The early growth of a seedling is marked by expansion of the cotyledon, particularly in species with epigeous germination. Not surprisingly, the expansion is accomplished by some combination of cell expansion and cell division. The cell expansion hormones (auxin and gibberellin) and the cell division hormones (cytokinins) could have some impact on cotyledon expansion. Since both processes occur simultaneously during expansion, we will use a measure of expansion as our dependent variable. Our independent variables will be the concentrations of hormones. Notice the plural construction. We will try to observe the impact on expansion by two hormones simultaneously applied to very young radish or squash cotyledons.
Each group will prepare a set of 16 Petri dishes each containing a disc of filter paper and 5 mL of a discrete combination of two hormones (IBA and Kinetin, IBA and Benzyl Adenine, GA and Kinetin, or GA and Benzyl Adenine). The four groups in the room will select one of these four hormone pairings for their study. Make sure no other group is duplicating your effort!
A dilution series (0.1, 0.01, 0.001 mM) will be prepared from 1 mM stock solutions and 0 M (distilled water). The volumes of each of the hormone solutions and water will be added to each dish according to the chart below. The volumes indicated in parentheses () are to be removed from the final mixture (why?):
| __________ Concentration (B) | ||||
|---|---|---|---|---|
| ___________ Concentration (A) | 0 M | 0.001 mM | 0.01 mM | 0.1 mM |
| 0 M | 5 mL water | 0.5 mL B2 4.5 mL water | 0.75 mL B1 6.75 mL water = B2 (-0.5 mL) | 0.75 mL B 6.75 mL water = B1 (-0.25 mL) |
| 0.001 mM | 0.5 mL A2 4.5 mL water | 0.5 mL A2 0.5 mL B2 4.0 mL water | 0.5 mL A2 0.5 mL B1 4.0 mL water | 0.5 mL A2 0.5 mL B 4.0 mL water |
| 0.01 mM | 0.75 mL A1 6.75 mL water = A2 (-0.5 mL) | 0.5 mL A1 0.5 mL B2 4.0 mL water | 0.5 mL A1 0.5 mL B1 4.0 mL water | 0.5 mL A1 0.5 mL B 4.0 mL water |
| 0.1 mM | 0.75 mL A 6.75 mL water = A1 (-0.25 mL) | 0.5 mL A 0.5 mL B2 4.0 mL water | 0.5 mL A 0.5 mL B1 4.0 mL water | 0.5 mL A 0.5 mL B 4.0 mL water |
You will be provided with a supply of 48-hr-old radish or squash seedlings, germinated in moderate light. Examine a seedling to note that there are two cotyledons present (radish is a dicot). If one cotyledon is larger than the other, use the smaller cotyledon. Remove the cotyledon from 10 seedlings and place these 10 cotyledons in one of the 16 Petri dishes you have prepared. Be careful to remove the cotyledon with as little damage as possible, and be sure you have not put any hypocotyl or root tissue in the Petri dish. Strive to select cotyledons of similar size. Continue in this way until all 16 dishes have been filled.
We will incubate these cotyledons for a few to several days (make note of how many) in light. At the end of the incubation, we will remove the cotyledons, blot them dry, and weigh them. We might also try an area determination using the photocopier or computer scanner. Put the resulting data in the chart:
| __________ Concentration (B) | ||||
|---|---|---|---|---|
| ___________ Concentration (A) | 0 M | 0.001 mM | 0.01 mM | 0.1 mM |
| 0 M | ||||
| 0.001 mM | ||||
| 0.01 mM | ||||
| 0.1 mM | ||||
As output for this project we will plot the hormone concentrations on the X and Z axis and the weight or area on the Y axis of a three-dimensional plot. This will demonstrate the relationship between the two independent variables upon the one dependent variable. Which of the hormone pairs are synergistic? Are any antagonistic? Do any pairs lack a relationship? Present your graph(s) and interpretation(s) in an amplified abstract.
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