AN INVESTIGATION INTO THE GRAVITROPIC RESPONSES OF PHASEOLUS

VULGARIS INTRODUCED TO SUPPLEMENTAL CALCIUM TREATMENTS

How can supplemental calcium affect the gravitropic response of “Provider” Phaseolus vulgaris over a

five-week period?

Subject: Biology

Word Count: 3748

ACKNOWLEDGEMENTS

I would like to thank my Extended Essay Advisor for her constant guidance and invaluable input. I

would also like to thank my family for their love and support throughout this experiment, with special

thanks to their allowance for me to use our backyard as a research space.

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 TABLE OF CONTENTS

INTRODUCTION………………………………………………………………………………………………………………….3
Relevance of Investigation………………………………………………………………………………………………...3
Gravitropism……………………………………………………………………………………………………………….4
The Sensory Apparatus………………………………………………………………………………………………….4
The Response and Mediation Apparatus………………………………………………………………………………..5
Calcium…………………………………………………………………………………………………………………….5
In Plants…………………………………………………………………………………………………………..5
In Gravitropic Responses ………………………………………………………………………………………..6
Calcium Hydroxide.……………………………………………………………………………………………...7
Phaseolus Vulgaris………………………………………………………………………………………………………….8
“Provider” Variety of Phaseolus vulgaris………………………………………………………………………..8
HYPOTHESIS……………………………………………………………………………………………………………………...9
METHODOLOGY………………………………………………………………………………………………………………...9
Rationale for Chosen Experiment…………………………………………………………………………………………9
Independent Variables……………………………………………………………………………………….….10
Dependent Variables……………………………………………………………………………………….……10
Controlled Variables ……………………………………………………………………………………………11
Materials………………………………………………………………………………………………………...12
Dosage Application Guidelines…..………………………………………………………………………..…13
Procedure…………………………………………………………………………………………………………………14
Part A: Preparation of the Experiment………………………………………………………………………….14
Part B: Transplant and Suspension ……………………………………………………………………………..14
Part C: Administering Calcium Hydroxide……………………………………………………………………..15
Data Collection and Processing……………………………………………………………………………………..……16
Q Qualitative Data………………………………………………………………………………………………….16
Q Quantitative Data………………………………………………………………………………………………...16
RESULTS………………………………………………………………………………………………………………………….18
Quantitative data………………………………………………………………………………………………………….18
Qualitative Data…………………………………………………………………………………………………………..18
Calculations and Processed Data…………………………………………………………………………………………20
ANALYSYS AND DISCUSSION….…………………………………………………………………………………………….21
Analysis and Discussion of Results………………………………………………………………………………………21
Statistical Analysis……………………………………………………………………………………………………….23
CONCLUSION…………………………………………………………………………………………………………………...25
EVALUATION……………………………………………………………………………………………………………………26
Research Question………………………………………………………………………………………………….….…26
Sources and Research…………………………………………………………………………………………………….26
Methodology……………………………………………………………………………………………………………..27
APPENDICES…………………………………………………………………………………………………………………….29
WORKS CITED………………………………………………………………………………………………………………….32

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INTRODUCTION

Relevance of Investigation

Understanding gravitropic responses and the utilization of calcium supplements in plants is

greatly beneficial to the horticulture-agriculture industries because of the potential economic

benefits it can provide. Reduction of space, increased durability, and increased growth rates of

plants in harsh or specialized environments becomes especially beneficial, as treatments may reduce

operating costs and produce uniform structures not wasted or impractical in desired environments.

This study aimed to investigate the question: “How can supplemental calcium affect the gravitropic

response of “Provider” Phaseolus vulgaris over a five-week period?”. This study deliberately

excluded the use of larger species and an extensive time period due to limited resources available.

In addition supplemental calcium was administered in the form of Ca(OH)2, as it is the most studied

compound containing calcium in agricultural research. Over the course of five weeks, specimens

were suspended upside-down and then administered one of three concentrations of Ca(OH)2

weekly. Every seven days the angle of the lowermost stem of specimens was measured. The goal of

this experiment was to identify any significant differences between the angle that lower stems

obtain when given different concentrations of Ca(OH)2. This study has the potential to provide a

greater understanding of the ways in which calcium can be utilized alongside gravitropism to

innovate current agricultural practices, especially in domesticated plant species such as “Provider”

Phaseolus vulgaris.

