Experiment With the Plant Growth Hormone Gibberellin
Transcript of Experiment With the Plant Growth Hormone Gibberellin
Southern Illinois University CarbondaleOpenSIUC
Honors Theses University Honors Program
12-1987
Experiment With the Plant Growth HormoneGibberellinJeanette A. BakerSouthern Illinois University Carbondale
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Recommended CitationBaker, Jeanette A., "Experiment With the Plant Growth Hormone Gibberellin" (1987). Honors Theses. Paper 282.
Botany 492
Jeanette Baker
Fall 1987
Table of Contents:
Introduction.
Purpose.
Design ..
Methods.
Results.
Discussion.
Key to Graphs.
Graphs.
Literature Cited.
. 1
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.. 7
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.. 10
. .18
Intr'oduction:
The plant growth hormone gibberellin caUSes cells in
the stem intern,~des of plants to elongate. Th i s hc:"\s
been demonstrated in both normal and dwarf plants
(:::~cott.. 1'315) . Cal:d:1al.;p.=s have been i nljuced to gr'ov.l to two
meters tall by the correct application of a gibberellin
Response to a treatmemt with
gibberellin has been shown to be quite rapid (Carr,
1'372:> .
The effect of gibberellin on plant growth was first
described in Japan in 1809 as a desease of rice plants
called I/bakanae" or foolish seedling. Th i S"f desease
caused affected seedlings to beccome tl~inl pale green l
and much taller than uninfected plants.
In 1895 Hori identified the cause of this problem to be
an imperfect form of the fungus Gibberella fujikuroi.
In 1926 Kurosawa grew G. fujikuroi and obtained a cell
free extract from the growth medium. He tr-eated )""·].ce
and maize seedlings with this extract and obtained the
same effect as rice growers had observed and called
!!!:Jakanae".
Since that. time , 24 gibberellins have been
isoli:":E.tt=::.-d fr'()f(l tJ·...e fungus G. fujikur'oi ..
gibbereJllic acid lGA7). In 1958 MacMillan and Suter
isolated GAl from immature seeds of Phaspnlus
coccineus. This was the first isolation of a
gibberellin from a higher plant. Now 43 of the known
57 gibberellins have been found in higher plants.
Also many gibberellins have been synthesized. The
first to be synthesized was GA] . This was done by
Cor't=y E·t al. til 1'37::;: (Macl'1illan 19 ). ("Iod,: is sti.ll1
continuing on the synthesis of gibberellins.
Purpose of this experiment:
The pLH'pose of t.h i s e}'.:per- i H,ent. was t;-, t.es t. t.he ef f ec ts
from treating g2-1 mutants of Arabidopsis thaliana with
GA , ~il~ .. and Gi', at differing concentrations and
differing time schedules with particular interest
directed toward finding how late treatments can be
delayed and how small amounts of Ga can be given
without decreasing height and seed production from that
which is ol~tair,ed from four weekly treatments of GA
at 10 molar concentration with the first treatment at
two weeks from the day the seeds were planted.
However! de four weeks into thiS experiment the
circulating fan on the growth chamber containing the
plants stopped functiofling. This allowed the chamber
t.o rleat up to about 90 degrees F. Af tel'" t.his t.he
plants began to mature ealier than is usual and
therefore there was not a measureable seed production
so I was not able to consider effects on seed
production at this time. Also these plants did not
attain the final height that is usually found with
these types of treatments, but comparisons can still be
made as to the differences in heights since all the
plant.s were subjected to the same heat. It would of
coarse have been best to begin the experiment over, but
time was not avai 12tble to do this.
Design of experment:
-rhis experiment was set up to be analyzed as a Model I
thl"'E'e-way ANOVA. The dependent var-illble to be measu)"'ed
was plant height after six weeks of growth from t.he
date the seeds were planted (seed weight would also
have been measured if this had been possible). The
alpl~a level chosen was 0.05. The analysis was done by
computer using SAS.
Fac t.O I"' S wel"'e:
GA
wi th levels ()f:
GA J
GA H •
GA, ..
