Germinator

Here we present the Germinator package: a simple, highly cost efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics.

The Germinator package contains three modules;

design of experimental setup with various options to replicate and randomize samples;
automatic scoring of germination based on the color contrast between the protruding radicle and seed coat;
curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters.

The curve fitting module enables analysis of general cumulative germination data and can be used for all plant species. We have shown that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well.

More information:

Joosen, R.V.L.; Kodde, J.; Willems, L.; Ligterink, W.; Plas, L.H.W. van der; Hilhorst, H.W.M. (2010) Germinator: A software package for high-throughput scoring and curve fitting of Arabidopsis seed germination The Plant Journal, (Online First, doi: 10.1111/j.1365-313X.2009.04116.x)

Click here to download a free copy of the Germinator Package

Updated curve fitter module! Version 1.29!
Added mean germination time (MGT) and t(x) calculations based on total amount of seeds or based on total germinated seeds and possibility to add t-test letters above bars in summary graphs.
Now with Office 2010 support.
Additional Germinator table module (version 2.01)
With functionality for different germination set-ups (eg 3, 20 or 24 germination tests per tray instead of 6). For these formats also specific photoshop actions have been added.

Germinator manual

Content
Curve fitting (standalone use of Germinator module III)
    Introduction
    Requirements
    Installation
    Video instruction

Automatic scoring of germination (Full Germinator package)
    Introduction
    Requirements
    Installation
    First steps
    Perform an experiment - Video instructions

Click here to download a free copy of the Germinator Package

Curve fitting (standalone use of Germinator module III)

Introduction

Using the visual basic module from the Microsoft Excel package we developed a script which performs automated curve fitting on cumulative germination data using the Solver add-in. The Solver is used in combination with the least sum of squares method to find the right parameters to fit the curves to the 4-parameter Hill function (El-Kassaby, et al. 2008):

y=y0+(axb/(cb+xb))

where:

y is the cumulative germination percentage at time x (hours),
y0 is the intercept on the y axis (≥ 0),
a is the maximum cumulative germination percentage (≤ 100)
b is controlling the shape and steepness of the curve
c is the time required for 50% of viable seeds to germinate (t50)

The user can define a threshold for the minimum number of germinated seeds, since curve fitting on very small amounts of germinated seeds won’t be very informative. In these situations the script returns a “False” and the data is not used in the statistical analysis. Uniformity (like U7525) of germination is the time interval between 75% and 25% of viable seeds to germinate. The Area Under the Curve (AUC) is the integration of the fitted curve between t = 0 and a user-defined endpoint, which results in a parameter that combines information on maximum germination, t50 and U7525. As described by (El-Kassaby, et al. 2008) the AUC can also be used to calculate a dormancy index (DI), by extracting the area under the curve after dormancy release by cold stratification, by the area under the curve without cold stratification. By the same analogy the AUC can be used to measure the effect of any stress treatment and calculate a stress index (SI). The Germinator script will summarize the results by calculating averages and standard errors for repeated samples, performing a student-t test, and provides a clearly formatted output including graphs for the different germination parameters.

Requirements

The whole Germinator package was tested on a standard desktop computer (Intel Core 2 Duo CPU, 2.33 GHz, 3.48 GB RAM) with Microsort XP, servicepack 2 and Microsoft Office 2003, service pack 3 installed.

Installation

Video instruction

This video contains detailed instructions for the Curve fitting module

Click here to download a free copy of the Germinator Package

Automatic scoring of germination (Full Germinator package)

Introduction

The Germinator package contains three modules;
1) design of experimental setup with various options to replicate and randomize samples;
2) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat;
3) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters.
We have shown that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well.

More information… (Refer to article)

Requirements

Hardware:

Computer:

Intel Core 2 Duo CPU
2.33 GHz
3.48 GB RAM

Camera:

Nikon D80
Nikkor AF-S 60 mm f/2.8 G Micro ED lens
Power adaptor
USB connector cable
Repro-stand

Germination trays:

15*21 cm, ref 109, DBP plastics, Belgium. www.dbp.be

Filter paper:

5.6”x8” Blue blotter paper, Anchorpaper company, St Paul; www.seedpaper.com

Incubator with light from the sides

Software:

Microsoft Windows XP professional, service pack 2
Microsoft Office 2003, service pack 3
ImageJ
Adobe Photoshop CS3
Nikon control pro version 2.0

Germinator module Installation

Microsoft Excel:

Open Microsoft Excel and check check if add in SOLVER is selected:

|tools -| add in -| SOLVER ADD IN)

and whether the reference SOLVER in visual basic is selected:

Next select:

Adobe Photoshop:

Select the window Actions with |Alt F9|. On the right top of this window you can open a menu |, |load actions|. Browse to the Germinator package, select folder |photoshop action| and select |germinator.atn|.

ImageJ:

Use the ImageJ version supplied with the Germinator package!

Install ij140-jdk6-setup.exe and extract mri-plugins-base.zip and mri-all-plugins.zip to the installation directory.

From the germinator package copy the directory: ..\Germinator files\imageJ applications\germinator to C:\Program Files\ImageJ\_applications.

First steps

Camera setup:
Attach the camera in the repro-stand and adjust height to ~53 cm. Switch to manual focus. Connect with USB to computer. Setup tl-tubes ~150 cm left and right from the camera. Place an A3 sheet of white paper around the repro-stand to prevent any direct light on the subject. Place a mask that fits the germination trays underneath the camera to fix the imaging position (Photo 1).

Adjust camera settings:
Take a picture of germinating seeds (some already germinated, others not) with the use of Nikon camera control Pro software. Set resolution to maximum, file format to jgp, normal compression. Set the camera to full manual control. We use ISO400, F/18, 1/3 sec, manual focus, but this settings have to be defined depending on your specific light conditions. It is advised to be very punctilious in optimizing the camera settings. Once the optimal settings are found they can be saved to a file (|save settings|). A sample image, indicating the desired image quality (Photo 2), is also available in the Germinator package download.

Photo 2: Example of the image quality needed for automatic scoring

Select |YUV| and play with color settings until you find a setting that selects seed + radicle but no background (Y100-255, U0-130, V120-255) for the example image. Write these settings down.
Again, play with color settings until you find a setting that selects the seed only (Y100-255, U0-80, V120-255) for the example image. Make sure that there is no size difference between both settings for non-germinated seeds but that there is an obvious size difference for germinated seeds. Write the settings down. Select |threshold| and close the color threshold window. Via menu |process|,|binary||make binary| followed by |edit|, |invert| you obtain a black/white image with black seeds and white background (photo 3).

Photo 3: example of a color thresholded image

Two tables will appear, the summary table shows the number of seeds |count| and the results table shows the parameters for the individual seeds. The area is a measure for size. This area can be used in the Germinator script to prevent counting of clustered seeds or other artifacts like remnants of siliques. To use the correct area threshold for your type of seeds goto “C:\Program Files\ImageJ\_applications\germinator” and open the text file “particle analysis macro.txt”:

run("Set Measurements...", "area centroid perimeter circularity display redirect=None decimal=4");
run("Analyze Particles...", "size=40-175 circularity=0.1-1.00 show=Nothing display exclude include summarize");
close();

Take a close look at your results table and adjust the size to a save limit (approx. 0.5xsmallest seed and 2x the biggest seed). Adjust the size in the text file in the position indicated above in bold and save.

That’s it! You are now ready to start your experiments!

Perform an experiment - Video instruction

This video instruction contains information about the different steps required for automated scoring with the Germinator Package

Click here to download a free copy of the Germinator Package