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Gravitropism

Gravitropism is the growth of a plant’s organs with or against the gravity gradient to re-align

displacement from the vertical, 90° (Wilkins 64). Positive gravitropism is typically seen in plant

shoots while negative gravitropism is typically seen in root systems, with orientation serving to best

fit the purpose of an organ’s function (Marschner 764). Plants may be disoriented at germination,

physically moved and damaged, or in angled terrain (Wilkins 64), and hence require a method in

which to ensure their display of reproductive organs, gathering of nutrients, and access to sunlight.

(Robinson 134). Tropic movements require three components to be effective: A sensory apparatus,

response apparatus, and an apparatus to connect and mediate the two on a cellular level (Wilkins

66).

The Sensory Apparatus

In animals, cellular chambers in which particles sediment on the lowermost portion with

respect to gravity influence growth orientation (Wilkins 68). In plants, a plastid organelle filled

with dense starch-granules serves this purpose (Marschner 765). These organelles, amyloplasts, are

located in most plant cells but only protrude in specialized locations such as root tips or plant shoots

(Robinson 134). Starch-filled amyloplasts detect disorientation upon a change in the symmetrical

pattern they sustain when upright, in pressuring the endoplasmic reticulum of affected cells upon a

change of orientation (Bailey 103). An asymmetrical collection of amyloplasts provides a plant with

the information, mediated through calcium-ion channels, to determine how to alter growth towards

the vertical (Wilkins 69).

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 The Response and Mediation Apparatus

Following disorientation from the vertical, the hormone Auxin is redistributed (Marschner

765). Auxin regulates plant growth on a cellular level, directing cells in which way to grow, by

inhibiting gene expression (Marschner 766), with respect to light and gravity based on location

(Allaby and Bailey 86). In gravitropic responses, auxin culminates on the adaxial (upper) side of

lower stems and stimulates rapid growth, resulting in upward curvature (Robinson 135). Once the

organ has re-obtained the vertical, detected by the sensory apparatus, the active re-distribution of

auxin is halted and the shoot’s cells begin to grow and divide without inhibited gene expression

(Wilkins 70). This redistribution of auxin is triggered by the sedimentation of cell amyloplasts

following disorientation, however, the biochemical responsible for mediating auxin-amyloplast

communication is not certain at present. However, there is evidence that both thickened

starch-bodies and calcium ion channels may assist in this communication (Marschner 766).

Calcium

In Plants

Within plants, calcium plays a role in the structural development of cellular membranes and

the signaling of intracellular messages prompted by various environmental changes (White and

Broadley 2). Calcium levels greatly affect the consistency of the middle-lamella layer between cells

(Robinson 94) as well as the rigidity of cell walls by interacting with membrane phospholipids to

provide stability (Thor 1). Calcium is distributed to cells via a Ca ion-pump gradient (White 1).

This network of Ca+ channels is the primary system in which calcium is facilitated during the

diffusion of nutrients (Bailey 34).

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In scope of cell-wall growth, calcium is believed to be a specialized cross-linker between adjacent

pectic acid chains between cells, notably catalyzing cross-linkages in higher concentrations

(Sinclair and Trewavas 86).

In Gravitropic Responses

There has been a long-held assumption among researchers that there is a close relationship

between calcium ions and gravitropic responses (Sinclair and Trewavas 85). However, there have

been few advancements in the full understanding of the independent role of calcium in tropisms

within the last century due to two main obstacles researchers still face.

1: Calcium is an essential element within plants, and therefore an experiment testing a

control group containing no calcium is not feasible as of this time.

2: The independent role of calcium in gravitropic responses is challenging to accurately

observe alongside so many variables with present technology, since calcium is commonly found

within elemental compounds (Sinclair and Trewavas 87).

