Experiment with the Plant Growth Hormone Gibberellin Jeanette A

Southern Illinois University Carbondale OpenSIUC

Honors Theses University Honors Program

12-1987 Experiment With the Plant Growth Hormone Gibberellin Jeanette A. Baker Southern Illinois University Carbondale

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Recommended Citation Baker, Jeanette A., "Experiment With the Plant Growth Hormone Gibberellin" (1987). Honors Theses. Paper 282.

This Dissertation/Thesis is brought to you for free and open access by the University Honors Program at OpenSIUC. It has been accepted for inclusion in Honors Theses by an authorized administrator of OpenSIUC. For more information, please contact [email protected]. Botany 492

Jeanette Baker

Fall 1987 Table of Contents:

Introduction. . 1

Purpose. .2

~ Design .. .. L

Methods. .4

Results. .5

Discussion. .6

.. 7

Key to Graphs. .8

Graphs. .. 10

Literature Cited. . .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 1 19 ). ("Iod,: is sti.ll 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.est. 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

1 O-~

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::

- 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·peat·ment.,

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 I 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)

, -. 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."eat.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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!J c D Trea1 fH~nt e~n r "'? 5 n '" 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

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. ";;,C

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