Effects of music on lettuce germination and growth in a hydroponic system. Preregistration (v2)

Study Information

1. Title (required) 

Effects of music on green towers romaine lettuce germination and growth in a hydroponic system.

2. Authors (required)

Cole Delyea & Krystle Ash

3. Description (optional)

The effect of music on green towers romaine lettuce (Lactuca sativa L. var. longifolia) in hydroponic systems has not been documented so far. We will measure the length of the cotyledon phase (number of seeds that emerge to stage, and time in stage) and plant growth (height, number of true leaves, length of true leaves, and width of true leaves) when green towers romaine lettuce plants are exposed to multiple different kinds of music. 

4. Impact/Significance (optional)

As urban centers and populations continue to expand, there is a growing demand for more resilient and cost-effective practices for food production to offset the loss of agricultural land and decrease the costs associated with food transport (1–4). Utilizing vertical urban farming which employs hydroponic growing systems has been identified as a promising solution to combat this growing pressure (5–7). Though the mechanism is not well understood, accumulating evidence suggests that certain sounds enable increased plant growth (8–27). It has been found that European classical music, Indian Vedic chants, and woodwind music have increased the growth of plants. In contrast, Rock and psychedelic music have mixed impact on the growth of plants (8–10,15,23,27). There has even been research performed on how specific frequencies could influence lettuce growth, which found that sound at 60 Hz increased the growth of lettuce (27). Research so far has been primarily focused on the growth of plants in soil (8–11,13,15,16,19–27). Therefore, the growth of green towers romaine lettuce in a hydroponic system is warranted. This research could inform urban vertical farms of ways to modify the yield of lettuce being grown, which could be utilized for financial benefit. 

5. Hypotheses (required)

If music influences green towers romaine lettuce length of the cotyledon phase, then lower time spent in the cotyledon stage, increased plants that grew out of those seeded, and increased plant growth will be correlated with European classical music and Indian Vedic chant compared to the no-music control.

Design Plan

In this section, you will be asked to describe the overall design of your study. Remember that this research plan is designed to register a single study, so if you have multiple experimental designs, please complete a separate preregistration.

6. Study type (required)

Observational Study - Data is collected from study subjects that are not randomly assigned to a treatment. This includes surveys, natural experiments, and regression discontinuity designs.

7. Blinding (required)

 No blinding is involved in this study.

8. Is there any additional blinding in this study?

No

9. Study design (required)

-plants will be grown in a controlled system (AeroGarden(TM)) which ensures that the same amount of light and nutrients are supplied to them. The light height will be kept at the minimum setting (8 inches) and be on for 16.5 hours a day with 7.5 hours of dark.

-Aerogarden liquid plant food (Nurtured by MiracleGro) will be used to fertilize the plants. 2 capfuls (8mLs) will be given to the plants on experiment start and every 2 weeks.

-Water levels will be documented with a ruler and will subsequently be filled to the fill line of the Aerogarden every week to ensure that plants are getting enough water.

-plants will be grown in a sound-proofed cabinet which is made of wood and covered by 2-inch acoustic foam inside the enclosure to block out any outside sounds and contain the sounds being applied to the plants within.  

-A fan will be placed in the enclosure to help circulate the air and simulate the fans in an industrial greenhouse setting.

-Sound treatments will be done sequentially because of a lack of space.

-Temperature and humidity will be kept constant (monitored and documented) across experiments at 23+/- 5^oC and ~50-70% relative humidity

-Sound level will be kept constant (and monitored) 55-70 dB for the music treatments

-Music will be played 24 hours a day for the plants over the course of the 28 experimental days.

-Music playlists will be recorded and played on loop for the full experiment. The BPM and frequency of the music will be determined as per music chosen.

-music will be played from one Bluetooth speaker that is kept at a fixed position from the aerogarden™ connected to an MP3 player.

-6 aeropods will be seeded with 5 seeds each for each music treatment.

-Data for each pod seeded will be recorded separately and averaged to generate one data point. Each treatment will have 6 data points.

-Plant measurements (number of plants to grow, time spent in the cotyledon stage, true leaf number, true leaf length, true leaf width) will be recorded every 2 days from the beginning of the experiment. This will be done by visual observation and measured with a ruler.

-Pictures will be taken every 2 days from the beginning of the experiment to record the progress of the plant growth and visually document how the plants are growing.

-At the end of the experiments (28 days/sound treatment), the plants will be photographed and weighed.

-Data will be analyzed sequentially and updated as the experiment progresses. This will be done in GraphPad.

-One-way ANOVA will be performed on data generated comparing treatments against the baseline (no music) and against different musical groups. Dunnett's test will subsequently be performed to determine which groups are significantly different. 

10. Randomization (optional)

There will not be randomization present in this study.

Sampling Plan

In this section, we’ll ask you to describe how you plan to collect samples, as well as the number of samples you plan to collect and your rationale for this decision. Please keep in mind that the data described in this section should be the actual data used for analysis, so if you are using a subset of a larger dataset, please describe the subset that will actually be used in your study.

