Plant Physiology

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Red Cabbage Protoplasts: Preparation & Appearance

Background Information:
Viable protoplasts are generally spherical, exclude the dye Evan's blue and exhibit cytoplasmic streaming (cyclosis). Protoplasts accumulate neutral red, a dye, in the vacuoles. The influence of osmotic environment can easily be observed by treating protoplasts with solutions of various osmolality - the protoplast volume changes accordingly.

Question(s): How are protoplasts obtained and purified from plant tissues such as from red cabbage? What is the appearance of a protoplast? Where is the anthocyanin pigment located in red cabbage protoplasts? What do leaf protoplasts from other species/tissues look like?

Hypotheses: Cell wall-digesting enzymes can be used to release protoplasts from cells of red cabbage and other species. Protoplasts will appear spherical with a large central vacuole. Vacuoles will be pigmented.

Protocol:

Slice the washed and gently dried leaves of red cabbage (Brassica oleracea, Brassicaceae) into thin strips approximately 0.5 - 1 mm wide using a sharp razor blade. Work in a petri dish or similar surface. To increase the yield of pigment-containing protoplasts, strip the epidermis from some of the cabbage leaves by bending and tearing the leaf.
Transfer the strips as they are cut to a flask containing 50 mL of digestion medium [0.7 M sorbitol (4.56 g), 2% cellulase (1.0 g), 0.3% Maceroenzyme (0.15), 5 mM CaCl2 (7.35 mg); 5 mM MES (49 mg); adjust to pH 5.5 with KOH before adding the enzymes].
Incubate the flask at 25 C overnight (statically or preferably with gentle shaking).
Carefully remove the digestion medium. Although this will contain some protoplasts that were released during the incubation, normally there are relatively few. Save this solution just in case. To free the protoplasts from the partially digested plant tissue, wash the plant tissue three times by shaking gently with 20 ml of wash medium [to make 100 ml - 0.7 M sorbitol (9.11 g), 1 mM CaCl2 (14.7 mg); 5 mM MES (98 mg); adjust to pH 6 with KOH].
Pour each wash through a tea strainer (0.5 - 1.0 mm pore size).
Filter the combined washes through a nylon mesh or other filter (100 - 200 �m pore size).
Transfer the protoplast suspension to a centrifuge tube and collect the protoplasts by gently centrifuging (50 - 100 x g for 3 minutes).
Pour off the supernatant, add 10 mL of fresh wash medium to the pellet and gently resuspend the protoplasts. This protoplasts can be further purified if necessary.
Place a drop of the red cabbage protoplast suspension in a depression slide and add a cover slip.
Observe the 3-D nature of the protoplast by focusing up and down through its entire depth. Look for cyclosis (cytoplasmic streaming - often easiest to see in the transvacuolar strands). On a separate sheet of paper, sketch several protoplasts from each sample. Alternately, take a digital photograph. Give your figure a caption and label it Figure 1. Label the structures that are visible such as vacuole, cell membrane, tonoplast, nucleus, trans-vacuolar strands, and/or chloroplasts. Indicate the plate magnification (size of image divided by the size of the sketch - same units).
Repeat with a drop of the protoplast suspension from geranium, paperwhite, amaryllis or other species available in lab.

Analysis & Conclusions:
Do you observe different types of protoplasts? How do they differ? Offer and explanation. Did the protoplasts appear as you expected? Explain. Did you observe tissue debris in your sample or was it reasonably pure? Explain. Did you observe cyclosis? Describe it. If not, offer a possible explanation(s) of why you didn't. How did the red cabbage protoplasts differ from the other species we studied?