Determining the Metabolism of GA3 in Four Genotypes of Brassica rapa
Anthonia George
Biology 240W
Section 5
February 13, 2013
ABSTRACT
The objective of this experiment was to determine how the different genotypes of
Brassica rapa differ in their GA3 metabolism; the different genotypes being, Wild type, petite,
elongate and rosette, (the wild type and petite versions have the normal amount of GA3, while
the elongate over produces it, and the rosette under produces it). GA3 promotes increased growth
rate in plants and internode elongation. Two other treatments were used, water (being the
control) and Cycocel which limits the effect of GA3. The experiment was placed in an apparatus
with a wicking system which keeps the plants moistened and the plants were kept under 24hr
illumination. Initial heights were recorded on the first day and final measurements on the last
day. In between this time, the plant had been treated and measured every day around the same
time for a whole week (excluding the weekend), using a ruler, spray bottles containing the
treatment and tubes. The class data was averaged. At the end of the experiment, the results
showed that the genotypes with abnormal amounts of GA3 (elongate and rosette) had the most
reaction to it. The results also showed that there was considerable growth in the GA3 treated
plants, and an insignificant amount of growth in the Cycocel treated plants since cycocel as
described before inhibits GA3 metabolism. The control grew, but not nearly as much as the
Gibberellic Acid treated plants, showing that GA3 was functioning.
INTRODUCTION
According to Campbell Biology 9th edition (Reese et al.,2011), a hormone is a signaling
molecule that is produced in tiny amounts by one part of an organism’s body and transported to
other parts, where it binds to a specific receptor and triggers responses in target cells and tissues.
Plant hormones help coordinate growth, development and responses to stimuli. They are also
active at very low concentrations. The major classes of plant hormones are abscisic acid, auxin,
cytokinin, gibberellic acid, ethylene, and brassinosteroids (McKeon and Warren, 2013).
Gibberellin (which is used in this experiment), also called GA3 promotes growth, by breaking
seed dormancy resulting in germination and elongation of internodes. GA3 also stimulates pollen
development, pollen tube and fruit growth, regulates sex determination and the transition from
juvenile to adult phase. The meristems of apical buds and roots, young leaves and developing
seeds are the primary sites for the production of GA3. Gibberellic Acid is responsible for the
elongation of stems and internodes in plants (Figure 1).
Figure 1- Chemical structure of GA3.
The plant used in this experiment is called Brassica rapa or the Wisconsin fast plant
(Figure 2).
Figure 2- Brassica rapa
Wisconsin Fast plants were developed by Dr. Paul William at the University of Wisconsin, and
have been bred by him for over 30 years now. Fast plants are a rapid-cycling member of the
mustard and cabbage family known as Cruciferae. The other members include Chinese cabbage,
canola, and turnips. (McKeon and Warren, 2013). These plants mature and reproduce quickly
(have a rapid life cycle), they also have a wide variety of recognized phenotypes and they
provide quick results. This plant is ideal for this experiment due to these advantageous qualities.
The genotypes of Brassica rapa used were wild type (Wt), rosette (Rs) – dwarf mutant, petite
(Pt) – dwarf mutant, and elongate (El) – tall mutant. Three treatments are used on the plants,
water, GA3 (described above) and cycocel. Cycocel is among the most reliable and widely used
plant growth regulators on the market today. It is a chemical inhibitor of endogenous gibberellic
acid synthesis.
The overall purpose of this experiment was to determine how these different genotypes of
Brassica rapa differ in their GA3 metabolism. This showed the difference in how the hormone
works in the different genotypes of the plant. It was predicted that the Gibberellin treated plants
would be much taller than the other samples (would have long thin stems) and Cycocel treated
plants would have thicker, shorter stems, since it inhibits stem elongation. Of the Gibberellin
treated the plants the elongate genotype was predicted to not grow that much because it is an
over producer of GA3 and it already has enough GA3, the dwarf and wild type are regular
producers of GA3 and was predicted to grow quite well but not as tremendous as the rosettes that
are under producers.
MATERIALS AND METHODS
Figure 3- Plant system setup
Each group in the class was given growing plants of all the four genotypes. These plants
were planted in a Styrofoam container that had one plant per square cell, and was on top of a
piece of wicking material that led into a water tank underneath the plants. The water that was
placed underneath contained copper sulfate, which was added to prevent mold growth on the
plants. Looking at Figure 3, the setup of the experiment can be further understood. The WT, PT,
EL and RS genotypes were grown in separate sections, and the fourth section remained empty.
Three different treatments (water, GA3- 100 parts per million, b-9 solution- 1:100 dilution of
cycocel) were administered. On the first day of the experiment, the plants were thinned using
forceps, leaving one plant to one cell. Each plant was then measured using a ruler and the results
gotten were converted from centimeters to millimeters. The different groups measured the plants
different ways even though they were not supposed to, some started measuring from the
styrofoam and some directly from the soil, so this could have made results vary. After the plants
were measured, they were sprayed using three different spray bottles, each contained a different
treatment and three different tubes, each labeled with one treatment.
To spray the plants, each plant was covered with a tube and about two squirts from the
spray bottle were squirted into the tube. Tubes were used to prevent the treatments from touching
the other plants. Another precaution taken was using three different tubes for the different
sections so as not to mix treatments. Every day after the first day, except the weekend, for one
week, someone from the group went to measure and spray each plant with the treatment needed
for it. These plants were kept under 24hr illumination and each groups tray was labeled so as not
to them mix up and confuse results. A week after the first day, the final measurements of the
plants were taken and all the class data was collected and entered into excel. The averages of the
replicates for each genotype and treatment was taken and entered into excel. The average of the
lab data was then calculated.