Despite these obstacles, researchers have been able to observe several characteristics and

patterns of calcium within plant organs during gravitropic responses. In these responses Ca+

culminates along the adaxial (upmost) side of root systems (Sinclair and Trewavas 86) and

lower-stems (Roblin and Fleurat-Lessard 243). These migrations are not only reversible (Roblin and

Fleurat-Lessard 248) but efficient, detected at these sites in high concentrations within the first

thirty minutes of inversion on average in comparison to the laggard migration of K+ and Cl+ (Roblin

and Fleurat-Lessard 247). Though there is little evidence suggesting that calcium initiates the

6
gravitropic signaling required to facilitate its rapid migration, there is evidence that the calcium

intervals themselves regulate the expansion-growth of adaxial cells, and are hence necessary at

these sites immediately following inversion (Sinclair and Trewavas 85). Also to consider are the

influences that calcium concentration has on membrane transport characteristics, such as membrane

permeability to H2O ions: an essential buffer for cell volume expansion (Roblin and Fleurat-Lessard

247).

Calcium Hydroxide
Calcium is found most abundantly within elemental compounds (Coombs et al. 182). In

agricultural practices, Ca(OH)2, is most commonly used to neutralize acidic soils and ease root

growth and plant development (Crozier and Hardy 1). Industries often need to reapply treatments in

periodic doses to maintain medium soil-Ph over extensive periods of time. Ca(OH)2 has been

observed to be non-toxic to plants in moderation, and an environmentally safe runoff product. In

excess however, Ca(OH)2 can damage roots and shoots due to the corrosive nature of concentrated

OH-, stunting growth (Jennings and Brown 506). Ca(OH)2 treatments typically apply concentrations

of 125g/100gal maximum per 100 ft2 in 4-week intervals due to the corrosiveness of OH- on organic

materials, specifically shoots and buds (Durant et al. 2). This ratio is adjusted for the experimental

study of more delicate species such as vegetables and grasses (Aurum and Trisnawati 3). In the

context of this investigation, it is prudent to suggest that Ca(OH)2 is a safe compound in which to

administer calcium to specimens, but must also be considered another variable due to excess OH- in

soil systems.

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Phaseolus Vulgaris

Phaseolus vulgaris is a vegetable within the Fabaceae (pea) family, most distinguishable by

its bean-filled fruit (Bumgarner 2) and a unique atmospheric nitrogen fixation. Phaseolus vulgaris

has an accelerated maturation period, with first yield typically seen 45-50 days after germination

(Beresford-Kroeger 1). This is vital in the context of the investigation’s small-scale observation

period as well as the limited time frame in which to collect data. The germination period is 5-10

days after planting, and this is accelerated by soaking seeds overnight in water (Westerfield et al. 1).

Bean species notably prosper in containers due to their shallow root system (Dillion and Purser 1).

This is a beneficial feature for this experiment, as containers will be utilized to manipulate a

specimen’s orientation.

“Provider” Variety of Phaseolus Vulgaris

The variety of Phaseolus vulgaris tested in this investigation is Provider, chosen for its

accelerated growth rate and viability within humid-subtropical climates (Valent 1). The variety’s

ability to grow within this climate is essential due to this investigation’s specific location (Gaben 3).

Bush beans, like Provider, typically grow in a compact shape, where they rarely require support to

display a negative gravitropic response (Bumgarner 1). The un-supported growth of bush-type

varieties is beneficial to this experiment, as thigmotropism (the response to a touch stimulus) will

not inhibit the natural gravitropic responses of the specimen (Scorza 2).

8
HYPOTHESIS

Table 1: List of Hypothesis for Investigation

Null Hypothesis There is no statistically significant difference between the angle that lower

‘Provider’ Phaseolus vulgaris stems obtain after five weeks when given

different Ca(OH)2 concentrations.

Alt Hypothesis There is a statistically significant difference between the angle that lower

‘Provider’ Phaseolus vulgaris stems obtain after five weeks when given

different Ca(OH)2 concentrations.

METHODOLOGY

Rationale for Chosen Experiment

The angle of lowermost stems was measured in order to compare the curvature of specimens

in the location where calcium deposits gather most, along adaxial tissues of lower-stems, during

gravitropic responses. This provides a comprehensive display of how lower stems adjust growth

and how many degrees they are from re-obtaining the vertical when compared to other groups. An

appropriate method for this investigation had to be viable with small quantities of specimens. This

experiment required precise substance concentrations and consideration for the rapid growth of

specimens and the effect on results.

9
 Independent Variables

The independent variable is concentration of Ca(OH)2. Concentrations tested in this

investigation were 0g/gal (none/control) , 0.00625g/gal (moderate), and 0.0125/gal (maximal).

