Solid Pollen Germination Medium (pgm): (Zhengbio Yang laboratory, University of California, Riverside)

Solid Pollen Germination Medium (PGM):

(Zhengbio Yang laboratory, University of California, Riverside)
18% Sucrose

0.01% Boric acid

1mM CaCl₂

1mM Ca (NO₃)₂

1mM MgSo₄

0.5% Noble agar (Difco)

pH the medium to 7.0. pH is important to get good pollen tube growth. Dissolve sucrose first, then add other ingredients. Finally add agar, place medium in a hot water bath and let the contents boil until agar completely dissolves (3-4 minutes). There is no need to autoclave the medium. Pour medium onto petri dishes (Fisher Brand small mini dishes,35 X 10 mm, catlog number 08-757-11YZ) or slides (in this case add medium to the slides by Pasteur pipettes).. Store plates at 4 ⁰ C. I have used plates for up to 6 weeks without any problem.
For GABA analysis:
The same medium as above, but drop out Ca (NO₃)₂, adjust calcium chloride to final concentration of 2mM, and replace Noble agar with ultra pure agarose from Bio-Rad (Reference: Palanivelu et al (2003), Cell: 114:47-59).
Procedure:

1. 
   Pick Arabidopsis flowers that are just open (option routinely used in Yang lab: leave plants at room temperature for 2 hrs before picking the flowers, this will increase synchronization of pollen germination).
   
2. 
   Dust pollen grains on medium (2) and allow them to germinate for 3-5 hours or overnight, at room temperature or on the bench.
   
3. 
   Observe under dissecting scope. Procedure works well for all ecotypes including Landsberg erecta that normally germinates poorly in vitro.
   
4. 
   Length of the tubes can be measured directly using a micrometer fitted on the microscope or from captured images using freely available NIH image software.
   

Liquid medium for pollen growth in vitro: (reference: Hicks et al, 2004; Plant Phys. 134: 1227-1239)

1. Prepare liquid medium as per receipe above (of course, omit the agar and related steps).

2. Place 30ul of liquid pollen growth medium on a glass slide (we use slides from eriesciences, www.eriesci.com; catlog #:10-175A-Black)

3. Touch pollen-containing anthers on the liquid meniscus.

4. Rapidly flip the slide upside down (do not worry, liquid won't fall; do not do the inverting process slowly, instead just flip the slide!) and let the slide remain in a wet chamber (tip box with wet kimwipes) for 6hrs-overnight. The drop should be

small enough that surface tension keeps it hanging there and it doesn't fall off,

yet not too small.

5. To have the slide in the inverted position, have them stand in between two raised surfaces, so that there is airspace below the liquid medium.

6. When done growing, reinvert the slide, add staining solution (optional), and place a cover slip and observe under microscope.