Wildtype
control
GA3
cycocel
day 1
20.5
19.4
22
day 2
24.8
28.5
27.5
day 3
30.1
33.6
25.8
day 4
day 5
day 6
38.4
41.1
28
day 7
40.8
47.5
29.6
day 8
46.6
62.3
28.5
Petite
control
GA3
cycocel
day 1
18.7
16.8
24.5
day 2
day 3
day 4
day 5
day 8
24
24.3
25.3
day 6
31.3
30.3
25.2
day 7
24
22.5
24
20
32.7
26.2
37
39.3
28.7
Elongate
control
GA3
cycocel
day 1
43.3
42.3
46.8
day 2
53.7
50.8
62.8
day 3
59.4
62.3
66
day 4
day 6
day 7
71.3
91.2
79.1
day 8
84.5
101
80
Rosette
control
GA3
cycocel
day 1
day 2
2.3
5.7
6.3
day 3
4.6
13.3
7.1
day 4
day 7
7.1
23.9
8.5
day 8
9.3
31.1
10.9
2
5.3
3.3
day 5
68
75.3
75.75
day 5
day 6
6
21.4
8.3
Figure 4- Averaged height data for lab section #5
The overall lab data was then used to create four graphs (average height in mm vs. days), which
showed the different growth comparisons.
RESULTS AND DISCUSSION
Figure 5 – plant set up on last day of experiment
Figure 5 above shows the plant set up on the last day. All the plants had grown, some
very tall and some not so much. The really tall plants had become droopy looking and they could
not support themselves. The following graphs were compiled from the data collected throughout
the experiment.
Wild Type Growth Comparison
Height vs. Time
70
Height (mm)
60
50
40
control
30
GA3
20
cycocel
10
0
day 1 day 2 day 3 day 4 day 5
day 6 day 7 day 8
Time (days)
Graph 1- Over the course of the experiment, the growth of the wild type plants treated
with GA3 seemed to have surpassed the other ones. The graph also shows that cycocel actually
inhibited the growth of plants and even made the plants shorter than those treated with just water
(control).
Petite Growth Comparison
Height vs. Time
50
Height (mm)
40
30
control
20
GA3
10
cycocel
0
day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8
Time (days)
Graph 2- The petite plant did not grow that tall overall, the tallest plant did not even reach
40mm. Although the plants were short, they still followed the same pattern of increased growth
in the plants treated with GA3, but in this case this did not start happening till the 7th day. As the
plants treated with cycocel decreased in growth rate, the more the plants treated with GA3
increased in growth rate, as time went by.
Elongate Growth Comaprison
Height vs. Time
120
Height (mm)
100
80
60
control
40
GA3
20
cycocel
0
day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8
Time (days)
Graph 3- This graph shows that the elongate genotype metabolized GA3 the most,
because these plants grew really tall, with the tallest being about 100mm. This genotype had the
tallest plants. The same trend followed here with plants treated with Cycocel having a decreased
growth rate and the plants treated with GA3 having an increased growth rate, as time went by.
Rosette Growth Comparison
Height vs. time
35
Height (mm)
30
25
20
control
15
GA3
10
cycocel
5
0
day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8
Time (days)
Graph 4- Although the rosette genotype had the absolute shortest plants in the whole
experiment, they did metabolize a whole lot of GA3, as there was a drastic change in height
between the plants treated with GA3 and the ones not treated.
Graph 1, 2, 3 and 4 support the hypothesis stated earlier. Gibberellic acid promotes and
increases the rate of growth in plants. Cycocel actually inhibited the growth (shorter stems), but
in some genotypes such as the elongate, this effect did not start showing till towards end. The
plants treated with the water acted as the control and served as a guide to which plants treated
with GA3 and cycocel could be compared too. Another thing noticed was that, even know the
Elongates grew the tallest, the rosettes which were much shorter, reacted to GA3 in the highest
degree (most change in height), probably because it usually under produces GA3 and now was
given a lot of GA3. The Elongates grew tall, but it seemed like at some point they reached a
maximum height because they are overproducers and have more than enough GA3. The wild
type genotype which is a normal producer of GA3 grew at a regular rate, but the petite plants
which are also regular producers did not really metabolize the GA3 because of possible
hormonal problems that don’t allow them get very tall, so it didn’t really matter how much GA3
it was going to get, nothing would have changed.
Possible sources of error would be the part of the experiment where the plants where
measured. The rulers were too big to fit in each cell, so people held the ruler at different angles
and from different heights, making it difficult to get an accurate reading.
In conclusion, in all of the experiments there was a large amount of growth in the GA3
treated plants, with the rosettes having the greatest growth spurts and the elongate plants growing
the tallest. The Cycocel treated plants did not show any great amount of growth, in comparison
to the control, in any of the experiments. All plants showed growth though, showing that the
overall experiment was a success. It was also noticed that GA3 increases growth in all of the
different genotypes of the plant Brassica rapa.
LITERATURE CITED
Cycocel®." OHP, Inc. 2005. Web. 13
Mckeon, Brian Sr., and Warren James Jr. 2013. Plant hormones. Biology 240: Form and
Function, A laboratory Manual (Penn State, The Behrend College)
Reece, Jane B., and Lisa A.. Urry. "Plant Responses to Internal and External Signals." Campbell
Biology. San Francisco, CA: Benjamin Cummings, 2011. 824-27. Print.
Reynolds, Laura. "Facts About the Wisconsin Fast Plant." GardenGuides. Demand Media, 1997.
Web. 13 Feb. 2013.
Williams, Coe. "The Story of Fast Plants." Wisconsin Fast Plants® Program. University of
Wisconsin- Madison, n.d. Web. 13 Feb. 2013. Feb. 2013.