Laboratory
Plant Biology 150L
Fall 2003
PROTOPLAST
Objectives of the laboratory exercise
I To observe enzymatic digestion of cell wall.
II To demonstrate the function of the cell wall in restricting cell shape and cell-cell adhesion in
leaf tissues.
III To demonstrate the hypotonic burst of protoplasts in water and in solutions.
Why are we doing this exercise?
Many of the differences between plants and animals --in nutrition, digestion, osmoregulation,
growth, reproduction, intercellular communication, defense mechanisms, and even morphology-can be traced to the CELL WALL. The cell wall provides a protective home for the protoplast
within. The wall of each cell binds to that of adjacent cells, cementing them together to form the
intact plant.
In this exercise, we will observe how a plant cell crawls out of plant tissues after the cell wall is
degraded by enzymes. We will see how a wall-less protoplast (a cell without wall) bursts in
solutions which harmlessly bathes a walled protoplast.
Relevant background information
The cellulosic walls of plant cells vary in thickness from 0.1 to many micrometers. In evolving
these relatively rigid cell walls, plant cells forfeited motility. This nonmotile life style has
persisted in multicellular plants.
It was the thick cell wall of cork (Quercus suber) that enabled Robert Hooke to distinguish cells
clearly and to name them as such. Even though each plant cell is encased within its own
“wooden box,” direct cell-to-cell communication is still possible through intercellular pores or
plasmodesmata, which cross the cell wall and link with adjacent cells, permitting movement of
molecules from cell to cell. In addition, fluids percolate along and through the cell walls. The
plant cell wall thus exhibits transport functions, protective functions and skeletal functions.
Newborn cells have a large potential for expansion. To accommodate expansion, the first wall is
thin and semirigid, and is referred to as the primary cell wall. The primary wall consists mainly
of cellulose, hemicellulose and pectin.
Once expansion stops, the cells often strengthen their walls by secondarily thickening the original
primary wall. This secondary thickening is accomplished by the deposition (inside the primary
wall) of another series of wall layers. These additional, strengthening wall layers are tougher
than the primary wall. They are composed of longer cellulose molecules and other specialized
components, such as lignin. These strengthening layers are called secondary cell walls.
The mechanical strength of the cell wall allows plant cells to survive in an extracellular
environment that is hypotonic with respect to the cell interior. This can be demonstrated by
removing the wall with cellulase and other wall-degrading enzymes, and observing the behavior
of wall-less cells (called protoplasts). If a spherical protoplast is exposed to the hypotonic fluid
that normally bathes the plant cell, it takes up water by osmosis, swells, and eventually bursts.
By contrast, a walled cell in the same solution still takes up water, but can swell to only within
the confines of the wall. The cell develops an internal hydrostatic pressure (or turgor pressure)
which pushes outward on the cell wall-- somewhat like an inner tube pressing against a bicycle
tire. This hydrostatic pressure brings the cell to osmotic equilibrium and prevents any further net
water influx of water and eventual rupture. Turgor pressure is the driving force for cell
expansion during plant growth and provides much of the mechanical rigidity of living plant
tissues.
Laboratory Procedure
Work in groups of 3-4 during this lab. You will need the following materials:
forceps, petri dishes, razor blades, small flasks, eppendorf tubes, 15 ml test tubes, centrifuge (low
speed clinical or hand-operated), enzyme solution (stock solution, containing 1.0% cellulase and
0.5% pectinase, and transmission microscopes.
Preparation of protoplasts
1 Remove carefully a half-expanded leaf from a fava bean plant (Vicia faba).
2 Put the leaf upside down in distilled water in a petri dish.
3 Using forceps, hold the leaf by the main vein and peel strips off the epidermal layer of the
lower leaf surface.
4 Cut out the area exposing the green mesophyll cells with a razor blade. Be careful not to
squeeze the leaf tissue. Slice them into small sections (0.5-1 mm wide).
5 Pipett 5 ml enzyme solution into a small 25 or 50 ml flask. Put the leaf pieces into the
enzyme solution in the flask.
6 Vacuum infiltrate the leaf for 30 seconds.
7 Leave the flask for 30 minutes at 28°C with gentle shaking provided by a shaking water bath.
8 Observe the release of protoplasts ("naked" plant cells) from the leaf pieces by taking a few
microliters of solution at different time intervals (e.g. 10', 20, 30', 60'). Also pay attention to
the morphology of the leaf pieces. You can take a slice of leaf tissue and observe under
microscope how it changes during enzymatic digestion.
NOTE: Some cells are released but they are still square-shaped. Why?
9 When a large number of protoplasts are observed in the solution, filtrate the leaf with 2 layers
of cheesecloth. Spin the solution by low speed centrifugation (100 xg) for five minutes.
10 Gently resuspend the cell pellet in 0.2 ml 0.6 M sorbital solution. Check under microscope
whether you can observe spherical cells in the solution. Those round cells are protoplasts.
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Burst of protoplasts in water or diluted salt solution
1 Prepare 3eppendorf tubes containing 0.5 ml of distilled water, 10 mM KCl, and 0.6 M
sorbital, respectively.
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slowly.
3 Leave the tubes for 10 minutes before observing the solutions under microscope.
4 Count the protoplasts in each tube.
Discussion
Discuss your observations according to the following questions:
What is the function of cell wall in cell shape, turgor, defense, and physiology?
What is the composition of cell wall?
How do the two enzymes remove the wall?
How can a walled cell survive while a wall-less cell ruptures in dilute solution?
How can we further demonstrate that cell wall is required for cell division?
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