Concentrations were calculated by reducing the maximal recommended concentration per 100 ft2

for approximately 1 ft2 of space (specimen). The maximal recommended concentration and ½ of the

maximal recommended concentration were suitable concentrations to test in reference to the Durant

and study.

Figure 1: Custom conversions to obtain maximal concentration and ½ maximal recommended concentration ratios in grams.

Dependent Variables

The dependent variable in this experiment is the angle of the first two millimeters of the

lowermost point of the lower specimen stem. The angle of the direction of the first two millimeters

of the lowermost point will be measured in order to maintain a uniform measurement method.

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 Controlled Variables

Table 2: Controlled Variables and Management Methods

Controlled Variable Effects on Results Management Method

Sunlight exposure Photosynthesis and growth rates All specimens in same area

Size and age of chosen specimens Similar specimens reduce outliers in Chosen subjects germinated same
results day, relatively same size

Same species/variety used Less chance for outliers to appear Variety verified at licensed seller

Time that dosages are administered Increases accuracy of results All dosages pre-measured for speed

Consistent dosage concentration less chance of outliers, false data Pre-measured and labeled

Size of containers Opportunity for growth Utilized same container

Amount of soil Nutrients given within soil Same amount of soil given

Soil type Nutrients difference, salinity/ph Used same brand

Distilled water Minerals administered Verified distilled, isolated with cap

Weather condition exposure same Same humidity and wind experienced Kept in same area

Initial suspension angle (90°) Speed and angle of growth change containers hung/adjusted using a
differentiates constructor-level

Concentration of Ca(OH)2 given to Amount of calcium received by plants Precise scale used, all doses
each specimen pre-measured, verified thrice

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 Materials

Table 3 : Materials List (Parts 1-2)

Part 1: Germination and Care of Phaseolus Vulgaris

Item Specification Quantity

Potting soil No additional Calcium As needed

Potting containers 4’-5’ diameter 30-35 (dependant)

Phaseolus vulgaris seeds Provider variety 35

Paper towel sheets Absorbent dependant

Distilled water n/a dependant

Part 2: Construction of Suspension Apparatus

Item Specification Quantity

Lumber Beams 2’x 4’ dimensions 8-9 (dependant)

Construction screws 3’/16’ screw 30-35 (dependant)

Power drill Fits 3’/16’ screw 1

Suspension Container 1-Gallon Jug 15

Cleaning Sponges Soft and cuttable 15

Nylon rope construction-grade As needed

Scissors Adult-intended size 1

Metal hooks Severely curved, sturdy 15

Ruler Must measure in cm and in 1

Permanent marker black 1

Plastic tarp Transparent, impermeable 2-3 (as needed)

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Table 4: Materials List (Parts 3-4)
Part 3: Equipment to Obtain Ca(OH)2

Item Specification Quantity

Resealable plastic bag 11’ x 8’ As needed

Specialized scale Must measure up to 0.00000 grams 1

Permanent marker Black, waterproof 1

Container of Ca(OH)2 Lab-grade, powdered, 30g 1

container

Part 4: Data Collection Materials

Item Specification Quantity

Protractor Transparent 1

Permanent marker Black, waterproof 1

Camera Digital or mobile 1

Dosage application guidelines

This investigation adhered to chemical and biological specimen use guidelines. All

hazardous items were handled with proper safety measures including but not limited to adult

supervision, gloves, facial-masks, safety-goggles, and close-toed footwear.

13
Procedure

Part A: Preparation of the Experiment

The construction of suspension-rigs began before germination. Suspension-rigs consisted of

three wooden beams upheld by lumber stilts, each endowed with five hooks. Fifteen containers

were cut along their tops, suspended through the use of macrame-netting, stuffed with “sponge

blockers” (to prevent soil drainage while the container was upside-down), and re-adjusted to a 90°

angle with a construction level. A specialized scale was not constantly accessible, so each

concentration dosage was pre-measured, packed, labeled, and stored prior to the experiment.

Thirty-five seeds of ‘Provider’ were gathered. To expedite germination, seeds were wrapped within

a dampened paper towel and stored within plastic bags in a dimly-lit area until roots sprouted.

Distilled water was used to prevent contamination or unaccounted nutrient intake.

