1. Plant materials and growth conditions
Although this method and tool can be used with many plant and root types, we developed it on tomato (Solanum lycopersicum var. St Pierre) seedlings that were used for all the experiments.
Seeds were surface sterilized with 70% (v/v) ethanol for 1 min followed by immersion in 0.87% (v/v) bleach for 1 min. After several washes in sterile distilled water, the seeds were stratified five days at 4°C to promote seed germination and finally sown on half-strength MS medium (Sigma). Agar 1% (Sigma) was used as a gelling agent. Media is disposed in 24 cm x 24 cm plates (Thermo Scientific™ Nunc™ Square BioAssay Dishes 500 cm²). Seeds were grown in a photoperiodic chamber under an 16 h daylight period at 25°C for ten days before treatment. 19.2 +/- 2.4 plants were analyzed per plates.
Each seedling was subjected to different treatments (one treatment per plate). Autoclaved mineral water (Cristalline® Emma source) was used as solvent and negative control. The bacterial peptide flagelline 22 (flg22) was used at 1µM (Millet et al. 2010; Plancot et al. 2013). Two algal extracts produced by the CMI (Roullier), referred to as AlgEx1 and AlgEx2, were diluted before use at three total carbon concentrations (ppm) AlgEx1: 2000, 6000 and 8000; AlgEx2: 1000, 4000 and 8000.
2. Computing environment
RootIX is an Excel file with a batch of macros developed in Visual Basic for Excel which is the well-known spreadsheet developed by Microsoft. This commonly used software is part of the Microsoft Office 365 Suit available on https://products.office.com for Windows and MACOS. Notice that it may be free for university members. Furthermore, RootIX is compatible with all Excel version since the 2007 version until the Microsoft Office 365 suit.
A macro is an automated program that can be launch from a simple button present in an Excel worksheet. Here, RootIX will analyze and organize the data obtained with ImageJ.
ImageJ is a public image processing program developed at the National Institute of Health. It allows to analyze images with a large variety of tools. ImageJ can be downloaded for free from http://rsbweb.nih.gov/ij/ and is available for Windows, Mac OS and Linux. Although ImageJ’s potential is almost unlimited (Schindelin et al. 2015), here we only require the simplest tool of “Segmented Line” measurement (right click on the line symbol).
3. Images acquisition
There is no precise images properties requirement if their quality is good enough to enable precise measurement and allow to distinguish roots that cross each other.
Here, images of plant were acquired with a scanner (Epson Expression 11000XL). Since the scans were conducted on sealed plates, the focus was manually adjusted to 5.0 in order to reach the best focus. Scan images were saved in “.tif” with the following routine properties: 800 dpi, 48-bit color depth.
4. Root sequential measurements
Roots are measured with the ImageJ segmented line tool. To get the appropriate length values, make sure to adjust the scale.
It is required to conduct the measurements in a specific sequence (Fig1):
- Choose a plant
- Measure the primary root length (See the “special cases” section below, for monocotyledons or indiscernible primary root).
- From the primary root apex, go back up to the hypocotyl measuring each lateral root on the way.
*Triple the record of a measurement for tertiary roots.
*Quaternary roots or higher order of ramification are not taken into account and should be recorded as tertiary roots.
- Choose a second plant and follow the same sequence.
- When the measurements of the picture are done, save the measurement results in “.tsv” (preferentially) or “.csv” file format. It is required to give a name that contains the condition (e.g. “water”), the time point (e.g. “t01h”) and the replicate (e.g. “R1”). In the examples given above the file name would be: “water-t01h-R1.tsv”. See other examples in the “legend and examples” sheet of the RootIX file.
It is recommended to use at least five decimals on the ImageJ records (on ImageJ: Analyze – set measurements… – decimal places (0-9): 5).
5. Data analysis
a. How to launch the analysis:
Data analysis can be launched in 3 simple steps.
- Fill in the “Parameters” sheet required cells (pale green cells) with their appropriate information following the instructions in red cells:
- File path in “B1”to access the folder that contain the data files.
- A giving name of the experiment in “B2”.
- Primary and lateral roots discrimination size in “E2” (primary root (RI) will be considered as higher and lateral root as lower than this size). See the “special cases” section below for size lower than 1cm and lateral roots longer than RI.
- Data name information of each file to analyze (“conditions” row 7 and when it is suitable “replicate”, “starting” and “ending time point” respectively rows 8, 9 and 10). This batch of information is required to find and work with the source data files.
- Launch the macro 1: “2. Macro 1 - Collect data” that collects and gathers the data present in the indicated folder in a new Excel workbook named with the “experiment name”.
- Launch the Macro 2: “3. Macro 2 -Analyse data” that processes the data.
b. Calibrate parameters:
Although RootIX is supplied already calibrated for tomato-seedling root analysis, it is obvious that all plant species have a different root system with different shapes and sizes. Thus, it is possible to personalize this calibration.
