Trent University

Department of Biology: 3180H, Plants in Action

Analysis of Plant Hormones GA3 and IAA on Seed Germination, Root Growth and

Hypocotyl Elongation using Lettuce (Lactuca sativa) and Cucumber (Cucumis

sativus) Seeds.

By: Winona Drouin (#0560422)

Lab Partners: Christy Dean, Megan Foster, Hillary LeFort & Terrence Grand

For: May Myklebust, Susan Chow and Thien Nguyen

April 4th, 2014

1.0 Introduction

Phytohormones are naturally occurring chemical signals that are responsible for plant functions such as

stimulating plant growth, regulating metabolism and morphogenesis (Raven P, 2013). The

communication between a plant‟s tissues and organs is possible due to the movement of these hormones.

Plant hormones are created in different tissues of the plant, as well as during different plant lifecycles, and

can be transported throughout in order to deliver a specific message (Raven P, 2013). Without the

encouragement of hormones, plants would be unable to differentiate growth in tissues or become dormant

(Raven P, 2013). Phytohormones are only successfully active when found in small quantities within the

plant tissue and if are too concentrated, may have detrimental effects (Raven P, 2013). It is important that

a plant maintains a balance between hormones present and concentration for optimal growth. Two

common phytohormones that are present within plants include auxins and gibberellins, which are both

responsible for the incentive of growth.

Auxins (IIA) are responsible for the stimulation of cell growth or cell elongation in the stem

region (Myklebust M, 2014; Raven P, 2013).

Phototropism is primarily due to the effects of IAA,

which causes the cells to elongate in the coleoptiles

giving the stem its “bent” shape. Auxins are produced

in the leaf primordial, shoot apical meristem and Figure 1. Auxin showing cell elongation/phototropism.
(Biology Beta, 2014)
young leaves in developing seeds (Raven P, 2013).

Gibberellins are the hormone group that terminates dormancy and stimulates germination. It is

also responsible for the elongation of the stem and leaf as well as promoting flowering and fruit growth

(Myklebust M, 2014; Raven P, 2013). Gibberellic acid (GA3) is the commonly produced hormone which

is found in the developing seed or in the young tissues of shoots (Raven P, 2013).
 The following experiment will research the affect on growth of two different hormones

commonly found in plants; Auxins and Gibberellins. Specifically, the auxin being examined in the

natural form of indole-3-acetic acid (IAA) and the gibberellin being gibberellic acid (GA3). It is

hypothesized that induced plant hormones will establish a response for growth however, it is predicted

that a minimal concentration of these hormones will establish optimal growth. By determining the optimal

concentration for plant growth, agricultural uses of phytohormones could be synthesized to create a faster

growing crop and furthermore, revenue. The following research paper will discuss the concentrations in

which provide optimal growth, the reason behind it and it‟s potentiality to be used in the agricultural

industry.

2.0 Methods and Materials

2.1 Natural Inhibitors of Seed Germination

Some species of plant have developed germination specific to light sensitivity, such as lettuce seeds, and

can only germinate when light is absent. Photodormancy is affiliated with the production of far-red

phytochrome within the seeds. Through the stimulation of gibberellins, far-red phytochrome becomes

produced which breaks the seeds photodormancy and causes germination (Myklebust M, 2014). The

following experiment is to replicate and determine the effects of GA3 and light on germination for photo-

dormant lettuce seeds.

A total of eight petri dishes (four dishes each with one replication), with filter paper, were used in

the beginning of the experiment. Approximately 40 lettuce seeds (var. Grand Rapids) were then added to

the individual petri dishes. Four petri dishes were brought to the dark room, while four remained in the

laboratory (Myklebust M, 2014).

In the dark room, a pipette of ~5mL of distilled water was added to two of the petri dishes and

another 5mL of 10 µM GA3 was added to the other two. The petri dishes were then sealed and wrapped

in aluminum foil so that no light was able to enter. In the laboratory, the same procedure was followed,
however the four petri dishes were not wrapped in aluminum foil as to allow light to enter. Afterwards,

the petri dishes were left in an incubator at 25°C for ~48hours (Myklebust M, 2014).

The percent germination was then calculated by adding the number of germinated seeds and

dividing it by the total number of seeds for each petri dish. With the two replicates, a mean of percent

germination was created (Myklebust M, 2014).

2.2 Effects of Auxin on the Growth of Cucumber Roots

The following experiment is to determine the optimal concentration of IAA needed for cucumber root

growth.