Part B: Transplant and Suspension

After germination, seeds were transplanted 1-inch below the soil surface using a ruler. All

were given two weeks to grow in identical conditions. The fifteen most visually-similar specimens

were selected for testing. Similarities included leaf size, stem length, height, and an approximate

90° stem-angle. Each was placed within an upside-down suspended container, with root systems

facing the open hood and the stem protruding out of the container’s lower narrow-region. Soil was

placed within and each container was suspended on one of three suspension-beams. It was vital to

maintain space between suspension rows to prevent any positive thigmotropism (touch-sensitive

growth) from specimens in proximity. To prevent excess rainwater and potential contamination, all

suspension beams were shielded by clear tarps overhead.

14
 Figure 2: Visual example of Suspension Rigs and Transplant Setup (photographed by author)

Part C: Administering Calcium Hydroxide

Each specimen was administered one of three pre-measured Ca(OH)2 concentrations every

seven days, beginning with the first day of suspension, for five weeks. Ca(OH)2 was evenly spread

atop specimen soil followed by distilled water. Ensuring consistency, each group of specimens was

clearly labeled.

15
Data Collection

Qualitative Data

Photographs of stems were taken to compare group-specimen curvatures when trying to

re-obtain the vertical. It is most efficient to observe such qualities at the end of the experiment, as

curvature is a ‘history’ of growth.

Quantitative Data

The angle of the lowermost point of specimen stems was measured with a transparent 360°

protractor (±0.5) weekly beginning seven days after suspension. To consistently measure the same

portion of the lowermost point, each measurement followed the direction of the first two

millimeters from the centermost point of the lowermost stem, as presented in figures 3-5. Each

angle was measured from the same side, where leaves and stem-tips were on the right to the

observer.

16
Figures 3-5: Visual Representation of Angle Data Collection Method and Growth progression, presenting the average angle of

specimens in group receiving 0.00625g Ca(OH)2 over a five week period.

17
RESULTS

Quantitative Data

Table 5: Sample of Raw Data of Lower-Stem Angles for one week (degrees +/- 0.5) (Cont. Appendix A)
Specimen Group receiving 0g Group receiving 0.00625g Group receiving 0.0125g
In Group Ca(OH)2 (deg +/- 0.5) Ca(OH)2 deg +/- 0.5) Ca(OH)2 deg +/- 0.5)

Week 1 1 -55.0 -49.5 -50.5

2 -40.5 -50.0 -49.0

3 -43.5 -34.5 -58.5

4 -49.5 -39.5 -52.0

5 -52.0 -29.0 -54.0

Qualitative Data

Table 6: Visual Curvature Observations of Groups 5 Weeks After Suspension

Group Group Specimen Curvation Observations

Receiving 0g/gal Ca(OH)2 ● No visual outliers

● Each fully re-obtained the vertical at stem-ends
● All demonstrated uniform curvature (symmetrical “U”-curve)
● No physical contact between stem organs.
● Moderate-curvature as compared to other groups.

Receiving 0.00625g/gal Ca(OH)2 ● No visual outliers

● Each fully re-obtained the vertical at stem-ends
● All with uniform curvature (“V”- curve of stems comparatively
smaller than other groups), less than one centimeter-diameter in
lower stems
● All had both portions of lower stems make physical contact.
● Most dramatic curvature as compared to other groups.

Receiving 0.0125g/gal Ca(OH)2 ● No visual outliers

● None re-obtained the vertical in any capacity
● Various curvatures, roughly similar askew ‘L” shape, notably
wider general lower-stem diameter.
● No physical contact between stem portions.
● Significantly wider curvature as compared to other group, large
space between lower stem portions

18
 Figures 6-7: Visual Samples of Curvature
of of specimens from group receiving 0g
C Ca(OH)2/gal 5 weeks after suspension.

Figures 8-9: Visual Samples of Curvature of

of specimens from group receiving 0.00625g C
Ca(OH)2/gal 5 weeks after suspension.

Figures 10-11: Visual Samples of

Curvature of specimens from
group receiving 0.0125g
Ca(OH)2/gal 5 weeks after
suspension.

19
 Calculations and Processed Data

I used my raw data (see Appendix A) to calculate the mean angle of each group’s specimens for

every week by following the standard mean equation. Error bars for the mean weekly angles seen on Graph

1 were set using the standard deviation (calculated in Excel) both above and below means.

