OF TESTING
SEED VIABILATY
By: Amanda A.
By: Amanda A.
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| Abstract |
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| Purpost |
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| Hypothesis |
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| Experimental Design |
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| Materials |
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| Procedures |
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| Research Report |
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| Results |
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| Graphs |
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| Conclusion |
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| Bibliography |
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The purpose of this experiment is to
find out what kind of solution will loosen the seed coat of a Kentucky
Blue Grass seed, enough to let the Triphenyl Tetrazolium solution soak
into the seed, with out causing damage to the seed. This process is done
to find out about what percent of a farmers crop will be able to germinate
and produce a product that the farmer will be able to sell. I think that
if we were able to find a solution, that would loosen the seed coat and
let the tetrazolium solution into the seed, that it would save the farmers
a lots of time, and help create a more efficient way of finding our what
percent of a farmers crop is growable.
My hypothesis is that hydrogen peroxide will loosen the
seed coat more efficiently than the hydrochloric acid and the isopropyl
alcohol, and that it will cause the lest amount of damage to the seed.
I base my hypothesis on the fact that hydrogen peroxide has a lower acidity
level than the hydrogen peroxide, but a higher acidity level than the isopropyl
alcohol.
The kind of experiment that I was dong was to find a
way to weaken the seed coats of Kentucky Bluegrass, by using an agent that
will weaken the seed coat enough to let the Triphenyl Tetrazlium solution
soak into the seed and dye the embryo red. I did my experiment by
using the traditional method for my control group and I tested a few new
ways of dyeing the seed coat. Some of the main steps that I used
to do my experiment were using the lab, getting all the chemicals and finally
getting to do my experiment. Some of the limitations on my experiment
were not being able to test all percentages of agents: I could have been
a little more precise with the amounts of liquids, when I was pouring them
into the beakers and not having enough time to do my experiment.
Manipulated Variables are the agents used to weaken the seed coat: Hydrogen
Peroxide, Isopropyl and Hydrochloric acid. The constants of this experiment
are: the seeds. From the results that I have gained through out my
experiment, I have learned that in fact the Isopropyl Alcohol was the best
choice for loosening the seed coats. In my original hypothesis, I
hypothesized that they hydrogen peroxide would have the best results with
out causing the most damage.
My results would indicate that I should reject my hypothesis
because, in the case of not being pierced, in all three-test groups, the
isopropyl alcohol had the best over all results. After seeing
the results of my experiment I wonder if it would be beneficial for the
seed annalists at the department of agriculture change form hydrogen peroxide
to isopropyl alcohol? If I continued experimentation I would test only
the isopropyl alcohol again using concentrations raging from seeds turned
red. But if the trend did continue once we reached 100% solution
the heights number of seeds able to turn red would only be 65 seeds.
The purpose of this experiment is to find out what kind of solution will loosen the seed coat of a Kentucky Blue Grass seed, enough to let the Triphenyl Tetrazolium solution soak into the seed, with out causing damage to the seed. This process is done to find out about what percent of a farmers crop will be able to germinate and produce a product that the farmer will be able to sell. I think that if we were able to find a solution, that would loosen the seed coat and let the tetrazolium solution into the seed, that it would save the farmers a lots of time, and help create a more efficient way of finding our what percent of a farmers crop is growable.
My hypothesis is that hydrogen peroxide will loosen the seed coat more efficiently than the hydrochloric acid and the isopropyl alcohol, and that it will cause the lest amount of damage to the seed. I base my hypothesis on the fact that hydrogen peroxide has a lower acidity level than the hydrogen peroxide, but a higher acidity level than the isopropyl alcohol.
The manipulated variables are the agents
used to waken the seed coat:
Hydrogen peroxide, hydrochloric acid and isopropyl alcohol.
The responding variable is: the percent of seeds that
are dyed red by the triphenyl tetrazlium solution.
The control group is done in the same traditional way,
as is done in the labs, with the hydrogen peroxide and triphenyl tetrazlium
solution.
The constants of this experiment are: the seeds, hydrogen
peroxide, isopropyl alcohol, hydrochloric acid and triphenyl tetrazlium.
