PROTOPLAST PRODUCTION
(Plant Cells Without Walls)

Karen Kyker
1993 Woodrow Wilson Biology Institute

INTRODUCTION

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.

TARGET GROUP:

CLASS TIME REQUIRED:

CONSUMABLE MATERIALS:

EQUIPMENT:

CHEMICALS

TEACHER GUIDE FOR PREPARATION OF MATERIALS

A. Buffer Solution
(0.625 M mannitol, 500 ml)

1. Dissolve 56.94 g of mannitol in 200 ml of distilled water. Add distilled water to bring the final volume to 500 ml.

2. 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.

Notes:

1. 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)

2. 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.

3. 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)

4. 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 volumes.

5. MES is a pH buffer.

B. Enzymes: macerase and cellulysin

Notes:

1. 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).

2. Macerase is Sigma's proprietary name for a crude pectinase obtained from the fungus Rhizopus; pectinase separates cells from one another.

3. Cellulysin is Sigma's proprietary name for a cellulase isolated from Trichoderma uride. This enzyme degrades cell walls.

TEACHER OUTLINE FOR PRESENTATION OF ACTIVITY

1. 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).

2. 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).

3. 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.

4. Students produce protoplasts and answer analysis questions.

5. Students review their proposed transformation methods, determining which might have worked.

ADDITIONAL TEACHER INFORMATION

About 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.

SAFETY PRECAUTIONS

1. 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.

2. Preparation of protoplasts for transformation requires use of sterile technique, which requires the use of ethanol and flame.

3. Ethanol is FLAMMABLE!

STUDENT SHEET

MAKING PROTOPLASTS (Cells Without Walls)

Day One - Preparations

1. 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.
2. 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 completely dissolved.
3. Carefully pour all 10 ml of enzyme solution into the bottom of the Petri dish.
4. 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.
5. 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 helpful.
6. Check to make certain that your teacher has prepared a dish of leaf squares in buffer solution WITHOUT the enzymes.

   Day Two - Observations

7. 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.)
8. Prepare a slide of an untreated leaf square (from step 6) and observe the appearance of its cells.
9. To observe protoplasts:
   a. Remove the parafilm and lid and set the Petri dish under a dissecting microscope at 50X. or

   b. Use a large-bore (wide opening) pipette to gently remove some of the green solution from the bottom of the Petri dish and place it on a microscope slide. Cover with a cover slip and observe under scanning or low power with a light microscope.

Analysis Questions

1. 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 level.

2. Sketch several protoplasts from your preparation (with enzymes), and describe their appearance (and note magnification level).

3. In what ways do the protoplasts differ from untreated plant cells?

4. How do the protoplasts compare to animal cells (e.g. cheek cells)?

5. Prepare a slide of one of the leaf squares found in your dish of enzyme solution. Sketch and describe the edges (and note magnification level).

6. 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.

7. Why did this procedure require scratching and cutting the leaves into smaller pieces?

8. Why is it necessary to be "gentle" with protoplasts?

9. 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.

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