Table 7: Average Lower-Stem Angles for five weeks (degrees +/- 0.5)
Group receiving 0g Group receiving 0.00625g Group receiving 0.0125g
Ca(OH)2 (deg +/- 0.5) Ca(OH)2 (deg +/- 0.5) Ca(OH)2 (deg +/- 0.5)

Week 1 Avg. -48.1 -40.5 -52.8

Week 2 Avg. -26.2 8.4 -29.9

Week 3 Avg. -5.1 15.2 -19.3

Week 4 Avg. 3.8 30.9 -7.8

Week 5 Avg. 18.6 44.8 5.7

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ANALYSIS AND DISCUSSION

Analysis and Discussion of Results

Graph 1 shows that specimens with lower-stem angles closest to the vertical after five weeks were

those receiving 0.00625g Ca(OH)2/gal, reaching 44.8° on average. Specimens receiving 0.0125g

Ca(OH)2/gal not only had lower-stem angles farthest from the vertical, 5.7° on average, but also did not

re-obtain the vertical with any portion of the stem as seen in figures 10-11. The control group, receiving 0g

Ca(OH)2/gal, obtained lower-stem angles second-closest to the vertical, 18.6°, on average, after five weeks.

Specimens receiving ½ the maximal-recommended Ca(OH)2 concentration per 1ft2, 0.00625g

Ca(OH)2/gal, displayed a significantly more rapid growth rate towards the vertical (increasing degrees of

lower-stem angles) than other groups between weeks 1-2, had a steadily increasing growth rate towards the

vertical between weeks 3-4, and displayed a final increasing growth rate towards the vertical between

weeks 4-5. The group receiving no supplemental Ca(OH)2 showed a sustained moderate growth rate

towards the vertical between weeks 1-3, a less aggressive growth rate between weeks 3-4, and a steadily

increasing growth rate towards the vertical between weeks 4-5. Specimens receiving the highest

recommended concentration of Ca(OH)2 per ft2, 0.0125g Ca(OH)2/gal, displayed a similar growth rate to

specimens receiving 0g Ca(OH)2/gal between weeks 1-2, a comparatively laggard growth rate towards the

vertical between weeks 2-4, and only showed a steadily increasing growth rate towards the vertical

between weeks 4-5.

As observed in Table 6, specimens receiving 0g Ca(OH)2/gal demonstrated uniform curvature five

weeks after suspension, where lower-stems formed symmetrical “U”-curvature, re-obtaining the vertical

with higher-stems. Specimens within this group sustained comparatively moderate-curvature as seen in

figures 6-7. Specimens receiving 0.00625g Ca(OH)2/gal re-obtained the vertical five weeks after

21
suspension with upper-stems. Specimens demonstrated uniform curvature, where lower-stem curvature was

comparatively smaller to other groups as seen in figures 8-9. No specimen receiving 0.0125g Ca(OH)2/gal

had re-obtained the vertical after five weeks of suspension. These specimens sustained a similar askew

“L”-shape and comparatively wider lower-stem diameters as seen in figures 10-11.

These results are likely due to the altered efficiency in which Ca+ facilitates both auxin signals and

nutrients throughout plants and lower adaxial cells as observed in the Bailey and White-Broadley studies. It

is also likely that more calcium was able to migrate to adaxial tissues faster when receiving supplemental

calcium which, in culminating intervals, regulates the expansion-growth of adaxial cells as mentioned by

Roblin and Sinclair. It is logical to assume these were the avenues in which specimens receiving 0.00625g

Ca(OH)2/gal were able to obtain angles so close to the vertical so rapidly. It is prudent to suggest that

specimens receiving higher concentrations, 0.0125g Ca(OH)2/gal, sustained damaged shoots from

excessive OH-, as outlined by Durant, and this is why askew curvature was the result.

22
 Statistical Analysis

I chose an one-way ANOVA (f)-test to deduce which concentration of Ca(OH)2 is more effective at

enabling specimens to obtain the lower-stem angle closest to the vertical after five weeks. With fifteen

trials and three sets of summary data, conducting an f-test to deduce whether there is a statistically

significant difference in the data from the final week of angle measurements was the most appropriate

method.

