Skip to content

Stage IV: Microgreen development and gravitropic response¶

Research Stage IV: Microgreen development and gravitropic response ¶

The second stage of this program focuses on observing gravity sensing mechanisms in microgreen root systems. This is an important concept to understand as it relates to how a critical element of plant biology develops. Data collection will focus on root directional growth and overall spread. Data is to be collected through the utilization of RootNav or SmartRoot software. Although this stage focuses on gravitropism, there is an extension available that expands on gravitropic and phototropic responses as well. EpiCollect5 Data collection templates are available for extensions and AstroBotany data viz assistance coming soon.

Length of Stage: \~14 Days

Supplies:

  • 100 x 15 mm square dish (4 per observation group), if this isn’t possible the clear boxes full of brownies at the supermarket can also work well.
  • Plant grow Media (Use one of these two options and note in data sheet)
  • Option 1: the Easy Filter paper and Water
  • Option 2: the Simple Agar Media
  • Selected Microgreen seeds (Mizuna is a suggested choice for first time observations but other options can be found online such as on this AstroBotany website
  • Computer with RootNav/SmartRoot installed
  • 70-100% Ethanol
  • Microcentrifuge Tubes (or small plastic containers/cups) if you don't have a centrifuge you either convert a salad spinner or even make one from string and cardboard.
  • Filter paper
  • Micropore Medical Tape (or electrical tape on 4 sides)

Protocols for root analysis ¶

Growth Media Preparation Protocol

Option 1: the Easy Filter paper and Water

  • Just add water to blue filter paper (link to product)

Option 2: the Simple Agar Media

  • Measure 100ml of distilled water and 2g of agar/phytogel/phytagel
  • Bring the water to a boil and add agar powder while stirring
  • Remove heat after 5 minutes.
  • Autoclave the bottles for 30 minutes. (if autoclave isn’t available then pressure cookers work well)
  • Let rest/cool for another 15 minutes.
  • Pour out onto dishes (in the lab we use 50ml falcon tubes to measure 30ml of media per square petri)
  • Let plates set for 20 minutes (Tap side of the plate to make sure it’s set).
  • Put lids back on plates and store in a cold room (4c refrigerator).

This seed sterilization technique is good for quickly sterilizing seed batches before putting them in a petri dish to grow. Keep in mind that although sterilization is best practice, the quality of the actual seeds play a big role in susceptibility to contamination.

Simple Sterilization Protocol¶

Purpose: to reduce contamination during experiments

Seed Ethanol Method:

Materials needed: 70% ethanol, microcentrifuge tubes, pipettes, filter paper

  • Carefully decant seeds into “microcentrifuge” or other small tubes.
  • Submerge seeds in 70-100% ethanol solution..
  • Let the seeds sit in ethanol for about 1-2 minutes, ideally shake or “buz” on a lab vortex.
  • Let the seeds settle to the bottom of the tube and either use the pipette to remove as much ethanol as you can, but not the seeds or pour out the ethanol.
  • If contamination is a persistent issue repeat this ethanol rinse.
  • Remove the seeds by sucking up the ethanol and seed solution.
  • Dispense the seeds and ethanol onto filter paper so they are ready to sow.

Simple Ethanol Rinse Sterilization Protocol for Petri Dishes:¶

  • Fill petri dish with ethanol
  • Close the lid shack it about, pour away the alcohol; let ethanol evaporate off (ideally in a flow hood).
  • Keep closed until it is time to add media

Image analysis Preparations to be conducted prior to starting

  • For MaxOS Download Fiji & Smartroot. Install Fiji and then the SmartRoot addon.
  • For Windows OS Download and install RootNav on designated devices

(Requires a computer with a window operating system.

See Getting Started on RootNav/SmartRoot section for details)

  • Select microgreen seeds to be utilized
  • Decide which growth media to be utilized (simple v. complex)
  • Decide what experimental variable of interest is going to be?
  • Such as genotype or light color
  • Pour a moderate amount of agar in selected plates. (If possible, keep the temperature of media under 40°C to minimize condensation)
  • Place lids on plates upside down and let sit for an 30min

Microgreen Seeding Protocol¶

After the media has settled, turn the lids over and close the plates.

  • Wipe condensation off with a sterile paper towel if needed
  • Sterilize tweezers with ethanol
  • Place six seeds in a straight line horizontally across each plate’s midway line. Make sure seeds are evenly spaced. (If plates have gridlines, utilize them for seed placement.
  • Gently press seed into the agar making sure the seed is embedded.

(Do not crush seeds. Use minimal pressure)

  • Cover plates with lids and seal with micropore medical tape.
  • Stand plates against a wall vertically
  • Place light source above plates (1-2 feet).
  • Record light intensity using the Google Science Journal application
  • Record day and time of seeding plates

Observational and Data Collection Protocol

  • Everyday record notes regarding seedling development in observation log:
  • How many seedlings have germinated?
  • Root growth patterns
  • Stem/leaf color and health
  • When the roots have emerged, root length
  • The emergence of cotyledons (first leaves)
  • Note any unusual root/stem growth patterns such as directional waving, skewing, curling or looping. Germination speed and totally rates of germination success can vary between specimens or treatments.
  • Epicollect5 questions coming soon

Photograph Analysis Protocol

  • Photograph all control and experimental plates at the same time everyday
  • Make sure that each photograph is saved with proper metadata which includes:
  • Plant Type
  • Control or Experimental Group
  • Plate Number
  • Data and Time of Photograph
  • Photographs are to be taken from the same distance and in the same resolution. (Preferably 300dpi)
  • Once completed, follow the included directions to upload and analyze root architectural patterns using RootNav or Smart Root.