11. Existing data (required)

 Registration prior to data creation: As of the date of submission of this research plan for preregistration, the data have not yet been collected, created, or realized.

12. Explanation of existing data (optional)

N/A

13. Data collection procedures (required)

-Plant measurements (number of plants to grow, time spent in the cotyledon stage, true leaf number, true leaf length, true leaf width) will be recorded every 2 days from the beginning of the experiment. This will be done by visual observation and measured with a ruler.

-Pictures will be taken every 2 days from the beginning of the experiment to record the progress of the plant growth and visually document how the plants are growing.

-water levels will be measured once a week with a ruler to gauge approximate water usage by the plants.

-Experiment will last for 28 days/sound treatment. At this point, the plants will be photographed and weighed.

-The tempo (BPM), and frequency (Hz) of the sounds will be recorded.

14. Sample size (required)

-6 aeropods will be seeded with 5 seeds each for each music treatment. 

15. Sample size rationale (optional)

-6 pods are being used because it is the size accommodated by the AeroGarden(TM) and because it should give sufficient statistical power.

-1 AeroGarden is being utilized because of space constraints

-sampling is done sequentially because of space constraints. 

16. Stopping rule (optional)

N/A

Variables

In this section you can describe all variables (both manipulated and measured variables) that will later be used in your confirmatory analysis plan. In your analysis plan, you will have the opportunity to describe how each variable will be used. If you have variables which you are measuring for exploratory analyses, you are not required to list them, though you are permitted to do so.

17. Manipulated variables (optional)

-16 sound treatments will be applied to the plants at 55-70 dB for 24 hrs a day over the course of a 28 day period.

1. no music
2. Nature sounds
3. Binaural beats
4. Music for plants (album)
5. European classical music
6. Indian classical music
7. Chinese classical music
8. Indian Vedic Chant (Gayatri Mantra)
9. Buddhist Mantra (om mani padme hum)
10. Mantra (mix)
11. Gospel music
12. Christian rock
13. Pop rock (1960s-1970s)
14. Latin jazz
15. Cool jazz
16. Spanish guitar

-The tempo (BPM), and frequency (Hz) of the music will be recorded.

18. Measured variables (required)

-number of plants to grow, 

-time spent in the cotyledon stage, 

-true leaf number (as determined by leaves appearing after the first leaves to sprout), 

-true leaf length, 

-true leaf width

-Wet plant weight after harvest

-Water used by plants each week

19. Indices (optional)

(from section 9)

-Data for each pod seeded will be recorded separately and averaged to generate one data point. Each treatment will have 6 data points.

Analysis Plan

You may describe one or more confirmatory analysis in this preregistration. Please remember that all analyses specified below must be reported in the final article, and any additional analyses must be noted as exploratory or hypothesis generating. A confirmatory analysis plan must state upfront which variables are predictors (independent) and which are the outcomes (dependent), otherwise, it is an exploratory analysis. You are allowed to describe any exploratory work here, but a clear confirmatory analysis is required. 

20. Statistical models (required)

(from section 9)

-One-way ANOVA will be performed on data generated comparing treatments against the baseline (no music) and against different musical groups. Dunnett's test will subsequently be performed to determine which groups are significantly different. 

21. Transformations (optional)

-Data for each pod seeded will be recorded separately and averaged to generate one data point. Each treatment will have 6 data points.

-The individual data will be marked as: Sound treatment ____. pod 1 (plant) (plant) (plant) (plant) (plant), pod 2 (plant) (plant) (plant) (plant) (plant), etc…

- The combined data will be marked as: Sound treatment ____. Pod 1 (measurements +/- SD), Pod 2 (measurements +/- SD).

• plants that did not grow will not be included in the data analysis.

22. Inference criteria (optional)

-I will use the standard p<.05 criteria for determining if the statistical tests suggest that the results are significantly different from those expected if the null hypothesis were correct. 

23. Data exclusion (optional)

A Grubb's test will be applied to identify if there are outliers present in the data. 

24. Missing data (optional)

-If time points are missed, the information will not be included in the final analysis and the data will not appear on the graph.

25. Exploratory analysis (optional)

N/A.

Other

--Costs-- (total: $1673.07)
 

Rulers  $17.7

Speaker           $27.99

MP3 Player     $24.99

Fan      $15.99

50-pack of pods          $35.83

Decibel sound meter   $39.99

Humidity and temperature sensor       $23.17

Foam panels    $38.99

Aerogarden     $159

Aerogarden nutrients  $23.42

Cabinet/hutch  $100

Seeds   $14

Time to do experiment            $1152 

--Links--

v1 of the preregistration: https://www.researchhub.com/post/1139/effects-of-music-on-lettuce-germination-and-growth-in-a-hydroponic-system-preregistration

OSF: https://osf.io/684yc/
 

--Acknowledgements---

Thank you, Jeffrey Koury, Patrick Joyce, Riccardo Goldoni, Tyler Diorio, and Sikander Shah, for reviewing this document and providing feedback before publishing.

---References---

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