sites.psu.edusites.psu.edu/.../12/bio-240-official-first-lab-report.docx · Web viewAccording to Campbell Biology 9th edition (Reese et al., 2011), a hormone is a signaling molecule

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 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8control 20.5 24.8 30.1 38.4 40.8 46.6GA3 19.4 28.5 33.6 41.1 47.5 62.3cycocel 22 27.5 25.8 28 29.6 28.5

Petite day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8control 18.7 24 24 31.3 20 37GA3 16.8 22.5 24.3 30.3 32.7 39.3cycocel 24.5 24 25.3 25.2 26.2 28.7

Elongate day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8control 43.3 53.7 59.4 68 71.3 84.5GA3 42.3 50.8 62.3 75.3 91.2 101cycocel 46.8 62.8 66 75.75 79.1 80

Rosette day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8control 2 2.3 4.6 6 7.1 9.3GA3 5.3 5.7 13.3 21.4 23.9 31.1cycocel 3.3 6.3 7.1 8.3 8.5 10.9

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.

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 80

10203040506070

Wild Type Growth Comparison Height vs. Time

controlGA3cycocel

Time (days)

Heig

ht (m

m)

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).

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 805

1015202530354045

Petite Growth Comparison Height vs. Time

controlGA3cycocel

Time (days)

Heig

ht (m

m)

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.

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 80

20

40

60

80

100

120

Elongate Growth Comaprison Height vs. Time

controlGA3cycocel

Time (days)

Heig

ht (m

m)

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.

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 805

101520253035

Rosette Growth Comparison Height vs. time

controlGA3cycocel

Time (days)

Heig

ht (m

m)

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.