Table 8: List of Hypothesis for Statistical Analysis

Null Hypothesis There is no statistically significant difference between the angle that lower

‘Provider’ Phaseolus vulgaris stems obtain after five weeks when given

different Ca(OH)2 concentrations.

Alt Hypothesis There is a statistically significant difference between the angle that

lower ‘Provider’ Phaseolus vulgaris stems obtain after five weeks when

given different Ca(OH)2 concentrations.

In this test, if f is more than the critical value, then the null hypothesis will be rejected (if |f| > f crit)

and the alternate accepted. The calculations to acquire the f-value and degrees of freedom are presented

below.

23
Figure 12: Steps to calculate degrees of freedom and identify f-value utilizing standard deviations (found through Excel application) in

ANOVA calculator (OW-ANOVA from Summary Data, danielsoper, Daniel Soper,

https://www.danielsoper.com/statcalc/references.aspx?id=43).

From the table of critical values for an f-test (See Appendix B) with the Df 1 being 2 and Df2 being

13, the f-critical value is 3.81.

From this f-test we can reject the null hypothesis that there is no statistically significant difference

in the angle that lower ‘Provider’ Phaseolus Vulgaris stems obtain after five weeks when given different

Ca(OH)2 concentrations. This suggests that the investigated concentrations were not equally effective in

enabling specimens to obtain the lower-stem angle closest to the vertical, in which case the alternate

hypothesis must be accepted. The confidence interval was calculated using an f-distribution calculator (See

Appendix B), where the probability of the null hypothesis was found to be 0.02% certain, meaning that

support of the Alt hypothesis is 99.98% certain.

24
 As expected from an ANOVA test, the most effective group cannot be identified solely based on

these calculations as simply as other data presentations, such as Graph 1. An alternative analysis-test that

could merge multiple data groups and identify those that are statistically significant simultaneously would

likely improve this analysis.

CONCLUSION

This experiment investigated the question: “How can supplemental calcium affect the gravitropic

response of “Provider” Phaseolus vulgaris over a five-week period?”

Generally, increasing the concentration of calcium, through Ca(OH)2, increased the growth rate and

angle of lower-specimen stems toward the vertical, except for the group receiving the

maximal-recommended concentration of Ca(OH)2, where growth rates and angles towards the vertical were

significantly slower and lower after five weeks. An ANOVA test deduced that there was a statistically

significant difference in these effectivenesses, but did not explicitly reveal which group was responsible

numerically. Based on my results I concluded that a moderate concentration of supplemental calcium in the

form of Ca(OH)2 is the most effective in increasing both the growth rate and angle of specimen stems of

‘Provider’ Phaseolus vulgaris toward the vertical after five weeks of suspension, no supplemental calcium

is the second most effective, followed by a high concentration of supplemental calcium in the form of

Ca(OH)2. Despite the ANOVA test not being able to identify which group was statistically significant, my

processed data and graphs supported this conclusion that the moderate concentration was indeed the most

effective.

25
EVALUATION

Research Question

Regarding my research question, it is prudent to say that my investigation thoroughly observed the

effects, quantitatively and qualitatively, of supplemental calcium on ‘Provider’ Phaseolus vulgaris over a

five-week period. Although physical gravitropic responses were measured referencing the vertical, the

affected cellular processes were not measured–only speculated based on research and data. Therefore my

research question is not entirely concise in focusing on the scope of the data being measured. It also does

not specify the calcium-containing compound or variety of Phaseolus vulgaris that was utilized, as

explained in my introduction. A question focused specifically on measuring the physical effects of

supplemental Ca(OH)2 on suspended ‘Provider’ variety when trying to reach the vertical would prove

more accurate.

Sources and Research

Many of the studies I utilized to present the holistic effect of supplemental calcium only surveyed

tree-like species, and therefore, not all articles from my bibliography directly related to my investigation

nor its studied species specifically. A strength of the sources I utilized are that they all contribute to at least

one aspect of the phenomenon I studied, ensuring my extensive research culminated in relation to my

research question. Another strength is that many of my sources studied part of the exact same phenomenon,

only measuring different factors, and therefore were excellent references on how to create a coherent and

logical investigation. A weakness is that a notable number of sources have not been updated within the last

three years, providing room for outdated information that could be recently amended. Some articles do not

explicitly state which species they reference in regard to gravitropism or calcium, and therefore much of

26
my research is speculated on the basis of what other plant species have shown in the past. Another

weakness was the lack of a study with a directly compatible ratio of Ca(OH)2/gal to give to Phaseolus

vulgaris, where the concentration ratio had to be manually reduced to conduct a comparative study.
.