Concentrations
with levels of:
10-3
1O-~
Tl"'eatrflent.s
wi th leve~ls of:
A One microliter at two weeks and each week after
for three more weeks
E: One micr'olitE.')"' at two wE'eks only
C One rni c \"'01 iter at two weeks anlj at. ttwee weeks
D One microl i t.E?l"' at thr·es y'leeks only
Sample size was 10 plants for each group making a total
of 240 plants for t.he experiment. I did a pilot study
earlier which indicated that due to the variance of
associated with these plants, a sample size ot 30 would
have been desirable. However, this was not possible
due to space limitations and the fact that this
experiment was designed to fit into a larger experiment
which is ongoing and has a sample size of 10.
ME·t-hods:
Gr'owth medium:
Hoagland's solution was prepared and pH adjusted with
sodium hydroxide to pH 5.6. This was solidified with
15 ml of this was added, to each 20 x 200 mm
culture tube which was capped with a plastic cap.
These were sterilized in an autoclave for 20 minutes.
Procedure fOl' startillg seeds:
Seeljs of ga-l ~\utant of Arabidopsis thaliana were
soaked for 10 minutes in 1/4 strength Clorox liquid
bleach with two or three drops of dishwashing liquid
added as wetting agent. Then these were rinsed three
times in sterile demineralized water. Ne::<t the seeds
- l"·N ,.-._." I· -.j J. II ('A ·1 (...)- ~ 'f ,._..,,' ,-_,,··,,0_., /-""_··'1 .."'.W~ ~ ~Ud~~l . J J I i This is
necessary for germination of these ga-1 mutant seeds_
These were again rinsed three times in sterile
demineralized water, The seeds were then planted with
a sterile pipette onto a petri dish containing 20 ml of
Hoagland's solution solidified with 2% agar.
CuI t.Ul'E.':
F'1C'.\"1\.'!=' 1,1,11.::'.'1"12 I;li"c,wn in a Shei"'el" Gi llett 9,"'owth chamber'
manufactured in Marshall, Michigan.
Tl"'eatrnent:
Treatments were administered with an Eppendorf digital
pipette 4710 micropipeter. GA,. and GA~f? were not
soluble at 10-1 molar concentration. Thel"'ef ore, in
order to administer a one microliter 1(-)-3 t·pea t·men t.,
the plants were given five microliters of the
appropriate GA at 104:lne day and five micl"'oliters mope
This was done in two fj.ve microliter
treatments illstead of one ten micpoliter treatment.
because of the difficu].ty in l.eepj.ng a t.er) ~ticroliter
drop from falling off the plants. All o'~her tr'eatments
wey'e one rnj.croliter.
Rl~sul ts:
The ANOVA showed a significant difference in the means
of the plants due to the diffepent gibberellins and due
to the different concentrations and due to the
different treatments. There was also a significant
interaction between concentration and treatment. This
was the only interaction found.
significant differences due to all three factors, the
DUllcan means comparison test was performed to find
where these differences were located.
Amon ';) the I;libber-ellins, GA.l and GA'T~ wer'e found t.o act.
wit.h no significant differ-ences. However, GA~o gave
means that. were signifcant.ly lower- t.han the other-so
Between the two differ-ent concentr-ations, there was a
significant difference with the 10- 1 molar giving
significantly higher- means than the lO-¥ molar
concent\"'at.ion.
Among the treatments, ther-e were found to be
significant differ-ences for each of the tr-eatments.
The highest nlean was obtained from treating the
plants with one microliter at two weeks and one
microliter each week after for three more weeks. The
next highest mean was obtained from treating with one
microliter at only the third week. The next highest
mean was from treating with one microliter at two and
at three 1,.I}ee~::::,. The lowest mean was frOM treating with
one microliter at two weeks only.