Quanity Product
18 .Petri dishes
900 .Kentucky Bluegrass seeds
1 .3%, 3 ounce, bottle of Hydrogen peroxide
1 .6%, 3 ounce, bottle of Hydrogen peroxide
1 ..12%, 3 ounce, bottle of Hydrogen peroxide
1 .3%, 3 ounce, bottle of Hydrochloric acid
1 .6%, 3 ounce, bottle of Hydrochloric acid
1 ..12%, 3 ounce, bottle of Hydrochloric acid
1 .3%, 3 ounce, bottle of Isopropyl Alcohol
1 .6%, 3 ounce, bottle of Isopropyl Alcohol
1 ...12%, 3 ounce, bottle of Isopropyl Alcohol
1..... .3%, 24 ounce, bottle of Triphenyl tetrazlium
1 High powered microscope
9 ..Sheets of blotter paper
1 .. ..Seed strainer
1 .. ..Seed piercer
18 .... .White labels
1
.......Warm oven
1. First get the 3%, 6% and 12% of the hydrogen peroxide,
hydrochloric acid & isopropyl alcohol.
2. Get 18-glass petri dishes-
3. Write up 18 labels.
4. You will need to label six petri dishes hydrogen peroxide,
then do the same for the other two substances.
5. For each substance, two of the six petri
dishes will need to be labeled as 3%, 6% & 12%
6. The two petri dishes that are labeled 3%, 6%, and
12%, one will need to be labeled pierce and the other one not pierced.
7. Count out 50 seeds, then put 50 seeds into each petri
dish.
8. Take the hydrogen peroxide solution that is labeled
3% and pour just enough solution into the petri dish to cover the seeds
completely.
9. Repeat this process for the 6% solution, the 12% solution,
and for the isopropyl alcohol and hydrochloric acid solutions.
10. Then put all petri dishes into the over at 35oC,
over night.
11. When you return you will want to take all petri dishes
that are labels Not pierced (one at a time) using a seed strainer,
pour out the solution into the sink.
12. Caution, not to let any seeds go down the drain
13. Dry the petri dish completely, then put the seeds
back into the petri dish.
14. Pour .3% triphenyl tetrazlium solution into each
petri dish. Using just enough to completely cover seeds.
15. Put back into the oven for 3 hours.
16. Take one sheet of blotter paper and run it under
the skink just enough to get it damp.
17. The petri dishes that are labeled pierced you will
want to take out (one at a time) using the seed strainer, pour out the
solution.
18. Cashion, not to let any seeds go down the drain.
19. Making sure that the blotter paper is still damp.
20. Use one sheet of blotter paper per sample.
21. Dry the petri dish completely, then put the seeds
on to the blotter paper.
22. Take the seeds (on the blotter paper) over to the
microscope, place sample under the microscope.
23. Bring the petri dish over to the microscope
also.
24. Adjust the microscope so that you can see each individual
seed clearly.
25. Once looking at the seeds under the microscope, use
your needle pen to pierce the seeds directly behind the embryo.
26. Once you have pierced an individual seed put it back
into the petri dish.
27. After you finish with each individual group pour
.3% triphenyl tetrazlium solution into the petri dish, using just enough
to cover the seeds.
28. Continue this process for all, hydrogen peroxide,
hydrochloric acid, isopropyl alcohol, 3%, 6% and 12% solutions, labeled
pierced.
29. Once you have done this to all groups, place all
groups back into the over, at 35oC for 3 hours.
30. Once seeds have set for 3 hours, you will want
to take a sample,
(One at a time) over to the microscope, place sample
under the microscope.
31. Bring the petri dish over to the microscope.
32. Adjust the microscope so that you can see each
individual seed
clearly.
33. Once looking at the seeds under the microscope,
look at each individual see to see weather or not it has turned red.
34. Now that the seed embryo is visible, separate
the seeds that have turned red form the seeds that are still white or yellow,
by putting them into two different groups in side the petri dish.
35. Count up the number of seeds that have turned
red out of 50.
36. Then find the percentage for each individual
sample, of seeds that turned red.
37. Make sure that after you have read the seed
sample that you record your results.
38. Continue this process for all hydrogen peroxide,
hydrochloric acid, isopropyl alcohol, 3%, 6%, 12%, pierced and not pierced
groups.
As I was finding information on the
viability of the seed coats, I decided to try and find out the most I could
about how seeds acting in normal conditions.
Most seeds remain quiescent during a cold or dry season
and germinate only with the coming of favorable growing conditions.
Seeds that require special treatment to germinate, even when presented
with adequate water and oxygen, and factorable temperatures, are said to
exhibit dormancy. Seeds with thick or waxy coats, which inhibit the
entry of water and oxygen, may remain in a prolonged quiescent state.
Most seeds loose their ability to germinate with in several years, of shedding
their coats.
Some kinds of seeds need different
kind of temperatures to grow or to be terminable. Temperatures between
20oC and 45oC, have no effect on accuracy of triphenyl tetrazlium tests,
but staining proceeds faster at the higher temperatures. Tests can
be performed satisfactorily at room temperature. If shorter
test periods are wanted, heat can be supplied by placing the tests in a
30*C or 35*C germinator, or some other kind of heating devise.
