The following descriptions are of protocols the authors
have tested and successfully run. They were all run with the Texas
Instruments' CBL probe system using the low pressure gas probe, and similar
exercises have been accomplished with the more traditional water-displacement
Certain features are common to each of the experimental systems and they are as follows:
The goal of this experiment is to demonstrate the
localization of primary differentiation in the radicle of the germinated
seed. To prepare for it, grow the seeds for approx. 60 hours prior
to the day of the lab and check that the radicles are approx. 4 cm in length
and that the hypocotyls have not yet emerged from the cotyledon.
Test the seeds under three conditions: entire seed intact with radicle attached, the removed radicle by itself, and the remaining seed minus the radicle. The whole seed group must be done first along with a control; then, intact seeds must be removed, the radicle sliced off, and two separate trials run with the radicles and seed remainders respectively. Be sure to control these two variables for the original volume of the whole seeds.
If done properly, there is a dramatic difference between the parts and the whole, with the radicle accounting for most of the energy produced and oxygen consumed as would be expected (sample data 1).
B. Proximal vs. Distal Radicles
The purpose of this experiment is to demonstrate
where inside the radical cellular growth and differentiation are taking
place. To prepare for it, grow the seeds for approx. 60 hours prior
to the lab and then remove the radicles from the cotyledons. Mass
the radicles and be sure that there is a total of at least 2.0-2.5 grams.
To test the hypothesis that the distal apical region of the radicle has the highest rate of cell respiration (and therefore is the region of maximum growth and differentiation), carefully slice the radicles in half and place the proximal halves in one respirometer and the distal ones with the apical meristems in the other. Then control for volume according to one of two conditions. If using radicles removed already from a previous experiment, use the original volume of that experiment (thus allowing one to compare data to the rates of respiration of intact seeds); if studying radicles only, control for an assigned absolute volume of 10 ml. Figure 4 shows these parts in a bean seedling; Figure 5 in corn.
When done properly, the data from an experiment like this one shows clearly a difference in rates of respiration between the distal and proximal radicles. The apical region's metabolic rate is dramatically higher (sample data 2).
C. Different Species of Plants
To see whether seeds from different species develop
at different rates, one can perform the following simple experiment.
Use germinated seeds that are at least 36 hours old and are all the same
age. Weigh out approx. 30 seeds of each of the kinds of plants that
will be analysed, and then mass each separate group, adding or subtracting
seeds one at a time, until all samples have equal masses. Then find
the volumes of each group and control the lower volume samples to the largest
volume (as well as preparing a separate control for atmospheric pressure
changes with glass beads alone). Place the samples in respirometers
and test for each group's separate metabolic rate.
The results one gets will obviously vary depending on the choice of plants to compare, and one interesting variation on this experiment is to compare monocots and dicots. There is a dramatic difference between the two (monocots respire much more slowly), and interesting evolution questions can branch off from this experiment (sample data 3).
D. Different Stages of Development
The goal of this experiment is to demonstrate changes in
the differentiation process itself. Start with germinated seeds that
are 36 hours old on the day of the lab and prepare 3 (or more) samples
of comparable mass and volume for studying. Set aside those samples
not being tested that day (store in moist paper towels in a dark, warm
place), and then follow the standard protocol discussed above in item A
for studying the rate differences between radicles and whole sprouting
Test a new sample each day for as many samples of the seeds as there are, and if done correctly, there is a steady change in respiration rates of the different parts of the plant as it alters which tissues are differentiating over the course of the developmental process.
One can also do a variation on this experimental system by studying the rates of the proximal and distal radicles alone over time. Be sure to allow the radicles to "age" while still attached to the seed rather than "aging" the radicles after they have been removed (they will not remain viable in isolation over the time frame of three or more days). When done carefully, it is possible actually to see the decrease in radicle apical dominance as the plant switches from growing the radicle to growing the hypocotyl (sample data 4).