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12月10日 通宵搞定的植物生理实验报告Plasmolysis and Deplasmolysis as a Viability Assay for Plant Cells and Measurement for Osmotic Potential of Plant Tissue
AUTHOR: Yi Yin STUDENT ID: 05221098 DEPARTMENT: Department of Bioscience and Biotechnology CLASS: 2005 DATE: 2006-12-04 PERSONNEL OF GROUP: Hongmei Chen; Yi Yin
ABSTRACT 本报告记录了“植物细胞死活的鉴定和植物组织渗透势的测定---质壁分离法”实验的原理、方法及结果，并对实验所用溶液的选择和实验结果的意义进行了进一步讨论。 本实验基于质壁分离及质壁分离复原现象发生的条件之一---质膜的半透性，将质壁分离法用于鉴定植物细胞的死活，并利用植物活细胞的质壁分离现象，测定等渗浓度，并进一步测定植物组织的渗透势。实验均以洋葱鳞叶为材料，以蔗糖溶液为实验试剂。
INTRODUCTION
The plant is a dynamic system that is normally not at equilibrium with its environment. Water, for instance, is constantly moving into the plant, within it, and out of it. These movements cannot, however, be detected by measurements of water content, for the plant is commonly in the steady state, the water content of its cells and tissues remaining essentially constant, because of approximately equal rates of absorption and loss of water. In order to understand and further to predict the direction and rate of such movements, the energy levels of the water must be known – in its individual cells, and in its environment. Plasmolysis is the separation of plant cell cytoplasm from the cell wall as a result of water loss, which is induced in the laboratory by immersing a plant cell in a strongly saline or sugary solution, so that water is lost by osmosis. If onion epidermal tissue is immersed in a solution of sucrose, cells rapidly lose water by osmosis and the protoplasm of the cells shrinks. This occurs because the molecules of sucrose freely permeate the cell wall and encounter the selectively permeable plasma membrane. The large vacuole in the center of the cell originally contains a dilute solution with much higher osmotic potential than that of the sucrose solution on the other side of the membrane. The vacuole thus loses water and becomes smaller. The space between the cell membrane and the cell wall enlarges and the plasma membrane and the protoplasm within it contract to the center of the cell. Strands of cytoplasm extend to the cell wall because of plasma membrane-cell wall attachment points. However, plasmolysis and deplasmolysis can only be observed in living cells because of its requirement of the semipermeability of the membrane. Thus, plasmolysis and deplasmolysis can be used as assay of viability of plant cells. The basic equation for calculating the osmotic potential in bars is: ψπ= -icRT (equation 1) where ψπ = osmotic potential (bar) i = isotonic coefficient (1 for sucrose solution) c = isotonic concentration(mol/L) R = gas constant (0.083 L·bar/mol·K) T = absolute temperature (K) 297K
MATERIALS AND METHODS
Experiment 1: 1. Prepare 1 or 2 temporal sections from the purple side of a scale of red onion, which are one-cell thick. Add 1 or 2 drops of distilled water and examine it with the light microscope. (Fig.1) 2. Prepare 50ml of 1.0 mol/L sucrose solution, add 2 or 3 drops to the same tissue and examine it with the light microscope. Plasmolysis is expected to be observed. (Fig.2) 3. Add distilled water from one side of the cover slip till the tissue is immersed in distilled water again and examine it with the light microscope. Deplasmolysis is expected to be observed. 4. Prepare another temporal section from the purple side of a scale of red onion, which is one-cell thick. Add 1 or 2 drops of distilled water and heat it with the alcohol burner so that the cells are killed and repeat step 2. 5. Analyze the different phenomena of step 2 and 4.
Experiment 2: 1. From the above stock solution of sucrose, prepare the following dilutions in culture dishes: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mol/L. 2. Prepare 24 or more temporal sections from the purple side of a scale of red onion, which are one-cell thick and from similar positions of the scale. Immerse 3 sections every 3 minutes in the sucrose solution (from higher concentration to the lower). 3. Examine each section for plasmolysis after 30 minutes. Identify the solution in which about 50% of these cells are just incipiently (slightly) plasmolyzed, the rest showing no detectable plasmolysis. This solution is isotonic for the epidermal cells. If all cells are plasmolyzed in one solution and none in the one just below it in concentration, the concentration halfway between the two is the isotonic solution. 4. Calculate the osmotic potential in bars by means of equation 1.
RESULTS
Results for Experiment 1:
Results for Experiment 2:
The concentration of the isotonic solution is: (0.4 mol/L + 0.5 mol/L) / 2 = 0.45 mol/L
ψπ = -icRT = -1*0.45mol/L*0.083 L·bar/mol·K*297K = -11.09bar
DISCUSSION
1. Comparison between the chosen solution sucrose and a suggested better one CaCl2 from further reading of Experimental Plant Physiology (See Reference 1): liquid exchange methods have been criticized both in the past and by modern investigators. These criticisms are mainly caused by the use of unphysiologic solutions, the solute consisting usually of a single pure sugar or sugar alcohol (sucrose, glucose mannitol, and so on) or of synthetic substances. The problems can be avoided by using a properly balanced solution (for example, nine parts of NaCl to one part of CaCl2) or more simply a pure CaCl2 solution. Both of these solutions tend to maintain the normal semipermeability of the cells and therefore prevent leakage of the cell contents. Maintenance of cell normalcy is also aided by the much shorter time that the cells or tissues need to be left in these salt solutions, because they come to equilibrium with the salt solutions about six times more rapidly than with the sugar solutions. This is because of the far lower viscosity of the solutions and the much higher diffusivity constants of the dissolved salts. 2. According to ψw = ψπ + ψp + ψg + ψm, (equation 2) where ψw = water potential ψπ = osmotic potential ψp = pressure potential ψg = gravitational potential ψm = matric potential and that water potential and osmotic potential are never greater than zero and are, therefore, normally negative quantities; the pressure potential is positive in turgid cells and zero or negative in flaccid cells; the gravitational potential and matric potential contribute too little to be concerned in cells with large vacuoles, the water potential of a cell or tissue is equal to the osmotic potential of a solution that neither gives water to nor removes water from the cell or tissue. Its osmotic potential is equal to the osmotic potential of a solution that reduces the turgor pressure of the cell or tissue to zero. So, the osmotic potential drawing out as the result is equal to the water potential of the cell.
CONCLUSION
1. Plasmolysis and deplasmolysis can be used as a viability assay for plant cells. 2. The osmotic potential of the experimental material is -11.09bar.
REFERNCES
1. Anthony San Pietro ed.1974. Experimental Plant Physiology. The C. V. Mosby Company, 155-161 2. Yidian Wang, Ning Liu ed. 2001. Experimental Guidance for Plant Biology. Beijing Higher Education Press, 40-42 3. Hans Mohr, Peter Schopfer ed. 1995. Plant Physiology. Springer-Verlag Berlin Heidelberg, 42-51 4. http://3e.plantphys.net/ |
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