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

Determining the Frequency & Volume of Pigmented and Unpigmented Red Cabbage Protoplasts

Questions
:  What is the volume of a red cabbage leaf protoplast? How much variation exists in the size of protoplasts? Are unpigmented protoplasts the same size as pigmented ones? What percent of the red cabbage protoplasts are pigmented? Unpigmented? Why are some protoplasts colorless (unpigmented)?

Hypotheses:  Red cabbage protoplasts will be similar in size (diameter = 80 �m; volume = 2.68 x 105 �m3) to those reported for tulip protoplasts (Wagner, 1979). The protoplasts will exhibit normal variation in size. There will be a greater number of unpigmented protoplasts since the pigment is restricted to the epidermal cells in the red cabbage leaf.

Protocol:

1. Mix the sample thoroughly and then place a drop of protoplast suspension on a slide. Randomly focus on one field.  Count the number of pigmented and unpigmented protoplasts. Repeat for a total of five fields or until you have counted at least 100 total protoplasts. Calculate the percent of pigmented vs. unpigmented protoplasts. (complete Table 1)
2. Record the calibration data for the ocular micrometer, or determine it using a stage micrometer.
3. @ 40x 1 omu = ________ �m
@100x 1 omu = ________ �m
@400x 1 omu = ________ �m

magnification used to measure protoplasts = ______________

4. Measure the diameter (in ocular micrometer units) of 30 randomly-selected, pigmented protoplasts in another drop of the protoplast suspension at 400x.  Record your data in Table 2.

5. Measure the diameter (in ocular micrometer units) of 30 randomly-selected, unpigmented protoplasts in another drop of the protoplast suspension. Record your data in Table 3.

Data, Analysis & Conclusions:

1. Calculate the mean volume (and standard deviation) for both for pigmented and unpigmented protoplasts.  What do you conclude about our hypothesis that there are more unpigmented protoplasts?

2. Perform a t-test to determine if there is a statistically significant difference in size between the pigmented and unpigmented protoplasts.
3. Question: Are the pigmented and unpigmented protoplasts the same size?
Ho
:
t =  ________________      p =  __________________

Conclusion:

4. Plot a frequency distribution for the pigmented protoplasts. Append your graph as Figure 1. What do you conclude from this graph?
5. Are protoplasts from cabbage and tulip (or other species) similar in size? Explain.

 Table 1:  Pigmented and Unpigmented Red Cabbage Protoplasts Field Total % Total 1 2 3 4 5 Pigmented Unpigmented

 Table 2: Diameter and volume of pigmented red cabbage protoplasts in 0.7 M mannitol Protoplast Diameter (omu) Diameter (�m) Radius (�m) Protoplast volume (�m3) Protoplast volume (pl) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Mean (+/- std dev)

 Table 3: Diameter and volume of unpigmented red cabbage protoplasts in 0.7 M mannitol Protoplast Diameter (omu) Diameter (�m) Radius (�m) Protoplast volume (�m3) Protoplast volume (pl) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Mean (+/- std dev)

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Last updated:  01/07/2009     � Copyright  by SG Saupe