Discussion:
The results from t.he gibberellins and from the
concentrations are hardly surprising since similar
results have been obtained in other experiments. The
concentration-treatment interaction should be
investigated in futher experiments. The results of the
compal~isons of the treatment means were very
interesting. It does not seem strange that the
treatment with one microliter at week two and each
week after for three more weeks gave the highest means
since this treatment supplied the plants with more
gibberellin than the other treatments. However} the
treatment which gave the next highest amount of
gibberellin did not give the next highest means. This
was the treatment with 0118 microliter at week two and
at week three. The means for this treatment were third
from the llighest I~eing preceded by means for the
treatment with one microliter at only the third week.
This seems very strallge since both of these groups of
plants had GA applied at week three, yet the group
which had the additional treatment at week two grew
less. The group with the lowest mean was the treatment
with one microliter at week two only. It is
interesting that this group did less well than the
plants treated only at three weeks. This certainly
should be investigated further in furture experiments.
Summary:
This experiment shows that the time and amount of
treatment as well as the type 0 do affect the height of
t.he plant.s. In t.he fut.ure I would like t.o repeat. t.his
experiment since it was adversely affected by the
failure of the growt.h chamber. Also I would like to
expand it in ~he area of treatment times and anlounts in
order to find the most effective tr~a~~e~t regime for
U',ese plant.s. A).so one very important. question which
was not able to address with this experi~ent. concel~ns
the most· effective time t.o t.reat· these plants in order
to attain seed production .. Flowel~ing a~d seed
prc,duction in these plants has been shown to be GA
dependent (Benzinger l 1983)
, -.
I
Literature cited:
Encyclopedia of Plant Physiology New Series, Vol. 10,
Ed. A. Pirson, Gottengen,M. H. Zimmermann, Havard,
Encyclopedia ot· Plant Physiology New Series, VOl. 9,
Ed. A. Pirson, Gotttengen, M.h. Zimmermann, Harvard,
lntroduct.ory Plant. Biology, JCingsley R. Stern, Wm, C.
Brown Publishers, Dubuque, Io~a , 1985
Plant Growth Substances 1970, Ed. D.J, Carr
Springer-Verlag, Berlin, Heidelberg, New, York, 1972
The Effects of Gibberellin, Brasinolide. and Other
Growth Regulators on a Gibberellin-Deficient Mutant of
Arabidopsis thaliana, Elizabeth A.benzinger, Bachelor
of Arts, Southern 111ino,i5 University,' Carbondale, IL,
1983
Key t.o Gl'aphs:
A :: One micr·olit.er' t:l"'ear-ment. at. t.wo weeks at. each week after for three"more weeks
E: = One mi·crol i te'.... t.r'eatment at. two weeks only
(: = One micl"'ol i tel" t."ea t.f1lent. at. two weeks and at. th''''ee weeks
o - One microliter ~reatment at. three weeks only
~ .. Vert.icle line = range
Horizontal line = mean
Shaded area = + or - standard deviation
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BOTANY 492
SPRING
1988
JEANETTE BAKER
This is a continuation of the experiment began in the Fall
of 1987. In that experiment, GA3 , GA4+7, and GA20 were
used in treating the plants. Since it was shown that GA3
and GA4+7 gave plant heights with no significant difference
in the means, and that GA20 gave heights with
significantly lower means, only GA3 was chosen as the GA
for this set of treatments.
Since the 10- 3 molar concentration gave plant heights with
significantly higher means than did the 10-4 molar
concentration, 10-3 was the concentration chosen for this
experiment.
For this experiment three different dosage levels were used.
Plants were treated with 1 microliter, 5 microliters, and 10
microliters to see if the amount of GA given at the various
treatment times would affect the mean height of the plants.
The treatment scheme used in this experiment was as follows:
Treatment A: Plants were given GA3 at 10-3 molar
concentration at six weeks from the
date the seeds were planted. This was
done at the 1, 5, and 10 microliter
dosage levels.
Treatment B Plants were given GA3 at 10-3 molar at
1, 5 and 10 microliters at five weeks
from the date the seeds were planted.
Treatment C Plants were given GA3 at 10-3 molar at
1, 5, and 10 microliters at both week
five and week six.