A total of three cucumber seeds (three replications) were placed in one of six petri dishes which

contained filter paper and a small amount of water was added to germinate the seeds. It is important to

note that each cucumber was placed strategically within the petri dish so that roots would not entangle

during growth. After ~2-3 days in the incubator, initial root length was measured. After the measurement,

each petri dish was given a corresponding concentration using a pipette. The concentrations were as

follows: control (0 mg/L), 0.01mg/L, 0.1mg/L, 1mg/L, 10mg/L and 100mg/L. Dishes were then placed

back into the incubator for ~45-50 hours. The final root growth was then measured for each seedling, and

a difference between final and initial growth provided the root elongation. A mean root elongation for

each concentration was then calculated by creating an average of the three seedling replicates (Myklebust

M, 2014).

2.3 The Effects of Auxin and Gibberellin on Hypocotyl Elongation

Because both auxin and gibberellins are responsible for inducing plant growth in above-ground plants, it

is important to define the differences and similarities of the two hormones using exogenous supplies

(Myklebust M, 2014). The following experiment will compare the cucumber hypocotyls growth to

exogenous amounts of auxin and gibberellins.

To begin the experiment, a total of 5 pots with water and soil were used to initially grow the

cucumber seedlings. Three cucumber seeds were placed (3 replicates) in each pot and left to germinate

and elongate in the incubator for ~7-8 days. Afterwards, the hypocotyls (~1.5cm below cotyledonary
node) of each seedling was marked using a permanent marker. Between the apical meristem and the

1.5cm is considered to be the hypocotyls unit. An initial length of the hypocotyls unit was recorded in

order to create a difference afterwards (Myklebust, 2014). During the same day, concentrations of IAA

and GA3 were added by placing a single drop on the apical meristem using an eyedropper. The

concentrations were as follows; 0(water), 10-3 M, 10-4 M, 10-5 M, 10-6 M. The plants were then left in the

incubator for 7 days. During this time, the plants were frequently watered. After the 7 days, the final

growth was measured and a mean increase in the hypocotyls unit was determined (Myklebust M, 2014).

3.0 Results

3.1 Natural Inhibitors of Seed Germination

The following results were obtained for experiment 1:

Figure 2. Percent germination for lettuce seeds treated within light/darkness, with either water of 10 um of GA3.
Figure 2 indicates that light/water (92.4%) and dark/GA3 (83.8%) had the highest percent

germination. The lowest percent germination was the seeds placed in the dark with water (68.8%). When

comparing the light variables, water increased percent germination and GA3 decreased percent

germination. With regards to darkness, the opposite effects were observed; GA3 increased percent

germination and water decreased percent germination. However, with the darkness variables, the errors

bars show some overlap which can indicate that the data was insignificant.
 3.2 Effect of Auxin on the Growth of Cucumber Roots

The following results were obtained from experiment 2:

Figure 3. Effects of auxin on the logarithmic growth (cm) of cucumber roots with decreasing concentrations of IAA (mg/L)
Figure 3 indicates the logarithmic growth for the decreasing concentrations of IAA. The 100mg/L

concentration of IAA showed the lowest root growth and the concentration of 1mg/L showed the highest;

indicating that medium amounts of IAA are needed for increasing root growth in cucumbers. With

reference to the control (0mg/L), 100mg/L actually proved to decrease or stunt the growth showing

detrimental affects to the root. However, it is important to note that the majority of the error bars are

overlapping which indicates the data may be classified as insignificant and that conclusions cannot be

justified.

3.3 The Effects of Auxin and Gibberellin on Hypocotyl Elgonation

The following results were derived from experiment 3:

0 10-3 10-4 10-5 10-6

Figure 4. Effects of various concentrations (M) of auxin and gibberellins on hypoctoyl mean length (cm).
4.0 Discussion

4.1 Natural Inhibitors of Seed Germination

As previously mentioned, gibberellic acid stimulates the germination process in unfavourable light

conditions associated with photodormant seeds. When red light is present, it increases the level of

gibberellic acid (GA3) present, causing germination (Cocucci S et al, 1981). This proves as an

explanation for the „dark‟ trials in experiment 1. When comparing the effects of GA3 for both light and

dark, it was concluded that the dark trial had the highest percent germination. This is because when GA3

was present in a light depleted environment; red light (or far-red phytochrome) became present causing

the GA3 levels to increase within the seedling. As the GA3 levels increased, germination was stimulated

much sooner than the trial involving GA3 and light (Cocucci S et al, 1981).

