPLASTS (Cells Without Walls)
Day One - Preparations
- Wash your hands. Obtain one or two leaves
from spinach, petunia or tobacco (or one whole
stalk of Elodea). Thoroughly rinse the leaves in
tap water then pat them dry. Lightly scratch
the undersides of the leaves with a pin or
needle. Then, cut or tear the leaves into
approximately one centimeter squares and place
them into the top half of a Petri dish. Obtain
enough leaf tissue to loosely cover the surface of
the top of the Petri dish.
- Measure 10 ml of buffer solution into a 15 ml
test tube or small beaker. Drop the pre-
measured, powdered enzymes into this solution.
Swirl the beaker or cap the test tube and shake
it back and forth until the enzymes are
- Carefully pour all 10 ml of enzyme solution into
the bottom of the Petri dish.
- Use forceps to float each leaf square on the
surface of the enzyme solution. Place the
LOWER EPIDERMIS in contact with the solution
(either surface of Elodea will work). The leaf
squares may overlap a bit.
- Seal the Petri dish with parafilm or tape and
leave it at room temperature (approximately
25deg.C) overnight. If proper equipment is
available, gentle agitation of the dishes will be
- Check to make certain that your teacher has
prepared a dish of leaf squares in buffer
solution WITHOUT the enzymes.
Day Two - Observations
- Gently swirl and shake the solution in the Petri
dish. (If no protoplasts are observed with the
microscope, let the solution stand for another
15-30 minutes then look again for protoplasts.)
- Prepare a slide of an untreated leaf square (from
step 6) and observe the appearance of its cells.
- To observe protoplasts:
- Sketch some cells from the leaf squares that
were soaked in buffer without enzymes. Label
all observable parts and describe the
appearance of the cells. Note the magnification
- Sketch several protoplasts from your
preparation (with enzymes), and describe their
appearance (and note magnification level).
- In what ways do the protoplasts differ from
untreated plant cells?
- How do the protoplasts compare to animal cells
(e.g. cheek cells)?
- Prepare a slide of one of the leaf squares found
in your dish of enzyme solution. Sketch and
describe the edges (and note magnification
- Based upon your observations of the protoplasts
and leaf squares, and your knowledge of plant
cell structure, hypothesize about the action of
the enzymes found in the enzyme solution.
- Why did this procedure require scratching and
cutting the leaves into smaller pieces?
- Why is it necessary to be "gentle" with
- DNA can be added to a solution that contains
specially-treated cells and under certain
conditions, the cells will "take-in" the DNA.
Such techniques are routinely used with
bacterial and yeast cells, without production of
protoplasts. Hypothesize about why protoplast
production is necessary before transformation
can take place with plant cells.