Arabidopsis Pollen Tube Germination

Materials and Reagents

1. Plant growth
   1. Stratify surface-sterilized seeds on 1x Johnson’s medium at 4 ˚C for at least 3 days to synchronize the flowering time. Transfer the plates to growth chamber with sufficient illumination (e.g. 12-16 h light/day, growth chamber on the 2nd or 3rd floor is OK).
   2. 1 week seedlings are transferred to soil pots. I usually grow up to 8 seedlings per standard pot. Mutant and wildtype should be put side by side to avoid position effect in the growth chamber. 
   3. Water the plants twice a week (Tuesday and Friday, for example), and apply 0.5x Johnson’s medium once every other week. The plants may begin to flower 3-4 weeks after transferring. The fresh flowers on the first inflorescence will be good for pollen germination experiments (note: The time to pick flowers is very critical).
      
      
2. Preparation of germination plates
   1. Prepare germination medium plates as following (Fan et al., 2001)
      

      	Stock	Final Conc.	V stock/40 ml
      MES-Tris (pH 5.8 adjusted with Tris base)	200 mM	5 mM	1 ml
      KCl	1 M	1 mM	40 μl
      MgSO4	0.5 M	0.8 mM	64 μl
      Boric acid	100 mM	1.5 mM	600 μl
      CaCl2	0.5 M	10 mM	800 μl
      Sucrose		5% w/v	2 g
      PEG4000		15% w/v	6 g

      Notes:
      
      1. Mix well when add MgSO₄ and CaCl₂ to avoid precipitation.
      2. 5 mM Ca might be better than 10 mM sometimes.
      3. You can make pollen germination medium (PGM) without Ca, use it to prepare the pollen resuspension. The unwanted germination before time 0 can be avoided this way.
         

Equipment

1. Incubator with humidity control or saturated humidity
2. Nikon microscope
3. Scion Image (free download from NCBI website)
4. 8-well chamber (Lab-Tek International, catalog number: 155411 or VWR International, catalog number: 43300-774)
   

Procedure