Some tests may be placed in subdued light or in
the dark for staining, however, light has little effect on the tetrazolium
test and may be of little use.
When you are conducting tests,
depending on what kind of seed you are using, there is different amounts
of seeds that should be used. For example at least 200 seeds should be
tested, in replicates of 100 seeds or less. The seeds should be randomly
selected and counted out in replicates before conditioning. Triphenyl
tetrazium tests require less replication than germination tests because
of less dependence on moisture, temperature, light, fungi, and other environmental
factors that may cause variation in results.
In the laboratory the seed analysis
use a .3% of the triphenyl tetrazlium solution. They use the testing
handbook to show, you how to make different kinds of percentages.
To make a 1.0% solution, dissolving 1 gram of triphenyl tetrazlium powder
in 100 ml. distilled or tap water. To make a 0.5% solution, mix 1
part of 1.0% solution with 1 part water, or dissolve 1-gram triphenyl tetrazlium
powder in 200 ml. water. To prepare a 0.1% solution, mix 1
part of 1.0% solution with 9 parts water, or dissolve 1-gram triphenyl
tetrazlium powder in 1000 ml. water.
Hydrogen Peroxide- H2O2.
An unstable, colorless, heavy, strongly oxidizing liquid, capable of reacting
explosively with combustibles and used chiefly in aqueous solution as an
antiseptic, bleaching or oxidizing agent, and laboratory reagent.
Isopropyl Alcohol-C3H80. A clear, colorless, flammable,
mobile liquid, used in antifreeze compound, lotions, and cosmetics and
as a solvent for shellac, gums, and essential oils.
Hydrochloric acid-HCL. A clear,
colorless, fuming, poisonous, highly acidic aqueous solution of hydrogen
chloride, used as a chemical intermediate, in petroleum production, and
in food processing, picking metal cleaning, and ore reduction.
Triphenyl Tetrazlium-(TZ) consists
of water, a buffer additive (if needed), and 2,3,5 triphenyl tetrazlium
chloride (TTZ), an oxidized, colorless, soluble salt. As the TZ solution
penetrates living tissue, hydrogen ions released by enzymes (dehydrogenases)
involved in the respiration process reduce TTC to form an insoluble reddish
compound. The TZ staining reaction rate is affected by seed physiology
related to respiration rate, cell and tissue composition, seed age health.
Other critical factors involved with the TZ.
Sodium Phosphate-Natt2P04, H20
and Na3P04. Any of the three-solium salts of phosphoric acid used
in industry, pharmaceutical manufacturing, medicine and chemistry
Lactophenol acid- an agent used to clean the triphenyl
tetrazlium solution off the seeds. (Mostly lactic acid)
My results would indicate that I should
reject my hypothesis because, in the case of not being pierced, in all
three-test groups, the isopropyl alcohol had the best over all results.
After seeing the results of my experiment I wonder if it would be beneficial
for the seed annalists at the department of agriculture change form hydrogen
peroxide to isopropyl alcohol? If I continued experimentation I would test
only the isopropyl alcohol again using concentrations raging from seeds
turned red. But if the trend did continue once we reached 100% solution
the heights number of seeds able to turn red would only be 65 seeds.
| Substances |
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Control Group
results |
| Hydrochloric acid
Not Pierced |
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| Hydrogen Peroxide
Not Pierced |
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| Isopropyl Alcohol
Not Pierced |
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| Control Group |
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Isopropyl Alcohol Results
Hydrogen Peroxide Results
Hydrochloric Acid Results
Control Group
From the results that I have gained through out my experiment, I have learned that in fact the Isopropyl Alcohol was the best choice for loosening the seed coats. In my original hypothesis, I hypothesized that the hydrogen peroxide would have the best results with out causing the most damage. My results would indicate that I should reject my hypothesis because, in the cases of not being pierced, in all three-test groups, the isopropyl alcohol had the best over all results. After seeing the results of my experiment I wonder if it would be beneficial for the seed annalists at the department of agriculture change for hydrogen peroxide to isopropyl alcohol?
If I continued experimentation I would test only
the isopropyl alcohol again, using concentrations ranging from 10-100,
because in my original experiment every time I increased the solution by
3%, 2 more seeds turned red. But if the trend did continue once we reached
100% solution the heights number of seeds able to turn red would only be
65 seeds.
1. Academic American Encyclopedia, Book S, Grulier Incorporated,
pg. 186-187. 1995
2. Academic American Encyclopedia, Book D Grulier Incorporated
pg. 242-243, 1995
3. Tetrazolium Testing Handbook, Don F. Grabe, pg. 3-47