- It is important to adjust the primary and lateral roots discrimination size in cell “E2”. Since RootIX assumes that lateral roots are shorter than primary roots, this whole number is used to discriminate between primary and lateral roots. In case the studied plants present shorter primary roots see “special cases” section below.
- RootIX determines different classes of plants based on two parameters: the number of lateral roots per plant and the size of lateral roots. To get the most suitable ranking, values used to generate it are adjustable in the “Optional” panel:
- Cells C14 to C16 determine the classes of root sizes (smaller size, middle size and large sizes). While the classes are automatically defined based on the discrimination size, they can be modified manually.
- Cells C18 to C20 determine the classes of lateral-root number per plant.
c. Several types of calculation:
RootIX collects and analyzes data from the folder whose path is filled in the worksheet (cell B1). Thus, it is required to store the files to compare in a same folder to make a comparison analysis.
The macro extracts, deduces and calculates several types of information from the data files and it presents them in up to six tables (named Table 1, Table 2, …, Table 6) that are disposed in a new sheet called “Analyse”. The following data are grouped in Table 1 located in the “Analyse” sheet:
- Number of primary and lateral roots per condition
- Different classes of number of lateral roots per plant
- Different classes of lateral root length per plant
- Total primary root lengths per condition (cm)
- Total lateral root lengths per condition (cm)
- Total primary root + lateral root lengths per condition (cm)
- Average total primary root + lateral root lengths per plant (cm)
- Average primary root length
- Average lateral root length
- Number of plants with tertiary root
- Average number of tertiary roots per plant.
When samples were measured at two different time points, RootIX calculates the percentage of increase of different parameters between the two time points in Table 2 below the previous one. Five parameters are evaluated:
1st, it indicates the elongation rate of the whole root system (primary and lateral roots taken together).
2nd, it estimates the part of elongation attributed to the primary root.
3rd, the elongation rate of the primary root is indicated.
4th, the rate of additional lateral roots is calculated.
5th it calculates the total lateral-root length’s increase.
6th it indicates the changes of ramification if new tertiary roots were formed.
A graph presenting these data is generated below Table 2.
When several replicates of a same condition are analyzed, average and standard deviation of these replicates are calculated for each condition in Table 3 below the second one. In this case, the graph generated present these averaged data below Table 3.
All the calculations in the “Analyse” sheet are linked to the source cells so the user can easily find how the results are calculated.
Furthermore, RootIX also gathered in the table 4, the cumulated lengths of RI and RII of a same plant, in table 5 the RI of each plant and in table 6 the RII of each plant.
It also offers the possibility to shift these tables in horizontal tables that can be used for ANOVA statistical analyses (i.e.: horizontal disposition required for GraphPad Prism one-way ANOVA analyses).
d. Pool the data of multiple replicates
If the experiment was conducted several times or if there are several files/replicates for a same condition, it could be really useful to pool all the data of each same condition together (without affecting individual files) with the “Optional 4. Pool data” macro. It combines all the data of the different replicates in a new sheet for each condition and then reconducts the analyses on the pooled data in the sheet named “Analyse Pooled Data”.
e. Special cases:
- Monocotyledons or indiscernible primary root measurements
If the analyzed plant is a monocotyledon with a fibrous root system, primary root may not be easy to reach. In that case, the first measured root of the plant may be considered as a primary root and the others as lateral roots. The discrimination size may be set at 0 and each new plant (and so primary root) may be recorded with at least four consecutive records of the measurement (eg. 2.43566 cm record like this “1) 2.43566; 2) 2.43566; 3) 2.43566; 4) 2.43566” on the result file of ImageJ). It will trigger the manual determination of the root type.
- One time point measurements (no growth calculations)
It may occur to compare root shapes between different conditions but regardless to time or growth evolution. In that case it is only required to complete the “conditions” information (row 7 in the “Parameters” sheet) regardless to the starting and ending time points (rows 9 and 10) that can be empty.
- Lateral roots longer than primary roots
In certain conditions, lateral roots could be longer than primary ones. In this particular case it is required to quadruple the record of a measurement thus RootIX will consider the root as a secondary root.
- Most of the roots are ≤ 1 cm or units lengths need to be modified
When studied roots are very small, the discrimination size could be smaller than one centimeter however RootIX requires a whole number. Then, the simplest solution is to convert the data in millimeters. A button named “Convert length unit of all data”, reachable below the “optional” parameters, launches a macro that convert all the data in the wanted unit. Notice that it also allows to convert the unit of the data from and into all the main length units used in the West (international units (m, dm, cm, mm, µm), American units (yd, ft, In)). For example, it can convert inches to cm or cm into mm and vice versa.