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

Protoplasts:  Pre-Lab Preparation

 I. Readings. To prepare for this lab you should read:

• Print copies of all materials for Lab 3.  Read these materials and bring copies to lab.
• Read any appropriate background information (text, articles) as necessary
• You may want to print copies of equations and conversions and exponential numbers

II. Additional Preparation. It would be helpful to review:

• the operation of a compound light microscope
• carbohydrate structure and chemistry
• the use of the spectrophotometer
• simple statistical procedures (i.e., mean, standard deviation). Visit the statistics web site for the Biology department, and/or check the articles in the filing cabinet.

III. Questions: (Answer on a separate sheet; show your work)

1. List the organelles you expect to see in a plant cell or protoplast with a light microscope.
   
   
2. What determines plant cell shape? 
   
   
3. What shape do you predict for the protoplasts? Why?
   
   
4. How many grams of sucrose (MW = 342) is required to make 1.0 liter of a 0.5 M sucrose solution?
   
   
5. How many grams of sucrose is required to make 1 liter of a 25% sucrose solution? What is the molarity of this solution?
   
   
6. What is cyclosis and do you expect to observe it in the protoplasts?
   
   
7. What types of contaminants do you expect to find in the initial protoplast suspension?
   
   
8. Draw the chemical structures of sucrose, mannitol and glucose.
   
   
9. Make a flow chart depicting, in general terms, a protocol that could be used to determine the concentration of nitrate in a leaf vacuole.
   
   
10. The protoplast membrane is permeable to neutral red (True/False)
    
    
11. The protoplast membrane is permeable to Evan's blue (True/False)
    
    
12. The tonoplast is permeable to neutral red (True/False)
    
    
13. Define: transvacuolar strand, cellulase, pectinase, hemicellulase.
    
    
14. If the objects in Figure 1 in the hemocytometer write-up are chloroplasts, what is their concentration (chloroplasts � mL^-1 )? Assume no dilution. SHOW YOUR WORK.
                                               
15. Wagner (Plant Physiol. 64:88; 1979) reports that the average diameters of tulip leaf protoplasts and vacuoles are 80 �m and 75 �m, respectively.  (SHOW YOUR WORK)

1. Calculate the volume (�m³) of a protoplast ______________
2. Calculate the surface area (�m²) of a protoplast _________
3. Calculate the volume (�m³) of a vacuole ________________
4. What percent of the protoplast volume does the vacuole occupy in tulip leaf cells? ____________
5. Imagine that the vacuoles are placed in a hypotonic medium and swell to a diameter of 90 �m. How much water (expressed in units of �m³ or nL) diffused into the vacuole? _______________
6. The average concentration of glucose in a vacuole is 150 mM. What will be the new concentration of glucose (mM) in the vacuole after they absorb water (see E above) ? ______________

16. Moskowitz and Hrazdina (Plant Physiol. 68:686; 1981) studied the vacuolar contents of cells from the skins of DeChaunac grapes. In their protocol, from 200 mature grape skins they obtained 1.5 X 10⁷ vacuoles which was a yield of approximately 5%.  SHOW YOUR WORK.

1. Calculate the number of cells per grape skin ___________
2. The average diameter of a vacuole was 32 �m. What is its volume? __________ (�m³) = ___________ (pL)
3. They homogenized a sample of containing 15.7 X 10⁶ vacuoles and found that it had an anthocyanin content of 2.62 x 10^-5 mol. What is the anthocyanin content (pmol) in each vacuole?____________
4. What is the vacuolar concentration (Molarity) of anthocyanin? __________ M or __________ mM
5. Of the eight cations studied, Moskowitz and Hrazdina observed low concentrations except for potassium which they determined to be 46.3 pmol/vacuole. What is the molar concentration (M) of potassium in the vacuole? ____________
6. Magnesium had the next highest concentration in the vacuole, but it was only 2% of the potassium concentration. What was its molar concentration (M) in the vacuole? _________

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