Software to assist with Root Analysis ¶

Goal: To create a folder of RSML files and a spreadsheet of root measurements

ImageJ / Smart Root analysis is the best software combination for MacOS and time series data. (Could also try RIA -> https://github.com/guillaumelobet/RIA)

  • Install Fiji
  • Install SmartRoot
  • Open Fiji
  • Open SmartRoot

  • Open images
  • Make use you have a light background (Sometimes the image colors need to be inverted).
  • Follow the SmartRoot instruction to measure the primary root.
  • Then measure the lateral roots.
  • The software automatically save the RSML files as you measure. At the end of your analysis you can export as .txt file / spreadsheet.

  • In addition, a range of characteristics can quickly be plotted within Fiji

Getting Started on RootNav¶

Goal: To create a folder of RSML files and a spreadsheet of root measurements

Note: Best for Window’s OS and a single time point

  • RootNav is a software used by astrobotany researchers to analyze root length and curvature for gravitropic assays (experiments that simulate components of microgravity). This invaluable tool provides a way to quantify data taken from these experiments.
  • The RootNavViewer Portal allows researchers to explore databases of root system architecture (Software still under development here). *
<figure><img src="https://lh7-us.googleusercontent.com/9OrwOTpwm7Cx-R4-yj80V8TP1QLNrNEWSg8zHYmMb8OxsPUbh2TckYPa86maRvw96ZfdPAsP61NqLAAF27MevyXcLn4iycmn8RAhWgdMHuwXZ-6-_LGfMpwhhiVY5J8-LFHZdCj6Xivr-I-yguVCTw" alt="" width="563"><figcaption></figcaption></figure>
  • RootNav2 is a new software that requires a very specific type of image acquisition. New protocol coming soon...

Installation:

  • Go to GitHub using this link:

https://github.com/dr-richard-barker/image-analysis-software-and-R-codes

  • Click the green button to “clone or download”. Click download ZIP.
  • Save the ZIP folder to a location of your choosing on your computer (ie. desktop).
  • Once the ZIP file is downloaded, click on the folder. Click onto the RootNav folder and click to extract all files.
  • After the extraction is complete, click on the RootNav application to open the software.
  • If you don’t have a window’s computer you can use one of these free cloud computer.
  • https://www.onworks.net/software/app-rootnav

🖥️ Run RootNav online (OnWorks)

AstroDart data visualization¶

  • Here’s an example of the ArchDart R-shiny package, this software allows you to vizulize modelled root system roots quickly and easily.
  • RSML data can be imported into this application and view easily but does require some experience with R-studio.
  • First down Anaconda and the latest version of R (or you can run it in the CyVerse cloud).
  • Then run the following lines of R code.
  • install_github("archiDART/archiDART", force = TRUE)
  • install.packages("shiny")
  • library(archiDART)
  • library(shiny)
  • shiny::runGitHub("archidart/astrodart", "astrodart")

AstroDart Shiny is designed to make plots of the data from the RSML files.

Data Analysis Protocol¶

Plant Root System Architecture data

RootSystem analysis, _“turning pictures into numbers then into graphs”_

Goal: Turn photos of roots into numbers for analysis

  • Images of the plant's root system growing on vertical petri dishes can be analyzed by the student researchers using RootNav in the class room (More detailed protocol below).
  • These data can be exported and analyzed using the methods above.

Student Data Entry

Goal: Observe a real experimental data matrix and then make your own

  • Students are given a example spreadsheet
  • Students can use these demo data to practice using these analysis tools.
  • Eventually the students will enter their own data into their classes shared spreadsheet and then analyse it using these applications.

Numbers entered into a google spreadsheet, attached link.

Goal: Student makes graphs using R-shiny applications.

Statistics

Goal: Introduction to statistics for teachers and students T-test

Summary and Application Discussion¶

Follow Up Inquiry:

  • Based on the observations made during this study, what conclusions can be made about the conditions that determine the direction of root growth in plants?
  • What direction do roots grow in? Why?
  • How would this change in an environment absent of, or that has reduced gravity?

Expansion:

  • Based on these observations, what directions do plants grow towards? (stems, leaves)
  • How can this idea be tested?

A new Machine learning approach known as Primal is worth further investigation

PRIMAL: Pipeline of Root Image analysis using MAchine Learning¶

DOIdx.doi.org/10.17504/protocols.io.h7bb9in

PRIMAL: Pipeline of Root Image analysis using MAchine Learning

🧪 PRIMAL: root image-analysis pipeline (protocols.io)