By inducing photodormancy seeds with small amounts of GA3, germination increases at a faster

rate. With regards to the agricultural industry, germinating seeds quicker with GA3, can allow for crops

that are photodormant (such as lettuce) to be planted later in the season and can allot for a higher percent

germination. However, when referring to Figure 2, it can be shown that just light and water had the

highest percent germination. In order to determine an optimal amount of GA3, perhaps a combination and

cooperation of light/water and dark/GA3 variables are needed to create a 100% germination.

With reference to Figure 2, some error bars are overlapping indicating that the data may be

insignificant. In order to correctly determine a difference between the two variables, more trials are

needed. It is also suggested for future studies, that different concentrations of GA3 are to be used as well

as different time intervals of dark and light to find the optimal percent germination.

4.2 Effect of Auxin on the Growth of Cucumber Roots

When referring to Figure 3, it can be concluded that the concentration of IAA which promoted the highest

growth of cucumber roots as 1mg/L. Auxin is found to promote root elongation only in minimal

concentrations which would clarify as to why the optimal concentration in experiment 2 was lower than

the majority. When concentrations of IAA are substantial, the production of ethylene is increased as a bi-

product (Myklebust M, 2014). Ethylene, similar to IAA, acts as another regulator of root growth and has
stimulatory effects in low concentrations. However when in high concentrations, root elongation is

directly inhibited (Eliasson L et al, 1989). As demonstrated in Figure 3, when the concentration of IAA

was greater than 10 mg/L, the logarithmic growth of cucumber roots were drastically decreased almost to

a halt. With the increased concentration of IAA, it is safe to assume that an increase in ethylene is present

as well. These two compounds cooperating together cause the cucumber root elongation to become

inhibited (Eliasson L et al, 1989).

There are some discrepancies with the data, as shown in Figure 3. The error bars, similar to

experiment 1, are overlapping with each other thus indicating that the data may be insignificant. For

example, the concentration of 0.1 mg/L has a large error bar and overlaps with the previous and preceding

concentration. To lower the error bars, it is suggested that more replicates/trials be used in the experiment

to create a smaller standard error value and furthermore, an accurate conclusion.

In determining a key concentration of IAA to be applied seedlings, the root and plant growth can

be accelerated. Specific derivatives and concentrations of auxins can be used as herbicides to promote

growth and crop yield. Since the late 1940‟s, auxins have been isolated and used for agricultural purposes,

such as the common herbicide 2,4-D (Overbeek J van, 1952). Because these compounds are effective in

small concentrations, quantities are readily available for the common agricultural practice (Overbeek J

van, 1952). Continuing to determine the optimal concentration and derivative of auxin is essential in

providing agricultural industries with the highest standards.

4.2 The Effects of Auxin and Gibberellin on Hypocotyl Elongation

When auxin is tested on plants in exogenous supply, the effects are found to be non-responsive and plant

growth is not stimulated. Cucumber seedlings are found to be an exception for exogenous auxin which

allows a comparison of auxin and gibberellins growth for the following experiment. Unlike auxins, when

gibberellins are placed exogenously, plant growth is found to be active (Little CH & MacDonald JE,

2003; Myklebust M, 2014).

 When comparing both IAA and GA3 for exogenous input, an increase in hypocotyls elongation

was demonstrated. However, in larger concentrations (10-3 M, 10-4 M) IAA was found to increase the

hypocotyls more rapidly than GA3 as observed in Figure 4. When concentrations became much lower,

such as 10-5 M, GA3 showed a gradual increase in elongation whereas IAA was found to drastically

decrease. Error bars were not added to the following chart due to being unable to distinguish which error

bars belonged to each variable. However, the standard error is calculated in Appendix 1, Table 3 and

shows to be minimal, indicating that the data can be concluded as significant.

The steep drop in hypocotyls elongation for IAA at a concentration of 10-4 M – 10-6 M could be a

response to endogenous auxin levels. The addition of exogenous auxin past these concentrations proves to

have inhibitory effects for hypocotyl elongation. As previously mentioned, high concentrations of auxin

could be inhibiting hypocotyl elongation through the production of ethylene (Collette C et al, 2000).

Throughout the experiments with GA3, an increase in hypocotyl elongation was shown in comparison to

the controlled variable. Gibberellins are known to increase cell growth exogenously only in light-grown

hypocotyls, such as the following experiment (Collette C et al, 2000). Even though some concentrations

of GA3 were more effective than others, an increase on hypocotyl elongation was still observed.

