2.1 Germination of spores

Yuji Hiwatashi

 

Introduction

A sporangium contains thousands of spores. A solid medium containing 4-10 mM Ca is used for spore germination.@@

 

Solution required

EBCD+10 mM Ca (germination medium)F1000 ml

H2O

900 ml

Stock B

10 ml

Stock C

10 ml

Stock D

10 ml

Alternative TES

1 ml

500mM Ammounim tartrate

10ml (= 5 mM)

CaCl2 2H2O

1.5g ( = 10 mM)

Agar (Sigma A6924)

8 g ( = 0.8%)

 

Fill up to 1000 ml with H2O

After autoclaved, pour into 9 cm-Petri dish.@

 

E Sterilized water

E Cellophane (autoclave) – Wash as the follows

1) Cut cellophanes to a little bit smaller than the size of a 9 cm-dish.

2) Place cellophane in a glass Petri dish and then add 5 mM EDTA solution (pH8.0). Autoclave.

3) Wash with MilliQ water several times.

4) Add MilliQ water in the glass plate, then autoclave

E Two tweezers (autoclave)

E Yellow tips and blue tips for pipetman (autoclave)

E 10% Sodium Hypocholorite Solution (Antiformin): Do not autoclave.

 

See 2.2 for the Stock solutions.

 

Procedure

@You should perform the following steps in a cleanbench.

One sporangium contains approximately thousands of spores. It may be better to see the sporangium by microscope or magnifying glass to confirm it is not empty.

 

1. Overlay agar medium with a sheet of cellophane. Do not include air between media and cellophane.

2. Put one or two sporangium in a 1.5ml microtube, then pour 1 ml of 10% Antiformin to sterilize.@

3. Mix gently for 5 min and discard supernatant using a pipetman. Usually the sporangium sink at the bottom of tube, but be careful not to discard the sporangium until step 7. Do not touch the sporangium with a tip of a pipettman.@ Otherwise, tiny spores are dispersed in a tube and it is not possible to recover.

4. Add 1ml of sterilized water into the tube and mix gently for few minutes.

5. Discard supernatant with a pipetman.

6. Repeat step 4 and 5 four times.

7. Add 1ml of sterilized water, crush the sporangium with the tip of a pipetman (yellow-tip), and mix gently. When you crush the sporangium, you can see spores disperse in the tube.

8. Pour 0.2ml of the spore-solution at the center of the agar medium, which had been overlayed with a cellophane.

9. Add 1 ml of sterilized water to the drop of the spore-solution on the agar medium. Spread the spore solution over the agar medium by gently shaking the plate.

10. Incubate at 25˚C under continuous light.

11. Spores will germinate in a couple of days and protonemata start to grow. After about 15 days, gametophores start to grow.