Materials and Reagents

1. Materials and Reagents1.Conical tubes (50 ml) (BD Biosciences, Falcon^®, catalog number: 352070 )
   
2. Miracloth (Calbiochem^®, catalog number: 475855 )
   Note: Currently, it is “EMD Millipore Corporation, catalog number: 475855”.
   
3. Labeling Tape (Shamrock, catalog number: ST20 )
   
4. Paper towels
   
5. Arabidopsis lines and Hpa isolate. Ws-2 eds1-1 mutants [the accession previously reported as Ws-0 is in fact Ws-2 (Parker et al., 1996)] and Col-0 plants were used as susceptible and resistant accessions of Arabidopsis for Hpa isolate Emoy2, respectively.
   
6. Ethanol (70%)
   
7. Plastic tray (270 x 270 x 60 mm) with a transparent lid
   
8. Sterile water
   
9. Bright-Line^TM Hemacytometer (Sigma-Aldrich, catalog number: Z359629 )
   
10. Trypan blue (Sigma-Aldrich, catalog number: T6146 )
    
11. Phenol (Wako Pure Chemical Industries, Siyaku, catalog number: 160-12725 )
    
12. Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
    
13. Glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 07500616 )
    
14. Trypan blue solution (see Recipes)
    
15. Chloral hydrate solution (see Recipes)
    

Equipment

1. Scissors
   
2. Biological safety cabinet (Labconco, model: Purifier Delta Series Class II Type A2 Cabinet )
   
3. Airbrush Kit (Airtex, model: ASCF4 and KIDS105 )
   
4. Growth chamber (NKsystem) (Nippon Medical & Chemical Instruments Co., catalog number: LPH410S )
   
5. Weight (Sartorius, model: Quintix 224-1S )
   
6. Water bath ( NE1-8 ) (Thermo Fisher Scientific, catalog number: 11459499)
   
7. Labo shaker (BIO CRAFT, catalog number: BC730 )
   
8. Stereomicroscope (Leica Microsystems, catalog number: M165FC )
   

Procedure