Methodology

A flaw within my experiment was the choice to test the gravitropic responses of specimens

suspended at 90°, whereas in horti-agriculture it is more common to bundle crops in closer proximity,

typically at 45°, to conserve space. Hence, my experiment is not entirely valid and applicable to the

horti-agricultural industries because it does not utilize gravitropism in the same way that they commonly

do. However, the decision to suspend specimens at 90° was made to only show an accelerated and dramatic

result from such procedures, and therefore was prudent under the limited time that I had to test my

experiment. Outside of my investigation, I observed that all of my specimens grew significantly smaller

than surplus saplings, which I decided to also grow as a reference in the same general area. This was

unexpected, as I had anticipated that at least the control group would grow as large as the reference

saplings. The general stunted growth of specimens was likely due to the limited space that suspension

containers had for roots to grow, as compared to reference-saplings in beds of free-soil. This factor is

important to note because horti-agricultural industries commonly utilize much larger containers or

extensive fields, demonstrating another avenue in which my experiment is not entirely valid and applicable

to these industries.

Perhaps the greatest factor affecting my results was the lack of excess nutrients given to selected

specimens. Distilled water enabled me to observe the effects of experimental Ca(OH)2 concentrations

independently, but this means that my study cannot claim to simulate realistic growing conditions of plants

27
in any horti-agricultural context. The introduction of common fertilizers, aside from the inherent nutrients

that the soil contained, may have produced alternative results. Such a complex investigation with a

multitude of variables simulating horti-agricultural environments would require more time that was not

available. Utilizing an isolated testing facility with controlled humidity, pressure, light, and wind conditions

would prove most effective in validating an experiment of this nature.

28
APPENDICES

Appendix A: Raw Data of Lower-Stem Angles Over Five Weeks

Table 9: Raw Data of Lower-Stem Angles (degrees +/- 0.5); Weeks 1-3
Specimen Group receiving 0g Group receiving 0.00625g Group receiving 0.0125g
In Group Ca(OH)2 (deg +/- 0.5) Ca(OH)2 deg +/- 0.5) Ca(OH)2 deg +/- 0.5)

Week 1 1 -55.0 -49.5 -50.5

2 -40.5 -50.0 -49.0

3 -43.5 -34.5 -58.5

4 -49.5 -39.5 -52.0

5 -52.0 -29.0 -54.0

Week 2 1 -33.5 19.5 -28.0

2 -29.5 14.0 -19.0

3 -13.0 17.5 -41.0

4 -31.0 -3.0 -29.5

5 -24.0 -6.0 -32.0

Week 3 1 -15.0 36.0 -18.5

2 -10.0 27.0 -12.5

3 5.0 19.0 -29.0

4 -17.5 -5.0 -17.0

5 -12.0 -1.0 -19.5

29
 Table 10: Raw Data of Lower-Stem Angles (degrees +/- 0.5); Weeks 4-5

Specimen In Group receiving 0g Group receiving 0.00625g Group receiving

Group Ca(OH)2 (deg +/- 0.5) Ca(OH)2 deg +/- 0.5) 0.0125g Ca(OH)2 deg
+/- 0.5)

Week 4 1 -2.5 45.5 -9.0

2 8.0 46.0 -7.5

3 15.0 27.5 -2.5

4 -6.5 18.0 -11.0

5 4.0 17.5 -9.0

Week 5 1 11.0 58.5 -1.0

2 23.5 61.0 -2.5

3 29.0 41.5 7.5

4 12.0 34.5 19.0

5 18.0 28.5 5.5

30
 Appendix B: Statistical Analysis Calculations Resources

Figure 12: F-table referenced to determine critical f-value for ANOVA test (F-ratio Table, SUSSEX AC.UK, Michael Burke,

http://users.sussex.ac.uk/~grahamh/RM1web/F-ratio%20table%202005.pdf).

Figure 13: Calculations to determine Confidence Interval (F-Distribution Calculator, STATOLOGY Statistics, Zach Stanford,

https://www.statology.org/f-distribution-calculator).

31
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