Scanner-based Time-lapse Root Phenotyping

Materials and Reagents

1. Seeds of the plants to be investigated
   Note: We used seeds of Brassica rapa L.
   
2. Nutrient solution (see Recipes)
   Note: Composition will depend on experimental requirements.
   

Equipment

1. Large (30 x 42 cm) and small (12 x 12 cm) steel blue Anchor seed germination paper (Anchor Paper Company, catalog number: SGB1924B) for phenotyping platform and seed germination, respectively
   
2. Duct tape (office DEPOT)
   
3. Ferrite magnetic strip with adhesive back (0.75 mm thick) (RS Components, catalog number: 297-9116)
   
4. Square bioassay or petri dishes (120 mm x 120 mm x 17 mm) (Sigma-Aldrich, catalog number: Z617679)
   Note: This could be improvised with empty compact disc (CD) cases or any kind of transparent box that meets the specified dimensions.
   
5. Black Perspex plates (21.5 x 30.0 cm) custom-made from opaque cast acrylic boards (Perspex Cast 9T30 Black Sheet) (plastock)
   
6. Computer
   Note: This should be Windows-based and should have a directory with sufficient space to save images. In our platform, a Canon flatbed scanner imaging a root system at 300 dpi produces a single image of 40.2 MB. With 24 scanners in operation and images taken every 12 h, a minimum free space of approximately 30.0 GB is required for an experiment lasting 14 days. We used a Dell computer Intel^® Core^TM i3-4150 Processor- Dual Core, 4 GB 1,600 MHz Memory and 500 GB Hard Drive for our experiments but images were saved on 2 TB WD external hard drive.
   
7. Canon flatbed scanners (CanoScan, model: 5600F)
   Note: Any scanner with a TWAIN driver can be used. A TWAIN driver is software that comes as part of the software package of digital imaging devices such as scanners and cameras. The TWAIN driver provides an interface for communication between the software and the hardware of digital imaging devices (http://www.twain.org/). In our platform, the TWAIN driver translated commands from our ArchiScan image acquisition software (http://www.archiroot.org.uk/) into instructions to control the hardware of the scanners.
   
   1. Setting up scanners
      
      1. Remove the cover of the scanner as shown in Figure 1.
         
      2. Attach magnetic strips to the short-edges of the scanner window (Figure 1B).
         Note: This is optional as duct tape could be used to hold plates to scanners.
         
         
         Figure 1. A. Original flatbed scanner. B. Modified flatbed scanner with cover detached.
   
   
8. Support for germination paper
   
   1. Glue to the edges of the Perspex plates strips of Perspex (0.3 x 0.3 x 30 cm and 0.3 x 0.3 x 21.5 cm for the long-edges and short-edges of the Perspex plate, respectively).
      Note: This is to provide some separation between the plate and the scanners.
      
   2. Allow two gaps in the Perspex strips, each approximately 3 cm wide, along the long edge of the Perspex plates to allow gas exchange with the surrounding atmosphere and unimpeded shoot growth (Figure 2).
      
   3.  Fix magnetic strips along the two short-edges of the Perspex plate to aid abutting of the Perspex plate to the scanner window (optional).
      
      
      Figure 2. (A) Schematic representation of the Perspex plate used as a scanner cover and also to attach germination paper onto scanners. (B) Black Perspex plates used as scanner covers and also to hold germination paper vertically on scanner surfaces

Software

1. ImageJ (http://imagej.nih.gov/ij/)
   
2. ArchiScan and ImageJ custom macros for convex hull and particle analyses (Adu et al., 2014; http://www.archiroot.org.uk/ )
   
3. SmartRoot (Lobet et al., 2011; http://www.uclouvain.be/en-smartroot)
   

Procedure