Treatment D Plants were given GA3 10-3 molar at 1,
5, and 10 microliters at week two.
Treatment E Plants were given GA3 10 -3 molar at
1, 5, and 10 microliters at weeks 2, 3,
4, and 5.
Treatment F Plants were given GA3 10-3 molar at
1, 5, and 10 microliters at weeks 2 and
3.
Treatment G Plants were given GA3 10-3 molar at
1, 5, and 10 microliters at week 3.
General methods and culture conditions were the same as set
up in the earlier part of the experiment.
Again the data were analysed using ANOVA to test for
differences in means and the Duncan means comparison test
was used to find where any observed differences were
located.
This time there was not the problem with growth chamber
failure causing the plants to suffer from high heat stress.
The plants were allowed to grow for 10 weeks to give good seed
production so the weights of seeds from the plants were
also analysed.
Results The dose ( 1, 5, or 10 microliters ) made no
significant difference in the mean height of the plants.
However, there was a significant difference due to the time
of treatment. Treatment E gave the highest mean and this
was significantly higher than that given by any of the other
treatments. Treatments D, G, and F gave the next highest
group of mean heights. Treatment A gave the lowest mean and
this was significantly lower than the other means. However,
Treatments Band C gave means which could not be separated
totally from all the others. These means were grouped with
the means from treatments D, G, and F and were also grouped
with the mean of treatment A. The interaction between dose
and treatment was significant.
The effect was sort of reversed for the means of the seed
weights. In this respect the different treatments produced
no significant differences, whereas, the dosage did produce
significantly different means in the seed weights. The 1
microliter dose gave the highest mean, and this mean was
significantly higher than the five microliter dose with the
5 microliter dose giving the lowest mean. The mean for the
10 microliter dose was grouped with both the 1 and the 5
microliter doses as it was between these two and could not
be placed absolutely in with either of them even though
likewise it could not be absolutely separated from either
of them. The interaction between dose and treatment was
also significant.
Discussion
Since the lowest dose of GA gave the highest mean for the
seed weight, it would seem that the amont of GA applied
at a treatment time is not directly proportional to the
weight of seed produced. In fact it would almost seem
that the opposite were true except that the 10 microliter
dose did not give lower means than the 5 microliter dose.
Altogether, this is a little puzzling and probably needs
further investigation in future experiments especially since
there was a significant interaction betweeen dose and
treatment.
It is very interesting that the week of treatment and the
number of weeks the treatment was given had no effect on the
mean weight of the seeds. This seems especially interesting
in light of the fact that these ga-1 mutant plants do not
produce seed at all if they are not treated with some amount
of GA.
Since the dosage made no significant difference in the mean
height of the plants it would seem that even 1 microliter of
GA 3 at 10 molar provides sufficient GA for elongation of
the plant stem. It also seems to show that additional GA of
at least up to the 10 microliter dose produces no
detrimental effect on the plant height.
Even though the treatment schedule did not affect the seed
production, it did influence plant height. Treatment E gave
the highest mean. This is also the treatment which gave the
plants the greatest number of treatments which started at
week two, which is the earliest treatment given. As far as
plant height is concerned, it seems that early treatment is
important since the second highest mean was the result of
treatment D which gave the plants one treatment of GA at
week two only. Again this time as in the earlier part of
the experiment, the height mean was greater for plants treated at
week three only than for plants treated at week two and week
three.
Summary
In this experiment seed production was not affected by the
various treatment schedules but was affected by dosage. The
fact that the higher dosages gave lower seed weights shows
that more experiments need to be done to further investigate
possible causes for this.
The interaction between dosage and treatment for both seed
weight and plant height needs further investigation.
The fact that treatment and dosage seem to have opposite
effects on plant height than they do on seed weight even
though GA is necessary for seed production and for stem
elongation in these plants needs further study.
The effect of the three week only treatment on plant height
is very interesting. Hore extensive experimentation in this
area might reveal some information as to the ideal time
schedule for treatment of these plants.
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