Based on the following experiment, it can be concluded that for the use of agricultural purposes,

GA3 is the compound in which produces optimal growth. GA3 could perhaps be isolated into herbicidal

sprays or become mixed with soil to allow farmers to increase their growing capacity. However, it is

important to further the study to determine the exact gibberellin compound and concentration in which

provides not only plant growth, but plant health.

5.0 Sources of Error

The following experiment exhibited a considerable amount of human error which potentially altered the

results. In the first experiment, the numbers of lettuce seeds were not counted as exactly 40 giving each

different trial a skewed average. In experiment 2, measurements for both final and initial root lengths

were done by different individuals; error could have been achieved due to reading measurements wrong,
or interpreting the initial length. For experiment 3, the addition of permanent marker caused the stems of

the cucumber seeds to wilt significantly. Because of this, many of the plants were unable to continue their

growth and were disposed of. Thus, other replicates from different variables were sacrificed so that each

variable had a minimum of 2 replicates instead of 3. Another source of error could have occurred when

using the pipettes. When initially adding the hormones with the pipette, the two stops were not correctly

achieved which could have potentially added a different volume of concentration and skewing the results.

In almost all results, the experimental data proved to be insignificant due to error bars

overlapping. As previously mentioned, to decrease the overlapping of error bars more replicates must be

preformed.

6.0 References

Biology Beta. 2014. [Online]. What chemicals and structures control the direction of plant growth in

roots. Biology Stack Exchange. Accessed on 20 March 2014. Available from:

http://biology.stackexchange.com/questions/8094/what-chemicals-and-structures-control-the-

direction-of-plant-growth-in-leaves

Cocucci S, Ranieri A, Morgutti S & Ciroli F. 1981. The role of darkness, GA and fusicoccin (FC) in

breaking photodormancy in Phacelia tanacetifolia seeds. Journal of Physiologia Plantarum. Vol.

52. pp. 177-180

Collett C, Harberd N, Leyser O. 2000. Hormonal interactions in the control of Arabidopsis on hypocotyls

elongation. Journal of Plant Physiology. American Society of Plant Biologists. Vol 124(2). pp.

553-562

Eliasson L, Bertell G, Bolander E. 1989. Inhibitory action of auxin on root elongation not mediated by

ethylene. Plant Physiology. Vol 91. pp. 310-14

Little CH, MacDonald JE. 2003. Effects of exogenous gibberelin and auxin on shoot elongation and

vegetative bud development in seedlings of Pinus sylyvestris and Picea glauca. Journal of Tree

Physiology. Vol (2). pp. 73-83

Myklebust M. 2014. Phytohormones. Trent University, Department of Biology. Plants in Action 3180H.

Accessed on Blackboard; Lecture Material.

Overbeek J van. 1952. Agricultural application of growth regulators and their physiological basis.

Annual Review of Plant Physiology. Vol 3. pp. 87-108

Raven P. 2013. Biology of plants. Regulating Growth and Development: The Plant Hormones. WH

Freeman and Company. New York, USA. pp. 638-660

 APPENDIX

Table 1. Data retrieved for Experiment 1: Natural Inhibitors of Seed Germination.

Light/Water Light/ GA3 Dark/Water Dark/GA3
Germinated Total Germinated Total Germinated Total Germinated Total
Trial 35 39 29 36 40 25 32 40
1
Trial 38 40 31 40 30 40 35 40
2
St. 0.026282051 0.015277778 0.0625 0.0375
Error

Table 2. Data retrieved for Experiment 2: Effect of Auxin on the Growth of Cucumber Roots
100 mg/L 10 mg/L 1 mg/L 0.1 mg/L 0.01 mg/L Water
Average Growth 1.53 6.93 7.60 5.57 6.80 4.03
(cm)
Standard Error 0.19 0.84 0.88 0.75 0.83 0.61

Table 3. Data retrieved for Experiment 2: Effects of Auxin and Gibberellin on Hypocotyl Elongation.
Water GA3 IAA St. Error - St. Error - St. Error -
Water GA3 IAA
10^-3 3.30 3.07 3.67 0.40 0.10 0.12
M
10^-4 3.30 4.25 0.05 0.18
M
10^-5 3.67 3.37 0.19 0.18
M
10^-6 3.17 2.83 0.17 0.34
M