Materials and Reagents

1. Rice (Oryza sativa L.) grain seeds
   
2. Mannitol (Sigma-Aldrich)
   
3. Morpholinoethane sulfonic acid (MES) (Sigma-Aldrich)
   
4. Cellulose R-10 (Yakult Honsha)
   
5. Macerozyme R-10 (Yakult Honsha)
   
6. Bovine serum albumin (BSA) (Sigma-Aldrich)
   
7. Carbenicillin/Ampicillin (AMRESCO)
   
8. β-Mercaptoethanol (β-ME) (Amresco)
   
9. Polyethtlene glycol (PEG) 4000 (Sigma-Aldrich)
   
10. Enzyme solution (see Recipes)
    
11. PEG4000 solution (see Recipes)
    
12. W5 solution (see Recipes)
    
13. MMG solution (see Recipes)
    

Equipment

1. Shaker P270 (Chinese Academy of Sciences, Wuhan Scientific Instrument Plant)
   
2. Collagen-coated 35-mm-diameter glass-base dish
   
3. Vortex XW-80A (JiaPeng Techno)
   
4. Nylon mesh (35 μm) (EMD Millipore)
   
5. Vacuum pump
   
6. 50 ml with round bottom centrifuge tube
   
7. 2 ml centrifuge tube
   
8. Tabletop centrifuges (Eppendorf 5810R and 5417R )
   
9. Collagen-coated 35-mm-diameter glass-base dish (Asahi Techno Glass Corporation)
   
10. Confocal microscopy (Olympus, model: FV1000 )
    
11. OLYMPUS FV1000 system (Fluoview Ver.1.7b Viewer) (Olympus)
    

Procedure